Bacteria from Fish and Other Aquatic Animals - N. Buller (2004) WW

Bacteria from Fish and Other Aquatic Animals

A Practical Identification Manual

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Bacteria from Fish and OtherAquatic Animals

A Practical Identification Manual

Nicky B. Buller

Senior MicrobiologistDepartment of Agriculture

South PerthWestern Australia

CABI Publishing



CABI Publishing is a division of CAB International

CABI PublishingCAB InternationalWallingfordOxfordshire OX10 8DEUK

Tel: +44 (0)1491 832111Fax: +44 (0)1491 833508E-mail: [email protected] site: www.cabi-publishing.org

CABI Publishing875 Massachusetts Avenue

7th FloorCambridge, MA 02139

USA

Tel: +1 617 395 4056Fax: +1 617 354 6875

E-mail: [email protected]

©N.B. Buller 2004. All rights reserved. No part of this publication may be reproducedin any form or by any means, electronically, mechanically, by photocopying, recordingor otherwise, without the prior permission of the copyright owners.

A catalogue record for this book is available from the British Library, London, UK.

Library of Congress Cataloging-in-Publication DataBuller, Nicky B.

Bacteria from fish and other aquatic animals : a practicalidentification manual / Nicky B. Buller.

p. cm.Includes bibliographical references and index.

ISBN 0-85199-738-41. Aquatic animals--Microbiology. I. Title.

QR106.B85 2004579.3′176--dc21 2003009624

ISBN 0 85199 738 4

Typeset by AMA DataSet, UK.Printed and bound in the UK by Biddles Ltd, King’s Lynn.



Администратор

librus

Contents

List of Tables and Figures vii

Foreword: J.A. Plumb ix

Acknowledgements x

Introduction xii

1 Aquatic Animal Species and Organism Relationship 11.1 Host Species, Bacteria and Disease 11.2 Bacterial Diseases 11.3 Bacteria and Relationship to Host 371.4 Taxonomy and Disease Status of Bacteria 75

2 Bacteriological Culture Techniques: Microscopy, Culture and Identification 832.1 Specimen Collection and Submission 842.2 Culture and Incubation 852.3 Examination of Culture Plates 852.4 Biochemical Identification Tests 1142.5 Inoculation of Biochemical Identification Sets 1152.6 API Identification Systems 116

3 Interpretation of Biochemical Identification Tests and Sets 1173.1 Conventional Media: ‘Biochem Set’ 1173.2 Identification Tests and their Interpretation 1173.3 Using the Biochemical Identification Tables 1233.4 Interpretation and Identification of Genera and Species 1233.5 Antisera Available 136

4 Biochemical Identification Tables 1374.1 Results for Conventional Biochemical Tests – ‘Biochem Set’ 1374.2 Results for API Kits 138

5 Technical Methods 2225.1 Total Bacterial Count (TBC) 222

v



5.2 Microscopy 2245.3 Storage of Isolates 224

6 Techniques for the Molecular Identification of Bacteria 2256.1 Molecular Identification by PCR Using Specific Primers 2256.2 PCR Protocols 2346.3 Molecular Identification by 16S rDNA Sequencing 2366.4 Fluorescence in Situ Hybridization (FISH) 242

7 Preparation of Media for Culture and Identification 2447.1 General Isolation and Selective Media 2447.2 Biochemical Test Media 261

Further Reading and Other Information Sources 278

Appendix: Common Name and Scientific Name of Aquatic Animals 281

Glossary of Terms 287

References 292

Index 329

vi Contents



List of Tables and Figures

Tables

Table 1.1. Host species and organism relationship.Table 1.2. Bacterial pathogens and saprophytes of fish and other aquatic animals.

Table 2.1. Outline of steps for culture and identification.Table 2.2. General culture.Table 2.3. Specific culture requirements of organisms.Table 2.4. Microscopic and cultural characteristics.

Table 3.1. Interpretation of tests for ‘biochem set’.Table 3.2. Carnobacterium, Lactobacillus, Vagococcus and Renibacterium differentiation.Table 3.3. Differential tests for some non-fastidious fish pathogenic Gram-positive cocci and rods.Table 3.4. Differential characteristics of the genera within the family Flavobacteriaceae.Table 3.5. Further differential characteristics for the genera in the family Flavobacteriaceae.Table 3.6. Additional tests for differentiation of biotypes of P. damselae.Table 3.7. Differentiation of V. alginolyticus and V. harveyi.

Table 4.1. Aeromonas salmonicida (non-motile Aeromonas species).Table 4.2. Aeromonas spp. Phenotypic tests according to DNA hydridization groups.Table 4.3. Aeromonas spp. – motile.Table 4.4. Anaerobes.Table 4.5. Brucella spp.Table 4.6. Cryptococcus.Table 4.7. Cytophaga–Flavobacteriaceae–Bacteroides group – pathogenic.Table 4.8. Cytophaga–Flavobacteriaceae–Bacteroides group – environmental.Table 4.9. Gram-negative coccobacilli and cocci.Table 4.10. Gram-negative, oxidase-negative rods.Table 4.11. Gram-negative, oxidase-positive rods.Table 4.12. Helicobacter spp.Table 4.13. Gram-positive cocci.Table 4.14. Gram-positive rods.Table 4.15. Mycobacterium and Nocardia spp.Table 4.16. Mycobacterium spp. – additional tests.Table 4.17. Mycoplasma spp.Table 4.18. Listonella spp.

vii



Table 4.19. Moritella spp.Table 4.20. Photobacterium spp.Table 4.21. Vibrio – pathogenic species.Table 4.22. Vibrio – environmental species.Table 4.23. API 20E database biochemical results.Table 4.24. API 20E database numbers (organisms listed alphabetically).Table 4.25. API 20E database numbers (numbers in ascending order).Table 4.26. API 20NE database biochemical results.Table 4.27. API 50CH database biochemical results.Table 4.28. API Coryne database biochemical results.Table 4.29. API 20 Strep database biochemical results.Table 4.30. API Rapid ID32 Strep database biochemical results.Table 4.31. API Zym database results.Table 4.32. Strains quoted in references.

Table 5.1. Total bacterial counts at sites in oyster hatcheries: indication of healthy and diseasedstates.

Table 6.1. List of specific primers available for PCR detection of aquatic organisms.Table 6.2. Standard PCR protocol.Table 6.3. Universal eubacterial primers for 16S rDNA sequencing.Table 6.4. Suggested primer pairs.Table 6.5. Universal primers for amplification of 16S rDNA from Vibrio spp.Table 6.6. Sequencing primers for the 16S rDNA from Vibrio spp.Table 6.7. Sequencing PCR master mix.

Figures

Figure 4.1. Schematic for using the biochemical identification tables.

Photographic section after p. xiv

viii List of Tables and Figures



Foreword

While aquatic animal diseases have been a concern for centuries, a profusion of marine and fresh-water aquaculture and environmental concerns across the globe in the last 75 years has promptedincreased interest in these diseases, particularly those caused by bacteria. As aquaculture continues tobecome more intensive and expands into new frontiers, fish health problems are likely to becomemore significant. No group of marine or freshwater animals, including mammals, fish, birds, molluscs,shellfish, reptiles and amphibians, have escaped the effects of bacterial diseases. Hundreds of bacterialspecies can be either pathogenic to wild and cultured aquatic animals or pose a potential disease threatunder favourable conditions. Furthermore, the costs incurred by governments, private aquacultureand the public, due to bacterial-related diseases and attempts to control them, totals millions of dollarsannually as a result of lost aquatic resources. In order to successfully cope with these disease-producingmicrobes in a cost-effective manner, prompt and accurate identification is essential.

Until now there has been no single source available for use in identifying bacterial microbes from somany diverse marine and freshwater animals. However, Bacteria from Fish and Other Aquatic Animals:a Practical Identification Manual now provides just such a source with global application. This practical,user-friendly identification manual will be of great value to inexperienced and experienced bacteriolo-gists, microbiology teachers and/or students, aquatic animal health researchers or diagnosticians, as wellas to workers in public health facilities or medical laboratories who work with marine and freshwater fish,birds, mammals, molluscs, shellfish, reptiles or amphibians. Aided by numerous tables and colourfigures the author discusses conventional bacterial identification procedures, commercially availabledata-based identification kits, molecularly based PCR and 16S rDNA sequencing, thus providing utilityto a broad scientific sector. In this single volume one can find biochemical, biophysical and molecularcharacteristics of nearly 400 species of aquatic bacteria, media on which they are cultured and a briefdiscussion of many diseases with which they are associated. Assembling this manual was a monumentaltask and its author, Nicky B. Buller, is to be highly commended for providing this invaluable addition toaquatic microbiology.

John A. PlumbDepartment of Fisheries and Allied Aquacultures

Auburn UniversityAlabama, USA

ix



Acknowledgements

I would like to acknowledge Dr Jeremy Carson (Department of Primary Industries, Water and Environ-ment, Tasmania), and Nick Gudkovs (Australian Animal Health Laboratories, Geelong, Victoria)for allowing me to visit their respective laboratories, particularly in the early part of my career, fordiscussions on fish bacterial diseases and isolation techniques. In addition, to Dr Jeremy Carson andhis laboratory who, over the years, have confirmed the identification of some of our Vibrio andFlavobacterium strains isolated from diagnostic cases. In particular, for confirmation of the identityof Animal Health Laboratory Department of Agriculture (AHLDA) diagnostic strains mentioned inthis manual (Flavobacterium columnare, Listonella anguillarum, Vibrio agarivorans, Vibrio halioticoli,and Vibrio mediterranei). To Dr Annette Thomas (Department of Primary Industries, Queensland),thank you for the gift of cultures of Vibrio alginolyticus, Vibrio (carchariae) harveyi, and Streptococcusiniae and for our many discussions over the years on veterinary and fish pathogens. Thank you toDr Bruno Gomez-Gil (CIAD/Mazatlán Unit for Aquaculture, Mexico) for test results on V. rotiferianusnot listed in the literature, and for supplying the paper on Vibrio pacinii, before publication, forinclusion in this manual. To Dr Fabiano Thompson (Laboratory for Microbiology, Ghent University,Belgium) for providing further API 20E reactions for Vibrio brasiliensis, Vibrio neptunius, Vibrio xuiinot listed in the journal article. To the fish pathologists who I have worked with, in particular Dr BrianJones and in memorandum Dr Jeremy Langdon, who have helped me to a better understanding offish diseases. I am also grateful to the Fisheries Research and Development Council for fundingto enable me to attend workshops on fish diseases held at the University of Tasmania in 1996 andattendance at the fish bacteriologists’ workshops held at the Australian Animal Health Laboratories,Geelong, in 2000 and 2001.

The photograph of Renibacterium salmoninarum was obtained from the Animal HealthLaboratory (AHL) archives. There was no documentation as to the source of this photograph andthus I am unable to acknowledge the origin. The rest of the photographs were taken by the authorand were of cultures obtained as diagnostic submissions at AHL, type strains, or from Dr A. Thomas. Thephotographs of Flavobacterium columnare adhering to gill tissue are courtesy of Dr Brian Jones.

A great many microbiologists and other scientists have been responsible over the years for develop-ing and refining the media and techniques used for isolation and identification of bacteria. The mediaquoted from the literature in this book are referenced to include the originator and refiners of thosemedia. My apologies if I am remiss in leaving anybody out.

Due to the expense of producing the photographic plates, assistance towards their costwas achieved through sponsorship from the following organizations. Their assistance is gratefullyacknowledged.

x



Principal sponsor:Agriculture, Fisheries and Forestry – Australia (AFFA), Aquatic Animal Health Subprogram, fundedthrough Fisheries Research and Development Corporation (FRDC).

Other sponsors:Department of Fisheries, Western AustraliaOxoid AustraliaAnimal Health Laboratories, Department of Agriculture, Western Australia.

The contribution by the Animal Health Laboratories, Department of Agriculture, Western Australia,is also gratefully acknowledged for the use of digital photographic equipment, culture media andbacterial cultures used for the photographic section.

I would also like to acknowledge Mr Tim Hardwick at CAB International, for his assistance inbringing this manuscript into publication.

Acknowledgements xi



Introduction

This manual attempts to provide a source that enables the identification of bacteria that may be foundin animals that inhabit the aquatic environment. The emphasis is on bacteria from farmed aquaticanimals. In the words of Louis Pasteur, ‘chance favours the prepared mind’; therefore, an informedmicrobiologist will have a better chance of identifying those bacteria.

Our knowledge about the isolation and identification of bacteria from aquatic animals and theaquatic environment is expanding at a rapid rate. New organisms, be they pathogens, environmental,normal flora or potential probiotics, are being described and reported each month. This has happeneddue to an increase in aquaculture research, an increase in intensive fish farming systems, an increase inthe international trade of live aquatic animals and products, and the emergence of new diseases. Moreand more laboratories are becoming involved in the isolation and identification of these bacteria ineither a diagnostic or research capacity. In this manual there is an emphasis on bacteria of interest to theaquaculture industry either as pathogens, normal flora or strains that may be used as probiotics. Somebacteria that have been isolated from diverse habitats are also included. This manual attempts to providethese laboratories with an up-to-date and standardized database of methods and biochemical identifica-tion tables that can be used to isolate and identify bacteria from aquatic sources. Molecular diagnostics isbecoming more routine in many laboratories and a section on molecular identification of bacteria usingthe PCR and 16S rDNA sequencing is also included in this manual.

Many laboratories receive samples not only from veterinary sources, but also samples from zooanimals such as penguins, seals, seabirds, and aquatic mammals both captive and wild. From otheraquatic sources, samples for analysis may come from fish, both wild and cultured, freshwater andmarine, aquarium fish, tropical fish, and cultured aquatic animals such as abalone, pearl oysters,seahorses, lobster, crayfish, yabbies, marron and prawns. All these hosts have their own microflora andpotential bacterial pathogens and are found in a diverse range of habitats from tropical to cool temperateclimates. This manual includes as many of those bacteria as possible that may be found during examina-tion of samples from this diverse range of host and habitat. Not only pathogens, but also environmentaland saprophytic organisms, are included to aid in the understanding of the microflora that may be foundin such samples. Many bacteria from the more extreme environments have also been included as, withthe increase in aquaculture throughout the world, and the increasing knowledge about the microflora ofsuch habitats, these organisms may find their way into some laboratories via the samples submitted.Therefore, those isolates that are capable of growing on the isolation media recommended for aquaticorganisms such as ZoBell’s or Marine agar 2216 (Difco) are included in this manual. In addition, I haveincluded some of the Antarctic organisms that have been suggested as a low-cost food source for somemarine finfish because they are rich in omega-3 polyunsaturated fatty acids significant in the diet (Nicolset al., 1996) and thus they may be cultured from samples that come into a laboratory.

xii



Medical laboratories are also required to identify an increasing number of bacteria from aquatichabitats that may be involved in clinical infections. This book may also assist in the identification of suchbacteria that are not normally listed in the commercial databases such as API (bioMérieux).

Results of phenotypic test results reported in the literature can be confusing. It is important toperform tests by the methods that have been used by the reporting literature. In this book, the majority ofbiochemical tests have been performed according to West and Colwell (1984) and Cowan and Steel(1970), and these methods are listed. Results are also included from the commercial identification kitsavailable, namely, API 20E, API 50CH and API-ZYM from bioMérieux. Results from these tests are listedin the appropriate tables. Phenotypic tests that may produce different results between biochemical tubemedia and commercial identification kits include citrate reaction, decarboxylases, indole, and somecarbohydrates. Where different strains have been used in the literature and different phenotypic resultsreported, the results of these organisms have been listed separately. This is an attempt to enablemicrobiologists to provide the best possible identification of an organism isolated from a diagnosticor research sample.

A clearly defined set of biochemical tests is used as much as possible in this manual and from this themajority of pathogens and non-pathogens encountered from aquatic sources can be identified to genuslevel and, and for the more commonly known bacteria, to species level. The aim of having a defined set isso that laboratories that make in-house media can prepare media that will enable as many bacteria aspossible to be cultured and identified in the routine laboratory without having to prepare an excessivenumber of test and growth media. Some of the problems with variations in biochemical reactionsreported in the literature are due to different methods being used. This manual provides a standardizedset of biochemical identification methods for aquatic organisms and the reactions reported here arebased on this defined set.

Bacteria that require specialized media and identification tests are included here to assistlaboratories in identification of these organisms. In the case of Brucella, Mycoplasma and Mycobacteria,these methods are intended only as a guide, as these organisms should always be sent to a laboratorythat specializes in their identification. Other media, such as alternative methods for the detection ofcarbohydrate fermentation by Flavobacterium species for example, are also included.

The terms fermentation and utilization are often confused in the literature, and in some cases ithas been difficult to assess by which method the test was done. It is important to distinguish betweenfermentation and utilization as they are separate methodologies and a bacterium may show a positivetest for fermentation of a carbohydrate, yet negative for utilization of that same carbohydrate whentested as a sole carbon growth source. Basically, fermentation refers to the fermentation or breakdownof a carbohydrate, commonly called a ‘sugar’. The breakdown products are detected by a pH change inthe medium indicated by a colour change in the pH indicator, usually phenol red. Utilization refers to atest where a carbon source is assessed as a sole growth source for that bacterium. There are no othernutrients in the medium and growth is observed macroscopically, seen as an increase in the turbidity ofthe test medium. There are no pH indicators in utilization tests. The exception to this is citrate utilizationusing the Simmons method.

Cryptococcus (a yeast) is also included in this manual, because although it is not a bacterium, it isa zoonotic hazard to fish pathologists, veterinary pathologists, microbiologists and other laboratorypersonnel who may be dealing with samples from aquatic mammals that are susceptible to thisorganism. It is therefore included so as to alert staff to the biological hazards of some samples. Otherzoonotic organisms include Brucella, Mycobacteria and Nocardia. Many of the bacteria from aquaticsources may cause infections in humans and these are listed in Table 1.1.

Layout of the Manual

This manual is divided into sections basically according to the steps taken to isolate and identify anunknown bacterium. Experienced microbiologists may find some sections basic, but these are includedfor the benefit of students and newly graduated microbiologists or for researchers who are unfamiliarwith the techniques of bacteriology, particularly those used in a diagnostic laboratory.

Introduction xiii



Thus the sections encompass the host and the microbe, isolation techniques, phenotypic(biochemical) identification techniques, molecular identification and a media section.

In the phenotypic identification section is a flow chart (see Fig. 4.1, p. 138) that directs themicrobiologist to the appropriate biochemical identification table required for the identification ofthe unknown organism. The biochemical identification tables are named according to genus such asAeromonas and Vibrio, or to Gram reaction and cell shape or oxidase reaction.

In the conventional identification tables, the organisms are listed in alphabetical order (with theexception of the Vibrio tables, 4.21 and 4.22) under headings of Pathogen or Environmental. ThePathogen and Environmental differences refer to the pathogenicity for fish and aquatic animals, andgenerally not to humans or terrestrial animals. The inclusion of biochemical reactions for saprophyticand other species is intended to assist with the identification and ensure that the correct identification ismade where species have similar results. In the Vibrio tables, the organisms are listed according to theirgroupings based on ODC, LDC and ADH reactions. The intention is to use these groupings as the start-ing point for identification, similar to a flow chart. Tables for the API databases have the organisms listedalphabetically.

Significance

Fish and other aquatic animals (farmed and wild) are prone to bacterial infections in the same way asland animals, especially when they are stressed. Disease may occur systemically or be confined toexternal surfaces such as the skin or gills. In many instances, the pathogenic bacteria are ubiquitousin the environment, or may form part of the normal internal bacterial flora of an aquatic animal.One study suggested that up to 28 different Vibrio species may be found in the hepatopancreas(104 CFU/g), intestine and stomach (106 CFU/g) of healthy shrimp. The Vibrio species identifiedincluded V. alginolyticus, V. parahaemolyticus, V. cholerae and P. damselae. In diseased states onlyone or two Vibrio species are found (Gomez-Gil et al., 1998). Therefore, many factors need to beconsidered in making a disease diagnosis, such as clinical signs and symptoms, pathology, amount ofgrowth of the cultured bacteria, the numbers of different species cultured, the tissue site from which itwas isolated, and sterility of the specimen collection (Lightner and Redman, 1998).

Bacterial microflora on the surface of fish are heterogeneous in their salt requirement for growth.This requirement for salt (halophilic) is usually retained after serial subculture. Likewise the gut of fishcan be composed almost exclusively of halophilic vibrios (Liston, 1957; Simidu and Hasuo, 1968).Therefore, when attempting culture for pathogens, their salt requirement needs to be taken into account.This also applies to the biochemical identification tests.

Bacterial diseases affecting aquatic animals are detailed in texts such as Austin and Austin (1999),and Woo and Bruno (1999) (see Chapter 8 ‘Further Reading and Other Information Sources’). In thismanual, the diseases and the bacterial cause are presented as a quick reference tabular format only.

xiv Introduction



Photographs of Culture and MicroscopicAppearance of Organisms

The microscopic and cultural appearance of 31 species of bacteria and some of the biochemical testresults are detailed here. Obviously not all bacteria can be presented, however, genera and specieshave been photographed on commonly used media to show their cultural appearance on thatmedium. Some species such as Vibrio mimicus and Vibrio cholerae are photographed to indicate howsimilar they can be by cultural appearance and how similar they may appear to motile Aeromonasspecies. Likewise the motile Aeromonas species all appear similar on blood agar media, whereas thenon-motile Aeromonas salmonicida has a distinctive colony appearance, slow-growing, with pigmentproduction after a few days incubation.

Reactions of some biochemical tests are included for those not familiar with these reactions.

15Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpTuesday, January 13, 2004 11:01:56 AM


Fig

. 1.A

eromonas hydrophila

on BA

, 24 h.

Fig

. 2.A

eromonas hydrophila on M

CA

, 24 h.

Fig

. 3.A

eromonas hydrophila on T

CB

S,

24 h.F

ig. 4.

Aerom

onas hydrophila,G

ram stain.

Fig

. 5.A

eromonas janadaeion B

A,

48 h.F

ig. 6.

Aerom

onas janadaeion MC

A,

3 days.F

ig. 7.

Aerom

onas janadaei,G

ramstain.

Fig

. 8.A

eromonas veroniissp. sobria

onB

A, 48 h, 25

oC.

Fig

. 9.A


onM

CA

, 24 h, 25oC

.F

ig. 10.A


on TC

BS

, 24 h, 25oC

.F

ig. 11.

Aerom

onas veronii ssp.sobria, G

ram stain.

Fig

. 12.Atypical A

eromonas salm

onicida(A

ustralian strain) on BA

, 3 days.F

ig. 13.A

typicalAerom

onas salmonicida

(Australian strain) on B

A, 7 days.

Fig

. 14.Atypical A

eromonas salm

onicida(A

ustralian strain) showing pigm

ent on NB

agar.

Fig

. 15.AtypicalA

eromonas

salmonicida

(Australian strain),

Gram

stain.

Fig

. 16.C

ryptococcus neoformans

var. gattiisubculture on BA

, 3 days.F

ig. 17.

Cryptococcus. O

riginal isolationplate (S

trep selective agar), 7 days.F

ig. 18.

Cryptococcus.

Gram

smear of tissue/culture, and w

ithin tissue/culture.

Fig

. 19.E

dwardsiella hoshinae

on BA

, 2days, 25

oC.

Fig

. 20.E

dwardsiella hoshinae

on MC

A,

24 h, 25oC

.F

ig. 21.

Edw

ardsiella hoshinae, G

ram stain.

Fig

. 22.E

dwardsiella tarda

on BA

, 24 h,25

oC.

Fig

. 23.E

dwardsiella tarda

on MC

A,

24h.

Fig

. 24.E

dwardsiella tarda, G

ram stain.

Fig

. 25.E

rysipelothrix rhusiopathiaeon B

A, 24 h, 25

oC.

Fig

. 26.E

rysipelothrix rhusiopathiae,G

ram stain.

Fig

. 27.F

lavobacterium johnsoniae

on BA

.F

ig. 28.

Flavobacterium

johnsoniaeon N

A, 48 h, 25

oC.

Fig

. 29.F

lavobacterium johnsoniae

on AO

, 48 h, 25oC

.

Fig

. 32.F

lavobacterium colum

naresubculture on A

O, 5 days.

Fig

. 33.F

lavobacterium colum

naresubculture on A

O, 5 days.

Fig

. 34.F

lavobacterium colum

narem

agnified colonies.F

igs 35 an

d 36.

Flavobacterium

columnare. H

E section. C

ellsattached end-on to skin tissue. G

ramsm

ear of cells from culture.

Fig

s 30 and

31.F

lavobacterium

johnsoniae.G

ram sm

ear of gill tissueand cells from

pure culture.

Fig

. 37.H

afnia alveion BA

, 48 h, 25oC

. F

ig. 38.

Hafnia alveion M

CA

, 48 h,25

oC.

Fig

. 39.H

afnia alvei, Gram

stain.

Fig

. 40.Listonella anguillarum

on BA

, 3days, 25

oC.

Fig


on M

SA

-B, 24 h, 25

oC.

Fig


on TC

BS

,48 h, 25

oC.

Fig


,G

ram stain.

Fig

. 44.M

ycobacterium m

arinumon

BA

, 5 days. F

ig. 45.

Mycobacterium

marinum

introut kidney, Z

N stain.

Fig

. 46.M

ycobacterium m

arinumin

trout kidney, ZN

stain. F

ig. 47.

Nocardia, G

ram stain.

Fig

. 48.P

hotobacterium dam

selaessp.

damselae

on MS

A-B

, 2 days, 25oC

.F

ig. 49.

Photobacterium

damselae

ssp. damselae

on BA

, 48 h.F

ig. 50.

Photobacterium

damselae

ssp. damselae

on TC

BS

, 2 days, 25oC

.F

ig. 51.

Photobacterium

damselae

ssp. damselae, G

ram stain.

Fig

. 52.P

hotobacterium dam

selaessp. piscicida, 13 days.

Fig

. 53.P

lesiomonas shigelloides

onB

A, 24 h, 25

oC.

Fig

. 54.P

lesiomonas shigelloides

onM

CA

, 24 h, 25oC

.F

ig. 55.

Plesiom

onas shigelloides,G

ram stain.

Fig

. 56.P

seudomonas fluorescens

onB

A, 24 h.

Fig

. 57.P

seudomonas fluorescens

onM

CA

, 2 days.F

ig. 58.

Pseudom

onas fluorescens,G

ram stain.

Fig

. 59.R

enibacterium salm

oninarum,

2–3 weeks.

Fig

. 60.S

treptococcus (difficile)agalactiae

Group B

on BA

, 3 days.F

ig. 61.

Streptococcus (difficile)

agalactiaeG

roup B, G

ram stain.

Fig

. 62.S

train of Streptococcus iniae

on BA

, 2 days with w

eak β-haemolysis.

Fig

. 63.S

train of Streptococcus iniae

on BA

, 2 days with stronger

β-haemolysis.

Fig

. 64.S

treptococcus iniae. Gram

smear.

Fig

. 65.V

ibrio agarivoranson M

SA

-B,

7 days, 25oC

.

Fig

. 66.V

ibrio alginolyticus on MS

A-B

,24 h. G

rowth has sw

armed across plate.

Fig

. 67.V

ibrio alginolyticus on BA

, 24 h. F

ig. 68.

Vibrio alginolyticus

on TC

BS

, 24 h.

Fig

. 69.V

ibrio alginolyticus,G

ram stain.

Fig

. 70.V

ibrio choleraenon-01 on B

A,

24 h. F

ig. 71.

Vibrio cholerae

non-01 on MC

A.

Fig

. 72.V

ibrio choleraenon-01 on

TC

BS

.F

ig. 73.

Vibrio cholerae, G

ramstain.

Fig

. 74.V

ibrio fluvialison B

A, 24 h,

25oC

.F

ig. 75.

Vibrio fluvialis

on MS

A-B

, 48 h,25

oC.

Fig

. 76.V

ibrio fluvialison M

CA

, 24 h,25

oC.

Fig

. 77.V

ibrio fluvialison T

CB

S, 24 h,

25oC

.

Fig

. 78.V

ibrio furnissiion BA

, 48 h. F

ig. 79.

Vibrio furnissiion M

CA

, 48 h.F

ig. 80.

Vibrio furnissiion T

CB

S, 48 h.

Fig

. 81.V

ibrio fluvialis, Gram

stain.

Fig

. 82.V

ibrio harveyion MS

A-B

, 48 h.F

ig. 83.

Vibrio harveyion B

A, 48 h.

Fig

. 84.V

ibrio harveyion TC

BS

, 48 h.F

ig. 85.

Vibrio harveyi, G

ramstain.

Fig

. 86.V

ibrio mim

icuson B

A, 24 h.

Fig

. 87.V

ibrio mim

icuson B

A, 48 h.

Fig

. 88.V

ibrio mim

icuson M

CA

.F

ig. 89.

Vibrio m

imicus

on TC

BS

.

Fig

. 90.V

ibrio mim

icus, Gram

stain.F

ig. 91.

Vibrio ordaliion M

SA

-B, 2 days.

Fig

. 92.V

ibrio ordaliion BA

, 3 days,25

oC. N

o growth on T

CB

S.

Fig

. 93.V

ibrio ordalii, Gram

stain.

Fig

. 94.V

ibrio parahaemolyticus

onM

SA

-B, 48 h. G

rowth has sw

armed

across plate.

Fig

. 95.V


on BA

,24 h, 25

oC.

Fig

. 96.V


onT

CB

S, 24 h.

Fig

. 97.V

ibrioparahaem

olyticus. Gram

smear.

Fig

. 98.V

ibrio proteolyticuson B

A, 24 h,

25oC

. Show

ing swarm

ing colonies.F

ig. 99.

Vibrio proteolyticus com

pletelycovers on M

SA

-B, 24

h.F

ig. 100.

Vibrio proteolyticus

on TC

BS

,24 h.

Fig

. 101.V

ibrio tubiashiion MS

A-B

, 2days, 25

oC.

Fig

. 102.V

ibrio tubiashiion TC

BS

, 3days, 25

oC.

Fig

. 103.V

ibrio tubiashii, Gram

stain.

Fig

. 104.V

ibrio vulnificuson M

SA

-B, 2

days, 25oC

. F

ig. 105.

Vibrio vulnificus

on TC

BS

, 2days, 25

oC.

Fig

. 106.V

ibrio vulnificus, Gram

stain.

Fig

. 107.Y

ersinia ruckerion BA

, 24 h,25

oC.

Fig

. 108.Y

ersinia ruckerion MC

A, 24 h,

25oC

.F

ig. 109.

Vibrio identification

discs. Top = 150 µg disc (sensi-

tive). Bottom

= 10 µg disc

(resistant).

Fig

. 110.M

otility positive at 25oC

(left), but negative at 37oC

(right)for Y

ersinia ruckeri.

Fig

. 114.D

ecarboxylase reactions. Left toright: arginine dihydrolase (positive); lysinedecarboxylase (positive); ornithine decar-boxylase (negative); and control tube(negative).

Fig

. 115.D

Nase reaction. N

egativereaction on left and positive reaction onright.

Fig

. 116.Indole reaction.

Negative on the left and posi-

tive on the right. Vibrio para-

haemolyticus

with 0.85%

NaC

l,and 2%

NaC

l, respectively.

Fig

. 117.P

late showing gelatin hydroly-

sis and growth on 3%

and 0% N

aCl.

Fig

. 118.M

ethyl Red reaction.

Positive.

Fig

. 111.C

arbohydrate fermen-

tation reaction. Sucrose-positive

(yellow) and sucrose-negative

(red), 24 h, 25oC

.

Fig

. 112.C

itrate test.Y

ersinia ruckeri, citrate-posi-tive (blue) at 24

oC, but cit-

rate-negative (green) at37

oC.

Fig

. 113.D

ecarboxylase reactions. Left toright: arginine dihydrolase (negative); lysinedecarboxylase (positive); ornithine decar-boxylase (positive); and control tube (nega-tive).

1Aquatic Animal Species and Organism Relationship

1.1 Host Species, Bacteria and Disease

This chapter deals with the relationship betweenthe host species and the bacterial flora thatmay be either part of the normal flora of thathost, or pathogenic for that host. This informationis presented in two formats. Table 1.1 liststhe aquatic animal hosts in alphabetical orderunder their common name. The scientific nameis in parentheses. Some hosts are groupedunder their Family name, which is in capitals.For example, trout and salmon are listedunder SALMONIDS; dolphin, porpoises, sealsand whales are listed under SEA MAMMALS;aquarium fish are listed under ORNAMENTALS.The adjacent columns in the table list the bacteriathat have been reported to be either pathogensof the host or that are considered part ofthe normal flora, the tissue site of infection,or the pathology presented and the diseasestate. Some organisms are considered to beopportunistic pathogens and in a healthy hostmay be part of the normal flora. In a stressedanimal, these same bacteria may overcomehost defence mechanisms and cause morbidityor infection in the animal. There are some organ-isms that have been identified and isolated from ahost, but the pathogenicity of the organism isunknown, as virulence studies were not carriedout.

In the second format, in Table 1.2, theinformation is presented by listing the bacteriain alphabetical order, with adjacent columnslisting the name of the disease, the tissuesite where the organism may be found, theaquatic animals where the organism has been

reported, and the geographical location of thedisease.

1.2 Bacterial Diseases

The following section provides more detail thanin the tables on some of the more commonlyrecognized fish bacterial diseases. The diseasesare described in a brief form, as there are othertexts available that provide more comprehensivedetail on diseases of fish. See recommendedtexts in Chapter 8, ‘Further Reading and OtherInformation Sources’.

Bacillary necrosis of Pangasius

This is a recently reported disease found infreshwater catfish (Pangasius hypophthalmusSauvage) in Thailand. The causative organismhas been identified as Edwardsiella ictaluri,which in catfish in America causes the diseaseknown as enteric septicaemia of catfish. Thedisease in Pangasius presents as multifocal, whitelesions, irregular in size and found in internalorgans, predominantly the kidney, liver andspleen. In histology the lesions appear as areasof necrosis and pyogranulomatous inflammation.The causative bacterium was identified as E.ictaluri biochemically; however, when examinedmicroscopically the bacterial cells showed agreater pleomorphism in length and size thannormally seen with other strains of E. ictaluri(Crumlish et al., 2002).

©N.B. Buller 2004. Bacteria from Fish and Other Aquatic Animals:a Practical Identification Manual (N.B. Buller) 1



2 Chapter 1

Abal

one

Pseu

doal

tero

mon

assp

p.,

Shew

anel

lasp

p.Vi

brio

agar

ivor

ans

(pat

hoge

nici

tyno

tdet

erm

ined

)Vi

brio

med

iterra

nei

Tiss

ueM

orta

lity,

lesi

ons

135

Hal

iotis

disc

usha

nnai

Vibr

ioha

liotic

oli

Gut

Nor

mal

mic

roflo

ra67

8

Red

abal

one

( Hal

iotis

rufe

scen

s)Vi

brio

algi

noly

ticus

Sick

larv

aear

eun

able

tosw

iman

dre

mai

nat

the

botto

mof

the

pond

Mor

talit

yin

larv

ae30

Hal

iotis

tube

rcul

ata

Vibr

io(c

arch

aria

e)ha

rvey

iW

hite

pust

ules

onfo

otM

orta

lity

576

Japa

nese

abal

one

( Sul

culu

sdi

vers

icol

orsu

prat

exta

)Vi

brio

harv

eyi(

stra

inw

asno

n-lu

min

esce

nt,O

DC

-neg

ativ

e,ur

ease

-neg

ativ

e)

Whi

tesp

ots

onfo

ot,t

issu

ele

sion

s,ne

crot

icde

gene

ratio

nin

mus

cle

fibre

sM

ass

mor

talit

y,lo

seab

ility

toad

here

581

Smal

laba

lone

( Hal

iotis

dive

rsic

olor

supe

rtext

a )Vi

brio

para

haem

olyt

icus

Org

anis

min

haem

olym

phW

ither

ing

synd

rom

e,m

ass

mor

talit

y49

9

Alga

Mar

ine

alga

( Ulv

ala

ctuc

a)Ps

eudo

alte

rom

onas

ulva

eH

asan

ti-fo

ulin

gpr

oper

ties

231

Re d

a lg a

( Del

esse

riasa

ngui

nea)

Zobe

lliaga

lact

anov

oran

s61

Allig

ator

Allig

ator

mis

siss

ippi

ensi

s1.

Edw

ards

iella

tard

a2.

Myc

opla

sma

allig

ator

is3.

Past

eure

llam

ulto

cida

4.St

aphy

loco

ccus

5.Ed

war

dsie

llata

rda,

Salm

onel

lam

iam

i ,S.

java

,S.h

artfo

rd1,

3.La

rge

inte

stin

e,cl

oaca

.Con

gest

ion

inki

dney

sw

ithne

crot

icfo

ci,p

erito

nitis

,ul

cers

inga

stric

muc

osa

2.Pu

lmon

ary

oede

ma,

inte

rstit

ial

pneu

mon

ia,p

eric

ardi

tis,m

yoca

rditi

s,m

enin

gitis

,syn

oviti

s3,

4.Lu

ngs

1,5.

Nep

hriti

s.Pa

thog

enic

ityno

tcon

clus

ive

2.Ac

ute

mul

tisys

tem

icin

flam

mat

ory

dise

ase

3,4.

Pneu

mon

ia

128

129

520

804

823

Ambe

rjack

.Jap

anes

eam

berja

ck.

See

Yello

wta

il

Anch

ovy

( Eng

raul

ism

orda

x)Te

naci

bacu

lum

mar

itim

umH

aem

orrh

agic

lesi

ons

onsn

out,

eye

and

mid

sect

ion

ofth

ebo

dyD

isea

se15

4

Arct

icCh

arr(

Salv

elin

usal

pinu

sLi

nnae

us).

See

unde

rSAL

MO

NID

S

Host

Path

ogen

Norm

alflo

raTi

ssue

site

Dise

ase

stat

usRe

f

Tab

le1.

1.H

osts

peci

esan

dor

gani

smre

latio

nshi

p.



Aquatic Animal Species and Organism Relationship 3

Arte

mia

spp.

See

unde

rshr

imp

–br

ine

shrim

p

Ayu

( Ple

cogl

ossu

sal

tivel

isTe

mm

inck

and

Schl

egel

)1.

Flav

obac

teriu

mps

ychr

ophi

lum

2.Li

ston

ella

angu

illaru

m01

and

02(E

urop

ean

desi

gnat

ion)

3.Ps

eudo

mon

asan

guilli

sept

ica

4.Ps

eudo

mon

aspl

ecog

loss

icid

a5.

Ren

ibac

teriu

msa

lmon

inar

um6.

Stre

ptoc

occu

sin

iae

7.Vi

brio

chol

era

non-

01(n

egat

ive

for

orni

thin

ede

carb

oxyl

ase)

4.H

aem

orrh

agic

asci

tes

5.W

hite

nodu

les

inki

dney

,abd

omen

swol

len

with

fluid

,exo

phth

alm

ia

1.C

old

wat

erdi

seas

e2.

Vibr

iosi

s3.

Dis

ease

4.M

orta

lity,

bact

eria

lha

emor

rhag

icas

cite

s(B

HA)

5.Ba

cter

ialk

idne

ydi

seas

e(B

KD)

6.M

orta

lity,

stre

ptoc

occo

sis

7.M

ass

mor

talit

y

434

442

561

564

568

582

712

722

757

803

Baitf

ish.

Amer

ican

baitf

ish

( Pim

epha

les

prom

elas

)Se

eM

inno

ws

Barr

amun

di( L

ates

calc

arife

rBl

och)

See

Bass

Bass

Euro

pean

seab

ass

( Dic

entra

rchu

sla

brax

Linn

aeus

)1.

Aero

mon

ashy

drop

hila

2.Ph

otob

acte

rium

dam

sela

ess

p.pi

scic

ida

3.Ps

eudo

mon

asan

guilli

sept

ica

4.M

ycob

acte

rium

mar

inum

and

Myc

obac

teriu

msp

p.5.

Stre

ptoc

occu

sin

iae

6.Te

naci

bacu

lum

mar

itim

um

1.En

larg

emen

tofs

plee

n,er

ythe

ma

and

swel

ling

ofan

us2.

No

obvi

ous

gros

spa

thol

ogy,

enla

rged

sple

enw

ithw

hite

nodu

les

3.O

rgan

ism

isol

ated

from

head

–kid

ney

and

sple

en4.

Deg

ener

atio

nin

eye,

exop

htha

lmia

,sk

inle

sion

s,ne

crot

icar

eas

ingi

lls4,

5.O

rgan

ism

sin

hear

tand

sple

en,

extre

me

sple

nom

egal

y6.

Pale

skin

zone

sw

ithye

llow

edge

s,le

adin

gto

necr

otic

lesi

ons

onbo

dyar

ound

fins,

oral

cavi

ty,e

yes

and

gills

1.M

orta

litie

s2.

Fish

past

eure

llosi

s3.

Hae

mor

rhag

icse

ptic

aem

ia4.

Myc

obac

terio

sis

5.Ex

udat

ive

men

ingi

tisan

dpa

noph

thal

miti

s6.

Der

mal

necr

osis

.Fin

gerli

ngs

very

susc

eptib

le.S

tress

-rela

ted

inol

derf

ish

60 91 96 140

183

209

227

Larg

emou

thba

ss( M

icro

pter

ussa

lmoi

des

Lace

pède

)Ed

war

dsie

llata

rda

Edw

ards

iella

icta

luri

Inte

stin

e,liv

er,s

plee

nIn

tern

alor

gans

pale

and

anae

mic

,blo

odw

ater

y,ha

emor

rhag

icno

dule

sin

stom

ach

wal

land

sero

saof

inte

stin

e

627

823

cont

inue

d



4 Chapter 1

Sea

bass

(Lat

esca

lcar

iferB

loch

)Al

sokn

own

asba

rram

undi

orba

rram

undi

perc

h

1.Fl

avob

acte

rium

john

soni

ae2.

Phot

obac

teriu

mda

mse

lae

ssp.

dam

sela

e3.

Phot

obac

teriu

mda

mse

lae

ssp.

pisc

icid

a4.

Stre

ptoc

occu

sin

iae

5.Vi

brio

harv

eyi

1.Su

perfi

cial

skin

eros

ion

onpo

ster

ior

flank

s,pe

ctor

alfin

san

doc

casi

onal

lyth

elo

wer

jaw

3.Ex

opht

halm

ia,h

aem

orrh

agic

skin

lesi

ons,

sept

icae

mia

4.O

rgan

ism

isol

ated

from

brai

n

1.D

isea

sein

juve

nile

s2,

3,4,

5.M

orta

litie

s12

713

514

573

4

Sea

bass

( Lat

eola

brax

japo

nicu

sC

uvie

r),Ja

pane

sese

aper

chN

ocar

dia

serio

lae

Whi

te-y

ello

wno

dule

sin

gill,

hear

t,ki

dney

,liv

er,s

plee

nN

ocar

dios

is15

5

Sea

bass

( Pun

tazz

opu

ntaz

zoC

uvie

r)Ae

rom

onas

hydr

ophi

laEn

larg

emen

tofs

plee

n,er

ythe

ma

and

swel

ling

ofan

usM

orta

litie

s22

7

Strip

edba

ss( M

oron

esa

xatil

isW

alba

um),

(Roc

cus

saxa

tilis

)1.

Car

noba

cter

ium

pisc

icol

a2.

Cor

yneb

acte

rium

aqua

ticum

3.M

ycob

acte

rium

mar

inum

and

Myc

obac

teriu

msp

p.4.

Phot

obac

teriu

mda

mse

lae

ssp.

pisc

icid

a5.

Serra

tiam

arce

scen

s6.

Stre

ptoc

occu

sin

iae

7.Vi

brio

chol

erae

non-

01an

dVi

brio

mim

icus

Did

notc

ause

deat

hsin

avi

rule

nce

assa

y;ho

wev

er,m

aybe

oppo

rtuni

stic

path

ogen

inst

ress

edfis

h

1.H

yper

aem

iaan

dha

emor

rhag

ein

liver

,ki

dney

,spl

een

and

brai

n2.

Org

anis

min

brai

ntis

sue,

exop

htha

lmia

3.N

odul

arle

sion

sin

allo

rgan

s4.

Org

anis

mis

olat

edfro

min

tern

alor

gans

,enl

arge

dki

dney

and

sple

en.

Abno

rmal

skin

pigm

enta

tion

5.N

ecro

sis

ofm

uscu

lart

issu

es

1.M

orta

lity

2.D

isea

se3.

Mor

talit

y4.

Fish

past

eure

llosi

s5.

Mor

talit

yin

finge

rling

s6.

Stre

ptoc

occo

sis

7.Pr

esen

tin

appa

rent

lyhe

alth

yw

ildfis

h.M

ayca

use

mor

talit

yin

stre

ssed

farm

edfis

h

73 75 76 235

333

337

339

474

507

708

Whi

tese

aba

ss( A

tract

osci

onno

bilis

A yre

s ).A

lso

k no w

na s

Whi

tew

eakf

ish.

Fam

ilySe

rrani

dae

Tena

ciba

culu

mm

ariti

mum

Lesi

ons

iney

e,op

ercu

lum

.Les

ions

onbo

dyra

ngin

gfro

msc

ale

loss

tode

epul

cers

inm

uscu

latu

re

Dis

ease

inju

veni

les

154

Gro

uper

Ora

nge-

spot

ted

grou

per

( Epi

neph

elus

coio

ides

Ham

ilton

,an

dE.

tauv

ina

Fors

skål

).Fa

mily

Serra

nida

e

1.St

rept

ococ

cus

inia

e2.

Vibr

ioha

rvey

i1.

Susc

eptib

leto

infe

ctio

n1.

Stre

ptoc

occo

sis

847

848

Blen

ny,V

ivip

arou

sbl

enny

( Zoa

rces

vivi

paru

sLi

nnae

us)

Atyp

ical

Aero

mon

assa

lmon

icid

aR

edul

cers

with

whi

tem

argi

ns.O

rgan

ism

also

isol

ated

from

inte

rnal

orga

nsSk

inul

cers

,sep

ticae

mia

832

Host

Path

ogen

Norm

alflo

raTi

ssue

site

Dise

ase

stat

usRe

f

Tab

le1.

1.C

ontin

ued.

20Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpTuesday, January 20, 2004 1:53:34 PM



Brea

m

Blac

kse

abr

eam

( Aca

ntho

pagr

usla

tus

Hou

ttuyn

).Kn

own

asYe

llow

finse

abre

am

1.Ps

eudo

mon

asan

guilli

sept

ica

1.H

aem

orrh

age

and

ulce

rativ

ele

sion

sN

otsu

scep

tible

toin

fect

ion

with

Stre

ptoc

occu

sin

iae

1.As

soci

ated

with

‘win

ter

dise

ase’

.Sep

ticae

mia

225

569

848

Com

mon

brea

m,C

arp

brea

m( A

bram

isbr

ama

Linn

aeus

)Ae

rom

onas

salm

onic

ida

ssp.

achr

omog

enes

Larg

eop

enle

sion

s,ar

eas

ofde

scal

atio

n.Ty

pica

l‘fu

runc

les’

nots

een

Mor

talit

ies

534

One

-spo

tsea

brea

m( D

iplo

dus

sarg

usko

tsch

yiSt

eind

achn

er)

Not

susc

eptib

leto

infe

ctio

nw

ithSt

rept

ococ

cus

inia

e84

8

Red

sea

brea

m,J

apan

ese

seab

ream

( Pag

rus

maj

orTe

mm

inck

and

Schl

egel

).Fa

mily

Spar

idae

1.Ed

war

dsie

llata

rda

2.Li

ston

ella

angu

illaru

m3.

Tena

ciba

culu

mm

ariti

mum

1.Se

ptic

aem

ia,f

ocal

supp

urat

ive

orgr

anul

omat

ous

lesi

ons,

cuta

neou

sul

cera

tions

1.Ed

war

dsie

llosi

s2,

3.In

fect

ion

Sea

brea

m( S

paru

sau

ratu

sLi

nnae

us).

Also

know

nas

Gilt

-hea

dse

abr

eam

.Fa

mily

Spar

idae

1.Li

ston

ella

angu

illaru

m2.

Phot

obac

teriu

mda

mse

lae

ssp.

dam

sela

e3.

Phot

obac

teriu

mda

mse

lae

ssp.

pisc

icid

a4.

Pseu

dom

onas

angu

illise

ptic

a5.

Stre

ptoc

occu

sag

alac

tiae

6.St

rept

ococ

cus

inia

e7.

Vibr

ioal

gino

lytic

us8.

Vibr

ioha

rvey

i9.

Vibr

iosp

lend

idus

2.Le

thar

gy,d

iste

nded

abdo

men

,ha

emor

rhag

eson

fins

and

tail,

pale

liver

3.N

oex

tern

alch

ange

sex

cept

anus

red

and

prot

rude

d,ab

dom

endi

sten

ded,

fluid

inab

dom

inal

cavi

ty,c

onge

sted

sple

en,

pete

chia

lhae

mor

rhag

eson

liver

,gr

anul

omat

ous

lesi

ons

insp

leen

and

kidn

ey,m

ultif

ocal

tissu

ene

cros

is4.

Erra

ticsw

imm

ing

atw

ater

surfa

ce,

sink

toth

ebo

ttom

ofth

eca

gean

ddi

e.As

cite

s,re

nalh

aem

orrh

age

5.H

aem

orrh

agic

area

son

body

,m

outh

,eye

,ope

rcul

uman

dfin

s1,

7,8,

9.U

lcer

s,ha

emor

rhag

es,

exop

htha

lmia

1,2.

Mor

talit

y3.

Fish

past

eure

llosi

s4.

Asso

ciat

edw

ith‘w

inte

rdi

seas

e’,h

aem

orrh

agic

sept

icae

mia

asso

ciat

edw

ithke

ratit

is5.

Stre

ptoc

occo

sis

–ep

izoo

ticw

ith10

0%m

orta

lity

6.Ex

udat

ive

men

ingi

tisan

dpa

noph

thal

miti

s1,

3,7,

8.9.

Prim

ary

path

ogen

sin

viru

lenc

est

udie

s

57 58 60 96 225

242

751

786

853

Silv

erbr

eam

,Whi

tebr

eam

( Blic

cabj

oerk

naLi

nnae

us)

Aero

mon

assa

lmon

icid

ass

p.ac

hrom

ogen

esLa

rge

open

skin

lesi

ons

surro

unde

dby

area

sof

desc

alat

ion.

Typi

cal‘

furu

ncle

s’no

tsee

n

Furu

ncul

osis

,mor

talit

y53

4

Carp

Bigh

ead

carp

( Aris

ticht

hys

nobi

lis)

Edw

ards

iella

icta

luri

627

cont

inue

d



6 Chapter 1

Com

mon

carp

,Koi

carp

( Cyp

rinus

carp

ioca

rpio

Linn

aeus

)

1.Ae

rom

onas

best

iaru

m2.

Atyp

ical

Aero

mon

assa

lmon

icid

a3.

A.ve

roni

issp

.ver

onii

4.C

itrob

acte

rfre

undi

i5.

Flav

obac

teriu

mco

lum

nare

6.Ye

rsin

iaru

cker

i

7.Ae

rom

onas

eucr

enop

hila

8.A.

sobr

iaw

ere

non-

path

ogen

icto

carp

invi

rule

nce

stud

ies

(452

)9.

Stre

ptoc

occu

sin

iae

and

S.ag

alac

tiae

(S.d

iffic

ile) w

ere

non-

path

ogen

icin

viru

lenc

est

udie

s(2

34)

1,3.

Hae

mor

rhag

e,ne

cros

is,u

lcer

s2.

Ulc

ers,

lesi

ons

3.Al

sobl

oody

asci

tes

fluid

4.In

finge

rling

s5.

Gills

6.C

anbe

infe

cted

with

orw

ithou

tclin

ical

sign

s7.

Asci

tes

1,3.

Path

ogen

icon

viru

lenc

est

udie

s2.

Ulc

erat

ing

derm

alle

sion

s4.

Hea

vym

orta

litie

s,se

ptic

aem

ia,o

ppor

tuni

stic

infe

ctio

n5.

Dis

ease

6.Ye

rsin

iosi

s7.

Non

-pat

hoge

nic

135

209

234

271

379

425

452

473

760

Cau

casi

anca

rp,C

ruci

anca

rp( C

aras

sius

cara

ssiu

sLi

nnae

us)

Esch

eric

hia

vuln

eris

Hae

mor

rhag

ein

eyes

,sw

olle

n,da

rkco

lour

edab

dom

en,y

ello

wliv

er,y

ello

wflu

idin

inte

stin

e

Mor

talit

y51

Iber

ian

toot

hcar

p,Sp

anis

hto

othc

arp

( Aph

aniu

sib

erus

Vale

ncie

nnes

)

Vibr

iopa

raha

emol

ytic

usEx

tern

alha

emor

rhag

e,ta

ilro

tM

orta

lity

10

Silv

erca

rp( H

ypop

htha

lmic

hthy

sm

olitr

ixVa

lenc

ienn

es)

1.Pr

ovid

enci

a(P

rote

us)r

ettg

eri

2.St

aphy

loco

ccus

aure

us1.

Larg

ere

dul

cera

tive

lesi

ons

onth

eab

dom

en,b

ase

ofth

epe

ctor

alfin

and

onth

ehe

ad.O

rgan

ism

isol

ated

from

inte

rnal

orga

ns.P

ond

had

been

ferti

lized

with

poul

tryfa

eces

from

whi

chP.

rettg

eri

was

isol

ated

2.R

edco

rnea

beco

min

gop

aque

Deg

ener

atio

nof

eye

tissu

e

1.M

ass

mor

talit

y2.

Eye

dise

ase

79 688

Catfi

sh

Cat

fish

spec

ies

( Icta

luru

ssp

p.R

afin

esqu

e)Fl

avob

acte

rium

colu

mna

reO

rgan

ism

inki

dney

88

Blac

kbu

llhea

d( A

mei

urus

mel

as–

valid

nam

e),(

Icta

luru

sm

elas

Raf

ines

que

–sc

ient

ific

nam

e)

1.Ed

war

dsie

llaic

talu

ri2.

Flav

obac

teriu

mco

lum

nare

2.Ye

llow

-edg

edsk

iner

osio

non

head

1.En

teric

sept

icae

mia

ofca

tfish

(ESC

),ed

war

dsie

llosi

s2.

Col

umna

risdi

seas

e

88

Blue

catfi

sh( Ic

talu

rus

furc

atus

Vale

ncie

nnes

)Ed

war

dsie

llaic

talu

riES

C33

4

Host

Path

ogen

Norm

alflo

raTi

ssue

site

Dise

ase

stat

usRe

f

Tab

le1.

1.C

ontin

ued.




Brow

nbu

llhea

d(Ic

talu

rus

nebu

losu

s )1.

Edw

ards

iella

icta

luri

2.Ed

war

dsie

llata

rda

1.In

fect

ion

inth

ebr

ain,

syst

emic

diss

emin

atio

nan

dlo

caliz

atio

nof

the

orga

nism

inth

evi

scer

alor

gans

and

mus

cula

ture

and

cuta

neou

sul

cers

2.Se

ptic

aem

ia,f

ocal

supp

urat

ive

orgr

anul

omat

ous

lesi

ons,

cuta

neou

sul

cera

tions

1.ES

C,e

dwar

dsie

llosi

s2.

Edw

ards

iello

sis,

oppo

rtuni

stic

infe

ctio

n

334

Cha

nnel

catfi

sh( Ic

talu

rus

punc

tatu

sR

afin

esqu

e)1.

Aero

mon

ashy

drop

hila

2.Ba

cillu

sm

ycoi

des

3.C

arno

bact

eriu

mpi

scic

ola

4.Ed

war

dsie

llaic

talu

ri,an

aero

bic

stra

ins

also

isol

ated

5.Ed

war

dsie

llata

rda

6.Ye

rsin

iaru

cker

i

2.U

lcer

ativ

esk

inle

sion

san

dfo

cal

necr

osis

ofep

axia

lmus

cle

3.H

yper

aem

iaan

dsl

ight

haem

orrh

age

inliv

er,k

idne

y,sp

leen

and

brai

n5.

Bact

eria

isol

ated

from

lesi

ons

onsk

inan

dsu

perfi

cial

mus

cle,

necr

osis

inor

gans

6.H

aem

orrh

agic

rings

arou

ndth

eey

esan

dfro

ntal

fora

men

s

1,2,

3.In

fect

ion

and

mor

talit

y4.

ESC

5.Ed

war

dsie

llosi

s,en

teric

sept

icae

mia

,opp

ortu

nist

icin

fect

ion

Yers

inio

sis

73 203

307

334

547

783

Fres

hwat

erca

tfish

,Sut

chic

atfis

h( P

anga

sius

hypo

phth

alm

usSa

uvag

e)

Edw

ards

iella

icta

luri

Mor

talit

y,m

ultif

ocal

irreg

ular

,whi

tele

sion

son

inte

rnal

orga

ns.N

ecro

sis

and

pyog

ranu

lom

atou

sin

flam

mat

ion

Baci

llary

necr

osis

ofPa

ngas

ius

194

Wal

king

catfi

sh( C

laria

sba

trach

usLi

nnae

us)

( Cla

rias

garie

pinu

s)

1.Ae

rom

onas

hydr

ophi

la2.

Edw

ards

iella

icta

luri

1.U

lcer

ativ

edi

seas

e,m

orta

lity

2.ES

C29 42

6

Whi

teca

tfish

( Am

eiur

usca

tus

Linn

aeus

)Ed

war

dsie

llaic

talu

riES

C33

4

Chub

,Eur

opea

nch

ub( L

euci

scus

ceph

alus

Linn

aeus

)At

ypic

alAe

rom

onas

salm

onic

ida

Skin

ulce

rsan

dfin

rot

Mor

talit

y83

7

Coal

fish

Phot

obac

teriu

milio

pisc

ariu

mIn

test

ine

Non

-pat

hoge

nic

599

767

Cod,

Atla

ntic

cod

( Gad

usm

orhu

aLi

nnae

us)

1.At

ypic

alAe

rom

onas

salm

onic

ida

2.Li

ston

ella

angu

illaru

mse

roty

pe02

3.Vi

brio

salm

onic

ida

4.C

arno

bact

eriu

msp

p.5.

Phot

obac

teriu

milio

pisc

ariu

m2.

Lesi

ons

4,5.

Inte

stin

e1.

Skin

ulce

rs2.

Infe

ctio

n,vi

brio

sis

4.N

on-p

atho

geni

c

186

232

599

712

767

cont

inue

d



8 Chapter 1

Cora

l

Ocu

lina

pata

goni

caVi

brio

shilo

nii(

sai d

t obe

al a

t er

subj

ectiv

esy

nony

mof

V.m

edite

rrane

i)Bi

nds

toco

ral

Cor

albl

each

ing

59 458

742

Poci

llopo

rada

mic

orni

sVi

brio

cora

lliily

ticus

Whi

tesp

ots

seen

at3–

5da

ysan

dco

mpl

ete

tissu

ede

stru

ctio

naf

ter2

wee

ksTi

ssue

lysi

san

dde

ath

83 84

Crab

Blue

crab

( Cal

linec

tes

sapi

dus)

Vibr

ioch

oler

ae-li

ke(2

%of

crab

s)Vi

brio

para

haem

olyt

icus

(23%

ofcr

abs)

Vibr

iovu

lnifi

cus

(7%

ofcr

abs)

Hae

mol

ymph

,dig

estiv

etra

ctPr

esen

tin

appa

rent

lyhe

alth

ycr

abs

212

Swim

min

gcr

ab( P

ortu

nus

tritu

berc

ulat

us)

Vibr

ioha

rvey

i(in

itial

lyca

lled

Vibr

iosp

.zo

ea)

Mas

sm

orta

lity

inzo

eall

arva

e39

0

Craw

fish

Amer

ican

fresh

wat

ercr

ayfis

hR

edsw

amp

craw

fish

( Pro

cam

baru

scl

arki

i)

Vibr

ioch

oler

aeVi

brio

mim

icus

Mor

talit

ies

507

Cray

fish

Aust

ralia

nfre

shw

ater

cray

fish

Yabb

y( C

hera

xal

bidu

s)R

edcl

aw( C

hera

xqu

adric

arin

atus

)

Vibr

iom

imic

usH

aem

olym

phM

orta

lity,

vibr

iosi

s13

523

084

0

Crev

alle

,Tre

vally

,Jac

kcr

eval

le( C

aran

xhi

ppos

Linn

aeus

)Vi

brio

harv

eyi

Der

mal

lesi

ons

Infe

ctio

n45

3

Croc

odile

Edw

ards

iella

tard

aPa

thog

enic

ityno

tkno

wn

Cro

codi

lus

acut

usEr

ysip

elot

hrix

rhus

iopa

thia

eBl

acke

ned

plaq

ues

onsc

ales

Cut

aneo

usle

sion

s40

8

Cai

man

croc

odile

( Cai

man

croc

odilu

s )Er

ysip

elot

hrix

rhus

iopa

thia

eN

ecro

ticfo

ciun

dert

hesc

ales

ofth

eab

dom

enan

dth

eja

wSe

ptic

aem

ia40

8

Cro

cody

lus

nilo

ticus

Myc

opla

sma

croc

odyl

iSw

olle

njo

ints

.Org

anis

mal

sofo

und

inlu

ngs

Exud

ativ

epo

lyar

thrit

is44

1

Host

Path

ogen

Norm

alflo

raTi

ssue

site

Dise

ase

stat

usRe

f

Tab

le1.

1.C

ontin

ued.




Dab

(Lim

anda

liman

daLi

nnae

us)

Atyp

ical

Aero

mon

assa

lmon

icid

aR

ound

,red

ulce

rsw

ithw

hite

mar

gin

ofne

crot

ictis

sue

Skin

ulce

rs83

2

Dace

,Com

mon

dace

( Leu

cisc

usle

ucis

cus

Linn

aeus

)1.

Aero

mon

assa

lmon

icid

ass

p.sa

lmon

icid

a2.

Atyp

ical

Aero

mon

assa

lmon

icid

a

1,2.

Skin

ulce

rs1.

Furu

ncul

osis

2.In

fect

ion

323

352

Dam

selfi

sh(F

amily

Pom

acen

trida

e)

Blac

ksm

ith(C

hrom

ispu

nctip

inni

sC

oope

r)Ph

otob

acte

rium

dam

sela

ess

p.da

mse

lae

Skin

ulce

rsFa

tali

nfec

tion

due

topr

oduc

tion

ofa

cyto

lysi

n50

4

Stag

horn

dam

selfi

sh,Y

ello

wta

ilcl

ownf

ish

( Am

blyg

lyph

idod

oncu

raca

oBl

och,

Amph

iprio

ncl

arki

iBe

nnet

t)

Pseu

doal

tero

mon

aspi

scic

ida

List

onel

laan

guilla

rum

and

Vibr

iopa

raha

emol

ytic

usdi

dno

tapp

ear

tobe

invo

lved

indi

seas

ein

expe

rimen

tali

nfec

tions

Dis

ease

deg

gs,s

een

asw

hite

brai

nan

dsp

inal

cord

,sun

ken

irreg

ular

eyes

,hea

rtre

cede

din

toyo

lksa

c

Infe

ctio

n,m

orta

lity

572

Dani

oSe

eun

derO

RN

AMEN

TAL

Disc

usfis

hSe

eun

derO

RN

AMEN

TAL

Dolp

hin

See

unde

rSEA

MAM

MAL

S

Eel

Amer

ican

eel(

Angu

illaro

stra

taLe

sueu

r)1.

Aero

mon

assa

lmon

icid

a1.

Foca

lles

ions

prog

ress

tola

rge

de-p

igm

ente

dne

crot

icpa

tche

sth

ende

tach

atde

rmo-

epid

erm

alju

nctio

nto

form

larg

eul

cers

that

expo

seun

derly

ing

mus

cle

1.U

lcer

ativ

esk

indi

seas

e,m

orbi

dity

584

Euro

pean

eel(

Angu

illaan

guilla

Linn

aeus

)1.

Atyp

ical

Aero

mon

assa

lmon

icid

a2.

List

onel

laan

guilla

rum

sero

type

053.

Pseu

dom

onas

angu

illise

ptic

a4.

Vibr

iofu

rnis

sii

5.Vi

brio

vuln

ificu

sse

rova

r04

6.Ye

rsin

iaru

cker

i

7.Ae

rom

onas

ench

elei

a1.

Skin

lesi

ons

2.Le

sion

s3.

Subc

utan

eous

haem

orrh

ages

.Ab

dom

inal

dist

ensi

on,o

rgan

ism

inin

tern

alor

gans

4.H

aem

orrh

ages

inin

test

inal

tract

5.O

rgan

ism

cultu

red

from

gills

,int

estin

alco

nten

ts,k

idne

y,sp

leen

6.C

anbe

infe

cted

with

orw

ithou

tclin

ical

sign

s

1.In

fect

ion

2.Vi

brio

sis

3.Se

ptic

aem

ia4.

Mor

talit

y.Vi

rule

ntin

path

ogen

icity

stud

ies

5.D

isea

se6.

Yers

inio

sis

7.N

on-p

atho

geni

c

201

209

240

241

271

323

356

541

712

cont

inue

d



10 Chapter 1

Japa

nese

eel(

Angu

illaja

poni

caTe

mm

inck

and

Schl

egel

)1.

Atyp

ical

Aero

mon

assa

lmon

icid

a2.

Edw

ards

iella

tard

a3.

Flav

obac

teriu

mco

lum

nare

4.Ps

eudo

mon

asan

guilli

sept

ica

5.Vi

brio

vuln

ificu

sbi

ogro

up2,

sero

var

Eco

ntai

nsvi

rule

ntan

dav

irule

ntst

rain

s

Oth

erbi

otyp

esof

V.vu

lnifi

cus

are

non-

path

ogen

icfo

reel

s2.

Sept

icae

mia

,foc

alsu

ppur

ativ

eor

gran

ulom

atou

sle

sion

s,cu

tane

ous

ulce

ratio

ns,a

bsce

ssed

orul

cera

tive

lesi

ons

inki

dney

orliv

er4.

Hae

mor

rhag

ican

dul

cera

tive

lesi

ons.

Lesi

ons

inm

outh

,ope

rcul

um,b

rain

,liv

eran

dki

dney

5.Le

sion

s

2.Ed

war

dsie

llosi

s,‘p

arac

olon

dise

ase’

4.‘S

ekite

n-by

o’(re

dsp

otdi

seas

e)5.

Vibr

iosi

s

26 506

746

757

799

800

830

Eel(

Angu

illare

inha

rdtii

)Ph

otob

acte

rium

dam

sela

ess

p.da

mse

lae

429

Eel

Lact

ococ

cus

garv

ieae

Eel(

Hyp

erop

lus

lanc

eola

tus

LeSa

uveg

e)At

ypic

alAe

rom

onas

salm

onic

ida

Hae

mor

rhag

eson

snou

t,ca

udal

finan

dta

il.Ty

pica

lum

bona

tefu

runc

les

onfla

nkSk

inul

cers

197

Elve

rsAe

rom

onas

allo

sacc

haro

phila

Dis

ease

527

Gre

enm

oray

eel(

Gym

noth

orax

fune

bris

)Sp

otte

dm

oray

eel(

G.m

orin

ga)

Myc

obac

teriu

m-tr

iple

x-lik

eFl

orid

skin

nodu

les

arou

ndth

ehe

adan

dtru

nkth

atar

eso

ftgr

ey,g

elat

inou

san

dta

n-co

lour

ed

Prol

ifera

tive

skin

dise

ase

345

Sand

eel(

Amm

odyt

esla

ncea

Cuv

ier)

Atyp

ical

Aero

mon

assa

lmon

icid

aH

aem

orrh

ages

onsn

out,

caud

alfin

and

tail.

Typi

calu

mbo

nate

furu

ncle

son

flank

Skin

ulce

rs19

7

Flou

nder

Flou

nder

( Pla

ticht

hys

flesu

sLi

nnae

us)

Atyp

ical

Aero

mon

assa

lmon

icid

aan

dox

idas

e-ne

gativ

est

rain

sEp

ider

mal

ulce

ratio

ns–

dark

,red

wou

nds

usua

llyro

und

buts

ome

irreg

ular

form

sse

en

Skin

ulce

rdis

ease

323

829

831

832

Gre

enba

ckflo

unde

r( R

hom

boso

lea

tapi

rina

Gün

ther

)At

ypic

alAe

rom

onas

salm

onic

ida

Ulc

erat

ive

derm

alle

sion

s82

6

Host

Path

ogen

Norm

alflo

raTi

ssue

site

Dise

ase

stat

usRe

f

Tab

le1.

1.C

ontin

ued.




Japa

nese

floun

der,

also

know

nas

Oliv

eflo

unde

r(Pa

ralic

hthy

sol

ivac

eus

Tem

min

ckan

dSc

hleg

el)

1 .At

ypic

alAe

rom

onas

salm

onic

ida

2.Ed

war

dsie

llata

rda

3.La

ctoc

occu

sga

rvie

ae4.

Noc

ardi

ase

riola

e5.

Phot

obac

teriu

mda

mse

lae

ssp.

pisc

icid

a6.

Stre

ptoc

occu

sin

iae

7.Te

naci

bacu

lum

mar

itim

um8.

Vibr

ioic

hthy

oent

eri

9.W

eiss

ella

helle

nica

stra

inD

S-12

2.Se

ptic

aem

ia,f

ocal

supp

urat

ive

orgr

anul

omat

ous

lesi

ons,

cuta

neou

sul

cera

tions

4.Tu

berc

ules

inki

dney

,gills

and

sple

en.

Absc

esse

son

epid

erm

is5.

Hae

mor

rhag

icse

ptic

aem

iaan

dw

hite

area

sof

gran

ulom

ain

the

kidn

ey,l

iver

,sp

leen

8.In

test

inal

necr

osis

,opa

que

inte

stin

es9.

Pres

enti

nin

test

ine

1.In

fect

ion

2.Ed

war

dsie

llosi

s3.

Stre

ptoc

occo

sis

4.N

ocar

dios

is5.

Fish

past

eure

llosi

s6,

7,8.

Mor

talit

y9.

Prob

iotic

pote

ntia

l

139

273

385

389

442

455

567

570

Sum

mer

floun

der(

Para

licht

hys

dent

atus

Linn

aeus

)1.

Myc

obac

teriu

msp

p.w

ithho

mol

ogy

toM

.mar

inum

and

M.u

lcer

ans

2.Vi

brio

(car

char

iae)

harv

eyi

1.G

ranu

lom

asin

the

kidn

ey,l

arge

red-

tan

colo

ured

mul

tilob

ulat

edm

ass

inki

dney

2.R

edde

ning

arou

ndan

alar

ea,

dist

ende

dab

dom

enfil

led

with

fluid

,en

terit

isan

dne

cros

is

1.M

ycob

acte

riosi

s2.

Flou

nder

necr

otiz

ing

ente

ritis

(FIN

E)

373

710

Gra

ylin

g( T

hym

allu

sth

ymal

lus

Linn

aeus

)1.

Aero

mon

assa

lmon

icid

ass

p.sa

lmon

icid

a2.

Atyp

ical

Aero

mon

assa

lmon

icid

a3.

Yers

inia

ruck

eri

1,2.

Skin

ulce

rs1.

Furu

ncul

osis

2,3.

Infe

ctio

n20

932

335

2

Gre

enkn

ifefis

h( E

igem

anni

avi

resc

ens

Vale

ncie

nnes

)Ed

war

dsie

llaic

talu

riEn

teric

sept

icae

mia

426

Gre

enlin

g–

ma r

ine

fish

( Hex

agra

mm

osot

akii)

Atyp

ical

Aero

mon

assa

lmon

icid

aIn

fect

ion

385

Gro

uper

See

Sea

bass

Hadd

ock

( Mel

anog

ram

mus

aegl

efin

usLi

nnae

us).

Fam

ilyG

adid

ae–

cod

and

hadd

ock

Atyp

ical

Aero

mon

assa

lmon

icid

aSk

inul

cers

Infe

ctio

n32

3

Halib

ut

Atla

ntic

halib

ut( H

ippo

glos

sus

hipp

oglo

ssus

Linn

aeus

)1.

Atyp

ical

Aero

mon

assa

lmon

icid

a2.

Tena

ciba

culu

mov

olyt

icum

1.Sk

inul

cers

2.D

isso

lves

chor

ion

and

zona

radi

ata

ofth

eeg

gsh

ells

1.In

fect

ion

2.O

ppor

tuni

stic

path

ogen

ofeg

gsan

dla

rvae

323

324

Gre

enla

ndha

libut

( Rei

nhar

dtiu

shi

ppog

loss

oide

sW

alba

um)

Arth

roba

cter

rhom

biO

rgan

ism

isol

ated

from

sple

enan

din

tern

alor

gans

Path

ogen

icity

notd

eter

min

ed60

0

cont

inue

d



12 Chapter 1

Herr

ing

Balti

che

rring

( Clu

pea

hare

ngus

mem

bras

Linn

aeus

)

1.Ps

eudo

mon

asan

guilli

sept

ica

2.Ph

otob

acte

rium

iliopi

scar

ium

1.H

aem

orrh

ages

inth

eey

e2.

Inte

stin

e1.

Dis

ease

2.N

on-p

atho

geni

c50

359

976

7

Hum

an( H

omo

sapi

ens)

1.A.

allo

sacc

haro

phila

2.Ae

rom

onas

cavi

ae3.

Aero

mon

ashy

drop

hila

ssp.

dhak

ensi

s4.

Aero

mon

ashy

drop

hila

5.A.

jana

daei

6.A.

schu

berti

i7.

A.so

bria

8.A.

trota

9.A.

vero

niis

sp.s

obria

10.A

erom

onas

vero

niis

sp.v

eron

ii11

.Bru

cella

abor

tus

12.B

ruce

llasp

ecie

s13

. Bur

khol

deria

(Pse

udom

onas

)ps

eudo

mal

lei

14.C

hrom

obac

teriu

mvi

olac

eum

15.C

rypt

ococ

cus

neof

orm

ans

16.E

dwar

dsie

llata

rda

17.E

rysi

pelo

thrix

rhus

iopa

thia

e18

.Gra

nulic

atel

lael

egan

s19

.Hal

omon

asve

nust

a20

.Myc

obac

teriu

mm

arin

um21

.Pho

toba

cter

ium

dam

sela

ess

p.da

mse

lae

22.R

ahne

llaaq

uatil

is23

.Rao

ulte

llapl

antic

ola

24.S

hew

anel

laal

gae

25.S

trept

ococ

cus

inia

e26

.Vag

ococ

cus

fluvi

alis

27.V

ibrio

chol

erae

01&

0139

28. V

ibrio

chol

erae

non-

01

1,2.

Faec

es3,

4.Fa

eces

5.W

ound

infe

ctio

n,st

ool,

bloo

d6.

Wou

nd,a

bsce

ss,b

lood

,ple

ural

fluid

7. 9,10

.Fae

ces

11.R

espi

rato

ryin

fect

ion,

abor

tion

12.H

eada

ches

,las

situ

de,s

inus

itis

13.W

ound

infe

ctio

ns,p

neum

onia

and

sept

icae

mia

14.W

ound

infe

ctio

ns15

.Cer

ebra

lspi

nalf

luid

16.I

ntes

tine

17.S

uppu

ratin

gsk

inle

sion

s18

.End

ocar

ditis

19.W

ound

with

wat

ery

disc

harg

efo

llow

ing

fish

bite

20.S

kin

lesi

ons

22.C

onta

min

ated

intra

veno

usflu

id24

.Leg

ulce

rs,s

eptic

aem

ia,o

titis

med

ia,

faec

es25

.Wou

nds

follo

win

gha

ndlin

gfis

h26

.Bite

-wou

nd,b

lood

cultu

reis

olat

esan

dfro

mpe

riton

ealf

luid

27.F

aece

s28

.Blo

odcu

lture

,fae

ces

29.B

lood

and

spin

alflu

id30

,31,

32,3

3.Fa

eces

34.W

ound

s,fa

eces

1.D

iarrh

oea,

rena

lfai

lure

inin

fant

2,3,

4,8.

Dia

rrhoe

a5.

Wou

ndin

fect

ion,

diar

rhoe

a6,

8.In

fect

ion

7.Ac

ute

rena

lfai

lure

inan

infa

nt9,

10.D

iarrh

oea

11,1

2.Br

ucel

losi

s13

.Mel

ioid

osis

14.I

nfec

tion

15.Z

oono

sis,

mor

talit

y17

.Ery

sipe

loid

18.E

ndoc

ardi

tis19

.Wou

ndin

fect

ion

20.I

nfec

tion

21.W

ound

infe

ctio

n22

.Sep

sis.

Also

infe

ctio

nin

imm

unoc

ompr

omis

edho

sts

23.S

eptic

aem

iain

new

born

s24

.Inf

ectio

n25

.Cel

lulit

is26

.Clin

ical

infe

ctio

ns27

.Cho

lera

28.C

ellu

litis

,m

enin

goen

ceph

aliti

s,se

ptic

aem

ia,d

iarrh

oea

29.M

enin

gitis

30,3

1,32

.Dia

rrhoe

a33

.Gas

troen

terit

is,f

ood

pois

onin

g34

.Inf

ectio

n,fo

odpo

ison

ing

120

123

125

142

143

151

177

196

255

259

276

310

346

347

348

378

383

392

482

527

555

588

653

732

792

816

822

Host

Path

ogen

Norm

alflo

raTi

ssue

site

Dise

ase

stat

usRe

f

Tab

le1.

1.C

ontin

ued.




29.V

ibrio

cinc

inna

tiens

is30

.Vib

rioflu

vial

is31

.Vib

riofu

rnis

sii

32.V

ibrio

hollis

ae33

.Vib

riopa

raha

emol

ytic

us34

.Vib

riovu

lnifi

cus

Japa

nese

med

aka

(Ory

zias

latip

esTe

mm

inck

and

Schl

egel

)M

ycob

acte

rium

absc

essu

sM

ycob

acte

riosi

s73

6

Lam

prey

Aero

mon

assa

lmon

icid

ass

p.sa

lmon

icid

aM

aybe

carri

ers

ofth

edi

seas

e.O

rgan

ism

foun

din

kidn

eySu

ccum

bto

infe

ctio

nfo

llow

ing

stre

ssin

stre

ss-te

stex

perim

ents

352

Lobs

ter.

Amer

ican

lobs

ter

( Hom

arus

amer

ican

us,H

.ga

mm

arus

L)

1.Ae

roco

ccus

virid

ans

var.

hom

ari

2.Li

ston

ella

angu

illaru

m1.

Org

anis

min

haem

olym

ph1.

Gaf

fkem

ia,h

igh

mor

talit

y2.

Vibr

iosi

s11

429

982

7

Mac

kere

l

Mac

kere

l(Sc

ombe

rsco

mbr

usLi

nnae

us)

Myc

obac

teriu

msp

p.G

rey-

whi

teno

dule

sin

sple

enan

dvi

scer

alor

gans

Myc

obac

terio

sis

515

Japa

nese

jack

mac

kere

l( T

rach

urus

japo

nicu

sTe

mm

inck

and

Schl

egel

)

1.La

ctoc

occu

sga

rvie

ae2.

List

onel

laan

guilla

rum

3.Vi

brio

trach

uri(

late

stin

form

atio

nsu

gges

tsth

isis

aju

nior

syno

nym

ofV.

harv

eyi)

3.H

aem

orrh

ages

inor

gans

and

exop

htha

lmia

3.D

isea

se40

074

3

Mah

i-mah

i(C

oryp

haen

ahi

ppur

usLi

nnae

us)a

lso

know

nas

Dol

phin

fish

Pant

oea

(Ent

erob

acte

r)ag

glom

eran

sH

aem

orrh

ages

iney

e,an

ddo

rsal

and

late

ralm

uscu

latu

reM

orta

lity,

oppo

rtuni

stic

path

ogen

325

Mak

onbu

( Lam

inar

iaja

poni

ca)

1.Ps

eudo

alte

rom

onas

bact

erio

lytic

a2.

Pseu

doal

tero

mon

asel

yako

vii

1.Pr

oduc

esa

red

pigm

ento

nth

eLa

min

aria

and

caus

esda

mag

eto

the

seed

supp

ly2.

Deg

rade

sfro

nds

1.R

edsp

otdi

seas

e2.

Spot

dise

ase

677

679

Milk

fish

( Cha

nos

chan

osFo

rssk

ål)

Vibr

ioha

rvey

iEy

ele

sion

s,ex

opht

halm

ia,o

paqu

enes

s,ha

emor

rhag

esin

eyes

Eye

dise

ase

and

mor

talit

y39

0

cont

inue

d



14 Chapter 1

Min

now

Baitf

ish

Amer

ican

baitf

ish,

Fath

ead

min

now

( Pim

epha

les

prom

elas

Raf

ines

que)

Yers

inia

ruck

eri

Yers

inio

sis

Bullm

inno

ws

(Fun

dulu

sgr

andi

sBa

ird)

Stre

ptoc

occu

ssp

p.N

on-h

aem

olyt

icgr

oup

BEx

opht

halm

ia,p

etec

hial

haem

orrh

age

inab

dom

en,o

edem

ain

liver

Mor

talit

y,st

rept

ococ

cosi

s63

7

Eura

sian

min

now

( Pho

xinu

sph

oxin

usLi

nnae

us)

Atyp

ical

Aero

mon

assa

lmon

icid

aEx

tens

ive

haem

orrh

agic

skin

lesi

ons

Mas

sm

orta

lity

331

Min

now

List

onel

laan

guilla

rum

Fish

kill

135

Men

hade

n( B

revo

ortia

patro

nus

Goo

de)

Stre

ptoc

occu

sag

alac

tiae

Stre

ptoc

occo

sis

MO

LLUS

CS

Biva

lve

( Nod

ipec

ten

nodo

sus)

Vibr

iobr

asilie

nsis

,V.n

eptu

nius

V.ne

ptun

ius

isol

ated

from

dise

ased

and

heal

thy

anim

als

Path

ogen

icity

notk

now

n74

0

Clam

Man

ilacl

am( R

udita

pes

philip

pina

rum

)and

(R.d

ecus

satu

s)Vi

brio

tape

tisIn

vasi

onof

pallia

l(m

antle

)cav

ityan

dad

here

nce

toth

epe

riost

raca

llam

ina

atth

egr

owin

ged

geof

the

shel

l.M

ain

char

acte

ristic

feat

ure

isa

brow

nco

nchi

olin

depo

sito

nth

ein

ners

urfa

ceof

the

shel

l

Brow

nrin

gdi

seas

e(B

RD

)14 10

814

661

061

1

Cla

ms

( Mer

cena

riam

erce

naria

),Bi

valv

em

ollu

scs

1.Vi

brio

tubi

ashi

i1.

Larv

aece

ase

swim

min

g,di

gest

ion

oftis

sue

1.Ba

cter

ialn

ecro

sis

and

syst

emic

dise

ase

321

762

Mus

sel(

Prot

otha

caje

doen

sis

Lisc

hke)

Shew

anel

laja

poni

ca39

7

Oys

ter

East

ern

oyst

er( C

rass

ostre

avi

rgin

ica )

1.R

oseo

bact

ersp

p.st

rain

CVS

P2.

Vibr

iotu

bias

hii

2.Sh

ewan

ella

colw

ellia

na1.

Lesi

ons,

man

tlere

tract

ion,

conc

hiol

inde

posi

tsin

side

shel

l2.

Prom

otes

settl

emen

tofo

yste

rlar

vae

1.Ju

veni

leoy

ster

dise

ase

(JO

D)

2.N

orm

alflo

ra10

410

581

481

5

Host

Path

ogen

Norm

alflo

raTi

ssue

site

Dise

ase

stat

usRe

f

Tab

le1.

1.C

ontin

ued.




Japa

nese

oyst

er(C

rass

ostre

agi

gas )

Paci

ficoy

ster

(Cra

ssos

trea

giga

s)

1.Al

tero

mon

assp

ecie

s(n

otsp

ecia

ted)

2.Li

ston

ella

angu

illaru

m3.

Noc

ardi

acr

asso

stre

ae4.

Phot

obac

teriu

mda

mse

lae

ssp.

dam

sela

e5.

Vibr

iosp

lend

idus

biov

arII

6.Vi

brio

sple

ndid

us7.

Vibr

iotu

bias

hii

8.Vi

brio

vuln

ificu

sse

rova

rE(s

ome

stra

ins

are

aviru

lent

forT

aiw

anes

eee

ls)

9.Ae

rom

onas

med

ia10

.Aer

omon

assp

p.11

. Alte

rom

onas

spp.

12. P

seud

omon

assp

p.13

. Vib

riosp

p.To

talb

acte

rialc

ount

inso

fttis

sue

=2.

9×

104

colo

nies

per

gram

,and

inha

emol

ymph

=2.

6×

104

colo

nies

perm

l(R

ef59

6)

1.La

rval

deat

hs2.

Failu

reof

velig

erla

rvae

tom

aint

ain

them

selv

esin

the

wat

erco

lum

n3.

Foca

lare

asof

brow

ndi

scol

orat

ion

onth

em

antle

,org

reen

-yel

low

nodu

les

onab

duct

orm

uscl

e,gi

lls,h

eart

and

man

tle5.

Org

anis

mis

olat

edfro

mgo

nads

ofbr

oods

tock

.Hig

hba

cter

iall

oad

inla

rvae

,ba

cter

iain

shel

lmar

gin,

loss

ofci

liaan

dve

lum

.7.

Larv

aece

ase

swim

min

g,di

gest

ion

oftis

sue

1,2,

4.M

orta

lity

3.N

ocar

dios

is5.

Mas

sm

orta

lity.

Dis

ease

rese

mbl

esba

cilla

ryne

cros

is6.

Mor

talit

y.D

isea

seis

stre

ss-a

ssoc

iate

d7.

Bact

eria

lnec

rosi

san

dsy

stem

icdi

seas

e9.

Prob

iotic

agai

nst V

.tub

iash

ii

26 222

270

280

294

321

466

467

596

721

762

798

Med

iterra

nean

oyst

ers

Vibr

iole

ntus

513

Scal

lop

Argo

pect

enpu

rpur

atus

Aero

mon

ashy

drop

hila

Vibr

ioal

gino

lytic

usLa

rval

deat

hs65

0

Pect

enm

axim

us1.

Vibr

iope

cten

icid

a2.

Ros

eoba

cter

galla

ecie

nsis

1.Af

fect

sla

rvae

1.Vi

brio

sis

662

470

Pike

,Nor

ther

npi

ke( E

sox

luci

usLi

nnae

us)

Yers

inia

ruck

eri

209

Mud

skip

per

List

onel

laan

guilla

rum

Fish

kill

135

Mul

let

Blac

km

ulle

t,G

rey

mul

let,

Flat

head

mul

let(

Mug

ilcep

halu

sLi

nnae

us)

1.Ed

war

dsie

llata

rda

2.Eu

bact

eriu

mta

rant

ella

e3.

Lact

ococ

cus

garv

ieae

(tent

ativ

eid

entif

icat

ion)

1.Se

ptic

aem

ia,f

ocal

supp

urat

ive

orgr

anul

omat

ous

lesi

ons,

cuta

neou

sul

cera

tions

2.O

rgan

ism

reco

vere

dfro

mbr

ain,

liver

,ki

dney

,blo

od3.

Fish

leth

argi

c,ex

opht

halm

ia,c

onge

stio

nan

dha

emor

rhag

ein

sple

enan

dki

dney

,w

hite

spot

s,as

cite

s,gr

anul

omas

,m

acro

phag

esan

dba

cter

iain

orga

ns

1.Ed

war

dsie

llosi

s2.

Mor

talit

y3.

Sept

icae

mia

,chr

onic

men

ingi

tis

157

343

764

cont

inue

d



16 Chapter 1

Born

eom

ulle

t,La

rges

cale

mul

let

( Liz

am

acro

lepi

sSm

ith)

Stre

ptoc

occu

sin

iae

Susc

eptib

leto

infe

ctio

nSt

rept

ococ

cosi

s84

8

Silv

erm

ulle

t(M

ugilc

urem

aVa

lenc

ienn

es)

Vibr

ioha

rvey

iO

rgan

ism

isol

ated

inpu

recu

lture

Hae

mor

rhag

icse

ptic

aem

ia23

Strip

edm

ulle

tPh

otob

acte

rium

dam

sela

ess

p.pi

scic

ida

Fish

past

eure

llosi

s

Wild

mul

let(

Liza

klun

zing

eriD

ay)

Stre

ptoc

occu

sag

alac

tiae

Hae

mor

rhag

eson

body

espe

cial

lyin

eye,

mou

th,o

perc

ulum

and

fins.

Org

anis

mcu

lture

dfro

mbr

ain,

eye

and

bloo

d

Stre

ptoc

occo

sis,

epiz

ootic

242

ORN

AMEN

TAL

FISH

Blac

kac

ara

( Cic

hlas

oma

bim

acul

atum

Linn

aeus

)M

ycob

acte

rium

chel

onae

absc

essu

sM

.for

tuitu

m,M

.sim

iae

Myc

obac

terio

sis

474

Com

ets

( Cal

lopl

esio

psal

tivel

isSt

eind

achn

er)

Flav

obac

teriu

mco

lum

nare

Ulc

ers

Mor

talit

y13

5

Dani

o,Si

ndda

nio

( Dan

iode

vario

Ham

ilton

)Ed

war

dsie

llaic

talu

riEn

teric

sept

icae

mia

Blue

disc

usfis

h( S

ymph

ysod

onae

quifa

scia

tus

Pelle

grin

)1.

Aero

mon

ashy

drop

hila

2.A.

jana

daei

3.M

ycob

acte

rium

fortu

itum

2.O

rgan

ism

inliv

er3.

Fin

lesi

ons

1,2.

Dea

ths

3.M

ycob

acte

riosi

s11

613

5

Elec

tric

blue

hap

( Sci

aeno

chro

mis

ahli T

rew

avas

)Ae

rom

onas

jana

daei

Dea

ths

135

Fire

mou

thci

chlid

( Tho

richt

hys

mee

ki,C

ichl

asom

am

eeki

Brin

d)M

ycob

acte

rium

chel

onae

absc

essu

sM

ycob

acte

riosi

s47

4

Flyi

ngfo

x( E

palz

eorh

ynch

oska

lopt

erus

Blee

ker)

Stre

ptoc

occu

sin

iae

Isol

ated

inpu

regr

owth

Dea

ths

135

Gol

den

shin

er( N

otem

igon

uscr

ysol

euca

sM

itchi

ll)1.

Stre

ptoc

occu

sag

alac

tiae

grou

pB

2.Ed

war

dsie

llaic

talu

ri1.

Stre

ptoc

occo

sis

627

Host

Path

ogen

Norm

alflo

raTi

ssue

site

Dise

ase

stat

usRe

f

Tab

le1.

1.C

ontin

ued.




Gol

dfis

h( C

aras

sius

aura

tus

Linn

aeus

)1.

Atyp

ical

Aero

mon

assa

lmon

icid

a2.

Aero

mon

assa

lmon

icid

ass

p.no

va3.

Flav

obac

teriu

mbr

anch

ioph

ilum

4.M

ycob

acte

rium

chel

onae

absc

essu

s5.

Vibr

ioch

oler

a(n

on-0

1)

1,2.

Ulc

erat

ive

skin

lesi

ons,

haem

orrh

ages

3.Ba

cter

iaon

gill

surfa

ce,f

lare

dop

ercu

la,

hype

raem

ic,s

wol

len

gill

tissu

e,ex

cess

muc

uspr

oduc

tion

5.Se

ptic

aem

ia

1,2.

Gol

dfis

hul

cerd

isea

se(G

UD

)3.

Bact

eria

lgill

dise

ase

4.M

ycob

acte

riosi

s5.

Mor

talit

ies,

oppo

rtuni

stic

infe

ctio

n,st

ress

-rela

ted

40 474

602

639

695

825

Gup

py( P

oeci

liare

ticul

ata

Pete

rs,

Lebi

stes

retic

ulat

us)

Myc

obac

teriu

mfo

rtuitu

mFi

nle

sion

s,em

acia

ted,

swam

inci

rcle

sM

ycob

acte

riosi

s11

6

Mol

ly

Ballo

onm

olly

( Poe

cilia

spp.

)Es

cher

ichi

avu

lner

isEx

opht

halm

iain

eyes

,pal

egi

lls,e

mpt

ydi

gest

ive

tract

,ope

ned

oper

culu

m,y

ello

wliv

er

Mor

talit

y51

Blac

km

olly

( Poe

cilia

sphe

nops

Vale

ncie

nnes

)Fl

avob

acte

rium

colu

mna

reW

hite

spot

son

the

back

,hea

dan

dsk

inul

cers

Mor

talit

y21

4

Silv

erm

olly

( Poe

cilia

spp.

)Es

cher

ichi

avu

lner

isPa

leliv

er,b

ento

rcur

ved

body

,thi

nnes

sM

orta

lity

51

Osc

ar( A

stro

notu

soc

ella

tus

Agas

siz,

Apis

togr

amm

aoc

ella

tus)

Myc

obac

teriu

mfo

rtuitu

mSk

inle

sion

son

head

Myc

obac

terio

sis

Ram

sSt

rept

ococ

cus

agal

actia

e(S

.diff

icile

)gr

oup

BO

rgan

ism

inpu

regr

owth

inliv

erD

eath

s13

5

Ros

yba

rbs

( Pun

tius

conc

honi

usH

amilt

on)

Edw

ards

iella

icta

luri

Mor

ibun

d,or

gani

smin

inte

rnal

orga

nsM

orta

lity

of40

%37

4

Siam

ese

fight

ing

fish

( Bet

tasp

lend

ens

Reg

an)

1.Ed

war

dsie

llata

rda

2.M

ycob

acte

rium

fortu

itum

1.M

orib

und,

sing

leor

mul

tiple

cuta

neou

sul

cers

of1

mm

diam

eter

.Org

anis

min

inte

rnal

orga

ns2.

Lesi

ons

1.M

orta

lity

of70

%2.

Myc

obac

terio

sis

374

633

Tetra

Blac

ksk

irted

tetra

( Hyp

hess

obry

con

spp.

)Ed

war

dsie

llata

rda

Sept

icae

mia

,foc

alsu

ppur

ativ

eor

gran

ulom

atou

sle

sion

s,cu

tane

ous

ulce

ratio

ns

Edw

ards

iello

sis

374

548

cont

inue

d



18 Chapter 1

Neo

nte

tra(H

yphe

ssob

ryco

nin

nesi

Mye

rs–

scie

ntifi

cna

me,

Para

chei

rodo

nin

nesi

Mye

rs–

valid

nam

e)

1.Fl

avob

acte

rium

colu

mna

re2.

Myc

obac

teriu

mfo

rtuitu

m1.

Skin

disc

olor

atio

n,w

hite

area

sof

necr

osis

.Org

anis

min

skin

,gills

,mus

cle

and

inne

rsur

face

ofsc

ales

2.Le

sion

s

1.M

uscl

ein

fect

ions

2.M

ycob

acte

riosi

s11

654

365

6

Serp

aete

traSt

rept

ococ

cus

agal

actia

e(S

.diff

icile

)gr

oup

BIs

olat

edin

pure

heav

ygr

owth

Dea

ths

135

Thre

e-sp

otgo

uram

i(Tr

icho

gast

ertri

chop

teru

sPa

llas)

Myc

obac

teria

spec

ies

Myc

obac

terio

sis

672

Otte

r,Eu

rope

anot

ter(

Lutra

lutra

)1.

Bruc

ella

pinn

iped

iae

2.St

aphy

loco

ccus

lutra

e3.

Vago

cocc

uslu

trae

1.O

rgan

ism

isol

ated

from

tissu

es2.

Org

anis

min

liver

,spl

een,

lym

phno

de3.

Org

anis

mfo

und

inbl

ood,

liver

,lun

gs,

sple

en

1.Br

ucel

losi

s2.

3.Pa

thog

enic

ityno

tkno

wn

262

264

267

477

Paci

ficst

agho

rnsc

ulpi

n( L

epto

cottu

sar

mat

usG

irard

)M

ycob

acte

rium

scro

fula

ceum

Live

rsw

hite

and

friab

leM

ycob

acte

riosi

s47

4

Perc

h

Euro

pean

perc

h( P

erca

fluvi

atilis

Linn

aeus

)1.

Aero

mon

assa

lmon

icid

ass

p.ac

hrom

ogen

es2.

Yers

inia

ruck

eri

1.La

rge

open

lesi

ons,

desc

alat

ion.

No

typi

cal‘

furu

ncle

s’se

en2.

Dis

ease

isas

soci

ated

with

poor

envi

ronm

enta

lcon

ditio

ns

1.M

orta

lity

2.Ye

rsin

iosi

s53

477

2

Silv

erpe

rch

(Bid

yan

perc

h)( B

idya

nus

bidy

anus

Mitc

hell)

Atyp

ical

Aero

mon

assa

lmon

icid

aC

utan

eous

ulce

rsSk

indi

seas

e,ul

cera

tive

derm

atiti

s82

5

Whi

tepe

rch

( Mor

one

amer

ican

a,G

mel

in, R

occu

sam

eric

anus

)Ph

otob

acte

rium

dam

sela

ess

p.pi

scic

ida

Org

anis

mis

olat

edfro

min

tern

alor

gans

Fish

past

eure

llosi

s,m

assi

vem

orta

lity

708

Yello

wpe

rch

( Per

cafla

vesc

ens

Mitc

hill)

Myc

obac

teriu

mch

elon

aeG

ranu

lom

atou

spe

riton

itis

and

hepa

titis

Myc

obac

terio

sis

204

Pike

No r

the r

np i

k e( E

sox

luci

usLi

nnae

us)

Atyp

ical

Aero

mon

assa

lmon

icid

aSk

inul

cers

Infe

ctio

n32

3

Host

Path

ogen

Norm

alflo

raTi

ssue

site

Dise

ase

stat

usRe

f

Tab

le1.

1.C

ontin

ued.




Pira

rucu

( Ara

paim

agi

gas

Cuv

ier),

agi

gant

ictro

pica

lfre

shw

ater

fish

Salm

onel

laar

izon

aeO

rgan

ism

isol

ated

from

liver

,spl

een,

hear

t,ki

dney

,blo

ody

exud

ate

inbo

dyca

vity

,cor

neal

opac

ity

Sept

icae

mia

447

Plai

ce,E

urop

ean

plai

ce( P

leur

onec

tes

plat

essa

Linn

aeus

)Li

ston

ella

angu

illaru

mse

roty

pe07

Lesi

ons

Vibr

iosi

s71

2

Plat

ies,

Sout

hern

plat

yfis

h( X

ipho

phor

usm

acul

atus

Gnt

her)

Flav

obac

teriu

mco

lum

nare

Whi

tesp

ots

onth

eba

ckan

dhe

ad,a

ndsk

inul

cers

Mor

talit

y21

4

Porp

oise

See

unde

rSEA

MAM

MAL

S

Praw

nSe

eun

derS

hrim

p

Rabb

itfis

h

Mar

bled

spin

efoo

t(Si

ganu

sriv

ulat

usFo

rssk

ål)

1.M

ycob

acte

rium

mar

inum

2.Ps

eudo

mon

aspu

trefa

cien

s3.

Stre

ptoc

occu

sin

iae

1.W

hite

-yel

low

nodu

les

inth

esp

leen

2.Sk

indi

scol

orat

ion,

and

foca

lnec

rosi

sbe

com

ing

haem

orrh

agic

,abd

omin

alas

cite

s,ex

opht

halm

ia,o

edem

aan

dne

cros

isin

kidn

eys

3.Sy

stem

icdi

seas

ew

ithdi

ffuse

visc

eral

haem

orrh

ages

1.M

ycob

acte

riosi

s–

infe

ctio

nsp

read

from

cage

fish

tow

ildra

bbitf

ish

2,3.

Mor

talit

y

218

260

666

853

Whi

te-s

potte

dsp

inef

oot(

Siga

nus

cana

licul

atus

Park

)St

rept

ococ

cus

inia

eAs

cite

s,he

pato

meg

aly,

sple

nom

egal

ySt

rept

ococ

cosi

s,m

ass

mor

talit

y84

8

Red

Sea

fish,

Wild

fish,

Strip

edpi

ggy

( Pom

adas

ysst

riden

sFo

rssk

ål),

Varie

gate

dliz

ardf

ish

( Syn

odus

varie

gatu

sLa

cepè

de)

Stre

ptoc

occu

sin

iae

Org

anis

mcu

lture

dfro

mbl

ood.

No

mar

ked

gros

ssi

gns

ofdi

seas

eM

orbi

dity

183

Redf

ish ,

Red

drum

( Sci

aeno

psoc

ella

tus

Linn

aeus

)1.

Euba

cter

ium

spec

ies

(tent

ativ

eid

entif

icat

ion)

2.St

rept

ococ

cus

inia

e

1.O

rgan

ism

isol

ated

from

brai

n,liv

er,

kidn

eyan

dbl

ood

2.Sk

inle

sion

s,ex

opht

halm

ia,e

yede

gene

ratio

n,ne

crot

icar

eas

ofgi

llro

t.O

rgan

ism

inhe

arta

ndsp

leen

1,2.

Mor

talit

y34

318

3

REPT

ILES

Snak

esEd

war

dsie

llata

rda

Isol

ated

from

faec

esC

arrie

rsta

tus

orpa

rtof

the

norm

alflo

ra39

9

Torto

ise

Cor

yneb

acte

rium

test

udin

oris

Mou

thle

sion

s18

0

cont

inue

d



20 Chapter 1

Turtl

es(C

aspi

ante

rrapi

n–

Mau

rem

ysca

spic

a,Ea

ster

nbo

xtu

rtle

–Te

rrape

neca

rolin

aca

rolin

a,M

issi

ssip

pim

aptu

rtle

–M

alac

lem

ysko

hni,

Nor

ther

ndi

amon

dbac

kte

rrapi

n–

Mal

acle

mys

terra

pin

terra

pin,

Pain

ted

turtl

e–

Chr

ysem

yspi

cta,

Red

-ear

edtu

rtle

–C

hrys

emys

scrip

tael

egan

s ,St

inkp

ottu

rtle

–St

erno

ther

usod

orat

us,T

rava

ncor

ecr

owne

dtu

rtle

–M

elan

oche

lys

triju

gaco

rona

ta)

Edw

ards

iella

tard

a,Sa

lmon

ella

durh

amC

loac

aeC

arrie

rsta

tus

606

Che

loni

am

ydas

,C.c

aret

ta,

Eret

moc

hely

sim

bric

ata

1.Ae

rom

onas

hydr

ophi

la2.

Der

mat

ophi

lus

chel

onae

3.Fl

avob

acte

rium

spp.

4.M

ycob

acte

rium

spp.

5.Ps

eudo

mon

assp

p.6.

Vibr

ioal

gino

lytic

us

7.Ac

inet

obac

terc

alco

acet

icus

8.Ba

cillu

ssp

p.,M

icro

cocc

ussp

p.,

Mor

axel

lasp

p.,P

rote

ussp

p.

2.Sk

in4.

Lung

lesi

ons

7.O

ralc

avity

8.Pa

rtof

norm

alflo

raon

skin

,ora

lcav

ityan

dtra

chea

1.As

soci

ated

with

bron

chop

neum

onia

,ulc

erat

ive

stom

atiti

s,an

dtra

umat

icul

cera

tive

derm

atiti

sca

used

bybi

ting

2.Sk

inle

sion

s,sk

inab

sces

s,sc

abs

3.As

soci

ated

with

traum

atic

ulce

rativ

ede

rmat

itis

caus

edby

bitin

g,ul

cera

tive

stom

atiti

s,br

onch

opne

umon

iaan

dke

rato

conj

unct

iviti

s–ul

cera

tive

blep

harit

is5.

Asso

ciat

edw

ithtra

umat

icul

cera

tive

derm

atiti

sca

used

bybi

ting,

ulce

rativ

est

omat

itis,

aden

itis,

perit

oniti

s,br

onch

opne

umon

iaan

dke

rato

conj

unct

iviti

s–ul

cera

tive

blep

harit

is

300

301

529

Host

Path

ogen

Norm

alflo

raTi

ssue

site

Dise

ase

stat

usRe

f

Tab

le1.

1.C

ontin

ued.




6.As

soci

ated

with

traum

atic

ulce

rativ

ede

rmat

itis

caus

edby

bitin

g,br

onch

opne

umon

iaan

dos

teom

yelit

is

Turtl

es( P

seud

emis

scrip

ta)

Aero

mon

ashy

drop

hila

Infe

ctio

n61

4

Haw

aiia

ngr

een

turtl

eC

itrob

acte

rfre

undi

i,H

afni

aal

vei,

Kleb

siel

laox

ytoc

a,Ph

otob

acte

rium

dam

sela

e ,Ps

eudo

mon

asflu

ores

cens

,Pse

udom

onas

putre

faci

ens ,

Pseu

dom

onas

stut

zeri ,

non-

haem

olyt

icSt

rept

ococ

cus

spp.

,Vib

rioal

gino

lytic

us,V

ibrio

fluvi

alis

Nas

alsw

abs

and

cloa

cals

wab

sfro

map

pare

ntly

heal

thy

turtl

esPa

thog

enic

ityno

tdet

erm

ined

.M

ostl

ikel

yno

rmal

flora

from

thes

esi

tes.

5

Leat

herb

ack

turtl

e( D

erm

oche

lys

coria

cea )

Phot

obac

teriu

mda

mse

lae

ssp.

dam

sela

eFl

uid

inbo

dyca

vity

,cal

care

ous

nodu

les

inlu

ngpa

renc

hym

a,lu

ngs

cong

este

dVa

lvul

aren

doca

rditi

san

dse

ptic

aem

ia59

0

Logg

erhe

adse

atu

rtle

( Car

etta

care

tta)

Aero

cocc

usvi

ridan

sG

asin

dive

rticu

lum

,with

mul

tifoc

algr

anul

omat

ous

sero

sitis

.Gre

enm

ucoi

dflu

idan

dfib

rinon

ecro

ticm

embr

ane

onm

ucos

a.

Oes

opha

geal

dive

rticu

lum

755

Rid

ley

sea

turtl

e( L

epid

oche

lys

oliv

acea

)Vi

brio

mim

icus

Red

uced

viab

ility

ofeg

gsC

ause

offo

odpo

ison

ing

inhu

man

s4

Rhyn

chop

elat

esox

yrhy

nchu

sTe

mm

inck

and

Schl

egel

,Coa

stal

fish

–Ja

pan

( The

rapo

nox

yrhy

nchu

s )

Vibr

ioho

llisae

Inte

stin

alco

nten

tsN

on-p

atho

geni

c58

0

Roac

h( R

utilu

sru

tilus

Linn

aeus

)1.

Aero

mon

assa

lmon

icid

ass

p.ac

hrom

ogen

es2.

Atyp

ical

Aero

mon

assa

lmon

icid

a3.

Yers

inia

ruck

eri

1.La

rge

open

lesi

ons,

area

sof

desc

alat

ion.

No

typi

cal‘

furu

ncle

s’se

en2.

Cut

aneo

usul

cers

3.D

isea

seis

asso

ciat

edw

ithpo

oren

viro

nmen

talc

ondi

tions

1.M

orta

litie

s2.

Ulc

erat

ive

derm

atiti

s3.

Yers

inio

sis

352

534

772

825

826

Rock

fish,

Schl

egel

’sbl

ack

rock

fish

( Seb

aste

ssc

hleg

eliH

ilden

dorf)

Atyp

ical

Aero

mon

assa

lmon

icid

aU

lcer

onth

etru

nk.O

rgan

ism

isol

ated

from

the

kidn

eyan

dbr

ain

Mor

talit

y38

540

3

cont

inue

d



22 Chapter 1

Rock

ling

Four

bear

dro

cklin

g(E

nche

lyop

usci

mbr

ius

Linn

aeus

)

Atyp

ical

Aero

mon

assa

lmon

icid

aSk

inul

cers

832

Rotif

er( B

rach

ionu

spl

icat

ilis)

1.Vi

brio

rotif

eria

nus

2.V.

nept

uniu

s1,

2.Is

olat

edfro

mro

tifer

flow

-thro

ugh

cultu

resy

stem

.Pat

hoge

nici

tyno

tkno

wn

305

740

Rudd

( Sca

rdin

ius

eryt

hrop

htha

lmus

Linn

aeus

)1.

Aero

mon

assa

lmon

icid

ass

p.ac

hrom

ogen

es2.

Yers

inia

ruck

eri

209

318

SALM

ONI

DS

Arct

icCh

ar( S

alve

linus

alpi

nus

Linn

aeus

)1.

Aero

mon

assa

lmon

icid

ass

p.sa

lmon

icid

a2.

Atyp

ical

Aero

mon

assa

lmon

icid

a3.

Serra

tialiq

uefa

cien

s

4.C

arno

bact

eriu

msp

p.1,

2.Sk

inul

cers

3.R

edne

ssan

dsw

ellin

gar

ound

anus

,as

cite

s,ha

emor

rhag

icin

tern

altis

sues

1.Fu

runc

ulos

is2.

Infe

ctio

n3.

Mor

talit

y4.

Non

-pat

hoge

nic

323

352

648

715

Salm

on

Atla

ntic

salm

on( S

alm

osa

lar

Linn

aeus

)1.

Aero

mon

assa

lmon

icid

ass

p.sa

lmon

icid

a2.

Atyp

ical

Aero

mon

assa

lmon

icid

a3.

Flav

obac

teriu

mps

ychr

ophi

lum

4.Li

ston

ella

angu

illaru

m5.

Mor

itella

visc

osa

6.M

ycob

acte

rium

chel

onae

7.N

ocar

dia

serio

lae

8.Pa

steu

rella

skye

nsis

9.Ph

otob

acte

rium

dam

sela

ess

p.da

mse

lae

10.P

seud

omon

asan

guilli

sept

ica

11.R

enib

acte

rium

salm

onin

arum

12.S

erra

tialiq

uefa

cien

s13

.Stre

ptob

acillu

sm

onilif

orm

is-li

keor

gani

sm14

. Ten

acib

acul

umm

ariti

mum

15.V

agoc

occu

ssa

lmon

inar

um

22.C

arno

bact

eriu

min

hibe

ns23

.Car

noba

cter

ium

spp.

24. L

acto

baci

llus

spp.

25. P

hoto

bact

eriu

milio

pisc

ariu

m26

.Vib

riopa

cini

i

1.Sk

inul

cers

2.O

rgan

ism

from

head

kidn

ey,c

utan

eous

lesi

ons

3.Er

osio

nof

fins

5.Sk

inul

cers

,int

erna

ldar

k-br

own

pete

chia

eor

ecch

ymot

icha

emor

rhag

e6.

Gra

nulo

ma-

like

nodu

les

intis

sues

7.G

ranu

lom

atou

sle

sion

son

body

8.M

ultif

ocal

area

sof

coag

ulat

ive

necr

osis

inki

dney

,liv

er,s

plee

n10

.Pet

echi

alha

emor

rhag

eson

skin

,m

outh

and

anus

,per

itone

uman

dliv

er11

.Whi

teno

dule

sin

kidn

ey12

.Sw

ellin

gof

kidn

eys,

blee

ding

atth

ean

usan

din

test

ine,

gast

roen

terit

is13

.Org

anis

min

trace

llula

rin

tissu

es,

endo

thel

ialc

ells

ofki

dney

glom

erul

ien

larg

ed

1.Fu

runc

ulos

is2.

Sim

ilart

ofu

runc

ulos

is3.

Mor

bidi

tyra

ther

than

mor

talit

y4.

Vibr

iosi

s5.

Win

teru

lcer

s6.

Myc

obac

terio

sis

7.N

ocar

dios

is8.

Mor

talit

y9.

Path

ogen

icin

chal

leng

eex

perim

ents

10.H

aem

orrh

agic

dise

ase

11.B

acte

rialk

idne

ydi

seas

e12

,13.

Mor

talit

y14

.Bac

teria

lsto

mat

itis

(mou

thro

t)in

smol

ts15

.Mor

talit

y16

.Blin

dnes

s17

.Pos

sibl

epa

thog

en

49 100

107

117

132

133

137

144

232

306

330

352

411

412

416

450

506

519

Host

Path

ogen

Norm

alflo

raTi

ssue

site

Dise

ase

stat

usRe

f

Tab

le1.

1.C

ontin

ued.




16.V

arra

calb

mi

17.V

ibrio

loge

i18

.Vib

rioor

dalii

19.V

ibrio

salm

onic

ida

20.V

ibrio

wod

anis

21.Y

ersi

nia

ruck

eris

erot

ype

I

14. Y

ello

w-c

olou

red

plaq

ueon

teet

han

dor

alca

vity

15.P

erito

nitis

,hae

mor

rhag

icas

cite

s,re

tain

edeg

gs,e

ngor

ged

test

es,l

angu

idsw

imm

ing

16.D

eep

skin

lesi

ons,

eye

lesi

ons,

haem

orrh

agic

and

pyog

ranu

lom

atou

sle

sion

sin

gills

,kid

ney,

liver

and

pseu

dobr

anch

17.S

kin

lesi

ons

21.H

aem

orrh

agic

mus

cula

ture

25.I

ntes

tine

18. M

orta

lity

20.O

ppor

tuni

stic

infe

ctio

nin

‘win

teru

lcer

’dis

ease

21.E

nter

icre

dmou

th(E

RM

)22

.Inh

ibits

grow

thof

L.an

guilla

rum

and

A.sa

lmon

icid

a23

.Pot

entia

lpro

biot

ic24

.Nor

mal

mic

roflo

ra25

.Non

-pat

hoge

nic

538

564

599

605

611

622

648

682

683

712

767

771

824

828

Chi

nook

salm

on( O

ncor

hync

hus

tsch

awyt

scha

Wal

baum

)1.

Edw

ards

iella

tard

a2.

Flav

obac

teriu

mbr

anch

ioph

ilum

3.F.

colu

mna

re4.

Myc

obac

teriu

mne

oaur

um5.

Myc

obac

teriu

msp

p.6.

Ren

ibac

teriu

msa

lmon

inar

um7.

Tena

ciba

culu

mm

ariti

mum

8.Ye

rsin

iaru

cker

i

2.Ba

cter

iaat

tach

edto

gill

epith

eliu

m3.

Gill

lesi

ons

4.O

rgan

ism

pres

enti

ney

es,

exop

htha

lmia

,and

ocul

arle

sion

s6.

Gill

lesi

ons

1.Ed

war

dsie

llosi

s2.

Bact

eria

lgill

dise

ase

(BG

D)

3.M

orta

lity

4.Pa

noph

thal

miti

s5.

Myc

obac

terio

sis

6.Ba

cter

ialk

idne

ydi

seas

e(B

KD)

7.M

orta

lity

8.ER

M

36 53 88 137

154

209

245

802

Paci

ficsa

lmon

,Coh

osa

lmon

( O.

kisu

tch

Wal

baum

)1.

Aero

mon

assa

lmon

icid

a(o

xida

se-n

egat

ive

stra

in)

2.Fl

avob

acte

rium

colu

mna

re3.

F.ps

ychr

ophi

lum

4.Li

ston

ella

angu

illaru

m01

5.R

enib

acte

rium

salm

onin

arum

1.Fi

nger

lings

affe

cted

.Sof

tkid

neys

,oc

casi

onal

fish

with

haem

orrh

age

onfin

,ot

herw

ise

noex

tern

alsi

gns

1.Fu

runc

ulos

is3.

Col

d-w

ater

dise

ase

4.Vi

brio

sis

5.BK

D

168

712

748

749

757

765

Che

rrysa

lmon

( Onc

orhy

nchu

sm

asou

mas

ouBr

evoo

rt).K

now

nas

Yam

ame

inJa

pan

Flav

obac

teriu

mbr

anch

ioph

ilum

BGD

802

Sock

eye

salm

on( O

.ner

kaW

alba

um)

1.Fl

avob

acte

rium

bran

chio

philu

m2.

Yers

inia

ruck

eri

1.Ba

cter

ium

atta

ches

togi

llep

ithel

ium

1.BG

D2.

ERM

137

802

cont

inue

d



24 Chapter 1

Salm

onid

s–

Trou

t

Broo

ktro

ut(S

alve

linus

font

inal

isM

itchi

ll)1.

Atyp

ical

Aero

mon

assa

lmon

icid

a2.

Edw

ards

iella

tard

a3.

Flav

obac

teriu

mbr

anch

ioph

ilum

4.N

ocar

dia

spp.

5.Ye

rsin

iaru

cker

i

1.Sk

inul

cers

3.Ba

cter

iaat

tach

togi

llep

ithel

ium

4.N

ecro

sis,

thro

mbo

sis

inki

dney

,spl

een,

gills

1.Sk

inul

cers

and

sept

icae

mia

2.Ac

ute

bact

eria

lsep

ticae

mia

3.BG

D4.

Noc

ardi

osis

5.ER

M

137

604

765

824

Brow

ntro

ut,S

eatro

ut,S

teel

head

trout

( Sal

mo

trutta

trutta

Linn

aeus

)1.

Aero

mon

assa

lmon

icid

ass

p.sa

lmon

icid

a2.

Atyp

ical

Aero

mon

assa

lmon

icid

a3.

Flav

obac

teriu

mbr

anch

ioph

ilum

4.Fl

avob

acte

rium

colu

mna

re5.

Haf

nia

alve

i6.

List

onel

laan

guilla

rum

027.

Pseu

dom

onas

angu

illise

ptic

a

1,2.

Skin

ulce

rs3.

Skin

arou

nddo

rsal

finbl

anch

ed,b

utno

ulce

ratio

nse

en5.

Org

anis

man

dle

sion

sin

the

kidn

ey6.

Lesi

ons

7.Pe

tech

ialh

aem

orrh

ages

onsk

in,m

outh

and

anus

,per

itone

uman

dliv

eran

dat

base

offin

s

1.Fu

runc

ulos

is2.

Skin

ulce

rsan

dse

ptic

aem

ia3.

Col

umna

risdi

seas

e,ch

roni

cm

orta

lity

4.BG

D5.

Opp

ortu

nist

icpa

thog

en6.

Vibr

iosi

s7.

Hae

mor

rhag

icdi

seas

e

88 352

652

712

802

824

828

Rai

nbow

trout

( Onc

orhy

nchu

sm

ykis

sW

alba

um)

Prev

ious

nam

e( S

alm

oga

irdne

riR

icha

rdso

n)

1.Ae

rom

onas

salm

onic

ida

ssp.

salm

onic

ida

2.At

ypic

alAe

rom

onas

salm

onic

ida

3.At

ypic

alAe

rom

onas

salm

onic

ida

(gro

wth

at37

°C)

4.Ae

rom

onas

sobr

ia5.

Car

noba

cter

ium

pisc

icol

a6.

Clo

strid

ium

botu

linum

7.Es

cher

ichi

avu

lner

is8.

Edw

ards

iella

tard

a9.

Flav

obac

teriu

mbr

anch

ioph

ilum

10.F

.psy

chro

philu

m11

.Jan

thin

obac

teriu

mliv

idum

12.K

lebs

iella

pneu

mon

iae

13.L

acto

cocc

usga

rvie

ae14

.Lac

toco

ccus

pisc

ium

15.L

isto

nella

angu

illaru

m16

.Mic

roco

ccus

lute

us17

.Mor

itella

visc

osa

31.C

itrob

acte

rfre

undi

i1,

2,3.

Skin

ulce

rs4.

Path

ogen

icfo

rfin

gerli

ngs

invi

rule

nce

assa

y(7

50)

5.Bi

late

rale

xoph

thal

mia

,per

iocu

lar

haem

orrh

ages

,asc

ites

fluid

and

haem

orrh

ages

inliv

er,s

wim

blad

der,

mus

cle

and

inte

stin

e6.

Toxi

nin

seru

man

din

test

inal

cont

ents

7.H

aem

orrh

agic

lesi

ons

onsk

in,b

lood

yex

udat

ein

dige

stiv

etra

ct,l

iver

yello

wan

dhy

pera

emic

,gon

ads

haem

orrh

agic

8. 9.Ba

cter

iaat

tach

togi

llep

ithel

ium

12.F

inan

dta

il15

.Les

ions

16.O

rgan

ism

isol

ated

from

kidn

ey,s

plee

nan

das

cite

sflu

id17

.Ski

nle

sion

s,pe

tech

ialh

aem

orrh

age

inliv

eran

dpe

riton

ealm

embr

anes

1.Fu

runc

ulos

is2,

3Sk

inul

cers

,sep

ticae

mia

4.H

aem

orrh

agic

sept

icae

mia

5.C

hron

icdi

seas

ew

ithlo

wle

vel

mor

talit

y6.

Botu

lism

7.M

orta

lity

8.Ed

war

dsie

llosi

s9.

BGD

10.

11.M

orta

lity

infry

,pos

sibl

eop

portu

nist

icin

fect

ion

12.F

inan

dta

ildi

seas

e13

.Mor

talit

y14

.Pse

udok

idne

ydi

seas

e,La

ctob

acillo

sis

15.V

ibrio

sis,

sept

icae

mia

16.I

sola

ted

from

mor

ibun

dfis

h17

.Mor

talit

y

296 40 43 48 51 73 76 81 82 107

135

137

141

195

205

233

234

352

268

Host

Path

ogen

Norm

alflo

raTi

ssue

site

Dise

ase

stat

usRe

f

Tab

le1.

1.C

ontin

ued.




18.M

ycob

acte

rium

mar

inum

19.N

ocar

dia

aste

roid

es20

.Pho

toba

cter

ium

dam

sela

ess

p.da

mse

lae

21.P

lesi

omon

assh

igel

loid

es22

.Pse

udom

onas

angu

illise

ptic

a23

.Ren

ibac

teriu

msa

lmon

inar

um01

,02

,03,

0424

. Ser

ratia

mar

cesc

ens,

S.pl

ymut

hica

25.S

taph

yloc

occu

sw

arne

ri26

Stre

ptoc

occu

sag

alac

tiae

(S.

diffi

cile

)27

Stre

ptoc

occu

sin

iae

28Va

goco

ccus

salm

onin

arum

29.V

ibrio

orda

lii30

.Yer

sini

aru

cker

i

18. L

esio

nsin

kidn

ey19

.Les

ions

inki

dney

20.O

rgan

ism

pres

enti

nhe

adki

dney

21.P

etec

hial

haem

orrh

ages

inin

test

ine

22.P

etec

hial

haem

orrh

ages

onsk

in,

mou

than

dan

usan

dba

seof

fins,

perit

oneu

man

dliv

er23

.Whi

teno

dule

sin

kidn

ey25

.Exo

phth

alm

ia,a

scite

sflu

id,l

esio

nson

fins,

orga

nism

inki

dney

and

liver

26,2

7.O

rgan

ism

sin

brai

nan

dey

e28

.Los

sof

equi

libriu

m,h

aem

orrh

age

arou

ndey

ean

dgi

ll,le

sion

son

body

,co

nges

tion

insp

leen

and

liver

.Per

itoni

tis,

haem

orrh

agic

asci

tes,

reta

ined

eggs

,en

gorg

edte

stes

,lan

guid

swim

min

g30

.Hae

mor

rhag

ear

ound

mou

than

din

test

ines

18. M

orta

lity,

myc

obac

terio

sis

19.N

ocar

dios

is20

.Pat

hoge

nic

inch

alle

nge

expe

rimen

tsan

dna

tura

lin

fect

ion

21.P

ossi

ble

oppo

rtuni

stic

path

ogen

22.H

aem

orrh

agic

dise

ase

23.B

KD24

.Mor

talit

yin

finge

rling

s25

.Dis

ease

dan

ddy

ing

trout

–op

portu

nist

icin

fect

ion

26,2

7.M

enin

goen

ceph

aliti

s,se

ptic

aem

ia28

.Chr

onic

dise

ase

with

mor

talit

y29

.Vib

riosi

s30

.ER

M31

.Opp

ortu

nist

icpa

thog

en

542

564

579

604

618

640

682

707

712

716

748

750

752

753

757

802

824

828

835

853

Amag

otro

utPs

eudo

mon

asch

loro

raph

isH

aem

orrh

ages

,inc

reas

edas

cite

sIn

fect

ion

332

Sard

ine

Paci

ficsa

rdin

e,So

uth

Amer

ican

pilc

hard

( Sar

dino

pssa

gax

Jeny

ns)

Tena

ciba

culu

mm

ariti

mum

Glid

ing

bact

eria

seen

asa

tan-

colo

ured

pseu

dom

embr

ane

over

the

body

Dis

ease

154

SEA

BIRD

S

Cran

e(S

andh

illcr

ane

–G

rus

cana

dens

is)

Edw

ards

iella

tard

a,Sa

lmon

ella

hartf

ord ,

S.ja

vaLa

rge

inte

stin

e82

3

Eagl

e,Ba

ldea

gle

( Hal

iaee

tus

leuc

ocep

halu

s )Ed

war

dsie

llata

rda

Clo

acal

swab

Bird

was

sick

butn

oot

her

evid

ence

ofin

fect

ion

823

Gul

l,R

ing-

bille

dgu

ll( L

arus

dele

war

ensi

s )Ed

war

dsie

llata

rda

Larg

ein

test

ine

823

Hero

n(G

reat

blue

hero

n–

Arde

ahe

rodi

as)

Edw

ards

iella

tard

aLa

rge

inte

stin

e82

3

Loon

(Com

mon

loon

–G

avia

imm

er)

Edw

ards

iella

tard

aIn

test

inal

cont

ent

Hae

mor

rhag

icen

terit

is,i

ntes

tinal

cont

entd

ark

and

tarry

823

cont

inue

d



26 Chapter 1

Pelic

anBr

own

pelic

an(P

elec

annu

soc

cide

ntal

isca

rolin

ensi

s )

Edw

ards

iella

tard

aIn

test

inal

cont

ent,

lung

,liv

erH

aem

orrh

agic

ente

ritis

,int

estin

alco

nten

tdar

kan

dta

rry82

3

Peng

uin

Gen

too

peng

uin

from

sub-

Anta

rctic

regi

onSa

lmon

ella

ente

ritid

is,S

.hav

ana,

S.ty

phim

uriu

mC

arrie

rsta

tus

612

Mac

aron

ipen

guin

( Eud

ypte

sch

ryso

loph

us)

Burk

hold

eria

(Pse

udom

onas

)ps

eudo

mal

lei

Pin-

poin

t,w

hite

lesi

ons

inliv

eran

dlu

ng,

fluid

inai

rsac

s,or

gani

smin

sple

en,l

iver

,he

artb

lood

Mel

ioid

osis

516

Peng

uins

( Apt

enod

ytes

pata

goni

ca,

Eudy

ptes

cres

tatu

s,Py

osce

lispa

pua ,

Sphe

nisc

usde

mer

sus,

Sphe

nisc

ushu

mbo

ldti)

Prov

iden

cia

fried

eric

iana

Isol

ated

from

faec

esof

capt

ive

peng

uins

559

Shad

Giz

zard

shad

( Dor

osom

ace

pedi

anum

Lesu

eur)

Aero

mon

asso

bria

Isol

ated

inpu

recu

lture

from

kidn

ey,l

iver

,sp

leen

Isol

ates

path

ogen

icfo

rfin

gerli

ngtro

utin

viru

lenc

eas

say

Epiz

ootic

insp

awni

ngfe

mal

es75

0

Sea

brea

mSe

eun

derB

ream

Sea

gras

sVi

brio

aero

gene

sVi

brio

diaz

otro

phic

usPr

esen

tin

sedi

men

t31

969

2

Seah

orse

( Hip

poca

mpu

sku

daan

dH

ippo

cam

pus

spec

ies)

Vibr

ioha

rvey

iEx

tern

alha

emor

rhag

es,h

aem

orrh

agic

liver

,asc

ites

fluid

Mor

talit

y11 13

5

Sea

drag

on,L

eafy

sea

drag

onPh

ycod

urus

equi

sVi

brio

harv

eyi

Isol

ated

inpu

recu

lture

from

hear

tblo

od,

liver

Dea

th.P

atho

geni

city

ofth

eor

gani

smno

tdet

erm

ined

135

Seal

.See

unde

rSEA

MAM

MAL

S

Sea

Lion

.See

unde

rSEA

MAM

MAL

S

SEA

MAM

MAL

S(O

rder

Cet

acea

)Se

am

amm

als

incl

ude

the

thre

em

amm

alia

nor

ders

cons

ider

edto

betru

eaq

uatic

mam

mal

s.Th

ese

are

Cet

acea

(wha

les,

dolp

hins

and

porp

oise

s),

Pinn

iped

ia(s

eals

,sea

-lion

san

dw

alru

ses)

,and

Sire

nian

s(s

eaco

ws)

(Fos

tere

tal.,

2002

).

Dolp

hin

Dol

phin

fish

–se

eM

ahi-m

ahi

Host

Path

ogen

Norm

alflo

raTi

ssue

site

Dise

ase

stat

usRe

f

Tab

le1.

1.C

ontin

ued.




Atla

ntic

whi

te-s

ided

dolp

hin

( Lag

enor

hync

hus

acut

us)

1.Br

ucel

lace

tace

ae2.

Hel

icob

acte

rcet

orum

1.O

esop

hage

alul

cera

tion,

abor

tion,

hepa

tican

dsp

leni

cco

agul

ativ

ene

cros

is2.

Org

anis

mfo

und

ingl

andu

larm

ucos

aof

mai

nst

omac

h

1.Br

ucel

losi

s2.

Gas

tric

ulce

ratio

n26

232

726

732

732

8

Amaz

onfre

shw

ater

dolp

hin

( Inia

geof

frens

is)

Stre

ptoc

occu

sin

iae

Subc

utan

eous

absc

esse

sG

olfb

alld

isea

se62

5

Turs

iops

adun

cas

Past

eure

llam

ulto

cida

Inte

stin

alha

emor

rhag

ean

dba

cter

aem

ia.

Sour

ceof

infe

ctio

nw

asco

ntam

inat

ion

from

loca

lbird

rook

ery

Ente

ritis

,mor

talit

y72

6

Turs

iops

geph

yreu

sH

elic

obac

ters

pp.

Pres

enti

nde

ntal

plaq

ueM

ayac

tas

are

serv

oirf

orga

stric

infe

ctio

nan

dga

stric

ulce

rs30

3

Atla

ntic

bottl

enos

edo

lphi

n( T

ursi

ops

trunc

atus

)1.

Bruc

ella

spec

ies

2.Bu

rkho

lder

iaps

eudo

mal

lei

3.C

lost

ridiu

mpe

rfrin

gens

4.Ed

war

dsie

llata

rda

5.Er

ysip

elot

hrix

rhus

iopa

thia

e6.

Hel

icob

acte

rcet

orum

6.M

annh

eim

iaha

emol

ytic

a8.

Phot

obac

teriu

mda

mse

lae

ssp.

dam

sela

e9.

Phot

obac

teriu

mda

mse

lae

ssp.

pisc

icid

a

1.Ab

ortio

n2.

Oed

ema,

haem

orrh

age

and

nodu

les

inlu

ngs

3.Ab

sces

sin

dors

alm

uscl

e4.

Puru

lent

mas

titis

5.U

lcer

son

skin

seen

asrh

ombo

idpl

aque

s,lu

ngs

cong

este

dan

doe

dem

atou

s.O

rgan

ism

inal

lorg

ans

6.O

rgan

ism

cultu

red

from

faec

es7.

Hae

mor

rhag

ictra

chei

tis

1.Br

ucel

losi

s2.

Mel

ioid

osis

3.C

lost

ridia

lmyo

sitis

4.M

astit

is5.

Erys

ipel

as,s

eptic

aem

ia6.

Ulc

ers

inoe

soph

agus

and

fore

stom

ach

–ga

strit

is7.

Sept

icae

mia

8.W

ound

infe

ctio

n9.

Past

eure

llosi

s

349

247

312

292

328

726

Com

mon

dolp

hin

( Del

phin

usde

lphi

s )1.

Bruc

ella

ceta

ceae

2.H

elic

obac

ters

pp.

3.Pa

steu

rella

mul

toci

da4.

Stap

hylo

cocc

usde

lphi

ni

1.Su

bcut

aneo

usle

sion

2.O

rgan

ism

sfo

und

ingl

andu

larm

ucos

aof

mai

nst

omac

h3.

Inte

stin

alha

emor

rhag

e4.

Puru

lent

skin

lesi

ons

1.Br

ucel

losi

s2.

Gas

tric

ulce

ratio

n3.

Ente

ritis

4.Sk

inin

fect

ion

327

267

404

658

726

778

Paci

ficw

hite

-sid

eddo

lphi

n( L

agen

orhy

nchu

sob

liqui

dens

)1.

Burk

hold

eria

pseu

dom

alle

i2.

Hel

icob

acte

rcet

orum

1.O

edem

a,ha

emor

rhag

ean

dno

dule

sin

lung

s2.

Org

anis

mcu

lture

dfro

mfa

eces

1.M

elio

idos

is2.

Ulc

ers

inoe

soph

agus

and

fore

stom

ach

–ga

strit

is

349

328

Strip

eddo

lphi

n( S

tene

llaco

erul

eoal

ba)

1.Br

ucel

lace

tace

ae2.

Cry

ptoc

occu

sne

ofor

man

s(y

east

)3.

Actin

obac

illus

delp

hini

cola

1.M

enin

gitis

2.Pu

lmon

ary

infe

ctio

n3.

Isol

ated

from

vario

ustis

sues

1.Br

ucel

losi

s2.

Pulm

onar

ycr

ypto

cocc

osis

3.Pa

thog

enic

ityno

tdet

erm

ined

278

262

263

267

cont

inue

d



28 Chapter 1

Porp

oise

Har

bour

porp

oise

(Pho

coen

aph

ocoe

na)

1.Br

ucel

lace

tace

ae2.

Erys

ipel

othr

ixrh

usio

path

iae

3.St

rept

ococ

cus

dysg

alac

tiae

ssp.

dysg

alac

tiae

Lanc

efie

ldG

roup

L4.

Vago

cocc

usfe

ssus

5.Ac

tinob

acillu

sde

lphi

nico

la6.

Actin

omyc

esm

arim

amm

aliu

m7.

Actin

obac

illus

scot

iae

8.Ar

cano

bact

eriu

mpl

uran

imal

ium

9.Ph

ocoe

noba

cter

uter

i

1.Ly

mph

node

s2.

Cut

aneo

usle

sion

s3.

Inte

stin

e,ki

dney

,lun

g,sp

leen

4,5.

Isol

ated

from

vario

ustis

sues

6.Lu

ng7.

Live

r,lu

ng,b

rain

and

sple

enof

stra

nded

porp

oise

9.U

teru

s

1.Br

ucel

losi

s2.

Erys

ipel

as3.

Sept

icae

mia

,br

onch

opne

umon

ia,m

yoca

rditi

s,py

elon

ephr

itis

4,5,

6,7,

8,9.

Path

ogen

icity

not

dete

rmin

ed

262

263

265

266

267

369

370

404

480

658

686

727

Sea

lion

Sea

lion

( Zal

ophu

sca

lifor

nian

us)

1 .Bu

rkho

lder

iaps

eudo

mal

lei

2.C

lost

ridiu

mpe

rfrin

gens

3.Ed

war

dsie

llata

rda

4.Es

cher

ichi

aco

li5.

Past

eure

llam

ulto

cida

6.Sa

lmon

ella

heid

elbe

rg,S

.ne

wpo

rt ,S.

oran

ienb

urg

1.O

edem

a,ha

emor

rhag

ean

dno

dule

sin

lung

s2.

Infe

ctio

nin

mus

cle

with

gas

and

pus

3.Ab

sces

ses

inlu

ngs,

sang

uino

-muc

opur

ulen

texu

date

intra

chea

and

term

inal

bron

chi

4.Ve

rruco

us,g

rey-

red

lesi

ons

inat

riove

ntric

ular

valv

es5.

Yello

w,p

us-fi

lled

fluid

inpl

eura

lcav

ity6.

Rec

over

edfro

mhe

alth

yan

imal

s

1.M

elio

idos

is2.

Clo

strid

ialm

yosi

tis3.

Bact

eria

lpne

umon

ia,

resp

irato

ryin

fect

ion

Path

ogen

icity

notc

oncl

usiv

e4.

Endo

card

itis

5.M

orta

lity

6.N

on-p

atho

geni

cor

carri

erst

atus

298

312

349

430

435

804

Seal

s( P

inni

pedi

a)

Com

mon

seal

orH

arbo

urse

al( P

hoca

vitu

lina)

1.Br

ucel

lapi

nnip

edia

e2.

Myc

opla

sma

phoc

icer

ebra

le3.

Myc

opla

sma

phoc

irhin

is4.

Stre

ptoc

occu

sph

ocae

5.Va

goco

ccus

fess

us

6.Ar

cano

bact

eriu

m(C

oryn

ebac

teriu

m)p

hoca

e7.

Arth

roba

cter

nasi

phoc

ae8.

Atop

obac

terp

hoca

e9.

Myc

opla

sma

phoc

idae

1.G

astri

cly

mph

node

,spl

een

2.Is

olat

edfro

mbr

ain,

hear

t,lu

ng,n

ose,

thro

at3.

Isol

ated

from

pus

inlu

ng4.

Lesi

ons

inlu

ng,e

xuda

tein

bron

chi

5.O

rgan

ism

isol

ated

from

liver

and

kidn

eyin

pure

grow

th6.

Org

anis

min

nasa

lpas

sage

1.Br

ucel

losi

s2,

3.As

soci

ated

with

resp

irato

rydi

seas

e4.

Pneu

mon

ia–

oppo

rtuni

stic

infe

ctio

nin

seal

mor

billiv

irus

infe

ctio

n5,

6,7,

8.Pa

thog

enic

ityno

tkn

own

404

182

295

449

267

369

479

613

Host

Path

ogen

Norm

alflo

raTi

ssue

site

Dise

ase

stat

usRe

f

Tab

le1.

1.C

ontin

ued.




7.O

rgan

ism

isol

ated

from

inte

stin

ean

dly

mph

node

s.Th

ean

imal

had

lym

phad

enop

athy

and

pulm

onar

yha

emor

rhag

e9.

Isol

ated

from

resp

irato

rytra

ct

9.Av

irule

nt65

866

070

0

Gre

yse

al( H

alic

hoer

usgr

ypus

)1.

Aero

mon

ashy

drop

hila

2.Br

ucel

lapi

nnip

edia

e3.

Burk

hold

eria

pseu

dom

alle

i4.

Stre

ptoc

occu

sph

ocae

5.Ac

tinom

yces

mar

imam

mal

ium

6.Ar

cano

bact

eriu

mph

ocae

1.O

rgan

ism

inlu

ngan

dliv

er.I

sola

tepo

sitiv

efo

raer

olys

inge

ne,c

ytot

oxin

and

haem

olys

in2.

Isol

ated

from

lung

3.O

edem

a,ha

emor

rhag

ean

dno

dule

sin

lung

s4.

Lesi

ons

inlu

ng,e

xuda

tein

bron

chi

5.Is

olat

edfro

min

test

ine

1.Se

ptic

aem

ia2.

Bruc

ello

sis

3.M

elio

idos

is4.

Pneu

mon

ia–

oppo

rtuni

stic

infe

ctio

nin

seal

mor

billiv

irus

infe

ctio

n5,

6.Pa

thog

enic

ityno

tcle

ar.

Isol

ated

from

seal

sw

ithse

ptic

aem

iaan

dpn

eum

onia

454

349

262

370

267

613

700

Har

pse

al( P

hoca

groe

nlan

dica

)Br

ucel

lasp

p.O

rgan

ism

inly

mph

node

sBr

ucel

losi

s26

1

Hoo

ded

seal

( Cys

toph

ara

cris

tata

)1.

Bruc

ella

pinn

iped

iae

2.Ac

tinom

yces

mar

imam

mal

ium

2.Is

olat

edfro

mlu

ng1.

Bruc

ello

sis,

stre

ss-re

late

d2.

Path

ogen

icity

notd

eter

min

ed26

237

026

7

Nor

ther

nfu

rsea

l(C

allo

rhin

usur

sinu

s )1.

Salm

onel

laad

elai

de,S

.he

idel

berg

,S.n

ewpo

rt,S.

oran

ienb

urg

2.Ac

inet

obac

terc

alco

acet

icus

,Ac

tinob

acillu

ssp

p.,A

erom

onas

eucr

enop

hila

,Alc

alig

enes

faec

alis

,E.

coli,

Ente

roba

cter

spp.

,Kl

ebsi

ella

spp.

,Mor

axel

lasp

p.,

Pseu

dom

onas

fluor

esce

ns,

Pseu

dom

onas

spp.

,S.e

pide

rmid

is,

Stre

ptoc

occu

ssp

p.3.

Baci

llus

spp.

,Cor

yneb

acte

rium

spp.

,E.c

oli,

List

eria

spp.

,M

orax

ella

spp.

,Nei

sser

iacu

nicu

li,Pr

oteu

sm

irabi

lis,S

taph

yloc

occu

sep

ider

mid

is,S

trept

ococ

cus

spp.

1.R

ecov

ered

from

heal

thy

anim

als

2.R

ectu

m3.

Oro

phar

ynx

1.N

on-p

atho

geni

cor

carri

erst

atus

2,3.

Nor

mal

flora

298

779

Rin

ged

seal

( Pho

cahi

spid

a)Br

ucel

lasp

ecie

sO

rgan

ism

inly

mph

node

sBr

ucel

losi

s26

1

cont

inue

d



30 Chapter 1

Sout

hG

eorg

ian

Anta

rctic

furs

eal

Salm

onel

laen

terit

idis

,S.h

avan

a,S.

new

port,

S.ty

phim

uriu

mC

arrie

rsta

tus

612

Seal

Myc

opla

sma

Seco

ndar

yin

fect

ion

due

tose

alm

orbi

lliviru

s

Sout

hern

elep

hant

seal

( Miro

unga

leon

ina )

Fack

lam

iam

iroun

gae

Nas

alsw

abIs

olat

edfro

map

pare

ntly

heal

thy

juve

nile

anim

al36

8

Wha

le

Belu

gaw

hale

,Whi

tew

hale

( Del

phin

apte

rus

leuc

as)

1.H

elic

obac

terc

etor

um2.

Myc

obac

teriu

mm

arin

um1.

Inap

pete

nce

and

leth

argy

.Oes

opha

geal

and

fore

stom

ach

ulce

rs2.

Pyog

ranu

lom

atou

sde

rmat

itis

and

pann

icul

itis

1.G

astri

tis2.

Myc

obac

terio

sis

329

327

328

111

Fals

eki

llerw

hale

( Pse

udor

cacr

assi

dens

)Bu

rkho

lder

iaps

eudo

mal

lei

Oed

ema,

haem

orrh

age

and

nodu

les

inlu

ngs

Mel

ioid

osis

349

Kille

rwha

le( O

rcin

usor

ca)

1.Bu

rkho

lder

iaps

eudo

mal

lei

2.C

andi

dasp

p.3.

Clo

strid

ium

perfr

inge

ns

1.O

edem

a,ha

emor

rhag

ean

dno

dule

sin

lung

s2.

Nec

rotic

skin

lesi

ons,

blow

hole

lesi

ons,

can

beco

me

syst

emic

3.To

xaem

ia,o

edem

atou

sly

mph

node

s,liq

uefie

dm

uscl

e

1.M

elio

idos

is2.

Infe

ctio

n3.

Clo

strid

ialm

yosi

tis

349

312

726

Min

kew

hale

( Bal

aeno

pter

aac

utor

ostra

ta)

1.Br

ucel

lasp

ecie

s2.

Gra

nulic

atel

laba

laen

opte

rae

1.Is

olat

edfro

mliv

eran

dsp

leen

2.Is

olat

edfro

mpu

regr

owth

from

liver

and

kidn

eyof

beac

hed

wha

le

1.D

isea

sest

atus

notk

now

n.W

hale

caug

htdu

ring

com

mer

cial

fishi

ng2.

Path

ogen

icity

notd

eter

min

ed

171

179

478

Paci

ficpi

lotw

hale

( Glo

bice

phal

asc

amm

oni )

Can

dida

spp.

Infe

ctio

nof

nare

sus

ually

asa

seco

ndar

yin

fect

ion

Infe

ctio

n72

6

Sow

erby

’sbe

aked

wha

leAc

tinob

acillu

sde

lphi

nico

laIs

olat

edfro

mva

rious

orga

nsPa

thog

enic

ityno

tdet

erm

ined

263

Sea

Urch

inVi

brio

diaz

otro

phic

usPa

rtof

gutf

lora

319

Shar

ks

Brow

nsh

ark

( Car

char

hinu

spl

umbe

us)

Phot

obac

teriu

mda

mse

lae

ssp.

dam

sela

e31

4

Host

Path

ogen

Norm

alflo

raTi

ssue

site

Dise

ase

stat

usRe

f

Tab

le1.

1.C

ontin

ued.




Nur

sesh

ark

(Ore

ctol

obus

orna

tus)

.An

imal

held

inca

ptiv

ityPh

otob

acte

rium

dam

sela

ess

p.da

mse

lae

Org

anis

mis

olat

edfro

mor

gans

Mor

talit

y61

8

Sand

bars

hark

Vibr

io(c

arch

aria

e)ha

rvey

iIs

olat

edfro

mki

dney

316

Smoo

thdo

gfis

h( M

uste

lus

cani

sM

itchi

ll)an

dSp

iny

dogf

ish

( Squ

alus

acan

thia

sL)

Alte

rom

onas

spp.

,Pho

toba

cter

ium

spp.

,Pse

udom

onas

spp.

,Sh

ewan

ella

putre

faci

ens,

Vibr

iosp

p.

Alli

sola

ted

from

lesi

ons

inth

ehe

adki

dney

ofhe

alth

ysh

arks

The

role

ofth

ele

sion

s,fro

mw

hich

thes

eor

gani

sms

wer

ecu

lture

d,in

mor

bidi

tyan

dm

orta

lity

isun

know

n

109

Blac

ktip

shar

k( C

arch

arhi

nus

limba

tus )

,Lem

onsh

ark

(Neg

aprio

nbr

eviro

stris

Poey

),N

urse

shar

k( G

ingl

ymos

tom

aci

rratu

m),

Tige

rsh

ark

( Gal

eoce

rdo

curv

ieri)

1.Ae

rom

onas

salm

onic

ida

Alte

rom

onas

spp.

Mor

axel

lasp

p.N

eiss

eria

spp.

Phot

obac

teriu

mda

mse

lae

ssp.

dam

sela

ePh

otob

acte

rium

dam

sela

ess

p.pi

scic

ida

Ples

iom

onas

shig

ello

ides

Vibr

ioal

gino

lytic

usVi

brio

harv

eyi

2.Vi

brio

harv

eyi

3.Ph

otob

acte

rium

dam

sela

ess

p.da

mse

lae

1.Is

olat

edfro

mva

rious

tissu

esi

tes

2.Ac

tive

sple

nic

and

hepa

ticdi

seas

eon

hist

olog

ical

exam

inat

ion

1.Pa

thog

enic

ityno

tass

esse

d2.

Isol

ated

from

expe

rimen

tally

infe

cted

Lem

onsh

arks

butd

idno

tsho

wcl

inic

aldi

seas

e.St

ress

-rela

ted

3.N

otre

cove

red

from

expe

rimen

tally

inoc

ulat

edLe

mon

shar

ks

315

316

Shea

tfish

,Wel

sca

tfish

( Silu

rus

glan

isLi

nnae

us)

1.Fl

avob

acte

rium

bran

chio

philu

m2.

F.co

lum

nare

1.Ba

cter

ium

atta

ches

togi

llep

ithel

ium

2.O

rgan

ism

isol

ated

from

kidn

ey,s

kin

blan

ched

and

ulce

rate

d

1.BG

D2.

Col

umna

risdi

seas

e88 25

180

2

Shrim

p

Blac

ktig

erpr

awn

( Pen

aeus

mon

odon

)1.

Vibr

ioha

rvey

i2.

Vibr

iopa

raha

emol

ytic

us3.

Vibr

iosp

lend

idus

II

1,2,

3.In

fect

she

pato

panc

reas

whe

reth

ere

isan

infla

mm

ator

yre

spon

sein

the

inte

rtubu

lars

inus

es

1,2,

3.M

orta

litie

s36

341

0

Brin

esh

rimp

( Arte

mia

spec

ies)

Vibr

iopr

oteo

lytic

usAf

fect

sm

icro

villi,

disr

upts

gute

pith

elia

lcel

lju

nctio

ns,d

evas

tate

sce

llsan

dtis

sues

inth

ebo

dyca

vity

Dea

ths

135

788

Chi

nese

shrim

p( P

enae

usch

inen

sis )

1.Vi

brio

algi

noly

ticus

2.Vi

brio

harv

eyi

3.Vi

brio

vuln

ificu

sse

rogr

oup

E

4.Vi

brio

paci

nii

1,2.

Affe

cts

larv

alde

velo

pmen

tfro

mzo

east

age

onw

ards

.Ina

ctiv

ity,a

nore

xia,

opaq

uene

ssan

dla

rvae

settl

eto

the

botto

mof

the

tank

1,2,

3.Vi

brio

sis,

mor

talit

y4.

Isol

ated

from

heal

thy

shrim

p98 30

677

7

cont

inue

d



32 Chapter 1

Fairy

shrim

p(B

ranc

hipu

ssc

haef

feri

Fish

er,C

hiro

ceph

alus

diap

hanu

sPr

évos

t,St

rept

ocep

halu

sto

rvic

orni

sW

aga)

Aero

mon

ashy

drop

hila

Blac

kno

dule

son

thor

acic

appe

ndag

es,

the

cerc

opod

san

dan

tenn

aeBl

ack

dise

ase

220

Gia

ntfre

shw

ater

praw

n( M

acro

bran

chiu

mro

senb

ergi

i)1.

Aero

mon

asca

viae

2.A.

vero

niis

sp.v

eron

ii3.

Lact

ococ

cus

garv

ieae

3.Ye

llow

ish-

whi

tesp

ots

inm

uscl

e,w

hitis

hm

uscl

ean

dsw

olle

n,ye

llow

hepa

topa

ncre

as,f

luid

accu

mul

atio

nbe

twee

ncu

ticle

and

mus

cle

tissu

e

1.2.

Path

ogen

icin

viru

lenc

est

udie

s3.

Mor

talit

y

156

723

Kuru

ma

praw

n( P

enae

us[M

arsu

pena

eus]

japo

nicu

s,Pe

naeu

sst

yliro

stris

)

1.Vi

brio

harv

eyi

2.Vi

brio

pena

eici

da3.

Acin

etob

acte

rspp

.,Al

tero

mon

assp

p.,B

acillu

ssp

p.,

Cor

yneb

acte

rium

spp.

,Fl

avob

acte

rium

spp.

,Mic

roco

ccus

spp.

,Mor

axel

lasp

p.,

Pseu

dom

onas

spp.

,St

aphy

loco

ccus

spp.

1.Bl

ack

spot

son

exos

kele

ton

2.Se

ptic

aem

ia3.

Isol

ated

from

appa

rent

lyhe

alth

ysh

rimp,

butn

otfro

mdi

seas

edsh

rimp

1.M

orta

lity

2.Sy

ndro

me

933.

Non

-pat

hoge

nic

187

388

198 23

Whi

tesh

rimp

( Pen

aeus

vann

amei

Boon

e)1.

Myc

obac

teriu

mpe

regr

inum

2.Vi

brio

harv

eyi

Vibr

ioxu

ii1.

Mul

tifoc

al,m

elan

ized

nodu

larl

esio

nson

cara

pace

2.Bl

ack

spot

son

exos

kele

ton

1.O

ppor

tuni

stic

infe

ctio

n2.

Mor

talit

y3.

Path

ogen

icity

notk

now

n

23 551

740

Snak

ehea

dfis

h( C

hann

ast

riatu

sFo

wle

r)M

ycob

acte

rium

porif

erae

,lat

erid

entif

ied

byPC

Rto

beM

.for

tuitu

mIn

tern

alno

dula

rles

ions

Myc

obac

terio

sis

633

756

Snoo

k( C

entro

pom

usun

deci

mal

isBl

och)

Vibr

ioha

rvey

iO

paqu

eco

rnea

Infe

ctio

n45

3

Sole

( Sol

ease

nega

lens

isKa

up)

Phot

obac

teriu

mda

mse

lae

ssp.

pisc

icid

aH

aem

orrh

agic

sept

icae

mia

and

whi

tear

eas

ofgr

anul

oma

inth

eki

dney

,liv

er,

sple

en

Fish

past

eure

llosi

s85

5

Dov

erso

le( S

olea

sole

a)Te

naci

bacu

lum

mar

itim

umBl

iste

ring

ofth

esk

inbe

twee

nth

eca

udal

and

mar

gina

lfin

s,lo

ssof

derm

altis

sues

,w

hich

prog

ress

esto

necr

otic

ulce

rs

Blac

kpa

tch

necr

osis

90 539

Spad

efis

hAt

lant

icsp

adef

ish

( Cha

etod

ipte

rus

fabe

r Bro

usso

net)

Vibr

ioha

rvey

iBi

late

rale

xoph

thal

mia

,hae

mor

rhag

esin

and

arou

ndey

es,c

orne

alop

acity

Mor

talit

y23

Host

Path

ogen

Norm

alflo

raTi

ssue

site

Dise

ase

stat

usRe

f

Tab

le1.

1.C

ontin

ued.




Spon

ge,M

arin

esp

onge

( Hal

icho

ndria

bow

erba

nki).

Also

know

nas

Cru

mb-

of-b

read

spon

ge

Myc

obac

teriu

mpo

rifer

ae60

8

Squi

d

Lolig

ope

alei

1.Sh

ewan

ella

peal

eana

2.Vi

brio

loge

i1.

Asso

ciat

edw

ithth

ere

prod

uctiv

eor

gan,

the

acce

ssor

yni

dam

enta

lgla

nd,i

nfe

mal

es2.

Sym

bion

tin

light

orga

n

1.2

Nor

mal

flora

257

492

Haw

aiia

nse

piol

idsq

uid

( Eup

rym

nasc

olop

es)

Vibr

iofis

cher

iLi

ghto

rgan

Ligh

torg

ansy

mbi

ont

257

Sepi

ola

affin

is,S

.rob

usta

Vibr

iofis

cher

iVi

brio

loge

iLi

ghto

rgan

Sym

biot

icba

cter

iain

light

orga

n25

7

Stin

gray

( Das

yatis

past

inac

a)An

imal

she

ldin

capt

ivity

Phot

obac

teriu

mda

mse

lae

ssp.

dam

sela

eO

rgan

ism

isol

ated

from

orga

nsM

orta

lity

618

Strip

edja

ck,W

hite

Trev

ally

( Pse

udoc

aran

xde

ntex

Bloc

han

dSc

hnei

der)

1.Ph

otob

acte

rium

dam

sela

ess

p.pi

scic

ida

2.Ps

eudo

mon

asan

guilli

sept

ica

1.H

aem

orrh

agic

sept

icae

mia

and

whi

tear

eas

ofgr

anul

oma

inth

eki

dney

,liv

er,

sple

en2.

Hae

mor

rhag

esin

mou

th,n

ose,

oper

culu

man

dbr

ain.

Org

anis

min

kidn

ey

1.Fi

shpa

steu

rello

sis

2.M

orta

litie

s46

556

7

Stur

geon

Adria

ticst

urge

on( A

cipe

nser

nacc

arii

Bona

parte

)La

ctoc

occu

sga

rvie

aeIn

appe

tenc

e,irr

egul

arsw

imm

ing,

som

ebi

late

rale

xoph

thal

mia

and

abdo

min

alas

cite

s

Mor

talit

y66

9

Sibe

rian

stur

geon

( Aci

pens

erba

erii

baer

iiBra

ndt)

Yers

inia

ruck

eri

Can

bein

fect

edw

ithor

with

outc

linic

alsi

gns

Yers

inio

sis

797

Tenc

h( T

inca

tinca

L.)

Myc

opla

sma

mob

ileIs

olat

edfro

mgi

llsPa

thog

enic

ityno

tsta

ted

439

440

Tila

pia

Nile

tilap

ia( O

reoc

hrom

issp

.,O

.ni

lotic

usni

lotic

usLi

nnae

us)

Also

know

nas

StPe

ter’s

fish

1.Ed

war

dsie

llata

rda

2.St

rept

ococ

cus

agal

actia

e3.

Stre

ptoc

occu

sin

iae

1.Se

ptic

aem

ia,f

ocal

supp

urat

ive

orgr

anul

omat

ous

lesi

ons,

cuta

neou

sul

cera

tions

3.C

entra

lner

vous

syst

emin

volv

emen

t,le

thar

gy,e

rratic

swim

min

g

1.Ed

war

dsie

llosi

s2,

3.St

rept

ococ

cosi

s23

344

2

cont

inue

d

49Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 3:31:34 PM


34 Chapter 1

Tila

pia

(Sar

athe

rodo

n(T

ilapi

a)au

reus

)1.

Edw

ards

iella

icta

luri

2.St

rept

ococ

cus

inia

e2.

Loss

ofor

ient

atio

n,ex

opht

halm

ia,

pete

chia

ear

ound

anus

,mou

than

dpe

ctor

alfin

s,flu

idin

perit

onea

lcav

ity,

enla

rged

orga

ns

1.Sl

ight

lysu

scep

tible

2.M

orta

lity

621

627

Tila

pia

( Sar

othe

rodo

nni

lotic

us)

Also

know

nas

Nile

tilap

ia1.

Lact

ococ

cus

garv

ieae

2.St

rept

ococ

cus

inia

e3.

Vibr

ioch

oler

aeno

n-01

1.D

erm

alha

emor

rhag

ean

dex

opht

halm

ia,e

pica

rditi

s,pe

riton

itis,

pale

-col

oure

dliv

er,s

plen

omeg

aly,

nodu

lefo

rmat

ion

ingo

nads

2.Lo

ssof

orie

ntat

ion,

exop

htha

lmia

,pe

tech

iae

arou

ndan

us,m

outh

and

pect

oral

fins,

fluid

inpe

riton

ealc

avity

,en

larg

edor

gans

1,2.

Syst

emic

infe

ctio

n–

stre

ptoc

occo

sis

3.Fa

rmm

orta

lity.

May

beop

portu

nist

507

550

621

Turb

ot( S

coph

thal

mus

max

imus

Linn

aeus

)1.

Atyp

ical

Aero

mon

assa

lmon

icid

a.Al

soan

oxid

ase-

nega

tive

stra

in2.

Chr

yseo

bact

eriu

msc

opht

halm

um3.

List

onel

laan

guilla

rum

01an

d02

b,02

a4.

Myc

obac

teriu

mch

elon

aean

dM

.m

arin

um5.

Phot

obac

teriu

mda

mse

lae

ssp.

dam

sela

e6.

Phot

obac

teriu

mda

mse

lae

ssp.

pisc

icid

a7.

Pseu

dom

onas

angu

illise

ptic

a8.

Serra

tialiq

uefa

cien

s9.

Stre

ptoc

occu

spa

raub

eris

10.V

ibrio

sple

ndid

usbi

otyp

eI

11.E

nter

ovib

riono

rveg

icus

12.V

ibrio

chol

erae

non-

01an

dVi

brio

mim

icus

did

notc

ause

deat

hsin

viru

lenc

eas

say

13. V

ibrio

nept

uniu

s14

.Vib

riosc

opht

halm

i

1.Sk

inul

cers

2.Sw

olle

nin

test

ines

,hae

mor

rhag

esin

eye,

skin

and

jaw

,gill

hype

rpla

sia

4.G

ranu

lom

asin

orga

ns7.

Org

anis

min

head

–kid

ney

and

sple

en8.

Swol

len

kidn

eyan

dsp

leen

,yel

low

nodu

les,

foci

ofliq

uefa

ctio

nne

cros

is9.

Lesi

ons,

haem

orrh

age

inth

ean

alan

dpe

ctor

alfin

san

dpe

tech

iae

onth

eab

dom

en,e

xoph

thal

mia

and

pus

inth

eey

es9.

Hae

mor

rhag

esin

mou

th,a

bdom

inal

dist

ensi

on,r

eddi

shflu

idin

perit

onea

lca

vity

11,1

3,14

.Par

tofg

utflo

ra

1.M

orta

lity

2.G

illdi

seas

e,ha

emor

rhag

icse

ptic

aem

ia3.

Vibr

iosi

s4.

Myc

obac

terio

sis

5.M

orta

lity

6.Fi

shpa

steu

rello

sis

7.H

aem

orrh

agic

sept

icae

mia

8.O

ppor

tuni

stpa

thog

en,

mor

talit

y9.

Stre

ptoc

occo

sis,

hepa

tom

egal

y,m

ucoh

aem

orrh

agic

ente

ritis

10.E

pizo

otic

inju

veni

les

11,1

4.N

orm

alflo

ra13

.Pat

hoge

nici

tyno

tkno

wn

268

281

224

149 31 96 475

507

557

617

673

712

740

741

748

749

751

754

791

832

Host

Path

ogen

Norm

alflo

raTi

ssue

site

Dise

ase

stat

usRe

f

Tab

le1.

1.C

ontin

ued.




Turb

ot(C

olis

tium

nudi

pinn

isW

aite

)Br

ill( C

.gun

ther

i).Bo

thsp

ecie

sar

eal

sokn

own

asfla

tfish

Vibr

ioca

mpb

ellii-

like,

Vibr

iosp

lend

idus

IO

rgan

ism

inbr

ain,

kidn

ey,l

iver

,whi

chsh

owed

lesi

ons

and

haem

orrh

ages

Acut

em

orta

lity

inju

veni

les,

oppo

rtuni

stic

infe

ctio

n22

1

Yello

wta

il( S

erio

laqu

inqu

erad

iata

and

S.pu

rpur

asce

nsTe

mm

inck

and

Schl

egel

)Al

soca

lled

Rud

derfi

shan

dJa

pane

seam

berja

ck

1.La

ctoc

occu

sga

rvie

ae2.

List

onel

laan

guilla

rum

3.M

ycob

acte

rium

spp.

4.N

ocar

dia

serio

lae

5.Ph

otob

acte

rium

dam

sela

ess

p.da

mse

lae

6.Ph

otob

acte

rium

dam

sela

ess

p.pi

scic

ida

7.St

rept

ococ

cus

inia

e

1.Er

osio

nof

tail

fin,r

edne

ssof

anal

fin,

pete

chia

ein

side

oper

culu

m,e

xoph

thal

mia

4.Ab

sces

ses

inep

ider

mis

,for

mat

ion

oftu

berc

ules

ingi

lls,k

idne

ys,s

plee

n5.

Org

anis

min

sple

enan

dki

dney

,tox

inpr

oduc

ed6.

Bact

eria

lcol

onie

sof

whi

te-g

rey

colo

urse

enon

sple

enan

dki

dney

7.O

rgan

ism

isol

ated

from

brai

n

1.St

rept

ococ

cosi

s3.

Myc

obac

terio

sis

4.N

ocar

dios

is5.

Mor

talit

y6.

Fish

past

eure

llosi

s,m

orta

lity

7.Se

ptic

aem

ia

459

462

424

455

464

236

233

235

Wha

le.S

eeun

derS

EAM

AMM

ALS

Whi

tefis

h( C

oreg

onus

sp.),

Cis

co( C

oreg

onus

arte

diLe

sueu

r),La

kew

hite

fish

( Cor

egon

uscl

upea

form

isM

itchi

ll),P

eled

(Cor

egon

uspe

led

Gm

elin

)Fa

mily

Salm

onid

ae

1.Ae

rom

onas

salm

onic

ida

ssp.

salm

onic

ida

2.At

ypic

alAe

rom

onas

salm

onic

ida

3.Ps

eudo

mon

asan

guilli

sept

ica

4.Ye

rsin

iaru

cker

i

3.Pe

tech

ialh

aem

orrh

ages

onsk

in,m

outh

and

anus

,per

itone

uman

dliv

er4.

Dis

ease

isas

soci

ated

with

poor

envi

ronm

enta

lcon

ditio

ns

1.Sk

inul

cers

,fur

uncu

losi

s2.

Skin

ulce

rs3.

Hae

mor

rhag

icdi

seas

e4.

Yers

inio

sis

323

352

772

828

Wol

f-fis

h( A

narh

icha

slu

pus

Linn

aeus

),Sp

otte

dw

olf-f

ish

(A.

min

orO

lafs

en)

1.At

ypic

alAe

rom

onas

salm

onic

ida

2.C

arno

bact

eriu

mdi

verg

ens

3.C

arno

bact

eriu

msp

p.1.

Atyp

ical

furu

ncul

osis

2,3.

Part

ofin

test

inal

mic

roflo

ra64

8

Wra

sse

( Lab

ridae

)C

lean

erfis

hAt

ypic

alAe

rom

onas

salm

onic

ida

Hae

mor

rhag

icin

tern

alor

gans

,blo

ody

exud

ate

Furu

ncul

osis

468



Bacterial gill disease (BGD)

Bacterial gill disease is caused by the bacteriumFlavobacterium branchiophilum, which are largefilamentous Gram-negative rods. The bacteriumattaches to the epithelial surface of the gill(Snieszko, 1981; Ostland et al., 1994).

Bacterial kidney disease (BKD)

The disease agent is Renibacterium salmoninarum,which affects salmonids. The disease begins in achronic form that develops full expression whenthe fish are 1 year old. Systemic granulomatouslesions are seen in all organs but particularly inthe kidney, where grey, necrotic abscesses occurthroughout the tissue, causing enlargement andnecrosis of the kidney (OIE, 2000a).

Bacterial stomatitis (mouth rot)

This condition is seen as yellow-coloured plaqueon the teeth and the oral cavity in Atlantic salmonsmolts in their first year in seawater. Thecausative organism was identified as Tenaci-baculum maritimum. Some differences werenoted between these strains and the referencestrains. The strains from mouth rot had anoptimum temperature of 18–25°C, which isslightly lower than the reference strains, and theyalso had an optimum for media containing 70%seawater. Many of the strains from mouth rot hada- and b-glucosidase activity, an unusual findingfor T. maritimum isolates (Ostland et al., 1999b).

Black patch necrosis

The disease affects Dover sole and is caused byT. maritimum. Initially there is a slight blisteringof the skin surface, which develops into loss ofepithelium and necrotic ulcers (Bernardet et al.,1990).

Brown ring disease (BRD)

This is a disease caused by Vibrio tapetis, andmay cause mass mortality in Manila clams

(Ruditapes philippinarum). It was first reportedin France in 1987 where it caused highmortality in cultured stocks. The disease ischaracterized by a ring of brown deposit ofseveral layers seen between the pallial lineand the edge of the shell. A shell repair processoccurs and this can be seen as white calcifiedareas that cover the brown deposit (Paillard andMaes, 1994).

Enteric redmouth disease (ERM)

This disease is caused by the Gram-negativebacterium Yersinia ruckeri. It is an economicallyserious disease in the rainbow trout farmingindustry of many countries. Clinical signs arehaemorrhagic areas around the mouth, intestinesand other organs.

There are a number of modes of transmissionof the disease, including birds, wild fish andcarrier fish (Willumsen, 1989). The organism alsoforms biofilms on fish tanks, which is a source ofreinfection (Coquet et al., 2002).

Enteric septicaemia of catfish (ESC)

The disease is caused by the bacteriumEdwardsiella ictaluri, and is a major diseaseproblem for the commercial channel catfishindustry. Ornamental species and salmonids aresusceptible and it has also been reported in seabass Dicentrarchus labrax (Hawke et al., 1981).Disease resistance is variable among channelcatfish species, with blue catfish (Ictalurusfurcatus) and Red River strain showing the mostdisease resistance (Wolters and Johnson, 1994).Outbreaks of the disease occur in the tempera-ture range of 18–28°C, although low-levelmortality and carrier status may be seen attemperatures outside this range.

In the acute form, the disease expressesas acute septicaemia. Petechial haemorrhagesoccur around the mouth, the throat and thefins, and internally in the liver and otherorgans. The organism crosses the intestinalmucosa into the internal organs. A chronic formof the disease occurs as a meningoencephalitiswith behavioural changes, and ulceration or‘hole-in-the-head’ (Hawke et al., 1981; OIE,2000b).

36 Chapter 1



Furunculosis

The causative organism is Aeromonassalmonicida spp. salmonicida, which causes highrates of mortality in salmonid fish. The disease ischaracterized by boil-like inflammatory lesions,known as furuncles, which can penetrate deepinto the musculature. These clinical signs are notalways present (OIE, 2000a).

Pasteurellosis

The disease known as fish pasteurellosis is causedby Photobacterium damselae spp. piscicida(previously Pasteurella piscicida). It can affectmany cultured fish species throughout Japan,USA and Europe. It usually causes high mortality,with very few external or clinical signs of disease,although a dark body colour may be seen. Thespleen usually shows white nodules or tuberclesfrom which the organism can be cultured(Kusuda and Yamaoka, 1972; Hawke et al.,1987; Toranzo et al., 1991; Baptista et al., 1996;Candan et al., 1996; Fukuda et al., 1996).

Petechiae

Petechiae (pinpoint-sized areas of haemorrhage)on the underside and on the abdomen of fishmay indicate septicaemia and generalizedbacteraemia.

Rainbow trout fry syndrome

This condition has been reported from theUK, where diseased fish have anaemia,exophthalmia, pale gills and increased pig-mentation of the skin. The abdomen is swollenwith ascites fluid in the peritoneal cavity and thekidney is swollen. As yet no definitive organismhas been deemed to be the causative organism.A number of bacteria have been suggestedto be associated with the condition. Theseinclude Flavobacterium columnare (previouslyCytophaga columnaris), Janthinobacterium spp.,Micrococcus luteus and Planococcus spp. (Austinand Stobie, 1992b).

Skin and tail rot

Skin and tail rot may be associated with bacteriasuch as Pseudomonas spp., Aeromonas spp.,Flavobacterium or Flexibacter spp. However, thecondition is usually due to poor water quality andis therefore regarded as primarily a managementproblem.

Skin ulcers

Skin ulcers are seen as boil-like or pimple-likeand convex. They may be caused by a varietyof bacteria including typical Aeromonassalmonicida and the many species of atypicalA. salmonicida. When the disease cause isListonella anguillarum, ulcers may be seenas boils with red putrefying and liquefying flesh.

Streptococcosis

This disease is caused by the Gram-positivecocci, Streptococcus agalactiae, a group B,b-haemolytic streptococcus. Clinical signsexhibited with this infection include abnormalbehaviour such as erratic swimming, whirling onthe surface, and C-shaped curvature of the bodywhile swimming at the surface. The eye may beopaque, exophthalmia may be seen and haemor-rhages may be present. Haemorrhagic areas arealso seen on the head and body, in particulararound the mouth, snout, operculum and fins.There may be a haemorrhagic enteritis.

Streptococcus iniae infection

Clinical signs of the disease may vary accordingto the species of fish infected. Chronic infectionseems to occur at a temperature of 25°C,whereas a more acute form of the disease is seenwhen the temperature ranges from 28 to 32°C(Yuasa et al., 1999).

1.3 Bacteria and Relationship to Host

Table 1.2 lists the bacteria that may be pathogensor saprophytes of fish and other aquatic animals.




Abio

troph

iaba

laen

opte

rae

spp.

nov.

See

Gra

nulic

atel

laba

laen

opte

rae

com

.nov

.17

9

Abio

troph

iael

egan

s(n

utrit

iona

llyva

riant

Stre

ptoc

occi

)Se

eG

ranu

licat

ella

eleg

ans

Achr

omob

acte

rxyl

osox

idan

sss

p.de

nitri

fican

s(p

revi

ousl

yAl

calig

enes

deni

trific

ans )

659

Acin

etob

acte

rbau

man

nii

Gen

ospe

cies

2H

uman

infe

ctio

nsIs

olat

edfro

mhu

man

patie

nts

and

envi

ronm

ent

110

Acin

etob

acte

rcal

coac

etic

usG

enos

peci

es1

1.En

viro

nmen

talo

rgan

ism

2.O

ralc

avity

flora

1.Is

olat

edfro

mso

il2.

Turtl

es2.

Part

ofno

rmal

flora

110

300

301

Acin

etob

acte

rhae

mol

ytic

usG

enos

peci

es4

Path

ogen

icity

notk

now

nfo

rfis

hLe

sion

sin

orga

nsIs

olat

edfro

mAt

lant

icsa

lmon

,cha

nnel

catfi

sh,e

nviro

nmen

tand

hum

ancl

inic

alsa

mpl

es

Nor

way

,USA

110

Actin

obac

illus

delp

hini

cola

Path

ogen

icity

notd

eter

min

edIs

olat

edfro

mva

rious

tissu

esSe

am

amm

als

–ha

rbou

rpor

pois

e,st

riped

dolp

hin,

Sow

erby

’sbe

aked

wha

leSc

otla

nd26

3

Actin

obac

illus

scot

iae

Path

ogen

icity

notd

eter

min

edO

rgan

ism

sin

liver

,lun

g,br

ain,

sple

enIs

olat

edfro

mst

rand

edpo

rpoi

seSc

otla

nd26

5

Actin

omyc

esm

arim

amm

aliu

mPa

thog

enic

ityno

tdet

erm

ined

Isol

ated

from

vario

ustis

sues

,(lu

ng,l

iver

,spl

een,

inte

stin

e)w

ithot

hero

rgan

ism

s

Dea

dho

oded

seal

,dea

dgr

eyse

al,d

ead

harb

ourp

orpo

ise

UK

370

Aequ

oriv

itaan

tarc

tica,

A.lip

olyt

ica,

A.cr

ocea

,A.s

ublit

hinc

ola

Envi

ronm

enta

liso

late

s(m

embe

rof

Flav

obac

teria

ceae

fam

ily)

Isol

ated

from

mar

ine

envi

ronm

ent–

seaw

ater

,sea

ice

Anta

rctic

a11

3

Aero

cocc

usvi

ridan

sAs

soci

ated

with

infe

ctio

nin

oeso

phag

eald

iver

ticul

umO

rgan

ism

inoe

soph

agea

lles

ions

.Lo

gger

head

sea

turtl

eSp

ain

755

Aero

cocc

usvi

ridan

sva

r.ho

mar

i(h

eavy

grow

th)

Hig

hm

orta

lity,

gaffk

emia

dise

ase,

fata

lsep

ticae

mia

May

see

pink

orre

dco

lora

tion

inth

eha

emol

ymph

.Org

anis

ms

mul

tiply

inth

ehe

pato

panc

reas

,ha

emol

ymph

and

toa

less

erex

tent

inth

ehe

arta

ndsk

elet

alm

uscl

e

Lobs

ter,

seaw

ater

and

sedi

men

tin

lobs

terp

onds

.Exi

stfre

e-liv

ing

inm

arin

ebe

ntho

s.C

rabs

and

othe

rcru

stac

eans

may

acta

sre

serv

oirs

orca

rrier

sof

the

orga

nism

Nor

way

,Can

ada

299

719

827

38 Chapter 1

Bact

eria

Dise

ase

Dise

ase

sign

sHo

st/is

olat

ion

site

Dist

ribut

ion

Ref

Tab

le1.

2.B

acte

rial p

atho

gens

and

sapr

ophy

tes

offis

han

dot

her

aqua

tican

imal

s.




Aero

mon

asal

losa

ccha

roph

ila(H

G15

)(p

revi

ousl

yH

G14

in19

95)

1.D

isea

sed

elve

rs2.

Dia

rrhoe

icst

ools

1.D

isea

sed

elve

rs2.

Faec

es1.

Elve

rson

eelf

arm

2.H

uman

1.Sp

ain

2.So

uth

Car

olin

a,U

SA52

7

Aero

mon

asbe

stia

rum

(HG

2)(fo

rmer

lyge

nom

ospe

cies

DN

Agr

oup

2,A.

hydr

ophi

la)

Path

ogen

icity

indo

ubt

Fish

,riv

erw

ater

,sea

wat

er,s

hellf

ish,

hum

anfa

eces

USA

13

Aero

mon

asca

viae

(HG

4)1.

Sept

icae

mia

,mor

talit

yw

hen

orga

nism

pres

enti

nhi

ghnu

mbe

rs2.

Infe

ctio

n,ga

stro

ente

ritis

1.D

erm

alul

cera

tion,

pres

enti

nhe

pato

panc

reas

2.In

fect

ion

1.Fr

eshw

ater

orna

men

talf

ish,

Atla

ntic

salm

on,o

ctop

us,g

iant

fresh

wat

erpr

awn,

turb

otla

rvae

2.H

uman

Ubi

quito

usin

the

envi

ronm

ent

Turk

ey,K

enya

,Tai

wan

21 723

Aero

mon

ascu

licic

ola

Pres

ence

inaq

uatic

spec

ies

notk

now

nIs

olat

edfro

mm

osqu

itoIn

dia

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Aero

mon

asen

chel

eia

(HG

16)

(pre

viou

sly

HG

11)

Non

-pat

hoge

nic

Hea

lthy

eels

,fre

shw

ater

Spai

n42

737

924

1

Aero

mon

aseu

cren

ophi

la(p

revi

ousl

yA.

punc

tata

ssp

punc

tata

)(H

G6 )

Non

-pat

hoge

nic

Asci

tes

ofca

rp,d

rinki

ngw

ater

,wel

lwat

erEu

rope

,Ger

man

y37

9

Aero

mon

asgr

oup

501

(form

erly

ente

ricgr

oup

501)

(HG

12)

See

A.sc

hube

rtii

Aero

mon

ashy

drop

hila

ssp.

hydr

ophi

la(H

G1)

(usu

ally

isol

ated

inhe

avy

reas

onab

lypu

regr

owth

fori

tto

beco

nsid

ered

the

prim

ary

path

ogen

)

1.H

aem

orrh

agic

sept

icae

mia

,pe

riton

itis,

reds

ore

dise

ase,

finro

t,re

d-fin

dise

ase,

mor

talit

y.As

soci

ated

with

the

fung

aldi

seas

eep

izoo

ticul

cera

tive

synd

rom

e,ca

used

byAp

hano

myc

esin

vada

ns,i

nca

tfish

and

snak

ehea

dfis

h(T

haila

nd,P

hilip

pine

s).

Opp

ortu

nist

icin

fect

ion

inse

alm

orbi

lliviru

sin

fect

ion

2.O

ppor

tuni

stic

and

prim

ary

path

ogen

3.Bl

ack

dise

ase

4.Pa

thog

enic

ityun

clea

r5.

Gas

troen

terit

is

1.Er

osiv

eor

ulce

rativ

ede

rmal

lesi

ons,

haem

orrh

age

onfin

san

dtru

nk,s

wel

ling

ofan

us,e

ryth

ema

2.R

ed-le

gfro

gdi

seas

e3.

Blac

kno

dule

son

thor

acic

appe

ndag

es4.

Isol

ated

inca

ses

ofab

ortio

n

1.Fr

eshw

ater

and

orna

men

talf

ish,

ayu,

chan

nelc

atfis

h,w

alki

ngca

tfish

,tila

pia,

trout

,tur

tles,

eels

,rep

tiles

,gre

yse

al,

occa

sion

ally

mar

ine

fish

–cu

lture

dAt

lant

icsa

lmon

,sea

bass

,sca

llop

larv

ae.I

ncre

ase

inor

gani

cm

atte

rand

tem

pera

ture

sab

ove

18°C

aid

prol

ifera

tion

ofA.

hydr

ophi

la.

Foun

din

fresh

,bra

ckis

han

dco

asta

lw

ater

s2.

Frog

s,fa

rm-ra

ised

bullf

rogs

3.Fa

irysh

rimps

4.C

attle

,hor

ses,

pigs

5.H

uman

s

1,2.

Ubi

quito

usin

the

envi

ronm

entw

orld

wid

e3.

Alge

ria,G

erm

any,

Spai

n

227

220 21 454 29 300

301

456

497

530

614

650

783

Aero

mon

ashy

drop

hila

ssp.

dhak

ensi

sgr

oup

BD-2

(HG

1)D

iarrh

oea

Cyt

otox

ican

dha

emol

ytic

prop

ertie

sIs

olat

edfro

mdi

arrh

oeal

child

ren

Bang

lade

sh38

3

cont

inue

d



Aero

mon

asja

ndae

i(H

G9)

(pre

viou

sly

HG

9A.

sobr

ia)

1.Pa

thog

en2.

Clin

ical

sign

ifica

nce

1.Ti

ssue

s2.

Isol

ated

from

bloo

d,w

ound

,di

arrh

oeal

stoo

ls

1.Ee

l2.

Hum

ans

1.Sp

ain

2.U

SA14

3

Aero

mon

asm

edia

(HG

5)1.

Envi

ronm

enta

lorg

anis

m2.

Clin

ical

,gas

troen

terit

is1.

Riv

erw

ater

.Pro

biot

icpr

oper

ties

agai

nstV

.tub

iash

ii2.

Hum

ans

1.U

K15 29

4

Aero

mon

aspo

poffi

iEn

viro

nmen

talo

rgan

ism

Drin

king

wat

erre

serv

oirs

Finl

and,

Scot

land

380

Aero

mon

assa

lmon

icid

ass

p.sa

lmon

icid

a(H

G3)

‘typi

cal’

A.sa

lmon

icid

a(p

rodu

ces

brow

npi

gmen

t)

Gol

dfis

hul

cerd

isea

se(G

UD

),fu

runc

ulos

isin

trout

and

salm

onD

erm

alul

cera

tion

show

ing

typi

cal

umbo

nate

furu

ncle

s.O

rgan

ism

spe

netra

teto

unde

rlyin

gtis

sues

,ki

dney

,coe

lom

icflu

id,s

plee

n,in

test

ine

Man

yfis

h;go

ldfis

h,ca

rp,s

ilver

perc

h,tro

ut,A

tlant

icsa

lmon

,mar

ine

floun

der,

gree

nbac

kflo

unde

r,ee

l,ca

tfish

,car

p,cl

eane

rfis

hFr

ogs,

afte

rfee

ding

from

cont

amin

ated

trout

Hig

hly

viru

lent

stra

infro

mN

orth

Amer

ica,

Euro

pe,U

K,U

SA.N

otpr

esen

tin

Aust

ralia

468

531

584

Aero

mon

assa

lmon

icid

ass

p.sa

lmon

icid

a(n

on-p

igm

ente

dst

rain

s)

Dis

ease

dsa

lmon

Hea

dki

dney

Farm

edAt

lant

icsa

lmon

Inje

ctio

nex

perim

ents

reve

alth

eno

n-pi

gmen

ted

stra

inpr

oduc

eda

high

erm

orta

lity

than

pigm

ente

dst

rain

s

Nor

way

450

Aero

mon

assa

lmon

icid

ass

p.ac

hrom

ogen

es‘A

typi

cal’

A.sa

lmon

icid

aVa

riety

ofpa

thol

ogie

s,ca

rper

ythr

oder

mat

itis,

gold

fish

ulce

rdi

seas

e,ul

cerd

isea

seof

floun

der

Larg

eop

ensk

inle

sion

ssu

rroun

ded

byar

eas

ofde

scal

atio

nw

ithso

ftene

dan

dha

emor

rhag

icde

rmis

Salm

onid

san

dno

n-sa

lmon

ids,

Atla

ntic

cod,

silv

erbr

eam

,per

ch,r

oach

Foun

din

fresh

wat

er,b

rack

ish

wat

eran

dm

arin

een

viro

nmen

ts

Wor

ldw

ide:

Aust

ralia

,Eng

land

,C

entra

land

Nor

ther

nEu

rope

,Ic

elan

d,Ja

pan,

Nor

thAm

eric

a,So

uth

Afric

a

186

534

830

Aero

mon

assa

lmon

icid

ass

p.m

asou

cida

1.‘A

typi

cal’

A.sa

lmon

icid

aSu

perfi

cial

skin

lesi

ons

1.Sa

lmon

ids

–(s

akur

amas

u–

Onc

orhy

nchu

sm

asou

and

pink

salm

on–

O.g

orbu

scha

)

Japa

n43

883

0

Aero

mon

assa

lmon

icid

ass

p.no

vaG

UD

.Cut

aneo

usul

cera

tive

dise

ase

ingo

ldfis

hC

utan

eous

ulce

rsG

oldf

ish,

salm

onid

s/no

n-sa

lmon

ids,

eel,

carp

,mar

ine

fish

Engl

and,

Japa

n,U

SA,

Wor

ldw

ide.

Aust

ralia

ngo

ldfis

hst

rain

sar

eth

ough

tto

belo

ngto

this

subs

peci

es.A

ustra

lian

salm

onid

sar

esu

scep

tible

144

535

695

761

824

825

Aero

mon

assa

lmon

icid

ass

p.pe

ctin

olyt

ica

Envi

ronm

enta

lorg

anis

mPo

llute

driv

erAr

gent

ina

615

Aero

mon

assa

lmon

icid

ass

p.sm

ithia

‘Aty

pica

l’A.

salm

onic

ida

Supe

rfici

alsk

inle

sion

sN

on-s

alm

onid

sEn

glan

d47 83

0

40 Chapter 1

Bact

eria

Dise

ase

Dise

ase

sign

sHo

st/is

olat

ion

site

Dist

ribut

ion

Ref

Tab

le1.

2.C

ontin

ued.




Aero

mon

assa

lmon

icid

a(a

typi

cals

train

s)M

orta

lity

Varie

tyof

path

olog

ies,

carp

eryt

hrod

erm

atiti

s,G

UD

,ulc

erdi

seas

eof

floun

der,

ulce

rhea

ddi

seas

eof

eels

,ulc

erat

ive

dise

ase

Skin

lesi

ons,

necr

osis

,ha

emor

rhag

icul

cers

,um

bona

tefu

runc

les

and

swel

ling

onhe

adof

eels

.Som

etim

esun

derly

ing

tissu

esaf

fect

ed,p

artic

ular

lyin

farm

edfis

h

Blen

ny,c

arp,

chub

,cle

aner

fish,

dab,

eels

,san

dee

ls,f

loun

der,

gold

fish,

min

now

,rai

nbow

trout

(sal

twat

er),

roac

h,ro

ckfis

h

Balti

cSe

a,D

enm

ark,

Engl

and,

Finl

and,

Japa

n,N

orw

ay,S

outh

Afric

a,U

SA

17 352

331

107

403

468

584

832

Aero

mon

assa

lmon

icid

aat

ypic

alst

rain

sO

xida

se-n

egat

ive

Dea

ths,

ulce

rativ

edi

seas

eLe

thar

gy,s

kin

ulce

rsTu

rbot

,and

floun

derf

rom

asa

lt-w

ater

fish

farm

,coh

osa

lmon

Den

mar

k,Ba

ltic

Sea,

USA

153

617

832

Aero

mon

assa

lmon

icid

aat

ypic

alst

rain

sG

row

that

37°C

Dea

than

dm

orbi

dity

Skin

ulce

rsC

arp,

gold

fish,

roac

hG

arde

npo

nds,

aqua

ria,r

iver

sEn

glan

d40

Aero

mon

assc

hube

rtii(

HG

12)

(pre

viou

sly

calle

den

teric

grou

p50

1)

1.En

viro

nmen

talo

rgan

ism

2.Se

ptic

aem

ia,g

astro

ente

ritis

,di

arrh

oea

2.Ab

sces

ses,

wou

nds,

pleu

ral

fluid

,blo

od2.

Hum

ans,

ofte

nim

mun

ocom

prom

ised

2.U

SA,P

uerto

Ric

oan

dso

uthe

rnco

asta

lsta

tes

ofU

SA34

8 2

Aero

mon

asso

bria

(HG

7)(n

owal

soca

lled

A.ve

roni

issp

.so

bria

)

1.Pe

riton

itis,

epiz

ootic

ulce

rativ

esy

ndro

me.

Path

ogen

icity

indo

ubt

2.D

iarrh

oea,

rena

lfai

lure

,cel

lulit

is,

necr

otiz

ing

gast

roen

terit

isin

adul

t

1.Pe

riton

itis

2.Fa

eces

.Pro

duct

ion

ofcy

toto

xin

1.Fr

eshw

ater

orna

men

talf

ish,

gizz

ard

shad

.May

befo

und

inth

ein

test

ines

ofhe

alth

yfis

h2.

Infa

nt,a

dult

with

alco

holic

liver

cirrh

osis

Ubi

quito

usin

the

envi

ronm

ent

USA

21 259

393

452

750

Aero

mon

astro

ta(H

G14

)(p

revi

ousl

yca

lled

A.en

tero

pelo

gene

s)

1.N

on-p

atho

geni

cfo

rfis

h2.

Dia

rrhoe

a2.

Stoo

lsam

ples

1.Fo

und

inm

arin

e,es

tuar

ine

and

fresh

wat

eren

viro

nmen

ts2.

Hum

ans

Sout

h-Ea

stAs

ia(B

angl

ades

h,In

dia,

Indo

nesi

a,Th

aila

nd),

wor

ldw

ide

142

178

382

Aero

mon

asve

roni

issp

.sob

ria(H

G8)

1.En

viro

nmen

talo

rgan

ism

2.H

uman

s1.

Ubi

quito

usin

the

envi

ronm

ent

2.H

uman

s–

mos

tpat

hoge

nic

ofAe

rom

onas

taxa

Wor

ldw

ide

259

393

Aero

mon

asve

roni

issp

.ver

onii

form

erly

know

nas

ente

ricgr

oup

7(H

G10

)(p

revi

ousl

yca

lled

A.ic

hthi

osm

ia)

1.M

orta

lity

whe

nor

gani

smpr

esen

tin

high

num

bers

2.D

iarrh

oea,

wou

nds,

chol

ecys

titis

1.H

epat

opan

crea

s2.

Faec

es,w

ound

site

1.G

iant

fresh

wat

erpr

awns

2.H

uman

sFo

und

infre

shw

ater

1.Ta

iwan

347 2

178

381

723

Alca

ligen

esfa

ecal

isho

mar

iSe

eH

alom

onas

aqua

mar

ina

cont

inue

d



42 Chapter 1

Allo

mon

asen

teric

aEn

viro

nmen

talo

rgan

ism

Isol

ated

from

cont

amin

ated

river

wat

eran

dhu

man

faec

esR

ussi

a41

8

Alte

rom

onas

spec

ies

Bact

eria

lnec

rosi

san

dse

ptic

aem

iaN

ecro

sis,

sept

icae

mia

Larv

alan

dju

veni

lem

ollu

scs

(oys

ter)

Alte

rom

onas

citre

aSe

ePs

eudo

alte

rom

onas

citre

a

Alte

rom

onas

colw

ellia

naSe

eSh

ewan

ella

colw

ellia

na

Alte

rom

onas

com

mun

isSe

eM

arin

omon

asco

mm

unis

Aqua

spiri

llum

spp.

Rep

orto

fan

asso

ciat

ion

inep

izoo

ticul

cera

tive

synd

rom

e–

caus

edby

the

fung

us, A

phan

omyc

esin

vada

ns

Aqua

spiri

llum

indu

ced

slig

htde

rmom

uscu

larn

ecro

ticle

sion

sC

atfis

h–

mild

viru

lenc

eon

ly.S

nake

head

fish

are

noti

nfec

ted

whe

nch

alle

nged

with

Aqua

spiri

llum

spec

ies

Thai

land

497

Arca

noba

cter

ium

bern

ardi

aeIs

olat

edfro

mcl

inic

also

urce

ses

peci

ally

bloo

dan

dab

sces

ses

Hum

an-d

eriv

edst

rain

s27

463

6

Arca

noba

cter

ium

(Cor

yneb

acte

rium

)pho

cae

Path

ogen

icity

notd

eter

min

edTi

ssue

san

dflu

ids

Seal

s.R

ecov

ered

inm

ixed

grow

thfro

mse

als

with

sept

icae

mia

and

pneu

mon

iaSc

otla

nd61

363

6

Arca

noba

cter

ium

plur

anim

aliu

mPa

thog

enic

ityno

tdet

erm

ined

Isol

atio

nsi

teno

tsta

ted

Dea

dha

rbou

rpor

pois

e,de

adsa

llow

deer

UK

480

Arca

noba

cter

ium

pyog

enes

Asso

ciat

edw

itha

varie

tyof

pyog

enic

cond

ition

sM

ucus

mem

bran

es,t

issu

esO

ccur

sin

hum

ans

and

anim

als

Wor

ldw

ide

636

641

Arth

roba

cter

agilis

Envi

ronm

enta

lorg

anis

mW

ater

,soi

l,hu

man

skin

Arth

roba

cter

nasi

phoc

aePo

ssib

lyno

rmal

flora

Nas

alca

vity

Com

mon

seal

(Pho

cavi

tulin

a)18

2

Arth

roba

cter

rhom

biPa

thog

enic

ityno

tdet

erm

ined

Org

anis

mis

olat

edfro

min

tern

alor

gans

Gre

enla

ndha

libut

(hea

lthy)

Gre

enla

nd60

0

Atop

obac

terp

hoca

ePa

thog

enic

ityno

tdet

erm

ined

Inte

stin

e,ly

mph

node

s,lu

ngIs

olat

edfro

mde

adse

alSc

otla

nd47

9

Baci

llus

cere

usBr

anch

io-n

ecro

sis

Foun

don

necr

otic

gills

ofca

rpC

arp,

strip

edba

ssR

ussi

a,U

SA74 63

4

Baci

llus

myc

oide

sM

orta

lity

Ulc

ers

ondo

rsum

,foc

alne

cros

isof

epax

ialm

uscl

eC

hann

elca

tfish

Org

anis

mis

ubiq

uito

usin

soil

and

has

been

impl

icat

edin

dise

ase

inhu

man

san

dpa

rrots

Pola

nd,U

SA30

7

Baci

llus

subt

ilisPa

rtof

bact

eria

lflo

rain

bran

chio

-nec

rosi

sC

arp

Pola

nd63

4

Bact

eria

Dise

ase

Dise

ase

sign

sHo

st/is

olat

ion

site

Dist

ribut

ion

Ref

Tab

le1.

2.C

ontin

ued.




Bene

chea

chiti

novo

ra(n

oton

the

listo

frec

ogni

zed

bact

eria

lnam

es)

prev

ious

lyca

lled

Baci

llus

chiti

novo

rus

Ulc

erat

ive

shel

ldis

ease

,she

llro

t,sp

otdi

seas

e,ru

stdi

seas

e.M

ildly

cont

agio

us,c

hron

icse

lf-lim

iting

dise

ase

Affe

cts

the

chiti

nous

plat

esof

the

cara

pace

(dor

sals

hell)

and

plas

tron

(ven

trals

hell)

.She

llbe

com

espi

tted

and

early

lesi

ons

have

blot

chy

dark

colo

ratio

n

Free

-rang

ing

and

capt

ive

turtl

es(s

piny

soft-

shel

led

turtl

es,r

ed-e

ared

slid

ers,

strip

ed-n

ecke

dm

usk,

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hAm

eric

ansi

de-n

eck

turtl

e,Ea

ster

npa

inte

dtu

rtle)

USA

806

Bord

etel

labr

onch

isep

tica

1.Br

onch

opne

umon

ia,s

econ

dary

path

ogen

inph

ocin

em

orbi

lliviru

s(d

iste

mpe

r)in

fect

ion

Lung

,tra

chea

1.Se

als

2.Br

onch

opne

umon

iain

dogs

,lab

orat

ory

anim

als,

cats

,rab

bits

,hor

ses,

turk

eys,

mon

keys

,hum

ans,

asso

ciat

edw

ithat

roph

icrh

initi

sin

pigs

Euro

pe,S

cotla

nd,D

enm

ark,

UK

642

Brev

undi

mon

as(P

seud

omon

as)

dim

inut

aEn

viro

nmen

talo

rgan

ism

685

Brev

undi

mon

as(P

seud

omon

as)

vesi

cula

risEn

viro

nmen

talo

rgan

ism

Foun

din

stre

ams

685

Bruc

ella

abor

tus

Bruc

ello

sis

Posi

tive

sero

logy

Wed

dell

seal

s(L

epto

nych

otes

wed

dellii

)An

tarc

tica

592

Bruc

ella

abor

tus,

B.m

elite

nsis

,B.

suis

and

rare

lyB.

cani

sBr

ucel

losi

sG

ener

ally

host

spec

ific

–B.

abor

tus

(cat

tle),

B.ca

nis

(dog

s),B

.mel

itens

is(g

oats

),B.

neot

omae

(des

ertw

ood

rats

),B.

ovis

(she

ep),

B.su

is(p

igs,

rein

deer

,ha

res)

Zoon

otic

.Use

biol

ogic

alsa

fety

cabi

net

Wor

ldw

ide

185

Bruc

ella

ceta

ceae

(pre

viou

sly

part

ofBr

ucel

lam

aris

s p.n

o v.b

iov a

rI&

II)

1.Br

ucel

losi

s,ab

ortio

n,in

fect

ion

2.Br

ucel

losi

s1.

Abor

ted

fetu

s,su

bcut

aneo

usle

sion

s,sp

leen

,lun

g2.

Illne

ssan

dpo

sitiv

ebl

ood

cultu

re

1.D

olph

ins

(Atla

ntic

whi

te-s

ided

dolp

hin,

com

mon

dolp

hin,

strip

eddo

lphi

n,bo

ttlen

ose

dolp

hin)

,har

bour

porp

oise

,w

hale

2.H

uman

infe

ctio

nZo

onot

ic.W

ork

with

alls

uspe

cttis

sues

and

Bruc

ella

cultu

res

ina

biol

ogic

alsa

fety

cabi

net

Can

ada,

Euro

pe,S

cotla

nd,

USA

261

404

125

267

172

658

Bruc

ella

pinn

iped

iae

(pre

viou

sly

part

ofBr

ucel

lam

aris

spp.

nov.

biov

arI&

II)

1.Br

ucel

losi

s,ab

ortio

n,in

fect

ion

1.Ab

orte

dfe

tus,

subc

utan

eous

lesi

ons,

sple

en,l

ung

1.Se

als

(com

mon

seal

,gre

yse

al,

hood

edse

al,h

arp

seal

,rin

ged

seal

),Eu

rope

anot

ter

Poss

ibly

zoon

otic

.Wor

kw

ithal

lsu

spec

ttis

sues

and

Bruc

ella

cultu

res

ina

biol

ogic

alsa

fety

cabi

net

Can

ada,

Euro

pe,S

cotla

nd,

USA

261

404

125

267

172

658

cont

inue

d



44 Chapter 1

Bruc

ella

spec

ies

Bruc

ello

sis

Org

anis

min

liver

and

sple

enof

wha

le,a

ndin

lym

phno

des

ofse

als

Har

pse

al,r

inge

dse

al,m

inke

wha

leC

anad

a,N

orw

ay26

117

1

Budv

icia

aqua

tica

Envi

ronm

enta

lorg

anis

mIs

olat

edfro

mriv

eran

ddr

inki

ngw

ater

Cze

chos

lova

kia,

Swed

en59

1

Burk

hold

eria

(Pse

udom

onas

)ce

paci

aEn

viro

nmen

talo

rgan

ism

Fres

hwat

er,s

oil

Ubi

quito

us29

8

Burk

hold

eria

(Pse

udom

onas

)ps

eudo

mal

lei

Mel

ioid

osis

Sept

icae

mia

,abs

cess

esin

lung

,sp

inal

colu

mn,

liver

,kid

ney

Cet

acea

nsin

ocea

nariu

m–

(fals

eki

ller

wha

le,b

ottle

nose

dolp

hins

,whi

te-s

ided

dolp

hins

,sea

lion,

grey

seal

),sh

eep,

peng

uin,

goat

s,do

g,ga

lah,

cock

atoo

,hu

man

sFo

und

inso

ilan

dw

ater

Zoon

otic

.Use

biol

ogic

alsa

fety

cabi

net

Hon

gKo

ng.

Dis

ease

oftro

pica

land

subt

ropi

calr

egio

ns(A

ustra

lia,

Sout

h-Ea

stAs

ia)

349

516

Can

dida

spp.

(yea

st)

Opp

ortu

nist

infe

ctio

nC

etac

eans

–pa

rticu

larly

dolp

hins

726

Car

noba

cter

ium

alte

rfund

itum

Envi

ronm

enta

lorg

anis

mLa

kew

ater

Anta

rctic

a41

2

Car

noba

cter

ium

dive

rgen

sN

orm

alin

test

inal

mic

roflo

raof

heal

thy

fish

Inte

stin

ean

ddi

gest

ive

tract

Atla

ntic

salm

onju

veni

les,

Atla

ntic

cod,

Arct

icch

arr,

saith

eFr

ance

,Nor

way

176

Car

noba

cter

ium

dive

rgen

sSt

rain

6251

Pote

ntia

lpro

biot

icag

ains

tA.

salm

onic

ida

ssp.

salm

onic

ida,

L.an

guilla

rum

,M.v

isco

sus

Pres

enti

nin

test

ine

Arct

icch

arr(

Salv

elin

usal

pinu

sL.

)64

9

Car

noba

cter

ium

fund

itum

Envi

ronm

enta

lorg

anis

mLa

kew

ater

Anta

rctic

a41

2

Car

noba

cter

ium

gallin

arum

Envi

ronm

enta

lorg

anis

mIs

olat

edfro

mic

esl

ush

arou

ndch

icke

nca

rcas

ses

176

Car

noba

cter

ium

inhi

bens

Stra

inK1

Nor

mal

inte

stin

alm

icro

flora

ofhe

alth

yfis

hFo

und

indi

gest

ive

tract

Inhi

bits

grow

thof

L.an

guilla

rum

and

A.sa

lmon

icid

ain

Atla

ntic

salm

on41

241

1

Car

noba

cter

ium

mob

ileEn

viro

nmen

talo

rgan

ism

Proc

esse

dch

icke

nm

eat

176

Car

noba

cter

ium

(Lac

toba

cillu

s)pi

scic

ola

Pseu

doki

dney

dise

ase,

Lact

obac

illosi

s.Po

st-s

tripp

ing

perit

oniti

s.Se

enin

fish,

1ye

aror

olde

r,fo

llow

ing

stre

sssu

chas

hand

ling

and

spaw

ning

.Mos

tst

rain

sar

eop

portu

nist

ican

dpo

sses

slo

wvi

rule

nce;

how

ever

,oth

erst

rain

sha

vehi

gher

viru

lenc

ean

dca

use

high

mor

talit

y

Epic

ardi

tis,p

erito

nitis

,vis

cera

lgr

anul

omas

,dis

tens

ion

ofab

dom

en,a

scite

sflu

id,b

lood

orbl

iste

rsun

dert

hesk

in.C

olle

ctsa

mpl

esfro

mki

dney

,spl

een,

swim

blad

der.

Viru

lent

stra

ins

caus

ebi

late

rale

xoph

thal

mia

,pe

riocu

lara

ndliv

erha

emor

rhag

es,

and

asci

tes

fluid

Salm

onid

fish,

cutth

roat

trout

,rai

nbow

trout

,chi

nook

salm

on,c

ultu

red

strip

edba

ss,c

hann

elca

tfish

,bro

wn

bullh

ead

catfi

shLe

ssvi

rule

ntin

strip

edba

ssan

dch

anne

lca

tfish

Aust

ralia

,Bel

gium

,Can

ada,

Fran

ce,U

K,U

SA35

3 73 176

752

Bact

eria

Dise

ase

Dise

ase

sign

sHo

st/is

olat

ion

site

Dist

ribut

ion

Ref

Tab

le1.

2.C

ontin

ued.




Cat

enib

acte

rium

spp.

See

Euba

cter

ium

spp.

Cel

lulo

phag

a(C

ytop

haga

)lyt

ica

Envi

ronm

enta

liso

late

Mar

ine,

beac

hm

udC

osta

Ric

a16

3

Chr

omob

acte

rium

viol

aceu

mPi

gmen

ted

and

non-

pigm

ente

dst

rain

s

1.En

viro

nmen

tali

sola

te2.

Wou

ndin

fect

ion,

sept

icae

mia

,ab

sces

ses

1.Fo

und

inso

ilan

dw

ater

2.H

uman

s1.

Trop

ical

and

subt

ropi

cal

regi

ons

2.Au

stra

lia,M

alay

sia,

Sene

gal,

Taiw

an,U

SA,V

ietn

am

482

599

Chr

yseo

bact

eriu

m(F

lavo

bact

eriu

m)b

alus

tinum

Flav

obac

terio

sis

Mar

ine

fish

USA

802

Chr

yseo

bact

eriu

m(F

lavo

bact

eriu

m)g

leum

Non

-pat

hoge

nic

tofis

hFo

und

inhu

man

clin

ical

spec

imen

s36

6

Chr

yseo

bact

eriu

min

dolo

gene

s(p

revi

ousl

ySp

hing

obac

teriu

man

dFl

avob

acte

rium

indo

loge

nes)

1.Sy

stem

icin

fect

ion

2.C

linic

also

urce

s1.

Torti

collis

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ssle

sion

s,gr

anul

omas

,enl

arge

dor

gans

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rmed

bullf

rogs

(Ran

aca

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na)

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uman

spec

imen

san

dho

spita

len

viro

nmen

t

USA

530

844

Chr

yseo

bact

eriu

m(F

lavo

bact

eriu

m)i

ndol

thet

icum

557

Chr

yseo

bact

eriu

m(F

lavo

bact

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m)

men

ingo

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icum

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on-p

atho

gen

forf

ish

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ricar

ditis

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ticae

mia

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stem

icin

fect

ion

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enin

gitis

inin

fant

s

2.Is

olat

edfro

mpe

ricar

dium

,liv

er,

eye

lesi

on3.

Torti

collis

,gro

ssle

sion

s,gr

anul

omas

,enl

arge

dor

gans

1.R

epor

ted

from

fish

bloo

dan

dm

arin

em

ud2.

Bird

s(c

hick

ens,

pige

on,f

inch

)3.

Farm

edbu

llfro

gs( R

ana

cast

esbe

iana

)4.

Hum

anpa

thog

en

USA

,wor

ldw

ide

530

773

Chr

yseo

bact

eriu

m(F

lavo

bact

eriu

m)s

coph

thal

mum

Gill

dise

ase,

haem

orrh

agic

sept

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mia

.100

%m

orta

lity

inju

veni

les

Swol

len

gill

lam

ella

e(h

yper

plas

ia),

haem

orrh

agic

sept

icae

mia

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olle

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test

ines

cont

aini

ngye

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fluid

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emor

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eye,

skin

,jaw

Hea

lthy

and

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ased

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otC

oast

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ater

sSc

otla

nd55

655

7

Citr

obac

terf

reun

dii

1.O

ppor

tuni

stic

infe

ctio

n,lo

wvi

rule

nce

fort

rout

2.Sy

stem

icin

fect

ion

1.H

aem

orrh

agic

spot

son

skin

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ean

dfin

s.O

rgan

ism

isol

ated

from

kidn

ey,l

iver

,spl

een

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rtico

llis,g

ross

lesi

ons,

gran

ulom

as,e

nlar

ged

orga

ns

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gelf

ish

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trout

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nfis

h,ca

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omm

only

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ated

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er,

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ased

and

heal

thy

anim

als

incl

udin

gm

amm

als,

bird

s,re

ptile

san

dam

phib

ians

2.Fa

rmed

bullf

rogs

( Ran

aca

stes

beia

na)

Indi

a,Ja

pan,

UK,

USA

,w

orld

wid

e42

553

067

575

3

cont

inue

d



46 Chapter 1

Clo

strid

ium

botu

linum

Type

EM

orbi

dity

and

mor

talit

yFa

rmed

trout

,sal

mon

,coh

osa

lmon

C.b

otul

inum

can

bea

com

men

sali

ntro

utin

test

ine

and

ongi

lls.T

oxin

usua

llyno

tpr

oduc

edin

live

fish,

buti

spr

oduc

edin

dead

fish

aspa

rtof

the

deco

mpo

sitio

npr

oces

s.Fo

und

inla

kese

dim

ent.

Bact

eriu

mpr

oduc

esto

xin

inan

anae

robi

cen

viro

nmen

t

Brita

in,C

anad

a,D

enm

ark,

USA

141

Clo

strid

ium

perfr

inge

nsTy

peA

1.En

tero

toxa

emia

2.M

yosi

tisat

inje

ctio

nsi

te1.

Gut

cont

ent

2.Ab

sces

sin

mus

cle

Cap

tive

wha

les,

dolp

hins

,sea

lsU

K31

2

Col

wel

liam

aris

prev

ious

lyVi

brio

stra

inAB

E-1

Envi

ronm

enta

lorg

anis

mPs

ychr

ophi

lic,f

ound

inse

awat

erJa

pan

849

Cor

yneb

acte

rium

aqua

ticum

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opht

halm

ia2.

Clin

ical

infe

ctio

n1.

Org

anis

mse

enin

brai

n,ha

emor

rhag

esin

eyes

1.St

riped

bass

,rai

nbow

trout

.Org

anis

mfo

und

inna

tura

lfre

shan

ddi

stille

dw

ater

2.R

epor

ted

toca

use

infe

ctio

nin

imm

unoc

ompr

omis

edpa

tient

s3.

Foun

din

fresh

wat

erso

urce

s

1.U

SA2.

Wor

ldw

ide

73 75

Cor

yneb

acte

rium

phoc

aeSe

eAr

cquo

bact

eriu

mph

ocae

Cor

yneb

acte

rium

test

udin

oris

Asso

ciat

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crot

icm

outh

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ons

Mou

thle

sion

sTo

rtois

eSc

otla

nd18

0

Cry

ptoc

occu

slu

pi(y

east

)En

viro

nmen

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rgan

ism

Soil

Anta

rctic

a55

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ptoc

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man

sva

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ttii

(yea

st)

Dea

th,s

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ism

isol

ated

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phno

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phin

Zoon

otic

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nism

Isol

ated

from

batg

uano

and

asso

ciat

edw

itheu

caly

ptus

trees

Aust

ralia

Trop

ics

and

sout

hern

hem

isph

ere

278

135

Cyt

opha

gaaq

uatil

isSe

eFl

avob

acte

rium

hyda

tis

Cyt

opha

gaar

vens

icol

aEn

viro

nmen

tali

sola

teSo

ilJa

pan

89

Cyt

opha

gaau

rant

iaca

Envi

ronm

enta

liso

late

Swam

pyso

ilG

erm

any

92

Cyt

opha

gaco

lum

naris

See

Flav

obac

teriu

mco

lum

nare

Cyt

opha

gafe

rmen

tans

Envi

ronm

enta

liso

late

Mar

ine

mud

Cal

iforn

ia89 16

2

Cyt

opha

gahu

tchi

nson

iiEn

viro

nmen

tali

sola

teSo

il89

Cyt

opha

gajo

hnso

nae

See

Flav

obac

teriu

mjo

hnso

niae

Cyt

opha

gala

terc

ula

Envi

ronm

enta

liso

late

Mar

ine

USA

163

Bact

eria

Dise

ase

Dise

ase

sign

sHo

st/is

olat

ion

site

Dist

ribut

ion

Ref

Tab

le1.

2.C

ontin

ued.




Cyt

opha

gam

arin

ofla

vaEn

viro

nmen

tali

sola

teSe

awat

erSc

otla

nd92

Cyt

opha

gaps

ychr

ophi

laSe

eFl

avob

acte

rium

psyc

hrop

hilu

m

Der

mat

ophi

lus

chel

onae

Der

mat

ophi

losi

sSk

inle

sion

s,sk

inab

sces

s,sk

insc

abs

Test

udin

es(C

helo

nian

s)–

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ean

dto

rtois

eAu

stra

lia52

9

Der

mat

ophi

lus

cong

olen

sis

Der

mat

ophi

losi

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ferre

dto

as‘lu

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dm

ycot

icde

rmat

itis

inin

fect

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Subc

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eous

nodu

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and

lesi

ons

cont

aini

ngca

seou

sm

ater

ial

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uatic

spec

ies

–cr

ocod

iles,

bear

ded

drag

on,p

olar

bear

s,se

als

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uman

s,ho

rses

,she

ep,b

lue-

tong

ueliz

ard,

cat,

cattl

e,go

ats,

deer

,mon

keys

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es,g

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Aust

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419

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is(p

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iella

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ofno

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fin,f

lam

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itor,

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317

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iella

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Stra

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have

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ated

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hell

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Goo

dwin

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teric

sept

icae

mia

ofca

tfish

2.En

viro

nmen

t1.

Initi

alin

fect

ion

inbr

ain.

Pete

chia

lha

emor

rhag

eun

derj

awan

dbe

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head

lesi

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idne

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fect

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eshw

ater

orna

men

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cultu

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chan

nelc

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tatu

s),

Dan

io,w

hite

catfi

sh,g

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knife

fish,

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Angu

illida

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atfis

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eth

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osts

usce

ptib

lesp

ecie

s2.

Isol

ated

from

orga

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llute

dw

ater

s,ur

ine

and

faec

esof

man

,and

inte

stin

alm

icro

flora

ofsn

akes

1.Th

aila

nd,U

SA,V

ietn

am19

433

437

442

650

054

762

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iella

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a(E

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uillim

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ese

nior

syno

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ever

, E.t

arda

isco

nser

ved

foru

se.O

rigin

ally

nam

edPa

raco

loba

ctru

man

guilli

mor

tifer

um)

1.Ed

war

dsie

llosi

s,re

dpes

t,em

phys

emat

ous

putre

fact

ive

dise

ase

ofca

tfish

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hga

ngre

ne2.

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emic

infe

ctio

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ancl

inic

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ptic

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ia,a

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374

530

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48 Chapter 1

Empe

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brev

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acte

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brev

e)1.

Envi

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als

2.H

uman

s

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and,

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nd,S

witz

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roba

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See

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alis

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rted

asE.

faec

alis

ssp.

lique

faci

ens)

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ssib

lepa

thog

en.I

dent

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doub

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Syst

emic

infe

ctio

n1.

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eria

inliv

eran

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dney

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nbow

trout

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fish,

brow

nbu

llhea

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bsPa

rtof

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alin

test

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flora

ofm

anan

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imal

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use

noso

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roat

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chM

edite

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ldw

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Ente

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ium

Nor

mal

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eno

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lin

fect

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ofno

rmal

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stin

alflo

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man

and

anim

als

Wor

ldw

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Ente

roco

ccus

serio

licid

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eLa

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rvie

ae73

1

Ente

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ay74

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rasi

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pigs

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ian

spec

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croc

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iona

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Wor

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ide

292

408

229

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eric

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coli

Endo

card

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435

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ptic

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ia,m

orta

lity

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ound

s1.

Abno

rmal

ities

and

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lls,l

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een

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eshw

ater

fish

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wtro

ut,b

allo

onm

olly

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erm

olly

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anca

rp2.

Hum

ans,

also

isol

ated

from

faec

es.

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din

wat

erof

fish

pond

san

dfis

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rkey

51

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cter

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dey

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77).

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Ref

Tab

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2.C

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50 Chapter 1

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m(C

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mR

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Req

uire

pyrid

oxal

hydr

ochl

orid

ean

alog

(Vita

min

B 6)o

rL-c

yste

ine

HC

l(G

.el

egan

s)fo

rgro

wth

421

179

653

Bact

eria

Dise

ase

Dise

ase

sign

sHo

st/is

olat

ion

site

Dist

ribut

ion

Ref

Tab

le1.

2.C

ontin

ued.




Gra

nulic

atel

laba

laen

opte

rae

com

.nov

.(p

revi

ousl

yAb

iotro

phia

bala

enop

tera

e )

Path

ogen

icity

notd

eter

min

edIs

olat

edin

pure

grow

thfro

mliv

eran

dki

dney

Beac

hed

min

kew

hale

Scot

land

179

478

Hae

mop

hilu

spi

sciu

mR

e-cl

assi

fied

as‘a

typi

cal’

A.sa

lmon

icid

a.M

ostc

lose

lyre

late

dto

A.sa

lmon

icid

aac

hrom

ogen

es50

Haf

nia

alve

i1.

Hae

mor

rhag

icse

ptic

aem

ia.

Mor

talit

ies

2.In

test

inal

diso

rder

s,pn

eum

onia

,m

enin

gitis

,abs

cess

esan

dse

ptic

aem

ia

1.C

herry

salm

on,r

ainb

owtro

ut,b

row

ntro

ut2.

Hum

ans

Ubi

quito

usin

the

envi

ronm

ents

ofso

il,se

wag

ean

dw

ater

1.Bu

lgar

ia,E

ngla

nd,J

apan

313

652

Hal

omon

asaq

uam

arin

a(s

ynon

ymou

sw

ithAl

calig

enes

faec

alis

hom

ari ,

Del

eya

aest

a,D

.aq

uam

arin

a ,A.

aqua

mar

inus

.Tr

ansf

erre

dto

genu

sH

alom

onas

asH

.aqu

amar

ina)

Mor

ibun

dSo

ftene

dsh

ells

,opa

que

area

son

cara

pace

.Org

anis

ms

isol

ated

from

haem

olym

ph

Lobs

ters

USA

45 719 8

Hal

omon

ascu

pida

(pre

viou

sly

Alca

ligen

escu

pidu

san

dD

eley

acu

pida

)

Mor

talit

ies

Blac

kse

abr

eam

fryJa

pan

463

Hal

omon

asel

onga

taEn

viro

nmen

talo

rgan

ism

Hyp

ersa

line

envi

ronm

ents

The

Net

herla

nds

795

Hal

omon

asha

lodu

rans

Envi

ronm

enta

lorg

anis

mH

yper

salin

een

viro

nmen

tsU

SA,T

heN

ethe

rland

s,Pa

cific

Oce

an33

6

Hal

omon

asm

arin

a(p

revi

ousl

yPs

eudo

mon

asm

arin

aan

dD

eley

am

arin

a)

Envi

ronm

enta

lorg

anis

mM

arin

een

viro

nmen

t66

Hal

omon

asve

nust

a(p

revi

ousl

yAl

calig

enes

venu

stus

)C

linic

alin

fect

ion

Hum

an–

caus

edby

fish

bite

.Pre

sent

inm

arin

ew

ater

sM

aldi

veIs

land

s66 31

0

Hel

icob

acte

rcet

orum

1.O

rgan

ism

foun

din

dent

alpl

aque

2.G

astri

cul

cera

tion

1.Po

tent

ialr

eser

voir

forg

astri

cin

fect

ions

2.O

rgan

ism

ingl

andu

larm

ucos

aan

dth

em

ain

stom

ach

1.C

aptiv

edo

lphi

ns( T

ursi

ops

geph

yreu

s)2.

Dol

phin

s,be

luga

wha

leU

SA30

332

732

9

Hyd

roge

noph

aga

(Pse

udom

onas

)pa

llero

nii

Envi

ronm

enta

lorg

anis

mPr

esen

tin

wat

erG

erm

any,

Rus

sia

834

cont

inue

d



52 Chapter 1

Hyd

roge

noph

aga

(Pse

udom

onas

)ps

eudo

flava

Envi

ronm

enta

lorg

anis

mPr

esen

tin

wat

er,m

ud,s

oil

Ger

man

y39 83

4

Iodo

bact

erflu

viat

ilis(p

revi

ousl

yC

hrom

obac

teriu

mflu

viat

ile)

Envi

ronm

enta

lorg

anis

mFo

und

infre

shw

ater

Anta

rctic

lake

s,En

glan

d,Sc

otla

nd,U

biqu

itous

502

Jant

hino

bact

eriu

mliv

idum

Anae

mia

Exop

htha

lmia

,pal

egi

lls,i

nter

nal

sym

ptom

sR

ainb

owtro

utFo

und

inso

ilan

dsp

ring

wat

erSc

otla

nd48

Kleb

siel

lapn

eum

onia

e1.

Fin

and

tail

dise

ase

2.M

icro

flora

1.R

ainb

owtro

ut2.

Mam

mal

ian

tissu

eU

KW

orld

wid

e20

5

Kleb

siel

lapl

antic

ola

Kleb

siel

latre

visa

nii

See

Rao

ulte

llapl

antic

ola

256

228

Kleb

siel

laor

nith

inol

ytic

aSe

eR

aoul

tella

orni

thin

olyt

ica

228

Kleb

siel

late

rrige

naSe

eR

aoul

tella

terri

gena

228

Kleb

siel

laox

ytoc

a22

8

Lact

obac

illus

spp.

,esp

ecia

llya

Lact

obac

illus

plan

taru

m-li

keis

olat

e

Nor

mal

inte

stin

alm

icro

flora

ofhe

alth

yfis

hIn

test

ine

and

dige

stiv

etra

ctAt

lant

icco

d,At

lant

icsa

l mon

,rai

nbow

trout

,wol

f-fis

h,Ar

ctic

char

rFr

ance

,Nor

way

Lact

obac

illus

pisc

icol

aSe

eC

arno

bact

eriu

mpi

scic

ola

Lact

ococ

cus

garv

ieae

Biot

ypes

1–13

(pre

viou

sly

Ente

roco

ccus

serio

licid

a,St

rept

ococ

cus

type

Ian

dSt

rept

ococ

cus

garv

ieae

)

1.La

ctoc

occo

sis,

haem

orrh

agic

sept

icae

mia

,hae

mor

rhag

icen

terit

is,

men

ingo

ence

phal

itis

2.Su

bclin

ical

mas

titis

3.In

fect

ion,

oste

omye

litis

1.Bi

late

rale

xoph

thal

mia

,da

rken

ing

ofsk

in,c

onge

stio

nof

inte

stin

e,liv

er,k

idne

y,sp

leen

,br

ain,

dist

ende

dab

dom

en,b

lood

yas

cite

sflu

idin

perit

onea

lcav

ity.

Org

anis

mse

enin

hear

t,gi

lls,s

kin,

sple

en,e

yes,

kidn

ey2.

Milk

3.Bl

ood,

skin

,urin

e,fa

eces

1.Fa

rmed

rain

bow

trout

,eel

,yel

low

tail,

praw

ns,t

urbo

t,Ad

riatic

stur

geon

Foun

din

seaw

ater

,mud

,int

estin

esof

wild

fish

e.g.

Span

ish

mac

kere

l,bl

ack

scra

per

(Bio

type

s1,

2,3,

4,5,

6,10

)2.

Cow

s,bu

ffalo

(Bio

type

s4,

7,8,

9)3.

Hum

ans

(Bio

type

s1,

2,10

,11,

12,1

3)

Aust

ralia

,Eur

ope,

Fran

ce,I

taly

,Is

rael

,Jap

an,N

orth

Amer

ica,

Saud

iAra

bia,

Spai

n,So

uth

Afric

a,Ta

iwan

,UK,

USA

236

238

237

156

174

157

464

669

731

780

Lact

ococ

cus

pisc

ium

Lact

obac

illosi

s,ps

eudo

kidn

eydi

seas

eR

ainb

owtro

utN

orth

Amer

ica

835

Bact

eria

Dise

ase

Dise

ase

sign

sHo

st/is

olat

ion

site

Dist

ribut

ion

Ref

Tab

le1.

2.C

ontin

ued.




List

onel

laan

guilla

rum

(Ser

ovar

s01

,02,

08,0

9)pr

evio

usly

Vibr

ioan

guilla

rum

biot

ype

I.M

osto

fthe

outb

reak

sar

eca

used

byse

roty

pes

01an

d02

Vibr

iosi

s,sy

stem

icdi

seas

e,ul

cera

tive

dise

ase,

necr

osis

Red

spot

son

vent

rala

ndla

tera

lar

eas

offis

h,ul

cera

tive

skin

lesi

ons.

Org

anis

ms

inbl

ood

and

haem

opoi

etic

tissu

es

1.Fi

sh,m

ollu

scs

–(la

rval

and

juve

nile

),ay

u,fla

tfish

(turb

ot,p

laic

efry

,win

ter

floun

der,

sole

,hal

ibut

),lo

bste

r,ee

l,sa

lmon

ids,

(rain

bow

trout

),se

abr

eam

,oc

topu

s2.

Shrim

ps3.

Cra

bs

1.W

orld

wid

e2.

Indo

-Pac

ific

&Ea

stAs

ia3.

UK

341

222

561

563

620

List

onel

lape

lagi

a(p

revi

ousl

yV.

pela

gia

I&II)

Mor

talit

ies

Eros

ion

onfin

san

dta

ilH

aem

orrh

ages

onfin

san

dor

gans

Juve

nile

farm

edtu

rbot

Spai

n

List

onel

laor

dalii

See

Vibr

ioor

dalii

Man

nhei

mia

haem

olyt

ica

(pre

viou

sly

Past

eure

llaha

emol

ytic

a )

1.U

lcer

ativ

est

omat

itis

2.D

isea

se3.

Hae

mor

rhag

ictra

chei

tis

1.R

eptil

es2.

Shee

p,go

ats,

cattl

e3.

Dol

phin

s

1.U

SA70

972

6

Mar

inila

bilia

salm

onic

olor

biov

arag

arov

oran

s(p

revi

ousl

yC

ytop

haga

salm

onic

olor

and

C.a

garo

vora

ns)

Envi

ronm

enta

lorg

anis

mM

arin

em

udC

alifo

rnia

92 89 162

Mar

inob

acte

rhy

droc

arbo

nocl

astic

us(P

seud

omon

asna

utic

a)

Foun

din

mar

ine

envi

ronm

ents

69

Mes

ophi

loba

cter

mar

inus

Envi

ronm

enta

lorg

anis

mFo

und

inse

awat

erJa

pan,

Indi

anO

cean

583

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roco

ccus

lute

usM

orta

lity

Pale

gills

,asc

ites

fluid

,ga

stro

ente

ritis

,int

erna

lha

emor

rhag

es

Rai

nbow

trout

fryEn

glan

d43

Mor

axel

lasp

p.As

soci

ated

with

mor

talit

ySt

riped

bass

USA

72

Mor

itella

japo

nica

Envi

ronm

enta

lorg

anis

mSe

abed

sedi

men

tJa

pan

585

Mor

itella

mar

ina

(Vib

riom

arin

us)

Skin

lesi

ons

Isol

ate

from

surfa

cesk

inle

sion

sAt

lant

icsa

lmon

,sea

wat

eran

dse

dim

ent

ofno

rthPa

cific

Oce

anIc

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d,N

orw

ay,P

acifi

cO

cean

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otla

nd82 99 76

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itella

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osa

(pre

viou

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iovi

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teru

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dise

ase

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lesi

ons,

haem

orrh

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ntic

salm

on,l

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ound

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ldw

ater

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dwat

erin

Icel

and,

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way

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otla

nd81 82 13

250

6

cont

inue

d



54 Chapter 1

Myc

obac

teriu

msp

p.M

ycob

acte

riosi

s,sy

stem

icdi

seas

eLe

sion

son

skin

and

kidn

eys.

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ules

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gans

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teni

ngof

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y,liv

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asin

tissu

es

Man

ysp

ecie

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fresh

wat

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altw

ater

and

orna

men

talf

ish,

fresh

wat

ersn

ails

,fro

gs,

rept

iles,

turtl

es,P

acifi

cgr

een

sea

turtl

es,

New

Zeal

and

furs

eals

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shw

ater

croc

odile

sin

Aust

ralia

Zoon

otic

Wor

ldw

ide

592

737

Myc

obac

teriu

mab

sces

sus

Gra

nulo

mas

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tem

icdi

seas

eN

oob

viou

scl

inic

alsi

gns

ofpi

scin

em

ycob

acte

riosi

s.O

ccas

iona

lext

erna

lgra

nulo

ma

arou

ndbu

ccal

cavi

tyan

dve

nt,

inte

rnal

gran

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as

Japa

nese

med

aka,

fresh

wat

ertro

pica

lfis

h,bl

ack

acar

as,g

oldf

ish,

firem

outh

cich

lid,o

scar

Zoon

otic

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474

736

Myc

obac

teriu

mch

elon

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orta

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gran

ulom

as,e

mac

iatio

n,ex

opht

halm

os,k

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skin

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rs,

abno

rmal

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min

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our

Mul

tiple

grey

ish-

whi

tem

iliary

gran

ulom

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tissu

es,

kidn

ey,l

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een

Atla

ntic

salm

on(S

alm

osa

lar),

yello

wpe

rch,

snak

e,tu

rbot

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tleZo

onot

ic

Aust

ralia

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ada,

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gal,

Shet

land

Isla

nds,

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land

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orld

wid

e

133

375

204

673

737

Myc

obac

teriu

mfo

rtuitu

m(p

revi

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cium

and

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alm

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m)

Sept

icae

mia

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hm

aybe

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iate

d,ex

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nfla

mm

atio

nof

skin

Gra

nulo

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onsk

inan

din

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es.S

een

asw

hitis

hsp

ots

onliv

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y,he

art,

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en

Orn

amen

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h–

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ckac

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com

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disc

usfis

h,go

uram

i,gu

ppy,

neon

tetra

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car,

Siam

ese

fight

ing

fish)

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ntic

salm

onZo

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skin

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ddi

ffuse

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seas

ein

hum

ans

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ralia

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thAf

rica,

Thai

land

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637

547

463

3

Myc

obac

teriu

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arin

umM

ycob

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uliti

sin

capt

ive

whi

tew

hale

Lesi

ons

inki

dney

and

onsk

in.

Nod

ular

lesi

ons

may

besy

stem

icin

allo

rgan

s.G

ranu

lom

asin

orga

ns

Fres

hwat

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ut,f

resh

wat

eror

nam

enta

lfis

h,m

arin

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h,ra

bbitf

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bass

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riped

bass

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tew

hale

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inle

sion

sin

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ans

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ralia

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ael,

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gal,

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iqui

tous

35 111

135

218

339

474

673

Myc

obac

teriu

mne

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noph

thal

miti

sO

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rles

ions

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ules

inm

uscl

ean

dor

gans

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anis

mis

olat

edw

itha

Rho

doco

ccus

spec

ies

Atla

ntic

salm

on,c

hino

oksa

lmon

.Zo

onot

icC

anad

a53

Myc

obac

teriu

mpe

regr

inum

Myc

obac

terio

sis.

Shrim

pap

pear

edhe

alth

yap

artf

rom

blac

kle

sion

son

cara

pace

Mul

tifoc

al,m

elan

ized

nodu

lar

lesi

ons

inca

rapa

cePa

cific

whi

tesh

rimp

(Pen

aeus

vann

amei

)C

ause

ssk

inin

fect

ion

inhu

man

s.Fo

und

inw

ater

and

soil

USA

551

Bact

eria

Dise

ase

Dise

ase

sign

sHo

st/is

olat

ion

site

Dist

ribut

ion

Ref

Tab

le1.

2.C

ontin

ued.




Myc

obac

teriu

mpo

rifer

aeTh

isis

olat

eha

ssi

nce

been

iden

tifie

dby

PCR

asM

.for

tuitu

m

Myc

obac

terio

sis

Inte

rnal

nodu

larl

esio

nsFr

eshw

ater

snak

ehea

dfis

h(C

hann

ast

riatu

s )Pr

evio

usly

repo

rted

from

am

arin

esp

onge

Thai

land

608

633

756

Myc

obac

teriu

msc

rofu

lace

umM

ycob

acte

riosi

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sion

sin

kidn

eyan

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er.L

iver

whi

tean

dfri

able

Paci

ficst

agho

rnsc

ulpi

nU

SA47

4

Myc

obac

teriu

msi

mia

eM

ycob

acte

riosi

sLe

sion

sin

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eyan

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erBl

ack

acar

aFo

und

inen

viro

nmen

talw

ater

Zoon

otic

USA

474

Myc

obac

teriu

msp

p.no

tide

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edto

know

nsp

ecie

sM

ycob

acte

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tern

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rmal

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dno

dule

sin

inte

rnal

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ons

Wild

strip

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ssC

hesa

peak

eBa

y(U

SA)

337

Myc

obac

teriu

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rium

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lom

atou

sde

rmat

itis

Flor

idsk

inno

dule

s–

soft,

gela

tinou

s,gr

ey-a

ndta

n-co

lour

edar

ound

head

and

trunk

Gre

enm

oray

eels

,spo

tted

mor

ayee

lsU

SA34

5

Myc

opla

sma

allig

ator

isEp

izoo

ticpn

eum

onia

,pol

yser

ositi

san

dm

ultif

ocal

arth

ritis

Org

anis

mfo

und

intra

chea

,lun

g,jo

intf

luid

,cer

ebro

spin

alflu

id(C

SF)

Amer

ican

allig

ator

USA

128

Myc

opla

sma

croc

odyl

iEx

udat

ive

poly

arth

ritis

Swol

len

join

ts.A

lso

foun

din

lung

sC

roco

dile

sZi

mba

bwe

441

Myc

opla

sma

mob

ileR

eddi

seas

eG

illsTe

nch

USA

439

440

Myc

opla

sma

phoc

icer

ebra

le(p

revi

ousl

yM

.pho

cace

rebr

ale)

Asso

ciat

edw

ithre

spira

tory

dise

ase

Isol

ated

from

,bra

i n,n

ose,

thro

at,

lung

s,he

art

Seal

sN

orth

Sea

295

449

Myc

opla

sma

phoc

idae

(Myc

opla

sma

phoc

ae–

nam

eco

rrect

ion

isno

tleg

itim

ate)

Aviru

lent

Res

pira

tory

tract

Har

bour

seal

sU

SA44

966

0

Myc

opla

sma

phoc

irhin

is(p

revi

ousl

yM

.pho

carh

inis

)As

soci

ated

with

resp

irato

rydi

seas

eIs

olat

edfro

mno

se,t

hroa

t,tra

chea

,lun

g,he

art

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orth

Sea

295

449

Myc

opla

sma

test

udin

isN

on-p

atho

geni

cC

loac

aof

torto

ise

UK

350

Myr

oide

s(F

lavo

bact

eriu

m)

odor

atim

imus

Clin

ical

spec

imen

s,ho

spita

lenv

ironm

ents

774

Myr

oide

sod

orat

us(p

revi

ousl

yFl

avob

acte

rium

odor

atum

)

Clin

ical

spec

imen

s(u

rine,

wou

ndsw

abs,

leg

ulce

r),ho

spita

lenv

ironm

ents

UK,

Cze

chos

lova

kia

362

774

cont

inue

d



56 Chapter 1

Noc

ardi

aas

tero

ides

Noc

ardi

osis

Neo

nte

tra,r

ainb

owtro

ut,l

arge

mou

thba

ss,F

orm

osa

snak

ehea

d.Al

soca

uses

infe

ctio

nsin

cats

,cat

tle,d

ogs,

fish,

goat

s,hu

man

s,m

arin

em

amm

als

Arge

ntin

a,Ta

iwan

155

Noc

ardi

abr

asilie

nsis

and

N.t

rans

vale

nsis

Actin

omyc

ete

myc

etom

a66

1

Noc

ardi

acr

asso

stre

aesp

.nov

.N

ocar

dios

isBr

own

disc

olor

atio

non

man

tle,

gree

nor

yello

wno

dule

son

abdu

ctor

mus

cle,

gills

,hea

rtan

dm

antle

Paci

ficoy

ster

sC

anad

a,U

SA27

0

Noc

ardi

afla

voro

sea

Envi

ronm

enta

lorg

anis

mSo

ilis

olat

eC

hina

165

Noc

ardi

ano

vaH

uman

path

ogen

805

Noc

ardi

asa

lmon

icid

aN

ocar

dios

isBl

ueba

cksa

lmon

391

Noc

ardi

ase

riola

e(p

revi

ousl

yN

.kam

pach

i)N

ocar

dios

isAb

sces

ses

and

light

-yel

low

-co

lour

edno

dule

sin

epid

erm

isan

dtu

berc

les

and

gran

ulom

asin

gills

,ki

dney

,liv

er,h

eart

and

sple

en

Cul

ture

dfis

h–

rudd

erfis

hes,

yello

wta

il,Ja

pane

seflo

unde

r,se

aba

ssJa

pan,

Taiw

an15

542

445

5

Noc

ardi

asp

p.Se

ptic

aem

ia,m

orta

lity

Smal

lwhi

tesp

otle

sion

son

derm

is,m

uscl

e,gi

llsan

dor

gans

.Al

sofo

und

inin

tern

alor

gans

Atla

ntic

salm

on,f

arm

edch

inoo

ksa

lmon

,m

arin

efis

h,fre

shw

ater

orna

men

talf

ish.

Isol

ated

from

soil

and

plan

ts

Wor

ldw

ide,

Aust

ralia

,Can

ada,

Japa

n,In

dia,

Taiw

an,U

SA11

7

Oce

anom

onas

baum

anni

iEn

viro

nmen

talo

rgan

ism

Deg

rade

sph

enol

Estu

ary

ofR

iver

Wea

rU

K13

0

Oce

anom

onas

(Pse

udom

onas

)do

udor

offii

Envi

ronm

enta

lorg

anis

mM

arin

een

viro

nmen

t69 13

0

Pant

oea

(Ent

erob

acte

r)ag

glom

eran

s1.

Path

ogen

icity

indo

ubt–

poss

ible

oppo

rtuni

st2.

Hum

ans

1.H

aem

orrh

ages

iney

es,d

orsa

lm

uscu

latu

re2.

Wou

nds,

bloo

d,ur

ine

1.D

olph

infis

h(m

ahi-m

ahi)

2.H

uman

s.Al

sofo

und

inen

teric

tract

3.Fo

und

onpl

ants

urfa

ces,

seed

s,w

ater

.Al

sore

porte

din

ente

rictra

ctof

deer

with

outd

isea

sesi

gns

USA

,ubi

quito

us32

529

124

9

Pant

oea

disp

ersa

Envi

ronm

enta

lorg

anis

mPl

ants

urfa

ces,

seed

s,so

il,en

viro

nmen

tU

biqu

itous

291

Past

eure

llam

ulto

cida

1.Pn

eum

onia

,dea

th,p

leur

isy

(fow

lch

oler

a)2.

Ente

ritis

(con

tam

inat

ion

from

near

bybi

rdro

oker

y)

1.Ex

udat

ein

lung

s,flu

idin

pleu

ralc

avity

2.In

test

inal

haem

orrh

age

1.Al

ligat

or,C

alifo

rnia

nse

alio

n,pe

ngui

ns2.

Dol

phin

sR

espi

rato

rydi

seas

ein

shee

p,go

ats,

cattl

e,ra

bbits

USA

Wor

ldw

ide

430

520

709

726

Bact

eria

Dise

ase

Dise

ase

sign

sHo

st/is

olat

ion

site

Dist

ribut

ion

Ref

Tab

le1.

2.C

ontin

ued.




Past

eure

llapi

scic

ida

See

Phot

obac

teriu

mda

mse

lae

ssp.

pisc

icid

a74

5

Past

eure

llask

yens

isM

orta

lity

(low

viru

lenc

e)Ex

hibi

tsig

nsof

loss

ofap

petit

e,m

orbi

dity

Sea-

farm

edAt

lant

icsa

lmon

(Sal

mo

sala

r L.)

Scot

land

100

416

Past

eure

llate

stud

inis

Mul

tifoc

albr

onch

opne

umon

iaan

dco

mm

ensa

lAb

sces

ses,

lung

lesi

ons

Cal

iforn

ian

dese

rtto

rtois

eU

SA70

9

Pedo

bact

erhe

parin

us(p

revi

ousl

yC

ytop

haga

and

Sphi

ngob

acte

rium

hepa

rinum

)( C

ytop

haga

hepa

rina)

Envi

ronm

enta

liso

late

Soil.

Deg

rade

she

parin

89 163

Pedo

bact

er(S

phin

goba

cter

ium

)pi

sciu

mEn

viro

nmen

talo

rgan

ism

Asso

ciat

edw

ithfro

zen

fish

Japa

n72

8

Phoc

oeno

bact

erut

eri

gen

nov.

spp.

nov.

Path

ogen

icity

unkn

own

Ute

rus

Har

bour

porp

oise

UK

266

Phot

obac

teriu

man

gust

umEn

viro

nmen

talo

rgan

ism

Mar

ine

envi

ronm

ent

67

Phot

obac

teriu

mda

mse

lae

ssp.

dam

sela

e(p

revi

ousl

yVi

brio

dam

sela

e,EF

-5)

1.Vi

brio

sis,

syst

emic

dise

ase,

gran

ulom

atou

sul

cera

tive

derm

atiti

s,de

aths

2.W

ound

infe

ctio

ns

1.Sk

inul

cers

inre

gion

ofpe

ctor

alfin

and

caud

alpe

dunc

le2.

Soft

tissu

ein

fect

ion

occu

rsdu

eto

prod

uctio

nof

cyto

lysi

n

1.Br

eam

,bar

ram

undi

,dam

selfi

sh,

dolp

hins

,eel

,oct

opus

,oys

ters

,pen

aeid

praw

ns,s

hark

s,sh

ellfi

sh,s

hrim

ps,

stin

gray

,tro

ut,r

ainb

owtro

ut,t

urbo

t,tu

rtles

,sea

hors

es,y

ello

wta

il.M

aybe

part

ofm

icro

flora

inhe

alth

yca

rcha

rhin

idsh

arks

and

mar

ine

alga

e.Au

stra

lian

nativ

ean

din

trodu

ced

fish

2.H

uman

s

Aust

ralia

,Den

mar

k,Eu

rope

,Ja

pan,

Spai

n,U

SA26

842

950

450

655

559

061

870

574

5

Phot

obac

teriu

mda

mse

lae

ssp.

pisc

icid

a(p

revi

ousl

yPa

steu

rella

pisc

icid

a,Fl

avob

acte

rium

pisc

icid

a,Ps

eudo

mon

aspi

scic

ida)

Past

eure

llosi

s,fis

hps

eudo

tube

rcul

osis

Bact

eria

lcol

oni e

san

dw

hite

nodu

les

insp

leen

,kid

ney

Bass

,Jap

anes

eflo

unde

r,gi

lt-he

adse

abr

eam

,sea

bass

,stri

ped

bass

,sol

e,w

hite

perc

h,ye

llow

tail.

Farm

edan

dw

ildfis

h

Euro

pe,F

ranc

e,G

reec

e,Ita

ly,

Japa

n,M

alta

,Por

tuga

l,Sc

otla

nd,S

pain

,Tai

wan

,Tu

rkey

,USA

Not

inAu

stra

lia

289 57 60 140

273

333

518

745

855

cont

inue

d



58 Chapter 1

Phot

obac

teriu

mfis

cher

iSe

eVi

brio

fisch

eri–

hom

otyp

icsy

nony

m

Phot

obac

teriu

mhi

stam

inum

Con

side

red

ala

ters

ubje

ctiv

esy

nony

mof

Phot

obac

teriu

mda

mse

lae

ssp.

dam

sela

e43

759

5

Phot

obac

teriu

milio

pisc

ariu

m(p

revi

ousl

yVi

brio

iliopi

scar

ius)

Non

-pat

hoge

nic

Inte

stin

eH

errin

g,co

alfis

h,sa

lmon

and

cod

livin

gin

cold

wat

ers

599

767

Phot

obac

teriu

mle

iogn

athi

Non

-pat

hoge

nic

Ligh

torg

anM

icro

flora

ofth

elu

min

ous

orga

nof

ase

afis

h,Le

iogn

athu

s64

3

Phot

obac

teriu

mlo

gei

See

Vibr

iolo

gei.

Phot

obac

teriu

mph

osph

oreu

mEn

viro

nmen

talo

rgan

ism

May

caus

esp

oila

geof

seaf

ood

Mar

ine

envi

ronm

ent.

Sym

biot

icas

soci

atio

nw

ithm

arin

ean

imal

sin

light

orga

nsof

tele

ostf

ishe

s

67

Phot

obac

teriu

mpr

ofun

dum

Envi

ronm

enta

liso

late

Isol

ated

from

deep

sea

sedi

men

t58

6

Plan

ococ

cus

citre

usEn

viro

nmen

tali

sola

teM

otile

Gra

m-p

ositi

veco

ccus

isol

ated

from

seaw

ater

,mar

ine

clam

and

froze

nbo

iled

shrim

p

326

Plan

ococ

cus

kocu

riiEn

viro

nmen

tali

sola

teSk

inof

Nor

thSe

aco

d,fis

hcu

ring

brin

e,fro

zen

boile

dsh

rimp,

froze

npr

awn

Japa

n32

6

Plan

ococ

cus

spp.

pres

umpt

ive

iden

tific

atio

nM

orta

lity

Pale

gills

,asc

ites

fluid

,ga

stro

ente

ritis

,int

erna

lha

emor

rhag

es

Rai

nbow

trout

fryEn

glan

d43

Plan

omic

robi

umok

eano

koite

s(p

revi

ousl

yPl

anoc

occu

sok

eano

koite

san

dFl

avob

acte

rium

okea

noko

ites )

Envi

ronm

enta

lorg

anis

mIs

olat

edfro

mm

arin

em

udJa

pan

566

Ples

iom

onas

shig

ello

ides

(pre

viou

sly

Aero

mon

assh

igel

loid

es)

Poss

ible

oppo

rtuni

stpa

thog

enEm

acia

tion,

pete

chia

lha

emor

rhag

esin

inte

stin

eAf

rican

catfi

sh,e

el,g

oura

mi,

rain

bow

trout

,stu

rgeo

n.C

aptiv

epe

ngui

n,aq

uatic

rept

iles,

ubiq

uito

usin

envi

ronm

ent

Aust

ralia

,Ger

man

y,Po

rtuga

l19

544

3

Prov

iden

cia

rettg

eri

(als

ok n

o wn

a sPr

oteu

sre

ttger

i)Se

ptic

aem

iaO

rgan

ism

isol

ated

from

inte

rnal

orga

ns,u

lcer

ativ

eex

tern

alle

sion

sSi

lver

carp

Asso

ciat

edw

ithpo

ultry

faec

esIs

rael

79

Bact

eria

Dise

ase

Dise

ase

sign

sHo

st/is

olat

ion

site

Dist

ribut

ion

Ref

Tab

le1.

2.C

ontin

ued.




Prov

iden

cia

rust

igia

nii

(pre

viou

sly

P.fri

eder

icia

na)

Nor

mal

flora

Faec

esPe

ngui

ns(A

pten

odyt

espa

tago

nica

,Eu

dypt

escr

esta

tus,

Pyos

celis

papu

a,Sp

heni

scus

dem

ersu

s,Sp

heni

scus

hum

bold

ti )

Peng

uins

inzo

oin

Ger

man

y55

9

Pseu

doal

tero

mon

as(A

ltero

mon

as)

anta

rctic

aEn

viro

nmen

talo

rgan

ism

Mud

dyso

ilsan

dse

dim

ents

inco

asta

lar

eas

Anta

rctic

a11

5

Pseu

doal

tero

mon

asba

cter

ioly

tica

Red

-spo

tdis

ease

Org

anis

mpr

oduc

esa

red

pigm

ent

onth

eLa

min

aria

and

indu

ces

dam

ages

toth

ese

edsu

pply

Cul

ture

beds

ofLa

min

aria

japo

nica

Japa

n67

7

Pseu

doal

tero

mon

as(A

ltero

mon

as)

citre

a1.

Envi

ronm

enta

liso

late

2.M

icro

flora

1.M

arin

esu

rface

wat

er2.

Far-e

aste

rnm

usse

l(C

reno

myt

ilus

gray

anus

and

Patin

opec

ten

yess

oens

is),

mol

lusc

s,as

cidi

ans,

spon

ges

1.M

edite

rrane

anSe

a,Fr

ance

2.Se

aof

Japa

n,Be

ring

Sea

285

396

Pseu

doal

tero

mon

as(A

ltero

mon

as)

deni

trific

ans

Envi

ronm

enta

lorg

anis

mN

orw

ayfio

rdco

ast

239

Pseu

doal

tero

mon

as(A

ltero

mon

as)

dist

inct

aM

icro

flora

Mar

ine

spon

geKo

man

dors

kie

Isla

nds,

Rus

sia

394

Pseu

doal

tero

mon

asel

yako

vii

(pre

viou

sly

Alte

rom

onas

elya

kovi

i)1.

Spot

dise

ase

2.M

icro

flora

1.Fr

onds

1.Sp

ot-w

ound

edfro

nds

ofLa

min

aria

japo

nica

2.Fa

r-eas

tern

mus

sel(

Cre

nom

ytilu

sgr

ayan

us)

1.Se

aof

Japa

n2.

Troi

tsa

Bay

(Rus

sia)

,Sea

ofJa

pan

679

Pseu

doal

tero

mon

as(A

ltero

mon

as)

espe

jiana

Envi

ronm

enta

liso

late

Mar

ine

envi

ronm

ent

Cal

iforn

ian

coas

t15

0

Pseu

doal

tero

mon

asfla

vipu

lchr

a(p

revi

ousl

yPs

eudo

alte

rom

onas

and

Alte

rom

onas

aura

ntia

)

Envi

ronm

enta

lorg

anis

mSu

rface

seaw

ater

Med

iterra

nean

,Fra

nce

398

286

Pseu

doal

tero

mon

as(A

ltero

mon

as)

lute

ovio

lace

aEn

viro

nmen

talo

rgan

ism

Med

iterra

nean

,Fra

nce

287

Pseu

doal

tero

mon

asm

aric

alor

isEn

viro

nmen

talo

rgan

ism

Mar

ine

spon

ge(F

asca

plys

inop

sis

retic

ulat

a )C

oral

Sea

398

Pseu

doal

tero

mon

as(A

ltero

mon

as)

nigr

ifaci

ens

Non

-pat

hoge

nic

Isol

ated

from

seaw

ater

and

mus

sels

Japa

n39

5

cont

inue

d



60 Chapter 1

Pseu

doal

tero

mon

aspi

scic

ida

(pre

viou

sly

Alte

rom

onas

pisc

icid

a,Ps

eudo

mon

aspi

scic

ida,

Flav

obac

teriu

mpi

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path

ogen

icLi

ver,

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en,l

ymph

node

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ated

from

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pean

otte

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K26

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hylo

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ussp

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pitis

,S.

cohn

ii,S.

epid

erm

idis

,S.

haem

olyt

icus

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apro

phyt

icus

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sim

ulan

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xylo

sus

Mic

roflo

raSk

inH

uman

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468

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Stap

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cocc

usw

arne

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Dis

ease

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ppor

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ctio

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Asso

ciat

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ithse

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ia,

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ytra

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1.U

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ated

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ons

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dex

opht

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sciti

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id.

Isol

ated

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and

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ey

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ainb

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ans

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ain

296

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pia

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rain

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bact

eriu

mst

ellu

latu

m)

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lpro

biot

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May

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entc

olon

izat

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veni

leoy

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USA

105

Stre

ptob

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sm

onilif

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isor

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ibly

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the

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tific

atio

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oncl

usiv

e(M

aher

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.,19

95)

Bact

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Intra

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ingu

inea

pigs

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lyar

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isin

mic

e,te

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ath

arth

ritis

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rkey

.Cau

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rin

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ans

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nd,w

orld

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961

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Bact

eria

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ase

Dise

ase

sign

sHo

st/is

olat

ion

site

Dist

ribut

ion

Ref

Tab

le1.

2.C

ontin

ued.




Stre

ptoc

occu

sty

peI

Now

reco

gniz

edas

Lact

ococ

cus

garv

ieae

Stre

ptoc

occu

sag

alac

tiae

grou

pB

Mor

talit

y,ex

opht

halm

ia,h

aem

orrh

age

Hae

mor

rhag

icar

eas

onbo

dy,

mou

th,f

ins.

Org

anis

min

inte

rnal

orga

ns

Blue

fish,

cultu

red

seab

ream

,wild

mul

let,

strip

edba

ss,s

eatro

ut,b

ullm

inno

ws.

Aqua

rium

fish

–ra

ms.

Cau

ses

mas

titis

inca

ttle,

infe

ctio

nsin

man

yot

hera

nim

alsp

ecie

s,an

dne

onat

alm

enin

gitis

inhu

man

s

Aust

ralia

,Kuw

ait,

Che

sape

ake

Bay,

Alab

ama

USA

71 135

242

637

Stre

ptoc

occu

sag

alac

tiae

grou

pB,

Type

1bca

psul

aran

tigen

( Stre

ptoc

occu

sdi

ffici

le)

Sept

icae

mia

,men

ingo

ence

phal

itis

Org

anis

mis

olat

edfro

mbr

ain

Car

p,ra

inbo

wtro

ut,t

ilapi

a,fre

shw

ater

orna

men

talf

ish,

frogs

,mic

e,bo

vine

s,hu

man

s

Aust

ralia

,Isr

ael

233

776

Stre

ptoc

occu

sdi

ffici

leC

onfir

med

asSt

rept

ococ

cus

agal

actia

egr

oup

B,ty

peIb

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ular

antig

en77

6

Stre

ptoc

occu

sdy

sgal

actia

ess

p.dy

sgal

actia

e,gr

oup

CAn

imal

path

ogen

Anim

als

–bo

vine

mas

titis

790

Stre

ptoc

occu

sdy

sgal

actia

ess

p.dy

sgal

actia

ese

rova

rLIn

fect

ions

and

sept

icae

mia

with

resu

lting

bron

chop

neum

onia

,m

yoca

rditi

s,os

teom

yelit

is,

pyel

onep

hriti

s,ab

sces

ses

Isol

ated

inal

mos

tpur

ecu

lture

from

lung

,kid

ney,

inte

stin

e,sp

leen

Isol

ated

from

harb

ourp

orpo

ises

stra

nded

orca

ught

infis

hing

nets

.Inf

ectio

nsin

cattl

e,do

gs,p

igs

and

othe

rani

mal

s

Balti

cSe

a,N

orth

Sea

727

790

Stre

ptoc

occu

sdy

sgal

actia

esu

bsp.

equi

sim

ilis,g

rou p

C,G

o rL

Anim

alan

dhu

man

path

ogen

Anim

als

and

hum

ans

790

Stre

ptoc

occu

sin

iae

(Stre

psh

iloiis

aju

nior

syno

nym

)1.

Dea

ths,

men

ingo

ence

phal

itis

2.G

olfb

alld

isea

sein

dolp

hins

3.Sy

stem

icin

fect

ion

4.W

ound

sw

hen

clea

ning

fish.

Dea

ths

1.C

ultu

reor

gani

smfro

mth

ebr

ain.

Also

kidn

eyan

dliv

er2.

Subc

utan

eous

absc

ess

3.To

rtico

llis,g

ross

lesi

ons,

gran

ulom

as,e

nlar

ged

orga

ns4.

Loca

lized

cellu

litis

,ulc

ers

1.Ba

rram

undi

,Eur

opea

nse

aba

ss,

gilth

ead

sea

brea

m,p

uffe

rfis

h,ra

bbit

fish,

reef

fish,

rain

bow

trout

,stin

gray

,tila

pia,

cultu

red

ayu,

flyin

gfo

xaq

uariu

mfis

h,fre

shw

ater

and

salt-

wat

erfis

h2.

Fres

hwat

erAm

azon

ian

dolp

hin

3.Fa

rmed

bullf

rogs

( Ran

aca

stes

beia

na)

4.H

uman

s–

espe

cial

lyel

derly

peop

lean

dfis

hha

ndle

rsM

arin

ean

dbr

acki

shw

ater

Aust

ralia

,Bah

rain

,Bar

bado

s,C

anad

a,C

hina

,Isr

ael,

Japa

n,So

uth

Afric

a,Th

aila

nd,U

SA

223

233

235

135

127

621

625

626

530

848

Stre

ptoc

occu

sm

illeri

(iden

tific

atio

nno

tcer

tain

.S.m

illeri

nota

reco

gniz

edna

me)

Dis

ease

Ulc

ers

onfla

nkan

dta

ilKo

icar

pU

K41 87

cont

inue

d



66 Chapter 1

Stre

ptoc

occu

spa

raub

eris

(pre

viou

sly

Stre

ptoc

occu

sub

eris

geno

type

II)

1.St

rept

ococ

cosi

s,m

ucoh

aem

orrh

agic

ente

ritis

2.M

astit

is

1.H

aem

orrh

ages

inan

alan

dpe

ctor

alfin

s,an

dey

es.O

rgan

ism

sis

olat

edfro

mliv

er,k

idne

y,sp

leen

2.M

ilk

1.C

ultu

red

turb

ot2.

Bovi

neSp

ain

224

754

Stre

ptoc

occu

sph

ocae

spp.

nov.

Opp

ortu

nist

icin

vira

linf

ectio

nsSe

als

Nor

way

700

Stre

ptoc

occu

spo

rcin

us(p

revi

ousl

ySt

rept

ococ

cus

infre

quen

s ,La

ncef

ield

grou

psE,

P,U

,V)

Infe

ctio

nsC

ervi

call

ymph

node

san

dm

ilkPi

gsW

orld

wid

e17

5

Stre

ptom

yces

salm

onis

(Stre

ptov

ertic

illium

salm

onis

baso

nym

)

Stre

ptom

ycos

isSa

lmon

ids

USA

41

Tena

ciba

culu

mm

aritim

um(p

revi

ousl

yC

ytop

haga

mar

ina

and

Flex

ibac

term

ariti

mus

)

Mar

ine

colu

mna

ris,e

rosi

vesk

indi

seas

e,gi

llle

sion

san

dul

cera

tive

derm

atiti

s,bl

ack

patc

hne

cros

is,

bact

eria

lsto

mat

itis

(mou

thro

t)

Erod

edm

outh

and

fins,

ulce

rate

dsk

inle

sion

sM

arin

efis

h,es

peci

ally

Atla

ntic

salm

on,

chin

ook

salm

on,s

eaba

ss,r

edse

abr

eam

,bla

ckbr

eam

,roc

kbr

eam

,nor

ther

nan

chov

y,st

riped

trum

pete

r,D

over

sole

,flo

unde

r,ye

llow

tail

Atla

ntic

,Can

ada,

Euro

pe,

Fran

ce,J

apan

,Sco

tland

89 90 91 154

551

605

725

801

Tena

ciba

culu

m(F

lexi

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er)

ovol

ytic

umO

ppor

tuni

stpa

thog

enLa

rvae

and

egg

mor

talit

yD

isso

lves

chor

ion

and

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radi

ata

ofth

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gsh

ells

Atla

ntic

halib

uteg

gsan

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rvae

Nor

way

324

725

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cocc

usfe

ssus

Path

ogen

icity

notd

eter

min

edO

rgan

ism

isol

ated

from

liver

,ki

dney

Isol

ated

from

ade

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rbou

rsea

land

porp

oise

Euro

pe36

9

Vago

cocc

usflu

vial

is1.

Envi

ronm

enta

lorg

anis

m2.

Clin

ical

sam

ples

2.Bl

ood,

perit

onea

lflu

id,w

ound

s1.

Riv

erw

ater

,chi

cken

faec

es,d

omes

tican

imal

s(p

ig,c

attle

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s,ho

rse)

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uman

s

UK

177

629

732

Vago

cocc

uslu

trae

Path

ogen

icity

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eter

min

edO

rgan

ism

foun

din

bloo

d,liv

er,

lung

s,sp

leen

Otte

rU

K47

7

Bact

eria

Dise

ase

Dise

ase

sign

sHo

st/is

olat

ion

site

Dist

ribut

ion

Ref

Tab

le1.

2.C

ontin

ued.




Vago

cocc

ussa

lmon

inar

umLa

ctob

acillo

sis,

pseu

doki

dney

dise

ase,

perit

oniti

s,se

ptic

aem

iaH

aem

orrh

ages

ongi

llsan

din

ocul

arre

gion

,per

itoni

tis,h

eart

lesi

ons,

enla

rged

sple

en,l

istle

ssbe

havi

our

Atla

ntic

salm

on,b

row

ntro

ut,r

ainb

owtro

utAu

stra

lia,F

ranc

e,N

orth

Amer

ica,

Nor

way

542

682

807

Varra

calb

mis

pp.n

ov.

Eye

lesi

ons

(blo

ody

eye)

and

blin

dnes

sLe

sion

sin

kid,

liver

,gills

,ps

eudo

bran

ch.O

rgan

ism

isol

ated

from

eyes

.Ski

nul

cers

Atla

ntic

salm

onN

orw

ay77

1

Vibr

ioae

roge

nes

Envi

ronm

enta

lorg

anis

mAs

soci

ated

with

sea

gras

sse

dim

ent,

foun

din

shal

low

coas

tala

ndoc

eani

cse

awat

ers

Taiw

an69

2

Vibr

ioae

stua

rianu

sEn

viro

nmen

talo

rgan

ism

Foun

das

soci

ated

with

shel

lfish

(oys

ter,

clam

,cra

b)an

des

tuar

ine

wat

ers

USA

747

Vibr

ioag

ariv

oran

sEn

viro

nmen

talo

rgan

ism

Seaw

ater

Abal

one

–pa

thog

enic

ityno

tkno

wn

Aust

ralia

,Med

iterra

nean

Sea

(Spa

in)

135

514

Vibr

ioal

gino

lytic

us1.

Syst

emic

dise

ase,

ulce

rativ

edi

seas

e,ne

cros

is,e

yedi

seas

e,vi

brio

sis,

scal

lop

larv

alm

orta

lity

2.W

ound

infe

ctio

ns,e

xter

nalo

titis

,ce

llulit

is

1.O

rgan

ism

isol

ated

from

orga

nsan

dey

e,sc

allo

pla

rvae

2.W

ound

son

expo

sure

tose

awat

er

1.Au

stra

lian

nativ

ean

din

trodu

ced

fish.

Mol

lusc

s(la

rval

and

juve

nile

),re

dab

alon

e,So

uth

Afric

anab

alon

e,sc

allo

pla

rvae

,eel

,rai

nbow

trout

,sea

brea

m,

turb

ot,t

urtle

s,se

ahor

se.S

hrim

ps2.

Hum

ans

Aust

ralia

,Chi

le,M

exic

o,w

orld

wid

eIn

do-P

acifi

c&

East

Asia

30 300

301

650

Vibr

ioan

guilla

rum

See

List

onel

laan

guilla

rum

Vibr

iobr

asilie

nsis

Path

ogen

icity

notk

now

n.Li

kely

tobe

norm

alflo

raIs

olat

edfro

mbi

valv

ela

rvae

(Nod

ipec

ten

nodo

sus )

Braz

il74

0

Vibr

ioca

lvie

nsis

Envi

ronm

enta

lorg

anis

mSe

awat

erW

este

rnM

edite

rrane

anSe

a,Fr

ance

216

Vibr

ioca

mpb

ellii

Envi

ronm

enta

lorg

anis

mSe

awat

erFl

orid

a,Pu

erto

Ric

o81

982

0

Vibr

ioca

mpb

ellii-

like

Mor

talit

ies

inju

veni

les

Org

anis

min

brai

n,ki

dney

,liv

er.

Org

ans

had

lesi

ons

and

haem

orrh

age

Hat

cher

y-re

ared

turb

otan

dbr

illN

ewZe

alan

d22

1

Vibr

ioca

rcha

riae

Isa

juni

orsy

nony

mof

V.ha

rvey

iSl

ight

diffe

renc

esof

phen

otyp

ebe

twee

nth

est

rain

sar

ere

porte

din

the

liter

atur

e61

9

cont

inue

d



68 Chapter 1

Vibr

ioch

oler

ae(n

on-0

1)H

eibe

rggr

oups

I,II

1.Se

ptic

aem

ia2.

Gas

troen

terit

is1.

Skin

haem

orrh

ages

2.D

iarrh

oea,

vom

iting

1.Ay

u,go

ldfis

h,sh

ellfi

sh.I

sola

ted

from

fresh

and

estu

arin

ew

ater

s2.

Hum

ans

–co

nsum

ptio

nof

raw

shel

lfish

1.Au

stra

lia,J

apan

2.Af

rica,

Asia

nco

ntin

ent,

Euro

pe,U

SA,U

K

196

255

275

434

507

Vibr

ioch

oler

aeEl

Tor

(ser

ogro

ups

01&

0139

)C

hole

raH

uman

sFo

und

inaq

uatic

envi

ronm

ent–

surfa

cew

ater

s

Sero

grou

p01

39in

Bang

lade

sh,

Indi

ansu

bcon

tinen

tonl

y.Pa

ndem

icst

rain

275

392 9

Vibr

ioch

oler

ae-li

keD

eath

Lobs

ters

USA

.(O

rgan

ism

does

n’tg

row

inte

mpe

ratu

res

grea

tert

han

25°C

)

Vibr

ioci

ncin

natie

nsis

1.En

viro

nmen

talo

rgan

ism

2.Ba

cter

aem

ia,m

enin

gitis

1.En

viro

nmen

t2.

Hum

anpa

thog

en2.

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120

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ioco

rallii

lytic

ussp

p.no

v.(p

revi

ousl

yV.

cora

lytic

usYB

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thog

enof

cora

lTi

ssue

lysi

san

dde

ath.

Smal

lwhi

tesp

ots

seen

onco

rala

t3–5

days

and

com

plet

ede

stru

ctio

nof

tissu

eaf

ter2

wee

ks

Cor

al( P

ocillo

pora

dam

icor

nis)

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ois

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edfro

moy

ster

larv

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mth

eAt

lant

icO

cean

Atla

ntic

Oce

an,I

ndia

nO

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and

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Sea

83 84

Vibr

iocy

clitr

ophi

cus

(orig

inal

lysp

eltV

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lotro

phic

us)

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ronm

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lorg

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gle

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bor,

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hing

ton,

USA

338

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iodi

abol

icus

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ronm

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lid(A

lvin

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pom

peja

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nde

ep-s

eave

ntPa

cific

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an63

5

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iodi

azot

roph

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ronm

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lorg

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mG

astro

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stin

altra

ctof

sea

urch

ins

and

mar

ine

anim

als,

surfa

ceof

reed

s,oc

curri

ngin

estu

arin

een

viro

nmen

ts,

seaw

ater

and

sedi

men

ts

Can

ada,

Scot

land

,Eng

land

319

Vibr

iofis

cher

i(s

ynon

ymou

sw

ithPh

otob

acte

rium

fisch

eri)

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thog

enic

ityin

doub

t2.

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bion

t1.

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teno

dule

son

skin

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emor

rhag

icul

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tion,

tum

ours

inpa

ncre

asan

dbi

ledu

ct2.

Pres

enti

nlig

htor

gan

ofse

piol

idsq

uid

1.Se

abr

eam

,tur

bot

2.Se

piol

idsq

uid

( Eup

rym

nasc

olop

es)

1.Sp

ain

2.H

awai

i10

626

734

248

6

Bact

eria

Dise

ase

Dise

ase

sign

sHo

st/is

olat

ion

site

Dist

ribut

ion

Ref

Tab

le1.

2.C

ontin

ued.




Vibr

ioflu

vial

is(p

revi

ousl

ygr

oup

F(F

urni

sset

al.,

1977

)and

grou

pEF

6(H

uqet

al.,

1980

))

1.M

orta

lity

2.En

viro

nmen

t3.

Acut

ew

ater

y,ch

oler

ae-li

kedi

arrh

oea.

Toxi

npr

oduc

ed

1.O

rgan

s2.

Non

-pat

hoge

nic

3.Fa

eces

1.Pl

aice

fry( P

leur

onec

tes

plat

essa

)2.

Foun

din

aqua

tic,p

artic

ular

lyes

tuar

ine

envi

ronm

ent.

Isol

ated

from

mar

ine

mol

lusc

san

dcr

usta

cea

3.H

uman

s

1.D

enm

ark

2.W

orld

wid

e3.

Bang

lade

sh,I

ndia

,Ken

ya,

Mid

dle

East

,Phi

lippi

nes,

Spai

n,Ta

nzan

ia,T

unis

ia

276

378

485

620

687

Vibr

iofu

rnis

sii

(form

erly

V.flu

vial

isbi

ovar

2(a

erog

enic

biog

roup

),gr

oup

F,gr

oup

EF-6

)

1.Pa

thog

enic

ityin

doub

t2.

Envi

ronm

enta

lorg

anis

m3.

Food

pois

onin

g,ga

stro

ente

ritis

1.H

aem

orrh

ages

inin

test

ine

3.D

iarrh

oea

1.Ee

l2.

Riv

erw

ater

,ani

mal

faec

es,m

arin

em

ollu

scs

and

crus

tace

a3.

Hum

ans

Foun

din

aqua

tic,p

artic

ular

lyes

tuar

ine

envi

ronm

ent

1.Sp

ain

2.U

K,w

orld

wid

e3.

Bahr

ain,

Bang

lade

sh,

Indo

nesi

a,Pe

ru,U

SA

123

276

240

485

687

Vibr

ioga

zoge

nes

Envi

ronm

enta

lorg

anis

m67

Vibr

ioha

liotic

olis

pp.n

ov.

Non

-pat

hoge

nic

Mai

ngu

tmic

roflo

raAb

alon

eJa

pan

678

Vibr

ioha

rvey

i(o

rgan

ism

prev

ious

lyid

entif

ied

asV.

carc

haria

eis

said

tobe

aju

nior

syno

nym

ofV.

harv

eyi)

1.M

orta

lity

insh

arks

and

abal

one,

necr

otiz

ing

ente

ritis

insu

mm

erflo

unde

r,ga

stro

ente

ritis

ingr

oupe

r,sy

stem

icdi

seas

e,ul

cera

tive

dise

ase,

necr

osis

,vib

riosi

s2.

Wou

nd(s

hark

bite

)

1.W

hite

spot

son

foot

ofab

alon

e.Lo

seab

ility

toad

here

.Nec

rotic

dege

nera

tion

and

vacu

olat

ion

inth

ele

sion

.Flo

unde

rhav

eex

tend

edab

dom

enan

dre

dan

us.F

ish

–or

gani

smfro

mki

dney

,eye

Shrim

ps–

blac

ksp

ots

onex

oske

leto

n.O

rgan

ism

inle

sion

s,ey

e,he

pato

panc

reas

1.Ab

alon

e,sh

ark,

floun

der,

grou

per,

milk

fish,

mol

lusc

s,oy

ster

s(la

rval

and

juve

nile

),bl

ack

tiger

praw

ns,p

enae

idpr

awns

and

shrim

ps,s

alm

onid

s,se

aba

ss,

sea

brea

m,s

eaho

rse,

shar

ks,o

ctop

us.

Aust

ralia

nna

tive

and

intro

duce

dfis

h2.

Hum

ans

1.Au

stra

lia,E

urop

e,In

done

sia,

Indo

-Pac

ific

&Ea

stAs

ia,

Japa

n,Ph

ilippi

nes,

Sout

hAm

eric

a,Ta

iwan

,Tha

iland

,Ve

nezu

ela,

USA

,wor

ldw

ide

23 11 410

576

581

619

710

734

735

847

Vibr

ioho

llisae

(pre

viou

sly

Gro

upEF

-13)

1.N

on-p

atho

geni

cfo

rfis

h2.

Food

pois

onin

g,ga

stro

ente

ritis

,w

ound

infe

ctio

n,ba

cter

aem

ia

2.D

iarrh

oea

1.Pr

esen

tin

fish

inte

stin

e2.

Hum

ans

Has

ther

mos

tabl

eha

emol

ysin

sim

ilart

oV.

para

haem

olyt

icus

1.Ja

pan

2.U

SA34

655

558

0

Vibr

ioic

hthy

oent

eri

Mor

talit

ies,

inte

stin

alne

cros

isO

paqu

ein

test

ines

Japa

nese

floun

derl

arva

eJa

pan

389

Vibr

iole

ntus

Path

ogen

icity

nots

tate

dAs

soci

ated

with

Med

iterra

nean

oyst

ers

Span

ish

Med

iterra

nean

coas

t51

3

Vibr

iolo

gei

1.Po

ssib

lepa

thog

en2.

Non

-pat

hoge

nic

1.Sk

inle

sion

s2.

Sym

bion

tin

light

orga

n1.

Atla

ntic

salm

on2.

Squi

d( S

epio

la)

1.Ic

elan

d2.

Wes

tern

Paci

fic25

7

cont

inue

d



70 Chapter 1

Vibr

iom

edite

rrane

i1.

Non

-pat

hoge

nic

2.Pr

obio

ticpr

oper

ties

3.C

oral

blea

chin

g–

see

unde

rV.

shilo

nii

Appe

ars

tobe

ahe

tero

gene

ous

spec

ies

1.Pl

ankt

on,s

ease

dim

ents

,sea

wat

er2.

Som

est

rain

sar

ea

prob

iotic

fort

urbo

tla

rvae

3.C

oral

blea

chin

g

Spai

n,M

edite

rrane

anco

ast

631

Vibr

iom

etsc

hnik

ovii

1.N

on-p

atho

geni

cfo

rfis

h2.

Perit

oniti

san

dba

cter

aem

ia1.

Isol

ated

from

fow

lwith

chol

erai

cdi

seas

e1.

Foun

din

mar

ine

and

fresh

wat

eren

viro

nmen

t,es

peci

ally

river

s,es

tuar

ies

and

sew

age.

Isol

ated

from

clam

s,co

ckle

s,oy

ster

s,lo

bste

r2.

Hum

ans

Wor

ldw

ide

483

Vibr

iom

imic

us(p

revi

ousl

yVi

brio

chol

erae

Hei

berg

grou

pV)

1.Sy

stem

icdi

seas

ean

dop

portu

nist

2.C

onta

min

anto

nse

atu

rtle

eggs

3.Fo

odpo

ison

ing,

gast

roen

terit

is,

otiti

sm

edia

1.H

aem

olym

ph,i

nfla

mm

atio

nof

peric

ardi

um3.

Dia

rrhoe

a,ea

rinf

ectio

n

1.C

rust

acea

ns–

pena

eid

praw

ns,

yabb

ies,

mar

ron,

fresh

wat

ercr

ayfis

h.Ba

rram

undi

2.R

educ

edvi

abilit

yof

turtl

eeg

gsan

dhu

man

path

ogen

follo

win

gco

nsum

ptio

nof

eggs

3.H

uman

s–

cons

umpt

ion

ofra

wsh

ellfi

sh.

Foun

din

fresh

and

brac

kish

wat

er

1,3.

Asia

,Aus

tralia

,Ba

ngla

desh

,Mex

ico,

New

Zeal

and,

Gua

m,C

anad

a,w

orld

wid

e2.

Cos

taR

ica

416

113

521

023

050

7

Vibr

iom

ytili

1,2.

Non

-pat

hoge

nic

1.N

orm

alflo

ra1.

Mus

sels

2.H

uman

sSp

ain

632

Vibr

iona

trieg

ens

Envi

ronm

enta

liso

late

Salt

mar

shm

ud,w

ater

,oys

ters

UK,

USA

820

Vibr

iona

varre

nsis

Envi

ronm

enta

liso

late

Envi

ronm

enta

l–se

wag

ean

dsu

rface

wat

erSp

ain

768

Vibr

ione

ptun

ius

Mic

roflo

raD

omin

antm

icro

flora

ina

reci

rcul

atin

gsy

stem

forr

otife

rs.I

sola

ted

from

heal

thy

and

dise

ased

biva

lve

larv

ae( N

odip

ecte

nno

dosu

s ),g

utof

turb

otla

rvae

,rot

ifers

305

Vibr

ione

reis

Envi

ronm

enta

liso

late

Seaw

ater

820

Bact

eria

Dise

ase

Dise

ase

sign

sHo

st/is

olat

ion

site

Dist

ribut

ion

Ref

Tab

le1.

2.C

ontin

ued.




Vibr

ioni

grip

ulch

ritud

oEn

viro

nmen

tali

sola

teSe

awat

er81

982

0

Vibr

ioor

dalii

(pre

viou

sly

Vibr

ioan

guilla

rum

biot

ype

2an

dLi

ston

ella

orda

lii)

Vibr

iosi

s,ba

cter

ialn

ecro

sis

and

syst

emic

dise

ase

Org

anis

ms

onm

uscl

e,sk

in,g

ills,

dige

stiv

etra

ct,h

eart,

liver

,kid

neys

,sp

leen

.Nec

rosi

san

dha

emor

rhag

e

Mol

lusc

s–

(larv

alan

dju

veni

le),

salm

onAu

stra

lia,J

apan

,Nor

th-W

est

Paci

fic,U

SA,w

orld

wid

e68

0

Vibr

ioor

ient

alis

Envi

ronm

enta

liso

late

Lum

inou

sm

arin

eba

cter

ium

Chi

na84

6

Vibr

iopa

cini

ispp

.nov

.Pa

thog

enic

ityno

tkno

wn

Isol

ated

from

shrim

pla

rvae

(Pen

aeus

chin

ensi

s )C

hina

306

Vibr

iopa

raha

emol

ytic

us(p

revi

ousl

yBe

neck

eapa

raha

emol

ytic

a )

1.Se

ptic

aem

ia,m

orta

lity

2.W

ither

ing

synd

rom

e3.

Food

pois

onin

g,ga

stro

ente

ritis

,w

ound

infe

ctio

n,ba

cter

aem

ia

1.Ex

tern

alha

emor

rhag

e,ta

ilro

t,or

gani

smin

inte

rnal

orga

ns2.

Org

anis

ms

foun

din

haem

olym

ph

3.D

iarrh

oea,

vom

iting

1.C

rust

acea

ns(s

hrim

ps,m

arro

n),m

ulle

t,oc

topu

s,Ib

eria

nto

othc

arp

2.C

ultu

red

smal

laba

lone

3.H

uman

s

1.In

do-P

acifi

can

dEa

stAs

ia,

Spai

n2.

Taiw

an

10 135

272

417

499

Vibr

iope

cten

icid

aPa

thog

enic

Mor

ibun

dsc

allo

pSc

allo

pla

rvae

,shr

imps

,pra

wn

East

Asia

,Ind

o-Pa

cific

,Fra

nce

470

Vibr

iope

lagi

usSe

eLi

ston

ella

pela

gia

Vibr

iope

naei

cida

(pre

viou

sly

Vibr

ioPJ

)Pr

opos

edca

usat

ive

agen

tof

Synd

rom

e93

1.Vi

brio

sis

ofpr

awns

2.Se

ptic

aem

ia,a

ssoc

iate

dw

ithSy

ndro

me

93

1.Br

own

spot

s(n

odul

es)i

nly

mph

oid

orga

nsan

dgi

lls2.

Hae

mol

ymph

1.Ku

rum

apr

awns

.Als

ois

olat

edfro

mhe

alth

ypr

awns

2.Sh

rimp

( Pen

aeus

styl

irost

ris)

1.Ja

pan

2.N

ewC

aled

onia

388

187

676

Vibr

iopr

oteo

lytic

us,s

train

CW

8T2

Path

ogen

Affe

cts

mic

rovi

lli,gu

tcel

ls,

deva

stat

esce

llsin

body

cavi

tyJu

veni

leAr

tem

iasp

p.(b

rine

shrim

p),

used

asliv

edi

ets

fora

quac

ultu

resp

ecie

sEu

rope

67 788

Vibr

ioro

tifer

ianu

sPa

thog

enic

ityno

tkno

wn

Isol

ated

from

rotif

erflo

w-th

roug

hcu

lture

syst

emBe

lgiu

m30

5

Vibr

ioru

moi

ensi

sEn

viro

nmen

tali

sola

teIs

olat

edfro

mdr

ain

pool

offis

hpr

oduc

tpr

oces

sing

plan

tJa

pan

850

Vibr

iosa

lmon

icid

aC

oldw

ater

vibr

iosi

s,H

itra

dise

ase

Sept

icae

mia

,hae

mor

rhag

esin

the

inte

gum

ents

urro

undi

ngth

eor

gans

.Org

anis

mfo

und

inbl

ood

and

kidn

ey

Atla

ntic

salm

on,d

isea

sed

cod

Can

ada,

Faro

eIs

land

s(D

enm

ark)

,Ice

land

,Nor

way

,Sc

otla

nd

198

232

Vibr

iosc

opht

halm

iN

on-p

atho

geni

cPa

rtof

gutm

icro

flora

Juve

nile

turb

otSp

ain

149

254

cont

inue

d



72 Chapter 1

Vibr

iosh

iloni

i(p

revi

ousl

yV.

shilo

i,Vi

brio

spec

ies

AK-1

).La

test

info

rmat

ion

sugg

ests

this

isa

late

rsyn

onym

ofV.

med

iterra

nei

(Tho

mps

onet

al.,

2001

a,b)

Cor

albl

each

ing

Adhe

res

and

pene

trate

sin

toth

eep

ider

mis

ofth

eco

ral,

Ocu

lina

pata

goni

ca.C

ultu

refro

mth

eco

ral

muc

us.T

heor

gani

smis

intra

cellu

lara

ndno

n-cu

ltura

ble

Bact

eriu

mbi

nds

toth

eco

rala

tele

vate

dte

mpe

ratu

res

and

disr

upts

the

sym

biot

icpr

oces

sw

ithth

eph

otos

ynth

etic

mic

ro-a

lgae

endo

sym

bion

ts(z

ooxa

nthe

llae)

.Hea

t-sta

ble

toxi

npr

oduc

ed

Med

iterra

nean

Sea

59 458

742

Vibr

iosp

lend

idus

Mor

talit

yin

stre

ssed

oyst

ers

Oys

terl

arva

e(C

rass

ostre

agi

gas)

Fran

ce46

646

7

1.Vi

brio

sple

ndid

usI.

2.Vi

brio

sple

ndid

us-li

ke1.

Sept

icae

mia

.Mor

talit

ies

inju

veni

les

2.M

orta

litie

s1.

Hae

mor

rhag

esat

mou

th,a

nus

and

fins.

Org

anis

ms

inbr

ain,

kidn

ey,l

iver

2.Sk

inha

emor

rhag

es,f

inan

dta

ilro

tero

sion

,dec

ayof

soft

tissu

ebe

twee

nfin

s.Sa

mpl

efro

mki

dney

and

sple

en

1.G

ilt-h

ead

sea

brea

m,r

ainb

owtro

ut,

turb

otla

rvae

,shr

imps

(pra

wns

).Ju

veni

leha

tche

ry-re

ared

turb

otan

dbr

ill2.

Plai

cefry

1.Fr

ance

,New

Zeal

and,

Scot

land

,Spa

in2.

Den

mar

k

281

254 31 221

620

Vibr

iosp

lend

idus

IIBa

cilla

ryne

cros

is,m

orta

lity

Larv

aebe

com

ein

activ

ean

dse

ttle

toth

ebo

ttom

ofth

eta

nkJa

pane

seoy

ster

larv

ae,c

uppe

doy

ster

(Cra

ssos

trea

giga

s)Ja

pan,

Fran

ce72

179

8

Vibr

iota

petis

(pre

viou

sly

Vibr

ioP1

orVP

1or

VTP)

Brow

nrin

gdi

seas

e,hi

ghm

orta

lity

Brow

nco

nchi

olin

depo

sito

nin

ner

surfa

ceof

shel

lbet

wee

npa

lliall

ine

and

shel

lmar

gins

Cul

ture

dM

anila

clam

sEn

glan

d,Fr

ance

,Por

tuga

l,Sp

ain

108

146 14 609

610

Vibr

iotra

chur

iLa

test

info

rmat

ion

sugg

ests

this

isa

juni

orsy

nony

mof

V.ha

rvey

i(T

hom

pson

etal

.,20

02b)

Dis

ease

Hae

mor

rhag

esin

orga

ns,

exop

htha

lmia

Hor

sem

acke

rel

Japa

n40

074

3

Vibr

iotu

bias

hii

Bact

eria

lnec

rosi

san

dsy

stem

icdi

seas

eLa

rvae

ceas

esw

imm

ing,

tissu

edi

gest

ion

Mol

lusc

s(o

yste

rand

clam

)–la

rval

and

juve

nile

,Pac

ific

oyst

erAu

stra

lia,U

K,U

SA32

129

4

Vibr

iovi

scos

usSe

eM

orite

llavi

scos

a82 50

6

Bact

eria

Dise

ase

Dise

ase

sign

sHo

st/is

olat

ion

site

Dist

ribut

ion

Ref

Tab

le1.

2.C

ontin

ued.




Vibr

iovu

lnifi

cus

biot

ype

I1.

Envi

ronm

enta

l2.

Infe

ctio

n,sy

stem

icdi

seas

e,ul

cera

tive

dise

ase,

necr

osis

3.W

ound

infe

ctio

n,se

ptic

aem

iafo

llow

ing

cons

umpt

ion

ofra

wse

afoo

des

peci

ally

oyst

ers.

Sept

icae

mia

has

a50

%fa

talit

yra

te

1.M

icro

flora

ofin

test

ines

orgi

llsof

fish,

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The information is presented in tabular form sothat it is readily accessible in a summarized form.

The bacterial names are listed in alphabeticalorder. The disease column indicates whether theorganism is a saprophyte, an environmentalorganism or is a pathogen of fish and other aquaticanimals. The next column indicates the diseasesigns that may be seen with an infection caused bythis organism, and then the aquatic species islisted, as is the geographical distribution of theorganism. The final column lists the referencenumber.

1.4 Taxonomy and Disease Statusof Bacteria

New bacteria from the aquatic world are beingdiscovered, identified and named at an increas-ing rate. Some are then reassigned to a differentor newly described genus, while others changetheir species name as more research and studyon the taxonomy follows. The following sectiongives brief details of the current taxonomic statusof a genus or species. Because some of thisinformation is likely to become out of date,microbiologists are encouraged to keep up withsuch information, which is found at taxonomicwebsites. Some addresses and suggested readingcan be found under Further Reading and OtherInformation Sources, p. 278. Refer to these andother texts for a comprehensive coverage ofdiseases.

Abiotrophia spp.

These bacteria, which are known as nutritionallyvariant streptococci (NVS), satellite aroundother bacterial growth, or require L-cysteineor pyridoxal hydrochloride (vitamin B6) forgrowth. A. elegans requires L-cysteine for growth.A. adiacens, A. defectiva and A. elegans areisolated as part of the normal flora from the oralcavity, intestinal tract and genital tract of humans.They have also been isolated in cases of endo-carditis, otitis media and post-surgery woundinfections. They have been included in this bookbecause an organism found in a minke whalewas originally placed into this genus. It has sincebeen placed into the genus Granulicatella alongwith some other previously named Abiotrophia

species. G. balaenopterae, found in the minkewhale, does not require additional growth factorsand grows in air or 5% CO2.

Aequorivita spp.

Strains from this genus belong to the familyFlavobacteriaceae and the order Cytophagales,and have been isolated from the marine environ-ment in Antarctica. They can grow on MA 2216.

Aeromonas spp.

The taxonomy of Aeromonas spp. (familyAeromonadaceae) is in a continual state of fluxas new species are described and the descriptionsof the existing taxa are refined. There is aphenotypic and a genotypic classification.Genotypic classification is based on DNA–DNAhybridization with 16 DNA HG recognized atpresent. The designation of the genospeciesis dependent on the type strain within the HG.Problems exist for the diagnostic microbiologist inthe phenotypic identification of strains. Pheno-typic diversity of strains occurs within a DNA HGand strains from different HG may be bio-chemically similar. In this book the phenotypictests are recorded for the type strains. A table(Table 4.2) is also presented of the phenotypictests according to HG (Abbott et al., 1992;Kaznowski, 1998). An average of the results fromthese two studies is reported. Results from differ-ent references can be difficult to assess. Abbottet al. (1992) stated that when using 22 strainsof HG1, including the type strain A. hydrophilaATCC 7966, 100% were positive for LDC. Thiswas also supported by research that recognizeda new subspecies within the A. hydrophilagroup, with the recognition of A. hydrophilassp. hydrophila and A. hydrophila ssp. dhakensis(Huys et al., 2002b). This study reported resultsfor A. hydrophila ssp. hydrophila of the typestrain LMG 2844, which is equivalent to ATCC7966. However, Kaznowski (1998), using onestrain only (the type strain ATCC 7966), statedthat 0% of HG1 are positive for LDC. Likewise,other researchers have found that the type strainATCC 7966 is negative for LDC (Nielsen et al.,2001). One of the reasons that there are variablereports for LDC may be due to misinterpretation




of the test, or the use of different methods todetect LDC. In the conventional tube test, LDC isa pale purple colour, particularly in comparisonwith the ADH tube. Also, a stronger result maybe obtained after 48 h incubation rather than at24 h incubation.

HG8 and HG10 can be separated by bio-chemical tests; they are genotypically identicaland are named A. veronii biogroup sobria and A.veronii biogroup veronii, respectively. The typestrain of the non-motile A. salmonicida belongs inHG3, even though HG3 corresponds mainly withthe phenospecies of A. hydrophila.

Aeromonas allosaccharophila and Aero-monas encheleia: The status of these speciesis controversial (Working group on Aeromonastaxonomy. International Journal of SystematicBacteriology, 1999, 49: 1946).

Aeromonas bestiarum was shown to bevirulent to the common carp in pathogenicitystudies (Kozinska et al., 2002).

A. caviae and A. veronii may cause 100%mortality in giant freshwater prawns when eitherorganism is present in high numbers; in the orderof 3.8 × 106 and 3.7 × 105 cells/g of body weight,respectively (Sung et al., 2000).

A. hydrophila is an opportunistic organismfollowing a primary stressor; however, the patho-genesis and virulence of this organism remainsunclear. A number of toxins are produced,including haemolysins, cytotoxins, enterotoxin, acytolytic haemolysin and aerolysin. Aerolysin isa channel-forming toxin that kills cells, is lethalto mice and possesses haemolytic and cytotoxicactivities. It is the main contributor to pathogenic-ity (Chopra et al., 1993). The aerolysin gene is notfound in non-haemolytic strains of A. hydrophila,non-haemolytic strains of A. caviae, or strains of A.sobria (Pollard et al., 1990).

A dominant A. hydrophila, which was nega-tive for the phenotypic tests of LDC and cellobiosefermentation, was found to be associated with dis-ease outbreaks in aquaculture in China (Nielsenet al., 2001). However, this is in contrast to otherfindings that have suggested that LDC-negativeisolates are less virulent than LDC-positiveisolates, where 78% (n = 23) of isolates fromdiseased mammals and 100% (n = 4) of isolatesfrom moribund fish were LDC-positive (Lallierand Higgins, 1988). Although the number of iso-lates tested from moribund fish was small (n = 4),strains of A. hydrophila in these cases were alsoindole-negative, did not produce enterotoxin,

and were negative for agglutination with 0.2%acriflavine.

Virulence studies in strains isolated fromwalking catfish suggested that there was a scale ofvirulence ranging from avirulent, weakly virulent,to strongly virulent. All virulent strains possessedhaemolysin genes, and were able to lyse red bloodcells of a wide variety of animal species. Themajority of avirulent or weakly virulent strainseither did not possess any of the three haemolysingenes tested (AHH1, AHH5, ASA) or had combi-nations of only one or two of the genes. Thesestrains were generally unable to lyse the redblood cells of as many animal species. Thevirulence strains were able to cause infection at adose rate of 104 CFU/ml, whereas avirulent strainsdid not cause disease at this dose (Angka et al.,1995).

A recent article reports a subgroup withinA. hydrophila HG1, and these are referred to asA. hydrophila ssp. dhakensis ssp. nov. Thesestrains were isolated from cases of diarrhoeain children from Bangladesh. They weredifferentiated from A. hydrophila ssp. hydrophilaby genetic tests and the following phenotypictests. A. hydrophila ssp. dhakensis is negative foracid production from L-arabinose, and negativefor utilization of methyl a-D-mannoside, L-fucose,and L-arabinose, whereas A. hydrophila ssp.hydrophila is positive for acid production fromL-arabinose and positive for utilization of methyla-D-mannoside, L-fucose, and L-arabinose (Huyset al., 2002b).

A. ichthiosmia is a later synonym ofAeromonas veronii (Huys et al., 2001).

Aeromonas sobria is now called Aeromonasveronii ssp. sobria. Some commercial systems stillrefer to it as A. sobria. Both terminologies arestated in the tables here.

Aeromonas trota is a junior synonym ofA. enteropelogenes. A. enteropelogenes waspublished first; however, because the nameA. trota is more widely used, this name hasbeen retained (Huys et al., 2002a).

Aeromonas veronii biovar sobria is beingretained so that there is continuity in the literatureand with biochemical identification schemes. Thisorganism was previously called A. sobria but isactually genetically A. veronii (Working groupon Aeromonas taxonomy, 1999, IJSB, 49:1946). This species has been shown to be non-pathogenic to the common carp when tested invirulence studies (Kozinska et al., 2002).

76 Chapter 1



Aeromonas salmonicida ssp. salmonicida

These strains are characterized by pigmentproduction and by being non-motile. Theybelong to Aeromonas DNA HG3. These virulentstrains are termed ‘typical’ A. salmonicida,possess an A-protein layer and autoagglutinate indistilled water. However, these phenotypic testsare not reliable indicators of the in vivo virulenceof A. salmonicida (Bernoth, 1990; Olivier, 1990).A. salmonicida ssp. salmonicida causes the dis-ease known as furunculosis, so called becauselesions in the dermis are seen as characteristicumbonate ulcers. The organism penetrates intothe underlying tissues and organs. Furunculosisaffects mainly salmonid fish.

The atypical Aeromonas salmonicida

The term ‘atypical’ is used to describe strains ofA. salmonicida that show variations in biochemi-cal reactions, may have slow growth, be slow toproduce pigment, be negative for oxidase, andthat may produce a variety of extracellular pro-teases. The pathogenicity of atypical strains showsgreat variability, and virulence mechanisms differfor those described for ‘typical’ Aeromonassalmonicida. An extracellular A-layer has beendetected, an extracellular metallo-protease anda different iron utilization mechanism has beendescribed for atypical strains compared to typicalstrains (Wiklund and Dalsgaard, 1998). Currentthought suggests that the skin ulceration seen inthe disease may be due to only one well-definedbiotype of atypical A. salmonicida, yet theseorganisms cannot be isolated consistently. Theorganisms are most commonly isolated in thehealing stage of the ulcer, suggesting thatthe infection is not lethal (Wiklund et al., 1999).

A number of subspecies have been named,including A. salmonicida ssp. masoucida, ssp.smithia, ssp. achromogenes, ssp. nova, the latterisolated from non-salmonid fish. The subspeciesof masoucida and smithia have not been reportedsince their initial isolation by Kimura (1969) andAustin et al. (1989). There are many strains ofA. salmonicida reported in the literature thatare atypical and do not fall into any of the above-mentioned subspecies (Wiklund and Dalsgaard,1998). There is some contention about the validityof retaining A salmonicida ssp. and research

continues in order to understand and enabledefinitive identification of this diverse species. Theatypical strains infect a wider variety of fish speciesthan does A. salmonicida ssp. salmonicida.

Alteromonas genus

The genus Alteromonas was divided intotwo genera: Pseudoalteromonas, containing11 species, and Alteromonas, which containsA. macleodii as the type strain. They grow onmarine agar 2216 (Difco).

Brucella spp.

Brucella species are small cocco-bacilli that areintracellular in the cells of mammals. Traditionallythey are classified according to host, as they aregenerally host-specific in their disease. Studiessuggest that the Brucella genus is monospecificand that only one species, Brucella melitensis,should be recognized in the genus. It is proposedthat the other six species be recognized as biovarsthus: B. melitensis biovar abortus, B. melitensisbiovar canis, B. melitensis biovar melitensis,B. melitensis biovar neotomae, B. melitensisbiovar ovis, and B. melitensis biovar suis (Vergeret al., 1985). However, it is practical to retain thecurrent species delineation to avoid confusion(Wayne et al., 1987).

Recent research into the Brucella speciesisolated from marine mammals indicates thatthe monospecific species theory does not applywhen these isolates are considered (Moreno et al.,2002). The Brucella strains isolated from marinemammals appear to be a distinct species based onIS711-based DNA fingerprinting (Bricker et al.,2000). The name Brucella maris, comprising threebiovars, was suggested for the isolates from seals,porpoises, dolphins, an otter and a whale (Jahanset al., 1997). PCR-restriction fragment lengthpolymorphism of two outer membrane proteingenes, omp2 and omp2b, indicates that the iso-lates from sea mammals are a separate group fromthose isolated from terrestrial mammals. The iso-lates from the sea mammals appear to be a moreheterogeneous group than those from terrestrialmammals and, therefore, instead of naming allaquatic isolates as B. maris, it is proposed that theisolates are named according to their host species




in a similar manner to terrestrial isolates. As yetthis has not been approved by the InternationalCommittee on Systematic Bacteriology. How-ever, the proposal is that the aquatic strains arenow named B. pinnipediae for strains isolatedfrom seals, and B. cetaceae for strains isolatedfrom cetaceans (whales, dolphins and porpoises)(Cloeckaert et al., 2001; Foster et al., 2002).

Carnobacterium spp.

Carnobacterium species can be differentiatedfrom Lactobacillus, as Carnobacterium speciesfail to grow on acetate agar (Rogosa medium,Oxoid; Rogosa et al., 1951). Discrepancies havebeen reported between conventional tube testsand the API 50CH system with results for inulin,lactose, sorbitol and galactose (Baya et al., 1991).The Carnobacterium species are phylogeneticallycloser to the genera Enterococcus and Vagococ-cus than to members of the genus Lactobacillus.

Carnobacterium (Lactobacillus) piscicola mayhave been reported as Corynebacterium spp.,Lactobacillus spp., Listeria spp. or Vagococcussalmoninarum in earlier reports in the literature.

Cytophaga spp.

This genus now contains only two cellulolyticspecies: C. hutchinsonii, the type species, andC. aurantiaca. Their closest relative is the genusSporocytophaga, containing S. myxococcoides,which is cellulolytic and produces microcysts(Bernardet et al., 1996).

Cytophaga aurantiacus, strain NCIMB 1382described by Lewin and Lounsbery (1969),is thought to be a Flavobacterium johnsoniae(Bernardet et al., 1996).

Edwardsiella tarda

This name is conserved for use, althoughEdwardsiella anguillimortifera is the more seniorsynonym. The organism can be found in theenvironment in pond water, lakes, rivers, soil andfrom the cloacae of crocodiles, lizards, seagulls,snakes, tortoises and waterfowl, and may be partof the normal flora or exist in a carrier status. It isa pathogen of many aquatic species including

catfish, trout, eels, and also causes enteritis inhumans. Because it is widespread in the environ-ment, the isolation of the organism from someaquatic animals may make it difficult to assess itspathogenic status. However, site of isolation, clin-ical signs, disease status and histopathologicalexamination should enable a correct diagnosis(Wallace et al., 1966; Otis and Behler, 1973;White et al., 1973; Miyashita, 1984; Humphreyet al., 1986; Reddacliff et al., 1996; Uhland et al.,2000).

Enterococcus spp.

Enterococcus faecalis was previously knownas Streptococcus liquefaciens and Streptococcusfaecalis.

Enterococcus seriolicida was previouslydescribed as Streptococcus species biotype 1 fromAustralian trout in Victoria and Tasmania, and isnow recognized as Lactococcus garvieae.

Enterovibrio norvegicus

This organism has recently been isolated andidentified to be a member of the Vibrionaceae. Itresembles a Vibrio, however, is resistant to thevibriostatic agent 0/129 but grows slowly after3 days on TCBS. It is part of the intestinal micro-flora of turbot larvae (Thompson et al., 2002a).

Flavobacterium/Flexibacter/Cytophaga

The Flavobacterium genus represents pre-dominantly gliding, yellow-pigmented bacteriathat belong to the Flexibacter–Bacteroides–Flavobacterium phylum. Flavobacterium is thetype genus of the Family Flavobacteriaceae, whichincludes the genera Bergeyella, Capnocytophaga,Chryseobacterium, Empedobacter, Gelidibacter,Myroides, Ornithobacterium, Polaribacter,Psychroflexus, Psychroserpens, Riemerella,Weeksella, and misclassified Cytophaga andFlexibacter species. Members of Flavobacteriumgenus are aerobic, Gram-negative rods, produceyellow-pigmented colonies, are motile by gliding,do not hydrolyse cellulose and are widely distrib-uted in soil and freshwater habitats. Some arepathogenic for fish (Bernardet et al., 1996).

78 Chapter 1



Flavobacterium columnare: Genetic diver-sity has been reported amongst strains of Flavo-bacterium columnare, which enables divisioninto three groups or genomovars. However, thephenotypic characteristics are identical for thethree genomovars and there is no evidence toclassify the groups as new species or subspecies(Triyanto and Wakabayashi, 1999). A genomovaris defined as phenotypically similar but geneticallydistinct (Ursing et al., 1995).

Flexibacter aurantiacus (Lewin and Louns-bery, 1969) strains NCIMB 1382 (previouslyCytophaga aurantiaca) and NCIMB 1455(Flexibacter psychrophilus) are now thought tobe Flavobacterium johnsoniae strains, and havebeen shown to be different from the bona fidestrains of Cytophaga aurantiaca and Flavobac-terium psychrophilum (Bernardet et al., 1996).

The Flexibacter genus contains one species,F. flexis. The only genuine members of theCytophaga genus are C. hutchinsonii (the typespecies), and C. aurantiaca (excluding strainsNCIMB 1382 and NCIMB 1455).

Hafnia alvei

Hafnia alvei is known to cross-react with Yersiniaruckeri antisera (Stevenson and Airdrie 1984;and personal observation).

Haemophilus piscium

Strains originally identified as H. pisciumhave been included with strains of Aeromonassalmonicida. Phenotypic evidence suggests thatthe strain is synonymous with A. salmonicida ssp.achromogenes, yet molecular studies involvingribotyping, RAPD and PCR grouping did notsupport this (Austin et al., 1998).

Helicobacter spp.

Since Helicobacter pylori was first isolated asa cause of gastric ulcer in humans (Marshalland Warren, 1984), the genus has expandedto include some 30 species isolated from thegastrointestinal tract of many different animals.The organisms are microaerophilic and the cellsare fusiform, curved or spiral, and possess flagella

in different numbers and location according tothe species.

Listonella spp.

There is contention in the scientific communityregarding the validity of placing V. anguillaruminto the genus Listonella as proposed byMacDonell and Colwell (1985). There is,however, agreement of the separation of V.anguillarum biotypes 1 and 2 into two species,as V. anguillarum and V. ordalii, respectively(Austin et al., 1997). In this book, V. anguillarumis referred to as Listonella anguillarum.

Listonella anguillarum

This pathogen causes disease in a wide range offish. It is of particular economic importance to thesalmonid industry, as salmonids are particularlysusceptible to disease when being transferredfrom freshwater to salt water.

Listonella anguillarum isolates can bedivided into ten serotypes (European serotypedesignation) based on the detection of the O anti-gen. Of these, only serotypes 01 and 02 have beenisolated from diseased fish, following outbreaks ata distribution rate of 70% and 15%, respectively.In 75% of vibriosis infections in feral fish, serotype02 is isolated. The other serotypes have onlybeen isolated from the environment (Sørensenand Larsen, 1986). Serotype 01 strains tend tobe arabinose-positive. Isolates from serotype 01are a homogeneous group, whereas isolates fromserotype 02 can be further divided into groups02a and 02b based on differences detected bydouble immunodiffusion, dot blot assay andenzyme-linked immunoabsorbant assay (Toranzoand Barja, 1990). Vibrio ordalii, previously classi-fied as L. anguillarum biotype II, cross-reacts withserotype 02 antiserum (Toranzo et al., 1987).

Listonella pelagia strain NCIMB and NCMB1900: Recent phylogenetic analysis of NCMB1900 has indicated that this strain is more likelyto be Vibrio natriegens, and that the originalstrain deposited at NCIMB may have beenlost. Phenotypic tests for strain NCIMB 1900corresponded to those expected for V. natriegens,which is positive for acid production fromL-arabinose, melibiose, and L-rhamnose, witha negative result for lactose fermentation andalginate degradation (Macián et al., 2000). The




phenotypic results recorded as L. pelagia NCIMB1900 by Lunder et al. (2000) have been recordedas V. natriegens in this book.

Listonella ordalii = Vibrio ordalii

Both names are used in the literature, althoughVibrio ordalii is officially used in taxonomydatabases.

Moritella marina and Moritella viscosa

Moritella marina and Moritella viscosa are closelyrelated and have a sequence similarity of 99.1%based on 16S rDNA sequencing (Benediktsdóttiret al., 2000).

Mycoplasma spp.

Mycoplasma phocicerebrale is a legitimatecorrection to the previously reported name of M.phocacerebrale. The correction of Mycoplasmaphocae to M. phocidae is not legitimate (de Vosand Truper, 2000). Mycoplasma phocirhinis is alegitimate correction from M. phocarhinis.

Nocardia spp.

Members of the Nocardia produce branchedsubstrate hyphae that fragment in situ into rod-shaped to coccoid non-motile elements. Aerialhyphae are numerous and are always present butsome may only be seen microscopically. Hyphaeon a 3-day-old colony may only be 1 mm inlength.

Photobacterium spp.

The species that should be contained in thisgenus is still in dispute. There is agreementof some species rightfully belonging to the genus(P. angustum, P. phosphoreum, P. leiognathi).However, other species are in dispute, such as V.fischeri, V. logei and V. damselae (Lunder et al.,2000) although placing V. damselae into thegenus Photobacterium is generally agreed upon.

It is agreed that Photobacteriumdamselae ssp. piscicida belongs to the genus

Photobacterium rather than Pasteurella whereit was first placed. However, there is stillcontroversy as to whether P. damselae ssp.piscicida is a subspecies of P. damselae or aspecies in its own right (Gauthier et al., 1995b;Thyssen et al., 1998).

Photobacterium histaminum is a later sub-jective synonym of P. damselae ssp. damselae(Kimura et al., 2000).

Photobacterium fischeri and Vibrio fischeriare homotypic synonyms.

Providencia rettgeri and Proteus rettgeri

Providencia rettgeri and Proteus rettgeri arehomotypic synonyms, which means they sharethe same type strain (Brenner et al., 1978).

Pseudoalteromonas species

See also under Alteromonas. The genus Pseudo-alteromonas was formed to include most of thepreviously named Alteromonas species withthe exception of A. macleodii. The majorityof Pseudoalteromonas are associated witheukaryotic hosts and are frequently isolated frommarine waters. The majority grow on MA 2216and show optimal growth at 23°C.

Shewanella putrefaciens

Strain NCIMB 400 has been reclassified asShewanella frigidimarina.

Sphingobacterium spp.

Members of the Sphingobacterium genus have alow G+C content (39–42 mol %) and containsphingolipids.

Streptococcus spp.

Streptococcus agalactiae is a Lancefield group Bstreptococcus and is non-haemolytic. There arenine serogroups of group B streptococcus basedon their capsular polysaccharide.

80 Chapter 1



Streptococcus dysgalactiae consists of twospecies. Streptococcus dysgalactiae ssp. dysga-lactiae strains are a- or non-haemolytic andare Lancefield group C. Strains belonging toStreptococcus dysgalactiae ssp. equisimilis areall b-haemolytic and may have the Lancefieldgroup C, G or L antigen (Vieira et al., 1998).

Streptococcus difficile has now beenidentified as Streptococcus agalactiae type Ib(Vandamme et al., 1997).

Vagococcus genus

The genus Vagococcus was proposed to accom-modate Gram-positive cocci that were motile andreacted with Lancefield group N antisera (Collinset al., 1989).

Vibrio spp.

Members of the Family Vibrionaceae areColwellia, Listonella, Moritella, Photobacteriumand Vibrio. Those isolated from fish grow well onordinary plate media.

Vibrio carchariae and V. harveyi: V.carchariae is a junior synonym of V. harveyiand the two organisms were indistinguishableby amplified fragment length polymorphisms,DNA : DNA hybridization, ribotyping (Pedersenet al., 1998) and 16S ribosomal DNA sequencing(Gauger and Gómez-Chiarri, 2002).

Vibrio cholerae: A biotyping scheme wasbased on the fermentation of sucrose, D-mannoseand L-arabinose. These biotypes are known asHeiberg types I–VIII. A classification scheme wassuggested where V. cholerae isolated from cholerapatients was classified as 01, and all otherstrains were classified as non-01. Non-01 strainscause gastroenteritis and septicaemia and otherinfections in humans, and have also been reportedto cause infections in terrestrial and aquaticanimals. There are two biotypes, El Tor andClassical; these cannot be differentiated sero-logically and belong to serovar 01. Serovar 01causes the classical cholera epidemics seen inhumans in some countries. Serotype 0139 Bengalis also an epidemic strain of cholera (Furniss et al.,1978; Albert et al., 1993).

Vibrio anguillarum: See notes on Listonellaanguillarum.

Vibrio natriegens: There are some reports inthe literature identifying strain NCIMB and NCMB1900 as Listonella pelagia. Sequencing has shownthat strain 1900 corresponds to Vibrio natriegens(Macián et al., 2000). See also notes underL. pelagia.

Vibrio trachuri: Isolated from Japanese horsemackerel (Trachurus japonicus) and identified asa new species of Vibrio (Iwamoto et al., 1995a).However, further testing has suggested that thisspecies is a junior synonym of Vibrio harveyi(Thompson et al., 2002b).

Vibrio vulnificus: Biotyping and serotyping ofVibrio vulnificus is a little confusing at present.Biotype 1 isolates are indole-positive and associ-ated with human infection, whereas biotype 2isolates are indole-negative and associated withinfection in eels (Tison et al., 1982). However, inDenmark and Sweden 85% of biotype 2 isolatesshowing pathogenicity for eels are indole-positive(Høi et al., 1998b). Biotype 2 strains are serovar Eand these can be further divided into 03 and 04based on LPS-O antigen. Some biotype 2, serovarE, strains from Taiwan have been found tobe avirulent for eels. These strains are positivefor mannitol fermentation (Amaro et al., 1999).Vibrio vulnificus biotype 3 has been put forwardby the Centre for Disease Control, USA, as apossible identification for Vibrio species isolatedfrom wound infections in humans in Israel(Bisharat et al., 1999). It is different from currentlyrecognized V. vulnificus species because ofnegative reactions in cellobiose, citrate, lactose,salicin and ONPG. It is positive by PCR for theV. vulnificus haemolysin gene (Bisharat et al.,1999; Nair and Holmes, 1999). In the conven-tional biochemical identification tables and theAPI 20E tables, the various V. vulnificus strainsand their different reactions are listed so as to assistthe microbiologist when making identification.

Vibrio wodanis has a sequence similarity of98.8% with Vibrio logei strain CIP 103204 basedon 16S rRNA sequencing (Benediktsdóttir et al.,2000).

Yersinia spp.

Yersinia enterocolitica: Y. enterocolitica wasdivided into the subspecies Y. enterocoliticassp. enterocolitica and Y. enterocolitica ssp. pale-arctica. Yersinia mollaretii and Y. bercovieri were




formerly Y. enterocolitica biogroups 3A and 3B,respectively (Wauters et al., 1988).

Yersinia ruckeri: An original serotypingscheme identified six serovars (Hagermanserovars). Most naturally occurring outbreaks indiseased fish and rainbow trout are caused byserovar I called the Hagerman strain (McCarthyand Johnson, 1982). Some Australian isolates ofY. ruckeri that cross-reacted with serovar I weretermed serovar I′. Serovar II may be significantwhen it is associated with ERM in chinook salmon(Cipriano et al., 1986). Serovar II and serovar Visolates are sorbitol-positive. Serovar III is foundin Australia. Initially it was suggested that sorbitolfermentation was associated with pathogenicity;however, this is not a reliable indicator of virulence(Stevenson and Airdrie 1984; Cipriano et al.,1986).

More recently it has been shown that Y.ruckeri can be divided into clonal groups based onbiotype, serotype and outer membrane proteins,and that clonal groups may relate to virulenceof the organism. There are two biotypes basedon hydrolysis of Tween 20 and Tween 80, andmotility. Biotype 2 strains are non-motile, and donot hydrolyse Tween 80 (Davies and Frerichs,1989). A serotyping scheme based on heat-stableO-antigens identified serotypes 01, 02, 05, 06 and07 in Europe, North America, Australia and South

Africa (Davies, 1990). All serotypes occur inEurope and North America, whereas in Australiaand South Africa only serotype 01 has beenidentified. This study suggested that the Australianisolate described as serotype III was serotype 01by their scheme. Five outer membrane proteinprofiles were identified, which enabled differentia-tion of isolates within a serotype. Serotype 01consisted of six clonal groups. Clone 2 was foundin disease outbreaks in the UK and clone 5 wasfound in disease outbreaks in mainland Europe,North America and South Africa. Clones 3, 4 and6 do not appear to be associated with diseaseoutbreaks. Clones 1 and 3 were found fromAustralian isolates, with clone 3 also being foundin Finland, France, West Germany and the USA.Clone 4 was from Norwegian isolates, and clone 6from isolates in Finland, France, Norway andCanada (Davies, 1991).

Hafnia alvei is known to cross-react withYersinia ruckeri antisera (Stevenson and Daly,1982; Stevenson and Airdrie 1984; and personalobservation).

ERM outbreaks caused by Yersinia ruckeriare generally a result of poor environmentalconditions, resulting in low oxygen level, highwater temperature and poor water quality, allof which stress the fish, making them moresusceptible to disease.

82 Chapter 1



2Bacteriological Culture Techniques: Microscopy,

Culture and Identification

Successful isolation and accurate identificationof a suspected pathogen depends upon the useof standardized bacteriological culture methods.It is important to work through these methodsin a precise and logical step-by-step manner.Basically the bacteria are grown on culture

media, then inoculated into biochemical identifi-cation tests and those results are recorded andcompared to standard results. Table 2.1 sets outthe steps for the isolation and identification of anorganism and also directs the reader to the loca-tion of the technique or method in this manual.


Day Activity Method or technique

Day 1 1. Sample collection and preparation2. Inoculation of sample to primary isolation plate media (or broths whereappropriate)3. Incubate at appropriate temperature and atmosphere

2.12.2, Table 2.2, Table 2.3

2.2, Table 2.2, Table 2.3

Day 2(24 h)

1. Examine culture plates2. Select suspect colonies and subculture to BA or MSA-B to obtain puregrowth (secondary plates)3. Re-incubate primary plates4. Incubate all plates at appropriate temperature and atmosphere as before

2.3Table 2.4 for cultural and microscopicappearance2.2, Table 2.2 and 2.3

Day 3(48 h)

1. Re-examine primary plates for slow-growing pathogens2. Check that subcultures on secondary plates are pure3. Perform primary identification tests4. Inoculate appropriate biochemical identification set5. Re-incubate primary plates as before

2.3

2.4, Chapter 3, and media (Chapter 7)2.5, 2.62.2, Table 2.2 and 2.3

Day 4(72 h)

1. Re-examine primary plates for growth of slow-growing pathogens.Re-incubate if disease suggests a pathogen that requires more than 3 daysfor growth2. Examine biochemical identification set/s and record results at 24 hincubation

2.3

Table 3.1

Day 5(96 h)

1. Examine biochemical identification set/s and record results for 48 hincubation. Add reagents for tests for indole, methyl red, nitrate,Voges-Proskaüer2. Interpret results from appropriate identification table

Table 3.1Chapter 3 and Tables 4.1 to 4.22 forbiochemical results (Biochem set), andTables 4.23 to 4.31 for results for API kits

BA, blood agar; MSA-B, marine salt agar.

Table 2.1. Outline of steps for culture and identification.



2.1 Specimen Collection and Submission

Samples should be collected from live sickanimals or from recently dead animals. Animalsthat have been dead for more than 6 h usuallyhave grossly contaminated organs due to theovergrowth of post-mortem invading bacteria. Itis very difficult for the laboratory to work withthese samples. Unfortunately, it is not alwayspossible to get the samples to the laboratorywithin the ideal time, due to large distancesbetween some laboratories and the aquaculturefarm.

Ideally all samples for bacteriologicalexamination should be collected before the start ofantimicrobial treatment. Samples collected fromaquatic animal species after commencement oftreatment are unlikely to grow any pathogenicbacteria.

Samples may be submitted as swabs, tissuesor whole fish. The most appropriate samples forthe identification of a systemic disease may bekidney, haemolymph or blood. For skin disease,swabs or samples of skin are appropriate. Referto Tables 1.1 and 1.2 for tissue sites where thebacteria are likely to be located.

Transport medium and transport tothe laboratory

If swabs have been collected, then they mustbe placed into a transport medium to preventthe swab drying out and for the organisms toremain viable. Amies transport medium isthe medium of choice for transport of swabsto the laboratory.

Fish, tissues and swabs in Amies transportmedium should be transported to the laboratoryas soon as possible after collection, and preferablyon ice or at 4°C.

Stuarts medium may be unsuitable for trans-port and survival of Vibrio species. Numbers ofviable cells of Vibrio alginolyticus, Vibrio harveyiand Photobacterium damselae ssp. damselaewere greatly reduced after 24 h and were almostnon-viable at 48 and 72 h when tested fromStuarts media kept at 4°C. However, there was noreduction of viable cells when all three organismswere tested from Amies transport media heldat 4°C for 24, 48 and 72 h (H. McLetchie andN. Buller, unpublished).

Sample preparation

Swabs

Swabs require no sample preparation, and areinoculated directly to the appropriate media.

Tissues

Aseptically remove a piece of infected tissuefrom the fish and place into a McCartney bottle.Macerate the tissue with flame-sterilized scissors,and use a sterile, cotton-tipped swab to inoculateto appropriate media. Tissues that have not beenaseptically removed from the fish need to besurface-sterilized. This can be done either byrinsing the piece of tissue in 3–4 washes of sterilewater or saline, or for larger pieces of tissue(walnut-sized), dip in 70% alcohol and flamebriefly for 3–5 s. The tissue is then placed into asterile McCartney bottle or appropriate containerand macerated with flame-sterilized scissors. Asterile, cotton-tipped swab is used to sample thetissue and inoculate to appropriate agar plates,and to make smears for Gram stain.

Lesions

Always collect material from the edge of thelesion, including some of the immediate tissue,to ensure sampling for true invading bacteria.Sampling from the middle of the lesion mayculture secondary invading bacteria that willconfuse the results. Also, the invading bacteriummay no longer be viable at the centre of thelesion.

Whole fish

For small fish, the whole fish is surface-sterilizedby rinsing the fish in 3–4 changes of steriledistilled water or normal saline. For larger fish,surface-sterilize by either flaming with 70%alcohol or washing the area with 3–4 washesof sterile distilled water or normal saline. Usingflamed-sterilized forceps and scissors, make anincision from behind the gills and to the mid-line.Next cut along the mid-line towards the anus. Liftback the flap of flesh to expose the internalorgans. Select the required organs for analysisand remove with flame-sterilized forceps andscissors. Chop tissues as before and inoculateto plates.

84 Chapter 2



2.2 Culture and Incubation

Culture media

All specimens should be cultured to a general-purpose medium. For freshwater specimens useBA and MCA. TSA can also be used; however,the addition of blood can improve the isolation ofsome organisms such as Aeromonas salmonicida,Carnobacterium and Streptococci. For marinespecimens, use a medium containing Na ions,BA, and MCA. Marine salt agar (MSA-B), whichis prepared from TSA with 5% horse blood and2% NaCl (see Media section), is a good general-purpose medium for marine organisms. Marine2216, commercially available from Difco, andbased on ZoBell’s (1941) original formula, is alsosuitable. MSA-B may give improved growth,particularly on primary culture with commonlyisolated Vibrio species. MA 2216 is more suitablewhen a complex mixture of electrolytes isrequired by some marine organisms.

Isolation and growth conditions: media,temperature and time of incubation

All specimens should be inoculated to thegeneral-purpose medium. This medium is thenincubated at 25°C for 2–5 days as indicated inTable 2.2 for general culture conditions. How-ever, specimens from a particular environmentsuch as a cold water environment or a marineenvironment may need a particular incubationtemperature or NaCl may be required in themedium. These variations are listed in Table 2.3.

Some organisms have special growth require-ments or may be better detected with the use of aselective and/or enrichment medium if they areavailable for that organism. Such requirements forspecific organisms are detailed in Table 2.3.

The preparation of all media mentioned inTables 2.2 and 2.3 is listed in Chapter 7.

2.3 Examination of Culture Plates

Many bacteria from fish and aquatic animalsneed 48 h before colonies appear, or before theyare of a suitable size for examination and subcul-ture. At 24 h incubation, the colony appearanceof different Vibrio species may not be distinguish-able; however, at 48 h incubation, the differencesin physical appearance between different speciesmay be more apparent. The addition of bloodinto a medium such as MSA-B, for Vibrio species,improves the ability to differentiate between thecolonies of different species, and to determinehaemolysis. Generally, a bacterium that is pres-ent in a moderate to heavy growth or as thepredominant organism will be significant. Bear inmind the age of the samples at the time they werereceived at the laboratory. Specimens older than24 h from the collection date may be overgrownwith commensal bacteria.

Select appropriate colonies for subculture.These may be given an individual number (e.g.#1, #2, etc). For example, selected colonies arecircled and numbered on the underside of thePetri dish using a felt pen, and then picked off andsubcultured to fresh plates. Freshwater samplesare subcultured to BA, MCA and TCBS (the latterfor suspect Vibrio species). Samples from marinesources are subcultured to BA, MCA, MSA-B andTCBS. Organisms that require special media forgrowth are subcultured to the appropriate media(see Table 2.3). Some organisms from marineenvironments may need to be cultured to MA2216 media. Incubate a further 24 h or as requiredfor sufficient growth. Pure subculture growthsare used for the inoculation of biochemicalidentification tests.

Microscopic and cultural characteristics

Table 2.4 details microscopic and colony appear-ance, plus the results of the primary tests of Gram

Bacteriological Culture Techniques 85

Fish, aquatic animals Media – freshwater animals Media – salt water animals Temperature optimum (range)

All specimens, tissues BA, MCA MSA-B, BA, MCA 20–25°C, 2–5 daysCold water BA, MCA MSA-B, BA, MCA 15–22°C, 3–5 daysTropical BA, MCA MSA-B, BA, MCA 25°C, 2–5 days

BA, blood agar; MCA, MacConkey agar; MSA-B, marine salt agar containing blood.

Table 2.2. General culture.



86 Chapter 2

Abio

troph

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e25

°C,2

–5da

ys.

Der

mat

ophi

lus

cong

olen

sis

BA,p

oly

plat

e37

°C,2

–3da

ys

Edw

ards

iella

icta

luri

BA,T

SA,E

IM25

–37°

C,a

erob

ican

dan

aero

bic.

Stra

ins

with

limite

dto

lera

nce

toox

ygen

have

been

isol

ated

(Mitc

hell

and

Goo

dwin

,20

00)

Edw

ards

iella

tard

aBA

,MC

A,D

CA,

SSag

ar,s

tront

ium

chlo

ride

Ben

richm

ent

brot

h37

°C,1

–2da

ys

Edw

ards

iella

spp.

BA,B

HIA

,NA

25–2

8°C

,2–5

days

Empe

doba

cter

(Fla

voba

cter

ium

)bre

vis

TSA

30°C

,2–3

days

Ente

roco

ccus

spp.

BA,T

SA,B

HIA

25°C

,48–

72h

Ente

rovi

brio

norv

egic

usM

A22

16or

NA

and

TSA

supp

lem

ente

dw

ith1.

5%N

aCl

27–2

8°C

,2da

ys

Erys

ipel

othr

ixrh

usio

path

iae

BA,W

oods

sele

ctiv

ebr

oth,

Pack

ers

plat

e37

°C,4

8h.

Subc

ultu

reW

oods

brot

hto

BAan

dPa

cker

spl

ates

at24

and

48h

Euba

cter

ium

tara

ntel

lae

BHIB

orBA

(ana

erob

e),A

NA

20–2

5°C

,7da

ys,a

naer

obic

cond

ition

s

Fack

lam

iam

iroun

gae

BAM

SA-B

37°C

,24–

48h

Flav

obac

teriu

mbr

anch

ioph

ilum

AO(n

ogr

owth

onm

edia

cont

aini

ng>0

.2%

NaC

l)18

°C,5

–7da

ys

Flav

obac

teriu

m(F

lexi

bact

er)c

olum

nare

AO,S

hieh

med

ium

with

adde

dto

bram

ycin

,TSA

,NA,

BA,

TYG

agar

,Hsu

-Sho

ttsag

ar22

–30°

C,5

–7da

ys

Flav

obac

teriu

m(C

ytop

haga

)ps

ychr

ophi

lum

TYG

agar

,Shi

ehm

ediu

m,A

Oag

ar(n

ogr

owth

onTS

A).

Sam

ple

from

muc

usan

dki

dney

.Cul

ture

dilu

tions

ofsa

mpl

etis

sue

toim

prov

eis

olat

ion

rate

.Use

ofFP

Mm

ediu

mm

ayim

prov

eis

olat

ion

rate

14–1

8°C

,5–7

days

cont

inue

d



88 Chapter 2

Flav

obac

teriu

mgi

llisia

e,F .

tege

tinco

la,F

.xan

thum

NA,

TSA,

R2A

MA

2216

20°C

,2–7

days

Flex

ibac

ters

pp.–

mar

ine

orig

inAO

-M(A

0m

ediu

mpr

epar

edw

ithar

tific

ials

eaw

ater

( Si g

ma)

at38

g/l )

25°C

,2–7

days

Flex

ibac

terp

olym

orph

usFl

exib

acte

rmed

ium

ofLe

win

(197

4)30

°C,2

–7da

ys

Flex

ibac

terr

oseo

lus

NA

25°C

,2–7

days

Flex

ibac

terr

uber

NA

25°C

,2–7

days

Hal

omon

asel

onga

taTS

A+8%

salt

30°C

,24–

48h

Hel

icob

acte

rspp

.TS

A+

5%bl

ood,

BA,B

ruce

llaag

ar,S

kirro

w’s

med

ium

(VPT

)H

omog

eniz

est

omac

htis

sue

in1

mlB

ruce

llabr

oth

(Difc

o)co

ntai

ning

5%fa

ecal

calf

seru

m.P

lace

100

mlto

p la t

e san

d/or

filte

rthr

ough

a0.

45or

0.8

mmfil

terb

efor

ein

ocul

atin

gto

plat

es(B

utzl

eret

al.,

1973

;Har

pere

tal.,

2000

)

37°C

mic

roae

roph

ilicat

mos

pher

eof

N2,

H2,

CO

2(8

0:10

:10)

for2

–4w

eeks

Com

mer

cial

syst

ems

are

avai

labl

efo

rge

nera

tion

ofco

rrect

atm

osph

ere

(e.g

.M

GC

Anae

roPa

k ,C

ampy

lofro

mM

itsub

ishi

Gas

Che

mic

alC

ompa

ny)

Lact

obac

illus

spp.

,Lac

toco

ccus

spp.

BA,T

SA,B

HIA

25–3

0°C

,48–

72h

List

onel

laan

guilla

rum

TSA

+1%

NaC

l,M

SA-B

,MA

2216

,VAM

22°C

,24–

48h

Mor

itella

visc

osa

TSA

+2%

NaC

l15

°C,4

–9da

ys

Myc

obac

teriu

mab

sces

sus

Mid

dleb

rook

7H10

-AD

Cm

ediu

m25

°C,7

–28

days

Myc

obac

teriu

mch

elon

aeBH

IA,T

SA,A

OM

SA-B

15–2

2°C

,5w

eeks

.(gr

owth

isus

ually

seen

with

in7

days

)

Myc

obac

teriu

mm

arin

um,

M.s

crof

ulac

eum

,M.i

ntra

cellu

lare

,M

.for

tuitu

m

BA,L

owen

stei

n-Je

nsen

(BBL

)20

–23°

C,7

–14

days

Will

grow

in5–

7da

ysat

room

tem

pera

ture

onbo

vine

BA

Myc

obac

teriu

mne

oaur

umBA

25°C

,3–4

days

Myc

obac

teriu

mpe

regr

inum

BA,M

iddl

ebro

ok7H

11ag

ar(D

ifco)

37°C

inC

O2,

4–7

days

Myc

opla

sma

spec

ies

Myc

opla

sma

agar

and

Myc

opla

sma

brot

h37

°Cin

CO

2.Su

bcul

ture

brot

hto

agar

med

ium

ever

y3–

4da

ysfo

r2w

eeks

.Ex

amin

epl

ates

at3–

4da

yin

terv

als

Myr

oide

s(F

lavo

bact

eriu

m)o

dora

tus

TSA

30°C

,48–

72h

Bact

eriu

mM

edia

forf

resh

wat

eran

imal

sM

edia

fors

altw

ater

anim

als

Tem

pera

ture

,atm

osph

ere,

time

Tab

le2.

3.C

ontin

ued.




Noc

ardi

asp

p.-g

ener

al( N

.ast

eroi

des,

N.b

rasi

liens

is,

N.n

ova,

N.p

seud

obra

silie

nsis

,N

.otit

idis

cavi

arum

,N.s

erio

lae,

N.v

acci

nii)

BA25

–30 °

C,1

4da

ys.C

olon

ies

begi

nto

appe

araf

ter5

days

Noc

ardi

acr

asso

stre

aeBH

IABH

IA+

1%N

aCl

28°C

,14

days

Noc

ardi

ase

riola

eBA

,BH

IA,L

owen

stei

n-Je

nsen

med

ium

25–3

7°C

for7

–30

days

Past

eure

llask

yens

isTS

A+

bloo

d+

1.5%

NaC

l.M

A22

16+

bloo

d(n

ogr

owth

with

outb

lood

or1.

5%N

aCl)

22–3

0°C

,in

air,

48h

Pedo

bact

erhe

parin

us,P

.pis

cium

NA,

PY28

°C,4

8h

Phot

obac

teriu

mda

mse

lae

ssp.

dam

sela

eBA

MSA

-B,T

SA+

2%N

aCl,

MA

2216

22–2

5°C

,24–

48h

Phot

obac

teriu

mda

mse

lae

ssp.

pisc

icid

aN

Bw

ith2–

3%N

aCl,

BHIA

with

2%N

aCl,

MA

2216

,or

MSA

-B22

°C,2

4–48

h

Pseu

doal

tero

mon

assp

p.M

A22

1615

–30°

C,1

–5da

ys

Pseu

doal

tero

mon

asan

tarc

tica

TSA,

TSB

MA

2216

,MSA

-B15

°C,5

days

Pseu

doal

tero

mon

asm

aric

alor

isTS

AM

A22

1625

–35°

C,5

days

Pseu

dom

onas

angu

illise

ptic

aBA

,NA,

TSA,

BHIA

.No

grow

thon

Pseu

dom

onas

isol

atio

nag

ar(D

ifco)

20–2

5°C

,7da

ys

Pseu

dom

onas

fluor

esce

nsBA

,Pse

udom

onas

sele

ctiv

eag

ar(B

ecto

n,D

icki

nson

Co)

10–2

5°C

,2–5

days

Ren

ibac

teriu

msa

lmon

inar

umKD

M2,

KDM

C,S

KDM

15–1

8°C

,20–

30da

ys

Ros

eoba

cter

stra

inC

VSP

SWT,

MA

2216

23°C

,5–7

days

Sale

gent

ibac

ters

aleg

ens

MA

2216

,NA,

TSA,

R2A

20°C

,2–7

days

Shew

anel

labe

nthi

ca,S

.col

wel

liana

,S.

hane

dai,

S.ge

lidim

arin

a,S.

peal

eana

,S.w

oody

i

MA

2216

,MSA

-B25

°C,4

8–72

h

Shew

anel

la(A

ltero

mon

as)h

aned

aiTS

A+

0.05

g/ly

east

extra

ct,M

A22

1615

°C,4

8–72

h

Sphi

ngob

acte

rium

(Fla

voba

cter

ium

)m

ultiv

orum

,S.s

pirit

ivor

umN

A,TS

A,PY

28–3

0°C

,48

h

Stap

hylo

cocc

uslu

trae

BA37

°C,2

4h

Stap

hylo

cocc

usw

arne

riBA

,TSA

22–2

5°C

,48–

72h

cont

inue

d



90 Chapter 2

Stap

pia

stel

lula

ta-li

kest

rain

M1

SWT,

MA

2216

23–2

5°C

,2–7

days

Stre

ptob

acillu

sm

onilif

orm

isBA

.Req

uire

sad

ditio

nof

20%

seru

mto

brot

hfo

rgro

wth

25–3

7°C

,5–7

days

Stre

ptoc

occu

sin

iae

BA25

°C,2

4–48

h

Stre

ptoc

occu

spa

raub

eris

BA,T

SA22

–37°

C,2

4h

Tena

ciba

culu

m(F

lexi

bact

er)m

ariti

mum

AOm

edia

cont

aini

ng30

%se

awat

er,A

Oco

ntai

ning

ASW

at38

g/l.

Will

notg

row

onAO

with

NaC

lalo

ne.

NaC

land

KCli

ons

requ

ired

forg

row

th.C

a2+, M

g2+

enha

nce

grow

th.I

sola

tion

ofor

gani

smm

aybe

impr

oved

bydi

lutin

gsa

mpl

em

ater

iali

nar

tific

ial

seaw

ater

and

cultu

ring

dilu

tions

toAO

+AS

Wm

ediu

m(O

stla

ndet

al.,

1999

).TY

G-M

,HSM

25°C

for2

–5da

ys(ra

nge

15–3

4°C

)

Tena

ciba

culu

m(F

lexi

bact

er)o

voly

ticum

Asfo

rT.m

ariti

mum

,MA

2216

19°C

,2–5

days

Vago

cocc

ussa

lmon

inar

umBA

,TSA

22–2

5°C

,48

h

Varra

calb

mi

MSA

-B4–

22°C

(opt

imum

15°C

),48

h

Vibr

ioag

ariv

oran

sM

SA-B

,MA

2216

25°C

,48

h

Vibr

ioco

rallii

lytic

usM

A22

16,T

SA+

2%N

aCl

30°C

,24–

48h

Vibr

ioha

liotic

oli

MA

2216

with

orw

ithou

t0.5

%so

dium

algi

nate

25°C

,24–

48h

Vibr

ioho

llisae

TSA

+1%

NaC

l,M

SA-B

,MA

2216

25°C

,37°

C,2

4–48

h

Vibr

iom

imic

usBA

,TSA

MSA

-B25

°C,3

7°C

,24–

48h

Vibr

ioor

dalii,

Vibr

iopa

raha

emol

ytic

usTS

A+

1%N

aCl,

MSA

-B,M

A22

1622

°C,2

4–48

h

Vibr

iosa

lmon

icid

aN

A+

5%bl

ood,

TSA

+1.

5%N

aCl,

MSA

-B15

°C,3

–5da

ys

Vibr

iovu

lnifi

cus

BAC

ello

bios

e-po

lym

yxin

Bco

listin

agar

(CPC

)se

lect

ive

agar

,CC

A,VV

M,M

A22

16,M

SA-B

,TSA

orN

Aw

ith0.

5%(w

/v)N

aCl.

AV.

vuln

ificu

s-sp

ecifi

cpr

obe

isav

aila

ble

25°C

,24

h

Yers

inia

ruck

eri

TSA,

BA,M

CA,

XLD

(Yer

sini

ase

lect

ive

agar

,(YS

A,O

xoid

)is

nots

uita

ble

for

Y.ru

cker

i)

25°C

,24–

48h

Not

eth

atm

otilit

yan

dci

trate

will

bene

gativ

eat

37°C

butp

ositi

veat

25°C

Bact

eriu

mM

edia

forf

resh

wat

eran

imal

sM

edia

fors

altw

ater

anim

als

Tem

pera

ture

,atm

osph

ere,

time

Tab

le2.

3.C

ontin

ued.




Acin

etob

acte

rspp

.N

egPr

edom

inan

tlydi

ploc

occa

lfor

ms

1.0

×0.

7mm

from

plat

egr

owth

–C

olon

ies

circ

ular

,sm

ooth

,tra

nslu

cent

tosl

ight

lyop

aque

,but

yrou

sto

muc

oid

non-

pigm

ente

d.0.

5–2

mm

at24

hat

30°C

+Bi

oche

mse

t

Acin

etob

acte

rha

emol

ytic

usN

egPr

edom

inan

tlydi

ploc

occa

lfor

ms

1.0

×0.

7mm

from

plat

egr

owth

bHC

olon

ies

onTS

Aar

eci

rcul

ar,c

onve

x,sm

ooth

,slig

htly

opaq

uean

dm

ayha

vea

stic

kyco

nsis

tenc

y

+−

Bioc

hem

set

Actin

obac

illus

delp

hini

cola

Neg

Pleo

mor

phic

rods

–w

NG

Col

onie

son

BAin

10%

CO

2ar

e0.

75–1

mm

at24

h,ro

und,

smoo

thgr

ey.B

lood

orse

rum

enha

nces

grow

th.G

row

that

42bu

tnot

at22

°C

−+

Bioc

hem

set,

API2

0E,A

PI20

NE,

API-Z

YM.U

sea

heav

yin

ocul

um

Actin

obac

illus

scot

iae

Neg

Pleo

mor

phic

rods

–N

GC

olon

ies

0.5

mm

roun

d,gr

ey,o

nBA

at24

h.R

equi

res

10%

CO

2.M

aybe

wea

kly

haem

olyt

icon

shee

pbl

ood

−+

Bioc

hem

set,

API2

0E

Actin

omyc

esm

arim

amm

aliu

mPo

sSt

raig

htor

slig

htly

curv

edro

ds,s

ome

bran

chin

g–

On

BA,c

olon

ies

are

0.5

mm

grey

,ent

ire,

conv

exat

48h.

Gro

wth

at37

°Cin

a irw

ith5 %

CO

2

−AP

ICor

yne,

APIr

apid

ID32

Stre

p

Aequ

oriv

itasp

p.N

egR

ods

0.5–

20×

0.2–

0.3

mmO

nM

A22

16,y

ello

wor

oran

geco

loni

es,

com

pact

,circ

ular

,con

vex,

smoo

thw

ithan

entir

eed

ge,n

on-s

prea

ding

,but

yrou

sco

nsis

tenc

y.G

row

thon

NA

+2.

5%N

aCl

Bioc

hem

set,

API2

0E,

API-Z

YM(s

ome

reac

tions

)

Aero

cocc

usvi

ridan

sva

r.ho

mar

iPo

sTe

trad-

form

ing

cocc

usa

NG

aH

o nB A

.Ex a

min

ew

ithIn

d ia

ink

for

enca

psul

ated

cocc

i+

Bioc

hem

set,

APIr

apid

ID32

Stre

p

Aero

mon

ascu

licic

ola

Neg

Rod

On

BAco

loni

essm

all,

grey

,met

allic

shee

nan

db

haem

olyt

icat

24h

++

Bioc

hem

set,

API2

0E,A

PI50

CH

Aero

mon

asen

chel

eia

Neg

Stra

ight

rods

bHN

GN

on-p

igm

ente

dco

loni

eson

TSA

at24

h+

+Bi

oche

mse

t

Aero

mon

aseu

cren

ophi

laN

egSt

raig

htro

dsN

G+

+Bi

oche

mse

t

Aero

mon

ashy

drop

hila

Neg

Smal

lrod

sbH

Wea

k3–

5m

msh

iny,

crea

m,b

ecom

ing

pale

brow

n/gr

een

with

age

++

Bioc

hem

set,

API5

0CH

,API

20E,

API2

0NE,

API-Z

YM

Bact

eriu

mG

ram

Mor

phol

ogy

bHTC

BSCo

lony

char

acte

ristic

sCa

tO

xId

entif

icat

ion

sets

Tab

le2.

4.M

icro

scop

ican

dcu

ltura

lcha

ract

eris

tics.

cont

inue

d



92 Chapter 2

Aero

mon

asm

edia

Neg

Rod

s1

×2

mmN

GO

nTS

A,cr

eam

,shi

ny,s

moo

th,r

ound

,rai

sed,

2m

maf

t er 2

days

at22

°C.A

diffu

sibl

ebr

own

pigm

enti

spr

oduc

ed

++

Bioc

hem

set

Aero

mon

assp

p.N

egR

ods

1–3

mmbH

Wea

kgr

owth

,ye

llow

2–3

mm

,gre

y,cr

eam

,shi

ny,r

ound

onBA

and

TSA

++

Bioc

hem

seto

rAPI

20E

Aero

mon

assa

lmon

icid

ass

p.sa

lmon

icid

a

Neg

Smal

lrod

sto

cocc

o-ba

cilli,

1–2

mmv

NG

Brow

ndi

ffusi

ble

pigm

ento

nFA

orTS

A.R

ough

and

smoo

thph

ase,

colo

nies

,0.

5–3

mm

at72

h.N

ogr

owth

at37

°C.F

Ais

pref

erre

dfo

rpig

men

tpro

duct

ion

++

Bioc

hem

set.

API2

0Egi

ves

varia

ble

resu

ltsat

25°C

,im

prov

edre

sults

at30

°C.

FAT

and

aggl

utin

atio

nte

stav

aila

ble

Aero

mon

assa

lmon

icid

ass

p.no

va

Neg

Coc

co-b

acilli

tosm

allr

ods

bHN

GO

nBA

,slo

wgr

owin

g,fri

able

colo

ny,0

.5m

mre

achi

ng4–

8m

mon

day

8.C

olon

ysl

ides

acro

sspl

ate

whe

npu

shed

with

loop

.Pr

onou

nced

zone

ofha

emol

ysis

,sim

ilar

toa

grou

pC

Stre

p.O

nTS

Aa

brow

n-w

ater

-sol

uble

pigm

enti

sno

tsee

nun

tilda

y8

at25

°C.N

ogr

owth

at37

°C

++ v

Bioc

hem

set,

API2

0E,

API-Z

YM.F

ATav

aila

ble,

may

cros

s-re

actw

ithAe

rom

onas

sobr

ia

Aero

mon

assa

lmon

icid

ass

p.pe

ctin

olyt

ica

Neg

Stra

ight

rods

sing

ly,i

npa

irsor

shor

tch

ains

bHN

DC

olon

ies

deve

lop

in24

hat

35°C

o nB A

a nd

TSA.

Prod

uctio

nof

brow

ndi

ffusi

ble

pigm

ent

++

Bioc

hem

set

Aero

mon

asve

roni

iN

egbH

NG

Col

onie

s2

mm

,bH

onBA

at24

h+

+Bi

oche

mse

t,AP

I20E

Allo

mon

asen

teric

aN

egSt

raig

htor

slig

htly

curv

edro

dsG

row

thon

med

iaco

ntai

ning

3–5%

NaC

l.Li

ght

brow

nno

n-di

ffusi

ngpi

gmen

tafte

r2–5

days

at25

or37

°C

++

Bioc

hem

set

Alte

rom

onas

spp.

Neg

Rod

sN

GG

row

thon

MA

2216

assm

allp

ale

yello

wco

loni

es+

+,v

Bioc

hem

set

Arca

noba

cter

ium

phoc

aePo

sC

occo

-bac

illian

dsh

ortr

ods,

sing

ly,

pairs

and

clus

ters

.Non

-aci

dfa

stbH

Col

onie

son

BAat

24h,

whi

te,t

iny,

roun

d,w

ithla

rge

zone

ofbH

vBi

oche

mse

t,AP

I50C

H,

API-Z

YM

Arca

noba

cter

ium

plur

anim

aliu

mPo

sSt

raig

htto

slig

htly

curv

ed,

non-

bran

chin

g,sl

ende

rrod

sa

Ha

haem

olys

ison

BA,s

mal

lcol

onie

s+

APIC

oryn

e,AP

Irap

idID

32St

rep

Arca

noba

cter

ium

pyog

enes

Pos

Coc

coba

cilli

and

shor

trod

s,si

ngly

,pa

irsan

dpa

lisad

efo

rmat

ion

with

shor

tdip

hthe

roid

form

sw

ithcl

ubs,

0.2–

0.9

×0.

3–2.

5mm

bHPi

npoi

nt,h

aem

olyt

icco

loni

esat

24h

onBA

.At

48h

colo

nies

0.5

mm

,circ

ular

,opa

que

whi

tew

ithzo

neof

haem

olys

is2–

3tim

esth

edi

amet

erof

the

colo

ny

−Bi

oche

mse

t.Li

tmus

milk

reac

tion

=ac

id,c

lota

ndre

duct

ion

at48

hus

ing

heav

yin

ocul

um

Bact

eriu

mG

ram

Mor

phol

ogy

bHTC

BSCo

lony

char

acte

ristic

sCa

tO

xId

entif

icat

ion

sets

Tab

le2.

4.C

ontin

ued.




Arth

roba

cter

agilis

Pos

Coc

ciin

pairs

and

tetra

ds,

0.8–

1.2

mmdi

amet

erN

HC

olon

ies

onag

arar

esm

ooth

, mat

t,en

tire,

with

aro

se-re

d-pi

gmen

ttha

tis

wat

er-in

solu

ble

++

Bioc

hem

set

Arth

roba

cter

nasi

phoc

aePo

sIrr

egul

ar-s

hape

dro

ds,n

on-s

pore

form

ing.

Som

eco

ccoi

dfo

rms

NH

Stric

taer

obe.

Gro

wth

onBA

asci

rcul

ar,

entir

e,co

nvex

,gre

yish

-whi

te,1

mm

at24

han

d37

°C

+Bi

oche

mse

t,AP

ICO

RYN

E,AP

I-ZYM

Arth

roba

cter

rhom

biPo

sSi

ngle

cory

nefo

rmro

ds,s

hort

orov

oid

insh

ape

Yello

w-w

hitis

hco

loni

es,1

mm

48h,

BHIA

with

1%N

aCl

++

Bioc

hem

set,

APIC

oryn

e

Atop

obac

ter

phoc

aePo

sSh

orti

rregu

larr

ods

NH

Col

onie

spi

n-po

int,

grey

,sm

ooth

onBA

at24

hat

37°C

i nC

O2

−−

Bioc

hem

set,

APIr

apid

ID32

Stre

p,AP

I-ZYM

Baci

llus

cere

usPo

sR

ods

bHO

nBA

colo

nies

are

whi

te-g

rey,

grou

nd-g

lass

appe

aran

ce,2

mm

at24

h.Be

com

esl

ight

lyye

llow

with

age

+Bi

oche

mse

t

Baci

llus

myc

oide

sPo

sR

ods

inch

ains

Rhi

zoid

colo

nies

with

coun

terc

lock

wis

efil

amen

tous

swirl

ing

patte

rnon

BA+

Bioc

hem

set

Bord

etel

labr

onch

isep

tica

Neg

Thin

rods

with

tape

red

ends

,sin

gly

and

pairs

.May

have

long

filam

ento

usfo

rms

NH

On

BAan

dC

FPA

colo

nies

are

1m

mat

48h

and

may

beha

emol

ytic

orno

n-ha

emol

ytic

.D

epen

ding

onph

ase

varia

tion,

roug

han

dsm

ooth

colo

nies

occu

rand

may

bero

ugh,

trans

luce

ntw

itha

rais

edce

ntre

and

undu

latin

gou

term

argi

n,or

smoo

th,o

paqu

ean

dpe

arl-l

ike

++

Bioc

hem

set

Brev

undi

mon

asdi

min

uta

Neg

Shor

trod

s1–

4×

0.5

mma

HG

row

thBA

,NA,

TSA,

30–3

7°C

,2da

ys.

Pant

othe

nate

,bio

tin,c

yano

coba

lam

inar

ere

quire

das

grow

thfa

ctor

s

++

Bioc

hem

set,

API5

0CH

,API

20N

E,AP

I-ZYM

Brev

undi

mon

asve

sicu

laris

Neg

aH

Yello

wco

loni

eson

BA,T

SA,N

A+

+Bi

oche

mse

t

Bruc

ella

abor

tus

Neg

Smal

lcoc

coid

rod

Gro

wth

at24

his

scan

t,oc

curri

ngw

here

inoc

ulum

ishe

avie

st+

+Se

ebe

low

Bruc

ella

spp.

Neg

Coc

coba

cilli.

May

stai

nfa

intly

Gro

wth

onpr

imar

ycu

lture

7–14

days

.10

%C

O2

requ

ired

fors

ome

stra

ins.

Gro

wth

onTS

Aor

FM.C

olon

ies

are

rais

ed,

conv

ex,e

ntire

edge

and

shin

ysu

rface

,ho

ney-

colo

ured

and

trans

pare

ntin

trans

mitt

edlig

ht

+Po

sitiv

eby

MAF

stai

n.Ph

enot

ypic

test

s,se

roty

ping

–be

stpe

rform

edby

asp

ecia

listl

abor

ator

yW

ork

with

susp

ectt

issu

esan

dBr

ucel

lacu

lture

sin

abi

olog

ical

safe

tyca

bine

t

cont

inue

d



94 Chapter 2

Budv

icia

aqua

tica

Neg

Stra

ight

rods

Smal

lcol

onie

son

NA,

0.5

mm

24h

at30

°C.

Tran

sluc

entw

ithsm

ooth

entir

eed

ges

+−

Bioc

hem

set

Burk

hold

eria

pseu

dom

alle

iN

egO

valt

oro

und

cells

with

bipo

lar

stai

ning

at3–

4da

ys.C

ells

may

bem

ista

ken

fors

pore

sas

only

the

perip

hery

stai

ns

Col

onie

s0.

5–1

mm

at24

h,an

d3–

4m

mat

3–4

days

.Whi

te,c

onve

xan

dsm

ooth

,with

whi

tesh

een,

beco

min

gun

even

and

umbo

nate

and

wrin

kled

.Bro

thcu

lture

istu

rbid

with

wrin

kled

pellic

le

++

Bioc

hem

set,

API2

0EW

ork

ina

biol

ogic

alsa

fety

cabi

net

Car

noba

cter

ium

inhi

bens

Pos

Rod

s.0.

2×

0.5–

1.2

mma

H1–

2m

mof

f-whi

teco

loni

eson

BA−

−Bi

oche

mse

t

Car

noba

cter

ium

(Lac

toba

cillu

s)pi

scic

ola

Pos

Very

smal

lrod

,1.1

–1.4

×0.

5–0.

6mm

.D

iplo

cocc

oid

intis

sues

,cha

ins

seen

inbr

oth

cultu

res

NG

1–1.

5m

m,g

rey/

whi

te,s

trep-

like

at25

°Cf o

r48

h.Ve

rylit

tlegr

eeni

ngof

the

agar

.Gro

wth

onBA

,BH

IA,T

SA

−−

Bioc

hem

set,

API2

0St

rep,

API5

0CH

Chr

omob

acte

rium

viol

aceu

mN

egN

Hor

bHPi

gmen

ted

stra

ins

are

deep

purp

le,r

ound

,sl

ight

lyra

ised

onBA

.Non

-pig

men

ted

stra

ins

show

b-ha

emol

ysis

onBA

++

Bioc

hem

set,

API2

0E,A

PI20

NE

Citr

obac

terf

reun

dii

Neg

Rod

sN

G2

mm

grey

colo

nies

at24

hon

BA+

−Bi

oche

mse

t,AP

I20E

Chr

yseo

bact

eriu

mba

lust

inum

Neg

Rod

s,0.

5×

1.0–

3.0

mm–

NG

Muc

oid,

yello

wco

loni

eson

AO+

+Bi

oche

mse

t,AP

I-ZYM

,API

20N

E

Chr

yseo

bact

eriu

m(F

lavo

bact

eriu

m)

gleu

m

Neg

Non

-spo

ring

rods

with

roun

ded

ends

NH

NG

On

NA

circ

ular

,ent

ire,v

isci

dbe

com

em

ucoi

dan

dtra

nslu

cent

afte

r5da

ys.B

right

yello

wpi

gmen

t

++

Bioc

hem

set,

API-Z

YM,A

PI20

NE

Chr

yseo

bact

eriu

min

dolo

gene

sN

egSt

raig

htro

ds0.

5×

1.3–

2.5

mmC

olon

ies

onhe

arti

nfus

ion

agar

are

1m

mat

2 4h

a t3 0

°C.O

n0.

3%ag

argr

owth

spre

ads

outi

na

flow

er-li

kegr

owth

patte

rn

++

Bioc

hem

set,

API2

0NE,

API-Z

YM

Chr

yseo

bact

eriu

mm

enin

gose

ptic

umN

egN

HC

olon

ies

onBA

are

abou

t1m

min

diam

eter

and

surro

unde

dby

azo

neof

gree

nish

disc

olor

atio

nat

24h

++

Bioc

hem

set,

API-Z

YM,A

PI20

NE

Chr

yseo

bact

eriu

msc

opht

halm

umN

egSh

ortr

ods,

2.0

×0.

8mm

NH

NG

Smoo

th,r

ound

,shi

ny,o

rang

epi

gmen

ted

colo

nies

,2–3

mm

afte

r2da

ysat

25o C

o nM

SA-B

,MA

2216

,TSA

.Fre

shis

olat

essh

owgl

idin

gm

otilit

yon

Anac

ker-O

rdal

med

ium

.G

lidin

gab

ility

lost

afte

rsto

rage

++

Bioc

hem

set,

API-Z

YM,A

PI20

NE

Bact

eriu

mG

ram

Mor

phol

ogy

bHTC

BSCo

lony

char

acte

ristic

sCa

tO

xId

entif

icat

ion

sets

Tab

le2.

4.C

ontin

ued.




Clo

strid

ium

botu

linum

Pos

3.4–

7.5

×0.

7mm

oval

,sub

term

inal

spor

esbH

Sem

i-opa

que

totra

nslu

cent

,mat

tcol

ony,

1–3

mm

,irre

gula

rlob

ate

mar

gin

and

rais

edce

ntre

−AP

I rap

i d32

A

Col

wel

liam

aris

Neg

Cur

ved

rods

,0.6

–1×

2–4

mmM

arin

e22

16ag

ar,o

ptim

algr

owth

at15

°C.

Gro

wth

rang

e0–

22°C

,but

nogr

owth

at25

°C+

+Bi

oche

mse

t

Cor

yneb

acte

rium

aqua

ticum

Pos

Rod

s0.

5–0.

8×

1–3

mm.S

light

pleo

mor

phis

mw

ithso

me

club

-sha

ped

form

san

dan

gula

rarra

ngem

ents

ofce

lls

bHO

nBA

and

TSA

colo

nies

roun

d,ra

ised

,ent

ire,

opaq

ue,s

light

lyvi

scid

with

yello

wno

n-di

ffusi

ble

pigm

enta

fter4

8h

at25

°C

+−

Bioc

hem

set,

APIC

oryn

e

Cor

yneb

acte

rium

phoc

oae

Pos

Cor

ynef

orm

rods

NH

Shin

y,ro

und

colo

nies

1m

mon

BA24

hr,

37°C

+Bi

oche

mse

t,AP

ICor

yne,

API5

0CH

,API

-ZYM

Cor

yneb

acte

rium

test

udin

oris

Pos

Dip

hthe

roid

rods

Col

onie

son

BAar

eye

llow

-pig

men

ted

+AP

ICor

yne,

API-Z

YM

Cry

ptoc

occu

sne

ofor

man

sva

r.ga

ttii

Pos

Budd

ing

yeas

tsN

HSl

owgr

owin

g.C

olon

ies

0.5–

1m

mcr

eam

dull,

3da

yson

BA.C

olon

ies

chan

gefro

mcr

eam

todi

rtyye

llow

,lig

htta

nco

lour

afte

r7da

yson

BA

Indi

ain

kfo

rcap

sule

stai

n,ur

ease

posi

tive.

Gro

wth

onSt

rep

sele

ctiv

em

edia

(Oxo

id),

and

SAB

plat

esUs

ebi

olog

ical

safe

tyca

bine

t

Der

mat

ophi

lus

Pos

Bran

chin

gfil

amen

tsco

ntai

ning

row

sof

cocc

i(zo

ospo

res)

.Zoo

spor

esar

em

otile

bHC

olon

ies

onBA

inC

O2

a re

g re y

-wh i

teto

grey

-yel

low

,adh

eren

tand

pitt

heag

ar.A

t48

h,co

loni

esar

etin

y,ro

ugh,

gran

ular

,ra

ised

.Fur

ther

incu

batio

npr

oduc

esco

loni

esup

to2–

3m

m,u

mbo

nate

,mol

arto

oth

crat

erfo

rms

and

beco

me

muc

oid

+Bi

oche

mse

t.M

ake

impr

essi

onsm

ears

from

unde

rsid

eof

fresh

lyre

mov

edsc

abs.

Stai

nw

ithG

iem

sast

ain.

Fila

men

tsar

ebe

stst

aine

dw

ithG

iem

sara

ther

than

Gra

mst

ain

Edw

ards

iella

hosh

inae

Stra

ight

rods

Gro

wth

onN

A,BA

,MC

A.C

olon

ies

flato

rsl

ight

lyco

nvex

,1–2

mm

at24

–48

h,30

and

37°C

+−

Bioc

hem

set,

API2

0E

Edw

ards

iella

icta

luri

Neg

Rod

toco

ccob

acilli

,0.

75×

1.5–

2.5

mmw

NG

Slow

grow

ing,

1–2

mm

,rou

nd,

non-

pigm

ente

d,pa

legr

ey,4

8h

at28

–30°

C.

May

bea

gree

nish

tinge

and

slig

htha

emol

ysis

unde

rthe

colo

ny(p

ale

lem

onco

lour

ona

loop

).M

usty

smel

l

+−

Bioc

hem

set,

API2

0E cont

inue

d



96 Chapter 2

Edw

ards

iella

tard

aN

egR

od.1

×1–

2mm

NG

0.5

mm

roun

d,gr

eyco

loni

esat

24–4

8h

(pal

ele

mon

colo

uron

alo

op).

Gro

wth

onBA

,MC

A(N

LF),

SSag

ar.C

olon

ies

will

besm

alle

rin

size

than

Salm

onel

laon

the

sele

ctiv

em

edia

+−

Bioc

hem

set,

API2

0E

Empe

doba

cter

brev

isN

egR

ods

Yello

wco

loni

eson

NA

and

BA,0

.2–2

.5m

mat

24h

and

30°C

,low

conv

ex,e

ntire

edge

.At

day

7on

BAm

ayse

esl

ight

a-h

aem

olys

is

++

Bioc

hem

set

Ente

rovi

brio

norv

egic

usN

egC

ells

0.8

×1.

0–1.

2mm

GO

nM

A22

16co

loni

esar

ebe

ige,

smoo

th,

roun

d,ra

ised

,1m

mat

48h

at28

°C+

+Bi

oche

mse

t,AP

I20E

,AP

I-ZYM

Erys

ipel

othr

ixrh

usio

path

iae

Pos

Gra

m-v

aria

ble

rods

,ple

omor

phic

,ta

ngle

dfo

rms,

2–5

mmN

Ha

HN

DG

row

thon

BAat

24–4

8h,

0.5

mm

grey

-gre

enco

loni

essm

allz

one

ofa

-hae

mol

ysis

−Bi

oche

mse

t,AP

Irap

idID

32St

rep,

APIC

oryn

e.N

ote:

add

afe

wdr

ops

ofst

erile

seru

mto

inoc

ulat

ing

med

ium

toim

prov

ere

actio

nsin

Bioc

hem

set

Euba

cter

ium

spp.

Pos

Long

chai

nsof

pleo

mor

phic

rods

.No

spor

es.I

nol

derc

ultu

res

cells

may

have

‘ovo

id’b

odie

s

bHO

nBA

unde

rana

erob

icco

nditi

ons,

colo

nies

are

haem

olyt

ic,t

rans

luce

nt,s

low

lysp

read

ing,

flat,

and

cont

oure

dw

ithfil

amen

tous

edge

s.An

aero

bic

atm

osph

ere

requ

ired

−−

Bioc

hem

set

Euba

cter

ium

limos

umPo

s0.

6–0.

9×

1.6–

4.8

mm.R

ods

may

have

swol

len

ends

NH

Punc

tate

,circ

ular

,con

vex,

entir

eco

loni

esth

atar

etra

nslu

cent

tosl

ight

lyop

aque

.Les

sth

an1

mm

at48

h.An

aero

bic

−Bi

oche

mse

t,AP

Irap

id32

A

Euba

cter

ium

tara

ntel

lae

Pos

Very

long

filam

ento

us,u

nbra

nche

dro

ds(1

0mm

)bH

NG

2–5

mm

,tra

nslu

cent

colo

nies

,slig

htly

rhiz

oid

and

muc

oid.

Anae

robi

c−

Bioc

hem

set

Fack

lam

iam

iroun

gae

Pos

Ovo

idce

lls(0

.8–0

.9mm

)in

pairs

and

shor

tcha

ins

NH

ND

Col

onie

son

BA0.

5m

mat

24h,

37°C

.C

ircul

ar,e

ntire

,shi

ny,c

onve

xan

dgr

ey-c

olou

red

−AP

Irap

idID

32,A

PI-Z

YM

Flav

obac

teriu

maq

uatil

eN

egC

olon

ies

onAO

med

ialo

w,c

onve

x,ro

und

++

Bioc

hem

set,

API-Z

YM

Flav

obac

teriu

mbr

anch

ioph

ilum

Neg

Long

thin

rods

0.5–

8×

10mm

.Sm

ears

from

colo

nies

may

show

circ

ular

,slig

htly

refra

ctile

,‘cy

sts’

NH

NG

On

AOm

edia

,lig

htye

llow

,sm

ooth

,rou

nd,

rais

ed,c

olon

ies,

0.5–

1m

m,5

days

at18

°C.

No

glid

ing

mot

ility.

Gro

wth

on20

-fold

dilu

teTS

Abu

tnot

on‘fu

ll-st

reng

th’T

SA

V+

Bioc

hem

set,

API-Z

YM,A

PI20

E

Bact

eriu

mG

ram

Mor

phol

ogy

bHTC

BSCo

lony

char

acte

ristic

sCa

tO

xId

entif

icat

ion

sets

Tab

le2.

4.C

ontin

ued.




Flav

obac

teriu

m(F

lexi

bact

er)

colu

mna

re

Neg

Long

thin

rods

(4–8

mm).

Fila

men

tous

NH

NG

Brig

htye

llow

,fla

t,dr

y,rh

izoi

d,sl

owsp

read

ing

grow

th5

days

20–2

5°C

.Ad

here

sst

rong

lyto

agar

V+

Bioc

hem

set,

API-Z

YM,A

PI20

E,AP

I50C

H.Y

ello

wpi

gmen

tcha

nges

topi

nkin

3%N

aOH

Flav

obac

teriu

mfle

vens

eN

egO

nAO

med

ia,l

owco

nvex

,rou

ndco

loni

essu

nken

into

agar

++

Bioc

hem

set,

API-Z

YM

Flav

obac

teriu

mfri

gida

rium

Neg

Rod

s0.

8–2

×0.

5–0.

7mm

,sin

gly

and

inpa

irsG

row

thon

AO,f

latr

ound

yello

wco

loni

esw

ithen

tire

mar

gins

.Gro

ws

onN

A,TS

Aan

dM

A22

16

++

Bioc

hem

set,

API-Z

YM

Flav

obac

teriu

mgi

llisia

eN

egR

ods

2–5

×0.

4–0.

5mm

Ora

nge-

pigm

ente

dco

loni

es,b

utyr

ous,

circ

ular

and

conv

exw

ithen

tire

edge

.Gro

wth

onM

A22

16,N

A,TS

A,R

2A

+−

Bioc

hem

set,

API2

0E

Flav

obac

teriu

mhi

bern

umN

egR

ods

0.7

×1.

8–13

mmG

row

thon

TSA.

Yello

w,m

ucoi

dco

loni

esat

25°C

,gel

atin

ous

at4°

C+

−Bi

oche

mse

t,AP

I20E

,API

20N

E

Flav

obac

teriu

mhy

datis

(Cyt

opha

gaaq

uatil

is)

Neg

Rod

s,8.

0×

0.5

mmN

HN

GM

ucoi

d,ye

llow

-ora

nge

colo

nies

with

flat

spre

adin

gfil

amen

tous

mar

gins

.Inc

ubat

e14

°Cfo

r14

days

+−

Bioc

hem

set,

API-Z

YM,A

PI20

E

Flav

obac

teriu

m(C

ytop

haga

)jo

hnso

niae

Neg

Long

thin

rods

NH

NG

On

AOm

edia

,pal

eye

llow

,5–1

0m

m,f

lat,

smoo

th,r

hizo

idw

ithsp

read

ing,

filam

ento

usm

argi

ns,5

days

.On

BA,y

ello

w,r

ound

,sm

ooth

,1–2

mm

,2da

ys

V+

Bioc

hem

set,

API-Z

YM,A

PI20

E

Flav

obac

teriu

mpe

ctin

ovor

umN

egO

nAO

med

ia,c

olon

ies

low

roun

dw

ithen

tire

mar

gins

+Bi

oche

mse

t,AP

I-ZYM

Flav

obac

teriu

m(C

ytop

haga

)ps

ychr

ophi

lum

Neg

Slen

der,

flexi

ble

rods

1–7

×0.

5mm

NG

Smoo

th,g

loss

y,br

ight

yello

wco

loni

esw

ithth

insp

read

ing

edge

s,5

days

at5–

25°C

.D

oes

nota

dher

eto

agar

V+

Bioc

hem

set,

API-Z

YM,A

PI50

CH

Flav

obac

teriu

msa

ccha

roph

ilum

Neg

On

AOm

edia

,col

onie

sfla

t,sp

read

ing,

sunk

enin

toag

ar+

−Bi

oche

mse

t,AP

I-ZYM

Flav

obac

teriu

msu

ccin

ican

sN

egO

nAO

med

ia,c

olon

ies

flat,

spre

adin

g,w

ithfil

amen

tous

mar

gins

Bioc

hem

set,

API-Z

YM

Flav

obac

teriu

mte

getin

cola

Neg

Rod

s2–

5×

0.4–

0.5

mmYe

llow

-pig

men

ted

colo

nies

,but

yrou

s,ci

rcul

ar,

conv

ex,e

ntire

.Gro

wth

onM

A22

16,N

A,TS

A,R

2A

+−

Bioc

hem

set,

API2

0E cont

inue

d



98 Chapter 2

Flav

obac

teriu

mxa

nthu

mN

egG

row

thon

MA

2216

,NA,

TSA,

R2A

++

Bioc

hem

set,

API2

0E

Flex

ibac

ter

poly

mor

phus

Neg

Fila

men

tsm

ultic

ellu

lar.

Aten

dof

each

cell

isa

refra

ctile

gran

ule

oflip

idm

ater

ial

Mar

ine

agar

.Cob

alam

inre

quire

dfo

rgro

wth

.Pe

ach-

colo

ured

pigm

ent

−+

Bioc

hem

set,

API-Z

YM,A

PI50

CH

Gra

nulic

atel

laad

iace

nsG

ranu

licat

ella

eleg

ans

Pos

Coc

ci,i

nclu

ding

pleo

mor

phic

ovoi

dce

lls,c

occo

-bac

illi,r

od-s

hape

dce

llsa

Stra

ins

are

nutri

tiona

llyde

ficie

nt,a

ndsa

tellit

ear

ound

othe

rbac

teria

lgro

wth

.Gro

won

BAw

ithSt

aph

stre

akor

pyrid

oxal

disc

,orB

Asu

pple

men

ted

with

20mg

pyrid

oxal

HC

lper

ml.

For G

.ele

gans

use

L-cy

stei

neH

Cl

(0.0

1%)

−−

Bioc

hem

set,

API2

0St

rep,

API r

api d

I D32

Str e

p

Gra

nulic

atel

laba

laen

opte

rae

Pos

Coc

cus,

sing

lece

llsan

dsh

ortc

hain

sa

NG

Gro

wth

onBA

,0.2

mm

colo

nyin

airo

rCO

2.Is

notn

utrit

iona

llyde

ficie

ntlik

eot

hers

train

sin

the

genu

s

−Bi

oche

mse

t,AP

I-ZYM

,API

20St

rep,

APIr

apid

ID32

Stre

p

Haf

nia

alve

iN

egSh

ortr

ods

Whi

teto

yello

wno

n-m

ucoi

dco

loni

es.G

row

thon

BA,N

A,M

CA,

DC

A+

−Bi

oche

mse

t,AP

I-ZYM

,API

20E

Hal

omon

asaq

uam

arin

a(p

revi

ousl

yAl

calig

enes

faec

alis

hom

ari )

Neg

Stra

ight

orcu

rved

rods

1.5

mm,

bipo

lars

tain

ing

Col

onie

son

MA

2216

,off-

whi

tetra

nslu

cent

,ra

ised

smoo

th2–

3m

mat

24h

at18

°Can

d37

°C.S

light

tend

ency

tosp

read

++

Bioc

hem

set

Hal

omon

asel

onga

taN

egR

ods

sing

lyor

paire

dC

olon

ies

2m

mat

24h,

smoo

th,g

liste

ning

,op

aque

whi

te.A

fter2

4h

colo

nies

spre

addu

eto

flexu

ous

filam

ents

,but

are

nola

rger

than

4m

m.R

equi

res

8%N

aClf

orgr

owth

Bioc

hem

set.

Add

8%sa

ltto

med

ium

Hal

omon

asha

lodu

rans

Neg

Rod

ssi

ngle

,pai

rsor

chai

nsO

nM

A22

16,c

olon

ies

are

smoo

th,g

liste

ning

,tra

nslu

cent

whi

te,c

onve

x,1–

2m

min

24h

at20

or30

°C

Bioc

hem

set

Hal

omon

asve

nust

aN

egR

odAt

48h

colo

urle

ss,m

ucoi

dco

loni

eson

BA,

MC

A+

+Bi

oche

mse

t,AP

I20N

E

Bact

eriu

mG

ram

Mor

phol

ogy

bHTC

BSCo

lony

char

acte

ristic

sCa

tO

xId

entif

icat

ion

sets

Tab

le2.

4.C

ontin

ued.




Hel

icob

acte

rcet

orum

Neg

Fusi

form

tosl

ight

lysp

iral

On

BA,S

kirro

w’s

med

ia(V

PT),

orTS

A+

bloo

d,co

loni

esar

epi

n-po

inta

ndm

aygr

owas

ath

insp

read

ing

film

acro

ssth

epl

ate,

5–14

days

37°C

++

Bioc

hem

ical

s,AP

ICam

py

Hyd

roge

noph

aga

(Pse

udom

onas

)ps

eudo

flava

Neg

Ovo

idce

lls.R

ods

of2.

5mm

inol

der

cultu

res

NG

On

NB

agar

yello

w-p

igm

ente

dco

loni

es2–

4m

mat

3da

ys,s

light

lyirr

egul

arsh

ape

with

anun

dula

ting

mar

gin

W+

Bioc

hem

set

Iodo

bact

erflu

viat

ileN

egSm

allr

od,0

.7×

3.0–

3.5

mmN

GVi

olet

-col

oure

dco

loni

es,t

hin

spre

adin

gm

argi

non

low

nutri

enta

gars

uch

as¼

stre

ngth

NA.

Non

-spr

eadi

ngon

NA

++

Bioc

hem

set

Lact

ococ

cus

garv

ieae

Pos

Coc

coba

cilli

0.7–

1.4

mm,p

aire

dco

cci,

shor

tcha

ins

aH

NG

1m

mgr

ey/w

hite

,rou

nd,w

ithgr

eeni

ngun

der

colo

ny.S

trep-

like

onBA

.Gro

wth

onN

A,TS

A,BA

−−

Bioc

hem

set,

API2

0Stre

p,AP

Irap

idID

32St

rep,

API

50C

H.S

trep

grou

p=

Dne

g

Lact

ococ

cus

pisc

ium

Pos

Coc

coba

cilli

−−

Bioc

hem

set

List

onel

la(V

ibrio

)an

guilla

rum

Neg

Shor

trod

s,cu

rved

orst

raig

ht,

roun

ded

ends

,occ

urrin

gsi

ngly

and

inpa

irs,p

leom

orph

ic.

0.5–

0.7

×1–

2mm

.Rap

idm

otilit

y

bHY

At2

days

colo

nies

are

2m

m,g

liste

ning

crea

m-c

olou

rin

youn

gco

loni

esan

dgr

eeni

sh-p

ale

brow

nin

olde

rcol

onie

son

MSA

-B.H

aem

olys

isun

derc

olon

y.O

nM

SA-B

,V.

chol

erae

and

V.m

imic

usha

vesi

mila

rap

pear

ance

butg

row

mor

equ

ickl

y.O

nN

Aco

loni

esar

eof

f-whi

teto

buff-

colo

ured

,tra

nslu

cent

orop

aque

,circ

ular

,shi

ny,1

–2m

m

++

Bioc

hem

set,

API-Z

YM,A

PI20

E,AP

I20N

E

List

onel

lape

lagi

aN

egN

HG

rey-

trans

luce

ntco

loni

eson

MSA

-B+

+Bi

oche

mse

t

Man

nhei

mia

haem

olyt

ica

Neg

Pleo

mor

phic

rods

,lon

gan

dsh

ort

form

sbH

Gre

yco

loni

es1–

2m

mon

BAat

24h.

b-ha

emol

ysis

seen

++

Bioc

hem

set,

API-Z

YM

Mar

inila

bilia

salm

onic

olor

Neg

Glid

ing,

yello

wto

pale

pink

-pig

men

ted

colo

nies

++

Bioc

hem

set,

API-Z

YM

Mes

ophi

loba

cter

mar

inus

Neg

Coc

coba

cilli,

pleo

mor

phic

NH

NG

Gro

wth

onM

A22

16an

dN

A.C

olon

ies

circ

ular

som

etim

esirr

egul

ar,c

onve

x,gl

iste

ning

,op

aque

,pal

eye

llow

-bro

wn.

Des

crib

esm

arin

eco

ccob

acilli

that

mor

phol

ogic

ally

rese

mbl

eth

eAc

inet

obac

ter-M

orax

ella

grou

p

++

Bioc

hem

set

Mor

itella

mar

ina

Neg

Cur

ved

orst

raig

htro

dsbH

NG

Gre

yish

-cre

am,t

rans

luce

nt,c

ircul

ar,c

onve

xco

loni

eson

MSA

-B+

+Bi

oche

mse

t,AP

I-ZYM

,API

20E

cont

inue

d

115Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpMonday, January 26, 2004 11:48:01 AM


100 Chapter 2

Mor

itella

visc

osa

Neg

Long

cells

whe

ngr

own

inliq

uid

med

ia.S

hort

orel

onga

ted

curv

edro

dson

solid

med

ium

bHN

GC

ream

y-ye

llow

colo

nies

onM

SA-B

and

TSA+

NaC

l,0.

5m

m24

hat

15–2

2°C

.C

olon

ies

are

visc

ous

and

adhe

reto

the

med

ium

.For

mlo

ngth

read

sw

hen

lifte

dfro

mth

epl

ate.

Ligh

thae

mol

ysis

seen

unde

rnea

thco

lony

++

Bioc

hem

set,

API-Z

YM,A

PI20

E,AP

I50C

H

Myc

obac

teriu

mab

sces

sus

AFB

Gro

wth

onM

iddl

ebro

ok7H

10-A

DC

med

ium

in7

days

.Gro

wth

onM

CA

+Bi

oche

mse

t,or

send

tosp

ecia

listl

abor

ator

y

Myc

obac

teriu

mch

elon

aePo

san

dAF

BPl

eom

orph

icro

ds,2

–7×

0.2–

0.5

mmN

HN

GC

olon

ies

onM

SA-B

are

circ

ular

,sm

ooth

,pa

le-c

ream

at7

days

and

15°C

.G

row

thon

MC

A,TS

A,BH

IA

+−

Bioc

hem

set,

orse

ndto

spec

ialis

tlab

orat

ory.

PCR

prim

ers

avai

labl

e.

Myc

obac

teriu

mm

arin

umPo

san

dAF

BPl

eom

orph

icro

ds,2

–7×

0.2–

0.5

mmN

HN

GC

olon

ies

onM

SA-B

are

circ

ular

opaq

ue,

0.2

mm

at7

days

,22 °

C.W

hen

grow

nin

the

light

,col

onie

sar

eye

llow

Bioc

hem

set,

orse

ndto

spec

ialis

tlab

orat

ory.

PCR

prim

ers

avai

labl

e

Myc

obac

teriu

mne

oaur

umG

ram

stai

n=

neg,

AFB

AFB

3–4

×0.

6mm

NH

Yello

wco

loni

eson

BAat

25°C

in5–

7da

ys.

No

grow

that

37°C

.At8

days

,col

onie

s0.

4m

m,s

moo

th,r

ound

clea

r,w

ithsl

ight

y ello

wc o

lou r

.No

g ro w

tho n

MS A

+Se

ndto

spec

ialis

tlab

orat

ory

Myc

obac

teriu

mpe

regr

inum

AFB

Acid

fast

rods

Buff-

colo

ured

colo

nies

onM

iddl

ebro

ok7H

11m

ediu

min

4da

ysun

derC

O2

Send

tosp

ecia

listl

abor

ator

y

Myc

obac

teriu

mpo

rifer

aeAF

BAc

idfa

stro

dsR

apid

grow

thw

ithsm

ooth

colo

nies

onM

iddl

ebro

ok7H

11m

ediu

mSe

ndto

spec

ialis

tlab

orat

ory

Myc

obac

teriu

msp

p.W

eak

pos

non-

stai

ning

,ac

id-fa

st

Pleo

mor

phic

med

ium

tolo

ngro

ds,

non-

bran

chin

g,1.

5–3.

0mm

NH

NG

0.5

mm

off-w

hite

,5–7

days

gran

ular

+Bi

oche

mse

t,or

send

tosp

ecia

listl

abor

ator

yUs

ebi

olog

ical

safe

tyca

bine

t

Myc

obac

teriu

mtri

plex

-like

AFB

Coc

coid

and

rod

form

s.Be

aded

AFB

rods

aH

Gro

wth

o nB A

a fte

r12

we e

k sa t

2 5°C

prod

uces

roug

h,dr

yco

loni

esw

itha

rais

edce

ntre

and

flatb

orde

r

Nes

ted

PCR

Myc

opla

sma

mob

ileC

ells

glid

ein

wet

prep

arat

ion

asse

enby

dark

grou

ndillu

min

atio

n.C

ells

are

elon

gate

d.O

ptim

umte

mpe

ratu

reis

25°C

Myc

opla

sma

set

Bact

eriu

mG

ram

Mor

phol

ogy

bHTC

BSCo

lony

char

acte

ristic

sCa

tO

xId

entif

icat

ion

sets

Tab

le2.

4.C

ontin

ued.




Myr

oide

s(F

lavo

bact

eriu

m)

odor

atim

imus

Neg

Rod

s0.

5×

1–4

mmN

HYe

llow

-pig

men

ted

colo

nies

.Gro

wth

onM

CA,

NA

and

TSA.

No

glid

ing

orsw

arm

ing.

Frui

tyod

our

++

Bioc

hem

set,

API-Z

YM,A

PI20

NE

Myr

oide

s(F

lavo

bact

eriu

m)

odor

atus

Neg

Rod

s0.

5×

1–4

mmN

HYe

llow

-pig

men

ted

colo

nies

,3–4

mm

onM

CA,

NA

and

TSA

at24

h.N

ogl

idin

gor

swar

min

g.Fr

uity

odou

r

++

Bioc

hem

set,

API-Z

YM,A

PI20

NE

Noc

ardi

acr

asso

stre

aePo

sBr

anch

edhy

phae

,fra

gmen

tint

oro

dsan

dco

cci

Dry

wrin

kled

,wax

yco

loni

es,p

ale

yello

w.N

oae

rialh

ypha

e+

Bioc

hem

set,

acid

fast

Noc

ardi

asa

lmon

icid

aPo

sBr

anch

edor

ange

,sub

stra

tem

ycel

ium

,with

whi

teto

pink

aeria

lgro

wth

.Fila

men

tous

colo

nym

argi

ns

Bioc

hem

set

Noc

ardi

ase

riola

ePo

sBr

anch

ing

vege

tativ

em

ycel

iath

atbr

eak

upin

tono

n-m

otile

rods

.C

occo

idan

dlo

ngsl

ende

rm

ulti-

sept

ate

rod

form

s

Mac

rosc

opic

aeria

lmyc

elia

are

notf

ound

.D

ry,w

axy,

wrin

kled

,col

onie

sth

atar

eye

llow

ish-

oran

geon

yeas

text

ract

–mal

tex

tract

agar

(ISP

no2

Difc

o).A

ppea

ras

whi

teco

loni

eson

BHIA

and

yello

wco

loni

eson

LJM

Bioc

hem

set,

wea

kly

acid

fast

Noc

ardi

asp

p.Po

sw

eak

acid

-fast

Coc

coid

toov

alce

lls,l

ong

slen

der

mul

ti-se

ptat

ero

ds,b

ranc

hing

.5–

50mm

NN

G<1

mm

,whi

te,c

ream

,rou

ghor

with

very

shor

tde

nse

myc

eliu

m.G

row

th3–

7da

ys.B

Aan

dN

A

V,+

Bioc

hem

set

Noc

ardi

asp

p.(A

ustra

lian

stra

in)

GPR

Bran

chin

gro

dsC

olon

ies

appe

arat

5da

ys.D

ry,r

ough

,ta

n/ye

llow

,adh

eren

t,1–

2m

mBi

oche

mse

t.M

odifi

edac

idfa

stst

ain

=po

sitiv

e

Pant

oea

(Ent

erob

acte

r)ag

glom

eran

s

Neg

Non

-enc

apsu

late

d,no

n-sp

ore

form

ing,

broa

d,st

raig

htro

d,0.

5–1.

0×

1.0–

3.0

mm

NH

NG

Col

onie

son

NA

are

smoo

th,c

onve

x,en

tire,

and

trans

luce

ntan

dm

ayha

vea

yello

wpi

gmen

t.G

row

thon

BA,M

SA-B

,and

MA

2216

.Gro

wth

at37

°Cbu

tnot

at44

°C

+−

Bioc

hem

set,

API2

0E,A

PI50

CH

Past

eure

llam

ulto

cida

Neg

Coc

co-b

acilli

NH

Gro

wth

onBA

,1–2

mm

at24

–48

h.C

olon

ysi

zean

dap

pear

ance

vary

from

diffe

rent

anim

alsp

ecie

s.C

olon

ies

may

besm

ooth

orm

ucoi

d,da

rkgr

eyw

ithgr

eeni

shap

pear

ance

.D

istin

ctiv

em

usty

swee

tish

smel

l

++

Bioc

hem

set,

API-Z

YM,A

PI20

E

Past

eure

llask

yens

isN

egN

Hor

wea

kG

row

thTS

A-B+

1.5%

NaC

l,at

48h

at22

°C.

Col

onie

sci

rcul

ar,e

ntire

,low

,con

vex,

grey

,0.

5m

m.N

ogr

owth

with

out1

.5%

NaC

lor

bloo

d.N

ogr

owth

at37

°C

−+

wea

kBi

oche

mse

t,AP

I-ZYM

.Add

1.5–

2%N

aClt

ose

tsan

dus

ehe

avy

inoc

ulum

inBi

oche

mse

t.R

ead

afte

r4da

ys cont

inue

d



102 Chapter 2

Past

eure

llate

stud

inis

NPl

eom

orph

icro

d0.

2×

1.5–

2mm

bHBA

whi

te,m

ucoi

d,0.

5–1

mm

24–4

8h,

20–3

7 °C

++

Bioc

hem

set

Pedo

bact

erhe

parin

usN

egN

on-s

porin

gro

ds,

0.4–

0.5

×0.

5–1.

0mm

bHsl

owG

row

thon

NA,

1–3

mm

at48

h,ci

rcul

ar,l

owco

nvex

,sm

ooth

,opa

que.

Aye

llow

orcr

eam

yw

hite

non-

fluor

esce

ntpi

gmen

tis

prod

uced

.C

ream

yw

hite

colo

nies

onPY

++

Bioc

hem

set,

API-Z

YM,A

PI50

CH

Pedo

bact

erpi

sciu

mN

egN

on-s

porin

gro

ds0.

4–0.

5×

0.5–

1.0

mmC

olon

ies

onN

Aar

eci

rcul

ar,e

ntire

,low

conv

exat

2da

ys.A

yello

wor

crea

my

whi

te,

non-

fluor

esce

ntpi

gmen

tis

prod

uced

++

Bioc

hem

set,

API-Z

YM,A

PI50

CH

Phoc

oeno

bact

erut

eri

Neg

Pleo

mor

phic

rods

NH

Gro

wth

onBA

with

and

with

outC

O2.

Col

onie

sen

tire,

circ

ular

,low

conv

ex,s

moo

th,g

rey

0 .5

mm

a t4 8

ha t

3 7°C

−+

Bioc

hem

set,

API2

0E,A

PI20

NE,

API-Z

YM

Phot

obac

teriu

man

gust

umN

egSh

ortr

ods

NH

Whi

teco

loni

eson

MA

2216

,MSA

-B+

+Bi

oche

mse

t,AP

I20E

,API

50C

H

Phot

obac

teriu

mda

mse

lae

ssp.

dam

sela

e

Neg

Rod

s,re

lativ

ely

pleo

mor

phic

bHG

Entir

e,sm

ooth

,gre

yish

-whi

te,t

rans

luce

ntco

loni

es,2

–3m

mon

MSA

-B+

+Bi

oche

mse

t,AP

I-ZYM

,API

20E,

API2

0NE,

API5

0CH

Phot

obac

teriu

mda

mse

lae

ssp.

pisc

icid

a

Neg

Smal

lrod

sto

cocc

obac

illi,1

–1.5

mm,

bipo

lars

tain

ing

bHN

G1–

2m

mgr

ey/y

ello

w,s

hiny

,72

h.G

row

thon

BAan

dN

Aw

ith0.

5%N

aCl,

MSA

-B,M

A22

16+

+Bi

oche

mse

t,AP

I-ZYM

,API

20E,

API2

0NE,

API5

0CH

Phot

obac

teriu

milio

pisc

ariu

mN

egPl

eom

orph

icro

ds,s

traig

htan

dcu

rved

NH

GC

olon

ies

smal

lcol

ourle

ssto

grey

ish

opaq

uew

ithen

tire

edge

.May

take

upto

14da

ysfo

rco

loni

esto

appe

ar

++

Bioc

hem

set,

API2

0E,A

PI50

CH

Phot

obac

teriu

mle

iogn

athi

Neg

Shor

trod

sor

cocc

obac

illi,

1–2.

5×

0.4–

1mm

NH

NG

Col

onie

son

MSA

-B,o

ff-w

hite

,tra

nslu

cent

,ci

rcul

ar,s

moo

th,s

hiny

.Lum

inou

sat

3da

ys+

−Bi

oche

mse

t,AP

I20E

,API

50C

H

Phot

obac

teriu

mph

osph

oreu

mN

egSh

ortr

ods

NH

Whi

teco

loni

eson

MSA

-Ban

dM

A22

16+

−Bi

oche

mse

t,AP

I20E

Plan

ococ

cus

kocu

riiPo

sSp

heric

alce

lls1.

0–1.

2mm

,sin

gly,

pairs

and

tetra

ds.M

otile

Col

onie

son

pept

one

yeas

text

ract

agar

are

circ

ular

,sm

ooth

,con

vex,

and

yello

w-o

rang

epi

gmen

ted

+−

Bioc

hem

set

Plan

omic

robi

umok

eano

koite

sN

eg,v

Rod

s0.

4–0.

8×

1–20

mmC

ells

brig

htor

ange

toye

llow

.Req

uire

s3%

NaC

l+

WBi

oche

mse

t.Ad

dN

aCl(

3%)

Bact

eriu

mG

ram

Mor

phol

ogy

bHTC

BSCo

lony

char

acte

ristic

sCa

tO

xId

entif

icat

ion

sets

Tab

le2.

4.C

ontin

ued.




Ples

iom

onas

shig

ello

ides

Neg

Stra

ight

,var

iabl

ele

ngth

rod

NH

W,G

Gro

wth

onBA

and

MC

A1–

2m

m24

hat

25–3

7 °C

.Pun

gent

smel

l+

+Bi

oche

mse

t,AP

I-ZYM

,API

20E

Prov

iden

cia

rettg

eri

Neg

Rod

0.6

×1.

0–1.

5mm

NH

Col

onie

son

BA,T

SAci

rcul

ar,d

iscr

ete,

conv

ex,g

liste

ning

,cre

amy-

whi

te24

hat

37°C

+−

Bioc

hem

set,

API2

0E

Prov

iden

cia

rust

igia

nii

Neg

Non

-spo

ring

rods

0.5

×1–

3mm

NH

On

BA,c

olon

ies

1–2

mm

at24

h,gl

ossy

,se

mitr

ansl

ucen

t,sm

ooth

.Ora

nge-

red

onM

CA

+−

Bioc

hem

set,

API2

0E

Pseu

doal

tero

mon

asge

nus

Neg

Stra

ight

orcu

rved

,non

-spo

refo

rmin

gro

ds0.

2–1.

5×

1.8–

3mm

.Sin

gle

flage

lla

Stric

taer

obes

,gro

wth

onM

A22

16at

20°C

.N

egat

ive

forA

DH

.All

posi

tive

forg

elat

inan

dD

Nas

e

+,w

,v

+Bi

oche

mse

t

Pseu

doal

tero

mon

asan

tarc

tica

Neg

Rod

s,0.

9×

1–3

mm,b

ecom

ela

rger

and

filam

ento

usw

ithag

e(1

0mm

)G

row

that

4–30

°Con

TSA,

MSA

-B,M

A22

16.

Col

onie

sro

und,

smoo

th,b

eige

,con

vex,

muc

oid,

1–2

mm

in5

days

at15

°C.G

row

thfa

ctor

sno

treq

uire

d.Ae

robi

c

++

Bioc

hem

set,

API2

0NE,

API-Z

YM

Pseu

doal

tero

mon

asfla

vipu

lchr

a(P

seud

oalte

rom

onas

aura

ntia

)

Neg

Stra

ight

rods

0.7–

1.5

×1.

5–4

mmO

nM

A22

16,c

olon

ies

1m

m,b

right

pale

yello

w24

h,23

°C.A

t5da

ys,o

rang

e-br

own

togr

een-

brow

n4

mm

.O

nM

SA-B

,brig

ht,m

ucoi

d,da

rk-b

row

nco

loni

es2

days

,the

nm

ediu

mtu

rns

blac

k,an

dha

emol

ysis

isse

en.P

oorg

row

thon

TSA

and

NA

even

with

2%N

aCl

++

Bioc

hem

set

Pseu

doal

tero

mon

asba

cter

ioly

tica

Neg

Rod

sw

ithro

unde

den

ds,

0.6–

0.9

×1.

9–2.

5mm

Gro

wth

onM

A22

16,r

edpi

gmen

tmay

orm

ayno

tbe

pres

ent

+w+

Bioc

hem

set,

API2

0NE

Pseu

doal

tero

mon

asci

trea

Neg

Stra

ight

rods

0.7–

1.5

×1.

5–4

mmbH sl

owO

nM

A22

16co

loni

es0.

5m

m24

hat

23°C

,br

ight

,whi

tish,

beco

min

g4

mm

and

lem

on-y

ello

wup

to4

days

.On

MSA

-B,

6–7

mm

brig

ht,m

ucoi

d,w

hitis

hco

loni

estu

rnin

gbl

ack

arou

ndth

eco

lony

afte

r2–3

days

.Rin

gof

haem

olys

isse

enaf

ter5

days

.Al

sogr

owth

onN

Aw

ithad

ded

NaC

l

++

Bioc

hem

set,

som

eAP

I-ZYM

reac

tions

Pseu

doal

tero

mon

asde

nitri

fican

sN

egR

ods

2–4

×0.

5–0.

7mm

On

MA

2216

,col

onie

sha

vepi

nkto

red

pigm

ent

−+

Bioc

hem

set

Pseu

doal

tero

mon

asel

yako

vii

Neg

Rod

sw

ithro

unde

den

ds,

0.5–

0.8

×1.

8–4.

0mm

Gro

wth

onM

A22

16as

beig

e-co

lour

edco

loni

es,r

ound

,circ

ular

,sm

ooth

,con

vex

++

Bioc

hem

set

cont

inue

d



104 Chapter 2

Pseu

doal

tero

mon

ases

pejia

naN

egSt

raig

htro

ds,0

.2–1

.0×

2.0–

3.5

mmG

row

thon

MA

2216

++

Bioc

hem

set

Pseu

doal

term

onas

flavi

pulc

hra

Neg

Rod

s,si

ngle

cells

,0.5

–1.5

mmG

row

thM

A22

16w

ithor

ange

colo

nies

++

Bioc

hem

set

Pseu

doal

tero

mon

aslu

teov

iola

cea

Neg

Gro

wth

onM

A22

16.C

olon

ies

3–5

mm

at4

days

at25

°C,r

egul

ar,c

onve

x,op

aque

,vi

olet

-col

oure

d

−+

Bioc

hem

set

Pseu

doal

term

onas

mar

ical

oris

Neg

Rod

s,si

ngle

cells

,0.

7–0.

9×

1.0–

1.2

mmG

row

thon

MA

2216

,as

roun

dco

loni

es2–

3m

m,c

ircul

ar,r

egul

ar,c

onve

x,tra

nslu

cent

,sm

ooth

,lem

on-y

ello

wpi

gmen

tat4

8h.

Opt

imum

25–3

5 °C

,0.5

–10%

NaC

l

++

Bioc

hem

set

Pseu

doal

tero

mon

aspi

scic

ida

Neg

Ovo

id,G

ram

-neg

ativ

ero

dsO

nM

A22

16co

loni

esar

e3–

6m

m,a

fter2

days

at28

°C.R

aise

dce

ntre

with

light

oran

geto

whi

tepi

gmen

tbec

omin

gda

rker

oran

geat

the

edge

s.W

ater

-sol

uble

pigm

entd

iffus

esin

toth

eag

ar

++

Bioc

hem

set,

carb

ohyd

rate

sus

ing

MO

F,AP

I50C

H

Pseu

doal

tero

mon

asru

bra

Neg

Stra

ight

orsl

ight

lycu

rved

rods

,2–

4×

0.8–

1.5

mmbH

Gro

wth

onM

A22

16br

ight

pink

ish-

whi

teat

2 4h

a t2 3

°C.C

entre

turn

sre

dan

dso

met

imes

blue

at4

days

,siz

e6–

7m

mw

ith2

or3

conc

entri

crin

gs.W

ithbl

ood

adde

dto

MA

2216

,col

onie

sar

ebr

ight

,muc

oid,

red

alm

ost

blac

kw

ithb-

haem

olys

is.C

olon

ies

prod

uce

adi

ffusi

ble

brow

npi

gmen

twith

anod

ouro

fhy

drog

ency

anid

e

++

Bioc

hem

set,

API-Z

YM

Pseu

doal

tero

mon

asul

vae

Neg

Rod

s1.

75–2

.5×

1–1.

5mm

Gro

wth

onTS

Aw

ith2%

NaC

l,M

SA-B

,MA

2216

.Col

onie

son

TSA

are

whi

te,w

here

ason

MA

2216

med

ium

colo

nies

are

purp

leat

48h,

23°C

++

Bioc

hem

set,

API2

0E

Pseu

doal

tero

mon

asun

dina

Neg

Cur

ved

rods

0.7–

0.9

×1.

8–3.

0mm

Gro

wth

onM

A22

16+

+Bi

oche

mse

t

Pseu

dom

onas

angu

illise

ptic

aN

egLo

ngro

ds,s

light

lycu

rved

,5–1

0mm

NH

NG

On

BA,T

SA,B

HIA

,NA

colo

nies

<1m

m,

roun

d,sh

iny,

pale

-gre

yat

4–7

days

.No

grow

thon

Pseu

dom

onas

isol

atio

nag

ar(D

ifco)

++

Bioc

hem

set

Bact

eriu

mG

ram

Mor

phol

ogy

bHTC

BSCo

lony

char

acte

ristic

sCa

tO

xId

entif

icat

ion

sets

Tab

le2.

4.C

ontin

ued.




Pseu

dom

onas

fluor

esce

nsN

egR

ods

Gro

wth

onBA

, lig

ht-g

rey

colo

nies

1.5

mm

24h

at25

°C.3

–5m

mat

48–7

2h

++

Bioc

hem

set,

API 2

0E

Pseu

dom

onas

plec

oglo

ssic

ida

Neg

Rod

s0.

5–1

×2.

5–4.

5mm

bHG

row

thon

TSA,

BA,M

SA-B

,25°

C+

+Bi

oche

mse

t,AP

I-ZYM

,API

20N

E

Pseu

dom

onas

pseu

doal

calig

enes

Neg

Shor

trod

sC

ream

-col

oure

dco

loni

es,‘

gum

my’

cons

iste

ncy

++

Bioc

hem

set

Pseu

dom

onas

stut

zeri

Neg

Stra

ight

and

slig

htly

curv

edro

ds,

som

ebi

pola

rsta

inin

gN

HN

GTh

ere

may

bero

ugh

and

smoo

thco

loni

es.

Col

onie

s0.

5m

m,g

rey,

wrin

kled

,dry

and

buff-

colo

ured

.May

besl

ight

lyye

llow

inco

lour

++

Bioc

hem

set

Ren

ibac

teriu

msa

lmon

inar

umPo

sSm

allb

acilli

,sin

gly

and

inpa

irs(0

.3–1

.5mm

)bH

NG

Col

onie

s2

mm

at20

days

at15

–18°

C,

smoo

th,c

ream

togr

anul

aron

KDM

2m

ediu

m.

On

cyst

eine

seru

mag

arco

loni

esar

eci

rcul

ar,

conv

ex,w

hite

tocr

eam

yye

llow

and

ofva

ryin

gsi

zes

On

Loef

flerc

oagu

late

dse

rum

acr

eam

ym

att

grow

this

seen

On

Dor

sete

ggm

ediu

m,g

row

thap

pear

sas

ara

ised

shin

yye

llow

laye

r

+−

Bioc

hem

set,

API-Z

YMH

aem

olys

isre

porte

dag

ains

tsa

lmon

idR

BCD

oes

notc

ross

-reac

twith

Lanc

efie

ldgr

oup

Gan

tiser

a.C

yste

ine

requ

ired

forg

row

th

Rho

doco

ccus

(lute

us)

fasc

ians

Pos

Stra

ight

orsl

ight

lycu

rved

rods

0.6–

1×

3–6

mmin

angu

laro

rpar

alle

lar

rang

emen

t

Wea

kgr

owth

onN

Aco

loni

esye

llow

,rai

sed,

glis

teni

ng,s

moo

th.G

row

thon

Low

enst

ein-

Jens

enm

ediu

mis

abun

dant

and

yello

w-o

rang

e

+Bi

oche

mse

t

( Rho

doco

ccus

mar

is)

Die

tzia

mar

isPo

sSh

orto

void

rods

0.6–

1×

1–2

mmW

eak

grow

thon

NA,

colo

nies

yello

w,r

aise

d,gl

iste

ning

,sm

ooth

+Bi

oche

mse

t

Rho

doco

ccus

spp.

Pos

Rod

s,sl

ight

lycl

ub-s

hape

d,2–

3×

0.6

mmG

row

thon

BAan

dM

SA-B

in3–

4da

ysat

25°C

.No

grow

that

37°C

(exc

eptf

orR

.equ

i).At

8da

ysco

loni

essl

ight

lydo

med

,rou

nd,

smoo

th,d

ry,d

eep

crea

my

yello

w

−−

Bioc

hem

set

Ros

eoba

cter

galla

ecie

nsis

Neg

Ovo

idro

d,0.

7–1

×1.

7–2.

5mm

On

MA

2216

at25

°C,c

olon

ies

are

circ

ular

0.5

mm

,sm

ooth

,con

vex,

brow

nish

colo

ur,

with

regu

lare

dges

.At7

days

,col

onie

sar

e2

mm

with

diffu

sibl

ebr

own

pigm

entp

rodu

ced

++

Bioc

hem

set

Ros

eoba

cter

spp.

CVS

PN

egR

ods,

0.25

×1.

0mm

Gro

wth

onSW

T,M

A22

16,1

mm

roun

d,no

n-m

ucoi

dat

5da

ys.A

ppea

ranc

eof

pink

pigm

enta

t7da

ys

w+

+Bi

oche

mse

t

cont

inue

d



106 Chapter 2

Sale

gent

ibac

ter

sale

gens

Neg

Rod

-sha

ped

cells

,sin

gly,

pairs

,oc

casi

onal

chai

nsG

row

thon

MA

2216

,NA,

TSA

++

Bioc

hem

set

Salin

ivib

rioco

stic

ola

Neg

Cur

ved

rods

0.5

×1.

5–3.

0mm

bHC

olon

ies

circ

ular

,con

vex,

crea

m-c

olou

red

at2

days

at37

°C.M

edia

with

0.5–

20%

NaC

l,M

A22

16,M

SA-B

++

Bioc

hem

set,

add

NaC

l

Serra

tiafo

ntic

ola

Neg

Rod

s0.

5×

3.0

mmG

r ow

t hon

NA

at30

and

37°C

+−

Bioc

hem

set,

API2

0E

Serra

tialiq

uefa

cien

sN

egR

ods

NH

NG

Gro

wth

onBA

,TSA

,MC

A,in

48h

at21

°C.

Non

-pig

men

ted

+−

Bioc

hem

set,

API2

0E

Serra

tiapl

ymut

hica

Neg

Rod

sN

HR

edco

loni

eson

TSA

24–4

8h

at22

°C+

−Bi

oche

mse

t,AP

I20E

Shew

anel

laal

gae

Neg

Shor

tstra

ight

rods

bHN

GC

olon

ies

yello

w-o

rang

eor

brow

naf

ter2

days

,37

°C.G

row

thon

SSag

ar,M

A22

16+

+Bi

oche

mse

t,AP

I20E

Shew

anel

laco

lwel

liana

Neg

Rod

1–3

mmlo

ng.O

nso

lidm

edia

and

inla

teph

ase

cells

beco

me

helic

al,

filam

ento

us–

20mm

NH

Col

onie

son

MA

2216

are

circ

ular

,1m

m,

conv

exw

ithun

dula

tem

argi

ns.A

t7da

ys,

5m

mw

ithirr

egul

arap

pear

ance

.In

brot

hcu

lture

(221

6)pr

oduc

esre

d-br

own

pigm

ent

++

Bioc

hem

set

Shew

anel

lafri

gidi

mar

ina

Neg

Rod

scu

rved

orst

raig

ht,

1–2.

5×

0.5–

0.8

mmG

row

thon

MA

2216

asta

n-pi

gmen

ted

colo

nies

,3–5

days

at10

°C.M

ucoi

dco

loni

esaf

ter1

0da

ysin

cuba

tion.

No

NaC

lre

quire

men

t.O

ptim

alte

mp

=20

–22 °

C

++

Bioc

hem

set,

API2

0E

Shew

anel

lage

lidim

arin

aN

egR

ods

curv

edor

stra

ight

,1.5

–2.5

mm,

sing

lyan

dpa

irsG

row

thon

MA

2216

asta

n-pi

gmen

ted

colo

nies

,3–5

days

at10

°C.M

ucoi

dco

loni

esaf

ter1

0da

ysin

cuba

tion.

NaC

lreq

uire

men

t.O

ptim

alte

mp

=15

–17 °

C

++

Bioc

hem

set,

API2

0E

Shew

anel

laja

poni

caN

egR

ods

1–2

×0.

6–0.

8mm

bHG

row

thon

MA

2216

asci

rcul

ar,s

moo

than

dsl

ight

lypi

nkco

loni

es,a

ndM

SA-B

at25

°C.

Deg

rade

sag

ar

++

Bioc

hem

set

Shew

anel

laon

eide

nsis

Neg

Rod

2–3

mm×

0.4–

0.7

mmN

HG

row

thon

MA

2216

,MSA

-B.P

ale

pink

tobe

ige-

colo

ured

colo

nies

,1–4

mm

,circ

ular

,sm

ooth

,con

vex

++

Bioc

hem

set

Shew

anel

lape

alea

naN

eg2.

0–3.

0×

0.4–

0.6

mmN

HG

row

thon

MA

2216

in2

days

at25

°C.

Col

onie

sop

aque

salm

on-c

olou

red

with

muc

oid

surfa

ce

++

Bioc

hem

set

Bact

eriu

mG

ram

Mor

phol

ogy

bHTC

BSCo

lony

char

acte

ristic

sCa

tO

xId

entif

icat

ion

sets

Tab

le2.

4.C

ontin

ued.




Shew

anel

lapu

trefa

cien

sN

egR

ods

NH

Are

ddis

h-br

own

orpi

nkpi

gmen

tis

prod

uced

++

Bioc

hem

set,

API-Z

YM,A

PI20

E,AP

I20N

E

Shew

anel

law

oody

iN

egN

on-s

poru

latin

gro

d0.

5–1.

0×

1.4–

2mm

NG

Gro

wth

onM

A22

16,a

spi

nk-o

rang

epi

gmen

tco

loni

esat

20°C

++

Bioc

hem

set,

API2

0E

Sphi

ngob

acte

rium

mul

tivor

umN

egN

on-s

porin

gro

ds1

mm,s

ingl

yor

pairs

Yello

wco

loni

eson

NA,

circ

ular

,low

,con

vex

++

Bioc

hem

set,

API-Z

YM

Sphi

ngob

acte

rium

spiri

tivor

umN

egN

on-s

porin

gro

ds1

mmN

HC

olon

ies

onN

A,ye

llow

,low

conv

ex,s

moo

th30

–37°

C+

+Bi

oche

mse

t,AP

I-ZYM

Sphi

ngom

onas

(Pse

udom

onas

)pa

ucim

obilis

Neg

Rod

s,0.

7×

1.4

mmN

HG

row

thon

NA

and

BA.Y

ello

w-p

igm

ente

dco

loni

esci

rcul

ar,l

ow,c

onve

xat

2da

ysat

22°C

++

Bioc

hem

set

Stap

hylo

cocc

usde

lphi

niPo

sC

occi

0.8–

1mm

,mos

tlyin

clus

ters

buta

lso

sing

lyan

dpa

ired

bHN

GO

nN

A,co

loni

es5–

7m

mci

rcul

ar,s

moo

th,

opaq

ueto

trans

luce

ntw

ithin

cuba

tion

+−

Bioc

hem

set.

Coa

gula

sete

st.

API-Z

YM

Stap

hylo

cocc

usho

min

isPo

sC

occi

inte

trads

,occ

asio

nally

pairs

NH

Col

onie

s3–

5m

m,s

moo

th,d

ull,

opaq

ue,

slig

htly

umbo

nate

with

beve

lled

edge

.Pi

gmen

tpal

eye

llow

togr

ey-w

hite

+Bi

oche

mse

t,C

oagu

lase

,D

Nas

e

Stap

hylo

cocc

uslu

trae

Pos

Coc

cisi

ngly

,pai

rsan

dcl

uste

rsbH

Col

onie

s1.

5–2

mm

,sm

ooth

,rou

nd24

h,37

°C+

−C

oagu

lase

test

,DN

ase

test

,Bi

oche

mse

t

Stap

hylo

cocc

usw

arne

riPo

sC

occi

0.5–

1.2

mmdi

amet

erin

pairs

and

sing

ly,o

ccas

iona

llyte

trads

NH

Col

ony

grow

thin

24h

at37

°C,3

–5m

m,

smoo

th,r

ound

,stic

ky.M

osts

train

sar

ebr

ight

yello

w-o

rang

eor

with

aye

llow

ring

arou

ndth

eed

geof

the

colo

ny.2

0%of

stra

ins

are

non-

pigm

ente

d

+Bi

oche

mse

t,C

oagu

lase

,D

Nas

e

Stap

pia

stel

lula

ta-

like

stra

inM

1N

egM

otile

rods

Gro

wth

onM

A22

16,S

WT

at23

°C.M

ucoi

dco

loni

esw

ithlig

ht-b

row

npi

gmen

tat3

days

.Po

orgr

owth

unde

rana

erob

icco

nditi

ons.

Form

sst

ar-s

hape

dag

greg

ates

whe

ngr

own

inliq

uid

med

ia

++

Bioc

hem

set,

API2

0NE

Stre

ptob

acillu

sm

onilif

orm

isN

egPl

eom

orph

icro

dsfre

quen

tlyin

chai

ns,t

angl

edfil

amen

tsw

ithbu

lbou

ssw

ellin

gs

NH

Cle

ar,n

on-h

aem

olyt

icco

loni

es,0

.5m

mat

4 8h

o nB A

inC

O2

and

37°C

.In

brot

hcu

lture

prod

uces

disc

rete

fluff-

ball-

like

colo

nies

−−

Bioc

hem

set

cont

inue

d



108 Chapter 2

Stre

ptob

acillu

sm

onilif

orm

is-li

keor

gani

sm.I

Dno

tco

nclu

sive

(Mah

eret

al.,

1995

)

Neg

Coc

coba

cilli

0.4–

0.6

mmin

tissu

es.

Old

ercu

lture

ssh

owfil

amen

tous

cells

with

poss

ible

bran

chin

gan

dso

me

term

inal

swel

lings

bHG

row

thon

BHIA

cont

aini

ngfe

talc

alfs

erum

and

NaC

l.C

olon

ies

0.1

mm

at4–

8da

ys,

aero

bica

llyat

15–2

2 °C

.Col

onie

sof

f-whi

te,

friab

le,c

onve

x,gr

anul

ar,‘

brea

d-cr

umb’

appe

aran

ce.O

lder

colo

nies

wer

eco

ncav

e‘m

olar

toot

h’ap

pear

ance

. bH

afte

r7–1

4da

ys

−−

Bioc

hem

set.

Wea

kly

acid

-fast

Stre

ptoc

occu

sag

alac

tiae

grou

pB

Pos

Coc

ciin

smal

lcha

ins

bHG

row

thon

BA.C

olon

ies

1m

m,p

ale

grey

at24

h,zo

neof

b-ha

emol

ysis

Bioc

hem

set,

API2

0St

rep,

APIr

apid

ID32

Stre

p,St

rep

grou

ping

antis

era

(gro

upB)

Stre

ptoc

occu

sag

alac

tiae

grou

pB,

Type

1b( S

.diff

icile

)

Pos

Coc

ciin

smal

lcha

ins

with

varia

ble

diam

eter

NH

Cel

lsad

here

stro

ngly

toag

ar.O

nBH

IAco

loni

es1

mm

at48

h.O

ptim

algr

owth

at30

°C.T

ests

mus

tbe

perfo

rmed

atth

iste

mpe

ratu

re.N

ogr

owth

at37

°Cun

less

grow

nin

mic

roae

roph

ilicat

mos

pher

e(5

%O

2,10

%C

O2,

85%

N2)

−St

rep

grou

ping

(gro

upB)

,AP

I20

Stre

p,AP

Irap

idID

32St

rep,

API5

0CH

,Bio

chem

set

Stre

ptoc

occu

sdy

sgal

actia

ess

p.dy

sgal

actia

ese

rova

rL

Pos

Coc

ciin

chai

nsbH

Col

onie

s1

mm

light

grey

onBA

with

zone

ofha

emol

ysis

at24

h−

APIr

apid

ID32

Stre

p.St

rept

exA-

G=

nega

tive.

Lanc

efie

ldgr

oup

L=

posi

tive,

Baci

traci

n=

sens

itive

Stre

ptoc

occu

ssp

ecie

s(G

roup

B)Po

sC

occi

inch

ains

bHN

G0.

5–1

mm

grey

-whi

te,z

one

ofha

emol

ysis

onBA

+Bi

oche

mse

t,AP

Irap

idID

32St

rep,

Stre

ptex

A-G

Stre

ptoc

occu

sin

iae

Pos

Coc

cus

0.3–

0.5

mm.l

ong

chai

nsbH

Gro

wth

onBA

,NA,

BHIA

,TSA

.Col

onie

son

BA1

mm

whi

te,u

mbo

nate

,opa

que

cent

resp

otat

24h.

b-ha

emol

ysis

surro

unde

dby

diffu

serin

gof

aha

emol

ysis

.Hae

mol

ysis

may

beva

riabl

e

−AP

Irap

idID

32St

rep,

API2

0St

rep,

API5

0CH

,Bio

chem

set.

Rea

ctio

nsat

25°C

a re

slow

erth

anat

37°C

Stre

ptoc

occu

spa

raub

eris

Pos

Shor

trod

sto

cocc

obac

illiin

pairs

orsh

ortc

hain

sa

HC

olon

ies

1.5–

2m

mat

24h,

roun

d,w

hitis

han

dsl

ight

lya

-hae

mol

ytic

−−

Bioc

hem

set,

API5

0CH

,API

rapi

dID

32St

rep.

Incu

bate

at37

°Cfo

r24

h

Stre

ptoc

occu

sph

ocae

Pos

Coc

ci1

mmin

diam

eter

,sin

gly,

pairs

orch

ains

bHO

nBA

colo

nies

circ

ular

,ent

ire,s

moo

th,

glis

teni

ngno

n-pi

gmen

ted.

0.8

mm

at24

h,37

°C

−Bi

oche

mse

t,AP

I20

Stre

p,AP

I50C

H

Stre

ptoc

occu

spo

rcin

usPo

sSp

heric

alto

ovoi

dce

llsin

smal

lch

ains

bHO

nBA

colo

nies

are

smal

l,el

evat

ed,e

ntire

and

haem

olyt

ic−

Bioc

hem

set,

API2

0St

rep,

API5

0CH

Bact

eriu

mG

ram

Mor

phol

ogy

bHTC

BSCo

lony

char

acte

ristic

sCa

tO

xId

entif

icat

ion

sets

Tab

le2.

4.C

ontin

ued.




Stre

ptom

yces

salm

onis

Pos

Myc

elia

ND

Bric

k-re

dto

oran

gesu

bstra

tem

ycel

iaan

dw

hite

topi

nkan

dye

llow

shad

esof

aeria

lm

ycel

ia

Bioc

hem

set

Tena

ciba

culu

m(F

lexi

bact

er)

mar

itim

um

Neg

Slen

der,

flexi

ble

rods

,0.5

×2–

30mm

,oc

casi

onal

lyup

to10

0mm

long

NG

On

AO-M

med

ium

,pal

eye

llow

oror

ange

,fla

t,th

inirr

egul

arco

loni

esw

ithun

even

edge

s.Sl

owsp

read

ing

grow

th,5

days

.Col

onie

sne

verl

arge

rtha

n5

mm

.Col

onie

sad

here

stro

ngly

toth

eag

ar.G

row

thva

riabl

eon

MA

2216

.Bro

wn

pigm

ento

nAO

-Mw

ith0.

5%ty

rosi

ne

V+

Bioc

hem

set,

API-Z

YM,A

PI20

E,AP

I50C

H

Tena

ciba

culu

m(F

lexi

bact

er)

ovol

ytic

um

Neg

Long

slen

derr

ods

0.4

×2–

20mm

NH

NG

Pale

yello

wco

loni

es,g

lidin

gm

otilit

y.O

nM

A22

16co

loni

eslig

htbr

owni

shye

llow

,fla

ttene

dan

del

evat

edw

ithre

gula

redg

es.R

apid

loss

ofvi

abilit

y,th

eref

ore

see

anar

eaof

lysi

sin

the

cent

reof

a5-

day-

old

colo

nyw

ithvi

able

cells

atth

eed

ge

++

Bioc

hem

set,

API-Z

YM

Vago

cocc

usfe

ssus

Pos

Cel

lsco

ccoi

d,el

onga

ted

indi

rect

ion

ofth

ech

ain,

sing

ly,p

airs

,sho

rtch

ains

aH

ND

Gro

wth

onBA

,37°

C,5

%C

O2,

s ma l

laH

colo

nies

.Cel

lsm

otile

−Bi

oche

mse

t,AP

Irap

idID

32St

rep,

API-Z

YM

Vago

cocc

usflu

vial

isPo

sC

ells

ovoi

d,el

onga

ted

indi

rect

ion

ofth

ech

ain,

sing

ly,p

airs

,sho

rtch

ains

aH

ND

On

BA,c

olon

ies

slig

htly

larg

erth

anEn

tero

cocc

ussp

ecie

san

da

H.I

mpr

oved

grow

thin

CO

2

−Bi

oche

mse

t,AP

Irap

idID

32St

rep,

API-Z

YM,L

ance

field

grou

pN

=po

sitiv

e

Vago

cocc

uslu

trae

Pos

Coc

cus,

sing

lean

din

chai

ns.C

ell

slig

htly

elon

gate

da

HSm

all0

.1–0

.2sm

ooth

colo

nies

onBA

afte

r2 4

ha t

3 7°C

and

5%C

O2.

Mot

ile−

Bioc

hem

set,

APIr

apid

ID32

Stre

p,AP

I-ZYM

Vago

cocc

ussa

lmon

inar

umPo

sC

occo

baci

lli.Fo

rms

chai

nsin

liqui

dm

edia

aH

NG

Col

onie

s0.

5–1

mm

,whi

te-g

rey,

glis

teni

ng.

Stre

p-lik

e.Sl

ight

gree

ning

ofag

araf

ter2

–3da

ys

−N

DBi

oche

mse

t,AP

I20

Stre

p,AP

Irap

idID

32St

rep,

API

50C

H,S

trep

grou

ping

Varra

calb

mi

Neg

Slen

der,

stra

ight

rods

1.7–

3.5

mmN

egor

aH

NG

1m

mop

aque

,gre

y,co

nvex

,adh

eren

tco

loni

esw

hich

leav

ean

impr

inti

nth

eag

ar.

aH

seen

afte

r1w

eek

−+

Bioc

hem

set

Vibr

ioae

roge

nes

Neg

Stra

ight

tosl

ight

lycu

rved

rods

0.6–

0.8

×2–

3mm

Col

onie

sfla

t,ci

rcul

ar,o

ff-w

hite

afte

r2da

yson

PYag

ar+

−Bi

oche

mse

t

Vibr

ioae

stua

rianu

sN

egSt

raig

htor

curv

edro

d,0.

5×

1.5–

2mm

bHY

Gro

wth

onM

SA,T

SAco

ntai

ning

0.5%

NaC

lBi

oche

mse

t.Ad

ditio

nof

NaC

lto

set,

optio

nal co

ntin

ued



110 Chapter 2

Vibr

ioag

ariv

oran

sN

egR

od2–

4×

0.4–

0.6

mmG

Non

-pig

men

ted

colo

nies

onM

SA-B

and

MA

2216

that

prod

uce

ash

allo

wpi

tin

the

med

ium

++

Bioc

hem

set,

API2

0E,

API-Z

YM.A

ddN

aClt

ose

t

Vibr

ioal

gino

lytic

usN

egN

HY

Gre

yco

loni

es.S

war

mac

ross

MSA

-Ban

dM

A22

16in

24h

at25

°C+

+Bi

oche

mse

t,AP

I20E

,AP

I-ZYM

,API

20N

E.Ad

dN

aClt

ose

ts

Vibr

iobr

asilie

nsis

Neg

Rod

,2.5

–3×

1mm

YG

row

thon

TSA+

2%N

aCl.

Col

onie

sbe

ige,

trans

luce

nt,c

onve

x,ro

und,

smoo

th,2

–3m

maf

t er 4

8h

at28

°C

++

Bioc

hem

set,

API2

0E,A

PIZY

M

Vibr

ioca

lvie

nsis

Neg

Slig

htly

curv

edor

stra

ight

rods

.0.

25–1

.0×

0.75

–2.5

mmG

Gro

wth

onM

A22

16.C

olon

ies

brow

nish

,tra

nslu

cent

,circ

ular

,sm

ooth

,con

vex,

entir

eat

25–3

0°C

.No

grow

that

37°C

++

Bioc

hem

set,

API-Z

YM.A

ddN

aClt

ose

t

Vibr

ioch

oler

aeN

egSl

ight

lycu

rved

rods

W, b

HY

Col

onie

sro

und

smoo

th2–

3m

mgr

eeni

sh-g

rey

onM

SA-B

++

Bioc

hem

set,

API2

0E,

API-Z

YM,A

PI20

NE

Vibr

ioch

oler

ae01

39N

egC

urve

dro

dsbH

YC

olon

ies

grey

ish-

opaq

uew

ithda

rker

cent

res

+Bi

oche

mse

t.R

esis

tant

tovi

brio

stat

icco

mpo

und

0/12

9

Vibr

ioci

ncin

natie

nsis

Neg

Rod

0.7–

2mm

YC

olon

ies

1–2

mm

crea

m,r

ound

,sm

ooth

,gl

ossy

,24

hat

25an

d35

°C+

+Bi

oche

mse

t.Ad

dN

aClt

ose

t

Vibr

ioco

rallii

lytic

usN

egR

ods

1.2–

1.5

×0.

8mm

YO

nM

A22

16,c

olon

ies

are

3m

mat

3da

ys,

crea

m-b

eige

,rou

nd,e

ntire

,sm

ooth

++

Bioc

hem

set,

API2

0NE.

Add

NaC

lto

sets

(3%

)

Vibr

iocy

clitr

ophi

cus

Neg

Rod

s0.

6×

1.5–

2.5

mmC

olon

ies

4m

m,c

ream

-col

oure

d,ci

rcul

ar,f

lat

onM

A22

16+

+Bi

oche

mse

t.Ad

dN

aClt

ose

t

Vibr

iodi

abol

icus

Neg

Stra

ight

rod

0.8

×2

mmY

Non

-pig

men

ted,

2m

mat

3da

yson

MA

2216

and

swar

min

g(9

mm

with

adde

dgl

ucos

e)+

+Bi

oche

mse

t,AP

I20E

,API

20N

E,AP

I50C

H,A

PI-Z

YM.

A dd

Na C

lto

s ets

Vibr

iodi

azot

roph

icus

Neg

Shor

trod

s,0.

5×

1.5–

2mm

NH

YC

olon

ies

onM

A22

16ar

efla

t,ci

rcul

arof

f-whi

te+

+Bi

oche

mse

ts.A

dditi

onof

NaC

lto

set,

optio

nal

Vibr

io(P

hoto

bact

eriu

m)

fisch

eri

Neg

Rod

s0.

5×

1–1.

5mm

.Sin

gly

orpa

irs,

roun

ded

ends

,stra

ight

orcu

rved

bH VN

Gor

GG

rey

orof

f-whi

te,t

rans

luce

ntco

loni

eson

MSA

-B,1

–2m

m.B

ecom

epa

leye

llow

,and

lum

inou

sat

3da

ys

++

Bioc

hem

set,

API-Z

YM,A

PI20

E,AP

I50C

H.A

dditi

onof

NaC

lto

set,

optio

nal

Vibr

ioflu

vial

isN

egSh

ortr

ods,

stra

ight

orcu

rved

,sin

gly

orpa

irs,m

aybe

pleo

mor

phic

VY

Col

onie

son

BHIA

are

opaq

ue,s

hiny

smoo

th,

roun

d,do

omed

,may

bem

ucoi

dan

dar

e2–

3m

mat

24h,

30°C

.Gro

wth

onBH

IA,

MSA

-B

++

Bioc

hem

set,

API2

0E.A

ddN

aClt

ose

ts

Bact

eriu

mG

ram

Mor

phol

ogy

bHTC

BSCo

lony

char

acte

ristic

sCa

tO

xId

entif

icat

ion

sets

Tab

le2.

4.C

ontin

ued.




Vibr

iofu

rnis

sii

Neg

Stra

ight

tolig

htly

curv

edro

dV

YC

olon

ies

onBH

IAar

eop

aque

,shi

nysm

ooth

,ro

und,

doom

ed,m

aybe

muc

oid

and

are

2–3

mm

at24

h,30

°C.G

row

thon

BHIA

,BA,

MSA

-B

++

Bioc

hem

set,

API2

0E,

API-Z

YM.A

ddN

aClt

ose

ts

Vibr

ioha

liotic

oli

Neg

Rod

s,0.

6–0.

8×

1.7–

2.0

mmN

DG

On

MA

2216

,bei

ge,c

ircul

ar,s

moo

th,c

onve

xco

loni

es+

+Bi

oche

mse

t.Ad

dN

aClt

ose

t

Vibr

ioha

rvey

iN

egSh

ortr

od,s

traig

htor

slig

htly

curv

ed,

roun

ded

ends

occu

rring

sing

lyor

inpa

irs

NH

YG

rey-

colo

ured

,off-

whi

te,r

aise

dsh

iny,

slow

spre

adin

ggr

owth

onM

SA-B

,may

bem

ucoi

d.Lu

min

ous

afte

r3da

ys

++

Bioc

hem

set,

API-Z

YM,A

PI20

E,AP

I20N

E,AP

I50C

H.

Add

NaC

l to

set

Vibr

ioho

llisae

Neg

Rod

,som

esl

ight

lycu

rved

W bHN

Gor

wea

kO

nBA

and

MSA

-B,c

olon

ies

1–2

mm

opaq

ue.

Hae

mol

ysis

afte

r7da

ys+

Bioc

hem

set.

Add

NaC

lto

set

Vibr

ioic

hthy

oent

eri

Neg

Shor

trod

s1.

6–2.

5×

0.6–

0.8

mmY,

wN

on-p

igm

ente

dco

loni

es+

+Bi

oche

mse

t.Ad

dN

aClt

ose

t

Vibr

iole

ntus

Neg

1.5–

3×

0.8–

1mm

GC

olon

ies

onM

A22

16at

24h

at22

°Ca r

e0.

3–0.

5m

m,r

ound

,opa

que,

non-

pigm

ente

d+

+Bi

oche

mse

t.Ad

dN

aClt

ose

t

Vibr

io(P

hoto

bact

eriu

m)

loge

i

Neg

NH

Yello

w-o

paqu

eco

loni

eson

MSA

-B+

+Bi

oche

mse

t,AP

I-ZYM

.Ad

ditio

nof

NaC

lto

sets

,op

tiona

l

Vibr

iom

edite

rrane

iN

egR

ods,

1–2

×0.

5mm

NH

YC

olon

ies

onm

arin

eag

arci

rcul

ar,t

rans

luce

nt,

non-

pigm

ente

d.O

nM

SA-B

,col

onie

sar

e2–

3m

mat

48h,

crea

my

and

muc

oid

++

Bioc

hem

set.

Add

NaC

lto

set

Vibr

iom

etsc

hnik

ovii

Neg

Shor

trod

s,cu

rved

orst

raig

ht,s

ingl

y,pa

irsor

shor

tcha

ins,

0.5

×1.

5–2.

5mm

bHY

orN

G+

−Bi

oche

mse

t.Ad

ditio

nof

NaC

lto

sets

,opt

iona

l

Vibr

iom

imic

usN

egC

urve

dro

dsbH

GC

olon

ies

roun

d,sm

ooth

,2–3

mm

gree

nish

-gre

yon

MSA

-Ban

dBA

++

Bioc

hem

set,

API2

0E,A

PI20

NE,

API-Z

YM.A

dditi

onof

NaC

l,op

tiona

l

Vibr

iom

ytili

Neg

Coc

coba

cilli.

YG

row

thM

A22

16,T

SAin

24h.

Col

onie

sro

und

and

non-

pigm

ente

d+

+Bi

oche

mse

t,AP

I-ZYM

,API

20E,

API2

0NE,

API5

0CH

.A d

dN

a Clt

os e

ts

Vibr

iona

varre

nsis

Neg

Rod

s1–

2×

0.8–

1mm

YC

olon

ies

2–3

mm

roun

dop

aque

non-

pigm

ente

d24

hon

NA

with

2%N

aCl

++

Bioc

hem

set,

API2

0NE,

API

20E.

Add

NaC

lto

sets

Vibr

ione

ptun

ius

Neg

Slig

htly

curv

edro

d,2.

3–3

×1

mmY

Gro

wth

onTS

A+

2%N

aCl.

Smoo

th,r

ound

ed,

beig

eco

loni

es3

mm

at48

han

d28

°C+

+Bi

oche

mse

t,AP

I20E

,API

ZYM

.Add

NaC

lto

sets co

ntin

ued



112 Chapter 2

Vibr

ioor

dalii

Neg

2.5–

3×

1mm

,cur

ved

bHN

GC

ircul

arco

nvex

off-w

hite

togr

eyco

loni

es,

1–2

mm

.4–6

days

,22°

C+

+Bi

oche

mse

t,AP

I-ZYM

,API

20E.

Add

NaC

lto

sets

Vibr

ioor

ient

alis

Neg

NH

YW

hite

-opa

que

colo

nies

onM

SA-B

++

Bioc

hem

set,

API-Z

YM.A

ddN

aClt

ose

ts

Vibr

iopa

cini

iN

egR

ods

YN

on-p

igm

ente

d,tra

nslu

cent

colo

nies

onM

A22

16Bi

oche

mse

t,AP

I20E

Vibr

iopa

raha

emol

ytic

usN

egPl

eom

orph

icw

ithst

raig

htor

curv

edro

dsbH

GG

rey

colo

nies

onBA

,MSA

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reaction, oxidase and catalase. The ‘Identifica-tion sets’ column lists the identification sets thatcan be performed for identification of thatorganism.

2.4 Biochemical Identification Tests

Biochemical identification of a bacteriumproceeds through a number of steps. A puresubculture of the organism is used to performprimary identification tests and to inoculatebiochemical identification sets (secondaryidentification tests), composed of either in-houseprepared media, or commercial identificationsets such as API, available from bioMérieux.The identification sets are incubated at theappropriate temperature and for the appropriatetime for reactions to occur. The results arerecorded on a laboratory work sheet or, in thecase of the commercial identification kits, onthe supplied recording sheets. See Chapter 3for the interpretation of biochemical identificationsets. The media and reagents used in the bio-chemical identification are detailed in Chapter 7,along with information on growth characteristicsand reagent reactions.

The tests described in this chapter and Table3.1 form the ‘Biochem set’.

Primary tests

The primary tests include microscopicexamination of smears, in particular the Gramstain, catalase, oxidase, presence of haemolysis,motility, and growth on MCA. More informationon the performance of these tests and theirinterpretation is found in Chapter 3, and methodsof media and reagent preparation are describedin Chapter 7.

Secondary tests: biochemicalidentification sets

Biochemical identification is achieved bysecondary identification tests, which are themain tests used to identify an organism to specieslevel.

Tubed media

CARBOHYDRATE FERMENTATION. L-arabinose,glucose, inositol, lactose, maltose, mannitol,mannose, salicin, sorbitol, sucrose, trehalose,xylose. The carbohydrates are commonly called‘sugars’.

DECARBOXYLASES. ADH, LDC, ODC, decar-boxylase control tube.

OTHER BIOCHEMICAL TESTS. Aesculin, motility,MRVP, nitrate, oxidative fermentative tubes,ONPG, TSI for H2S, indole, urea. See next sectionfor inoculation methods.

GROWTH AT DIFFERENT TEMPERATURES. U s eeither a TSB or glucose tube and incubate at 37°C,40°C or desired test temperature.

GROWTH IN 10% NaCL. To test an organism’sability to grow in the presence of 10% NaCl, dis-pense equal volumes of TSB and 20% NaCl into asterile McCartney bottle or bijou bottle to give afinal concentration of 10% NaCl. Inoculate withthe organism to a concentration of McFarlandopacity tube 1. Incubate at 25°C or optimumtemperature and after 24 h incubation observe forevidence of growth as seen by an increase in theturbidity of the broth.

Plate media

DNase plate, Gelatin/salt plate, MCA, TCBS. Usea MSA-B or BA plate for purity check of theinoculum.

Discs

Ampicillin 10 mg, ‘Vibrio discs’ 0129 10 mg and150 mg. The discs are placed on a lawn inoculumof the bacterium on either a BA or MSA-B plateaccording to the organism’s growth requirements.The inoculum should be prepared to a density ofMcFarland tube 1. Use for Listonella, Moritella,Photobacterium, Vibrio and Aeromonas species.The Vibrio discs (vibrio static agent pteridine0/129) differentiates between Vibrio species andAeromonas species, the latter being resistant toboth concentrations. A zone of 9 mm is classifiedas susceptible for the 0/129 150 mg disc(Bernardet and Grimont, 1989). Also seeChapter 7, ‘Preparation of Media for Cultureand Identification’.

114 Chapter 2



2.5 Inoculation of BiochemicalIdentification Sets

Examine the subculture plate and, if the growth ispure, use to inoculate biochemical identificationsets. For marine samples, NaCl needs to beadded to the set at a final concentration of 2%.Many enzymes from a variety of Vibrio specieswill not be active at lower NaCl concentrationsand false negative results will be obtained. Thismay apply to indole, VP, ADH, ODC and LDCreactions. Some tests need a heavy inoculum.These are the decarboxylases and ADH, ureaand citrate. Incubate the set at the appropriatetemperature for a minimum of 48 h. ODC, LDC,ADH, MR, VP, and indole usually require48 h incubation as a minimum for a positivereaction even if reasonable growth is seen inthe tubes.

To inoculate the tubes, plunge a sterilePasteur pipette three-quarters of the way into thetubed media, releasing 3–4 drops of inoculum asthe pipette is withdrawn.

After inoculating all the tubes, place one dropof the inoculum on to a purity plate and streak outfor isolated colonies. After suitable incubation,check that the growth is pure.

Freshwater isolates

For bacteria isolated from freshwater sources,inoculate bacterium into sterile normal saline orsterile distilled water (usually 10 ml) to opacityMcFarland 3. Add 3–5 drops of inoculum to eachtube. Some media require a heavy inoculum(opacity approx McFarland 6). These areaesculin, ADH, LDC, ODC, the decarboxylasecontrol, MRVP, citrate and urea.

Marine isolates and addition of NaCl

Fish organs are at physiological saline level.Therefore, be aware that some bacteria mayadapt to this NaCl concentration. By subculturingan organism to BA and MSA-B, or culturing to a0% and 3% salt plate, the salt requirement orpreference can be determined. However, manyof these organisms produce more accurate results

of biochemical tests when the NaCl is added tothe media, usually at a final concentration of 2%.Therefore check the optimal NaCl concentrationrange of the suspect organism from the NaClcolumn in the tables for biochemical results(Tables 4.1 to 4.22).

For bacteria isolated from the marineenvironment, the optimal final salt concentrationin the medium is all-important. Insufficient saltconcentration will lead to false negative resultseven though good growth may be seen in the tube.An example of this is V. parahaemolyticus, whichwill give a negative indole result when aninoculum of physiological saline is used, but apositive result when a final concentration of 2%NaCl is achieved in the tryptone water (indole test)medium (See photographic section).

Tubed media can be prepared with orwithout added NaCl. However, a laboratory maywant to keep media preparation to a minimumand prepare all media without additional NaCl. Atthe time of inoculation, 500 ml of a 20% sterileNaCl stock solution is added to all liquid media(5 ml) (aesculin, indole test, ADH, ODC, LDC,nitrate, MRVP). A sterile 20% salt stock solutioncan be prepared in a Schott bottle to which anautoclavable 2 ml volumetric dispenser is added,and set to 500 ml. An example of a volumetricdispenser is a Socorex Calibrex 520, 2 ml with0.05 ml divisions.

Paraffin oil overlay

ADH, LDC, ODC and control and one tubeof the OF media is overlaid with sterile paraffinoil, to approximately 5 mm depth. For easeof use, paraffin oil can be sterilized in a Schottbottle to which a 2 ml volumetric dispenser isadded (Socorex, Calibrex 520, 2 ml with 0.05 mldivisions).

Incubation

Plate and tubed media are incubated at theappropriate temperature and duration for theappropriate bacterium; 25°C and 2–5 daysis used as a general rule. Refer to incubationguidelines in Table 2.3.




2.6 API Identification Systems

API 20E bioMérieux, Marcyl’Etoile, France

API 20NE bioMérieux, Marcyl’Etoile, France

API 50CH bioMérieux, Marcyl’Etoile, France

API rapid A bioMérieux, Marcyl’Etoile, France

API rapid ID 32 STREP bioMérieux, Marcyl’Etoile, France

API 20 Strep bioMérieux, Marcyl’Etoile, France

API Coryne bioMérieux, Marcyl’Etoile, France

API ZYM bioMérieux, Marcyl’Etoile, France

Identification using API 20E

There have been a number of reports in theliterature about the failure of the API 20E systemto identify many Aeromonas and Vibrio species(Santos et al., 1993). Some of this is due in partto the lack of such information in the API data-base. However, it is well known that differencesin reactions occur between the API system andconventional biochemical tests. This is especiallytrue of the decarboxylases, citrate, urea, indoleand VP. Where such differences have beenreported in the literature, these have beenindicated in the conventional tables (Biochemset). When using the biochemical identification

tables in this book, make sure the correctdatabase is referred to, i.e. the conventional data-base (Tables 4.1 to 4.22), or the API 20Edatabase (Tables 4.23 to 4.25).

MacDonell et al. (1982) recommends usinga diluent containing 20‰ salt. They usedmarine salts mix (Instant Ocean) purchasedfrom Aquarium Systems, Mentor, Ohio, which iscomposed of marine salts with salinities adjustedto 20‰. Kent (1982) recommended suspendingbacterial cells in 50% sterile artificial seawater forinoculating the API 20E strips. Artificial seawatersalts can also be purchased from Sigma (seeChapter 7, ‘Preparation of Media for CulturalIdentification’). Alternatively, an inoculum with2% sterile NaCl works well for the commonlyisolated Vibrio, Photobacterium and Listonellaspecies. The optimal NaCl concentration isextremely important for tests such as citrate,urea, MR, VP and indole. For example,V. parahaemolyticus gives a negative result forindole when an inoculum of 0.85% NaCl is used,but a positive indole result is achieved when 2%NaCl is used.

Adjust the suspension of cells to an opacityof McFarland number 1 and inoculate the set.It is recommended that the API 20E system isincubated at 25°C for 48 h. The sugars are read at24 h and the remaining tests are read at 48 h. Thedecarboxylases (LDC, ODC) and ADH reactionsmay not be detected until 48 h. As recommendedby the manufacturer, a negative nitrate reactionshould always have zinc dust added to the reactioncupule to determine a true negative result (seeTable 3.1). See Chapter 4 for interpretation andresults from the API kits.

116 Chapter 2



3Interpretation of Biochemical Identification Tests and Sets

3.1 Conventional Media: ‘Biochem Set’

Positive sugar fermentation results may berecorded at 24 h. All liquid media must be incu-bated for at least 48 h, and sometimes longer, forpositive results to be achieved. This applies inparticular to ADH, LDC, ODC, aesculin, citrateand MRVP. For interpretation see Table 3.1.

3.2 Identification Tests and theirInterpretation

This section contains notes on the interpretationof identification tests and any problems that mayoccur. A description of each test is also found inChapter 7 ‘Preparation of Media for Culture andIdentification’.

Aeromonas salmonicida fluorescent antibodytest (FAT)

A FAT for the Australian strain of Aeromonassalmonicida has been noted to cross-react withAeromonas sobria.

Aesculin

A positive result is recorded when the colouris pitch-black and when half or more of thetube is blackened (MacFaddin, 1980). Shades ofgrey should not be recorded as positive. Resultsshould be read up to 3 days. Some organisms will

not grow in the aesculin medium despite theoptimal salt concentration. These are Listonellaanguillarum, L. pelagia and Vibrio ordalii. Theseorganisms are all negative for aesculin production.

Some bacteria have been reported toproduce melanin (Coyne and Al-Harthi, 1992)and in the aesculin medium will cause blackeningof the medium (Choopun et al., 2002). Therefore,it is recommended that an aesculin test showingblackening of the medium be tested for truehydrolysis of aesculin by determining the loss offluorescence in long-wave UV light at 354 nm.Aesculin will fluoresce and therefore the presenceof fluorescence will indicate that the aesculin hasnot been hydrolysed (MacFaddin, 1980).

Some plastic tubes are UV-opaque; there-fore, test for fluorescence by holding a Wood’s UVlamp over the top of an opened tube, or pour thecontents into a Petri dish to observe presence orabsence of fluorescence. When using UV light,protect the eyes with UV-opaque glasses.

ADH

Some strains of Vibrio may show contradictoryresults for ADH when using Møller’s or Thornley’smedium. These are V. mediterranei, V. mytili,V. orientalis, most strains of V. splendidus I, andsome strains of V. tubiashii. They are all positiveaccording to Thornley’s method, but negative inMøller’s ADH. The glucose in Møller’s mediumappears to inhibit the reaction due to cataboliterepression of the inducible ADH system (Maciánet al., 1996). Photobacterium species were found




118 Chapter 3

Test

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Interpretation of Biochemical Identification Tests 119

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to produce alkaline products in Thornley’smedium, yet none possessed a constitutive ADHsystem when tested with more sensitive analyticalmethods (Baumann et al., 1971; West andColwell, 1984). Other Vibrio species such asListonella (Vibrio) anguillarum, Photobacterium(Vibrio) damselae ssp. damselae, V. fluvialisand V. furnissii are positive for ADH byboth methods. Thornley’s method is therecommended method for Vibrio species (Maciánet al., 1996).

Carbohydrate fermentation

In some reports in the literature, the termsutilization and fermentation have been usedinterchangeably and it has been difficult toascertain which result was reported from whichtest method. Fermentation should be used todescribe a result from the breakdown of acarbohydrate or ‘sugar’ reaction such as used inthe conventional biochemical media and the API20E. A pH indicator in the medium is able todetect the acid change caused by the breakdownproducts. Utilization should only refer to tests thatdetermine an organism’s ability to use a sub-stance as a sole carbon source. There is usuallyno pH indicator in the medium and growth isobserved as an increase in opacity or turbidityof the media. The exception is Simmons citratemedium, which determines an organism’s abilityto use citrate as a sole carbon source. There areno other nutrients in this medium. On the otherhand, Christensen’s citrate method containsother nutrients and is not a utilization test forcitrate as a sole carbon source (Cowan and Steel,1970; MacFaddin, 1980).

Carotenoid pigment detection

A few drops of 0.01% aqueous CR (Congo Red)is placed on a few isolated colonies growing ona medium such as AO agar. After 2 min thecolonies are rinsed with water. The coloniesdevelop a red colour if the test is positive and thecolour can last for a number of hours. CR detectsthe presence of extracellular galactosamineglycan (Johnson and Chilton, 1966, citing, E.J.Ordal, personal communication).

Catalase

A loopful of bacterial growth is removed from theculture plate and smeared to a glass slide. A dropof 30% hydrogen peroxide is placed on to thebacterial cells and the appearance of bubblesindicates a positive test. When removing cellsfrom an agar medium that contains blood, caremust be taken not to transfer any of the blood-containing medium with the bacterial growth, asthe presence of blood may lead to a false-positiveresult.

CR

Used for the detection of carotenoid pigment.See under carotenoid pigment detection.

Decarboxylases

ADH, ODC and LDC should be inoculated with aheavy inoculum of the organism. Incubate for aminimum of 2 days and maximum of 14 days.Add salt to give a final concentration of 2% formarine organisms. Without NaCl many tests mayproduce a false-negative reaction.

Flexirubin pigment

Flexirubin pigment may be produced by someof the members of the Flavobacteriaceae family.For its detection, growth is taken from a cultureplate (usually AO medium) and smeared toa glass slide, which is placed on a white back-ground such as a piece of paper. The colonymass is flooded with 20% KOH and examined foran immediate colour change to reddish purple orbrown. It may be helpful to place two bacterialmasses on the glass slide, one that is floodedwith KOH and the other to act as a controlfor colour differentiation. The KOH can also beused directly on the colony growth on a cultureplate; however, the colour change may notbe noticeable if there is only a thin layer ofgrowth (Bernardet et al., 2002). A reversion tothe original colour occurs when the bacterial cellsare flooded with an acidic solution (Reichenbachet al., 1989).

120 Chapter 3



Gliding motility

The demonstration of gliding motility of theCytophaga–Flavobacterium–Bacteroides groupmay be difficult as much by choice of culturetechnique and as by the definition of glidingmotility. The organism needs to be grown ona low-nutrient-concentration agar such as thatof AO, which contains 1.5% w/v agar. Glidingis affected by the amount of surface moistureand best results are obtained with freshly pouredplates and incubation in a humid atmosphere.Gliding is observed by direct microscopic obser-vation of the swarming edge of bacterial growthon a thinly poured plate after overnight incuba-tion. A high-powered dry lens is used. Glidingis defined as ‘movement that is continuous andregularly follows the long axis of the cells thatare predominantly organized in bundles duringmovement’. It should be noted that spreadingcolonies are not necessarily an indication ofgliding motility, as spreading may result fromother forms of surface translocation mechanisms(Henrichsen, 1972). Gliding can usually besuspected if a colony has a rhizoid shape onAO agar; however, this will not be seen if theagar surface is too dry. Nor will gliding motilitybe seen if bacterial growth is suspended insaline. Gliding motility can also be observed ina hanging drop prepared from a liquid culture(Bernardet et al., 2002).

Haemolysis

Haemolysis is observed on BA. A clear zoneof lysis of the red cells around a bacterial colonyis referred to a b-haemolysis (bH). A greenishtinge seen around some colonies such assome of the Streptococci species, is referredto as a-haemolysis (aH). Streptococcus iniaeshows complete b-haemolysis on sheep bloodagar, but only partial haemolysis when themedium is supplemented with human or bovineblood.

Some Vibrio species are haemolytic onBA without salt, but non-haemolytic on salt-containing media such as MSA-B, even thoughtheir preferred growth medium is with Na ions. Itis suggested that haemolysins may be producedwhen the organism is under stress.

Indole

A false-negative result may be recorded forsalt-requiring organisms if there is an insufficientfinal concentration of NaCl in the test medium.Growth may be seen in the test medium;however, the organism may not express theenzyme unless the NaCl concentration is optimal.To achieve a 2% NaCl final concentration that isoptimal for the majority of marine organisms, add500 ml of 20% NaCl stock solution to 5 ml of testmedium. Refer also to section 2.5 (Inoculation ofBiochemical Identification Sets).

KOH

Used for the detection of flexirubin pigment.See under flexirubin pigment.

Luminescence

Luminescence may be detected by growing theorganism on an appropriate growth medium.Nutrient broth No. 2 (Oxoid) (25 g), NaCl(17.5 g), KCl (1.0 g), MgCl2.6H2O (4.0 g), agar(12.0 g), distilled water (1000 ml) (Furniss et al.,1978). However, reliance on luminescence needsto be treated with caution, as expression of lumi-nescence appears to be dependent on a numberof factors, including media, and ideally should bemeasured with a luminometer rather than by eye(J. Carson, Department of Primary Industries,Water and Environment, Tasmania, 2003, per-sonal communication). Luminescence is optimalafter 18–24 h of incubation at 25°C. It is alsodependent upon aerobic conditions, so that brothcultures need to be shaken to aerate the mediumbefore luminescence can be detected. Lumines-cence is detected by observing the plates or brothculture in a dark room and allowing the eyes toadjust to the dark for 5 min. However, if possible,luminescence should be measured from a brothculture using an instrument such as a WallacMicrobeta Plus liquid scintillation counter, whichmeasures relative light units (Manefield et al.,2000).

Organisms that are positive for luminescenceinclude Photobacterium leiognathi, P. phos-phoreum, Vibrio fischeri, V. logei, V. orientalis,




V. splendidus biovar I (Furniss et al., 1978; Lunderet al., 2000).

Organisms that are negative for luminescenceinclude Aeromonas salmonicida, Enterovibrionorvegicus, Listonella anguillarum, L. pelagia,Moritella marina, M. viscosa, Photobacteriumangustum, Photobacterium damselae ssp.damselae, Plesiomonas shigelloides, Vibrioagarivorans, V. alginolyticus, V. brasiliensis,V. calviensis, V. campbellii, V. coralliilyticus, V.diazotrophicus, V. fluvialis, V. furnissii, V.gazogenes, V. halioticoli, V. ichthyoenteri,V. lentus, V. metschnikovii, V. natriegens, V.navarrensis, V. neptunius, V. nereis,V. nigripulchritudo, V. ordalii, V. pacinii, V.parahaemolyticus, V. penaeicida, V. proteolyticus,V. rotiferianus, V. salmonicida, V. splendidusbiovar II, V. tapetis, V. tubiashii, V. vulnificus,V. wodanis and V. xuii (Furniss et al., 1978;Lunder et al., 2000; Gomez-Gil et al., 2003a,b).Vibrio fischeri NCMB 1281T was reported tobe negative for luminescence (Lunder et al.,2000).

Strains of V. cholerae, V. logei, V. salmon-icida and V. harveyi are variable for luminescence(Furniss et al., 1978; Lunder et al., 2000).

MR

The MRVP medium (Difco) should be inoculatedwith a heavy inoculum of the organism. Saltneeds to be added if required as a growth factorby the organism under identification. Incubate ateither 25 or 37°C according to the organism’srequirements (see Tables 2.2, 2.3, 2.4). Incubatefor 48 h, and then detect the presence ofacetylmethylcarbinol by adding 3–5 drops ofMR. Continuation of a red colour indicates apositive result. For a negative reaction the redcolour disappears as the MR reagent is addedto the reaction tube. The success of this testfor a positive result is dependent on incubationtime, not the amount of growth, althoughobviously growth must be seen in the tubeto indicate that the organism has grown in themedium. The test can be carried out at 24 h ifdesired, by removing an aliquot (200 ml) to amicrofuge tube and testing for MR by the additionof 1 drop of MR reagent. A negative reactionmust be incubated for a further 24 h and re-tested after this time before being classified asnegative. MR may take 3 days for a positivereaction.

Oxidase test

Oxidase test strips are available commercially,and these are recommended because ofstandardization of the reagent. For the manualoxidase test use the oxidase reagent tetramethyl-p-phenylenediamine. Prepare a 1% solution indistilled water and in a light-protecting bottle andstore at 4°C. For use, place a few drops of thereagent on to some filter paper. Using a woodenmatchstick such as an orange stick or a platinumloop, smear bacterial growth on to the moistenedfilter paper. Development of a purple colourwithin 10–30 s denotes a positive.

A nichrome loop should not be used, as itcauses false-positive reactions. This test should notbe performed from organisms grown on mediathat contain sucrose or nitrates. Therefore TCBS isan unsuitable medium from which to determinean oxidase reaction (Furniss and Donovan, 1974;Jones, 1981).

TCBS

This medium is used to identify an organism as aVibrio species. However, there are some Vibriospecies that do not grow on TCBS. These areMoritella species, Photobacterium leiognathi,Vibrio hollisae and Vibrio ordalii. Listonella(Vibrio) anguillarum may show slow growth onTCBS. Strains of Vibrio fischeri may show weakor negative growth.

TCBS is not strictly selective and some otherorganisms will grow, although at a reduced colonysize. Species of Aeromonas and Enterococcus willgrow as small (1 mm) yellow colonies on TCBSplates. Proteus species may grow as 1 mm yellow/green colonies on TCBS. Plesiomonas does notgrow well on TCBS. Some yellow colonies ofVibrio species may revert to green colonies after afew days growth as they use up the sucrose in themedium (Oxoid manual).

VP

The MRVP medium (Difco) should be inoculatedwith a heavy inoculum of bacterium. Salt needsto be added if required by the organism. Incubateat either 25 or 37°C according to the organism’srequirements (see incubation Table 2.3). As ageneral rule use an incubation temperature of25°C for all bacteria isolated from aquatic envi-ronments and aquatic animals. Incubate for 48 h,and then detect acetoin production using the

122 Chapter 3



reagents. VP can be tested at 24 h if there issufficient growth in the test medium. A negativeresult, however, should be re-incubated for48–72 h. Test an aliquot by placing 200 ml of theincubated medium into a 0.6 ml microfuge tube.Add one drop each of VP reagent A and VPreagent B. Commercial reagents from the API20E kit work well with the Difco MRVP medium.Examine for red colorization up to 20 min.Although the two tests can be performed in thesame tube, it is preferable to test the reactions at24, 48 and 72 h. The incubation time influencesthe production of acetoin.

Vibriostatic agent

Resistance to the vibriostatic agent pteridine(0/129) has been noted in some developingcountries. Microbiologists are advised to continueusing 0/129 as a means of differentiating Vibriospecies from Aeromonas species, but cautionmay be required in some countries (Huq et al.,1992; Nair and Holmes, 1999). A zone of 9 mmis classified as susceptible for the 0/129 500 mgdisc for Vibrio species (Bernardet and Grimont,1989). A zone size of 22 mm is considered sensi-tive for Photobacterium damselae ssp. damselae(Love et al., 1981). Vibrio cholerae strain 0139 isresistant to the 0/129 500 mg disc (Albert et al.,1993; Islam et al., 1994).

3.3 Using the BiochemicalIdentification Tables

The following sections are devoted to the identifi-cation of an unknown organism using phenotypicor biochemical tests. The variations in the litera-ture for different strains are also recorded, so thatthe microbiologist will be aware of the difficultiesencountered when identifying particular species.Hopefully this system should not prove tootedious for the user of the manual; it is intendedto give the users more confidence in namingan organism and to alert them to potentialdifficulties.

In some cases, species are relatively newlydescribed and different researchers have obtaineddifferent biochemical reactions. Rather than pre-senting a consensus result, both results are listed.It must be stressed that difficulties arise when

assessing results of some phenotypic tests whentwo different versions of a biochemical test havebeen used. Where differences were noted fromdifferent journal articles, these are either stated inthe tables, or are detailed under the notes on inter-pretation and the problems encountered, section3.4. Other notes are from the author’s experiencewith particular species. Phenotypic tests are stillthe primary identification system, and thereforemicrobiologists must be aware of the difficultiesencountered with some tests for particular organ-isms. Some species have been newly described onone strain only and as more information is gath-ered about this species it may become evident thatthe type strain is not in actual fact representative ofthe species (Janda and Abbott, 2002). Thereforesubsequent strains isolated and identified asbelonging to this species may show slightly differ-ent results in the biochemical tests. Therefore,reporting these differences may assist the user ofthis manual with identification. For example thetype strain of Vibrio lentus is negative for mannitolfermentation, yet all other species reported thusfar are positive. Likewise with Vibrio penaeicida,where the type strain is negative for indole and50% of other strains are positive. The atypicalAeromonas salmonicida group is another exam-ple. In this case, different biochemical results arereported from strains isolated from different fishspecies. For the diagnostic laboratory trying toidentify an unknown isolate, it may be difficultto say for certain that the isolate is an atypicalA. salmonicida. However, with the phenotypicdetails listed of all isolates from different fishspecies, it may enable a more definitiveidentification, or at least an identification can bemade with slightly more confidence.

There are limitations to all phenotypic(and genotypic) identification systems (Janda andAbbott, 2002). By detailing the biochemical varia-tions it is hoped that the user of the manual will beassisted in making a more informed identification.

3.4 Interpretation and Identification ofGenera and Species

In the biochemical identification tables (Tables4.1–4.31), where authors have recorded a differ-ent result for the same type strain, then theseresults are listed separately. Rather than present aconsensus from the literature, listing the differentresults provides an indication of those tests that




are likely to produce disparate results for the indi-vidual organisms. However, for some results a ‘v’or variable reaction is recorded. Variable resultsmay have been reported in the literature or theorganism itself may be variable in its fermentationof the biochemical.

This next section provides informationabout discrepancies found in the literature orinformation that is specific to the identification of acertain organism.

Aeromonas spp.

Aeromonas spp. – motile strains

All strains of motile Aeromonas species arepositive for the fermentation of glucose andmaltose, and ONPG. All strains are negative forurease and fermentation of inositol and xyloseand are resistant to the vibriostatic reagent 0/129.

Aeromonas hydrophila ssp. hydrophila andA. hydrophila ssp. dhakensis

A subspecies of A. hydrophila, A. hydrophila ssp.dhakensis, has been described (Huys et al.,2002b). The subspecies is differentiated fromA. hydrophila ssp. hydrophila by the followingtests. A. hydrophila ssp. dhakensis is negative foracid production from L-arabinose, and negativefor utilization of methyl a-D-mannoside, L-fucoseand L-arabinose, whereas A. hydrophila ssp.hydrophila is positive for acid production fromL-arabinose and positive for utilization of methyla-D-mannoside, L-fucose and L-arabinose (Huyset al., 2002b).

There are many reports in the literature ofvariable results for LDC. Some reports state thatA. hydrophila (the type strain ATCC 7966) isnegative for LDC (Kaznowski, 1998; Nielsen et al.,2001). However, there are other reports of thetype strain ATCC 7966 being positive for LDC(Abbott et al., 1992; Huys et al., 2002b). One ofthe reasons that there are variable reports for LDCmay be due to misinterpretation of the test, orthe use of different methods to detect LDC. Inthe conventional tube test, LDC is a pale purplecolour, particularly in comparison with the ADHtube. Also, a stronger result may be obtained after48 h incubation than after 24 h incubation.

There are also varying reports for utilizationof citrate and reaction for MR. Citrate is said to be

positive for 60% of strains of A. hydrophila, andMR is positive for 53% of strains (Abbott et al.,1992).

Aeromonas popoffii

Type strain LMG 17541 is indole-negative, butother strains are indole-positive (Huys et al.,1997b).

Aeromonas salmonicida: non-motileAeromonas – general

Identification of an isolate as A. salmonicida isbased on Gram-negative rod, negative motility,catalase positive, oxidase positive, production ofacid from glucose, resistance to vibriostatic agent0/129, and lack of growth at 37°C (Shotts et al.,1980). The initial division for A. salmonicida wasbased on production of a brown water-solublepigment on media containing tryptone. However,these criteria are not necessarily reliable as thereare many reports of atypical strains producingpigment, and strains that grow at 37°C (Austin,1993).

Pigment production occurs under aerobicconditions, whereas no pigment production isseen under anaerobic conditions (Donlon et al.,1983). It has also been shown that pigmentproduction can be reduced in the presence ofD-glucose. In the case of atypical A. salmonicidastrains from goldfish in Australia, which arethought to belong to the subspecies nova, pigmentproduction was intense after 3 days on Columbiaagar, and light-brown at 6 days on TSA. Additionof 0.1% w/v of glucose delayed pigment produc-tion, and 0.15% (w/v) of glucose completely inhib-ited pigment production (Altmann et al., 1992).

Differences in biochemical reactions havebeen noted when Aeromonas salmonicida strainsare incubated at different temperatures such as11, 18 and 28°C (Hahnel and Gould, 1982). Forconsistency, an incubation temperature of 22°Cis recommended and was used in the study byKoppang et al. (2000).

The A-protein layer, a major virulence factorfor A. salmonicida ssp. salmonicida, can be detec-ted using TSA supplemented with CBBA wherebacterial colonies containing the A-protein layerare seen as blue-coloured colonies, and A-protein-negative colonies appear white (Evenberg et al.,1985). Autoagglutination in distilled water alsoindicates the presence of the A-protein layer.

124 Chapter 3



However, these tests are not reliable indicators ofthe in vivo virulence of A. salmonicida (Bernoth,1990; Olivier, 1990).

Aeromonas salmonicida – atypical strains

The term ‘atypical’ is used to describe isolatesthat are slow-growing, have slow or no pigmentproduction, and biochemical characteristicsdifferent from A. salmonicida ssp. salmonicidaand the subspecies achromogenes, masoucida,salmonicida and smithia. The atypical strainsrepresent a diverse group of organisms, and aredifficult to identify to species level because of therange of phenotypes. A recent study highlightedthe need for standardized phenotypic tests toreduce inter-laboratory variation, and alsosuggests that ‘atypical’ isolates should be definedas any isolate that does not fit the existingclassification of A. salmonicida ssp. salmonicida(Dalsgaard et al., 1998). Therefore, to assistthe microbiologist in the identification of theseorganisms, biochemical tests are reported in thismanual from a number of different isolates fromdifferent fish species (although the list is notextensive). Results that have been achievedusing the standardized recommended methodsare indicated in the Table 4.1. See Chapter 7 formedia composition.

Pigment production can vary and maydepend on the media used. In a study of atypicalstrains by Hänninen and Hirvelä-Koski (1997),FA detected the highest number of strains withpigment (100%), followed by BHIA (86%) andTSA (74%). A. salmonicida ssp. achromogenesproduced pigment on FA, TSA and BHIA, but noton nutrient agar. The addition of L-tyrosine to TSAor BHIA leads to an increase in pigment detection;however, pigment is still detected earlier on FA,within 3–7 days at 20°C. TSA, BHIA and FA arenot recommended for primary isolation, as thenumber of organisms detected is not as high aswhen BA is used. They are, however, suitablefor subculture (Bernoth and Artz, 1989; Austin,1993).

Brucella spp.

The Brucella species isolated from marinemammals are zoonotic (Brew et al., 1999) and assuch should be dealt with in a class III biologicalsafety cabinet.

Isolates from seal and an otter, and someof the strains from cetaceans, have an absoluterequirement for 10% CO2. Most of the strains fromcetaceans do not require CO2 atmosphere forgrowth (Foster et al., 2002).

Brucella species from aquatic mammals givea numerical profile of 1200004 in the API 20NE.The manufacturer’s database will identifythis as ‘good identification for Moraxellaphenylpyruvica’ (Foster et al., 1996a).

Carnobacterium spp.

Carnobacterium is distinguished from Lacto-bacillus by its ability to grow at pH 9.0 but noton acetate agar (pH 5.4) or at pH 4.5.

When testing Carnobacterium piscicolafor carbohydrate fermentation, discrepanciesbetween results for some carbohydrates havebeen reported in the literature. In particular, aweak reaction for a positive fermentation of acarbohydrate tested using Phenol Red Broth basehas been reported as negative when Purple Brothbasal medium is used in the test. Sorbitol andlactose may be positive when Phenol Red Brothbase is used, but negative when Purple Brothbasal medium is used (Toranzo et al., 1993).

Carnobacterium, Lactobacillus, S. iniae,Vagococcus and Renibacterium differentiation

The following characteristics in Tables 3.2 and3.3 give a guide to the differentiation of thesegenera and species. Renibacterium salmoninarumis differentiated mainly by its slow growth andspecific growth requirements. Use KDM2 andKDMC media for culture and isolation. Plates areincubated at 15°C for up to 2 months with initialgrowth of pin-point colonies visible between 2and 8 weeks. The other genera all grow within1–3 days of incubation on general-purpose agarsuch as BA.

Table 3.3 was kindly supplied by Dr JeremyCarson (Department of Primary Industries, Waterand Environment, Tasmania).

Citrobacter freundii

Strains of Citrobacter freundii show variationin reactions for ADH, ODC, fermentation of




126 Chapter 3

Test

Lact

obac

illus

spp.

Car

noba

cter

ium

pisc

icol

aC

arno

bact

eriu

mdi

verg

ens

Lact

ococ

cus

garv

ieae

Lact

ococ

cus

pisc

ium

Vago

cocc

usflu

vial

isVa

goco

ccus

lutra

eVa

goco

ccus

salm

onin

arum

Ren

ibac

teriu

msa

lmon

inar

um

Gro

wth

onac

etat

eag

ar(R

AA)

+−

−−

−−

−−

ADH

v+

++

−−

−−

Acid

from

man

nito

lv

+−

++

+−

−−

H2S

−−

−−

−−

ND

+La

ncef

ield

grou

pD

reac

tion

Posi

tive

Neg

ativ

eW

eak,

dela

yed

reac

tion

ND

Wea

k,de

laye

dre

actio

nLa

ncef

ield

grou

pN

reac

tion

Posi

tivea

Posi

tiveb

Posi

tive

ND

Neg

ativ

eSt

rept

ococ

calg

roup

ing

kitg

roup

sA-

G(O

xoid

)D

Neg

ativ

eN

egat

ive

Dw

eak

reac

tion

ND

Dw

eak

reac

tion

Neg

ativ

e

a Elda

reta

l.(1

999)

repo

rted

apo

sitiv

ere

sult.

Teix

eira

etal

.(19

96)r

epor

tsas

nega

tive.

b Schm

idtk

ean

dC

arso

n(1

994)

repo

rted

ane

gativ

ere

sul t.

Tab

le3.

2.C

arno

bact

eriu

m,L

acto

baci

llus,

Vag

ococ

cus

and

Ren

ibac

teriu

mdi

ffere

ntia

tion.



sucrose, melibiose, amygdalin and salicin(API 20E), regardless of source or geographicallocation (Toranzo et al., 1994).

Cryptococcus spp. (yeasts)

All Cryptococcus species are positivefor urease, whereas Candida species arenegative.

Edwardsiella spp.

Edwardsiella hoshinae may smell like Plesio-monas shigelloides, which has a strong, pungent,sweetish smell. Vibrio (carchariae) harveyi ATCC35084 has a similar pungent smell, but notas strong as P. shigelloides. E. hoshinae wasreported as indole-positive by Grimont et al.(1980), but negative or weak results werereported by Farmer and McWhorter (1984).Likewise for TSI, Farmer and McWhorter (1984)


Test L. garvieae* C. piscicola V. salmoninaruma L. piscium S. iniae

Gram + + + + +Cell shape ec sr sr/cb c (sr/cb)b cHaemolysis a − a − ac, bc,d

Catalase − − − − −VP (plate) 89%+ + −# + −H2S − − + − −Bile-aesculin + + 62%+ − −PYR + + + − 95%+e

ADH + + − − 71%+e

Aesculin + + + + +Glucose + + + + +Galactose + 65%+ − + +Lactose − 35–60%+ − + −Maltose 90%+ 94%+ 17%+ + +Mannitol 90%+ 88%+ 4%+ + +Raffinose − 29%+ − + −Salicin 98%+ + 87%+ + +Sucrose 7%+ + 52%+ + +Sorbitol − − 4%+ − −Trehalose + + 87%+ + +Glycerol − 40–100%+ − − −Inulin − 0–100%+ − − −L-arabinose − − − − (+)b −Dulcitol − − − − −Fructose + + + + +Starch − 0–100%+ 13%+ − +Xylose − − − − −Adonitol − − − − −Melibiose − 30%+ − + (−)b −

All reactions determined by miniaturized microtitre tray tests (Schmidtke and Carson, 1994) or conventional tests. #Type strainis positive; boriginal description reaction in brackets; ca on bovine blood, b on sheep blood. ec, Elongated coccus; sr, short rod;cb, cocco-bacilli; c, coccus; PYR, L-Pyrrolidonyl-b-naphthylamide; ADH, arginine dihydrolase. *Lactococcus garvieae (syn.Enterococcus seriolicida).aSchmidtke and Carson, 1994; bEldar et al., 1994; cWeinstein et al., 1997; dVuillaume et al., 1987; eDodson et al., 1999.

Table 3.3. Differential tests for some non-fastidious fish pathogenic Gram-positive cocci and rods.



reported a negative result, whereas a positiveresult was recorded by Grimont et al. (1980).

Edwardsiella ictaluri will grow on BrilliantGreen agar, and Salmonella–Shigella agar. MRand VP are positive and negative, respectively, atboth 37 and 20°C. There is no growth at 42°C.The organism grows in 0–1.5% NaCl, but not in2% NaCl.

Enterococcus spp.

Enterococcus faecalis and E. faecium are positivefor Lancefield group D antigen, grow at 45°C,and grow in the presence of 6.5% NaCl. Reliabletests for differentiation between the two speciesare acid production from L-arabinose (E. faecalisis negative and E. faecium is positive) andpyruvate utilization (E. faecalis is positive andE. faecium is negative).

Enterococcus seriolicida is now known asLactococcus garvieae.

Family Flavobacteriaceae

Many samples from the aquatic environment, bethey freshwater or marine, will produce yellow-pigmented colonies on culture. It is important toidentify whether these colonies are pathogensor saprophytes. Clinical information will beextremely useful in knowing how much time andeffort to put into their identification. Thus, ifbacterial cells, usually long and thin, are seenadhering to the surface or epithelium of aquaticanimals, then genera of the FlavobacteriaceaeFamily may be suspected. To aid in their identifi-cation, it is important for the microbiologist tounderstand the complexities of this family. Thefollowing information will assist in this regard.Much of this information has been taken from apaper by Bernardet et al. (2002), setting out theirproposed guidelines for describing members ofthe family Flavobacteriaceae.

The Cytophaga–Flavobacterium–Bacteroidesphylum contains the family Flavobacteriaceae,which consists of 18 genera and two unaffiliatedorganisms – Cytophaga latercula and C. marino-flava – that remain genetically misclassified at thisstage. At various times, this group has been knownas the yellow-pigmented rods, Flavobacterium-like, or Cytophaga group or Flexibacter group,

and the genera and species within this grouphave undergone numerous name changes andreclassifications in recent years. The type genus isFlavobacterium.

Genera of the Family Flavobacteriaceae con-sist of a group of halophilic organisms (toleratingsaline conditions), some of which are psychro-philic (tolerating cold temperatures). They aredescribed as having cells that are Gram-negative,1–10 mm long and 0.3–0.6 mm wide, with somespecies forming filamentous flexible cells and oth-ers coiled or helical cells. Some members exhibit agliding motility and the rest are non-motile. Theoptimum temperature for growth for all genera is25–35°C, with some species being psychrophilicor psychrotolerant. Growth is aerobic for most ofthe genera; however, microaerophilic or anaero-bic conditions are required for some genera.

Sphingolipids are absent and this featuredifferentiates the Family Flavobacteriaceae fromthe Family Sphingobacteriaceae (Tables 3.4 and3.5). None of the genera in the Family Flavo-bacteriaceae digests crystalline cellulose whentested with filter paper, and this characteristicdistinguishes them from the genus Cytophaga,which only contains species that digest crystallinecellulose. It is important to differentiate the pres-ence of the enzyme cellulase from other enzymesthat can degrade cellulose derivatives, such ascarboxymethylcellulose or hydroxyethylcellulose,which may be present in some species ofthe Flavobacteriaceae Family. Only cellulase iscapable of degrading crystalline cellulose, whichis tested using filter paper.

A number of other families are con-tained under the Cytophaga–Flavobacterium–Bacteroides phylum. Like the Family Flavo-bacteriaceae these include genera that produceyellow-pigmented colonies that may be culturedfrom samples received from the aquatic environ-ment. Some of these species include Cyclo-bacterium marinum, Cytophaga hutchinsonii,Flexibacter flexilis, Marinilabilia salmonicolor,Pedobacter heparinus, Sphingobacteriumspiritivorum and others.

Most of the members of the Flavo-bacteriaceae Family are aerobic except for Cap-nocytophaga, Coenonia, Ornithobacterium andRiemerella. An appropriate isolation technique isculture on to BA and incubation in a carbon-dioxide enriched atmosphere with 5–10% CO2

or a commercial gas generated atmosphere thatachieves 5% O2, 10% CO2 and 85% N2. Growth is

128 Chapter 3



either poor or absent under anaerobic conditions(Bernardet et al., 2002).

Agar digestion is positive for Cellulophaga,Zobellia and variable for Flavobacterium. None ofthe other genera in the Family Flavobacteriaceaedigests agarose.

Researchers describing new species in theFamily Flavobacteriaceae are recommended tofollow the guidelines for description and identifica-tion of new taxa and species in Bernardet et al.(2002).

Flavobacterium columnare

Genetic diversity is reported amongst strains;however, phenotypically they are similar.Genomic group I, which contains the type strainIAM 14301T, are 50% positive for the nitrate test.Nitrate reductase is also variable for genomicgroup II, whereas strains in genomic group III arenegative for nitrate. Strains for genomic group II

and III are able to grow at 37°C but not at 15°C,whereas strains of genomic group I are variable,with 85% showing growth at 15°C and only 75%at 37°C. The genomovars are distinguishable byPCR (Triyanto and Wakabayashi, 1999). Seesection 6.1, Molecular Identification by PCRusing Specific Primers.

Tenacibaculum maritimum

Reactions for T. maritimum in the API ZYMare reasonably consistent in the literature. TheAPI ZYM should be incubated at 22–25°C withovernight incubation.

Hafnia alvei

H. alvei may be differentiated from Yersiniaruckeri, as H. alvei is positive for xylosefermentation whereas Y. ruckeri is negative.


Genus Host Pigment typeSeawaterrequirement

Glidingmotility Atmosphere

Growthon MCA

Aequorivita Sea water, sea ice Carotenoid Variable Negative Aerobic Not testedBergeyella Human None Negative Negative Aerobic No growthCapnocytophaga Human and dog Flexirubin Negative Positive Microaerophilic No growthCellulophaga Marine alga and beach mud Yes Variable Positive Aerobic Not testedChryseobacterium Fish, marine mud, human,

cow’s milk, soilFlexirubin Negative Negative Aerobic Variable

Coenonia Peking duck Not tested Negative Negative Microaerophilic No growthEmpedobacter Human Flexirubin Negative Negative Aerobic PositiveFlavobacterium Fish, water, sea ice, soil, mud,

marine lake, AntarcticaYes Negative 11 species,

positiveAerobic Not tested

Gelidibacter Sea ice Carotenoid Positive Positive Aerobic NegativeMyroides Human Flexirubin Negative Negative Aerobic PositiveOrnithobacterium Turkey None Negative Negative Microaerophilic NegativePolaribacter Seawater, sea ice, marine lake Carotenoid Positive Negative Aerobic Not testedPsychroflexus Antarctica Carotenoid Variable Variable Aerobic NegativePsychroserpens Antarctica Carotenoid Positive Negative Aerobic NegativeRiemerella Duck, pigeon Variable Negative Negative Microaerophilic NegativeSalegentibacter Antarctica Carotenoid Negative Negative Aerobic Not testedTenacibaculum Marine algae, marine sponge,

fishCarotenoid, orweak reaction

Variable Positive Aerobic Not tested

Weeksella Human None Negative Negative Aerobic PositiveZobellia Red marine alga, marine

sedimentFlexirubin Positive Positive Aerobic Not tested

Some genera only produce one type of pigment, others none, whereas other genera contain species that produce either aflexirubin-type pigment, a carotenoid-type pigment or both. A ‘yes’ in this column indicates that both or one type of pigmentmay be produced. MCA, MacConkey agar.

Table 3.4. Differential characteristics of the genera within the family Flavobacteriaceae.



Lactococcus spp.

The Lactococcus genus was created to accommo-date ‘lactic’ or group N streptococci. There arecurrently four species, L. garvieae, L. lactis,L. piscium, L. plantarum and L. raffinolactis.There are varying reports in the literature forgroup N results for L. garvieae and L. piscium.

Lactococcus garvieae

Different results have been noted for ribose,depending on the identification system used.Ribose is positive when using the API 50CH sys-tem, but negative with the API Rapid ID 32 Strepsystem (Vela et al., 2000). L. garvieae may be bio-chemically indistinguishable from Streptococcusparauberis (Doménech et al., 1996). Lactococcusgarvieae (Enterococcus seriolicida) is phenotypi-cally similar to L. lactis; however, they can be dif-ferentiated by sensitivity testing to clindamycin.L. garvieae is resistant to clindamycin, whereasL. lactis is sensitive (Elliott and Facklam, 1996).L. lactis is now known as L. delbrueckii ssp. lactis.

L. garvieae was divided into biotypes (Velaet al., 2000); however, these divisions have been

disputed. Further research indicates that L. garvi-eae strains are relatively homogeneous regardlessof geographical location or aquatic host. What isimportant in biochemical identification is that thedensity of the inoculum be standardized whenusing the API Rapid ID 32 Strep system and thatthe bacterial cells to be used for the inoculum aretaken from BA plates. Reliable, repetitive resultswere only obtained when the inoculum densitywas adjusted using a spectrophotometer, toan optical density of 0.8 at wavelength of 580.Also, cells grown on BA produced reliable resultsfor biochemical tests, whereas growth taken fromTSA (Oxoid) produced variability in biochemicaltests. However, slight variability was still observedfor biochemical tests such as b-galactosidase,hippurate, b-mannosidase, acid from melezi-tose, N-acetyl-b-glucosaminidase and acid frompullulan (Ravelo et al., 2001).

Listonella anguillarum

Some strains are negative for citrate in both theconventional tube test and in the API 20E. TheMR tube may show weak growth and a weak

130 Chapter 3

Genus Aesculin Catalase DNase Gelatin Glucose acid Indole ONPG Nitrate Sucrose Urease

Aequorivita + + 75% Neg + Neg NT NT Neg Neg VBergeyella Neg + Neg + Neg + Neg Neg Neg +Capnocytophaga V V NT V + Neg V V + VCellulophaga NT + V V V NT NT V V VChryseobacterium + + + + + + V V V VCoenonia + + NT Neg + Neg + Neg Neg NegEmpedobacter Neg + + + + + Neg Neg Neg VFlavobacterium 10/14 =+ + or weak V 11/14 =+ V Neg V V V VGelidibacter + + + V + Neg Neg Neg Neg NegMyroides Neg + + + Neg Neg Neg Neg Neg +Ornithobacterium Neg Neg Neg Neg V Neg + Neg Neg +Polaribacter V + or weak NT V + Neg V Neg V NegPsychroflexus V + + V V Neg Neg Neg Neg VPsychroserpens Neg + Neg V Neg Neg V Neg Neg NegRiemerella V + NT + + V Neg Neg Neg VSalegentibacter + + + + V NT + + V VTenacibaculum Neg + + + Neg NT NT V NT NTWeeksella Neg + Neg + Neg + Neg Neg Neg NegZobellia + + + + + + + + + Neg

V, variable; NT, not tested; Neg, negative; +, positive; ONPG, o-nitrophenyl b-D-galactopyranoside.10/14 =+ means that 11 of the 14 species in the genus are positive.

Table 3.5. Further differential characteristics for the genera in the family Flavobacteriaceae.



positive reaction at 48 h after incubation andsome strains may be negative. Growth on TCBStends to be slow and there is substantially lessgrowth when compared with the amount ofgrowth on MSA-B or BA plates. Strains isolatedfrom an estuarine environment may grow betterin 0.85% NaCl, whereas those strains from amarine environment show optimal growth with2–3% NaCl, and this can be seen clearly on a gel-atin-salt plate. Use growth on this plate or growthon a BA plate, as opposed to growth on MSA-B,as a guide to the salt requirement of the organismwhen preparing the NaCl for the inoculating fluidfor either the conventional biochemical set orthe API 20E. Strain NCIMB 2129 is reported tobe negative for fermentation of sorbitol and tre-halose (Benediktsdóttir et al., 1998). Differencesin citrate, indole and MR are reported for NCIMB2129, NCIMB 6 and ATCC 14181 (Myhr et al.,1991; Benediktsdóttir et al., 1998; Lunder et al.,2000). Serotype 01 strains tend to be positive forL-arabinose (Toranzo and Barja, 1990).

Mesophilobacter marinus

Mesophilobacter marinus describes marinecoccobacilli that morphologically resemble theAcinetobacter – Moraxella group.

Moritella spp.

Moritella (Vibrio) viscosus often needs prolongedincubation times for growth in test media. Moststrains require 1% peptone for optimal growth.Results for salt requirements and temperaturegrowth limits are inconsistent in the literature.One study suggests 1–4% NaCl and a tempera-ture of 25°C (Lunder et al., 2000). A secondstudy suggests 2–3% NaCl with no growth at 4%NaCl, and a temperature range of 4–21°C withno growth at 25°C (Benediktsdóttir et al., 2000).

Photobacterium spp.

Photobacterium damselae ssp. damselae

There are some biochemical differences accord-ing to the biochemical method used and theamount of salt in the inoculum. The tube ureaseis positive; however, in the API 20E the ureaseresult seems to depend on the salt concentrationin the inoculum. When an inoculum of 0.85%NaCl is used, then the urea gives a positive result,but at an inoculum concentration of 2% NaCl, anegative urea may be obtained. Time of incuba-tion is important also, as urea may be weak ornegative at 24 h incubation, but a strong positiveat 48 h incubation. P. damselae ssp. damselaehas been grouped into biotypes (Pedersen et al.,1997) and many of these are listed in the con-ventional biochemical table for Photobacterium.Table 3.6 lists other biochemical tests not listed inthe conventional biochemical table 4.20.

Photobacterium damselae ssp. piscicida

The fermentation reactions may be improvedunder anaerobic conditions. This is easilyachieved by overlaying the medium with sterileparaffin oil. The glucose reaction in the API 20Ekit may be very weak or negative. Likewise, theVP reaction may be variable, and research intothe biochemical properties of isolates fromFrance, Greece, Italy and Japan found that allisolates tested were negative for VP in the API20E system (Bakopoulos et al., 1995).

Pseudoalteromonas spp.

Pseudoalteromonas citrea

First described by Gauthier (1977), the typestrain is ATCC 29719. Similar strains have beendescribed by Ivanova et al. (1998). Some pheno-typic tests are slightly different, which is thought


Biotypes 1 2 3 4 5 6 7 8 9

TestLipase + − + + + + + + +Cellobiose + + + + − − + + +

Taken from Pedersen et al. (1997).

Table 3.6. Additional tests for differentiation of biotypes of P. damselae



to be due to the different ecological niches. Thephenotypic reactions described in this manual arethose of the type strain.

Pseudomonas anguilliseptica

Finnish isolates and the type strain are positivefor gelatin, whereas Japanese isolates are nega-tive. Motility is only seen when the organism isgrown below 20°C (Michel et al., 1992). Thereare three serotypes of P. anguilliseptica based ona heat-labile surface K antigen. Serotypes fromeels in Japan are K− and K+; ayu have K+serotype as do the Finnish isolates (Wiklund andBylund, 1990). Antisera against P. anguillisepticamay show cross-reaction against Pseudomonasputida and Listonella anguillarum; however, if itis used diluted at 1:5 to 1:10, this may overcomethe cross-reaction (Toranzo et al., 1987).

Renibacterium salmoninarum

This organism is the causative bacterium of BKD.It is a fastidious and slow-growing organismand the culture medium used for isolation andgrowth, KDM2, may give inconsistent perfor-mance due to lot-to-lot variations in peptone(Evelyn and Prosperi-Porta, 1989). Consistencyin growth can be improved either by using anurse culture technique, or by supplementationof the growth medium with spent culturemedium. The nurse culture technique involvesplacing a 25 ml drop of nurse culture (preparedfrom cells suspended in saline or peptone) intothe centre of a plate inoculated with the testsample. Once the drop is dry the plates areinverted, sealed to prevent drying, and incubatedat 15°C for 21 days. The supplementation withspent culture broth method can be achievedby preparing new KDM2 medium with 1.5%(v/v) of spent culture medium (Evelyn et al.,1990).

Streptococcus spp.

Streptococcus agalactiae

Lancefield group B. Different reactions are repor-ted for S. agalactiae isolated from human, fish andanimal sources, and this may in part be due to theoptimum incubation temperature for enzyme

reactions. For example, hippurate may be positiveat an incubation temperature of 25°C but nega-tive at 37°C for isolates from fish. The conven-tional VP method shows a negative reactionfor S. agalactiae, but it is positive in the APIrapid ID 32 (Vandamme et al., 1997). Strains ofS. agalactiae from mammalian sources are b-haemolytic; however, most of the strains isolatedfrom fish were reported to be non-haemolyticand were initially identified as S. difficile. Theyare now identified to be S. agalactiae group B,capsular type Ib (Vandamme et al., 1997). Theseisolates are slow-growing and adhere strongly tothe agar plate, which makes it difficult to resus-pend the cells in an inoculating fluid. Conven-tional biochemical tests may need to be incu-bated for more than 48 h for sufficient growth tooccur in the tubes. MR and VP reactions may beweak, and poor growth is seen in the tryptonewater (indole test). The ADH test using con-ventional media may take more than 3 days’incubation before a slight colour change is seen.

For the S. agalactiae strains that were isolatedfrom a disease outbreak in seabream and wildmullet in Kuwait, there are some differencesin the biochemical reactions compared with theATCC type strain (Evans et al., 2002). Therefore,the results of the S. agalactiae isolates from theseabream and the wild mullet are listed separatelyin the API rapid ID 32 Table 4.30.

Streptococcus iniae

The organism shows complete b-haemolysis onsheep BA, but only partial haemolysis when themedium is supplemented with human or bovineblood. Variations in haemolysis may be seenbetween strains.

Strains isolated from either fish or humanshave a slightly different biochemical profile.

S. iniae strains isolated from culturedand wild fish in the Red Sea were similar in bio-chemical profile to other S. iniae strains, exceptfor a negative ADH in the majority of strains, andlate fermentation of galactose and amygdalin asdetected in the API 50CH, which was incubatedfor 72 h before results were recorded (Colorniet al., 2002). The biochemical profile achievedwith the API 20 Strep agrees with other resultsrecorded for fish isolates where 30% of strains maybe negative for ADH, and that variation may beseen in the fermentation of mannitol (Dodsonet al., 1999).

132 Chapter 3



Streptococcus parauberis

This may be biochemically indistinguishablefrom Lactococcus garvieae (Doménech et al.,1996).

Streptococcus uberis

There are different reports for the reactionto pyrrolidonylarylamidase. Bergey’s Manualof Determinative Bacteriology (1994) reports anegative reaction, whereas Collins et al. (1984)and Doménech et al. (1996) report a positivereaction.

Tenacibaculum maritimum

See under Flavobacteriaceae.

Vagococcus salmoninarum

The genus Vagococcus was proposed to describemotile, Lancefield group N, Gram-positivecocci; however, Schmidtke and Carson (1994)described V. salmoninarum as negative for the Nantigen.

Vibrio spp.

Vibrio agarivorans

These strains degrade agar and the effect can beseen as small depressions on an agar plate after3 days’ incubation at 25°C. This effect increasesover time, and by about 7 days the effect isvery marked. This was particularly the case withisolates from abalone in Western Australia. Thecarbohydrate fermentation reactions may be dif-ficult to read and this effect has been noticed withall strains tested. All carbohydrate fermentationtubes should be compared alongside the glucosereaction, as this will be a bright yellow colour.

The positive reactions are easy to determine, butthose that appear as a weak reaction are, in fact,negative reactions. These may appear as a paleyellow colour in the middle of the tube. However,after 48 and 72 h incubation these reactions arequite clearly negative. V. agarivorans strains firstisolated and reported from the MediterraneanSea show a positive reaction for aesculin anda negative reaction for gelatin (Macián et al.,2001b). Strains from Western Australia all showa negative reaction for aesculin using the con-ventional tube test, and a positive reaction forgelatin when the plate method is used. However,the gelatin result in the API 20E is negative after48 h incubation at 25°C.

Vibrio alginolyticus and Vibrio harveyi

These may be difficult to differentiate (Table 3.7).The VP test differentiates them, as does theadditional test for fermentation of D-glucuronate(Baumann et al., 1984). V. alginolyticus swarmsand completely covers an MSA-B plate in 24 hat 25°C, whereas V. harveyi has a slow orspreading type of growth. V. alginolyticus isurease-negative, whereas V. harveyi is usuallyurease-positive (50% reported as positive).

Vibrio cholerae

Strains of Vibrio cholerae are negative foraesculin; however, some strains cause blackeningof the medium due to the production of melanin(Coyne and Al-Harthi, 1992). To determine truehydrolysis as opposed to melanin production,the aesculin tube must be tested for loss of fluo-rescence (Choopun et al., 2002). The aesculinsolution will fluoresce under long-wave UV light(354 nm), therefore a loss of fluorescenceindicates hydrolysis of aesculin and a positive testresult (MacFaddin, 1980). The plastic tubes thatthe aesculin medium is in may be UV opaque.Therefore the test medium can be poured intoa Petri dish and a Wood’s UV light held overthe dish in a darkened room. An uninoculated


VP 10% NaCl Growth at 42°C Urease D-Glucuronate

Vibrio alginolyticus + + + − −Vibrio harveyi − − − v +

Table 3.7. Differentiation of V. alginolyticus and V. harveyi.



control and a negative result will show a ringof fluorescence around the edge of the liquid,whereas a true positive aesculin will not show thefluorescence. Protect the eyes with UV-opaqueglasses.

VIBRIO CHOLERAE NON-01. Strains isolated fromayu in Japan were found to be negative forODC (Kiiyukia et al., 1992). Normally, V. choleraeisolates are positive for ODC.

Vibrio coralliilyticus

Six strains of this newly described Vibrio specieshave been characterized. There is some bio-chemical variation between strains, with two ofthe six strains positive for ADH as detected in theAPI 20NE, and four strains negative (Ben-Haimet al., 2003).

Vibrio fischeri

A positive result is obtained for VP in the API 20Esystem, but VP is negative in MRVP medium(Difco).

Vibrio fluvialis and Vibrio furnissii

Both species are indole-negative when tested forindole production in peptone water containing1% NaCl. When indole is tested using heartinfusion broth, 14% of V. furnissii are positive,and 4% of V. fluvialis are positive. Both speciesare positive for indole in the API 20E system. TheAPI 20E database may identify these speciesas Aeromonas species (Brenner et al., 1983). V.furnissii may show a weak pink indole reactionwhen approximately 12 drops of Kovács reagentis added to a 48 h tryptone water (5 ml). Thecolour disappears after a minute and appearsas a dirty orange-brown colour. V. furnissiiwill show a negative ADH reaction if no NaCl isadded to the reagent tube. ADH will be positivewhen the final salt concentration is 2% (Buller,2003). V. furnissii strains produce gas in theglucose tube, whereas V. fluvialis strains donot produce gas. V. furnissii strains (57%) maybe positive for L-rhamnose, whereas V. fluvialisstrains are negative, and 63% of V. fluvialisare positive for cellobiose, whereas V. furnissiiis negative (Lee et al., 1981; Brenner et al.,1983).

Vibrio (carchariae) harveyi ATCC 35084

Citrate and gelatin (plate method) are negativeat 24 h and need to be read at 48 h or longer.The culture has a pungent smell similar toPlesiomonas shigelloides.

Vibrio harveyi

The strain ATCC 14126 is positive for lumines-cence and negative for urease, whereas ATCC35084 is negative for luminescence and positivefor urease (Alcaide et al., 2001). Strains NCIMB1280, ATCC 14126 and ATCC 14129 arereported to be gelatin-positive (Baumann et al.,1984; Benediktsdóttir et al., 1998). Strains ATCC14126 and ATCC 14129 are reported to benegative for fermentation of sorbitol, whereasstrain ATCC 35084 is positive after 2 days(Alcaide et al., 2001; Buller, 2003). Haemolysisis variable, and is reported to be positive againstsheep red blood cells (Alcaide et al., 2001).See Table 3.7 for further tests to differentiateV. harveyi and V. alginolyticus.

Vibrio neptunius

The type strain is positive for ADH; however,other strains are reported to be negative for ADH(Thompson et al., 2003).

Vibrio parahaemolyticus

In the API 20E, strain ATCC 43996 shows a moreclearly positive reaction in the arabinose testwhen incubated for 48 h and prepared with a 2%NaCl inoculum. However, ADH shows a weakpositive reaction with a 2% NaCl inoculum andincubation at 48 h. With the conventional tests,indole will record a negative result if normalsaline is used as the inoculum. A positive resultis recorded with 1.5% and 2% NaCl as a finalconcentration.

Vibrio mediterranei

At 48 h these are large, creamy, mucoid colonieson MSA-B. After 24 h incubation and aninoculum of 2% NaCl, the ADH in the API 20E ispositive and at 48 h incubation LDC may also bepositive. However, in the conventional tube testsonly LDC is positive after 48 h incubation.

134 Chapter 3



Vibrio proteolyticus

The growth swarms quickly across an agar plate,within 24 h, and could be mistaken for V.alginolyticus. In the conventional citrate testa positive result may not be seen until 48 h.A positive urea is seen in the conventional tubetest, but is negative in the API 20E at 48 h andwith an inoculum of 2% NaCl.

Vibrio scophthalmi

The type strain in positive for chitin hydrolysis,whereas other strains are negative (Farto et al.,1999).

Vibrio scophthalmi and V. splendidus biovar Imay be difficult to differentiate based on pheno-typic tests and this is compounded due to thevariation of phenotypic test results between strainsin each species (Farto et al., 1999). Indole, ONPGand fermentation of mannitol appear to bethe best differentiating phenotypic tests.V. scophthalmi is negative for all three, whereasV. splendidus biovar I is positive for all three.

Vibrio splendidus

Variations in phenotypic tests are reportedbetween different research groups. One of thetests that seems to be a problem for consistency isADH, although the majority of reports suggestthat ADH is positive. Most V. splendidus I strainswere found to be ADH-positive when tested usingThornley’s method for ADH, yet negative whentested by the more commonly used Møller’sarginine method (Macián et al., 1996). V.splendidus I type strain (NCMB 1, ATCC 33125)is reported to be positive for ADH by bothLunder et al. (2000), and Benediktsdóttiret al. (1998). However, the results vary forV. splendidus biovar 2 type strain NCMB 2251,which is reported to be negative by Lunder, yetpositive by Benediktsdóttir. Both papers reporta negative sucrose, whereas the same type strainis reported positive by Farto et al. (1999). Thestrains are also variable for ONPG. Variationssuch as these make it difficult for the diagnosticlaboratory to identify an unknown.

There are two biovars and both have beenimplicated in disease. Biovar 1 isolates are positivefor luminescence and fermentation of mannose,ribose and melibiose, whereas biovar 2 isolatesare negative for these tests. In addition, biovar 1

is negative for fermentation of glycerol, anddegradation of chitin, with biovar 2 being positive(Benediktsdóttir et al., 1998).

There are reported differences in virulencebetween strains of V. splendidus and it issuggested that valine aminopeptidase as testedby API ZYM is a virulence factor becausepathogenic strains produced the enzyme whereasnon-pathogenic isolates did not (Gatesoupe et al.,1999).

Vibrio vulnificus

The division of Vibrio vulnificus into two mainbiovars is under contention. The original classifi-cation by Tison (1982) was that biovar 1 isolateswere positive for indole and ODC and weremainly isolated from human clinical sources.Biovar 2 strains were isolated mainly fromdiseased eels and were indole- and ornithine-negative. However, there is some overlapbetween these divisions, and therefore the reac-tions for the different strains and the geographicalsite and source of isolation have been included inthe conventional identification tables, and in theAPI 20E tables for profile number. It has beenreported that approximately 20% of V. vulnificusstrains are sucrose-positive (Arias et al., 1998).Note that sucrose medium that is autoclaved forsterility may give false-positive reactions. Filter-sterilized sucrose medium is preferred for moreaccurate fermentation results.

Vibrio wodanis

A range of salt requirements, 0.5–5% NaCl, existswith these strains (Lunder et al., 2000). Onestudy found that 1 of 16 isolates showed growthat 0.5% NaCl, and 16 of 23 isolates showedgrowth at 4% (Benediktsdóttir et al., 2000).

Yersinia spp.

Yersinia frederiksenii

These are rhamnose-positive strains, formerlycalled atypical Y. enterocolitica. Y. inter-media comprises rhamnose-positive, melibiose-positive, raffinose-positive strains, formerlycalled Y. enterocolitica or Y. enterocolitica-like.Y. kristensenii refers to sucrose-negative strains.




Yersinia ruckeri

Strains of Y. ruckeri may be divided into motileand non-motile types, a finding that is geo-graphically biased. Strains from the UK tend tobe non-motile, with occasional non-motile strainsreported from Canada and Norway. Non-motilestrains lack lipolytic activity when tested usingthe Tween 20 and Tween 80 tests. Therefore,Shotts-Waltman medium is inappropriate forthe differentiation isolation of non-motile strainsof Y. ruckeri as it contains Tween 80 (Daviesand Frerichs, 1989). These authors suggestedthat non-motile, Tween 80 hydrolysis-negativestrains should be referred to as Y. ruckeri biotype2. Results between conventional tube tests andthe API 20E system may give variable results.These include citrate utilization, gelatin hydroly-sis, VP and nitrate. The nitrate test in the API 20Esystem may give unreliable results for Y. ruckeri,thus a conventional tube nitrate test is recom-mended. After 24 h incubation at 25°C, citrateand gelatin may be falsely negative, therefore48 h incubation is recommended. Motility andcitrate are negative at 37°C but positive at 25°C.The API 20E may show more VP-positive resultsthan the conventional tube test (Davies andFrerichs, 1989).

Y. ruckeri may be differentiated fromHafnia alvei by xylose fermentation. Y. ruckeri isnegative, whereas H. alvei is positive for xylosefermentation.

3.5 Antisera Available

The following are some of the antisera that areavailable commercially.

Aeromonas salmonicida ssp. salmonicida,specific monoclonal antibody (BIONOR MonoAS, BIONOR Aqua, Skien, Norway).

Brucella abortus antiserum (Difco).Listonella anguillarum, specific monoclonal

antibody for serotypes 01, 02, 03 and 04, 05and 07 for environmental serotypes (BIONORMono-Va, BIONOR Aqua, Skien, Norway).

Photobacterium damselae ssp. piscicida,specific monoclonal antibody (BIONORMono-Pp, BIONOR Aqua, Skien, Norway).

Renibacterium salmoninarum, specificmonoclonal antibody (BIONOR Mono-Rs,BIONOR Aqua, Skien, Norway).

Salmonella O and Salmonella H antiserumand specific antiserum for species (Difco).

Staphylase test for identification of Staphylo-coccus aureus (Oxoid).

Streptococcus group A, B, C antiserum(Difco). Antiserum for groups A, B, C, D, F, G(Oxoid).

Vibrio cholerae. Anti-V. cholerae 01 serum(Denka Seiken). Bacto-Vibrio-Cholerae anti-serum for detecting three serotypes in group 01.These are serotype Ogawa (AB O antigen factors),Inaba (AC O antigen factors) and Hikojima (ABCO antigen factors) (Difco).

Yersinia ruckeri specific monoclonal anti-body (BIONOR Mono-Yr, BIONOR Aqua, Skien,Norway).

The BIONOR Mono-Va kit detected serotypesof 01, 02 and 03 of Listonella anguillarum andalso detected the environmental serotypes of 04,05 and 07. However, non-specific agglutinationagainst strains of V. splendidus and motileAeromonas species occurred. A cellular concen-tration of 108/ml is recommended for positiveagglutination (Romalde et al., 1995).

Strains of Photobacterium damselae ssp.piscicida were detected by the BIONOR Mono-Ppkit, with no cross-reactions detected for Aero-monas salmonicida. However, non-specificagglutination occurred from strains of Actino-bacillus pleuropneumoniae, Haemophilusparasuis, Mannheimia haemolytica andPasteurella multocida (Romalde et al., 1995).

Renibacterium salmoninarum was success-fully detected using the BIONOR Mono-Rs kit;however, strains that lacked the p57 surfaceprotein were not detected (Romalde et al., 1995).No cross-reaction with other Gram-positiveorganisms such as Corynebacterium aquaticum,Carnobacterium piscicola, Enterococcus faecalisand Lactococcus garvieae were detected(Romalde et al., 1995).

The BIONOR Mono-Yr kit detected classicalserotypes 01 and 03 of Yersinia ruckeri. For thedetection of 02 serotypes the test needed tobe performed at either 48 h culture growth orfrom subcultures. The kit is unable to detect theY. ruckeri serotypes 05 and 06 because of thecomposition of the serotypes included in the kit. Acell concentration of 108/ml is needed for positiveagglutination. No cross-reaction with other bacte-rial pathogens was found (Romalde et al., 1995).

136 Chapter 3



4Biochemical Identification Tables

4.1 Results for Conventional BiochemicalTests – ‘Biochem Set’

The following tables (Tables 4.1 to 4.22) are tobe used for interpreting the results obtainedfrom in-house or conventional biochemicalmedia. The tables are listed in alphabetical orderof the table heading. The organisms in the tablesare grouped into those that are consideredto be either pathogens in aquatic animals orenvironmental and saprophytic organisms. Thesaprophytic, environmental and other speciesof bacteria are included under ‘environmental’so as to assist in differentiating a pathogenfrom a closely related species and thus obtain-ing a definitive identification of the unknownorganism.

In a number of cases, different or variablebiochemical tests were recorded in the literatureby different laboratories for the same type strain.Also, differences in a biochemical were recordedfor different strains. These results are includedhere so that the reader will be aware that some testresults may not be reliable and that different resultsmay be recorded by different laboratories. In thecases reported in this Manual, the tests that gavevariable results appeared to be performed usingthe same method. It is known that differentmethods can give different results for the sameorganism, and therefore it is important to be con-sistent with the methods used. Where a differencebetween the conventional biochemical test andthe API 20E was reported, this is recorded in thetables, so that the reader is aware of variations for

different test methods for that particular organism.The numbers in the tables report the percentagesof positives as reported in the literature whena number of strains were tested. The range ofnumbers in the ‘temp’ and ‘NaCl’ columns referto the temperature range (°C) and range of saltconcentrations (%) at which the organism cangrow. A single number indicates the optimumgrowth temperature or NaCl concentration,respectively.

With the exception of the Vibrio tables(Tables 4.21 and 4.22), all other tables list theorganisms alphabetically within the groupings of‘pathogenic’ or ‘environmental’ organisms.

The species in the Vibrio tables are groupedaccording to their ODC–LDC–ADH reaction.Therefore all species with a ODC+, LDC+, ADH-result are grouped together. Likewise otherODC–LDC–ADH reaction combinations aregrouped together. Use these groupings as astarting point to identify an unknown.

Use the tables in conjunction with thesection on interpreting test results and specificreactions noted for some genera and species (seeChapter 3).

An example of a Laboratory Worksheet forrecording results is included on page 177.

Use the following schematic (Fig. 4.1) asa guide to which table or tables (Conventionalmedia, ‘Biochem set’) to use when identifyingan unknown. Start with the result for the Gramstain, then cell morphology and, for the Gram-negatives, a further division based on oxidaseresult.




4.2 Results for API Kits

Interpretation of API 20E profile

Use the profile index provided by the manu-facturer as a guide only. Kent (1982) does notrecommend using the profile index provided withthe kit because of misidentifications, especiallywhen attempting to identify an organism notfound in the commercial database. The API 20Esystem will even misidentify organisms whenthe identification is given at the 99% confidence

level. For example, Dalsgaard et al. (1996)found that the API 20E system misidentifiedV. vulnificus. The system gave a greater than98% confidence level as Aeromonas hydrophila,Flavobacterium meningosepticum, Burkholderiacepacia, and between 90 and 95% confidencelevels as V. alginolyticus and V. parahaemolyti-cus. All these ‘isolates’ were in fact V. vulnificus,which they proved using a species-specific DNAprobe.

It is recommended that the API 20E systembe used with caution. Some additional tests may

138 Chapter 4

Fig. 4.1. Schematic for using the biochemical identification tables.



be required to give a definitive answer. Thisis particularly so for the identification of speciesfrom the genus Aeromonas. Adding conventionalmedia of aesculin, MR, and salicin in addition tothe API 20E system will improve the likelihood ofthe correct identification.

The API 20E database listed in this book is acollation from the literature. For some organismssuch as Vibrio vulnificus, many profile numberswere obtained, indicating the phenotypic strainvariation amongst species.

The reactions for the API 20E are listed inthree different formats (see Tables 4.23, 4.24 and4.25). The first format, in Table 4.23, indicates thereactions obtained for the different tests in thesame order as the API 20E test strip. The organ-isms are listed in alphabetical order. The secondformat, in Table 4.24, lists all organisms in alpha-betical order and then the corresponding API 20Eprofile number. The third format, in Table 4.25,lists the API 20E profile numbers in numericalorder. Use this table first when identifying anunknown. Then use Table 4.23 to check the posi-tive and negative reactions obtained. Tables 4.24and 4.25 should be checked to assess the similaritywith close profile numbers and to determinewhether additional tests should be done to con-firm this initial identification. In some cases thecomplete 9-digit API 20E profile number was notreported in the journal article, as the tests forgrowth on MCA and OF were not reported.In these cases the API 20E profile number isrecorded in the manual as a 7- or 8-digit profilenumber.

The numbers in the cells of the tables are thepercentage of positive strains recorded with thattest result.

Identification using API ZYM

The API ZYM tables are included because manynewly described species are tested for the

production of enzymes using this kit. It is not gen-erally considered a routine diagnostic kit; how-ever, it is useful for some bacteria from veterinaryisolations such as the species from Pasteurella,Actinobacillus and Histophilus ovis and is able todifferentiate between species when phenotypictests may be in doubt (Cousins and Lloyd, 1988).For these organisms the intensity of the colourreactions is consistent for a species and allowsspecies identification. Therefore, the correctinoculum concentration is important. It can alsobe used to confirm or back-up phenotypic testswhen identifying an unknown bacterium isolatedfrom an aquatic animal species.

Species within a genus give very similarresults for the API ZYM; however, there are oftendifferences with one or two enzymes betweenspecies that can give an indication of theidentification of the species. The intensity of thecolour reaction is important. Thus it is importantto be consistent with the inoculum density.The manufacturer recommends a density ofMcFarland tube 6.

For Flavobacterium, Flexibacter and Cyto-phaga species, an incubation time of 12 h isrecommended when incubating at a temperatureof 18–30°C (Bernardet and Grimont, 1989).For an incubation temperature of 37°C use anincubation time of 4 h as recommended by themanufacturer. In the author’s experience isolatesfrom an aquatic environment give better reactionswhen incubated for 24 h at a temperature of 25°C.However, it must be stressed that if the incubationtime and temperature is stated in the tables thenuse these parameters because the results arereported on these. Some references did not statethe time and temperature of incubation thatwas used, and therefore it is assumed that themanufacturer’s recommendations were used.

Improved results may also be obtainedif ammonium salt sugar broth is used asthe inoculating fluid for the Flavobacterium/Cytophaga group from marine and freshwatersources (Bernardet and Grimont, 1989).

Biochemical Identification Tables 139



140

Test

Gm

Ox

cat

bHm

otPi

gO

DC

LDC

ADH

Nit

Ind

Cit

urea

mr

vpae

sG

onpg

OF

arab

glu

inos

lac

mal

tm

anm

ano

sal

sor

suc

treXy

lH

2SM

CA

TCBS

DN

ase

tem

pN

aCl

0129 10

KfAm

pR

ef

Org

anis

m

Path

ogen

ic

Typi

cal

A.sa

lmon

icid

ass

p.sa

lmon

icid

aN

CM

B11

02H

G3

−+

++

−+

−+

++

−−

−+

−+

++

F+

+ g+

−−

++

++

−−

+−

−+

−+

4–30

0–3

RS

S32

2,45

0

No

grow

th37

°C,A

PI20

ELD

Cm

aybe

neg

200,

475

A.sa

lmon

icid

ass

p.sa

lmon

icid

aAT

CC

1417

4

−+

−+

−+

+−

−−

+F

−+

−+

+−

−−

−−

186

A.sa

lmon

icid

ass

p.sa

lmon

icid

a−r

++

+−

−*−

−+

+−

−−

++

−F

++ g

+−

++

+−

−+

−−

+−

+4–

300.

5–3

R45

0

Non

-pig

men

ted

stra

in

‘Aty

pica

l’

A.sa

lmon

icid

ass

p.ac

hrom

ogen

esN

CM

B11

10T

−r+

++

−+ft

−−

++

+−

−−

+−

++

F−

+ g−

−−

++

+−

−+

−−

v+

−+

4–30

0–4

RR

R45

0,53

4

No

grow

th37

°C.A

PIIn

dm

aybe

neg

200,

322

A.sa

lmon

icid

ass

p.m

asou

cida

−+

++

−−

−+

++

+−

−+

++

++

F+

+ g+

−+

++

−−

++

−+

+−

+30

0–2

RR

322,

450

No

grow

th37

°C.I

nAP

I20E

LDC

,VP

&H

2Sm

aybe

neg

A.sa

lmon

icid

ass

p.no

va−

++

+−

−−

−−

++

v−

−−

−−

F−

+ g−

−−

++

v−

−+

−−

−18

–25

RR

695

Hae

min

requ

irem

ent

A.sa

lmon

icid

ass

p.sm

ithia

−+

+25

−−

−−

−−

v−

−−

−+

+F

+−

−−v

−v

−+

−−

+5–

200–

2R

R47

,450

No

grow

th37

°CAm

eric

anee

l−

+−

−b−

−v

+−

−−

+v

F−

+−

+−

−v

−−

584

Aust

ralia

ngo

ldfis

h−r

++

−−

+b−

v+

+−

−−

−−

−v

F−

+ g−

−−

++

+−

−+

−−

−0.

5–3

R14

4,82

5

Aust

ralia

ngo

ldfis

h−

++

−−

+bt13

5

Balti

cse

atu

rbot

−r−

++

−−b

−−

v−

−−

vv

+−

F+ g

−−

−−

+R

RS

200,

832

Balti

cse

aflo

unde

r−r

−+

+−

−b−

−v

v−

−+

++

vF

+ g−

++v

++

RS

S20

0,83

2

Balti

cse

a(F

inni

sh)

floun

der

−−

++

−h−

−+

v−

−−

++

+F

++ g

−−

−+

−−

−+

−+

RS

S83

1

Balti

cse

ada

b−r

++

−−

−b−

−−

−+

−+

++

−F

+ g−

−+

++w

RS

S83

2

Balti

cse

abl

enny

−r+

+−

−+b

−−

++

+−

+−

+−

F+ g

−+

−+

+R

RR

832

Can

adia

nAt

lant

icco

d−

+−

+s+

+−

−F

−+

−+

++

Can

adia

nAt

lant

icsa

lmon

Keij

stra

in−

+−

−b−

+−

−F

−+

−+

++

186*

Tab

le4.

1.A

erom

onas

salm

onic

ida

(non

-mot

ileA

erom

onas

spp.

).



141

Can

adia

nsa

blef

ish

AS2

−+

−+b

++

−−

F−

+−

+−

+18

6*

Dan

ish

cod

−+

−+

−v

+−

−+

F+g

v+

−20

0–2

197

Dan

ish

sand

eel

−+

−+b

−−

−+

+−

F+ g

−+

+20

0–2

197

Dan

ish

turb

ot−

−+

−−

−−

−+

−+

++

F−

+ g−

−+

−−

RR

617

Engl

ish

non-

salm

onid

−+

++

−+t

−−

+−

v−

−−

v+

F+

−v

v+

++

−+

++

4–37

0–3

R40

Nor

way

min

now

−+

−−b

+−

+−

F−

−−

+w+

+w−

−+

−−

−R

S33

1

Sout

hAf

rican

rain

bow

trout

−r+

+−

+b+

−+

+F

++

−+

++

−+

2210

7

pigm

entp

ositi

ve+

−+

−+

+−

63

323

pigm

entn

egat

ive

5672

323

Envi

ronm

enta

l

A.sa

lmon

icid

ape

ctin

olyt

ica

−+

++

−+

−−

−v+

++

−+

−+

+F

++ g

+−

++

−+

++

−−

+35

0R

R61

5

VPat

25°C

.Bro

wn

pigm

ent

Furu

ncul

osis

agar

(FA)

ism

ore

sens

itive

topi

gmen

t pro

duct

ion

than

TSA,

TSA

+t.

P=

pigm

entp

rodu

ced,

b=

Pigm

entt

este

don

BA,f

=pi

gmen

ton

FA,h

=pi

gmen

ton

BHIA

,s=

pigm

entp

rodu

ctio

nsl

ow,t

=Pi

gmen

ttes

ted

onTS

A,*

=Pi

gmen

ttes

ted

onTS

Aco

ntai

ning

L-ty

rosi

ne(T

SA+

t).Th

efo

llow

ing

met

hods

are

reco

mm

ende

dso

asto

stan

dard

ize

test

ing

and

redu

cedi

scre

panc

ies

betw

een

diffe

rent

labo

rato

ries.

Car

bohy

drat

efe

rmen

t atio

nis

perfo

rmed

inph

enol

red

brot

h(D

ifco)

with

1%ca

rboh

ydra

te.H

aem

olys

isis

test

edus

ing

BAco

ntai

ning

hors

ebl

ood,

othe

rmet

hods

are

from

Cow

anan

dSt

eel(

1970

)(an

din

this

Man

ual).

Form

arin

eis

olat

esth

ead

ditio

nof

NaC

lto

afin

alco

ncen

tr atio

nof

1.5%

in‘liq

uid’

tube

ssu

chas

MR

VP,a

escu

lin,n

itrat

e,AD

H,L

DC

,OD

Cm

ayim

prov

eco

nsis

tenc

yof

resu

lts.Z

ones

of20

mm

and

grea

t era

reco

nsid

ered

sens

itive

forA

mp

and

Kf.I

ncub

ate

at20

°Can

dre

adat

7–14

days

,Dal

sgaa

rdet

al.(

1998

)(re

f200

).R

efs

197,

450,

831

and

832

also

use

thes

em

etho

ds.

Test

Gm

Ox

cat

bHm

otpi

gO

DC

LDC

ADH

Nit

Ind

Cit

urea

vpae

sG

OF

arab

glu

inos

lac

mal

tm

anm

ano

sal

sor

suc

treXy

lD

Nas

e40

°C01

29 1001

2915

0Am

p

DNA

hybr

idiz

atio

ngr

oup

HG1

−+

++

+−

−96

++

+−

−91

96+

F87

+ g+

−−

+96

++

−+

+−

++

RR

R

HG2

−+

++

+−

−59

94+

++

−83

++

F+

+ g+

−6 7

++

++

++

+−

+−

RR

R

HG3

−+

+9 2

+−

−55

90+

+46

−90

++

F+

+ g+

−5 4

6 7+

++

++

+−

+−

RR

R

HG4

−+

+1 0

+−

−−

++

9770

−−

97+

F+

+−

87+

+31

+7

++

−+

87R

RR

HG5

−+

+−

−50

−−

9 2+

++

−−

92+

F+

+−

++

++

+−

++

−+

−R

RR

HG6

−+

+−

+−

−−

8 7+

++

−−

++

F8 7

+−

++

++

+−

++

−+

−R

RR

HG8/

10−

++

9 0+

−−

57+

++

91−

9010

+F

14+ g

+−

10+

++

−−

++

−+

+R

RR

Gm

=G

ram

reac

tion,

Ox

=ox

idas

e,ca

t=ca

tala

se,b

H=

bha

emol

ysis

,mot

=m

otilit

y,pi

g=

brow

npi

gmen

t,O

DC

=or

nith

ine

deca

rbox

ylas

e,LD

C=

lysi

nede

carb

oxyl

ase,

ADH

=ar

gini

nedi

hydr

olas

e,N

it=

nitra

tere

duct

ion,

Ind

=in

dole

,Cit

=ci

trate

,ur

ea=

urea

hydr

olys

is,v

p=

Voge

s-Pr

oska

uer,

aes

=ae

scul

inhy

drol

ysis

,G=

gela

tinhy

drol

ysis

,OF

=O

xida

tive

Ferm

enta

tive,

arab

=L-

arab

inos

efe

rmen

tatio

n,gl

u=

gluc

ose

ferm

enta

tion,

inos

=in

osito

lfer

men

tatio

n,la

c=

lact

ose

ferm

ent a

tion,

mal

t=m

alto

sefe

rmen

tatio

n,m

an=

man

nito

lfer

men

tatio

n,m

ano

=m

anno

sefe

rmen

tatio

n,sa

l=sa

licin

ferm

enta

tion,

sor=

sorb

itolf

erm

enta

tion,

suc

=su

cros

efe

rmen

tatio

n,tre

=tre

halo

sefe

rmen

tat io

n,Xy

l=xy

lose

ferm

enta

tion,

Dna

se=

hydr

olys

isof

DN

A,40

°C=

grow

that

40°C

,012

9/10

=se

nsiti

vity

tovi

brio

stat

icag

ent0

129

at10

mgco

ncen

tratio

n,01

29/1

50=

sens

itivi

tyto

vibr

iost

atic

agen

t012

9at

150

mgco

ncen

tatio

n,Am

p=

sens

itivi

tyto

ampi

cillin

.Num

bers

refe

rto

perc

enta

geof

posi

tive

stra

ins,

−=

nega

tive,

+=

posi

tive

reac

tion.

HG

=hy

brid

izat

ion

grou

p.D

ata

from

Abbo

ttet

al.(

1992

),Ka

znow

ski(

1998

).

Tab

le4.

2.A

erom

onas

spp.

Phe

noty

pic

test

sac

cord

ing

toD

NA

hybr

idiz

atio

ngr

oups

.



142

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Org

anis

m

Path

ogen

ic

A.al

losa

ccha

roph

ilaH

G15

−+

++

+−

−+

++

++

−−

++

+F

++ g

+−

−+

++

−−

++

−−

++

4–4

0–3

RR

R52

7

A.be

stia

rum

HG

2−

++

++

−−

++

++

+−

++

++

+F

++ g

v−

++

++

+−

++

−+

+4–

42R

R42

7

A.ca

viae

HG

4−

++

10+

−30

−+

++

+−

+−

++

+F

++ g

−−

87+

+31

+−

++

−−

++

4–37

RR

R21

,14

2

A.hy

drop

hila

dhak

ensi

s−

++

++

−−

++

++

+−

−+

++

+F

−+ g

+−

−+

++

+−

++

−−

+−w

25–4

2R

RR

383

A.hy

drop

hila

LMG

2844

TH

G1

−+

++

+−

−+

++

+6 0

−53

++

++

F+

+ g+

−−

++

++

−+

+−

−+

−w+

4–42

0–2

RR

R1, 383,

427

A.ja

ndae

iH

G9

−+

++

+−

−+

++

+7 5

−+

+−

++

F−

+ g+

−−

++

+−

−−

+−

−+

−w+

4–42

0–3

RR

R13

5,14

2,14

3

A.ve

roni

issp

.ver

onii

HG

10−

++

++

−+

+−

++

+−

++

++

+F

−+ g

+−

−+

++

+−

++

−−

+−

+4–

420–

5R

RR

142,

347

Envi

ronm

enta

l

A.cu

licic

ola

−+

++

+−

−+

++

++

−+

−+

+F

−+ g

+−

−+

++

−−

+−

+4–

37R

RR

624

A.en

chel

eia

HG

16−

++

21+

−−

−+

++

−−

v−

++

+F

−+ g

+−

−+

++

+−

75+

−−

+−

+4–

370–

3R

RR

241,

379

A.eu

cren

ophi

laH

G6

−+

+50

+−

−−

++

+v

−+

−+

++

F+

+ g+

−+

++

++

−+

+−

−+

25–3

70–

2R

RR

1, 379,

420,

427

A.m

edia

HG

5A/5

B−

++

25−

−−

−+

++

5 5−

+−

−+

+F

++ g

−−

++

++

9 4−

++

−−

+−

+4–

370–

3R

RR

15,

294

A.po

poffi

i−

++

−w+

−−

−+

+−

70−

+−

++

F50

+ g+

−−

++

+−

−−

+−

50−

+28

–37

0–2

RR

R38

0

The

type

stra

inis

indo

le-n

egat

ive,

othe

rstra

ins

a re

indo

le-p

ositi

ve

A.so

bria

HG

7−

++

++

−−

++

++

+−

45−

−+

+F

−+ g

v−

++

++

v−

++

−+

+−

30–4

20–

5R

RR

21,

420

88%

VPpo

sin

API2

0E

Tab

le4.

3.A

erom

onas

spp.

–m

otile

.



143

A.ve

roni

ispp

.sob

riaH

G8

−+

++

+−

−+

++

+50

−+

+−

++

F−

+ g+

−5

++

+−

35+

+−

++

+4–

42R

RR

21,

142,

427,

456

A.sc

hube

rtii

HG

12−

++

++

−−

++

+−

+−

++

−+

+F

−+ g

−−

−+

−+

−−

−+

−−

−+

4–42

0–3

RR

R14

2,34

8

gela

t22 °

C,N

on-h

aem

olyt

icsh

eep

bloo

d

A.tro

taH

G14

−+

++

+−

−+

++

++

−+

−−

++

F−

+ g+

−−

++

+−

−−

+−

−+

−4–

42R

RR

142

Aero

mon

asgr

oup

501

HG

13−

++

+−

++

+−

−+

F−

+ g+

−+

23+

−+

0–3

RR

R

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Org

anis

m

Path

ogen

ic

Clo

strid

ium

botu

linum

E+r

−+

+−

−−

−−

−−

+−

−−

−−

141

Rod

sw

ithov

alsu

bter

min

alen

dosp

ores

with

appe

ndag

esan

dex

ospo

ria

Clo

strid

ium

perfr

inge

ns+r

−v

−v

−v

++

−+

−v

+v

−

Edw

ards

iella

icta

luri

oxyg

enin

tole

rant

stra

in

−−

+−

−+

+−

+−

−−

−−

−−

−F

−+ g

−−

++

−+

−−

−−

−−

+25

–37

547

Non

-mot

ileat

37°C

Euba

cter

ium

spp.

841

+r−

−−

−−

++ g

++

+−

−−

+20

0–1

343

Euba

cter

ium

spp.

1065

+r−

−−

−+

++ g

++

+−

−−

−20

0–1

343

Euba

cter

ium

tara

ntel

lae

+r−

+−

−−

−−

−−

++

−−

−−

−−

−−

+25

–37

0–1

764

Long

unbr

anch

edfil

amen

tous

rods

Tab

le4.

4.A

naer

obes

.



144

Test

Gm

Ox

cat

bHm

otC

O2

Nit

cit

ind

MR

VPH

2Sur

eaBF

SOTH

Ala

Asp

Glu

tAr

gO

rnLy

sG

alR

ibXy

lEr

yU

roA

MR

Ref

Hos

t

Org

anis

mSu

rface

Antig

ens

Path

ogen

ic

Bruc

ella

abor

tus

−+

v−

+−

−−

−v

++

++

+−

−−

++++

+++

+++

++

185,

404

Cat

tle

Bruc

ella

cani

s−

++

−−

−−

−+

−−

−+

+++

++−

+++

−−

−18

5,40

4D

ogs

Bruc

ella

ceta

ceae

−+

−−

−+

++

+−v

−+

−−

−+

++

+−

++

−26

7,40

4U

Kdo

lphi

n,po

rpoi

se

Bruc

ella

mel

itens

is−

++

−−

−−

−+

+++

++++

+−

−−

++

+++

++

+18

5,40

4Sh

eep,

goat

Bruc

ella

neot

omae

−−

++

−−

−−

++

−−

+++

−−

+++

−+

+++

+18

5,40

4D

eser

twoo

dra

t

Bruc

ella

ovis

−−

+−

−−

−−

++

+−

++

−−

−−

−+

−+

185,

404

Shee

p

Bruc

ella

pinn

iped

iae

−+

−+

−+

++

+−v

−+

−−

−−

++

+−

+−

−26

7,40

4Se

al,o

tter

Bruc

ella

spp.

−+

+−

++

++

++

−−

261

Rin

ged

seal

,har

pse

al

Bruc

ella

spp.

−+

+−

−+

+−

++

171

Min

kew

hale

Bruc

ella

suis

1−

+−

−−

−+

+−

−−

−++

++++

++++

+++

+++

+++

++

185,

404

Pigs

Bruc

ella

suis

2−

+−

−−

−−

+−

−−

+++

++

++++

+++

+++

+++

++

185,

404

Pigs

,har

e

Bruc

ella

suis

3−

+−

−−

−−

++

−−

++++

++++

−++

++

+++

+++

+18

5,40

4Pi

gs

Bruc

ella

suis

4−

+−

−−

−−

++

−−

++++

++++

−++

+−

+++

−+

+18

5,40

4R

eind

eer

Bruc

ella

suis

5−

+−

−−

−+

−+

+ +−

−+

−++

+−

+++

++

+40

4

Ala

=L-

alan

ine,

Arg

=L-

argi

nine

,Asp

=L-

aspa

ragi

ne,B

F=

grow

thon

med

iaco

ntai

ning

basi

cfu

chsi

n20

mg/m

l(1/

5000

0),b

H=

bha

emol

ysis

,CO

2=

carb

ondi

oxid

ere

quire

men

t,ca

t=ca

tala

se,c

it=

citra

te,E

ry=

mes

oery

thrit

ol,G

al=

D-g

alac

tose

,G

lut=

L-gl

utam

icac

id,G

m=

Gra

mre

actio

n,H

2S=

hydr

ogen

sulp

hide

,ind

=in

dole

,Lys

=L-

lysi

ne,M

R=

Met

hylR

ed,N

it=

nitra

tere

duct

ion,

Orn

=D

L-or

nith

ine,

Ox

=ox

idas

e,R

ib=

D-ri

bose

,SO

=gr

owth

onm

ediu

mco

ntai

ning

safra

nin

O10

0mg

/ml

(1/1

000

0),T

H=

grow

thon

med

ium

cont

aini

ngth

ioni

n20

mg/m

l(1/

5000

0),U

ro=

uroc

anic

acid

,VP

=Vo

ges

Pros

kaue

r,Xy

l=D-x

ylos

e,Su

rface

antig

ens,

A,M

&R

=ag

glut

inat

ion

with

mon

ospe

cific

antis

era.

Tab

le4.

5.B

ruce

llasp

p.

Test

Gm

Ox

cat

bHm

otC

apO

DC

LDC

ADH

Nit

Ind

Cit

urea

mr

vpae

sG

onpg

OF

arab

glu

inos

lac

mal

tm

anm

ano

sal

sor

suc

treXy

lG

Tte

mp

NaC

l

Org

anis

m

Path

ogen

ic

Cry

ptoc

occu

sne

ofor

man

sva

r.ga

ttii

+−

−+

+−

−+

++

+−

−−

+−

−20

–42

Envi

ronm

enta

l

C.l

upi

++

−−

++

−+

++

++

+4–

250–

2

No

ferm

enta

tion,

only

assi

mila

tion.

Anta

rctic

habi

t at.

Gro

wth

ongl

ucos

epe

pton

eye

aste

xtra

ct

Can

dida

spp.

+−

−

Cap

=ca

psul

e;G

T=

germ

tube

.

Tab

le4.

6.C

rypt

ococ

cus.



145

Test

Gm

Ox

cat

bHm

otG

lidO

DC

LDC

ADH

Nit

Ind

Cit

urea

mr

vpae

sG

onpg

OF

arab

glu

inos

lac

mal

tm

anm

ano

sal

sor

suc

treXy

lH

2SM

CA

CR

KOH

DN

ase

tem

pN

aCl

0129

150

NA

TSA

Ref

Org

anis

m

Path

ogen

ic

Chr

yseo

bact

eriu

mba

lust

inum

−+

+−

−v−

−−

−+v

+−

−v+

+−v

O−

+−

−−

−−

−−v

−v−

−+

−+

+v15

–35

0–2

++

89,8

02

Prev

ious

lyFl

avob

acte

rium

balu

stin

um.Y

ello

wpi

gmen

t

C.s

coph

thal

mum

−+

++

−v

−−

−−

−−

+−

−+

+F

−+

−−

+−

−−

−−

−−

++

15–2

50–

4+

556,

557

Prev

ious

lyFl

avob

acte

rium

scop

htha

lmum

.Ora

nge

pigm

ent

Flav

obac

teriu

mbr

anch

ioph

ilum

ATC

C35

035

−+

+−

−−

−−

++

O−w

+ g−

−−

+−

−v+v

−+

+−w

−−

−−

−10

–25

0–0.

2S

−−

92,8

02

Yello

wpi

gmen

t,no

agar

hydr

olys

is.G

row

thon

1/20

TSA

603

F.co

lum

nare

IAM

1430

1T

−+

+−

−+

−−

−80

−−

−−

−−

+−

O−

−−

−−

−−

−−

−v

++

++

10–3

70–

0.5

S−

−14

5,60

3

Adhe

res

stro

ngly

toag

ar.H

2Sfro

mle

adac

etat

em

ediu

m(o

ther

refe

renc

es11

1,13

9,29

8,29

9)13

5,75

9

F.hy

datis

−−v

++

−+

−−

−+

−−

−−

−+

++

F+

+−

++

++

−−

+−

+−

−−

++

20–3

00–

1R

++

92,7

20

Prev

ious

lyC

ytop

haga

aqua

tilis

.Muc

oid,

oran

ge-y

ello

wco

loni

es89

F.jo

hnso

niae

AHLD

A17

14−

++

−+

−−

−+

−+

+s−

−+

++

F−

w−

−−

−−

−−

−−

−−

−+

−S

++

135

Fjo

hnso

niae

ATC

C17

061

−+

+−

+−

−−

+−

++

++

O+

++

+−

++

−+

+w−

++

+5–

300–

1S

++

145,

603

F.jo

hnso

niae

DSM

2064

−+

++

−−

−−

−+

++

O+

−−

−+

10–3

00–

2S

+89

F.jo

hnso

niae

DSM

2958

5*−

++

+−

−−

+−

++

+O

+−

−+

+10

–30

0–1

S+

+89

F.ps

ychr

ophi

lum

−+v

+70

−w

−−

2−

−−

−−

−+

−O

−−

−−

−−

−−

−−v

−−

−−

−+

+w4–

200–

0.8

S−

−89

,92

Yello

wco

loni

es.G

row

then

hanc

edin

AOm

edia

with

0.5%

trypt

one

90,1

68

Tena

ciba

culu

m(F

lexi

bact

er)m

ariti

mus

−+

+−

++

−−

−−

−+

−I

−−

−−

−−

−−

−−

−−

−+

−+w

14–3

42+

S−

89,8

01

Pale

yello

wco

loni

es.N

oag

arhy

drol

ysis

.Pre

pare

med

iaw

ithAS

Wba

se90

T.(F

lexi

bact

er)

ovol

ytic

us−

++

−+

−−

−+

−−

−+

−O

−−

−−

−−

−−

−−

+4–

253+

−32

4

Pale

yello

wco

loni

es.N

Gon

TCBS

*Thi

sor

gani

smis

nolo

nger

inth

eD

SMZ

data

base

.N

umbe

rsre

fert

ope

rcen

tage

ofpo

sitiv

est

rain

s.1/

20TS

A=

TSA

prep

ared

ata

1:20

dilu

tion,

+s=

slow

reac

tion,

−w=

nega

tive

orw

eak

reac

tion,

Ov

=O

xida

tive

varia

ble

resu

lts,v

=va

riabl

ere

actio

n,N

G=

nogr

owt h

.

Tab

le4.

7.C

ytop

haga

–Fla

voba

cter

iace

ae–B

acte

roid

esgr

oup

–pa

thog

enic

.



146

Org

anis

m

Aequ

oriv

itaan

tarc

tica

−+

−−

−−

−−

−v+

+O

−−

−−

−−

−−

−−

−−

−0–

250.

5–6

113

Ora

nge-

pigm

ente

dco

loni

es

Aequ

oriv

itacr

ocea

−+

−−

−−

−−

−+

+O

−−

−−

−−

−−

−−

−−

+v0–

250.

5–6

113

Yello

w-p

igm

ente

dco

loni

es

Aequ

oriv

italip

olyt

ica

−+

−−

−−

−−

+v+

+O

−−

−−

−−

−−

−−

−−

−0–

250.

5–6

113

Yello

w-p

igm

ente

dco

loni

es

Aequ

oriv

itasu

blith

inco

la−

+−

−−

−−

−+

−+

O−

−−

−−

−−

−−

−−

−−

0–25

1–6

113

Ora

nge-

pigm

ente

dco

loni

es

Cel

lulo

phag

aly

tica

−+

+−

+−

−−

−−

+O

−+

−−

−15

–37

1–2

89

Chr

yseo

bact

eriu

m(F

lavo

bact

eriu

m)

gleu

m

−+

+−

−−

−−

−v

−−

v+

+I

+−

−−

−−

−−

++

+25

–37

+36

6

Indo

lepo

sw

ithEh

rlich

sbu

tneg

ativ

eby

K ová

cs

Chr

yseo

bact

eriu

min

dolo

gene

s−

++

−+

−−

−v

+−

++

vO

+−

++

−−

+−

++

+36

557

Chr

yseo

bact

eriu

min

dolth

etic

um−

−+

−+

+−

++

3655

7

Chr

yseo

bact

eriu

mm

enin

gose

ptic

um−

++

−−

−−

−−

−+

vv

−−

++

+O

−+

−+

+v

+−

−−

v−v

−w

−−

+37

–42

0–2

++

89,8

02

prev

ious

lyFl

avob

acte

rium

men

ingo

sept

icum

Cyt

opha

gaal

lerg

inae

−+

+−

−−

−−

++

O−

−+

15–2

50–

1+

89

Cyt

opha

gaar

vens

icol

a−

++

−−

−−

−+

++

O−

−+

15–3

70–

2+

89

Cyt

opha

gafe

rmen

tans

−+

v−

−−

−−

−+

+F

++

−−

−15

–25

1–2

89,1

62

Cyt

opha

gahu

tchi

nson

ii−

++

−+

−−

−−

−+

v−

O+

−−

+−

15–2

50–

189

pigm

ente

dco

loni

es.D

iges

tsce

llulo

se

Cyt

opha

gala

terc

ula

−+

−−

−−

−+

+−

O+

++

+15

–30

1–2

89,4

94

Brig

htre

dpi

gmen

t

Cyt

opha

ga-li

ke−

++

++

−−

−−

−26

+−

−+

+O

+−

−−

−−

+15

–25

0–4

556

Ora

nge

colo

nies

.No

agar

dige

stio

n

Cyt

opha

gam

arin

ofla

va−

++

+F

++

−16

2

Test

Gm

Ox

cat

bHm

otG

lidO

DC

LDC

ADH

Nit

Ind

Cit

urea

mr

vpae

sG

onpg

OF

arab

glu

inos

lac

mal

tm

anm

ano

sal

sor

suc

treXy

lH

2SM

CA

CR

KOH

Das

ete

mp

NaC

l01

2915

0N

ATS

AR

ef

Tab

le4.

8.C

ytop

haga

–Fla

voba

cter

iace

ae–B

acte

roid

esgr

oup

–en

viro

nmen

tal.



147

Empe

doba

cter

(Fla

voba

cter

ium

)br

evis

−+

+−

−−

−−

−−

+−

−−

+−

O−

−v−

−+

−−

−−

−−

−−

+−

++

28–3

70–

2+

+89

,363

Pigm

ente

dco

loni

es,f

ruity

odou

r80

2

Flav

obac

teriu

maq

uatil

e−

++

−+

−−

−+v

−−

−+v

+v+v

O−

+−

++

−−

−+

−−

−−

−−

−5–

300–

0.5

R−v

+92

,802

89,6

03

Flav

obac

teriu

mfle

vens

e−

++

−+

−−

−−v

+−

+O

+−

−−

−15

–22

0–2

S+

+89

,533

Hyd

roly

ses

agar

92

Flav

obac

teriu

mfri

gida

rium

−+

+−

−−

−−

−−

−+

+−

O−

−−

−−

−+

−−

15–2

80–

5+

+37

6

Flav

obac

teriu

mgi

llisia

e−

−+

−−

−+

−−

−−

−+

−−

O−

+−

−+

++

−+

+−

−−

−−

0–27

0–5

R+

+53

3

Flav

obac

teriu

mhi

bern

um−

−+

+−

−−

+−

−−

−+

++

F+

+−

−−

+−

−+

+0–

300–

2+

+53

2,53

3

Flav

obac

teriu

mm

izut

aii

−+

+−

−v−

+v−

+−

O+

++

−+

++

−−

30–3

7+

+84

4

Flav

obac

teriu

mpe

ctin

ovor

um−

++

++

++

+v

−+

+25

0–1

S+

+92

,533

Flav

obac

teriu

msa

ccha

roph

ilum

−−

++

++

++

−+

−+

−4–

300–

2S

++

92

Hyd

roly

ses

agar

Flav

obac

teriu

msu

ccin

ican

s−

++

+v

++

+F

+w

−−

−+

250

S+

+92

,162

,53

3

Flav

obac

teriu

mte

getin

cola

−−

++

−−

−−

−−

−−

−−

−O

−+

−−

+−

−−

−−

−−

−0–

271–

5R

++

533

Flav

obac

teriu

mxa

nthu

m−

++

−−

−−

+−

−−

−+

+−

O−

+−

−+

++

−+

+−

+−

−−

0–20

2R

++

533

Flex

ibac

tera

ggre

gans

−+

−−

+−

−−

−−

+O

++

+−

−22

–37

1–2

89

Hyd

roly

ses

agar

Flex

ibac

terc

anad

ensi

s−

++

−−

+−

−−

+−

−−

−+

+F

++

−−

−v−

−−

+10

–40

0–1

+89

Flex

ibac

tere

lega

ns−

+−

−−

+O

−−

−22

–25

0–2

162

Flex

ibac

terf

lexi

lis−

+−

−+

−−

−−

−+

−O

−+

−+

−−

+−

−−

−−

5–30

0–0.

2−

−89

,603

Cel

ls15

–20

mm,O

rang

e-pi

gmen

ted

colo

nies

Flex

ibac

terl

itora

lis−

+−

−−

−−

−+

−O

−−

+−

−15

–25

2–5

89,1

62

Pink

pigm

ent

cont

inue

d



148

Flex

ibac

ter

poly

mor

phus

−+

−−

+−

−−

+v

+O

−−

−22

–32

289

Cel

ls>1

00mm

.Hyd

roly

ses

agar

.Pea

ch-c

olou

red

pigm

ent

Flex

ibac

terr

oseo

lus

−+

−−

−−

−−

+−

O−

−−

−−

22–3

70–

2+

+89

,162

Cel

ls50

–100

mm.B

right

-red

oran

gepi

gmen

t

Flex

ibac

terr

uber

−+

−−

−−

−−

+−

O+

+−

−−

22–3

70–

2+

+89

,162

Brig

htre

d-or

ange

pigm

ent.

Cel

ls50

–100

mm

Flex

ibac

ters

anct

i−

++

−−

−+

+O

−+

+15

–37

0–1

+89

Flex

ibac

tert

ract

uosu

s−

++

−−

−−

−+

−O

−/+

−−

22–3

70–

289

,162

Ora

nge

pigm

ent

Mar

inila

bilia

salm

onic

olor

−+

+−

−−

−−

++

F+

++

−−

15–3

71–

289

,162

Salm

on-p

ink

colo

ur

Mar

inob

acte

rhy

droc

arbo

nocl

astic

us−

++

−+

+−

−40

–45

269

Prev

ious

lyPs

eudo

mon

asna

utic

a

Myr

oide

sod

orat

imim

us−

++

−−

−−

−−

−+

−+

−O

−+

18–3

789

,362

Myr

oide

sod

orat

us−

++

−−

−−

−−

−−

−+

−+

−al

k−

−−

−−

−−

−−

−−

−+

−−

+18

–37

0–2

++

89,3

62

Prev

ious

lyFl

avob

acte

rium

odor

atum

,fru

ityod

our

802

Pedo

bact

erhe

parin

us−

++

+s−

−−

−−

v−

−v

−−

+−

+O

−+s

++

−−

++

−+

−−

−−

+5–

370–

3+

+89

,163

,72

8

Pedo

bact

erpi

sciu

m−

++

−−

−−

−+

−O

−−

−+

−−

+−

+5–

28+

+72

8

Sale

gent

ibac

ter

sale

gens

−+

+−

−O

−−

−−

−−

0–30

0–20

R53

3

Sphi

ngob

acte

rium

mul

tivor

um−

++

−−

−−

−−

−−

++

14+

O+

+v−

++

−+

+−

++

+−

+−

−60

15–3

70–

2+

+89

,802

Sphi

ngob

acte

rium

spiri

tivor

um−

++

−−

−−

−−

−−

−+

++

+O

27+v

−+v

++

++

−+

++

−+

−−

+15

–37

0–2

++

89,3

65,

802

Zobe

lliaga

lact

aniv

oran

s−

++

−+

−+

−−

−−

++

+O

++

++

+−

+−

++

13–4

50.

5–6

61

Zobe

lliaul

igin

osa

−+

+−

+−

+−

−+

++

O+

+−

++

+13

–30

0.5–

261

alk

=al

kalin

ere

actio

n,C

R=

Con

goR

ed,G

lid=

glid

ing

mot

ility,

KOH

=po

tass

ium

hydr

oxid

e,N

A=

grow

thon

nutri

enta

gar,

TSA

=gr

owth

ontry

pton

eso

yag

a r.

Test

Gm

Ox

cat

bHm

otG

lidO

DC

LDC

ADH

Nit

Ind

Cit

urea

mr

vpae

sG

onpg

OF

arab

glu

inos

lac

mal

tm

anm

ano

sal

sor

suc

treXy

lH

2SM

CA

CR

KOH

Das

ete

mp

NaC

l01

2915

0N

ATS

AR

ef

Tab

le4.

8.C

ontin

ued.



149

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Org

anis

m

Path

ogen

ic

Acin

etob

acte

rsp

p.−c

r+

++

−−

−−

−−

−−

−−

−F

−−

−−

+−

+−

−−

−41

Not

e:Ac

inet

obac

terd

ono

tusu

ally

prod

uce

oxid

ase

Mor

axel

lasp

p.cb

++

+−

−−

−−

−−

−−

−−

−−

−−

−+w

−−

−−

72

Hae

mol

ysis

test

edon

shee

pbl

ood

Taxo

nom

icpo

sitio

nof

both

thes

eor

gani

sms

isin

doub

t

Envi

ronm

enta

l

Acin

etob

acte

rba

uman

nii

−−

+−

−−

−v

−−

−O

+−

−15

–37

110

Acin

etob

acte

rca

lcoa

cetic

us−

−+

−−

−−

+−

−−

O+

−−

15–3

711

0

Acin

etob

acte

rha

emol

ytic

us−

−+

+−

−−

+−

+−

O52

−−

15–3

711

0

Mes

ophi

loba

cter

mar

inus

−cb

++

−−

−+

v+

v+

−v

O+

+−

5–37

1–6

583

Gro

wth

onN

A,M

A22

16

Tab

le4.

9.G

ram

-neg

ativ

eco

ccob

acill

i and

cocc

i.

Org

anis

m

Path

ogen

ic

Citr

obac

terf

reun

dii

−−

++

−12

−v

+−

+−

+−

−−

+F

++ g

+−

++

+−

+10

++

++

−20

–37

0–2

414,

425

Edw

ards

iella

hosh

inae

−−

+−

+−

++

−+

w+

−−

+−

−−

−F

30+ g

v−

−+

++

7 5−

++

−+

+−

25–4

00–

1.5

S31

7

Mot

ileat

25°C

and

35°C

Edw

ards

iella

icta

luri

−−

+−

+−

++

−+

−−

−+

−−

−−

F−

+ g+

−−

+−

+−

−−

−−

−+

−20

–37

0–1.

533

4,37

4

Wea

kly

mot

ileat

25°C

butn

otat

35°C

.Rea

dM

Rat

48h

808

Edw

ards

iella

icta

luri

oxyg

enin

tole

rant

stra

in

−−

+−

−+

+−

+−

−−

−−

−−

−F

−+ g

−−

++

−+

−−

−−

−−

+25

–37

547

Cul

ture

anae

robi

cally

.Non

-mot

ileat

37°C

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Tab

le4.

10.

Gra

m-n

egat

ive,

oxid

ase-

nega

tive

rods

.

cont

inue

d



150

Edw

ards

iella

tard

a−

−+

−+

−+

+−

++

−−

+−

−−

−F

−+ g

+−

−+

−+

−−

v−

−+

+−

420–

237

4,64

0

Mot

ileat

25°C

and

35°C

Esch

eric

hia

vuln

eris

−−

++

+−

++

+−

−−

+−

+−

F+

+ g+

−+

++

+−

−+

+−

+−

−20

–37

RR

51

Haf

nia

alve

i−s

r−

+−

+−

++

−+

−−

−21

87−

−+

F+

+ g+

−−

++

+−

−−

++

−+

−−

4–40

0–4

RPS

313,

652

Hal

omon

ascu

pida

−−

+−

++

++

+−

−−

I+

++

++

+ w+

−10

–25

S

Kleb

siel

lapn

eum

onia

e−

−+

−−

−−

+v−

+−

++

6040

+−

+F

++ g

++

++

++

++

++

−+

−35

0–5

RR

414

Muc

oid

Pant

oea

aggl

omer

ans

−−

+−

+−

−−

−+

−+

−−

++

++

F+

+ g−

−−

++

++

−+

++

−+

−−

4–37

0–6

249,

325

Opp

ortu

nist

,yel

low

pigm

ent,

MR

+ve

with

2%N

aCl

Prov

iden

cia

(Pro

teus

)ret

tger

i−

−+

+−

−−

−+

++

++

−+

−−

F−

++

−−

++

v−

−−

−−

++

18–3

70–

379

Salm

onel

laar

izon

ae−

−+

−+

−+

+−

+−

+−

−−

−F

−+ g

++

++

−+

++

++

+15

–41

0–6

RR

447

Serra

tialiq

uefa

cien

s−

−+

++

−+

+−

−−

+−

−+

++

+F

++ g

++

−+

++

++

++

+−

+−

+4–

370–

5R

R25

0,29

0

Ref

290

isM

R+8

6%at

37°C

,17%

at30

°C71

5

Serra

tialiq

uefa

cien

s−

−+

++

−+

+−

−−

+−

−+

++

+F

++ g

+−

−+

++

++

++

+−

+−

+4–

370–

5R

R71

5

Arct

icch

aris

olat

esar

ein

dole

nega

tive,

inos

nega

t ive

Serra

tiam

arce

scen

s−

−+

v+

−+

+−

+−

+−

−+

+v

+F

−+ g

++

−+

++

++

++

−−

+4–

450–

8R

RR

76

Red

pigm

ent

Serra

tiapl

ymut

hica

−−

+−

−−

31−

−+

−+

−−

++

+F

++ g

−+

−+

++

+v

++

+−

−+

4–37

0–8

RR

R76

,57

9

Red

pigm

ent.

MR

+ve

at37

°Con

ly

Stre

ptob

acillu

sm

onilif

orm

is−

−−

−−

−−

+−

v−

−−

−−

−F

+−

+−

−−

−v

10–2

51–

451

9,61

1

IDof

orga

nism

was

notc

oncl

usiv

e

Stre

ptob

acillu

sm

onilif

orm

is−

−−

−−

−−

−−

−−

−−

−−

F−

w−

w−

−−

−−

−25

–35

169

Rea

ctio

nsof

type

stra

in.R

equi

res

addi

tion

of20

%se

rum

forg

row

th

Yers

inia

inte

rmed

ia−

−+

++

−−

++

++

++w

+−

+F

++

+−

++

++

++

+50

−−

402

Mot

ileat

25°C

butn

otat

35°C

.MR

,VP,

cit+

veat

35°C

,−ve

at25

°C

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Tab

le4.

10.

Con

tinue

d.



151

Yers

inia

ruck

eri

−−

++

+−

++

−85

−+

−+

−−

52+

F−

+ g−

−−

++

97−

−−

97−

−+

−37

0–3

137,

250

Mot

ileat

25°C

butn

otat

35°C

.Citr

ate

+ve

at25

°Cbu

t−ve

at37

°C50

0,65

7

Y.ru

cker

i−

−+

−+

−+

+−

++

+−

+−

++

F−

+−

−+

++

−+

−+

−+

−0–

550

0

Aust

ralia

nst

rain

repo

rted

byLl

ewel

lyn

(198

0)

Y.ru

cker

iser

ovar

I−

Y.ru

cker

iser

ovar

II+

Y.ru

cker

iser

ovar

III−

Y.ru

cker

iser

ovar

IVTh

ese

are

nots

train

sof

Y.ru

cker

i+

+

Y.ru

cker

iser

ovar

V+

Y.ru

cker

iser

ovar

VI

Fish

isol

ates

that

ferm

enta

rab

&rh

amno

sear

eno

tY.r

ucke

ri,bu

tmay

beH

afni

aal

vei

Envi

ronm

enta

l

Budv

icia

aqua

tica

−−

++

−−

−−

+−

−+

v−

−−w

F+

+−

−+ s

−−

−−

−+

++

−4–

370–

4

Citr

obac

terd

iver

sus

−−

++

−+

−60

++

++

+−

−−

+F

++ g

+−

33+

+10

+17

++

−R

R24

8

Ente

roba

cter

aero

gene

s−

−+

+−

++

−−

−−

++

−+

+ g+

++

+40

++

++

−−

0–5

414

Ente

roba

cter

cloa

cae

−−

+−

+−

+−

++

−+

−−

++

++

F+

+ g+

70+

++

++

++

+−

+−

−0–

5R

R41

4

Ente

roba

cter

lique

faci

ens

−−

++

−+

65−

−−

25+

++

++ g

++

60+

++

++

+−

+0–

541

4

Esch

eric

hia

coli

−−

+v

50−

6050

−+

+−

−+

−40

−+

+ gv

−55

+30

+55

++

−−

25–4

40–

541

4

Hal

omon

asel

onga

ta−

+v+

+−

++

+22

6611

1156

4433

F+

67+

+−

300.

1–20

R79

5

Hal

omon

asha

lodu

rans

−−

+−

++

−−

+−

+−

F+

+−

−+

−22

–37

0.1–

2033

6

Hal

omon

asm

arin

a−

Kleb

siel

laor

nith

inol

ytic

a−

−+

−+

+v

F10

–37

228

Kleb

siel

laox

ytoc

a−

−+

−−

++

++

F10

–41

228

Kleb

siel

lapn

eum

onia

eoz

aena

e−

−+

−−

−−

−+

−+

−+

−+

−+

+ g−

−+

++

+−

++

−+

−25

–37

0–5

414

Kleb

siel

larh

inos

cler

omat

is−

−+

−−

−−

−−

+−

+−

+−

−−

F+

+ g−

+−

++

++

++

−+

−25

–37

0–5

414

Kluy

vera

asco

rbat

a−

−+

+−

++

−+

++

−+

−−

+F

+ g+

+41

+v−

5−Kl

uyve

racr

yocr

esce

ns−

−+

+−

++

−+

++

−+

−−

+F

+ g+

+41

+v−

5+

cont

inue

d



152

Pant

oea

disp

ersa

−−

+−

10−

−+

−+

−+

−+ s

+F

++ g

−+

−+

++

−−

++

+−

−30

–41

0R

R29

1

Yello

wpi

gmen

t.Ty

pest

rain

isO

DC

+ve

Prov

iden

cia

fried

eric

iana

−−

+−

+rt

−−

−−

++

w−

+−

−−

−F

−+ g

+−

−−

−+

−−

+ s−

−−

+−

10–4

00

559

Mot

ileat

25°C

butn

egat

37°C

Rah

nella

aqua

tilis

−−

+−

−−

−+

−+

−89

++

−+

F+

+ g+

−+

++

++

++

++

−+

−−

4–37

0R

R12

4

Mot

ileat

25°C

butn

otat

35°C

.Man

=−v

eby

API2

0E

R.a

quat

ilisG

enos

peci

es2

−−

31−

−−

−+

−79

−+

++

34+

F+

+ g+

−+

++

++

++

++

−+

−−

124

R.a

quat

ilisG

enos

peci

es3

−−

−−

−−

−+

−−

−52

72+

−+

F+

+ g+

−+

++

++

++

++

−+

−−

124

Rao

ulte

llapl

antic

ola

−−

+−

−−

−+

−+

++

++

++

−+

F+

+ g+

++

++

++

++

++

−+

−−

4–41

022

8

Rao

ulte

llate

rrige

na−

−+

−−

−−

+−

+−

++

++

+−

+F

++ g

++

++

++

++

++

+−

+−

4–35

0R

R22

8,40

2

Sim

ilarm

orph

olog

ical

lyto

K.pn

eum

onia

e

Serra

tiafo

ntic

ola

−−

++

−+

+−

+−

+−

+−

+−

+F

++ g

++

++

++

++

+ s+

70−

+−

4–37

290,

558

Serra

tialiq

uefa

cien

s−

−+

−+

−− v

+−

+−

+v

37+

++

F+

+ g+

6416

++

++

++

++

−+

−+

500

Serra

tiam

arce

scen

s−

−+

−+

−+

++

4018

+73

70F

27−

7717

++

+−

+29

0

Serra

tiaru

bida

ea−

−+

+−

−+

−+

2 4+

++

F+

−35

++

−+

++

250,

290

Yers

inia

aldo

vae

−−

+−

+ v−

+−

−+

−+

++

++

−+

F+

++

− v+

++

−+

++

+−

85

Yers

inia

berc

ovie

ri−

−+

−+

−+

−−

+−

−+

+−

+ s−

+F

++

−−

++

++ s

++

++

−+

−81

2

Yers

inia

ente

roco

litic

a−

−+

−−

−v

−+

v−

++

+15

−+

F+

−−

++

+36

250

Yers

inia

frede

rikse

nii

−−

+−

+−

+−

−+

+−v

++

++

−+

F+

+44

22+

++

++

++

20−

−25

–37

042

3

Mot

ility

&VP

posi

tive

at25

°C,b

utne

gativ

eat

37°C

Yers

inia

kris

tens

enii

−−

+−

+−

+−

−+

43−

++

−−

−+

F+

+ g−

2760

++

+−

+−

++

−−

4–41

038

3,40

2

Mot

ileat

25°C

butn

otat

35°C

.VP

−ve

at25

°CYe

rsin

iam

olla

retii

−−

+−

+−

+−

−+

−−

++

−+ s

−F

++

+ s−

++

++ s

++

++

−+

−81

2

Yers

inia

pseu

dotu

berc

ulos

is−

−+

+−

−−

−+

−−

++

−+

−F

++

−−

++

++

−−

++

−v

121

Yers

inia

rohd

ei−

−+

−+

−+ s

−−

+−

++ s

+−

−−

F+

+−

++

++

−+

++

+−

+−

−25

0R

R12

All Y

ersi

nia

and

Haf

nia

spec

ies

shou

ldbe

incu

bate

dat

25–2

8°C

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Tab

le4.

10.

Con

tinue

d.



Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Tab

le4.

11.

Gra

m-n

egat

ive,

oxid

ase-

posi

tive

rods

.

153

Org

anis

m

Path

ogen

ic

Bene

chea

chiti

novo

ra−

?+

+−

++

++

−+

F+ g

++

+20

–25

1–4

806

Bord

etel

labr

onch

isep

tica

−+

++

+−

−−

−+

−+

+−

−−

−O

−−

−−

−−

−−

−−

−+

25–3

70–

419

9,64

2

Bruc

ella

abor

tus

−+

+v

−+

−−

+s−

−O

v−

−−

−+

−v

350

169

Burk

hold

eria

pseu

dom

alle

i−

++

v+

−−

−+

+−

v−

−−

+v

−O

++

++

++

+−

+−

++

−+

20–4

238

,62

3

APIr

eact

ions

may

bene

gfo

rure

a&

Nit,

18%

pos

forc

it

Del

eya

aqua

mar

inus

−+

+−

+−

+−

+−

−+

−−

−−

−O

++s

−4–

421–

10S

8,45

Prev

ious

lyAl

calig

enes

faec

alis

hom

ari

Jant

hino

bact

eriu

mliv

idum

−+

++

−+

+−

+−

++

+−

O+s

−+

−+

++

++

+−

+s4–

300–

249

6

Purp

lepi

gmen

t

Past

eure

llam

ulto

cida

−+

+−

−−

+−

−+

+−

−−

−−

−−

Fv

+−

v+

−v

+v

v−

−25

–37

016

9,56

2

Past

eure

llask

yens

is−

+w−

−w−

−+

+−

−+

−−

−F

+−

++

++

−−

−+

−−

14–3

21.

5–2

100

Past

eure

llate

stud

inis

−+

++

−−

−−

−+

+−

−+

+F

v+ g

−+s

−85

+−

−v

+v

+v

709

Ples

iom

onas

shig

ello

ides

−+

+9 0

+−

++

9 5+

5 0−c

−v

−−

−+

F−

+ gv

50+

+50

+−

50−

+−

−+

−G50

10–4

20–

3R

SS

169

Pseu

doal

tero

mon

asba

cter

ioly

tica

−+

+w+

−−

−+

O+

15–3

53

677

Red

pigm

ent

Pseu

doal

tero

mon

aspi

scic

ida

−+

++

−−

+−

+O

+−

+−

+−

+25

–40

5–10

134,

572

Pseu

dom

onas

angu

illise

ptic

a−

++

−+

−−

−−

−−

+−

−v

−I

−−

−−

−−

−−

−−

−+

−v

5–30

0–3

SR

Pen

=R

96,

541

Finn

ish

isol

ates

are

gelp

os,J

apan

ese

isol

ates

are

geln

eg82

8

Pseu

dom

onas

chlo

rora

phis

−+

++

−v

v+

++

+O

+−

+−

++

4–35

Gre

enco

loni

es.5

days

forg

row

th

Ps.f

luor

esce

nsbi

ovar

I−

++

+−

−+

−+

+−

O+

+−

++

+−

++

++

4–37

cont

inue

d



154

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Tab

le4.

11.

Con

tinue

d.

Ps.f

luor

esce

nsbi

ovar

II−

++

+−

−+

++

+−

O+

+−

++

+−

++

++

4–37

Ps.f

luor

esce

nsbi

ovar

III−

++

+−

−+

++

+−

O+

+−

+v

+−

v−

++

4–37

Ps.f

luor

esce

nsbi

ovar

IV−

++

+−

−+

++

+−

O+

+−

++

+−

++

++

4–37

Ps.f

luor

esce

nsbi

ovar

V−

++

+−

−+

−+

+−

O+

+−

+v

+−

vv

++

4v−3

7

Pseu

dom

onas

fluor

esce

ns−

++

++

−−

+v

−+

v−

−−

+−

O+

+v

−+

++

−v

vv

+−

+4–

30R

R62

3

Pseu

dom

onas

plec

oglo

ssic

ida

−+

++

+−

−−

++

−+

−−

−O

−10

–30

0–5

RR

582

Clo

sely

rela

ted

toPs

eudo

mon

aspu

tida

Pseu

dom

onas

pseu

doal

calig

enes

−+

+−

+−

v−

+v

−+

−−

−v

−O

A+

+−

−14

−−

−−

11−

+−

15–4

10

297

Pseu

dom

onas

putid

a−

++

v+

−−

−+

−−

+44

−−

−O

++

−−

2119

+−

−v9

−+

−+

4–35

0–5

297,

623

Pseu

dom

onas

stut

zeri

−+

+−

+−

−−

−+

−+

14−

v−

Ov

+−v

−+

68+

−−

v−

+−v

+−

25–4

10–

629

7,35

9

Wrin

kled

colo

nies

,slig

htly

yello

w62

3

Ros

eoba

cter

stra

inC

VSP

−+

w+

−I

232

104

Shew

anel

lapu

trefa

cien

s−

++

−+

−+

−−

+−

+−

−−

−+

−O

50+s

−−

70−

9−

−35

−−

++

−+

4–37

0–3

433

Varra

calb

imi

−+

−−a

−−

−−

+−

−−

−−

F−

+ g−

++

++

+4–

222

SS

771

Envi

ronm

enta

l

Actin

obac

illus

delp

hini

cola

−+

−−w

−−

++

−+

−−

−+

−−

F+

−−

−−

+−

−−

−−

−30

–42

263

Stra

infro

mM

esop

lode

nbi

dens

.10%

CO

2re

quire

dfo

rgro

wth

Actin

obac

illus

delp

hini

cola

−+

−−w

−−

70+

21+

−−

−+

−−

F+

−−

−−

+−

−−

−−

−30

–42

263

Stra

ins

from

Phoc

oena

phoc

oena

.10%

CO

2re

quire

dfo

rgro

wth

Actin

obac

illus

delp

hini

cola

−+

−−w

−−

−−

++

−−

−+

−−

F+

−−

−−

+−

−−

−−

−30

–42

263

Stra

ins

from

Sten

ella

coer

uleo

alba

.CO

2re

quire

dfo

rgro

wth

Actin

obac

illus

scot

iae

−+

−−w

−−

70−

−+

−−

++

−+

F−

+−

+−

−+

−−

−−

−−

−25

–37

026

5

10%

CO

2re

quire

dfo

rgro

wth

.Nit

=ne

gus

ing

Ros

cota

blet

s



155

Achr

omob

acte

rxy

loso

xida

nsde

nitri

fican

s

−+

+v

+−

−−

−+

−+

−−

−−

−O

−−

−−

−−

−−

+−

10–3

70–

429

7

Prev

ious

lyAl

calig

enes

deni

trific

ans

Allo

mon

asen

teric

a−

++

++v

70−

80+

+−

+−

+F

++ g

−−

++

−+

+−

20–3

73–

5

Alte

rom

onas

mac

leod

ii−

++

+−

−−

−+

O+

++

−−

+−

++

2035

–40

2R

R28

6,81

5

Aqua

spiri

llum

spp.

−+

++

−−

−+

−−

+−

−−

I−

−−

+−

−+

−−

−25

0–2

RR

497

Brev

undi

mon

asdi

min

uta

−+

+a

+−

−−

−−

−−

−−

−−

O−

−v−

−−

−−

−−

−−

−−

++1

430

–37

0–5

Brev

undi

mon

as(P

seud

omon

as)

vesi

cula

ris

−+

+a

+−

−−

−−

−−

−+

5852

O+

−46

−−

−−

−−

16−

20−

250–

529

7

Yello

wco

loni

es

Burk

hold

eria

(Pse

udom

onas

)ce

paci

a

−v+

+−

+−

66+

−−

−+

4228

7379

O+v

++

++

−81

+−

+−

25–4

00

RR

297

Fres

hwat

er,y

ello

wpi

gmen

t

Chr

omob

acte

rium

viol

aceu

m−

++

+ v+

−−

−+

+2 0

+−

37−

−86

−F

−+ g

−−

−−

−+

−−

+−

−25

–37

0R

R48

2

Viol

etpi

gmen

t.So

me

non-

pigm

ente

dst

rain

s

Col

wel

liam

aris

−+

++

−+

−−

−+

O−

+−

−−

+0–

223–

4S

S

Hal

omon

asaq

uam

arin

a−

++

−+

−−

−−

−−

+−

−−

−−

−O

−−

−−

−−

−+

−25

–37

0–4

169,

297

Prev

ious

lyAl

calig

enes

faec

alis

Hal

omon

asve

nust

a−

++

−+

−−

−−

+−

++

+−

O−

++

−v

−−

vv

v−

+−

4–37

0–6.

5R

S66

,31

0

Hyd

roge

noph

aga

(Pse

udom

onas

)fla

va

−+

w−

−−

−−

−O

++ g

−+

++

++

+−

−39

Hyd

roge

noph

aga

(Pse

udom

onas

)pa

llero

nii

−+

w+

−−

−−

−+

−−

O−

−−

−−

−−

−−

−−

+30

39,

834

Yello

wpi

gmen

t

Hyd

roge

noph

aga

pseu

dofla

va−

+w

++

+−

−+

−−

−O

++ g

−−

++

+−

++

++

35–4

139

,83

4

Yello

wpi

gmen

t.N

itrat

epo

sw

ithye

aste

xtra

ct

Iodo

bact

erflu

viat

ilis−

++

+ w+

−−

−+

85−

−+

F−

+ g−

−−

+−

+−

−−

+−

−+

4–25

0–1

RR

R50

2

Viol

eton

MC

A.Sp

read

ing

colo

nies

on1/

4N

A

Man

nhei

mia

haem

olyt

ica

−+

++

−−

−−

−+

−v−

−−

−+

F−

++

+−

+−

++w

−20

–37

cont

inue

d



156

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Tab

le4.

11.

Con

tinue

d.

Mar

inom

onas

com

mun

is−

+−

+−

O+

−+

++

−+

−25

+35

–40

286

Mar

inom

onas

vaga

−−

−−

+−

+−

40+

+−

+−

2060

3528

6

Oce

anim

onas

baum

anni

i−

++

−−

+−

+−

−−

−O

−10

–41

1–7

130

Oce

anim

onas

doud

orof

fii−

++

−−

+−

+−

−−

−O

−10

–41

269

,13

0

Nitr

ate

may

bene

gby

tube

test

,pos

byAP

I20N

E

Phoc

oeno

bact

erut

eri

−+

−−

−−

−−

−+

−−

−+

−+

F−

+−

−−

−−

−−

−−

−−

22–3

726

6

Pseu

doal

tero

mon

asan

tarc

tica

−+

++

−−

−−

−−

+−

+−

I−

+−

++

+−

−−

−+

−4–

300.

1–9

115

Pseu

doal

tero

mon

asci

trea

−+

++

+−

−−

−−

−−

−−

−+

−O

+−

−−

+−

−−

+−

+10

–30

1–10

RR

285

Pseu

doal

tero

mon

asde

nitri

fican

s−

+−

+−

+−

+O

+4–

221.

5–5

RR

239

Red

pigm

ent

Pseu

doal

tero

mon

asdi

stin

cta

−+

+

Pseu

doal

tero

mon

asel

yako

vii

−+

++

−−

++

O+

10–3

72

679

Pseu

doal

tero

mon

ases

pejia

na−

++

−−

+35

215

0

Pseu

doal

tero

mon

asfla

vipu

lchr

a−

++

++

−−

−−

−−

−−

−−

+−

O+

−−

+−

−−

+−

+10

–44

0.5–

10R

R28

6,39

8

Pseu

doal

tero

mon

asha

lopl

ankt

isha

lopl

ankt

is

−+

+w+

−−

−+

++

O+

−+

50+

−−

++

−+

4–15

228

5

Pseu

doal

tero

mon

asha

lopl

ankt

iste

traod

onis

−+

++

−−

+O

4–15

228

5

Pseu

doal

tero

mon

aslu

teov

iola

cea

−+

−−

−−

−−

−+

+−

O+ v

−+

−−

−+

−−

+10

–30

2–4

RR

287

Prod

uces

avi

olet

pigm

ent

Pseu

doal

term

onas

mar

ical

oris

−+

++

−−

−+

O10

–37

0.5–

10R

R39

8



157

Pseu

doal

tero

mon

asni

grifa

cien

s−

++

−−

−+

+O

4–28

239

5

Prod

uces

blac

kpi

gmen

t

Pseu

doal

tero

mon

asru

bra

−+

++

+−

−−

−−

−−

++

O+

−−

−+

−−

−+

+25

–35

2R

R28

3

Red

pigm

ent

Pseu

doal

tero

mon

asul

vae

−+

++

−−

−−

+−

+−

I−

4–25

1–2

SS

S23

1

Pseu

doal

tero

mon

asun

dina

−+

−−

+O

−−

−−

−+

25–3

52

150

Pseu

dom

onas

acid

ovor

ans

−+

+−v

+−

−−

−+

−+

−−

−v−

I−

−−

+w−

−−

v+

−25

–35

029

7

Pseu

dom

onas

aeru

gino

sa−

++

−+

−−

−+

+−

++v

−+

−O

++v

−−

−+

80−

−−

v83

−+

−25

–41

0–5

RR

623

Gre

enpi

gmen

t.M

ayha

vebr

ick-

red

pigm

ent

Pseu

dom

onas

alca

ligen

es−

++

14+

−−

−+

+−

v−

−−

O−

−−

−−

−−

−−

−−

−+

−25

–41

0–5

169

Rod

orfil

amen

t

Pseu

dom

onas

aure

ofac

iens

−+

++

−+

−+

+v

−+

−v

v4–

37

Ora

nge

pigm

ent

Pseu

dom

onas

men

doci

na−

++

++

−−

−+

+−

+50

−−

−O

+−

−−

+−

−−

−+

++

−20

–41

0–6

297

Yello

wpi

gmen

t

Pseu

dom

onas

mes

ophi

lica

−+

+−

+−

v−

−v

−−

O+ w

−−

−−

−−

v25

–35

0

Fatc

ells

,ple

omor

phic

,vac

uola

ted.

Cor

al-p

ink

pigm

ent

Pseu

dom

onas

perfe

ctom

arin

a−

+−

−+

+−

+40

2

Pseu

dom

onas

stan

ieri

−+

+−

−+

−−

402

Ros

eoba

cter

galla

ecie

nsis

−+

++

−−

−−

−−

−−

O+

++

++

15–3

72

662

Ovo

idro

ds,b

row

ndi

ffusi

ble

pigm

ent

Salin

ivib

rioco

stic

ola

ssp.

cost

icol

a

−cv

++

++

−−

−+

12−

20−

53+

++

−F

−+ g

−−

−+

+50

20−

++

−80

+5–

450.

5–20

RS

S27

9

Prev

ious

lyVi

brio

cost

icol

a.Fo

und

inhy

pers

alin

eha

bita

ts37

1

Salin

ivib

rioco

stic

ola

NC

MB

701T

−cv

++

++

−+

−−

−−

−+

+−

−−

F−

+ g−

−−

++

−+

+−

−+

5–45

0.5–

20R

SS

279,

371

Shew

anel

laal

gae

−+

+7 5

+−

+−

−+

−−

−v−

−−

+−

I−

−−

−−

−−

−−

−+

+−

+25

–42

0–10

433,

588,

851

Shew

anel

laam

azon

ensi

s−

++

++

−−

−−

+−

+−

+O

−+

4–30

0–3

Anta

rctic

mar

ine

orga

nism

cont

inue

d



158

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Tab

le4.

11.

Con

tinue

d.

Shew

anel

laba

ltica

−+

+−

+−

+−

−+

−+

−−

−−

−−

O+

++

+4–

3085

1

Shew

anel

labe

nthi

ca−

++

+−

−−

−+

−−

−O

+4–

150–

2

Shew

anel

laco

lwel

liana

−+

+−

+−

−−

−+

+−

−+

−O

−−

−−

−−

−−

−−

−+

8–30

1–5

RR

112,

814

Adhe

res

tosu

rface

s.Su

gars

goal

kalin

e.O

lder

brot

hcu

lture

sha

vere

d-br

own

pigm

ent

815

Shew

anel

lafri

gidi

mar

ina

−+

++

−−v

−−v

+−

+v−

−v

+−v

O−

+−

−+

+−v

−+

+v−v

+v+

0–28

0–8

112

Anta

rctic

mar

ine

orga

nism

Shew

anel

lage

lidim

arin

a−

++

+−

−−

−+

−−

−−

−+

−O

−−

−−

−−

−−

−−

−+

+v4–

151–

611

2

Anta

rctic

mar

ine

orga

nism

Shew

anel

laha

neda

i−

++

+−

−−

−+

−−

−+

O−

−−

−−

−−

−−

−−

+4–

252

409

Shew

anel

laja

poni

ca−

++

++

−+

F+

+10

–37

0–3

397

Deg

rade

sag

ar

Shew

anel

laon

eide

nsis

−+

+−

+−

−−

−+

−−

−+

F+

+15

–40

0–3

782

Shew

anel

lape

alea

na−

++

−+

−+

−+

−−

O−

+−

−−

−−

−−

+4–

300.

1–0.

749

2

Shew

anel

law

oody

i−

++

+−

−−

−+

−−

−+

O−

−−

−−

−+

4–25

211

2

Sphi

ngom

onas

pauc

imob

ilis−

++

−+

−−

−−

−−

−−

+−

+O

−+

+−

++

+−

−+

30–3

729

7

Yello

wpi

gmen

t

Stap

pia

stel

lula

ta-

like

M1

−+

++

−−

++

++

F25

1.5

105

Sten

otro

phom

onas

(Pse

udom

onas

)m

alto

philia

−−

+a

+−

−+

−−

−+

−vv

++

O−

+w−

+v+

−+

+−

+v+

54+

++

350–

562

3

Yello

wco

loni

es

S ee

a ls o

T ab l

e4 .

5fo

rBru

cella

spec

ies.

−c=

nega

tive

inC

hist

ense

n’s

citra

tem

etho

d;O

A=

Oxi

dativ

e;I=

Iner

trea

ctio

n,m

aysh

owan

alka

line

reac

tion;

−a=

nega

tive

oral

pha

haem

olys

is;n

umbe

rsre

fert

ope

rcen

tage

ofpo

sitiv

est

rain

s;nu

mbe

rsin

‘tem

p’an

d‘N

aCl’

colu

mns

refe

rto

rang

e( °

Cor

%N

aCl)

atw

hich

the

orga

nism

will

grow

.



159

Test

Gm

Ox

cat

mot

Nal

KfG

lyIA

1.5

NaC

l25

°C37

°C42

°Cur

eaN

ithi

pG

GT

APH

2SR

ef

Org

anis

m

H.a

cino

nych

is−

+R

S−

−−

+−

++

327

H.a

cino

nyx

−+

RS

−−

+−

++

540

H.b

ilis−

++

RR

+−

−−

++

++

+54

0

H.c

anis

−−

SI

++

−−

+54

0

H.c

etor

um−

++

+I

S−

−−

++

+−

+−

327,

328,

329

H.c

hole

cyst

us−

++

IR

+−

−−

++

−+

−−

+−

540

H.c

inna

edi

−+

SI

+−

−−

+−

−+

−−

−−

540

H.f

elis

−+

++

RS

−−

−−

++

++

−+

+−

327,

540

H.f

enne

liae

−+

SS

++

−−

−−

+54

0

H.h

epat

icus

−+

+R

R+

++

−+

−+

+−

+54

0

H.m

urid

arum

−+

+R

R−

+−

−+

−+

−−

++

+54

0

H.m

uste

lae

−+

+S

R−

+−

−+

++

+−

++

−32

7,54

0

H.n

emes

trina

e−

+R

S−

−+

+−

+32

7,54

0

H.p

amet

ensi

s−

++

SS

+−

−+

+−

+−

−+

−54

0

H.p

ullo

rum

−+

SR

−+

−+

−54

0

H.p

ylor

i−

++

RS

−−

−−

+−

+−

−+

+−

327,

540

H.t

rogo

ntum

−+

++

RR

++

+−

+−

+54

0

AP=

alka

line

phos

phat

ase

hydr

olys

is;c

at=

cat a

lase

;GG

T=

gam

mag

luta

myl

trans

pept

idas

eac

tivity

;Gly

=gr

owth

in1%

glyc

ine;

Gm

=G

ram

reac

tion;

Hip

=hi

ppur

ate

hydr

olys

is;H

2S=

prod

uctio

nof

hydr

ogen

sulp

hide

;I=

inte

rmed

iat e

susc

eptib

ility;

IA=

indo

xyla

ceta

tehy

drol

ysis

;Kf=

sens

itivi

tyto

ceph

alot

hin

30mg

;Mot

=m

otilit

y;N

al=

susc

eptib

ility

tona

ladi

xic

acid

30mg

;1.5

%N

aCl=

grow

thin

the

pres

ence

of1.

5%N

aCl;

Nit

=ni

trate

redu

ctio

n;O

x=

oxid

ase;

S=

sens

it ive

;R=

resi

stan

t;U

rea

=ur

ease

activ

ity;2

5 °C

,37°

C,4

2°C

=gr

owth

atth

ese

tem

pera

ture

s.Te

sts

fora

lkal

ine

phos

phat

ase

and

hydr

olys

isof

indo

xyl a

ceta

tew

ere

done

usin

gan

ANI-I

dent

disc

from

bioM

erie

ux,G

GT,

AP,h

ip,N

it,H

2Sw

ere

done

usin

gth

eAP

ICam

pyid

entif

icat

ion

syst

em(b

ioM

erie

ux).

Ure

ahy

drol

ysis

was

test

edus

ing

ara

pid

urea

test

from

Rem

el.G

row

thon

1%gl

ycin

eca

nbe

test

edon

agr

owth

med

ium

such

asbl

ood

agar

(BA)

,con

tain

ing

1%gl

ycin

e.Se

nsiti

vity

toN

aan

dKf

wer

edo

neon

BAus

ing

stan

dard

antib

iotic

sens

itivi

tyte

stm

etho

ds.

Tab

le4.

12.

Hel

icob

acte

rsp

p.



160

Org

anis

m

Path

ogen

ic

Aero

cocc

usvi

ridan

s+

−a

−−

−−

+75

5

Aero

cocc

usvi

ridan

sva

r.ho

mar

i+

−w

a−

−−

−−

+−

F50

++

+50

+60

50+

+−

10–3

70–

10+

299,

827

Ente

roco

ccus

faec

alis

var.

lique

faci

ens

+c−

−−

+−

+−

−+

−+

Gra

nulic

atel

laba

laen

opte

rae

+c−

−−

−+

−w

+−

+−

−F

−+

−+

−−

−+

−−

179,

478

Lact

ococ

cus

garv

ieae

+−

−a

−−

−−

+−

−−

−+

++

−−

F−

+−

−+

++

+−

V+

−−

v10

–45

0–6.

5−

−46

4,73

1

Gro

wth

in6.

5%N

aCl,

optim

um0%

NaC

l,37

°C.R

esis

tant

tocl

inda

myc

in63

8,78

0

biot

ype

1,2,

12+

−a

−+

++

−+

++

−+

+−

10–4

50–

6.5

780

b io t

y pe

3 ,4 ,

5 ,6 ,

7 ,10

,11,

13+

−a

−+

++

−+

++

−−

+−

10–4

50–

6.5

780

biot

ype

8,9

+−

a−

++

+−

++

−−

−+

−10

–45

0–6.

578

0

Lact

ococ

cus

pisc

ium

+c−

−−

−−

−−

−+

+F

++

−+

++

++

−+

++

−+

5–30

−−

835

Mic

roco

ccus

lute

us+

++

−−

−−

v−

−−

v−

+−

O−

−−

v−

v−

10–4

50–

1043

Plan

ococ

cus

spec

ies

+−

++

−−

−−

+−

−+

−+

O−

+−

−−

−−

372

43

Stap

hylo

cocc

usau

reus

+−

++

−−

++

++

F−

++

++

++

+−

++

+29

6

Stap

hylo

cocc

usde

lphi

ni+

−+

+−

−+

++

−−

F−

++

++s

++

−−

++w

37–4

50–

15−

264,

778

Stap

hylo

cocc

usep

ider

mid

is+

−+

−−

−+

80+

+F

−+

−+

+−

+−

−+

−−

−−w

25–4

50–

729

6

Stap

hylo

cocc

uslu

trae

+c−

++

−−

−+

+−

+F

++

++

v+

++

++

+ w25

–42

0–10

264

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl H

2SM

CA

Coa

gD

Nas

ete

mp

NaC

lMan An

RAA

Hip

Ref

Tab

le4.

13.

Gra

m-p

ositi

veco

cci.



161

Stap

hylo

cocc

usw

arne

ri+

−+

−w−

−+

−+

+F

−+

−−

+ s63

−−

−+

+−

−−

25–4

00–

1029

6

Stre

ptoc

occu

sag

alac

tiae

+c−

−−

−−

−−

+−

−−

++

−−

−Fw

−+

−v

+−

+−

−+

+−

−+

25–3

70–

3+

71,

242

Gro

upB,

No

grow

that

10°C

or45

°C.V

Pm

aybe

nega

tive

Stre

ptoc

occu

sag

alac

tiae

(S.d

iffic

ile)

+−

−−

−−

−−

−−

−−

−+

+−

−−

F−

+−

−+

−+

−−

v−

−−

300–

1+

135,

776

Gro

upB,

Type

Ib.H

ip+v

eat

30°C

,−ve

at37

°CSt

rept

ococ

cus

dysg

alac

tiae

ssp.

dysg

alac

tiae

+−

−+

−−

+−

−v

F−

++

−v

−+

0–4

−72

7,79

0

Posi

tive

toLa

ncef

ield

grou

pL

antis

era.

Baci

traci

n=

S

Stre

ptoc

occu

sdy

sgal

actia

ess

p.dy

sgal

actia

e

+−

−v

−−

+−

−F

++

−−

−+

−0–

4−

135,

776

Gro

upC

Stre

ptoc

occu

sin

iae

ATC

C29

177

+−

−+

−−

−−

+−

−−

+−

−−

F−

+−

++

++

−−

++

−−

−w+

10–3

52–

4−

574

No

grow

that

6.5%

NaC

l,or

10°C

o r4 5

°C.I

sola

tes

from

fish

Stre

ptoc

occu

sin

iae

ATC

C29

178

+−

−+

−−

+−

−−

+−

−F

−+

−−

++

++

−+

+−

−w10

–37

2–4

−22

3,62

5

No

grow

that

6.5%

NaC

l,or

10°C

o r4 5

°C.I

sola

tes

from

fish

Stre

ptoc

occu

sin

iae

+−

−+

−−

−−

−+

−−

F−

+−

−+

++

+−

++

−37

2–4

−22

3

Stra

ins

from

hum

ans

Stre

ptoc

occu

sm

illeri

+−

−+

−−

+−

−+

−−

+−

+−

++

−+

−+

+−

++

+−

+−

87

Stre

ptoc

occu

spa

raub

eris

+c−

−a

−−

+−

++

−F

−+

−+

++

++

++

+−

v10

–37

0–4

v17

5,22

4

No

grow

that

6.5%

NaC

l,or

4 °C

o r4 5

°C.N

egfo

rstre

pgr

oup

D

Stre

ptoc

occu

sph

ocae

+−

−+

−−

−−

F−

+−

−+

−+

−−

−−

−25

–37

0−

700

Sens

itive

toba

citra

cin

disc

,som

est

rain

sre

actw

ithst

rep

grou

pC

antis

era

Vago

cocc

usfe

ssus

+cr

−a

+−

−−

−−

F−

+ g−

−−

−−

−−

−−

369

Vago

cocc

uslu

trae

+cr

−a

+−

−−

−−

+−

+F

−+

+−

++

+−

370

−47

7

Vago

cocc

ussa

lmon

inar

um+c

b−

−a

−−

−−

−−

−−

+−

−F

−+ g

−−

−+

−+

+−

++

−w+

v−

5–30

0–6.

5−

−54

2,68

2

May

grow

onTC

BS,w

eak

dela

yed

reac

tion

wit h

stre

pgr

oup

Dan

tiser

a.H

ip&

VPpo

sw

ithAP

I20

STR

EP73

2,80

7

Vago

cocc

usde

scrib

esm

otile

Lanc

efie

ldgr

oup

N.V

.sal

mon

inar

umha

sbe

enre

porte

das

nega

tive

forL

ance

field

grou

pN

cont

inue

d



162

Envi

ronm

enta

l

Abio

troph

iade

fect

iva

+−

−a

−−

−−

−+

F+

++

20–4

2−

421

Abio

troph

iapa

ra-a

diac

ens

+−

−a

−−

−−

F+

−42

1

Ente

roco

ccus

faec

alis

+−

−a

−−

−−

+−

−−

++

v−

F−

+−

++

++

+v

++

−+

10–4

50–

6.5

+63

8

Ente

roco

ccus

faec

ium

+−

av

−+

−+

+−

+F

++

−+

++

++

−+

+−

10–5

00–

6.5

+

Fack

lam

iam

iroun

gae

+−

a−

−+

−+

−−

−F

−−

−−

−−

+25

–42

0–5

−36

8

Gra

nulic

atel

laad

iace

ns+

−−

a−

−−

−−

−F

−+

−20

–42

−42

1

Gra

nulic

atel

lael

egan

s+

−−

a−

−+

v−

−F

−+

−27

–37

+65

3

Lact

ococ

cus

lact

isSe

rogr

oup

N+

−−

−+

++

F−

++

−−

−+

10–4

50–

2−

731

Phen

otyp

ical

lysi

mila

rto

E.se

riolic

ida.

Sens

itive

tocl

inda

myc

in

Plan

ococ

cus

citre

us+

−+

+−

−−

−−

−v

−−

−−

+−

+−

−−

−−

−−

−−

−−

+5–

300–

15v

326

Plan

ococ

cus

kocu

rii+

−+

+−

−−

−−

−v

−−

−−

v−

v−

−−

−−

−−

−−

−−

+5–

300–

10v

326

Plan

omic

robi

umok

eano

koite

s−v

w+

+−

++

+−

−−

−−

−+

O−

−−

−−

−−

20–3

73–

556

6

Stap

hylo

cocc

usau

reus

anae

robi

us+

−+

−−

++

264

Anae

robi

cgr

owth

only

Stap

hylo

cocc

usca

pitis

++w

−w−

+v

F−

+−

−−

++

−−

+s−

−−

w30

–45

0–10

Stap

hylo

cocc

usca

rnos

us+

−−

−+

+F

+v

−+

+−

v−

−15

–45

0–15

Stap

hylo

cocc

usch

rom

ogen

es+

v−

Fv

+−

−−

Stap

hylo

cocc

usco

hnii

++

−w−

−+w

F−

+−

+80

8080

−−

−+

−−

2015

–45

0–10

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl H

2SM

CA

Coa

gD

Nas

ete

mp

NaC

lMan An

RAA

Hip

Ref

Tab

le4.

13.

Con

tinue

d.



163

Stap

hylo

cocc

usco

hnii

urea

lytic

um+

−+

−−

2815

+25

28+

F−

+−

22+

+−

−+

−−

−

Stap

hylo

cocc

usha

emol

ytic

us+

++w

−+

+F

−+

−50

+50

−−

−+

+−

−−

20–4

50–

10

Stap

hylo

cocc

usho

min

us+

+−

−+

wF

−+

−60

+−

−−

−+

+−

−W

20–4

50–

7

Stap

hylo

cocc

ushy

icus

++

−+

−F

−+

+−

v+

−26

4

Stap

hylo

cocc

usin

term

ediu

s+

++

+−

F−w

++

−+

+−

264

Stap

hylo

cocc

ussa

prop

hytic

us+

+−

−−

+F

−+

−+

++

−−

−+

+−

−−

15–4

00–

15

Stap

hylo

cocc

ussc

hlei

feri

coag

ulan

s+

++

F−

−−

++

264

Stap

hylo

cocc

ussc

hlei

feri

schl

eife

ri+

++

F−

v−

−+

264

Stap

hylo

cocc

ussi

mul

ans

++

−w−

+−

F−

+−

+−

+7 7

−−

+77

−−

v15

–45

0–10

+

Stap

hylo

cocc

usw

arne

ri+

+w−

−−

+F

−+

−−

+s+

−−

−+

+−

−−w

25–4

00–

10

Stap

hylo

cocc

usxy

losu

s+

+−

−80

−F

80+

−80

++

+−

−80

80+

−−

15–4

00–

10

Stre

ptoc

occu

sdy

sgal

actia

eeq

uisi

milis

+−

+−

−+

−F

−−

+−

790

Stre

ptoc

occu

spo

rcin

us+

++

++

−F

−+

−+

++

++

++

+−

10–3

70–

6.5

−17

5,62

5

Stre

ptoc

occu

sub

eris

+−

−−

−+

++

−F

−+

−+

++

++

++

+−

10–3

70–

6.5

+17

5,62

5

Vago

cocc

usflu

vial

isAT

CC

4951

5

+cr

−a

+−

−+

−−

+−

vF

−+ g

−−

−+

++

+−

−+

−−

5–40

0–6.

5−

−17

7,73

2

Wea

kde

laye

dre

actio

nw

ithst

rep

grou

pD

ant is

era

629

The

listo

fStre

ptoc

occi

and

Stap

hylo

cocc

iis

not c

ompr

ehen

sive

,and

read

ers

are

urge

dto

use

ate

xtsu

chas

Berg

ey’s

Man

ual,

ifa

Stre

ptoc

occu

ssp

ecie

sdo

esno

tfit

with

the

spec

ies

liste

dhe

re.

Coa

g=

coag

ulas

ere

actio

n;H

ip=

hydr

olys

isof

hipp

urat

e;M

anAn

=fe

rmen

tatio

nof

man

nito

lund

eran

aero

bic

cond

ition

s;R

AA=

grow

thon

Rog

osa

Acet

ate

agar

.



164

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

AD

Nas

ete

mp

NaC

lZN

MAF

RAA

Hip

Ref

Org

anis

m

Path

ogen

ic

Arca

noba

cter

ium

phoc

ae+

10+

−−

−−

−−

F−

+ g−

++

+40

60−

−+

+10

−−

636

Baci

llus

cere

us+

++

+−

++

O+

25–4

5−

−74

,30

7

Baci

llus

myc

oide

s+

++

−−

++

O+

25–3

7−

−30

7

Car

noba

cter

ium

pisc

icol

a+s

r−

−−a

−−

−−

+−

−−

−+

++

−+

F−

+ g−

−40

*+

++

+−*

++

−−

−−

0–37

0–6

−−

−−

73,

176

*W

eak

reac

tions

inph

enol

red

med

ium

,neg

ativ

ein

purp

leba

sem

ediu

m75

2

Car

noba

cter

ium

spp.

+r−

−−

−−

−−

+−

−−

−+

++

−v

F+ g

−+v

++

++

v+

+−

−−

10–3

70–

6−

−−

73

Cor

yneb

acte

rium

aqua

ticum

+−

++

+−

−−

−+

−−

−+

+v

+I

−−

−−

−−

−−

4–42

0–5

75

Cor

yneb

acte

rium

test

udin

oris

++

−−

+−

+−

F+

−+

−+

−37

018

0

Der

mat

ophi

lus

chel

onae

++

++

−+

−−

−−

+F

+−

v−

−−

−−

−25

−52

9

Der

mat

ophi

lus

cong

olen

sis

++

++

−−

−+

−−

+F

+−

+v−

−−

−−

−37

−30

8

Erys

ipel

othr

ixrh

usio

path

iae

+r−

−a

−−

−−

−−

−F

++

−+

+−

−w−

−−

−−w

+5–

3729

2

Ren

ibac

teriu

msa

lmon

inar

um+

−+

+−

−−

−−

−−

I−

−−

−−

−−

−15

–18

0−

671

Rho

doco

ccus

spp.

+r−

−−

+F

−+

−−

−−

−+

250–

5−

53

No

grow

th37

°C.G

row

thin

5%

NaC

l

Stre

ptom

yces

salm

onar

is+

+O

−+

+−

w+

−−

+12

–37

41

Bric

k-re

dto

oran

gepi

gmen

ted

myc

elia

Envi

ronm

enta

l

Actin

omyc

esm

arim

amm

aliu

m+

−−

−−

−−

−F

−+

++

−−

−−

−−

−37

0

Arca

noba

cter

ium

bern

ardi

ae+

−v

−−

−−

F+

+−

−−

636

Arca

noba

cter

ium

haem

olyt

icum

+

Tab

le4.

14.

Gra

m-p

ositi

vero

ds.



165

Arca

noba

cter

ium

plur

anim

aliu

m+

+a

−−

−−

−−

+w+

F−

+−

v−

−−

−−

+

Arca

noba

cter

ium

pyog

enes

+−

+−

−−

−−

−−

−−

++

F−

+ g−

++

+v−

+−

v+v−

++

20–4

0−

−+,

−63

6

Acid

,clo

t,re

duct

ion

inlit

mus

milk

Arth

roba

cter

agilis

+c+

+−

+v−

−−

−−

−−

−−

++

+O

−−

−−

v20

–30

0–1

Arth

roba

cter

nasi

phoc

ae+

+−

−−

−−

+O

−−

−−

−−

25–4

05

+8

Arth

roba

cter

rhom

bi+c

++

−−

−−

+−

+O

−+

−+

++

++

−+

+−

4–30

1–10

600

Atop

obac

terp

hoca

e+

−−

+−

−−

−−

F−

+v

+−

−v

v−

25−

479

Car

noba

cter

ium

alte

rfund

itum

+r−

−−

+−

−nt

−−

F−

+−

−w

−w

nt−

w−

−nt

0–20

1–6

−−

−41

2

Car

noba

cter

ium

dive

rgen

s+r

−−

−−

−−

+−

−−

++

+F

−+

−−

+−

++

−+

+−

−0–

400–

7−

−−

176

Car

noba

cter

ium

dive

rgen

s62

51+r

−−

−−

−−

−−

F−

0–35

0–6

−64

9

Car

noba

cter

ium

fund

itum

+r−

−−

+−

−nt

−−

F−

+−

−+

++

+−

++

−nt

0–20

1–6

−−

−41

2

Car

noba

cter

ium

gallin

arum

+r−

−−

−−

−−

+−

−−

++

+F

−+ g

−−

++

−+

+−

++

+−

0–35

−17

6

Car

noba

cter

ium

inhi

bens

+r−

−+

−−

−−

−−

−+

−−

F+

+ g−

−w

++

++

−+

+−

−0–

301–

6−

−−

+41

2

Car

noba

cter

ium

mob

ile+r

−−

+−

−−

+−

−−

−+

+F

−+g

−−

+−

++

−+

+−

0–35

−17

6

Cor

yneb

acte

rium

phoc

ae+r

+−

−−

−v

−−

F−

+−

+−

+−

−v

−−

613

Cor

yneb

acte

rium

pseu

dodi

phth

eriti

cum

+r−

++

−+

−−

+−

−−

−I

−−

−−

−−

−20

–42

0–5

133

Cor

yneb

acte

rium

pseu

dotu

berc

ulos

is+r

−+

+−

−−

−−

−−

+−

−−

+F

+ g−

−−

−−

−−

20–4

20–

313

3

Cor

yneb

acte

rium

xero

sis

+r−

++

−−

−−

+−

−−

−−

−−

F+ g

−−

−−

−−

−20

–35

0–3

−−

133

Die

tzia

mar

is+

+−

−+

−−

30−

−−

O−

+−

−−

−−

−−

−−

−25

0–7

−−

573

Rho

doco

ccus

equi

++

−+

−−

−−

++

−+

−−

0–7

−−

573

Rho

doco

ccus

fasc

ians

++

−−

15−

++

−−

36O

++

−−

−+

+−

++

+40

250–

5−

−57

3

Wei

ssel

last

rain

DS-

12+c

r

Car

noba

cter

ium

spec

ies

are

very

sim

ilart

oLa

ctob

acillu

ssp

p.Va

goco

ccus

desc

ribes

mot

ileLa

ncef

ield

grou

pN

stra

ins.

+c=

gram

posi

tive

cocc

us;+

cb=

gram

posi

tive

cocc

o-ba

cillu

s;+c

r=gr

ampo

sitiv

eco

ccoi

dro

d;+r

=gr

ampo

sitiv

ero

d;H

ip=

Hip

pura

tehy

drol

ysis

;MAF

=M

odifi

edAc

idFa

stst

ain

reac

tion;

nt=

nott

est e

d;R

AA=

Rog

osa

Acet

ate

Agar

;v=

varia

ble

reac

tions

repo

rted;

w=

wea

kre

actio

n;ZN

=Zi

ehl-N

eils

on.

Also

see

tabl

esfo

r Noc

ardi

aan

dM

ycob

acte

ria.



166

Test

Gm

Ox

cat

bHm

otO

DC

LDC

ADH

Nit

Ind

Cit

urea

mr

vpae

sG

onpg

arab

glu

inos

lac

mal

tm

anm

ano

sal

sor

suc

treXy

lH

2Ste

mp

NaC

lN

aCl

450

°CH

ipR

ef

Org

anis

m

Myc

obac

teriu

mab

sces

sus

AFB

+−

+6.

5+

736

Myc

obac

teriu

mav

ium

AFB

+−

+−

37–4

55−

737

Myc

obac

teriu

mch

elon

aeAF

B−

+−

−−

+−

+−

−+

−−

−−

15–2

50–

3−

−13

3

Myc

obac

teriu

mfo

rtuitu

mAF

B+

−+

+28

–37

0–5

+56

9

Myc

obac

teriu

mm

arin

umAF

B+

−−

+28

–30

011

1

Rou

gh&

smoo

thco

loni

es.C

ells

may

belo

ngan

dcr

oss-

band

ed

Myc

obac

teriu

mne

oaur

umAF

B25

5−53

Myc

obac

teriu

mpe

regr

inum

AFB

+−

++

+25

–37

+55

1

Myc

obac

teriu

mpo

rifer

aeAF

B−

+25

–37

5+

756

Myc

obac

teriu

msc

rofu

lace

umAF

B−

+56

9

Myc

obac

teriu

msi

mia

eAF

B−

+56

9

Myc

obac

teriu

msp

ecie

sAF

B−

−−

+28

337

Myc

obac

teriu

mtri

plex

-like

AFB

25–3

034

5

Noc

ardi

aas

tero

ides

+−

+−

+−

++ w

−−

+−

+−

+−

−−

−−

−−

−+

−−

20–3

70–

4+

+v

270,

455,

457

Noc

ardi

abr

asilie

nsis

++

+−

++

−−

++

+−

++

−−

+−

−−

−−

30–4

50–

2+

−−

270,

457

Noc

ardi

abr

evic

aten

a+

−−

−+

−−

−−

−+

165

Noc

ardi

aca

rnea

++

−−

+−

+v

++

+16

5

Noc

ardi

aca

viae

+−

+−

+−

v+ w

−−

+−

+−

++

−−

+−

−−

−v

−0–

4+

v45

7

Noc

ardi

acr

asso

stre

ae+

−+

−−

++

−−

+−

−−

−+

+−

22–3

00–

2−

270

Tab

le4.

15.

Myc

obac

teriu

man

dN

ocar

dia

spp.



167

Noc

ardi

afa

rcin

ica

+−

++

−+

+−

−+

−−

v−

−−

v−

20–4

50–

4+

455

Noc

ardi

afla

voro

sea

+−

−−

−−

−−

−−

−−

−−

−−

−−

−+

−16

5

Noc

ardi

ano

va+

+−

++

−+

−−

−−

−−

−−

−20

–40

0–4

−+

455,

805

Noc

ardi

aot

itidi

scav

iaru

m+

+−

++

v+

+v

−v

165

Noc

ardi

aps

eudo

bras

iliens

is+

+−

−+

++

−+

+−

−+

++

−20

–37

165,

661

Noc

ardi

asa

lmon

icid

a+

−+

++

++

−−

+−

−−

−−

−−

−10

–30

0–4

−+

455,

391

Noc

ardi

ase

riola

e+

−+

−+

−+

−+

−−

+−

−−

−v

−−

−−

+20

–30

0–2

+−

455

Noc

ardi

atra

nsva

lens

is+

++

−+

vv

−v

165

Noc

ardi

ava

ccin

ii+

−+

++

+v

+−

v16

5

Noc

ardi

asp

p.(A

ust)

+−

+−

++

−−

117

5−=

nogr

owt h

i n5%

NaC

l .

Test

niac

inN

itrat

eTw

een

80ca

tar

ylur

ease

pyr

Feth

io5%

NaC

lM

CA

Ref

Org

anis

m

Myc

obac

teriu

mab

sces

sus

−−

++

++

+−

++

+73

6

Myc

obac

teriu

mav

ium

−−

−v

−−

++

−73

7

Myc

obac

teriu

mch

elon

aev

−−

++

+−

+13

3,73

7

Myc

obac

teriu

mfo

rtuitu

m−

+v

++

++

+11

6,73

7

Myc

obac

teriu

mm

arin

um−

−+

−−

++

+−

−73

7,11

1

Myc

obac

teriu

mne

oaur

um+

+−

53

Myc

obac

teriu

mpe

regr

inum

−+

−+

++

++

551

Myc

obac

teriu

mpo

rifer

ae−

−+

+−

++

++

−75

6

Myc

obac

teriu

msc

rofu

lace

um−

−−

+−

++

+−

737

Myc

obac

teriu

msi

mia

ev

−−

++

++

+−

737

Myc

obac

teriu

msp

p.(n

ew)

−−

−−

++

−33

7

aryl

=ar

ylsu

lfata

se;p

yr=

pyra

zina

mid

ase;

thio

=2-

thio

phen

ecar

boxy

licac

idhy

draz

ide.

Tab

le4.

16.

Myc

obac

teriu

msp

p.–

addi

tiona

ltes

ts.



168

Test

Gm

Ox

cat

bHm

otPO

4O

DC

LDC

ADH

Nit

Ind

Cit

urea

mr

vpae

sG

TTC

OF

arab

glu

inos

lac

mal

tm

anm

ano

sal

CS

treXy

lF&

Ste

mp

NaC

lPe

nR

ef

Org

anis

m

Path

ogen

ic

Myc

opla

sma

allig

ator

is+

−−

−+

++

−30

–34

128

Myc

opla

sma

croc

odyl

i+

+−

−−

++

++

++

−25

–42

R44

1

Myc

opla

sma

mob

ile+

++

++v

−−

−+

++

++

++

+4–

30R

440

Cel

lsad

here

stro

ngly

togl

ass

orpl

astic

.Cel

lsgl

ide.

Wea

kgr

owth

onBA

.F&S

with

hors

ese

rum

,not

bovi

nese

rum

Myc

opla

sma

phoc

icer

ebra

le+

++

−−

−+

+−

3729

5

Myc

opla

sma

phoc

idae

a+

−−

660

Myc

opla

sma

phoc

irhin

is+

+−

−−

−+

++

3729

5

Myc

opla

sma

test

udin

is+

−−

−−

−−

+−

−+

++

−−

−F

350

F&S

=fil

man

dsp

ots;

C=

chol

este

rolr

equi

rem

ent f

orgr

owth

;S=

seru

mre

quire

dfo

rgro

wth

;Pen

=Se

nsiti

vity

tope

nici

llin10

Uni

ts.S

eeC

hapt

er7

fors

p eci

ficbi

oche

mic

alte

sts

forM

ycop

lasm

a.

Tab

le4.

17.

Myc

opla

sma

spp.

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Org

anis

m

Path

ogen

ic

List

onel

laan

guilla

rum

ATC

C14

181

−+

++

+−

−−

++

−+

−−

+−

++

F−

+ g−

−−

++

+−

++

+−

−−

Y+

10–3

70.

5–7

SS

R50

6

Prev

ious

lyVi

brio

angu

illaru

m

List

onel

laan

guilla

rum

NC

IMB

6

−+

++

+−

−−

++

+−

−+

+−

++

F+

+ g−

−−

++

+−

++

+−

−−

wG

+4–

300.

5–8

SS

81,

563

MR

may

bene

gativ

eor

wea

k.C

itrat

eis

posi

tive

orne

gativ

e

Sero

type

01st

rain

ste

ndto

bepo

sitiv

efo

rara

bino

se

List

onel

lape

lagi

aN

CM

B22

53

−+

+v

+−

−−

−+

++

−+

−−

++

F−

+ g−

−−

++

+−

−+

+−

−v

−10

–37

0.5–

7S

SS

506,

563

Prev

ious

lyVi

brio

pela

gius

II

wG

=w

eak

grow

thof

gree

n-co

lour

edco

loni

es; v

=va

riabl

ere

actio

n.Al

soch

eck

Tabl

e4.

19( M

orite

llasp

p.),

Tabl

e4.

20( P

hoto

bact

eriu

msp

p.),

Tabl

e4.

21( V

ibrio

–pa

thog

enic

spec

ies)

, Tab

le4.

22(V

ibrio

–en

viro

nmen

tals

peci

es).

Tab

le4.

18.

List

onel

lasp

p.



169

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Org

anis

m

Path

ogen

ic

Mor

itella

mar

ina

ATC

C15

381,

NC

MB

1144

T

−+

++

+−

−−

−+

−−

+−

−+

+F

−+ g

−−

−−

−−

−−

−−

−−

−+

0–20

3–5

RS

S50

6,76

6

Man

o=

pos,

DN

ase

=ne

g(R

ef50

6)

Mor

itella

mar

ina

ATC

C15

381,

NC

MB

1144

T

−+

−+

−+

−−

+−

+F

−+ g

−−

−−

−−

−−

−−

+4–

203

SS

81,

82

Mor

itella

visc

osa

NC

IMB

1358

4−

++

++

−−

+−

+−

−+

5−

−+

−I

−+ g

−−

−−

−−

−−

−−

−−

+4–

211–

4R

SS

506

Isol

ates

from

Scot

land

,Nor

way

.May

need

1%pe

pton

ead

ded

tom

edia

forg

row

th.N

ogr

owth

at25

°CM

orite

llavi

scos

a−

++

−+

−+

I−

+−

−+

−+

−−

−−

−4–

212–

3R

S82

Isol

ates

from

sout

h-w

estI

cela

ndon

ly.

No

grow

that

25°C

Mor

itella

visc

osa

−+

+−

−−

+I

−+

−−

−−

−−

−−

−−

4–21

2–3

RS

82

Isol

ates

from

Nor

thIc

elan

d

Envi

ronm

enta

l

Mor

itella

japo

nica

−+

+R

R58

5

Also

chec

kTa

ble

4.18

( Lis

tone

llasp

p.),

Tabl

e4.

20( P

hoto

bact

eriu

msp

p.),

Tabl

e4.

21( V

ibrio

–pa

thog

enic

spec

ies)

, Tab

le4.

22(V

ibrio

–en

viro

nmen

tals

peci

es).

Tab

le4.

19.

Mor

itella

spp.

Org

anis

m

Path

ogen

ic

P.da

mse

lae

ssp.

dam

sela

eAT

CC

NC

IMB

2184

T

−+

w+

+ w−

−−

++

−−

++

+−

−−

F−

+ g+

−−

+−

+−

−−

−−

−+

G+

10–3

70.

5–5

SS

S26

8,50

6,61

8

80%

ofP.

dam

sela

est

rain

sm

aybe

posi

tive

fort

reha

lose

(ref7

45)

81,

289,

705

P.da

mse

lae

ssp.

dam

sela

ebi

otyp

es

biot

ype

1−

++

++

−−

−+

+−

++

+−

−−

F−

+ g+

−+

−−

−G

0.5–

7S

S61

8

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Tab

le4.

20.

Pho

toba

cter

ium

spp.

cont

inue

d



170

biot

ype

2−

++

++

−−

−+

+−

++

+−

−−

F−

+ g+

−+

−−

−G

0.5–

7S

S61

8

biot

ype

3−

++

++

−−

−+

+−

++

+−

−−

F−

+ g+

−+

−−

−G

0.5–

5S

S61

8

biot

ype

4−

++

++

−−

−+

+−

++

−−

−−

F−

+ g+

−+

−−

−G

0.5–

7S

S61

8

biot

ype

5−

++

++

−−

++

+−

++

+−

−−

F−

+ g+

−+

−−

−G

0.5–

7S

S61

8

biot

ype

6−

++

++

−−

−+

+−

++

+−

−−

F−

+ g+

−+

−−

−G

0.5–

7S

S61

8

biot

ype

7−

++

++

−−

++

+−

++

+−

+−

F−

+ g+

−+

−−

−G

0.5–

5R

R61

8

biot

ype

8−

++

++

−−

−+

+−

++

+−

+−

F−

+ g+

−+

−−

−G

0.5–

7R

R61

8

biot

ype

9−

++

++

−−

−+

+−

++

+−

+−

F−

+ g+

−+

−−

−G

0.5–

7S

S61

8

Hae

mol

ysis

may

bem

ore

appa

rent

onBA

than

MSA

-B

P.da

mse

lae

ssp.

pisc

icid

aAT

CC

1791

1

−cb

++

−−

−−

−+

−−

−−

w+

−−

−F

−+ g

−−

−−

−+

−−

+w−

−−

−−

7315

–35

0.5–

3S

SS

518,

745

Shor

trod

s,bi

pola

rsta

inin

g.VP

resu

ltsva

riabl

eor

neg

byAP

I20E

289,

459

Envi

ronm

enta

l

P.an

gust

umN

CIM

B18

95−

++

−−

−−

−+

+−

−−

++

++

+F

−+ g

−−

v+

−+

−−

+v

+−

−−

+4–

370.

5–6

SS

S74

5

P.ilio

pisc

ariu

mAT

CC

5176

0−

++

−+

−+

++

−−

−+

+−

F−

+ g+

−−

+−

+−

−v

−G

+4–

250.

5–2

SS

81

P.le

iogn

athi

ATC

C25

521

−−

++

−+

−v

−+

++ s

F−

+ g−

−−

−+

−−

−20

–30

0.5–

534

0,74

5

P.le

iogn

athi

LMG

4228

−−

−−

−−

−−

+−

−−

−−

+−

−+

F−

+ g−

−v

−−

+−

−−

−−

−+

−−

20–3

50.

5–6

SS

S74

5

P.ph

osph

oreu

mN

CIM

B84

4−

−+

−v

−−

++

+−

−−

++

v−

+F

−+ g

+−

−+

++

−−

−−

−−

+4–

300.

5–8

RS

81

P.ph

osph

oreu

mN

CIM

B12

82−

−+

−v

−−

−+

+−

−−

++

v−

+F

−+ g

+−

−+

−+

−−

−−

−−

−4–

211–

8S

S81

P.pr

ofun

dum

−−

−+

+−

+−

F+ g

++

−+

−−

−−

5–20

586

Also

chec

kTa

ble

4.18

( Lis

tone

llasp

p.),

Tabl

e4.

19( M

orite

llasp

p.),

Tabl

e4.

21( V

ibrio

–pa

thog

enic

spec

ies)

, Tab

le4.

22(V

ibrio

–en

viro

nmen

tals

peci

es).

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Tab

le4.

20.

Con

tinue

d.



171

Org

anis

m

V.al

gino

lytic

us−

++

+v+

+53

+−

++

60−

+83

−+

−F

−+ g

−−

−+

++

33−

++

−−

+Y

+15

–42

1–10

RS

R55

2,82

1

V.al

gino

lytic

usN

CIM

B19

03−

+−

+−

++

++

F−

+ g−

−−

++

−−

−+

−+

15–4

21–

10S

S81

V.ch

oler

ae01

−+

++

+−

++

−+

++v

−−

−−a

++

F−

+ g−

−−

++

80+v

−+

+−

−+

Y+

4–42

0–6v

SS

R50

7,82

1

V.ch

oler

aeno

n01

−+

++

+−

++

−+

++

−−

+−a

++

F−

+ g−

−−

++

70−

−+

+−

−+

Y+

10–4

20–

3S

SSv

507

V.ch

oler

ae01

39−

++

+−

++

−+

−75

+F

−+ g

−−

−+

++

−Y

0–3

RR

S9, 39

2

V.m

imic

us−

++

++

−+

+−

++

9 5−

−−

−+

+F

−+ g

−−

−+

++

−−

−+

−−

+G

+4–

420–

6S

SR

210,

230,

507

V.pa

raha

emol

ytic

us−

++

++

++

+−

62+

63−

+−

−+

−F

80+ g

−−

−+

++

5 3−

−+

−−

+G

+20

–40

3–8

RS

V27

2,55

2

V.pa

raha

emol

ytic

usAT

CC

4399

6−

++

++

++

+−

++

−+s

+−

−+

−F

++ g

−−

−+

++

−−

−+

−−

+G

w20

–40

0–3

RPS

135

V.pa

raha

emol

ytic

usAT

CC

2796

9−

++

++

++

−+

−+

−−

+−

F−

+ g−

−+

+−

−G

S49

9

V.(c

arch

aria

e)ha

rvey

iAT

CC

3508

4

−+

+−

++s

++

−+

++ s

++

−−

v−

F−

+ g−

−−

++

+−

+s+

+−

−+

Y/G

+10

–40

0.5–

8R

S11

,13

5,84

7

Col

onie

sof

V.ha

rvey

islo

wly

spre

adac

ross

the

plat

e.C

itm

ayta

ke3–

5da

ys70

,81

V.ha

rvey

iAT

CC

1412

6,AT

CC

1412

9

−+

+w

+−

++

−+

++ w

−−

+−

F−

+ g−

−+

+−

−+

−Y

12–4

03–

6R

S11

Hae

mol

ysis

agai

nsts

heep

RBC

V.vu

lnifi

cus

ATC

C27

562

biot

ype

1,se

rova

rnon

-E

−+

++

+−

++

−+

++

−+

−−

++

F−

+ g−

−+

+−

−−

−−v

+−

−+

G−

20–4

23–

6S

S98

Hum

anst

rain

,USA

–no

n-vi

rule

ntfo

reel

s.C

it=

neg

inAP

I20E

butp

osin

Sim

mon

stu

beci

trate

V.vu

lnifi

cus

ATC

C27

562

biot

ype

1

−+

++

+−

89+

−+

++

−+

−+

+F

−+ g

−−

++

44+

+44

−+

−−

G37

0.5–

7S

S74

6

Asor

igin

ally

desc

ribed

byTi

son

etal

.(19

82)u

sing

conv

entio

nalm

edia

cont

inue

d

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Tab

le4.

21.

Vib

rio–

path

ogen

icsp

ecie

s.



172

V.vu

lnifi

cus

ATC

C33

184

biot

ype

1,se

rova

rnon

-E

−+

++

+−

++

−+

++

−+

−−

++

F−

+ g−

−+

++

+−

−+

−−

+G

370.

5–6

SS

S98

Hum

anst

r ai n

–U

SA

V.vu

lnifi

cus

biot

ype

1,se

rova

rnon

-E

−+

+v

201

Isol

ated

from

dise

ased

eels

.Bel

gium

(man

=ne

g),S

wed

en(m

an=

pos)

V.vu

lnifi

cus

ATC

C33

187

biot

ype

2,se

rova

rE

−+

++

+−

++

−+

−+

−+

−−

++

F−

+ g−

−+

+−

−−

−−

+−

−+

G+

20–3

53–

6S

S98

Hum

anst

rain

,USA

–vi

rule

ntfo

reel

s

V.vu

lnifi

cus

ATC

C33

149

biot

ype

2,se

rova

rE,0

4

−+

++

+−

−+

−+

−+

−+

−−

++

F−

+ g−

−+

+−

−−

−−

+−

−+

G+

20–3

53–

6S

S98

Viru

lent

fore

els

–Ja

pane

sest

rain

V.vu

lnifi

cus

biot

ype

2se

rova

rE−

++

++

−20

+−

+−

+−

+−

−+

+F

−+ g

−−

++

+−

−−

−+

−−

+G

+20

–35

3–6

SS

26

Taiw

anes

est

rain

s–

aviru

lent

fore

els

V.vu

lnifi

cus

ATC

C33

148

biot

ype

2

−+

++

+−

−+

−+

−+

−+

−+

+F

−+ g

−−

++

−+

+−

−+

−−

G37

0.5–

7S

S74

6

Asor

igin

ally

desc

ribed

byTi

son

etal

.(19

82)u

sing

conv

entio

nalm

edia

V.vu

lnifi

cus

biog

roup

3−

++

++

−+

+−

++

−−

+−

−+

−sF

−+ g

−−

−+

−−

−−

+−

−−

GS

S10

1,56

5

Hu m

a ns t

rain

–Is

rae l

V.tra

chur

i−

++

−+

−+

+−

−−

F−

+−

++

++

−Y

303–

7S

S40

0

V.sh

iloni

i−

++

++

−+

−+

+−

++

F+ g

++

Y16

–37

2–4

SS

59,

458

V.fis

cher

iAT

CC

7744

−+

+−v

+−

−+

−+

−v

++

−−

−20

F−

+ g−

−−

++

−60

−40

−−

−,G

+10

–30

0.5–

6S

SS

Yello

w-o

rang

epi

gmen

t.W

estd

escr

iptio

n=

man

o+v

e

V.fis

cher

iN

CM

B12

81,

ATC

C77

44

−+

++

−+

−+

−+

−+

F−

+ g−

−−

v+v

++v

−−

+v−

v+

0.5–

5S

S81

,34

2

V.fis

cher

iAT

CC

2591

8−

++

−+

−−

+−

+−

−−

−−

−−

−F

−+

−−

+−

−+

−−

−−

−−

−+

10–3

01–

6R

SR

745

VP+v

ein

API2

0E, −

vein

MR

VPm

ediu

m

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Tab

le4.

21.

Con

tinue

d.



173

V.fis

cher

iN

CM

B12

81−

++

++

−−

+−

−−

−+

+−

F−

+ g−

−−

+−

+−

+−

−v

+10

–30

0.5–

7S

SS

506

Gre

yco

loni

es

V.lo

gei

ATC

C29

985T

−+

+−

+−

−+

−−

−+

−+

−F

−+ g

−−

−+

+−

−−

+−

+4–

220.

5–5

SS

S50

6

V.lo

gei

ATC

C15

382

−+

+−

+−

−−

−−

++

−+

−F

−+ g

−−

+−

+−

−+

+−

+4–

250.

5–5

SS

S50

6

V.lo

gei

NC

MB

1143

−+

+−

+−

−+

−+

−+

v−

+−

F−

+ g−

−v

+−

++

−+

+−

+4–

300.

5–5

SS

S81

,74

5

V.lo

gei

NC

IMB

2252

−+

−+

+−

+−

+−

+F

−+ g

−−

+−

+−

−−

+4–

220.

5–5

S81

V.lo

gei

Yello

w-o

rang

epi

gmen

t.St

rain

sar

eci

trate

nega

tive

V.pr

oteo

lytic

us−

++

++

+−

++

++

+33

++

−+

−F

−+ g

−−

−+

+−

−50

76+

−Y/

G+

20–4

01–

10R

SS

67,

788

V.pr

oteo

lytic

usAH

LDA

1735

−+

++

++

−+

++

++

++

+−

+−

F−

+ g−

−−

++

w−

+−

+−

−w

G−

42−

1–10

PSS

135

V.flu

vial

isAT

CC

3380

9−

++

v+

−−

−+

++

+−

+−

72+

+F

++ g

−−

−+

++

−−

++

−−

+Y

+10

–35

1–6

RS

V81

,62

0

V.flu

vial

isN

CTC

1132

7−

++

v+

−−

−+

+−

+−

+−

−+

+sF

++ g

−−

−+

++

−−

++

−−

+Y

+10

–35

1–6

RS

V12

3,48

5

API2

0E

pos

forV

Pan

din

dole

V.fu

rnis

sii

ATC

C35

016T

−+

+v

+−

−−

++

−+

−+

−−

+−

F+

+ g+

−−

++

+−

−+

+−

−+

Y+

20–3

71–

8R

SR

123,

485

API2

0E

pos

forV

Pan

din

dole

V.fu

rnis

sii

ATC

C11

218

−+

++

+−

−−

++

−+

−+

−−

++

F+

+ g+

−−

++

+−

−+

+−

−+

Yw

20–3

71–

8R

S13

5

ADH

=ne

gat

0%N

aCl.

Pos

ADH

at2%

NaC

l

V.sp

lend

idus

IAT

CC

3312

5−

++

v+

−−

−90

++

43−

+−

++

+F

−+ g

−−

−+

++

−−

−v+

−−

−G

−4–

371–

6S

SS

281,

506,

620

Type

stra

inis

citra

tene

gativ

e81

,25

4

V.sp

lend

idus

IIN

CM

B22

51−

++

++

−−

−v

++

+−

+−

−+

+F

−+ g

−−

−+

+−

−−

−+

−G

+4–

300.

5–7

SS

R81

,50

6,81

9

V.tu

bias

hii

ATC

C19

109

−+

+−

+−

−−

83+

+v

30+

−−

++

F−

+ g−

−−

++

−−

++

−−

Y+

10–3

70.

5–6

SS

S10

8,32

1

cont

inue

d



174

V.tu

bias

hii

NC

MB

1340

T

−+

+w

+−

−−

+s+

+−

−+

−−

++

F−

+ g−

−−

++

+−

−+

+−

−−

Yw+

10–3

00.

5–7

SS

S13

5,50

6

V.ic

hthy

oent

eri

−+

++

−−

−−

+−

−−

−−

F−

+ g−

−−

+−

+−

−+

+−

−Yw

15–3

01–

6S

S38

9

V.ta

petis

−cb

++

+−

−−

−+

+−

−−

−+

+F

−+ g

−−

−+

−+

−−

+−

−G

4–22

1–5

SS

S10

8,14

6

Aesc

ulin

repo

rted

posi

tive

(ref6

10)

587

V.or

dalii

ATC

C33

934

−cv

++

++

−−

−−

−−

−v−

−−

−+

−F

−+ g

−−

−+

+−

−−

+−

−−

−+

4–30

0.5–

7S

SR

v50

6,68

0,81

9

V.or

dalii

DF-

3K−c

v+

++

+−

−−

−−

−−

−−

−−

+−

F−

+ g−

−−

+−

−−

−+

−−

−−

+4–

300.

5–7

SS

Rv

198,

680

V.or

dalii

NC

IMB

2167

−+

−−

−−

−−

−−

−F

−+ g

−−

−+

+−

−−

++

+21

–37

0.5–

5S

S81

V.pe

cten

icid

a−

++

++

−−

−+

−+

−+

−F

−+ g

−−

+−

−−

−−

+4–

301–

6S

S47

0

V.pe

naei

cida

−+

++

−−

−−

+50

67−

+−

++

F−

+ g−

−50

+−

+−

−−

+−

−G

20–3

01–

3S

S18

7,38

8

Type

stra

in=

indo

lene

g

V.sa

lmon

icid

a−

++

−+

−−

−−

−−

−−

−−

−−

−F

−+ g

v−

−−

++

−−

−+

−−

G+

1–22

0.5–

4S

SS

198,

232,

506

V.sa

lmon

icid

aN

CIM

B22

62−

+−

−−

−−

−−

+F

−+ g

−−

−+

−−

−−

−+

1–22

1–4

SS

81

V.co

rallii

lytic

usYB

1T

−+

++

++

++

−+

+F

++

Y25

–30

1–7

SR

83,8

4

4of

6st

rain

sar

eAD

Hne

gativ

e

−aV.

chol

erae

isne

gativ

efo

raes

culin

,but

som

est

rain

sca

use

blac

keni

ngof

the

med

ium

due

topr

oduc

tion

ofm

elan

in. T

ode

term

ine

atru

ene

gativ

eth

elo

ssof

fluor

esce

nce

mus

tbe

mea

sure

dat

354

nm.S

eeun

der‘

aesc

ulin

’ in

inte

rpre

tatio

nof

test

s.In

this

tabl

e,Vi

brio

sar

egr

oupe

dac

cord

ing

toth

eirO

DC

,LD

C,A

DH

reac

tions

.Use

this

asa

star

ting

poin

tin

iden

tific

atio

ns.4

2 −=

nega

tive

grow

that

42°C

.Al

soch

eck

Tabl

e4.

18( L

isto

nella

spp.

),Ta

ble

4.19

( Mor

itella

spp.

),Ta

ble

4.20

( Pho

toba

cter

ium

spp.

),Ta

ble

4.22

( Vib

rio–

envi

ronm

enta

lspe

cies

).

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Tab

le4.

21.

Con

tinue

d.



175

Org

anis

m

Envi

ronm

enta

l

V.di

abol

icus

−+

++

++

+−

++

−−

−−

+−

F−

+−

++

+−

−+

+−

−20

–45

2–5

SS

635

V.ro

tifer

ianu

s−

++

−+

+−

+−

v−

++

F+

+ g−

−+

−+

−Y

28–4

02–

6S

S30

5

V.ci

ncin

natie

nsis

−+

++

−−

+−

+−

+−

+−

+F

++ g

−+

−−

++

−+

+−

Y+

22–3

71–

6R

SS

120

V.m

edite

rrane

i−

++

−+

−−

+−

++

−−

+−

75−

+F

−+ g

−−

−+

++

25+

++

−−

−Y

+20

–30

3–6

SS

S63

1

V.m

etsc

hnik

ovii

−cr

−+

++

−−

++

−20

30−

++

40+

+F

−+ g

−−,

v59

++

+−

30+

+−

−v

Y,−

+4–

400.

5–7

SS

V48

3,81

9

V.br

asilie

nsis

LMG

2054

6T

−cr

++

+−

−−

++

+−

++

++

F−

+−

+−

+−

Y20

–40

2–6

SS

740

V.ne

ptun

ius

LMG

2053

6T

−cr

++

+−

−−

++

++

−+

++

−F

+−

−−

+−

Y20

–35

2–6

SS

740

V.pa

cini

iLM

G19

999T

−+

++

−−

−+

+−

w−

+66

vF

++

−+

−Y

4–35

1.5–

6S

S14

7

V.xu

iiLM

G21

346T

−cr

++

+−

−−

++

+−

++

−−

F+

+−

+−

+−

Y20

–40

2–8

SS

740

V.or

ient

alis

NC

MB

2195

T

−+

+−

+−

−−

++

++

−+

−−

+F

−+ g

−−

−+

+−

−+

+−

Y+

4–35

0.5–

8S

SS

506

Som

eLD

Cpo

sitiv

est

rain

s

V.ae

roge

nes

−−

++

−−

−+

++

+−

+F

−+ g

++

−+

+−

+−

++

20–3

51–

7R

RS

692

V.ae

stua

rianu

s−

++

++

−−

−+

++

+−

−+

++

F−

+ g−

−+

++

−v+

++

−−

+Y

+4–

370.

5–6

SS

S74

7

V.di

azot

roph

icus

−+

+−

+−

−−

++

++

1 6+

−+

−+

F+

+ g−

−+

++

−50

−+

++

−Y

−10

–35

0.5–

6R

SS

319,

819,

820

V.le

ntus

−+

++

−−

−+

++

+−

+F

−+ g

−−

++

+−

−−

+−

G+

4–30

2–6

RR

513

Thor

nley

’sAD

H=

pos,

Møl

lerA

DH

=ne

g.Ty

pest

rain

=m

anne

g

V.m

ytili

−+

++

−−

−+

+−

+v−

−+

−F

++ g

+−

++

−+

−+

++

−−

Y−

10–3

71–

10R

S63

2

V.ne

reis

−+

−+

+−

−−

++

++

−+

−−

+−

F−

+ g−

−−

+−

+−

−+

+−

−Y

+20

–35

3–6

RS

S81

9

Ente

rovi

brio

norv

egic

us−

++

+−

−−

+−

+−

−−

−−

+F

−+

−−

+−

−−

+−

G−

20–2

81.

5–6

RR

741

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Tab

le4.

22.

Vib

rio–

envi

ronm

enta

lspe

cies

.

cont

inue

d



176

V.sc

opht

halm

i−

++

−+

−−

−90

+−

−−

+−

+−

−F

−+ g

−−

−+

−+

−−

+−

−−

−Y

−22

–35

0.5–

3S

S14

9,25

4

V.cy

clitr

ophi

cus

−+

++

−−

+w−

−−

−+

−F

−+ g

−−

+−

−−

++

+−

4–37

2–10

S33

8

V.ag

ariv

oran

s−

++

−+

−−

−−

+−

−−

+−

−+

+F

−+ g

−−

−+

w−

−−

−−

+−

−G

−20

–37

1–6

SS

135,

514

Hyd

roly

ses

agar

.Gel

atin

neg

inAP

I20E

V.ca

mpb

ellii

−+

+−

+−

−−

−+

+58

−50

−50

+−

F−

+ g−

−−

+48

5082

−−

+−

−G

+20

–35

3–6

RS

R82

0

V.ga

zoge

nes

−cr

−+

−−

−−

−−

+−

−+

F+

+ g+

−−

++

++

++

+Y

20–4

23–

6S

S67

Red

toor

ange

colo

nies

V.ha

liotic

oli

−+

+−

−−

−−

−+

+v−

−+

−−

−+v

F−

+ g−

−−

++

+ v−

−−v

−−

−−

G−

15–3

02–

3S

S67

8

V.ho

llisae

−cv

+w

+w−

−−

−+

+−

−−

−−

−−

F+

+ g−

−−

−−

+−

−−

−−

−−

−−

30–3

71–

6R

SS

346,

580

Hae

mol

ysis

wea

kat

24h,

posi

tive

afte

r7da

ys

V.na

trieg

ens

−+

+−

+−

−−

−+

−+

62−

++

67F

++ g

−60

−+

+3 0

+−

++

−−

−Y+

10–4

03–

6R

SS

44,

820

V.na

trieg

ens

NC

MB

1900

−+

+−

+−

−−

−+

−+

−−

−+

−+

F+

+ g−

−−

++

++

−+

+−

−Y

+4–

370.

5–7

SS

S50

6,56

3

V.na

varre

nsis

−+

++

−−

−−

++

+−

−+

+F

−+ g

−−

++

+−

+−

Y10

–40

0.5–

7S

S76

7

V.ni

grip

ulch

ritud

o−

+−

−−

−+

++

−−

50+

+F

−+ g

−+

++

+−

−v+

−+

−G

+20

–30

3–5

RS

S27

7

Col

onie

sbl

ue-b

lack

V.w

odan

isN

CIM

B13

582

−+

++

+−

−−

−89

20+

−−

+F

−+ g

−−

−+

43+

−−

83+

−30

+4–

251–

4S

SS

506

Col

onie

sbl

ue-b

lack

V.ru

moi

ensi

s−

++

−+

−+

+−

−F

++

−+

++

++

−+

2–34

3–6

SS

850

V.ca

lvie

nsis

−+

++

−+

−+

++

−+

+F

+ g−

+G

4–30

1.5–

12S

SS

216

V.na

trieg

ens

stra

inN

CM

B19

00w

asin

itial

lyre

cord

edas

the

type

stra

info

rLis

tone

llape

lagi

aI(

Ma c

ián

etal

.,20

00).

Inth

ista

ble,

Vibr

ios

are

grou

ped

acco

rdin

gto

thei

rOD

C,L

DC

,AD

Hre

actio

ns.U

seth

isas

ast

artin

gpo

inti

nid

entif

icat

ions

.Al

soch

eck

Tabl

e4.

18( L

isto

nella

spp.

),Ta

ble

4.19

( Mor

itella

spp.

),Ta

ble

4.20

( Pho

toba

cter

ium

spp.

),Ta

ble

4.21

( Vib

rio–

path

ogen

icsp

ecie

s).

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l01

29 1001

2915

0Am

pR

ef

Tab

le4.

22.

Con

tinue

d.



177

Case

Num

ber.

......

......

......

......

.

Test

Gm

Ox

cat

bHm

otSW

OD

CLD

CAD

HN

itIn

dC

itur

eam

rvp

aes

Gon

pgO

Far

abgl

uin

osla

cm

alt

man

man

osa

lso

rsu

ctre

Xyl

H2S

MC

ATC

BSD

Nas

ete

mp

NaC

l0/

301

29 1001

2915

0Am

pId

entif

icat

ion

Isol

ate

No.

Gm

=G

ram

;Ox

=ox

idas

e;ca

t=ca

tala

se;b

H=

bha

emol

ysis

;mot

=m

otilit

y;SW

=sw

arm

ing;

OD

C=

orni

thin

ede

carb

oxyl

ase;

LDC

=ly

sine

deca

rbox

ylas

e;AD

H=

argi

nine

dihy

drol

ase;

Nit

=ni

trate

;Ind

=in

dole

;Cit

=ci

trate

;ure

a=

urea

;mr=

met

hyl

red;

vp=

Voge

s-Pr

oska

uer;

aes

=ae

scul

in;G

=ge

lat in

;onp

g=

o-ni

troph

enyl

-b-g

alac

topy

rano

side

(b-g

alac

tosi

dase

test

);O

F=

Oxi

dativ

efe

rmen

tativ

e;ar

ab=

arab

inos

e;gl

u=

gluc

ose;

inos

=in

osito

l;l a

c=

lact

ose;

mal

t=m

alto

se;m

an=

man

nito

l;m

ano

=m

anno

se;s

al=

salic

in;s

or=

sorb

itol;

suc

=su

cros

e;tre

=tre

halo

se;x

yl=

xylo

se;H

2S=

hydr

ogen

sulp

hide

;MC

A=

Mac

Con

key

agar

; TC

BS=

thio

sulp

hate

-citr

ate-

bile

-suc

rose

agar

;DN

ase

=de

oxyr

ibon

ucle

ase

agar

;tem

p=

tem

pera

ture

;N

aCl0

/3=

grow

thin

0%an

d3%

salt;

0129

10=

sens

it ivi

tyto

vibr

iost

atic

disc

10mg

;012

915

0=

sens

itivi

tyto

vibr

iost

atic

disc

at15

0mg

;Am

p=

sens

itivi

tyto

ampi

cillin

10mg

.

Lab

ora

tory

Wo

rksh

eet



178

Acin

etob

acte

r/Mor

axel

la−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−+

41

Actin

obac

illus

delp

hini

cola

−+

−−

−−

−−

−+

−+

−−

−−

−−

−−

++

−−

++

3748

0.85

263

Dol

phin

Actin

obac

illus

delp

hini

cola

−21

+70

−−

−−

−+

−+

−−

−−

−−

−−

++

−−

++

3748

0.85

263

Porp

oise

Actin

obac

illus

delp

hini

cola

−+

−−

−−

−−

−+

−+

−−

−−

−−

−−

++

−−

++

3748

0.85

263

Wha

le

Actin

obac

illus

scot

iae

+−

−+

−−

+−

−+

−+

−−

−−

−−

−−

++

−−

++

3748

0.85

265

Aero

mon

asal

losa

ccha

roph

ila+

v+

vv

−−

−+

−+

++

−−

v+

v−

v+

++

++

+41

Aero

mon

asbe

stia

rum

++

+−

−−

−−

++

++

+−

−+

+−

++

++

++

++

2724

0.85

452

Car

p

Aero

mon

asca

viae

++

−−

10−

−−

+8

80+

+−

−−

+−

−+

++

++

++

21

Aero

mon

asca

viae

++

−−

+−

−−

+−

++

+−

−−

+−

−+

++

++

++

2548

0.85

240

ATC

C15

468T

Aero

mon

ascu

licic

ola

++

+−

+−

−+

++

++

−−

−+

+−

−+

++

++

3024

0.85

624

NC

IM51

47T

Aero

mon

asen

chel

eia

++

−−

−−

−−

+−

++

+−

−75

75−

+−

++

++

++

2724

0.85

241,

452

Aero

mon

aseu

cren

ophi

la+

+−

−−

−−

−+

−+

++

−−

−+

++

++

+

Aero

mon

ashy

drop

hila

++

+−

−−

−−

++

++

+−

−−

+−

−+

++

++

++

2548

0.85

240

ATC

C79

66T

Aero

mon

ashy

drop

hila

++

−−

−−

−−

++

−+

+−

−−

+−

−+

++

++

++

674b

ATC

C79

66T

Aero

mon

ashy

drop

hila

++

−−

v−

−−

++

++

+−

−+

+−

++

++

++

++

322

ATC

C14

715

Aero

mon

ashy

drop

hila

++

−−

+−

−−

+−

++

+−

−−

+−

++

++

++

++

2.85

509

WFM

504

Aero

mon

ashy

drop

hila

++

−−

−−

−−

++

++

+−

+−

+−

++

++

++

+2 3

4 82.

8522

7a

Aero

mon

asja

nada

ei+

++

−+

−−

−+

++

++

−−

−−

50−

−+

++

++

+14

3

Aero

mon

asja

nada

ei+

++

−+

−−

−+

++

+w

−−

−−

+−

−+

++

++

+25

480.

8513

5AH

LDA

1718

Aero

mon

aspo

poffi

i+

+−

−+

5 0−

−+

++

++

−−

−+

++

++

Aero

mon

assa

lmon

icid

a−

+−

−−

−−

−−

−+

++

−−

−−

−−

−+

+−

++

+45

0

A.sa

lmon

icid

asa

lmon

icid

a+

++

−−

−−

−−

−+

++

−−

−−

−+

−+

+−

++

+46

8N

CIM

B11

02

A.sa

lmon

icid

aac

hrom

ogen

es−

−−

−−

−−

−−v

−−

++

−−

−+

−−

−+

+−

++

+32

2

A.sa

lmon

icid

aac

hrom

ogen

es−

−−

−−

−−

−+

+−

++

−−

−+

−−

−+

+−

++

+46

8N

CIM

B11

10

A.sa

lmon

icid

am

asou

cida

++

−−

−−

−−

++ v

−+

+−

−−

+−

+−

++

−+

++

322

A.sa

lmon

icid

asa

lmon

icid

a+

+−

−−

−−

−−

−+

++

−−

−−

−+

−+

+−

++

+32

2

A.sa

lmon

icid

asa

lmon

icid

a−

+−

−−

−−

−−

−+

++

−−

−−

−−

−+

+−

++

+67

4

A.sa

lmon

icid

asa

lmon

icid

a−

++

−−

−−

−−

−+

++

−−

−−

−−

−+

+−

++

+67

4

A.sa

lmon

icid

aat

ypic

al+w

−−

−76

1Au

stra

lian

stra

in

A.sa

lmon

icid

aat

ypic

al−

+w−

−−

−−

−+

−−

+−

−−

−w

−−

−+

+−

ww

2572

0.85

135

AHLD

A13

34

Aero

mon

asso

bria

++

+−

−−

−−

++

++

+−

−−

+−

−−

++

++

++

2548

0.85

240

CIP

74.3

3T

Aero

mon

asso

bria

++

+−

50−

−−

++

++

+−

−−

+−

−28

++

++

++

21

Test

son

pgAD

HLD

CO

DC

cit

H2S

ure

TDA

ind

vpG

glu

man

inos

sor

rha

suc

mel

amy

arab

oxN

O2

N2

mot

MC

AO

FTe

mp

Tim

eIn

ocR

efSt

rain

Org

anis

m%

NaC

l

Tab

le4.

23.

AP

I20E

data

base

bioc

hem

ical

resu

lts.



179

Aero

mon

astro

ta+

++

−+

−−

−+

−+

++

−−

−−

−+

−+

++

++

+37

240.

8514

2AT

CC

4965

7T

Aero

mon

asve

roni

iver

onii

+−

++

+−

−−

++

++

+−

−−

+−

+−

++

++

++

2724

0.85

347,

2

Allo

mon

assp

p.+

+−

−−

−−

−+

−+

++

−−

−+

−+

−2.

8550

9W

FM40

1

Burk

hold

eria

pseu

dom

alle

i−

+−

−63

−−

−−

−+

++

++

−43

−60

++

++

++

−37

480.

8538

Car

noba

cter

ium

dive

rgen

s+

+−

−−

−−

−+

−C

itrob

acte

rfre

undi

i+

v−

−−

+−

−−

−−

−−

−+

++

+−

+−

41

Edw

ards

iella

icta

luri

−−

++

−−

−−

−−

−+

−−

−−

−−

−−

−+

++

+33

4,41

Edw

ards

iella

tard

a−

−+

w+

+−

−+

−−

+−

−−

−−

−−

−−

++

++

2548

0.85

135

AHLD

A13

5

Edw

ards

iella

tard

a−

−+

+−

+−

−+

−−

+−

−−

−−

−−

−−

41

Edw

ards

iella

tard

a–

atyp

i cal

stra

ins

−−

+−

++

−−

+−

−+

+−

−−

−−

−−

−25

480.

8584

5

Ente

rovi

brio

norv

egic

us+

+−

−−

−−

−+

−−

+−

−−

−−

−−

−+

−+

++

2848

2.85

741

LMG

1983

9T

Flav

obac

teriu

mbr

anch

ioph

ilum

−−

−−

−−

−−

−−

++

−−

−−

++

−−

−41

Flav

obac

teriu

mco

lum

nare

−−

−−

−+

−−

−−

+−

−−

−−

−−

−−

+41

Flav

obac

teriu

mco

lum

nare

+−

−−

+−

−−

−+

−−

−−

−−

−−

−+

+−

−−

−25

480.

568

9

Flav

obac

teriu

mgi

llisia

e−

+−

−−

−−

−−

−−

++

−−

−+

−−

−−

2048

1.5

533

Flav

obac

teriu

mhi

bern

um+

−−

−−

−−

−−

−+

+−

−−

++

−−

+−

+25

481.

553

2

Flav

obac

teriu

mhy

datis

−−

−−

−−

−−

−−

++

+−

−−

+−

−+

−41

Flav

obac

teriu

mjo

hnso

niae

+−

−−

+−

−−

−+

+−

−−

−−

−−

−−

++

2572

0.85

135

AHLD

A17

14

Flav

obac

teriu

mps

ychr

ophi

lum

−−

−−

−−

−−

−−

+−

−−

−−

−−

−−

+41

Flav

obac

teriu

mte

getin

cola

−−

−−

−−

−−

−−

−+

+−

−−

−−

−−

−−

2048

1.5

533

Flav

obac

teriu

mxa

ntha

m−

−−

−−

+−

−−

++

+−

−−

+−

−−

++

2048

1.5

533

Haf

nia

alve

i−

−+

++

−−

−−

−−

++

−−

−−

−−

+−

+41

Haf

nia

alve

i+

−+

+−

−−

−−

−−

++

−−

+−

−−

+−

++

++

+65

2

Haf

nia

alve

i+

−+

++

−−

−−

+−

++

−−

+−

−+

+−

++

++

+25

480.

8513

5AH

LDA

1729

Haf

nia

alve

i+

−+

++

−−

−−

−−

++

−−

+−

−−

+−

++

++

+25

480.

8520

3AT

CC

5187

3

Hal

omon

ascu

pida

−+

++

−−

−−

−−

−−

+−

++

+−

−−

−41

Jant

hino

bact

eriu

mliv

idum

−+

−−

+−

−−

−+

++

+−

−−

−−

−−

+41

Kleb

siel

lapn

eum

onia

e+

−v

−+

−v

−−

+−

++

++

++

++

+−

++

++

+41

List

onel

laan

guilla

rum

++

−−

+−

−−

++

++

+−

+−

+−

−−

++

+v

++

2548

0.85

240

NC

MB

6

List

onel

laan

guilla

rum

++ v

−−

+−

−−

++

++

+−

+−

+−

++v

++

++

428,

41

List

onel

laan

guilla

rum

++

−−

−−

−−

++

++

w−

+−

w−

−−

++

−+

+25

482.

85

List

onel

laan

guilla

rum

++

−−

+−

−−

++

++

+−

+−

+−

−+

++

−+

+25

482.

8513

5AH

LDA

1730 co

ntin

ued



180

Mor

itella

mar

ina

−−

−−

−−

−−

−−

++

−−

−−

−−

−−

++

+−

++

766

Mor

itella

visc

osa

−−

+−

−−

−−

−−

v+

−−

−−

−−

−−

++

++

+13

2

Pant

oea

aggl

omer

ans

+−

−−

+−

−−

−+

+s+

+−

−+

+−

−+

−+

++

+29

1

Pant

oea

aggl

omer

ans

+−

−−

+−

−−

−+

++

+−

−−

+−

−−

−41

Pant

oea

disp

ersa

+−

−−

+−

−−

−+

+s+

+−

−+

+v

++

−+

++

+29

1

Past

eure

llam

ulto

cida

−−

−−v

−−

−−

+−

−+

+−

+−

+−

−−

++

−+

+42

8

Past

eure

llate

stud

inis

+−

−−

−−

−−

+−

−+

+70

−60

+30

−30

++

−+

++

709

Phoc

oeno

bact

erut

eri

−−

−−

−−

−−

−+

−+w

−−

−−

−−

−−

++

−−

++

266

Phot

obac

teriu

man

gust

um−

+−

−−

−−

−−

++

+−

−−

−+

−−

−+

−−

−+

+26

721.

574

5N

CIM

B18

95

P.da

mse

lae

ssp.

dam

sela

e−

+−

−−

−+

−−

+−

+−

−−

−−

−−

−+

++

++

+26

721.

574

5AT

CC

3353

9T

P.da

mse

lae

ssp.

dam

sela

e−

++

−−

−−

−−

−−

+−

−−

−−

−−

−+

++

++

+25

480.

8524

0AT

CC

3353

9T

P.da

mse

lae

ssp.

dam

sela

e−

+−

−+

−−

−−

−−

+−

−−

−−

−+

−−

++

++

+26

721.

574

5N

CIM

B21

84

P.da

mse

lae

ssp.

dam

sela

e−

+5 5

−15

−85

−−

+20

+−

−−

−−

−15

−75

++

++

+26

721.

574

5

P.da

mse

lae

ssp.

pisc

icid

a−

+−

−20

−−

−−

+−

+−

−−

−−

−−

−+

−−

46+

+26

721.

574

5AT

CC

1791

1

P.da

mse

lae

ssp.

pisc

icid

a−

−−

−−

−+

−−

+−

−−

−−

−−

−−

−+

−−

++

+26

721.

574

5Ab

erra

ntst

rain

P.da

mse

lae

ssp.

pisc

icid

a−

+−

−−

−−

−−

−−

+w−

−−

−−

−−

−+

−−

++

428

ATC

C17

911,

2968

7

P.da

mse

lae

ssp.

pisc

icid

a−

+−

−−

−−

−−

+−

+−

−−

−−

−−

−+

−−

++

2248

1.5

#AT

CC

2969

0,17

911

Phot

obac

teriu

milio

pisc

ariu

m−

++

−−

−−

−−

−−

+−

−−

−−

−−

−+

++

++

599

Phot

obac

teriu

mle

iogn

athi

−+

−−

−−

−−

−+

−+

−−

−−

−−

−−

−+

−+

++

2672

1.5

745

LMG

4228

Phot

obac

teriu

mph

osph

oreu

m−

+w

−−

−−

−+

−+

+−

−−

−+

−+

−2.

8550

9IB

39

Ples

iom

onas

shig

ello

ides

++

++

−−

−−

+−

−+

−+

−−

−−

−−

+41

Prov

iden

cia

(Pro

teus

)ret

tger

i−

−−

−−

−+

++

−−

++

+−

+−

−−

−−

41

Prov

iden

cia

rust

igia

nii

−−

−−

+w−

−+

+−

−+

−−

−−

+w−

−−

++

++

+55

9

Pseu

doal

tero

mon

asul

vae

−−

−−

+−

−+

+−

++

−−

2348

1.5

231

Pseu

dom

onas

angu

illise

ptic

a−

−−

−−

−−

−−

−+

−−

−−

−−

−−

−+

41

Pseu

dom

onas

fluor

esce

ns−

+−

−+

−−

−−

−+

++

++

−+

−−

++

4 1

Pseu

dom

onas

fluor

esce

ns/p

utid

a−

75−

−75

−−

−−

1027

25−

−−

−−

251

20+

26−

++

+−

*

Pseu

dom

onas

plec

oglo

ssic

ida

−−

−−

−−

+−

+−

−+

−42

8

Pseu

dom

onas

putid

a−

−vR

ahne

llaaq

uatil

is+

−−

−+

−−

−−

+−

+−

−+

++

++

−+

++

++

151

Test

son

pgAD

HLD

CO

DC

cit

H2S

ure

TDA

ind

vpG

glu

man

inos

sor

rha

suc

mel

amy

arab

oxN

O2

N2

mot

MC

AO

FTe

mp

Tim

eIn

ocR

efSt

rain

%N

aCl

Tab

le4.

23.

Con

tinue

d.



181

Sale

gent

ibac

ters

aleg

ens

−20

481.

553

3

Salm

onel

lach

oler

asui

sar

izon

ae+

−+

−+

+−

−−

−+

+−

+−

+−

−−

−−

41

Serra

tiafo

ntic

ola

+−

++

+−

−−

−+

−+

+−

++

−+

+−

++

++

+55

8

Serra

tiafo

ntic

ola

+−

++

+−

−−

−−

−+

++

++

20+

+−

++

++

+29

0

Serra

tialiq

uefa

cien

s+

−+

++

−v

−−

v+

++

v+

−+

v+

−−

+41

Serra

tiapl

ymut

hica

+−

−−

+−

−−

−+

++

++

−−

+−

−+

−+

41

Shew

anel

laal

gae

−−

−+

−+

−−

−−

+−

−−

−−

−−

−−

++

++

+−

433

Shew

anel

lafri

gidi

mar

ina

−−v

−−

+v+

−−

−−

++

+−

−−

+−

−−

++

++

+11

2

Shew

anel

lage

lidim

arin

a−

−−

−−

+−

−−

−+

−−

−−

−−

−−

−+

++

112

Shew

anel

lapu

trefa

cien

s−

−−

++

+−

−−

−+

−−

−−

−35

−−

49+

++

++

−43

3

Shew

anel

law

oody

i−

−−

−−

−−

−−

−+

−−

−−

−−

−−

−+

++

+−

112

Tena

ciba

culu

mm

ariti

mum

−−

−−

−−

−−

−−

+−

−−

−−

−−

−−

+41

Vibr

ioae

stua

rianu

s+

+v

−+

++

++

++

Vibr

ioag

ariv

oran

s+

−−

−−

−−

−−

−−

++

−−

−−

−+

−+

+−

++

2548

2.85

135

AHLD

A17

32

Vibr

ioal

gino

lytic

us−

−+

5 3v

−−

−+

8 3v

++

−−

−+

−67

−+

++

++

552

Vibr

ioal

gino

lytic

us−

−+

−−

−−

−+

w+

++

−−

−+

−−

−+

++

−+

+13

5

Vibr

ioal

gino

lytic

us−

−+

+−

−−

−+

++

++

−−

−+

vv

−+

++

++

650

Vibr

iobr

asilie

nsis

++

−−

+−

−−

++

++

+−

−−

+−

+−

++

+n t

++

2 54 8

2.85

740

LMG

2054

6T

Vibr

ioch

oler

aeno

n-01

+−

++

v−

−−

++

++

+−

−−

+−

−−

++

++

+

Vibr

ioch

oler

ae01

+−

++

v−

−−

+−

++

+−

−−

+−

−−

++

++

+

Vibr

ioch

oler

ae+

−+

+w

−−

−+

+w

++

−−

−+

−+

−+

++

++

+2.

8550

9,55

2W

F110

r

Vibr

iodi

abol

icus

−−

++

−−

−+

+−

++

+−

−−

+−

+−

++

++

+63

5

Vibr

iofis

cher

i−

−+

−−

−−

−−

+−

+−

−−

−−

−+

−+

++

−+

+26

721.

574

5AT

CC

2591

8

Vibr

ioflu

vial

is+

+−

−+

−−

−+

−+

++

−−

−+

−−

++

++

++

+25

480.

8524

0M

EJ31

1

Vibr

ioflu

vial

is+

+−

−81

−−

−+

5 6+

++

−−

−+

−−

++

++

++

+37

480.

8548

5,68

7

Vibr

iofu

rnis

sii

++

−−

63−

−−

8875

++

+−

−−

+−

−+

++

++

++

3 74 8

0 .8 5

6 87

Vibr

iofu

rnis

sii

++

−−

w−

−−

+w

−+

+−

−−

+−

−+

++

++

++

2548

0.85

135

ATC

C11

218

Vibr

iofu

rnis

sii

++

−−

+−

−−

+−

++

+−

−−

+−

−+

++

++

++

2548

0.85

240

ATC

C35

016T

Vibr

ioha

liotic

oli

−−

−−

−−

−−

−−

−w

+−

−−

−−

−−

++

−w

w25

482.

8513

5AH

LDA

1734

Vibr

ioha

rvey

i−

−+

++ w

−−

−+

−+

++

−−

−+

−+

−+

++

++

2 54 8

2.85

581

ATC

C14

129

Vibr

io(c

arch

aria

e)ha

rvey

i−

−+

++

−+

−+

−+

++

−−

−+

−+

−+

++

++

+25

482.

8513

5,58

1,84

7AT

CC

3508

4

Vibr

ioic

hthy

oent

eri

−−

−−

−−

−−

−−

−+

−−

−−

+w−

−−

++

+38

9

cont

inue

d



182

Vibr

iom

edite

rrane

i+

++

−−

−−

−+

−+

++

++

−+

−+

−+

++

−+

+25

482.

8513

5AH

LDA

1733

Vibr

iom

imic

us+

−+

++

−−

−+

−+

++

−−

−−

−−

−+

++

++

+16

1,21

0

Vibr

iom

imic

us+

−+

+−

−−

−+

−+

++

−−

−−

−−

−+

++

++

+16

1

Vibr

iom

ytili

+−

−−

−−

−+

+−

−+

+−

−−

+−

+−

++

+63

5

Vibr

iona

varre

nsis

−−

−−

−−

−+

−+

++

−−

−+

−−

++

++

+76

8

Vibr

ione

ptun

ius

−+

−−

+−

−+

++

++

−−

−−

+−

−−

++

+nt

++

2548

2.85

740

LMG

2053

6T

Vibr

ione

resi

s−

−−

−−

−−

++

−−

+−

−−

−+

−−

−+

++

635

Vibr

ioor

dalii

−−

−−

−−

−−

−−

++

+−

−−

+−

−−

+−

428

DF

3K

Vibr

iopa

cini

iv

+−

−w

−−

+−

66v

+−

−−

−+

6 6+

−+

++

++

2848

1.5

306

LMG

1999

T

Vibr

iopa

raha

emol

ytic

us21

−+

+−

−−

−−

++

+−

−−

−+

7 7+

++

+*

,552

Vibr

iopa

raha

emol

ytic

us−

−+

+−

−−

−+

−+

++

−−

−−

−−

w+

++

+w+

+48

1.5

135

ATC

C43

996

Vibr

iopa

raha

emol

ytic

us−

−+

++

−−

−+

−+

++

−−

−−

−−

++

++w

++

428

Vibr

iopa

raha

emol

ytic

us−

−+

+−

−+

−+

−+

++

−−

−−

−−

++

++

++

+10

ATC

C17

802

Vibr

iope

cten

icid

a−

−−

−+

−−

−−

−+

+−

−−

−−

−−

−+

++

n t+

+2.

8547

0

Vibr

iope

naei

cida

+−

−−

+−

nt−

50−

++

−−

−−

−+

+−

++

+nt

++

2538

8

Vibr

iopr

oteo

lytic

us−

++

−+

−−

−+

++

++

−+

−−

−−

−+

+w

++

2 54 8

2.85

135

AHLD

A17

35

Vibr

ioro

tifer

ianu

s+

−+

+−

−83

++

−+

++

−−

−+

++

++

nr+

++

2548

2.85

305

LMG

2146

0T

Vibr

iosa

lmon

icid

a−

−−

−−

−−

−−

−−

+−

−−

−−

−−

−+

41

Vibr

iosc

opht

halm

i−

90−

−−

−−

−−

−−

+−

−−

−+

−−

−+

++

nt+

+25

481.

514

9,25

4C

ECT

4638

Vibr

iosp

lend

idus

biov

arI

++

−−

−v−

−−

+−

++

+−

−−

+−

−−

++

++

+22

480.

8525

4

Vibr

iosp

lend

idus

++

−−

−−

−−

+−

++

+−

−−

v+

+−

++

++

2 52.

8528

1

Vibr

iosp

lend

idus

−−

−−

−−

−−

+−

−+

+−

−−

++

+−

++

−−

++

2548

2.85

466

Vibr

iota

petis

+−

−−

−−

−−

+−

++

−−

−−

−−

+−

++

++

+2 5

2.85

108,

146

Vibr

iotu

bias

hii

+−

−−

−−

−+

+−

++

+−

−−

+−

+−

++

++

+32

1,63

5

Vibr

iotu

bias

hii

++ s

−−

−−

−−

+−

−+

+−

−−

+−

+−

++

++

+2 5

4 82.

8513

5

Vibr

iovu

lnifi

cus

biot

ype

1+

−+

+−

−−

−+

−+

++

−−

−−

−+

−+

+2 6

f

V.vu

lnifi

cus

biot

ype

2+

−+

−+

−−

−−

−+

+−

−−

−−

−+

−+

26

V.vu

lnifi

cus

biot

ype

2+

−+

++

−−

−−

−+

+−

−−

−−

−+

−+

26

Test

son

pgAD

HLD

CO

DC

cit

H2S

ure

TDA

ind

vpG

glu

man

inos

sor

rha

suc

mel

amy

arab

oxN

O2

N2

mot

MC

AO

FTe

mp

Tim

eIn

ocR

efSt

rain

%N

aCl

Tab

le4.

23.

Con

tinue

d.



183

V.vu

lnifi

cus

biot

ype

2se

roE

+−

++

+−

−−

−−

++

+−

−−

−−

+−

++

2548

26g

V.vu

lnifi

cus

biot

ype

2se

roE

+−

++

+−

−−

−−

++

−−

−−

−−

+−

++

98AT

CC

3318

7a

V.vu

lnifi

cus

biot

ype

2se

roE

+−

+−

−−

−−

−−

++

−−

−−

−−

+−

++

98AT

CC

3314

9b

V.vu

lnifi

cus

biot

ype

2se

roE

+−

+−

+−

−−

−−

++

−−

−−

−−

+−

++

98c

V.vu

lnifi

cus

biot

ype

2se

roE

+−

+−

−−

−−

−−

++

−−

−−

−−

−−

++

98d

V.vu

lnifi

cus

biot

ype

2se

roE

−−

+−

−−

−−

−−

++

−−

−−

−−

+−

++

98e

V.vu

lnifi

cus

+−

++

+−

−−

+−

++

+−

−−

−−

w+

4850

9W

F8A1

110

V.xu

ii−

+−

−−

−−

++

+−

++

−−

−+

−+

++

++

n t+

+2 5

4 82.

8574

0LM

G21

346T

Yers

inia

inte

rmed

ia+

−−

+−

−+

−+

−−

++

v+

++

++

v−

41

Yers

inia

ruck

eri

+−

++

+−

−−

−−

++

+−

−−

−−

−−

−41

Yers

inia

ruck

eri

+−

−+

−−

−−

−−

−+

+−

−−

−−

−−

−67

4,71

7

Yers

inia

ruck

eri

+−

−+

−−

−−

−−

−+

+−

+−

−−

−−

−71

7

Yers

inia

ruck

eri

+−

−+

−−

−−

−+

−+

+−

+−

−−

−−

−71

7

Yers

inia

ruck

eri

+−

++

−−

−−

−+

−+

+−

−−

−−

−−

−+

++

++

2548

0.85

135,

203

AHLD

A13

13

Yers

inia

ruck

eri

+−

++

−−

−−

−−

−+

+−

−−

−−

−−

−+

++

++

674

Yers

inia

ruck

eri

+−

++

−−

−−

−+

−+

+−

+−

−−

−−

−+

++

++

2548

0.85

184

Yers

inia

ruck

eri

+−

++

−−

−−

−+

++

+−

+−

−−

−−

−+

++

++

2548

0.85

184

Yers

inia

ruck

eri

+−

++

−−

−−

−−

−+

+−

−−

−−

−−

−+

++

++

2548

0.85

184

Zobe

lliaga

lact

aniv

oran

s+

−−

−−

−−

−−

−+

++

−−

++

−−

++

+−

+−

307d

1.5

61

Zobe

lliaul

igin

osa

+−

−−

−−

−−

−+

+−

−−

++

+−

+−

307d

1.5

61

#=

Ref

eren

ces:

149,

289,

518,

674,

751,

855.

onpg

=b-

gala

ctos

idas

e;AD

H=

argi

nine

dihy

rola

se;L

DC

=ly

sine

deca

rbox

ylas

e;O

DC

=or

nith

ine

deca

rbox

ylas

e;ci

t=ci

trate

;H2S

=pr

oduc

tion

ofhy

drog

ensu

lphi

de;u

re=

urea

se; T

DA

=try

ptop

hane

deam

inas

e;in

d=

indo

le;v

p=

Voge

s-Pr

oska

uer;

G=

gela

tin;g

lu=

gluc

ose;

man

=m

anni

tol;

inos

=in

osito

l;so

r=so

rbito

l;rh

a=

rham

nose

;suc

=su

cros

e;m

el=

mel

ibio

se;a

my

=am

ygda

lin;a

rab

=ar

abin

ose;

ox=

oxid

ase;

NO

2=

nitra

tere

duct

ion;

N2

=re

duct

ion

toni

trite

gas;

mot

=m

otilit

y;M

CA

=gr

owth

onM

acC

onke

y;O

=ox

idat

ive

ferm

enta

tion;

F=

ferm

enta

tion;

Tem

p=

tem

pera

ture

ofin

cuba

tion;

Tim

e=

time

ofin

cuba

tion;

h=

hour

s;d

=da

ys;I

noc

=in

ocul

um;%

NaC

l=fin

alN

aClc

once

ntra

tion

inin

ocul

um;w

=w

eak;

+=

posi

tive

reac

tion;

−=

nega

tive

reac

tion;

s=

slow

reac

tion;

num

bers

indi

cate

perc

enta

geof

posi

tive

stra

ins.

a=

note

that

A.hy

drop

hila

stra

ins

are

cons

ider

edto

bene

gativ

efo

rsor

bito

l.b

=Th

ein

cuba

tion

time

was

nots

tate

dfo

rthi

sre

sult .

Itis

impo

rtant

toin

cuba

tefo

r48

hto

ensu

reco

rrect

LDC

resu

lt.s

=sl

owre

actio

n;V

=va

riabl

ere

actio

n;*

from

APId

atab

ase;

Ref

=re

fere

nce

num

ber.



184

Acinetobacter/Moraxella 0000004 1 41

Actinobacillus delphinicola 200500416 2 Dolphin, whale 263

Actinobacillus scotiae NCTC 12922 111500416 1 265

Aeromonas allosaccharophila 724613657 1 41



Aeromonas bestiarum 704713757 5 Carp 452

Aeromonas caviae ATCC 15468T 324612657 21,240

Aeromonas encheleia 304613457 241

Aeromonas hydrophila ATCC 7966T 704712657 1 240

Aeromonas hydrophila ATCC 7966T 304512657 1 Important to incubate for 48 h forcorrect LDC result

674

Aeromonas hydrophila ATCC 14715 324713757 1 322

Aeromonas janadaei 724714457 2 135,143

Aeromonas salmonicida 200610417 450

Aeromonas salmonicida AHLDA 1334 204402417 1 ‘Atypical’ strain, imported goldfish(Western Australia)

135

Aeromonas salmonicida achromogenes 004412417 322

Aeromonas salmonicida achromogenes NCIMB 1110 004512417 468

Aeromonas salmonicida masoucida 304512517 322

Aeromonas salmonicida salmonicida 200610417 674



Aeromonas salmonicida salmonicida NCIMB 1102 700610517 468

Aeromonas sobria CIP 74.33T 704712457 240

Aeromonas sobria 724712457 21

Aeromonas trota ATCC 49657T 724610557 142,560

Aeromonas veronii veronii ATCC 35623 114612557 1 347

Aeromonas veronii veronii ATCC 35604 114712457 1 Human, USA 347


Aeromonas veronii veronii ATCC 35605 134712557 1 Wound isolate, human, USA 347


Aeromonas veronii veronii 534712557 12 Various strains 2,347,452

Burkholderia cepacia 0004004 3/10 38




Burkholderia pseudomallei 0006727 1/91 38




Burkholderia pseudomallei NCTC8016 2206707 1/91 38










Edwardsiella hoshinae 454412057 *

Edwardsiella hoshinae 474412057 *

Edwardsiella ictaluri 410400057 41,334

Edwardsiella tarda 454400057 41

Edwardsiella tarda AHLDA 135 474400057 135

Organism Strain API 20 E number No of strains Notes Ref

Table 4.24. API 20E database numbers (organisms listed alphabetically).



185

Edwardsiella tarda – atypical 4644100.. 4 Atypical strains from red sea bream,Japan

845

Enterobacter agglomerans 120712057 41

Enterovibrio norvegicus 30440044. 741

Flavobacterium branchiophilum 0006060.. 41

Flavobacterium columnare 0402004.. 41

Flavobacterium columnare 140200410 689

Flavobacterium gillisiae 200412000 533

Flavobacterium hibernum 100603210 532

Flavobacterium hydatis 000612210 41

Flavobacterium psychrophilum 0002004.. 41

Flavobacterium tegetincola 00041000. 533

Flavobacterium xantham 04061241. 533

Hafnia alvei 430410257 41



Hafnia alvei ATCC 51873 5304112.. 1 203

Halomonas cupida 6100530.. 41

Janthinobacterium lividum 2207104.. 41

Klebsiella pneumoniae 521577357 41

Listonella anguillarum V10 304452456 1 Japan 49


Listonella anguillarum PT-87050 304652456 2 Japan 49

Listonella anguillarum 304752456 1

Listonella anguillarum V244 to V246 304752476 5 Japan 49

Listonella anguillarum HT-77003 304752657 1 Japan 49



Listonella anguillarum ET-78063 324632657 1 Japan 49

Listonella anguillarum UB 4346, 434 324712677 2 Spain 49

Listonella anguillarum NCMB 6 3247524.. 1 240

Listonella anguillarum UB (ET-1) 324752557 1 Japan 49

Listonella anguillarum V318, AHLDA 1730 324752656 2 Japan, Australia 49,135

Listonella anguillarum RH-8101, AVL 324752657 6 France, Japan 49

Listonella anguillarum 3247527.. 41



Listonella anguillarum LMG 3347 324752757 4 Japan, Norway, Spain 49

Listonella anguillarum UB A078 324752777 1 Spain 49

Listonella anguillarum V320 324772656 1 Spain 49

Listonella anguillarum UB A054–56 324772657 4 Japan, Spain 49

Listonella pelagia 100412456



Moritella marina 000600456 766

Moritella viscosa 400400456 132

Moritella viscosa 400600456 132

Pantoea agglomerans CDC 1429–71 100577357 291

Pantoea agglomerans ATCC 14589 120516357 291

Pantoea agglomerans NCPPB 2285 120517257 291

Pantoea agglomerans DNA HG 14589 120517357 291

Pantoea agglomerans 120713257 291

Pantoea dispersa 120713357 291

Pantoea dispersa 120717357 291


Table 4.24. Continued.

continued



186

Pasteurella multocida 014452456 428

Phocoenobacter uteri 000100416 266

Phocoenobacter uteri 000500416 266

Photobacterium angustum NCIMB 1895 200702406 745

Photobacterium damselae damselae LMG 7892 200500457 49

Photobacterium damselae damselae 201500457 41

Photobacterium damselae damselae ATCC 33539T 201500457 745

Photobacterium damselae damselae NCIMB 2184 220400157 745

Photobacterium damselae damselae ATCC 33539T 600400457 240

Photobacterium damselae damselae LMG 13639 600500457 6 Various sources 49

Photobacterium damselae piscicida P90029 001100407 1 Atypical strain 745

Photobacterium damselae piscicida ATCC 17911 200400406 ATCC 29687 428

Photobacterium damselae piscicida ATCC 17911 200500406 ATCC 29690, and other strains.Refs: 149, 518, 855, 751

289,674,745

Photobacterium damselae piscicida ATCC 17911 200500407 Majority NCIMB 2058 745

Photobacterium damselae piscicida 220500407 745

Photobacterium iliopiscarium 600400457 1 599

Photobacterium leiognathi LMG 4228 200500017 745

Photobacterium phosporeum IB39 6046021.. 509

Plesiomonas shigelloides 714420457 41

Providencia rettgeri 007431057 41

Providencia rustigianii 026402057 559

Pseudomonas aeruginosa 0206006 1/18 38





Pseudomonas anguilliseptica 000200440 41

Pseudomonas anguilliseptica 020000440 96,541,828

Pseudomonas anguilliseptica 020200440 96,541,828

Pseudomonas fluorescens 0204004.. 2/8 38

Pseudomonas fluorescens 2000004.. 5/8 38

Pseudomonas fluorescens 220000443 1/8 38

Pseudomonas fluorescens 220000453

Pseudomonas stutzeri 0000004.. 1/9 38



Rahnella aquatilis 100557317 *

Rahnella aquatilis 120547257 151

Rahnella aquatilis 120557317 *

Salmonella cholerasuis arizonae 560621057 41

Serratia fonticola 530475257 290



Serratia plymuthica 120732257 41

Shewanella algae 050200453 433

Shewanella frigidimarina 06061245. 112

Shewanella frigidimarina 26061245. 112

Shewanella gelidimarina 04020045. 112

Shewanella putrefaciens 070200453 433



Shewanella woodyi 000200452 112

Tenacibaculum maritimum 000200410 41

Vibrio agarivorans AHLDA 1732 100410556 2 From abalone. Pathogenicityunknown

135





187

Vibrio alginolyticus 404712456 135






Vibrio brasiliensis LMG 20546T 32471255. 740

Vibrio cholerae LMG 16741 204612457 1 Shrimp, Thailand 49

Vibrio cholerae LMG 16742 324602557 1 Pe. orientalis, PRC, Thailand 49

Vibrio cholerae 01 514612457

Vibrio cholerae non 01 514712457

Vibrio cholerae PS-7701 524712457 1 Pl. altivelis, Japan 49

Vibrio cholerae PS-7705 524712476 1 Pl. altivelis, Japan 49

Vibrio cholerae 91/1198 534612457 1 Carassius auratus, Australia 49

Vibrio cholerae 01 534612457

Vibrio cholerae non 01 LMG 16743 534712457 1 49

Vibrio cholerae WF 110r 534712557 509,552

Vibrio diabolicus 416612557 635

Vibrio fischeri ATCC 25918 400500556 745

Vibrio fluvialis 304612657 485,687



Vibrio fluvialis and MEJ 311 324612657 2 240,485,687


Vibrio furnissii ATCC 35016T 324612657 240

Vibrio furnissii 304712657 687

Vibrio furnissii 324712657 687

Vibrio furnissii ATCC 11218 324512657 Citrate, indole, VP, weak reaction 135

Vibrio (carchariae) harveyi 415412557 135,581,847


Vibrio harveyi ATCC 14129 434612557 581


Vibrio (carchariae) harveyi ATCC 35084 435612557 135,581,847

Vibrio halioticoli AHLDA 1734 000410456 1 Abalone 135

Vibrio ichthyoenteri 00040245. 389

Vibrio mediterranei AHLDA 1733 704672556 1 Abalone 135

Vibrio mimicus 514610457 1 161

Vibrio mimicus 534610457 1 161,210

Vibrio mytili 106412556 635

Vibrio neptunius LMG 20536T 22670245. 740

Vibrio nereis 00640245. 635

Vibrio ordalii LMG 13544 000402446 1 49

Vibrio ordalii LMG 10951 000402476 3 Salmon 49

Vibrio ordalii V-306 000402556 1 Amago trout, Japan 49

Vibrio ordalii RF-2, PT-81025 000402576 8 O. mykiss, Pl. altivelis, Japan 49

Vibrio ordalii V-11 000412446 1 O. mykiss, Japan 49

Vibrio ordalii F378, F380, V-250 000602546 4 Salmon, Australia; O. mykiss, Japan 49

Vibrio ordalii F379, F381 000612446 2 Salmon, Australia 49

Vibrio ordalii DF 3K 000612446 1 Denmark 428

Vibrio ordalii V-302 304752476 4 O. mykiss, Japan 49

Vibrio parahaemolyticus 410610657 552


Vibrio parahaemolyticus ATCC 43996 414610657 1 135




continued



188


Vibrio parahaemolyticus ATCC 17802 434610657 10

Vibrio pectenicida 020600456 470

Vibrio penaeicida 12060455. 388

Vibrio penaeicida 12460455. 388

Vibrio proteolyticus AHLDA 1735 624750456 1 Artemia 135

Vibrio rotiferianus LMG 21460T 5176167.. 305

Vibrio salmonicida RVAU 890206–1/12 000410040 1 Salmo salar, Faroe Islands 49





Vibrio scophthalmi 20040245. 149

Vibrio splendidus 004416516 466

Vibrio splendidus UB S292 204610456 1 Seawater, Sweden 49

Vibrio splendidus 90–0652 224614446 1 Striped trumpeter, Australia 49

Vibrio splendidus RVAU 88–12–686 304610456 3 Sa. Salar, Norway 49

Vibrio splendidus UB S236 304610556 1 Seawater, Sweden 49

Vibrio splendidus RVAU 88–12–711 304612456 1 Sp. auratus, Norway 49

Vibrio splendidus 89–1638 304612556 1 O. mykiss, Australia 49



Vibrio splendidus RVAU 88–12–717 324410556 1 Sc. maximus, Norway 49

Vibrio splendidus LMG 16752 324414557 1 Oyster, Spain 49

Vibrio splendidus RVAU 88–12–712 324610456 1 Solea solea, Norway 49

Vibrio splendidus UB S308, LMG 16747 324610556 2 Seawater, Sweden; oyster, Spain 49

Vibrio splendidus LMG 16745 324610557 1 Sp. auratus, Greece 49

Vibrio splendidus LMG 16744, 16750 324612557 2 Sp. auratus, Greece; oyster, Spain 49




Vibrio tapetis 10460055. 108,146

Vibrio tubiashii 106612556 321,635

Vibrio tubiashii 304412556 321,635

Vibrio vulnificus biovar 1 non-serovar E M06–24 434610557 1 Human blood, USA 98

V. vulnificus biovar 1 non-serovar E 532 504600557 2 Diseased eel, Belgium, avirulent foreels

33,98

V. vulnificus biovar 1 non-serovar E 628 514412757 1 Paguara fish, Venezuela 98

V. vulnificus biovar 1 non-serovar E 530 514602557 7 Diseased eel, Belgium, virulent 33,98

V. vulnificus biovar 1 non-serovar E ATCC 27562 514610557 1 Human strain, USA 26,98

V. vulnificus biovar 1 non-serovar E 514610557 23 Human (USA), eels (Spain,Sweden), shrimp (Thailand)

33,98

V. vulnificus biovar 1 non-serovar E B9629, C7184 514610557 Human wound infection, USA 33,98

V. vulnificus biovar 1 non-serovar E UMH1, 374 514610557 Human wound infection, USA 33,98

V. vulnificus biovar 1 non-serovar E ATCC 27562 534600557 Human strain, USA 33

V. vulnificus biovar 1 non-serovar E E109 534600557 Healthy eel, Spain, avirulent for eels 98

V. vulnificus biovar 1 non-serovar E C7184 534610557 1 Human blood, USA, avirulent foreels

98

V. vulnificus biovar 1 serovar E 534 514410557 2 Diseased eel, Sweden, avirulent foreels

33,98

V. vulnificus biovar 1 serovar E ATCC 33186 514610557 Human blood, USA, avirulent foreels

98

V. vulnificus biovar 1 serovar E 521 534600557 Unknown, Australia, avirulent foreels

98

V. vulnificus biovar 1 LMG 12092 104602557 1 Eel, Belgium 33





189

V. vulnificus biovar 1 169 104610557 1 Eel, Belgium 33

V. vulnificus biovar 1 E109 114000557 1 Eel, Spain 33


V. vulnificus biovar 1 M626 410610457 1 Eel, Spain 33

V. vulnificus biovar 1 Vv1 504600557 Human wound infection, USA 33

V. vulnificus biovar 1 UMH1 504610557 1 Human wound infection, USA 33

V. vulnificus biovar 1 M631 510610557 1 Eel, Spain 33


V. vulnificus biovar 1 VIB 521 516600557 1 Unknown 33

V. vulnificus biovar 1 524610557 97

V. vulnificus biovar 2 serovar E 171 100600557 1 Diseased eel, Sweden 98


V. vulnificus biovar 2 serovar E NCIMB 2136 400600557 1 Diseased eel, Japan 98

V. vulnificus biovar 2 420600557 97

V. vulnificus biovar 2 serovar E 520 500400557 1 Shrimp, Taiwan, virulent for eels 98

V. vulnificus biovar 2 serovar E NCIMB 2137 500600457 1 Diseased eel, Japan 33,98

V. vulnificus biovar 2 serovar E ATCC 33149 500600557 1 Diseased eel, Japan 33,98,356

V. vulnificus biovar 2 NCIMB 2138 500600557 1 Diseased eel, Japan 98



V. vulnificus biovar 2 serovar E 524 510600557 Diseased eel, Norway 98

V. vulnificus biovar 2 NCIMB 2136 510600557 1 Diseased eel, Japan 33

V. vulnificus biovar 2 510600557 14 Eels (Japan, Norway – serovar E,Spain, Sweden), shrimp, human

33,98

V. vulnificus biovar 2 serovar E 523 514600557 5 Eels (Belgium – serovar E, Spain,Sweden), sea bream (Spain)

33,98

V. vulnificus biovar 2 serovar E NCIMB 2138 520600557 1 Diseased eel, Japan 98

V. vulnificus biovar 2 serovar E E86 520600557 2 Diseased eel, (Japan, Spain) 26,98


V. vulnificus biovar 2 serovar E ATCC 33187 530600557 1 Human strain, USA 98

V. vulnificus biovar 2 serovar E E105 530600557 Diseased eel, Spain 26,98

V. vulnificus biovar 2 serovar E 530600557 2 Human (USA), eel (Spain) 26,98

V. vulnificus biovar 2 serovar E 530610557 1 Taiwanese strains, avirulent for eels 26

V. vulnificus 818 414412557 1 Unknown, France 98

V. vulnificus 822 504410557 1 Shrimp, Senegal 98

V. vulnificus WF8A1110 534610357 509

Vibrio xuii LMG 21346T 20651275. 740

Yersinia intermedia 115457157 41




Yersinia ruckeri 110410057 674,717

Yersinia ruckeri 110450057 717


Yersinia ruckeri ATCC 29473 510410057 2 184,674

Yersinia ruckeri AHLDA 1313 510510057 1 135

Yersinia ruckeri 5105500 20 Environmental strains 184

Yersinia ruckeri 5107500 1 Environmental strains 184


Zobellia galactanovorans 10061361. 61

Zobellia uliginosa 100.0..5. 61

*from API database.To be used in conjunction with the API database supplied by bioMerieux.





190

0000004.. Acinetobacter/Moraxella 1 41

0000004.. Pseudomonas stutzeri 1/9 38

000100416 Phocoenobacter uteri 266

0002004.. Flavobacterium psychrophilum 41

000200410 Tenacibaculum maritimum 41

000200440 Pseudomonas anguilliseptica 41

000200452 Shewanella woodyi 112


0004004.. Burkholderia cepacia 3/10 38

000410456 Vibrio halioticoli AHLDA 1734 1 Abalone 135

000402446 Vibrio ordalii LMG 13544 49

00040245. Vibrio ichthyoenteri 389

000402476 Vibrio ordalii LMG 10951 Salmon 49

000402576 Vibrio ordalii RF-2, PT-81025 O. mykiss, Pl. altivelis, Japan 49

00041000. Flavobacterium tegetincola 533

000410040 Vibrio salmonicida RVAU 890206–1/12 Salmo salar, Faroe Islands 49





000500416 Phocoenobacter uteri 266

000600456 Moritella marina 766

000602546 Vibrio ordalii F378, F380 Salmon, Australia 49

0006060.. Flavobacterium branchiophilum 41

000612210 Flavobacterium hydatis 41

000612446 Vibrio ordalii F379, F381 Salmon, Australia 49

000612446 Vibrio ordalii DF 3K 428

0006727.. Burkholderia pseudomallei 1/91 38

001100407 Photobacterium damselae piscicida P90029 1 Atypical strain 745

004412417 Aeromonas salmonicida achromogenes 322

004416516 Vibrio splendidus 466

004512417 Aeromonas salmonicida achromogenes NCIMB 1110 468

00640245. Vibrio nereis 635

007431057 Providencia rettgeri 41

014452456 Pasteurella multocida 428

020000440 Pseudomonas anguilliseptica 96,541,828

020200440 Pseudomonas anguilliseptica 96,541,828

0204004.. Pseudomonas fluorescens 2/8 38

020600456 Vibrio pectenicida 470


0206006.. Pseudomonas aeruginosa 1/18 38


026402057 Providencia rustigianii 559

0402004.. Flavobacterium columnare 41

04020045. Shewanella gelidimarina 112

04061241. Flavobacterium xantham 533

050200453 Shewanella algae 433

06061245. Shewanella frigidimarina 112

070200453 Shewanella putrefaciens 433


API 20 E number Organism Strain No of strains Notes Ref

Table 4.25. API 20E database numbers (numbers in ascending order).



191


100.0..5. Zobellia uliginosa 61

100410556 Vibrio agarivorans AHLDA 1732 2 From abalone, pathogenicity unknown 135

100412456 Listonella pelagia

100577357 Pantoea agglomerans CDC 1429–71 291

100600557 Vibrio vulnificusbiovar 2 serovar E

171 Diseased eel, Sweden 98

100603210 Flavobacterium hibernum 532

10061361. Zobellia galactanovorans 61

10460055. Vibrio tapetis 108,146

104600557 Vibrio vulnificus biovar 2 171 Eel, Belgium 33

104602557 Vibrio vulnificus biovar 1 LMG 12092 Eel, Belgium 33



106412556 Vibrio mytili 635

106612556 Vibrio tubiashii 321,635

110410057 Yersinia ruckeri 674,717

110450057 Yersinia ruckeri 717


111500416 Actinobacillus scotiae NCTC 12922 1 265

114000557 Vibrio vulnificus biovar 1 E109 Eel, Spain 33


114612557 Aeromonas veronii veronii ATCC 35623 1 347

114712457 Aeromonas veronii veronii ATCC 35604 1 Human, USA 347

115457157 Yersinia intermedia 41



120516357 Pantoea agglomerans ATCC 14589 291

120517257 Pantoea agglomerans NCPPB 2285 291

120517357 Pantoea agglomerans DNA HG 14589 291

120547257 Rahnella aquatilis 151

12060455. Vibrio penaeicida 388

120712057 Enterobacter agglomerans 41

120713257 Pantoea agglomerans 291

120713357 Pantoea dispersa 291

120717357 Pantoea dispersa 291

120732257 Serratia plymuthica 41

12460455. Vibrio penaeicida 388



134712557 Aeromonas veronii veronii ATCC 35605 1 Wound isolate, human, USA 347

140200410 Flavobacterium columnare 689



200400406 Photobacterium damselae piscicida ATCC 17911 ATCC 29687 428

20040245. Vibrio scophthalmi 149

200412000 Flavobacterium gillisiae 533

200500017 Photobacterium leiognathi LMG 4228 745



continued



192

200500406 Photobacterium damselae piscicida 149,289,751,855

200500406 Photobacterium damselae piscicida ATCC 17911 ATCC 29690 674,745

200500407 Photobacterium damselae piscicida ATCC 17911 Majority NCIMB 2058 745

200500416 Actinobacillus delphinicola 2 Dolphin, whale 263

200500457 Photobacterium damselae damselae LMG 7892 49


200610417 Aeromonas salmonicida 450

200610417 Aeromonas salmonicida salmonicida 674






200702406 Photobacterium angustum NCIMB 1895 745

201500457 Photobacterium damselae damselae 41

201500457 Photobacterium damselae damselae ATCC 33539 745

204402417 Aeromonas salmonicida AHLDA 1334 1 ‘Atypical’ strain, goldfish 135

204610456 Vibrio splendidus UB S292 Seawater, Sweden 49

204612457 Vibrio cholerae LMG 16741 Shrimp, Thailand 49

20651275. Vibrio xuii LMG 21346T 740




220400157 Photobacterium damselae damselae NCIMB 2184 745

220500406 Photobacterium damselae piscicida 745





2206707.. Burkholderia pseudomallei NCTC8016 1/91 38



2207104.. Janthinobacterium lividum 41

224614446 Vibrio splendidus 90–0652 Striped trumpeter, Australia 49

22670245. Vibrio neptunius LMG 20536T Rotifers, bivalve larvae 740

26061245. Shewanella frigidimarina 112


324612657 Aeromonas caviae ATCC 15468T 240

30440044. Enterovibrio norvegicus LMG 19839T 741

304412556 Vibrio tubiashii 321,635

304452456 Listonella anguillarum 49

304512517 Aeromonas salmonicida masoucida 322

304512657 Aeromonas hydrophila ATCC 7966 1 674

304512657 Vibrio furnissii ATCC 11218 1 Citrate, indole, VP weak reaction 135


304610456 Vibrio splendidus RVAU 88–12–686 Sa. Salar, Norway 49


304612456 Vibrio splendidus RVAU 88–12–711 Sp. auratus, Norway 49

304612556 Vibrio splendidus 89–1638 O. mykiss, Australia 49





193

304612657 Vibrio fluvialis 485,687

304613457 Aeromonas encheleia 241





304712657 Vibrio furnissii 687

304752476 Listonella anguillarum



324410556 Vibrio splendidus RVAU 88–12–717 Sc. maximus, Norway 49


324414557 Vibrio splendidus LMG 16752 Oyster, Spain 49


324512657 Vibrio furnissii ATCC 11218 1 Citrate, indole, VP weak reaction 135



324602557 Vibrio cholerae LMG 16742 Pe. orientalis, PRC 49

324610456 Vibrio splendidus RVAU 88–12–712 Solea solea, Norway 49


324610557 Vibrio splendidus LMG 16745 Sp. auratus, Greece 49

324612557 Vibrio splendidus LMG 16744 Sp. auratus, Greece 49

324612657 Aeromonas caviae 21

324612657 Vibrio fluvialis and MEJ 311 3 485,687,240

324612657 Vibrio furnissii ATCC 35016T 240



32471255. Vibrio brasiliensis LMG 20546T Bivalve larvae 740



324712657 Vibrio furnissii ATCC 11218 135,687


324713757 Aeromonas hydrophila ATCC 14715 1 322


324752656 Listonella anguillarum and AHLDA 1730 2 Japan, Australia (freshwater fish) 49,135

324752756 Listonella anguillarum 41,49,428





400400456 Moritella viscosa 132

400500556 Vibrio fischeri ATCC 25918 745

400600456 Moritella viscosa 132

400600557 V. vulnificusbiovar 2 serovar E

NCIMB 2136 Diseased eel, Japan 98

404712456 Vibrio alginolyticus 135


410400057 Edwardsiella ictaluri 41,334

410610457 V. vulnificus biovar 1 M626 Eel, Spain 33



continued



194

410610657 Vibrio parahaemolyticus 552


414412557 V. vulnificus 818 Unknown, France 98

414610657 Vibrio parahaemolyticus ATCC 43996 135




415412557 Vibrio (carchariae) harveyi 135,581,847


416612557 Vibrio diabolicus 635

420600557 V. vulnificus biovar 2 97


430410257 Hafnia alvei 41


434610557 V. vulnificusbiovar 1 non-serovar E

M06–24 Human blood, USA 98


434610657 Vibrio parahaemolyticus ATCC 17802 10

434612557 Vibrio harveyi ATCC 14129 581



435612557 Vibrio (carchariae) harveyi ATCC 35084 135,581,847

454400057 Edwardsiella tarda 41

4644100.. Atypical Edwardsiella tarda 4 Atypical strains from red sea bream,Japan

845

474400057 Edwardsiella tarda AHLDA 135 ATCC 15947T 135,845


520 Shrimp, Taiwan, virulent for eels 98


NCIMB 2137 Diseased eel, Japan 33,98


ATCC 33149 Diseased eel, Japan 33,98,356

500600557 V. vulnificus biovar 2 NCIMB 2138 Diseased eel, Japan 98

504410557 V. vulnificus 822 Shrimp, Senegal 98


532 Diseased eel, Belgium, avirulent foreels

98

504600557 V. vulnificus biovar 1 Vv1 Human wound infection, USA 33

504610557 V. vulnificus biovar 1 UMH1 Human wound infection, USA 33





510410057 Yersinia ruckeri ATCC 29473 184,674

510411257 Hafnia alvei 652

510510057 Yersinia ruckeri AHLDA 1313 2 Channel catfish 135,203

510550057 Yersinia ruckeri 20 Environmental isolates 184

510600557 V. vulnificus biovar 2 NCIMB 2136 Diseased eel, Japan 33


524 Diseased eel, Norway 98

510610557 V. vulnificus biovar 1 M631 Eel, Spain 33

510710057 Yersinia ruckeri YR55, YR80 2 Environmental isolates 184

510750057 Yersinia ruckeri 1 Environmental isolates 184





195


534 Diseased eel, Sweden, avirulent foreels

98


628 Paguara fish, Venezuela 98


523 Diseased eel 98


530 Diseased eel, Belgium, virulent 98

514610457 Vibrio mimicus 161


ATCC 27562 Human strain, USA 26,98


B9629, C7184 Human wound infection, USA 33,98


UMH1, 374 Human wound infection, USA 33,98


ATCC 33186 Human blood, USA, avirulent for eels 98

514612457 Vibrio cholerae 01

514650557 V. vulnificus biovar 1 167 Eel, Belgium 33

514712457 Vibrio cholerae non 01


516600557 V. vulnificus biovar 1 VIB 521 Unknown 33

5176167.. Vibrio rotiferianus LMG 21460T Rotifer 305



NCIMB 2138 Diseased eel, Japan 98


E86 Diseased eel, Spain 26,98

521577357 Klebsiella pneumoniae 41

524610557 V. vulnificus biovar 1 97

524712457 Vibrio cholerae PS-7701 Pl. altivelis, Japan 49

524712476 Vibrio cholerae PS-7705 Pl. altivelis, Japan 49


5304112.. Hafnia alvei ATCC 51873 1 203

530475257 Serratia fonticola 290


530510057 Yersinia ruckeri AHLDA 1313 135

530511357 Hafnia alvei AHLDA 1729 135





ATCC 33187 Human strain, USA 98


E105 Diseased eel, Spain 26,98



Taiwanese strains, avirulent for eels 26


ATCC 27562 Human strain, USA 33


E109 Healthy eel, Spain, avirulent for eels 98



continued



196


521 Unknown, Australia, avirulent for eels 98

534610357 V. vulnificus WF8A1110 509

534610457 Vibrio mimicus 161,210


C7184 Human blood, USA, avirulent for eels 98

534612457 Vibrio cholerae 91/1198 Carassius auratus, Australia 49

534612457 Vibrio cholerae 01

534712457 Vibrio cholerae non 01

534712557 Aeromonas veronii veronii 2,347

534712557 Vibrio cholerae WF 110r 509,552

560621057 Salmonella cholerasuis arizonae 41

600400457 Photobacterium iliopiscarium 599

600400457 Photobacterium damselae damselae ATCC 33539T Usually VP positive 240

600500457 Photobacterium damselae damselae LMG 13639 6 Various sources 49


6046021.. Photobacterium phosphoreum IB39 509

6100530.. Halomonas cupida 41

624750456 Vibrio proteolyticus AHLDA 1735 1 Artemia 135

700610517 Aeromonas salmonicida salmonicida NCIMB 1102 468

704672556 Vibrio mediterranei AHLDA 1733 1 Abalone 135

704712457 Aeromonas sobria CIP 74.33T 240

704712657 Aeromonas hydrophila ATCC 7966T 1 240

704713757 Aeromonas bestiarum 5 Carp 452

714420457 Plesiomonas shigelloides 41

724610557 Aeromonas trota ATCC 49657T 1 142,560

724613657 Aeromonas allosaccharophila 1 41



724712457 Aeromonas sobria 21

724714457 Aeromonas janadaei 2 135,143

Not all references reported the 9-digit profile number, hence some species only have a 7- or 8-digit profile number with no results for growth on MCA orOF results.





197

Org

anis

m

Acin

etob

acte

rcal

coac

etic

us−

−−

−−

−−

−−

−−

−−

−−

+−

−+

+−

2441

Actin

obac

illus

delp

hini

cola

+−

−−

−−

−−

−−

−−

−−

−−

−−

−−

+26

3D

olph

in

Actin

obac

illus

delp

hini

cola

+−

−+

−−

−−

−−

−−

−−

−−

−−

−−

+26

3Po

rpoi

se

Actin

obac

illus

delp

hini

cola

+−

−−

−−

−−

−−

−−

−−

−−

−−

−−

+26

3W

hale

Aero

mon

ashy

drop

hila

+v

++

−+

++

+v

++

++

++

−+

v−

+41

A.sa

lmon

icid

aac

hrom

ogen

es/m

asou

cida

+v

−−

−v

v−

v−

vv

−−

−−

−−

−−

+41

A.sa

lmon

icid

asa

lmon

icid

a+

vv

v−

++

−+

−v

++

++

−−

+−

−+

41

Aero

mon

asso

bria

+v

++

−−

++

+−

++

++

++

−+

+−

+41

Alte

rom

onas

nigr

ifaci

ens

−−

++

+−

++

++

++

395

Bord

etel

labr

onch

isep

tica

−−

−−

+−

−−

−−

−−

−−

−−

+−

++

+37

0.85

102

Type

stra

in

Bord

etel

labr

onch

isep

tica

+−

−−

+−

−−

−−

−−

−−

−−

+−

++

+37

0.85

102

Type

stra

in

Bord

etel

labr

onch

isep

tica

+−

−−

+−

−−

−−

−−

−−

−−

+−

+−

+37

0.85

102

Type

stra

in

Brev

undi

mon

asdi

min

uta

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

+68

5

Bruc

ella

spp.

+−

−−

+−

−−

−−

−−

−−

−−

−−

−−

+37

0.85

262

Chr

omob

acte

rium

viol

aceu

m−

−+

+−

−+

−+

−−

−+

−+

+−

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−+

482

Pigm

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d

Chr

omob

acte

rium

viol

aceu

m+

−+

+−

−+

−+

−−

−+

−+

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482

Non

-pig

men

ted

Chr

yseo

bact

eriu

mba

lust

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−−

++

−−

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−−

w−

−−

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−+

3748

0.85

773

Chr

yseo

bact

eriu

mgl

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−−

++

++

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+−

−−

−+

−+

3748

0.85

773

Chr

yseo

bact

eriu

min

dolo

gene

s−

+−

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w−

w−

−w

−−

−−

w−

+37

480.

8577

3

Chr

yseo

bact

eriu

min

dolth

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um−

+−

−−

++

w−

w−

−w

−−

−−

−w

+37

480.

8577

3

Chr

yseo

bact

eriu

mm

enin

gose

ptic

um−

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−18

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+−

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+−

−−

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27−

+37

480.

8577

3

Chr

yseo

bact

eriu

msc

opht

halm

um−

−w

−+

++

+−

−+

−+

557

Flav

obac

teriu

mhi

bern

um+

−+

−−

++

++

+−

++

−−

−−

−−

−53

2

Hal

omon

ascu

pida

++

++

++

+−

69

Hal

mon

asve

nust

a+

−−

−+

+−

++

−−

−+

++

+−

++

++

310

Jant

hino

bact

eriu

mliv

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+−

−−

−+

−−

++

++

v+

−v

v+

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+49

6

NO

3TR

PG

luAD

HU

reEs

cG

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gG

luAr

aM

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Tim

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Tab

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26.

AP

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Eda

taba

sebi

oche

mic

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sults

.

cont

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198

List

onel

laan

guilla

rum

++

++

−−

++

++

++

++

++

+10

8

Myr

oide

sod

orat

imim

us−

−−

−+

−+

−−

−−

−−

−−

−+w

−−

+77

4

Myr

oide

sod

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us−

−−

−+

−+

−−

−−

−−

−−

−+w

−−

+24

774

Oce

anom

onas

baum

anni

i+

−−

−−

−−

−−

−−

−−

−−

+−

++

−+

2548

1.5

130

Oce

anom

onas

doud

orof

fii+

−−

−−

−−

−−

−−

−−

−−

+−

++

−+

2548

1.5

130

Phoc

oeno

bact

erut

eri

+−

+w−

−−

−−

−−

−−

−−

−−

−−

−−

+26

6

Phot

obac

teriu

mda

mse

lae

+−

++

+−

−−

−−

−−

−−

−−

−v

−−

+41

P.da

mse

lada

mse

la+

−+

++

v−

−+

−+

−+

+−

−+

−−

++

289

P.da

mse

lapi

scic

ida

−−

++

−−

−−

+−

+−

+−

−−

+−

−−

+28

9

Ples

iom

onas

shig

ello

ides

++

++

−−

−+

v−

−−

vv

vv

−v

−−

+41

Pseu

doal

tero

mon

asan

tarc

tica

−−

+−

−−

+−

+−

++

++

+−

−−

+−

+5

5d11

5

Pseu

doal

tero

mon

asba

cter

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tica

−+

++

+−

−−

−−

−+

+67

7

Pseu

doal

tero

mon

asha

lopl

ankt

is+

++

−−

−28

8

Pseu

dom

onas

angu

illise

ptic

a−w

−−

−−

−+

−−

−−

−v

+−

−+

−−

−+

541

Pseu

dom

onas

chlo

rora

phis

−−

−−

−v

−−

++

++

−−

+v

−+

+−

v41

Pseu

dom

onas

fluor

esce

nsv

−−

v−

−v

−+

v+

+v

−+

+−

++

−+

41

Pseu

dom

onas

plec

oglo

ssic

ida

+−

−+

−−

−−

+−

−−

−−

++

−+

++

+25

4858

2

Pseu

dom

onas

putid

a+

+−

vv

vv

+58

2

Shew

anel

laal

gae

LMG

2265

+−

−−

−−

+−

−−

−−

+−

−+

−+

−−

+85

1

Shew

anel

laba

ltica

NC

TC10

735

+−

+−

−−

+−

+−

−−

++

+−

−+

+−

+85

1

Shew

anel

lape

alea

na−

−+

−+

+49

2

Shew

anel

lapu

trefa

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s+

−+

−−

−+

−−

+−

−+

+−

+−

++

−+

433

Sphi

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onas

suba

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a−

−−

−−

+−

++

+−

−+

+−

−−

++

−

NO

3TR

PG

luAD

HU

reEs

cG

elPn

gG

luAr

aM

neM

anN

agM

alG

ntC

apAd

iM

ltC

itPa

cO

xTe

mp

Tim

eIn

ocR

efSt

rain

Tab

le4.

26.

Con

tinue

d.



199

Stap

pia

stel

lula

ta- l i

kest

r ai n

M1

+−

++

−+

++

++

++

−+

++

−+

2548

1.5

105

Vago

cocc

ussa

lmon

inar

um−

−+

−−

+−

+−

542

Vibr

ioal

gino

lytic

us+

++

−−

v+

−v

−v

vv

vv

−−

+−

−+

41

Vibr

ioal

gino

lytic

us+

++

−−

−+

+−

++

++

+nt

nt+

+nt

+10

8

Vibr

ioal

gino

lytic

us+

++

−−

−+

++

++

++

++

−−

+−

−+

Vibr

ioco

rallii

lytic

us+

−+

−+

++

−+

83+

++

83+

3048

3.5

83,8

4YB

Vibr

io(c

arch

aria

e)ha

rvey

i+

++

−+

++

++

−+

+−

++

−−

++

−+

11AT

CC

3508

4

Vibr

ioch

oler

ae+

++

−−

−+

++

−v

vv

++

−−

++

−+

41

Vibr

iodi

abol

icus

++

+−

−−

++

−−

−−

−+

−+

−+

+−

+63

5

Vibr

ioha

rvey

i+

++

−−

++

++

++

++

++

−−

++

−+

11AT

CC

1412

6

Vibr

ioha

rvey

i+

++

−−

++

++

−−

+−

−+

−−

++

−+

11

Vibr

ioic

hthy

oent

eri

+−

+−

−33

50−

+38

9

Vibr

iom

imic

us+

++

−−

++

−+

++

+−

+23

0

Vibr

iom

ytili

++

++

−+

+−

−+

+−

635

Vibr

iona

varre

nsis

++

+−

−+

++

−+

++

+−

+60

−+

768

Vibr

ione

reis

++

+−

−−

+−

−+

+−

635

Vibr

iope

naei

cida

+−

+−

++

++

++

388

Vibr

iosh

iloni

i+

++

−−

++

++

+45

8

Vibr

iota

petis

++

−−

−+

+−

+−

+−

−−

−10

8

Vibr

iotu

bias

hii

++

++

−+

+−

−+

−−

635

Zobe

lliaga

lact

aniv

oran

s+

−+

−−

++

++

w+

++

+−

−−

−−

−+

307d

2.5

61

Zobe

lliaul

igin

osa

+−

+−

−+

++

++

++

++

w−

−−

−−

+30

7d2.

561

NO

3=

nitra

te;T

RP

=in

dole

;Glu

=gl

ucos

efe

rmen

tatio

n;AD

H=

argi

nine

dihy

drol

ase;

Ure

=ur

ease

;Esc

=ae

scul

in;G

el=

gela

tinhy

drol

ysis

;Png

=p-

nitro

phen

yl-b

-D-g

alac

topy

rano

side

;Glu

=gl

ucos

eas

sim

ilatio

n;Ar

a=

L-ar

abin

ose

assi

mila

tion;

Mne

=m

anno

seas

sim

ilatio

n;M

an=

man

nito

lass

imila

tion;

Nag

=N

-ace

tylg

luco

sam

ine;

Mal

=m

alto

seas

sim

ilatio

n;G

nt=

gluc

onat

eas

sim

ilatio

n;C

ap=

capr

ate

assi

mila

t ion;

Adi=

adip

ate

assi

mila

tion;

Mlt

=m

alat

eas

sim

ilatio

n;C

it=

citra

teas

sim

ilatio

n;Pa

c=

phen

ylac

etat

e;O

x=

oxid

ase;

*AP

Idat

abas

e.



200

Org

anis

m

Aero

cocc

usvi

ridan

sva

r.ho

mar

iw

++

++

+v

v+

++

++

++

Aero

mon

asbe

stia

rum

HG

2+

−−

++

−−

−−

++

++

−−

−−

++

−+

+−

++

+67

6767

Aero

mon

asca

viae

HG

4+

−−

++

−−

−−

++

+31

−−

−−

+7

−−

+−

++

+90

+87

Aero

mon

ascu

licic

ola

+−

−−

+−

−−

−+

++

+−

−−

−+

−−

−+

−−

−−

−+

−Ae

rom

onas

ench

elei

a+

−−

−+

++

−−

+−

−+

−+

−Ae

rom

onas

eucr

enop

hila

HG

6+

−−

++

−−

−−

++

++

−−

−−

+−

−−

+−

++

++

++

Aero

mon

ashy

drop

hila

HG

1+

−−

++

−−

−−

++

++

−−

−−

+−

−−

+−

++

+−

+−

Aero

mon

ashy

drop

hila

HG

3+

−−

++

−−

−−

++

++

−−

−−

++

−+

+−

++

+62

+54

Aero

mon

asm

edia

HG

5+

−−

++

−−

−−

++

++

−−

−−

+−

−−

+−

++

++

++

Aero

mon

aspo

poffi

i+

−−

−+

++

−−

−+

−−

−+

−A.

salm

onic

ida

‘aty

pica

l’

A.sa

lmon

icid

aac

hrom

ogen

es+

−+

++

++

−−

−−

−Ba

ltic

sea

turb

ot−

−−

−−

−−

−−

−+

+−

−−

−−

−−

−−

−−

−v

−−

−−

Balti

cse

aflo

unde

r−

−−

−v

−−

−−

−+

+v

−−

−−

v−

−−

−−

−+

v−

+−

Balti

cse

ada

b+

−−

−−

−−

−−

v+

+v

−−

−−

−−

−−

+w−

−+

+−

−−

Balti

cse

abl

enny

+−

−−

+−

−−

−+

++

+−

−−

−+

−−

−+

−−

−−

−+

−‘A

typi

cal’

Pigm

entp

os+

−−

−+

−−

−−

++

+−

−−

−+

−−

17−

‘Aty

pica

l’Pi

gmen

tneg

44−

−−

+−

−−

−44

++

−−

−−

+−

−88

−‘A

typi

cal’

Pigm

entn

eg44

−−

−+

−−

−−

44+

+−

−−

−−

−−

88−

Aust

ralia

nst

rain

−−

−−

−−

−−

−A.

salm

onic

ida

typi

cal

++

++

++

+−

++

++

+−

+

A.sa

lmon

icid

asa

lmon

icid

a+

++

++

−+

++

++

−

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

29

Test

Gly

Ery

Dar

aLa

rarib

dXyl

lXyl

ado

mdx

gal

glu

frum

neLs

orrh

adu

lin

om

anso

rm

dmm

dgna

gam

yar

bes

csa

lce

lm

alla

c

Tab

le4.

27.

AP

I 50C

Hda

taba

sebi

oche

mic

alre

sults

.



201

Org

anis

m

Aero

cocc

usvi

ridan

sva

r.ho

mar

iv

++

++

+v

+v

827

Aero

mon

asbe

stia

rum

HG

2−

++

−−

−+

+−

−−

−−

−−

−−

−30

480.

8542

7C

DC

9533

–76

Aero

mon

asca

viae

HG

4−

++

−−

−+

+−

36−

−−

−−

−−

+−

−30

480.

8542

7,21

ATC

C15

468T

Aero

mon

ascu

licic

ola

++

−−

−−

+−

−−

−−

−−

−−

−−

3024

0.85

624

NC

IMB

5147

T

Aero

mon

asen

chel

eia

+−

−Ae

rom

onas

eucr

enop

hila

HG

6−

++

−−

−+

+−

−−

−−

−−

−−

−30

480.

8542

7AT

CC

2330

9T

Aero

mon

ashy

drop

hila

HG

1−

++

−−

−+

+−

−−

−−

−−

−−

+−

−30

480.

8542

7AT

CC

7966

T

Aero

mon

ashy

drop

hila

HG

3−

++

−−

−+

+−

−−

−−

−−

−−

−30

480.

8542

7C

DC

0434

–84

Aero

mon

asm

edia

HG

5−

++

−−

−+

+−

−−

−−

−−

−−

−30

480.

8542

7C

DC

0862

–83

CD

C90

72–8

3

Aero

mon

aspo

poffi

i−

−+

−−

A.sa

lmon

icid

a‘a

typi

cal’

A.sa

lmon

icid

aac

hrom

ogen

es+

+−

++

−−

+s20

7d0.

583

1

Balti

cse

atu

rbot

−−

−−

−−

−−

−−

−−

−−

−−

−−

−+

2014

d83

2

Balti

cse

aflo

unde

r−

v+

−−

−v

+−

−−

−−

−−

−−

−−

+20

14d

832

Balti

cse

ada

b−

+−

−−

−−

−−

−−

−−

−−

−−

−−

+20

14d

832

Balti

cse

abl

enny

−+

+−

−−

++

−−

−−

−−

−−

−−

−+w

2014

d83

2

‘Aty

pica

l’Pi

gmen

tpos

+68

−−

100

3−

−−

−−

−20

7d32

3,35

2

‘Aty

pica

l’Pi

gmen

tneg

+27

−−

6375

−−

−−

−−

207d

323

‘Aty

pica

l’Pi

gmen

tneg

+−

−63

75−

−−

−−

−20

7d32

3

Aust

ralia

nst

rain

+−

−76

1

A.sa

lmon

icid

aty

pica

l−

−−

++

−+

−20

–22

7d35

2

A.sa

lmon

icid

asa

lmon

icid

a−

−−

++

−+

+s20

7d0.

583

1

3031

3233

3435

3637

3839

4041

4243

4445

4647

4849

Test

mel

suc

trein

um

lzra

fam

dgl

ygxy

ltge

ntu

rly

xta

gD

fuc

Lfuc

Dar

lLa

rlgn

t2k

et5k

etTe

mp

Tim

eIn

ocR

efSt

rain

Stra

in cont

inue

d



202

Aero

mon

asso

bria

+−

−28

+−

−−

−+

++

+−

−−

−+

−−

24+

−−

16−

52+

16

Aero

mon

astro

ta+

−−

−+

−−

−−

++

++

−−

−−

+−

−−

++

−−

−+

+−

Aero

mon

asve

roni

isob

riaH

G8/

10+

−−

14+

−−

−−

++

++

−−

−−

+−

−29

+−

−10

−57

+10

Aero

mon

asve

roni

iver

onii

−−

−−

−+

++

−−

+−

++

++

+−

Arca

noba

cter

ium

bern

ardi

ae+

vv

−+

++

−−

−−

−+

Arca

noba

cter

ium

phoc

ae+

−−

−+

−−

−−

++

+70

−−

−+

30−

−−

+−

−−

−−

++

Arca

noba

cter

ium

phoc

ae−

−−

−−

−−

−−

v+

−+

−−

−−

−−

−−

+−

−−

−−

+v

Arca

noba

cter

ium

pyog

enes

−+

−+

++

++

++

−+

−+

−−

−+

++

Arth

roba

cter

rhom

bi+

−−

−−

−−

−−

++

++

−v

−−

+−

−−

−+

++

++

++

Brev

undi

mon

asdi

min

uta

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−Br

evun

dim

onas

vesi

cula

ris−

−−

−−

+−

−−

−+

−−

−−

−−

−−

−−

−−

−−

−−

+−

Car

noba

cter

ium

dive

rgen

s−

−−

+−

−−

−−

++

+−

−−

−−

−−

−+

++

++

−C

arno

bact

eriu

mga

llinar

um−

−−

++

−−

−+

++

−−

−−

−−

−+

++

++

+

Car

noba

cter

ium

mob

ile−

−−

+−

−−

−+

++

−−

−−

−−

−−

+−

++

+

Car

noba

cter

ium

pisc

icol

a+

−−

−+

−−

−−

+w+

++

−−

−−

+v

++

++

++

++

++w

Chr

yseo

bact

eriu

msc

opht

halm

um−

−−

−−

−En

tero

cocc

usfa

eciu

mv

+−

−+

−−

−Fl

avob

acte

rium

colu

mna

re−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

Flav

obac

teriu

mps

ychr

ophi

lum

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−Fl

exib

acte

rlito

ralis

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−Fl

exib

acte

rpol

ymor

phus

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−Fl

exib

acte

rros

eolu

s−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

Flex

ibac

terr

uber

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−Ja

nthi

noba

cter

ium

livid

um+

−+

++

+−

−−

++

++

−v

−+

++

−−

v−

+−

++

+−

Kleb

siel

lapl

antic

ola

+−

++

++

++

++

++

−+

++

++

++

++

Lact

ococ

cus

garv

ieae

−−

−−

+−

−−

−+

++

+−

−−

−+

−−

−+

++

++

++

−

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

29

Test

Gly

Ery

Dar

aLa

rarib

dXyl

lXyl

ado

mdx

gal

glu

frum

neLs

orrh

adu

lin

om

anso

rm

dmm

dgna

gam

yar

bes

csa

lce

lm

alla

c

Tab

le4.

27.

Con

tinue

d.



203

Aero

mon

asso

bria

12+

+−

−−

++

−−

−−

−−

−−

−+

−−

2948

21

Aero

mon

astro

ta−

−+

−−

−+

+−

−−

−−

−−

−−

−−

−37

240.

8520

ATC

C49

657T

Aero

mon

asve

roni

isob

riaH

G8/

10−

++

−−

−+

+−

−−

−−

−−

−−

−30

480.

8542

7C

DC

0437

–84

Aero

mon

asve

roni

iver

onii

−+

+−

−Ar

cano

bact

eriu

mbe

rnar

diae

−+

v+

+v

−+

3748

274

Arca

noba

cter

ium

phoc

ae−

++

−70

−+

+−

−+

−30

−−

−−

−−

+37

2463

6

Arca

noba

cter

ium

phoc

ae−

v−

−−

v−

−−

−−

−−

−−

−−

−−

−37

4861

3

Arca

noba

cter

ium

pyog

enes

−−

+−

+−

++

3748

641

Arth

roba

cter

rhom

bi+

++

−−

−−

−−

++

−−

−−

+−

+−

−25

4860

0

Brev

undi

mon

asdi

min

uta

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

284

685

Brev

undi

mon

asve

sicu

laris

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

284

685

Car

noba

cter

ium

dive

rgen

s−

++

−+

−−

−−

+−

−−

−−

−−

+−

−25

7d17

6

Car

noba

cter

ium

gallin

arum

++

−+

−−

−+

−+

−−

−−

+−

176

Car

noba

cter

ium

mob

ile+

++

−−

−−

−−

−v−

−−

−−

−17

6

Car

noba

cter

ium

pisc

icol

a+

++

++

v−

−−

++w

−−

−−

−−

+−

−25

7217

6,73

Chr

yseo

bact

eriu

msc

opht

halm

um−

−En

tero

cocc

usfa

eciu

mv

v−

−−

−−

−v

Flav

obac

teriu

mco

lum

nare

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

2212

88,8

9,21

1

Flav

obac

teriu

mps

ychr

ophi

lum

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

2212

88,8

9,21

1

Flex

ibac

terl

itora

lis−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−89

Flex

ibac

terp

olym

orph

us−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−89

Flex

ibac

terr

oseo

lus

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

89

Flex

ibac

terr

uber

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

89

Jant

hino

bact

eriu

mliv

idum

−+

−v

−v

−−

+−

−+

−−

++

−−

+−

496

Kleb

siel

lapl

antic

ola

++

+−

+−

−La

ctoc

occu

sga

rvie

ae−

−+

−−

−−

−−

+−

−−

−−

−−

−−

−28

24–9

617

4,23

6AT

CC

4915

6

3031

3233

3435

3637

3839

4041

4243

4445

4647

4849

Test

mel

suc

trein

um

lzra

fam

dgl

ygxy

ltge

ntu

rly

xta

gD

fuc

Lfuc

Dar

lLa

rlgn

t2k

et5k

etTe

mp

Tim

eIn

ocR

efSt

rain

Stra

in cont

inue

d



204

Lact

ococ

cus

garv

ieae

−−

−−

+−

−−

−+

++

+−

−−

−+

−−

−+

++

++

++

−

Biot

ypes

1,2,

12+

Biot

ypes

3,7,

11,1

3+

Biot

ypes

4,5,

6,10

,+

Biot

ypes

8,9

−La

ctoc

occu

spi

sciu

m−

−−

++

+−

−−

++

++

−−

−−

+−

−−

++

++

++

++

Mor

itella

visc

osa

−−

−−

+−

−−

−−

++

−−

−−

−−

−−

−+

−−

−−

−+

−Pe

doba

cter

hepa

rinus

−−

−+

−+

−−

−+

++

+−

−−

−−

+−

−−

−−

+−

++

+

Pedo

bact

erpi

sciu

m−

−−

−v

−−

++

++

−−

−−

++

++

++

++

+

Phot

obac

teriu

man

gust

um−

−−

−+

−−

−−

++

++

−−

−−

−−

−−

+−

−−

−−

+−

P.da

mse

lae

dam

sela

e+

−−

−+

−−

−−

++

++

−−

−−

−−

−−

+−

−−

−+

+−

P.da

mse

lae

pisc

icid

a−

−−

−+

−−

−−

+w+

++

−−

−−

−−

−−

+−

−−

−−

−−

P.da

mse

lae

pisc

icid

a−

−−

−+

−−

−−

++

++

−−

−−

−−

−−

+−

−−

−−

++

Phot

obac

teriu

milio

pisc

ariu

mv

−−

−+

−−

−−

++

++

−−

−−

−−

−−

+−

−−

−+

−Ph

otob

acte

rium

leio

gnat

hi+

−−

−+

−−

−−

++

++

−−

−−

−−

−−

+−

−−

−−

−−

Stre

ptoc

occu

s(d

iffic

ile)a

gala

ctia

e−

−−

−+

−−

−−

−+

++

−−

−−

−−

−−

+−

−−

−−

+−

Stre

ptoc

occu

s(d

iffic

ile)a

gala

ctia

e−

−−

−+

−−

−−

−+

++

−−

−−

−−

−+

+−

−−

−−

+−

Stre

ptoc

occu

s(d

iffic

ile)a

gala

ctia

e−

−−

−+

−−

−−

−+

++

−−

−−

−−

−+

+−

−−

−−

+−

Stre

ptoc

occu

sin

iae

−−

−−

+−

−−

−?

++

+−

−−

−+

−+

−+

−+

++

++

−St

rept

ococ

cus

inia

e−

−−

−+

−−

−−

−+

++

−−

−−

+−

−+

+−

++

++

+−

Stre

ptoc

occu

sin

iae

−−

−−

+−

−−

−−

++

+−

−−

−+

−−

−+

−+

++

++

−St

rept

ococ

cus

inia

e−

−−

−+

−−

−−

++

++

−−

−−

+−

−−

++

++

++

+−

Stre

ptoc

occu

spa

raub

eris

−−

−−

+−

−−

−+

++

+−

−v

−+

+−

−+

++

++

++

+

Stre

ptoc

occu

sph

ocae

−−

−−

+−

−−

−−

++

+−

−−

−−

−−

−+

−−

−−

−+

−St

rept

ococ

cus

porc

inus

+−

−−

+−

−−

−+

++

+−

−−

−+

+−

−+

v+

++

+v

Stre

ptoc

occu

sub

eris

−−

−−

+−

−−

−+

++

+−

−v

−+

+−

−+

++

++

++

+

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

29

Test

Gly

Ery

Dar

aLa

rarib

dXyl

lXyl

ado

mdx

gal

glu

frum

neLs

orrh

adu

lin

om

anso

rm

dmm

dgna

gam

yar

bes

csa

lce

lm

alla

c

Tab

le4.

27.

Con

tinue

d.



205

Lact

ococ

cus

garv

ieae

−−

+−

−−

−−

−+

−−

−−

−−

−−

−−

2824

–96

174,

211,

236,

237,

780

ATC

C49

156

Biot

ypes

1,2,

12+

+78

0

Biot

ypes

3,7,

11,1

3−

+78

0

Biot

ypes

4,5,

6,10

,−

−78

0

Biot

ypes

8,9

−−

780

Lact

ococ

cus

pisc

ium

++

+−

++

+w−

−+

+−

−−

−−

−+

−−

2548

0.85

835

NC

FB27

78

Mor

itella

visc

osa

−−

−−

−−

++

−−

−−

−−

−−

−−

+−

1572

132

NC

IMB

1348

4

Pedo

bact

erhe

parin

us−

+−

−−

−+

−−

−+

−−

+−

−−

−+

2848

0.5

728

Pedo

bact

erpi

sciu

m+

+−

+−

++

−−

−−

+−

−−

−28

480.

572

8

Phot

obac

teriu

man

gust

um−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−26

721.

574

5N

CIM

B18

95

P.da

mse

lae

dam

sela

e−

−+

−−

−−

−−

−−

−−

−−

−−

−−

2672

1.5

745

ATC

C35

083

ATC

C33

539

P.da

mse

lae

pisc

icid

a−

+w−

−−

+wv

−−

−−

−−

−−

−−

−−

−26

721.

528

8,74

5N

CIM

B20

58N

CIM

B25

918,

ATC

C17

911

P.da

mse

lae

pisc

icid

a−

−−

−−

−−

−−

−−

−−

−−

−−

−−

2672

1.5

745

P90

029

Atyp

ical

stra

in

Phot

obac

teriu

milio

pisc

ariu

m−

−v

−−

−−

−−

−−

−−

−−

−−

v−

−22

2.85

599

Phot

obac

teriu

mle

iogn

athi

−−

−−

−−

−−

−−

−−

−−

−−

−+

−−

2672

1.5

745

LMG

4228

Stre

ptoc

occu

s(d

iffic

ile)a

gala

ctia

e−

+−

−−

−−

−−

−−

−−

−−

−−

−−

−30

Stre

ptoc

occu

s(d

iffic

ile)a

gala

ctia

e−

+−

−−

−−

−−

−−

−−

−−

−−

−−

−30

7223

3N

D2–

22

Stre

ptoc

occu

s(d

iffic

ile)a

gala

ctia

e−

+−

−−

−−

−−

−−

−−

−−

−−

−−

−30

7223

3N

D2–

22

Stre

ptoc

occu

sin

iae

++

−+

−+

+−

+−

−−

−−

−−

−−

−St

rept

ococ

cus

inia

e−

++

−+

−+

+−

+−

−−

−−

−−

−−

−30

7223

3N

D2–

16

Stre

ptoc

occu

sin

iae

−+

+−

+−

++

−+

−−

−−

−−

−−

−−

3072

233,

235

ND

2–16

Stre

ptoc

occu

sin

iae

−+

+−

+−

++

−+

−−

−−

−−

−−

−−

2472

0.85

183

Stre

ptoc

occu

spa

raub

eris

−+

+v

−−v

−v−

−+

−−

+−

−−

−−

−−

3724

224

Stre

ptoc

occu

sph

ocae

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

3724

700

Stre

ptoc

occu

spo

rcin

us−

++

−−

v−

−−

−−

−−

−−

−−

−37

24–4

80.

517

5

Stre

ptoc

occu

sub

eris

−+

++

−−

−−

−+

−−

+−

−−

−−

−−

3724

224

3031

3233

3435

3637

3839

4041

4243

4445

4647

4849

Test

mel

suc

trein

um

lzra

fam

dgl

ygxy

ltge

ntu

rly

xta

gD

fuc

Lfuc

Dar

lLa

rlgn

t2k

et5k

etTe

mp

Tim

eIn

ocR

efSt

rain

Stra

in cont

inue

d



206

Tena

ciba

culu

mm

ariti

mum

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

−Va

goco

ccus

fluvi

alis

−−

−−

+−

−−

−−

++

+−

−−

−+

+−

++

++

++

++

−Va

goco

ccus

fluvi

alis

−−

−−

+−

−−

−−

++

+−

−−

−+

−−

+−

++

++

++

−Va

goco

ccus

salm

onin

arum

−−

−−

60+w

−−

−−

++

+90

−−

−−

70−

−+

++

++

6070

−Vi

brio

carc

haria

eAT

CC

3508

4−

−−

−+

−−

−−

++

++

−−

−−

+−

−−

+−

−+w

−+

+−

Vibr

iodi

abol

icus

+−

−−

+−

−−

−+

++

+−

−−

−+

−−

−+

+−

−−

−+

−Vi

brio

fisch

eri

−−

−−

+−

−−

−+

++

+−

−−

−−

−−

−+

−−

−+

++

−Vi

brio

harv

eyi

ATC

C14

129

−−

−−

+−

−−

−+

++

+−

−−

−+

−−

−+

−−

+w−

++

−

Vibr

iom

ytili

+−

−+

++

−−

−+

++

+−

−−

−+

−−

−+

++

++

++

−Vi

brio

nere

is−

−−

−+

−−

−−

−+

++

−−

−−

−−

−−

+−

−−

−−

+−

Vibr

iope

naei

cida

−−

−−

−+

++

−−

−−

−−

++

−Vi

brio

salm

onic

ida

+sl

−−

−+

−−

−−

++

+−

−−

−−

+−

−−

+−

−−

−−

+−

Vibr

iosp

lend

idus

v−

−−

+−

−−

−+

++

+−

−−

−+

−−

−+

−−

+−

++

v

Vibr

iotu

bias

hii

−−

−−

+−

−−

−+

++

+−

−−

−+

−−

−+

+−

−−

++

−Ye

rsin

iakr

iste

nsen

ii+

−−

++

+−

−−

++

++

+−

−−

++

−−

+v

+−

−+

++

Zobe

lliaga

lact

anov

oran

s−

++

++

++

+−

++

+

Zobe

lliaul

igin

osa

−+

−−

++

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

29

Test

Gly

Ery

Dar

aLa

rarib

dXyl

lXyl

ado

mdx

gal

glu

frum

neLs

orrh

adu

lin

om

anso

rm

dmm

dgna

gam

yar

bes

csa

lce

lm

alla

c

Tab

le4.

27.

Con

tinue

d.



207

Tena

ciba

culu

mm

ariti

mum

−−

−−

−−

−−

−−

−−

−−

−−

−−

−−

2212

ASW

88,8

9,21

1

Vago

cocc

usflu

vial

is−

++

−−

−−

++

−−

−−

−−

−−

177

Spec

ies

Vago

cocc

usflu

vial

is−

−+

−−

−−

−−

+v

−−

−−

−−

−−

−17

7,49

8N

CD

O24

97

Vago

cocc

ussa

lmon

inar

um−

++

−−

−−

−−

50−

−+

−60

−−

−+w

−25

2–7d

542,

807

NC

FB27

77

Vibr

ioca

rcha

riae

ATC

C35

084

−+

+−

−−

++

−−

−−

−−

−−

−+

−−

581

Vibr

iodi

abol

icus

−+

+−

−−

++

−−

−−

−−

−−

−+

−−

2548

2.85

635

Vibr

iofis

cher

i−

−−

−−

−+

−−

−−

−−

−−

−−

−−

−26

721.

574

5AT

CC

2591

8

Vibr

ioha

rvey

iAT

CC

1412

9−

++

−−

−+

+−

−−

−−

−−

−−

+−

−25

242.

8558

1

Vibr

iom

ytili

−+

+−

+−

++

−+

v−

−−

−−

−+

−−

2548

2.85

635

Vibr

ione

reis

−+

+−

−−

+−

−−

v−

−−

−−

−+

−−

2548

2.85

635

Vibr

iope

naei

cida

−−

+−

+25

7d38

8JM

C91

23

Vibr

iosa

lmon

icid

a−

−+

−−

−−

−−

−−

−−

−−

−−

+−

−23

2

Vibr

iosp

lend

idus

+−

+−

−−

++

−−

−−

−−

10−

−+

−−

252.

8528

1

Vibr

iotu

bias

hii

−+

+−

−−

++

−−

−−

−−

−−

−+

−−

2548

2.85

635

Yers

inia

kris

tens

enii

−−

+−

−−

+−

2848

0.85

86

Zobe

lliaga

lact

anov

oran

s+

−−

−30

7d2.

523

DSM

1280

2T

Zobe

lliaul

igin

osa

+w

++

307d

2.5

61D

SM20

61T

Gly

=gl

ycer

ol;E

ry=

eryt

hrito

l;D

ara

=D-a

rabi

nose

;Lar

a=

L-ar

abin

ose;

rib=

ribos

e;dX

yl=

D-x

ylos

e;ad

o=

adon

itol;

mdx

=b-

met

hyl-D

-xyl

osid

e;ga

l=ga

lact

ose;

glu

=gl

ucos

e;fru

=f ru

ctos

e;m

ne=

man

nose

;Lso

r=L-

sorb

ose;

rha

=rh

amno

se;

dul=

dulc

itol;

ino

=tin

osito

l;m

an=

man

nito

l;so

r =so

rbito

l;m

dm=

a-m

ethy

l-D-m

anno

side

;mdg

=a

-met

hyl-D

-glu

cosi

de;n

ag=

N-a

cety

lglu

cosa

min

e;am

y=

amyg

dalin

;arb

=ar

butin

;esc

=ae

scul

in;s

al=

salic

in;c

el=

cello

bios

e;m

al=

mal

tose

;la

c=

lact

ose;

mel

=m

elib

iose

;suc

=su

cros

e;tre

=tre

halo

se;i

nu=

inul

in;m

lz=

mel

izito

se;r

af=

raf fi

nose

;am

d=

star

ch;g

lyg

=gl

ycog

en;x

ylt=

xylit

ol;g

en=

gent

iobi

ose;

tur=

D-tu

rano

se;l

yx=

D-ly

xose

;tag

=D-ta

gato

se;D

fuc

=D-fu

cose

;Lf

uc=

L-fu

cose

;Dar

l=D-a

rabi

tol;

Larl

=L-

arab

itol;

gnt=

gluc

onat

e;2k

et=

2-ke

togl

ucon

ate;

5ket

=5-

keto

gluc

onat

e.N

umbe

rsin

dica

teth

epe

rcen

t age

ofst

rain

sw

itha

posi

tive

resu

lt.

3031

3233

3435

3637

3839

4041

4243

4445

4647

4849

Test

mel

suc

trein

um

lzra

fam

dgl

ygxy

ltge

ntu

rly

xta

gD

fuc

Lfuc

Dar

lLa

rlgn

t2k

et5k

etTe

mp

Tim

eIn

ocR

efSt

rain

Stra

in



208

Nit

Pyz

Pyra

Pal

bgu

rb

gal

agl

ubN

AGes

cur

ege

lgl

urib

xyl

man

mal

lac

sac

gly

cat

Ref

Actin

omyc

esm

arim

amm

aliu

m−

−−

v−

++

−−

−+

370

Arca

noba

cter

ium

bern

ardi

ae−

−−

−+

−−

+−

480

Arca

noba

cter

ium

haem

olyt

icum

−−

−−

+−

v48

0

Arca

noba

cter

ium

phoc

ae−

−+

+−

++

−−

−−

+v

30+

v+

+v

480,

636

Arca

noba

cter

ium

phoc

ae−

−+

+−

−+

−−

v−

−−

−+

vv

−+

613

Arca

noba

cter

ium

plur

anim

aliu

m−

−v

−+

−−

−w

+−

++

−−

+−

−−

+48

0

Arth

roba

cter

nasi

phoc

ae−

−+

+−

−+

−−

−+

−−

−−

−−

−−

+18

2

Arth

roba

cter

rhom

bi−

−−

−−

+−

−+

−−

−−

++

++

−60

0

Cor

yneb

acte

rium

aqua

ticum

−+

++

−+

+−

+−

+v−

133

Cor

yneb

acte

rium

pseu

dodi

phth

eriti

cum

++

++

−−

−−

−+

−−

133

Cor

yneb

acte

rium

pseu

dotu

berc

ulos

is−

−−

+−

+−

−−

+−

−13

3

Cor

yneb

acte

rium

test

udin

oris

+−

−−

−−

v−

+−

−+

−−

+−

+−

+18

0

Cor

yneb

acte

rium

xero

sis

+−

++

−−

−−

−−

−−

133

Nit

=ni

trate

;Pyz

=py

rrolid

onyl

acry

lam

idas

e;Py

ra=

pyra

zina

mid

ase;

Pal=

alka

line

phos

phat

ase;

bgu

r=b

gluc

uron

idas

e;b

gal=

bga

lact

osid

ase;

agl

u=

agl

ucos

idas

e;bN

AG=

N-a

cety

l-bgl

ucos

amin

idas

e;es

c=

aesc

ulin

;ure

=ur

ease

;ge

l=ge

latin

;glu

=gl

ucos

e;rib

=rib

ose;

xyl=

xylo

se; m

an=

man

nito

l;m

al=

mal

tose

;lac

=la

ctos

e;sa

c=

sucr

ose;

gly

=gl

ycog

en;c

at=

cata

lase

.Ref

=re

fere

nce;

+=

posi

tive

reac

tion;

−=

nega

tive

reac

tion;

num

bers

indi

cate

perc

enta

geof

posi

tive

stra

ins;

w=

wea

kre

actio

n.

Tab

le4.

28.

AP

ICor

yne

data

base

bioc

hem

ical

resu

lts.



209

vphi

pae

spy

raa

-gal

b-gu

rb-

gal

Pal

Lap

ADH

ribar

am

anso

rla

ctre

inu

raf

amd

glyg

Hem

Ref

Abio

troph

iade

fect

iva

−−

−+

+−

+−

+−

−−

−−

++

−+

+−

Car

noba

cter

ium

inhi

bens

−+

+−

−−

−−

−−

+−

+−

+w+

+w−

−−

412

Car

noba

cter

ium

pisc

icol

a+

−+

+nt

++

−+

+v

++

v−

+54

2

Ente

roco

ccus

aviu

m+

−+

+−

−−

−−

−+

++

++

++

++

−a

156

Ente

roco

ccus

dura

ns+

−+

++

−+

−−

++

−−

−+

−−

−+

−a

156

Ente

roco

ccus

faec

alis

+−

++

−−

+−

++

+−

++

++

−−

+−

g15

6

Ente

roco

ccus

faec

ium

+−

−+

++

+−

−−

−v

Gra

nulic

atel

laad

iace

ns−

−−

+−

+−

−+

−−

−−

−−

−+

−−

−G

ranu

licat

ella

bala

enop

tera

e−

−+

−−

−−

−+

+−

−−

−−

+−

−−

−47

8,77

3

Gra

nulic

atel

lael

egan

s−

+−

+−

−−

−+

+−

−−

+−

−a

653

Lact

ococ

cus

garv

ieae

+−

++

−−

−−

+−

+−

++

−+

−−

−−

a15

6

Lact

ococ

cus

garv

ieae

+−

++

−−

−−

++

+−

+−

−+

−−

−−

a23

7

Lact

ococ

cus

lact

isss

p.cr

emor

is+

−+

−−

−+

−+

++

−−

−−

+−

−−

−g

156

Lact

ococ

cus

lact

isss

p.la

ctis

+−

++

−−

−−

++

+−

−−

−+

−−

+−

g15

6

Lact

ococ

cus

raffi

nola

ctis

+−

+−

+−

−−

+−

−−

−−

++

−+

+−

g15

6

Stre

ptoc

occu

sin

iae

Fish

stra

ins

−−

++

−+

−+

+7 0

+−

46−

−+

−−

++

ab

183,

223

Stre

ptoc

occu

sin

iae

Hum

anst

rain

s−

−+

−−

−−

++

−−

−+

−−

+−

−+

−a

b22

3

Stre

ptoc

occu

sin

iae

−−

++

−+

−+

++

+−

+−

−+

−−

++

b23

5,84

8

Stre

ptoc

occu

s(d

iffic

ile)a

gala

ctia

e+

−−

−−

−−

++

++

−−

−−

−−

−−

+23

3

Stre

ptoc

occu

s(d

iffic

ile)a

gala

ctia

e+

+−

−−

+−

++

−+s

−−

−−

−−

−−

+77

6

Stre

ptoc

occu

sph

ocae

−−

−−

−−

−+

−−

+−

−−

−−

−−

−−

700

Stre

ptoc

occu

spo

rcin

us+

−+

−−

+−

++

++

−+

++

−−

v−

b17

5

Vago

cocc

usflu

vial

is+v

−+

+−

−−

−v+v

−+

−+

+−

+−

++

+a

629

Vago

cocc

ussa

lmon

inar

um+

++

+−

−−

++

−70

−−

70−

+−

−−

−a

542

VP=

Voge

s-Pr

oska

uer;

hip

=hi

ppur

ate;

aes

=ae

scul

in;p

yra

=py

rrolid

onyl

aryl

amid

ase;

a-g

al=

aga

lact

osid

ase;

bgur

=b

gluc

uron

idas

e;b-

gal=

bga

lact

osid

ase;

Pal=

alka

line

phos

phat

ase;

Lap

=le

ucin

ear

ylam

idas

e;AD

H=

argi

nine

dihy

drol

ase;

rib=

ribos

e;ar

a=

arab

inos

e;m

an=

man

nito

l;so

r=so

rbito

l;la

c=

lact

ose;

tre=

treha

lose

;inu

=in

ulin

;raf

=ra

ffino

se;a

md

=am

ygda

lin;g

lyg

=gl

ycog

en;H

em=

haem

olys

is;R

ef=

refe

renc

e;nt

=no

tte s

ted.

Num

bers

show

perc

enta

geof

posi

tive

stra

ins.

+=

posi

tive

resu

lt;−

=ne

gativ

ere

sult;

w=

wea

kre

actio

n;s

=sl

owre

act io

n.a

=a

haem

olys

is;b

=b

haem

olys

is;g

=no

haem

olys

is.

Tab

le4.

29.

AP

I20

Str

epda

taba

sebi

oche

mic

alre

sults

.



210

Org

anis

m

Abio

troph

iade

fect

iva

−−

50−

+−

−−

−50

+50

−50

+75

−−

−−

++

−−

+−

−−

v−

−−

*,4

21

Abio

troph

iapa

ra-a

diac

ens

−+

−50

−−

−50

−+

−42

1

Actin

omyc

esm

arim

amm

aliu

m−

−+

−−

v−

−−

+−

−−

++

−+

+−

v−

+−

−−

−−

−−

−−

−37

0

Aero

cocc

usvi

ridan

s−

703

3060

−28

7525

7991

42−

−10

83−

−92

1010

95−

−+

−−

65−

−−

−*

Arca

noba

cter

ium

bern

ardi

ae−

−−

+−

−−

−+

480

Arca

noba

cter

ium

haem

olyt

icum

−v

−−

+−

v−

−48

0

Arca

noba

cter

ium

phoc

ae+

−+w

+v

+−

−+

480

Arca

noba

cter

ium

plur

anim

aliu

m−

−−

+−

−+

−−

−−

−−

+−

+−

v+

−−

−−

−−

−−

−−

−−

−48

0

Arca

noba

cter

ium

pyog

enes

++

−+

++

−v

v48

0

Atop

obac

terp

hoca

e+

−−

−−

++

−−

vv

−−

w−

−w

−−

−+

++

−−

v−

−−

−−

+−

479

Car

noba

cter

ium

alte

rfund

ium

−−

−−

−−

−+

+18

1

Car

noba

cter

ium

dive

rgen

s+

+−

++

++

++

181

Car

noba

cter

ium

fund

itum

−−

−−

−−

++

+18

1

Car

noba

cter

ium

gallin

arum

++

−+

++

++

+18

1

Car

noba

cter

ium

inhi

bens

−+

−+

−−

++

−+w

+−

−−

−−

−−

+−

−+

−−

++

++

−−

−−

412

Car

noba

cter

ium

mob

ile+

−−

−+

−−

−+

181

Car

noba

cter

ium

pisc

icol

a+

−−

+−

++

v−

+w

++

++

−−

−+

ww

+−

w+

−47

9

Ente

roco

ccus

faec

alis

++

−−

−−

++

−+

+−

++

−+

+−

−−

−+

−−

+−

−+

−+

−63

8

Erys

ipel

othr

ixrh

usio

path

iae

42−

+−

−28

−−

−75

−−

−64

−+

8085

−−

−−

−−

−−

−−

−−

−−

*

Fack

lam

iam

iroun

gae

+−

−−

−−w

nr−

−−

+−

−+

−+

−ww

−−

−−

−−

−−

−−

−−

−+

368

Gra

nulic

atel

laad

iace

ns−

+−

30−

−−

−−

−−

−−

+−

7025

−−

−−

+−

−+

−−

−+

−−

−*

,421

Gra

nulic

atel

laba

laen

opte

rae

+−

−−

−−

−−

−−

+−

−−

−+w

+−

−−

++

−−

−−

−−

−−

−+w

478,

479

Gra

nulic

atel

lael

egan

s+

−−

−−

−−

−+

−−

+−

−+

−−v

421

Lact

ococ

cus

garv

ieae

++

−−

−−

++

−−

+−

++

−+

+−

−−

−+

−−

+−

−+

−+

−63

8

Lact

ococ

cus

garv

ieae

++

−−

−−

3575

−50

+−

++

−74

10−

−−

−75

−−

50−

−85

50−

50−

*

Biot

ype

1+

+−

++

−*

,780

Biot

ype

2+

+−

++

+*

,780

Biot

ype

3+

+−

−+

−*

,780

Biot

ype

4+

−−

−−

−*

,780

Biot

ype

5+

−+

−−

−*

,780

11.

11.

21.

31.

41.

51.

61.

71.

81.

91A

1B0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0A0B

1C1D

1E0C

0D0E

1F0F

Ref

ADH

bglu

bgar

bgur

aga

lpa

lrib

man

sor

lac

trera

fvp

appa

bgal

pyrA

bnag

gta

hip

glyg

pul

mal

mel

mlz

suc

lara

darl

mbd

gta

gbm

ancd

exur

e

Tab

le4.

30.

AP

IRap

idID

32S

trep

data

base

bioc

hem

ical

resu

lts.



211

Biot

ype

6+

−+

−−

+*

,780

Biot

ype

7+

+−

−+

−*

,780

Biot

ype

8−

+−

−−

−*

,780

Biot

ype

9−

++

−−

−*

,780

Biot

ype

10+

−−

−−

+*

,780

Biot

ype

11+

+−

−+

+*

,780

Biot

ype

12+

−−

++

−*

,780

Biot

ype

13+

−−

−+

−*

,780

Lact

ococ

cus

lact

isla

ctis

++

5−

−−

9526

−50

75−

74+

26−

50−

26−

−+

−−

26−

−85

350

95−

*

Stre

ptoc

occu

sag

alac

tiae

+−

−50

10+

85−

−30

74−

90+

−−

−−

+4

++

−−

+−

−90

26−

−−

*

Stre

ptoc

occu

sag

alac

tiae

+−

−+

31+

+−

−−

++

+−

−−

−+

−+

+−

−+

−−

+−

−−

−g2

42

Stre

ptoc

occu

sag

alac

tiae

(diff

icile

)+

−−

−−

−−

−−

−−

−+

−−

−−

−−

−−

+−

−−

−−

−−

−−

−13

5

Stre

ptoc

occu

sdy

sgal

actia

ess

p.dy

sgal

actia

e+

−−

+−

++

−−

++

−−

+−

−−

−−

++

+−

−+

−−

−−

−40

−72

7

Lanc

efie

ldgr

oup

L=

posi

tive.

Stre

ptex

grou

psA–

G=

nega

tive

Stre

ptoc

occu

sin

iae

–fis

h6 7

+−

83+

−−

+−

−+

−+

−St

rept

ococ

cus

inia

e–

hum

an−

−−

−−

−+

−−

−−

−−

Stre

ptoc

occu

sin

iae

–fis

h+ s

+−

+−

w−

+−

−+

−−

+−

+−

−−

−−

−−

−−

−−

−−

−−

−13

5s

Stre

ptoc

occu

sin

iae

–fis

h+

+−

+−

−+

+−

−+

−−

+−

+−

−−

+−

+−

−+

−−

−−

−−

−13

5#a

Stre

ptoc

occu

sin

iae

–fis

h+

+−

−−

−−

+−

−+

−−

+−

+−

−−

−−

+−

−+

−−

−−

−−

−13

5#b

Stre

ptoc

occu

spa

raub

eris

++

−−

15−

++

++

+1 0

++

−10

−−

50−

−+

−10

+−

−+

3 03 0

−−

*,2

24,

745

Stre

ptoc

occu

sub

eris

++

30+

155

++

++

+20

++

−30

−−

905

−+

−−

+−

−+

5−

−−

*

Vago

cocc

usfe

ssus

−v

v−

−−

−−

−−

−−

−−

−+

−v

−−

−−

−−

−−

−−

−−

v−

369

Vago

cocc

usflu

vial

is−

+−

−−

−+

++

−+

−−

−−

++

+−

−−

+−

−+

−−

−−

v++

−17

7,36

9,62

9

Vago

cocc

uslu

trae

−+

−w−

+−w

+−

+−

+−

−−

−w+

++

−−

−+

−−

+−

−+

−+

+−

477

Vago

cocc

ussa

lmon

inar

um−

++

+−

+−

−−

+−

−+

−−

+−

682,

732

Vago

cocc

ussa

lmon

inar

um−

++

+−

+−

−+

−+

−−

+−

−+

−68

2,73

2

ADH

=ar

gini

nedi

hydr

olas

e;bg

lu=

bgl

ucos

idas

e;bg

ar=

bga

lact

osid

ase;

bgur

=b

gluc

uron

idas

e;a

gal=

aga

lact

osid

ase;

pal=

alka

line

phos

phat

ase;

rib=

ribos

e;m

an=

man

nito

l;so

r=so

rbito

l;la

c=

lact

ose;

tre=

treha

lose

;raf

=ra

ffino

se;

vp=

acet

oin

prod

uctio

n(V

oges

-Pro

skau

er);

appa

=al

anin

e-ph

enyl

alan

ine-

prol

ine

aryl

amid

ase;

bgal

=b

gala

ctos

idas

e;py

rA=

pyro

glut

amic

acid

aryl

amid

ase;

bnag

=N

-ace

tyl-b

gluc

osam

inid

ase;

gta

=gl

ycyl

-tryp

toph

ane

aryl

amid

ase;

hip

=hi

ppur

ate;

glyg

=gl

ycog

en;p

ul=

pullu

lan;

mal

=m

alto

se;m

el=

mel

ibio

se;m

lz=

mel

ezito

se;s

uc=

sucr

ose;

lara

=L-

arab

inos

e;da

rl=

D-a

rabi

nose

;mbd

g=

met

hyl-b

-D-g

luco

pyra

nosi

de;t

ag=

taga

tose

;bm

an=

bm

anno

sida

se;c

dex

=cy

clod

extri

n;ur

e=

urea

se.

Ref

=re

fere

nce;

w=

wea

kre

actio

n;+

=po

sit iv

ere

actio

n;−

=ne

gativ

ere

actio

n;s

=sl

owre

actio

n.N

umbe

rsin

dica

tepe

rcen

tage

ofpo

sitiv

est

rain

s.*

=fro

mAP

Idat

abas

e;#

=AH

LDA

1722

stra

infr o

mim

porte

daq

uariu

mfis

h(a

=re

sults

at37

°Cin

cuba

tion;

b=

resu

ltsfro

m25

°Cin

cuba

tion)

;s=

stra

infro

mQ

ueen

slan

d;g

=st

rain

sfro

mm

ulle

tand

seab

ream

from

Kuw

ait.



212

Actin

obac

illus

delp

hini

cola

−5

13

−3

−−

−−

50–

2−

−−

−−

−−

−26

3

Actin

omyc

esvi

scos

us−

−−

+−

++

−−

−+

++

+−

−+

−−

+41

Aequ

oriv

itaan

tarc

tica

−+

+−

2011

3

Aero

cocc

usvi

ridan

sva

r.ho

mar

i−

++

+−

+−

−−

−−

+−

−−

−−

−−

−41

Aero

mon

ashy

drop

hila

−5

−4

24

1−

3−

51

−3

−1

−4

−−

254

0.85

NC

TC78

1013

5

Aero

mon

asja

nada

ei−

12

3−

2w

−−

−2

2−

2−

w−

2−

−25

40.

85AH

LDA

1718

135

Aero

mon

assa

lmon

icid

a−

3−

1−

2w

−1

−5

1−

−−

−−

2−

−25

200.

85AH

LDA

1334

135

Aero

mon

asve

roni

isob

ria−

5−

41

3w

−3

−5

11

4−

1−

5−

−25

40.

85AH

LDA

1684

135

Alte

rom

onas

mac

leod

ii−

4−

2−

22

−4

−4

1−

−−

−−

−−

−28

3

Arca

noba

cter

ium

phoc

ae−

42

2−

3−

1v

−5

−3

1−

5−

−−

−63

6

Arth

roba

cter

aure

scen

s−

−−

+−

+−

++

−+

+−

++

++

−+

−41

Arth

roba

cter

nasi

phoc

ae−

+1

−−

+−

−−

−+

−−

−+

−−

−−

MI

MI

MI

CC

UG

4295

318

2

Atop

obac

terp

hoca

e−

+1

1−

−w−

−−

−+

−−

v−

−−

−−

−47

9

Bord

etel

labr

onch

isep

tica

−w

−11

1−

3–4

w−

−−

2w

−2−

−−

−−

−−

−7

40.

85Va

rious

135

Brev

undi

mon

asdi

min

uta

−+

+−

+−

−+

++

−−

−−

−−

−68

5

Brev

undi

mon

asve

sicu

laris

−+

+−

+v

−+

−+

−−

+−

−−

−68

5

Car

noba

cter

ium

alte

rfund

itum

−+

+−

−−

−−

−−

++

−−

−−

+−

−−

250.

85C

CU

G34

643

649

Car

noba

cter

ium

dive

rgen

s−

++

−−

−−

−−

−w

+−

−−

−−

−−

−25

0.85

CC

UG

3009

464

9

Car

noba

cter

ium

dive

rgen

s62

51−

−+

w−

−−

−−

−−

w−

−+

−−

−−

−25

0.85

6251

649

Car

noba

cter

ium

fund

itum

−+

+−

−−

−−

−−

++

−−

−−

+−

−−

250.

85C

CU

G34

644

649

Car

noba

cter

ium

gallin

arum

−+

+−

−−

−−

−−

−w

−−

−−

−−

−−

250.

85C

CU

G30

095

649

Car

noba

cter

ium

inhi

bens

−−

−−

−−

−−

−−

−−

−−

−−

+−

−−

412

Car

noba

cter

ium

inhi

bens

−−

−−

−−

−−

−−

−+

−−

−−

+−

−−

250.

85C

CU

G31

728

649

Car

noba

cter

ium

mob

ile−

++

−−

−−

−−

−−

w−

−−

−−

−−

−25

0.85

CC

UG

3009

664

9

Car

noba

cter

ium

pisc

icol

a−

−−

+−

479

Cel

lulo

phag

aly

tica

−5

34

15

42

31

55

11

−3

25

2−

2212

ASS

89

Chr

yseo

bact

eriu

mba

lust

inum

−5

12

25

53

21

55

−−

−3

32

−−

2512

ASS

89,5

57

Wel

l1

23

45

67

89

1011

1213

1415

1617

1819

20Te

mp

Tim

eIn

ocSt

rain

Ref

Enzy

me

con

alk

C4

C8

C14

aryl

val

cys

trych

rac

pnp

a-g

alb-

gal

b-gl

ucr

a-g

lub-

glu

N-a

bglu

man

fuc

°CH

%N

aCl

Org

anis

mes

tes

tlip

lip

Tab

le4.

31.

AP

IZY

Mda

taba

sere

sults

.



213

Chr

yseo

bact

eriu

mgl

eum

−5

23

15

52

22

55

−−

−4

54

−−

2212

557

Chr

yseo

bact

eriu

min

dolo

gene

s−

52

31

54

22

25

5−

−−

5−

4−

−22

1255

7

Chr

yseo

bact

eriu

min

dolth

etic

um−

52

31

54

1−

−4

4−

−−

4−

3−

−22

1255

7

Chr

yseo

bact

eriu

mm

enin

gose

ptic

um−

54

43

55

53

44

53

−−

42

52

237

4AS

S89

Chr

yseo

bact

eriu

msc

opht

halm

um−

53

41

54

24

15

5−

−−

31

2−

−55

7

Cor

yneb

acte

rium

phoc

ae−

+−

+−

v−

−−

−+

−−

−−

+−

−−

−61

3

Cor

yneb

acte

rium

test

udin

oris

−−

ww

−+

−−

−−

+w−

−−

−v

+−

−−

180

Cyt

opha

gaal

lerg

inae

−5

24

15

52

13

55

33

25

54

−−

2212

ASS

89

Cyt

opha

gaar

vens

icol

a−

52

31

55

24

14

43

4−

43

52

322

12AS

S89

Cyt

opha

gafe

rmen

tans

−5

13

−5

2−

2−

55

−5

12

55

−−

2212

ASS

89

Cyt

opha

gahu

tchi

nson

ii−

42

41

44

3−

−3

2−

−−

−−

−−

−22

12AS

S89

Cyt

opha

gala

terc

ula

−5

24

15

53

55

44

−−

−−

−−

−−

2212

ASS

89

Edw

ards

iella

tard

a−

5−

−w

51

−−

−4

2−

−−

−−

3−

−25

200.

85AH

LDA

135

135

Empe

doba

cter

brev

is−

53

4−

55

33

25

5−

−−

4−

−−

−22

12AS

S89

Ente

rovi

brio

norv

egic

us−

++

++

+−

−−

−+

+−

nr−

nrnr

nrnr

nr28

482.

85LM

G19

839T

741

Fack

lam

iam

iroun

gae

−−w

−−

−+

ww

−w

ww

−−

−−

−−w

−−

368

Flav

obac

teriu

maq

uatil

e−

42

41

55

2−

−1

2−

−−

5−

−−

−22

12AS

S89

,92

Flav

obac

teriu

maq

uatil

e−

++

+−

++

++

−+

+−

−−

+−

+−

−18

48AT

CC

1194

760

3

Flav

obac

teriu

mbr

anch

ioph

ilum

−5

23

−5

42

2−

44

−−

−1

−−

−−

2512

ASS

92

Flav

obac

teriu

mbr

anch

ioph

ilum

−+

++

−+

++

+v−

++

v−

−+

+−

−−

1848

0.85

603

Flav

obac

teriu

mbr

anch

ioph

ilum

−+

++

−+

++

−−

++

−−

−−

−−

−−

802

Flav

obac

teriu

mco

lum

nare

−5

23

−4

41

31

33

−−

−−

−−

−−

2512

ASS

88,9

2

Flav

obac

teriu

mco

lum

nare

−5

−2

−5

53

−−

54

−−

−3

−1

−−

376

Flav

obac

teriu

mco

lum

nare

−+

++

−+

++

−−

++

−−

−−

−−

−−

1848

NC

MB

2248

603

Flav

obac

teriu

mco

lum

nare

−5

−2

−5

22

−−

11

−−

−−

−−

−−

254

0.85

AHLD

A14

6813

5

Flav

obac

teriu

mfle

vens

e−

51

21

51

1−

−3

31

5−

3−

4−

−22

12AS

S89

,92

Flav

obac

teriu

mfri

gida

rium

−5

−2

−5

51

−1

55

−−

−−

−−

−−

376

Flav

obac

teriu

mhy

datis

−5

24

14

52

−−

45

−−

−4

−2

−−

2212

ASS

89,9

2

Flav

obac

teriu

mhy

datis

−5

−1

−5

53

−−

55

−−

−5

−5

−−

376

Flav

obac

teriu

mjo

hnso

niae

−5

13

15

52

11

45

−3

−4

14

−−

2512

ASS

89,9

2

Flav

obac

teriu

mjo

hnso

niae

−5

34

14

53

12

54

55

15

25

−−

2512

ASS

89

cont

inue

d



214

Flav

obac

teriu

mjo

hnso

niae

−5

23

15

53

12

54

33

15

25

−−

2512

ASS

89

Flav

obac

teriu

mjo

hnso

niae

−5

−1

−3

11

−−

43

21

−3

32

−−

2537

6

Flav

obac

teriu

mjo

hnso

niae

−+

++

++

++

++

++

++

−+

++

−−

1848

ATC

C17

061

603

Flav

obac

teriu

mpe

ctin

ovor

um−

53

3−

44

2−

−4

4−

21

45

3−

−22

12N

CIM

B90

5992

Flav

obac

teriu

mps

ychr

ophi

lum

−5

23

15

1−

−−

33

−−

−−

−−

−−

2012

NC

IMB

1947

89,9

2

Flav

obac

teriu

mps

ychr

ophi

lum

−+

−+

−+

+−

−−

++

−−

−−

−−

−−

1848

NC

MB

1947

603

Flav

obac

teriu

msa

ccha

roph

ilum

−5

34

−4

42

−−

53

24

−5

−4

−−

2212

ASS

NC

IMB

2072

92

Flav

obac

teriu

msa

ccha

roph

ilum

−5

−1

−2

1−

−−

52

−1

−1

−1

−−

NC

IMB

2072

376

Flav

obac

teriu

msu

ccin

ican

s−

53

3−

44

21

−5

5−

−−

42

4−

−22

12AS

SN

CIM

B22

7792

Flex

ibac

tera

ggre

gans

−5

34

15

43

3−

55

13

−4

25

33

2212

ASS

NC

MB

1443

89

Flex

ibac

tera

uran

tiacu

s*−

52

41

55

3−

−5

3−

1−

53

3−

−22

12AS

SN

CM

B13

8289

Flex

ibac

tera

uran

tiacu

s*−

52

41

55

3−

−5

4−

−−

53

4−

−22

12AS

SN

CM

B14

5589

Flex

ibac

terc

anad

ensi

s−

52

31

55

34

15

54

1−

53

4−

322

12AS

SAT

CC

2959

189

Flex

ibac

terf

lexi

lis−

53

4−

54

31

23

1−

−−

3−

−−

−22

12AS

SN

CM

B13

7789

Flex

ibac

terf

lexi

lis−

+−

+−

++

+−

++

+−

−−

++

−−

−AT

CC

2307

960

3

Flex

ibac

terl

itora

lis−

52

41

55

35

15

5−

−−

−−

−−

−22

12AS

SN

CM

B13

6689

Flex

ibac

terp

olym

orph

us−

52

31

55

34

12

2−

−−

2−

−−

−30

12AS

SAT

CC

2782

089

Flex

ibac

terr

oseo

lus

−4

23

13

32

13

22

−−

−−

−−

−−

2512

ASS

NC

MB

1433

89

Flex

ibac

terr

uber

−5

34

23

32

13

33

−−

−−

−−

−−

2512

ASS

NC

MB

1436

89

Flex

ibac

ters

anct

i−

5−

2−

41

−4

−4

54

4−

43

4−

222

12AS

SN

CM

B13

7989

Flex

ibac

tert

ract

uosu

s−

52

31

54

31

45

4−

−−

51

−−

−22

12AS

SN

CM

B14

0889

Gra

nulic

atel

laba

laen

opte

rae

−−

−+

−+

−−

−−

−−

−−

−−

−+

−−

478

Haf

nia

alve

i−

5−

w1

32

−w

−4

1−

2−

21

w−

−25

40.

85AH

LDA

1729

135

List

onel

laan

guilla

rum

−+

++

−+

++

−−

++

−−

−−

−+

−−

2224

ATC

C14

181

506

List

onel

laan

guilla

rum

−4

−2

−w1

−−

−−

−−

−−

−−

−−

−−

2524

2.85

NC

MB

681

List

onel

laan

guilla

rum

−3

12

22

11

−−

−w

−w

−−

2−

−−

2524

2.85

AHLD

A17

3013

5

List

onel

lape

lagi

aII

−+

++

−+

−−

+−

++

−−

−−

−−

−−

2224

NC

MB

2253

506

Man

nhei

mia

haem

olyt

ica

−5

11−

w−

1–3

0–1

−−

−5

1–2

−1–

2−

−−

−−

0–2

374

0.85

Vario

us13

5

Wel

l1

23

45

67

89

1011

1213

1415

1617

1819

20Te

mp

Tim

eIn

ocSt

rain

Ref

Enzy

me

con

alk

C4

C8

C14

aryl

val

cys

trych

rac

pnp

a-g

alb-

gal

b-gl

ucr

a-g

lub-

glu

N-a

bglu

man

fuc

°CH

%N

aCl

Org

anis

mes

tes

tlip

lip

Tab

le4.

31.

Con

tinue

d.



215

Mar

inila

bilia

salm

onic

olor

−5

12

−−

−−

−4

21

3−

15

55

−−

2512

ASS

NC

MB

2216

89

Mor

itella

mar

ina

−+

++

−+

−−

−−

++

−−

−−

−−

−−

2224

NC

MB

1144

506

Mor

itella

visc

osa

−+

++

++

−−

−−

++

−−

−−

−−

−−

2224

NC

MB

1358

450

6

Myr

oide

sod

orat

imim

us−

++

+−w

3−w

−w−

−w+

+−

−−

−−

−−

−N

CTC

1118

077

4

Myr

oide

sod

orat

us−

++

+−w

1−w

−w−

−w+

+−

−−

−−

−−

−N

CTC

1103

677

4

Myr

oide

sod

orat

us−

52

3−

2−

−1

−5

5−

−−

−−

−−

−30

12N

CTC

1103

689

Past

eure

llam

ulto

cida

−5

1−w

1–2

−2

0−w

−−

−5

1−

−−

−−

−−

−37

40.

85Va

rious

135

Past

eure

llask

yens

is−

51

2−

2−

−−

−5

5−

−−

−−

−−

−25

4d1.

5N

CTC

1320

410

0

Pedo

bact

erhe

parin

us−

++

+−

+−

−−

−+

++

+−

++

++

−28

40.

85N

CIB

9290

728

Pedo

bact

erhe

parin

us−

52

4−

42

1−

−4

2−

−−

1−

41

−22

12N

CIB

9290

89

Pedo

bact

erpi

sciu

m−

++

++

++

++

++

−+

−+

++

+−

284

0.85

728

Phoc

oeno

bact

erut

eri

−5

15

−1

−−

−−

55

−−

−−

−−

−−

266

Phot

obac

teriu

mda

mse

lae

−+

++

−+

−−

−−

++

−−

−−

−+

−−

2224

NC

MB

2184

506

Phot

obac

teriu

mda

mse

lae

−5

−2

−4

11

−w

51

−1

−−

−4

−−

2520

2.85

AHLD

A16

8313

5

Phot

obac

teriu

mpi

scic

ida

−+

++

−+

−−

−−

++

−−

−−

−+

−−

855

Ples

iom

onas

shig

ello

ides

−4

−1

w4

1−

−−

51

−w

−2

−5

−−

254

AHLD

A19

213

5

Pseu

doal

tero

mon

asan

tarc

tica

−+

++

−+

++

++

++

−−

−+

−+

+−

115

Pseu

doal

tero

mon

asci

trea

−+

++

−−

−w

−−

285

Pseu

doal

tero

mon

asru

bra

−4

−2

12

1−

41

42

−−

−−

−2

−−

283

Pseu

dom

onas

angu

illise

ptic

a−

+−

++

+−

−−

−+

+−

−−

−−

−−

−N

CIM

B19

49T

96

Pseu

dom

onas

plec

oglo

ssic

ida

−+

−+

−+

+−

+−

++

−−

−−

−−

−−

582

Ren

ibac

teriu

msa

lmon

inar

um−

+−

+−

+−

−+

−+

+−

−−

+−

−+

−41

Shew

anel

laal

gae

−+

−+

−+

13−

++

++

−−

−−

−+

−−

433

Shew

anel

laba

ltica

−−

−−

−−

−−

−w

−−

−−

−−

−+

−−

851

Shew

anel

lapu

trefa

cien

s−

+−

+−

+67

−78

89+

+−

−−

−−

+−

−43

3

Sphi

ngob

acte

rium

mul

tivor

um−

+−

+−

++

−−

−+

−−

+−

+−

+−

−36

4

Sphi

ngob

acte

rium

mul

tivor

um−

54

41

51

1−

−5

52

4−

43

53

−37

4AS

SN

CTC

1134

389

Sphi

ngob

acte

rium

spiri

tivor

um−

+−

−−

−−

−+

−+

1−

1−

+−

+−

−N

CTC

1138

636

5

Sphi

ngob

acte

rium

spiri

tivor

um−

52

4−

51

15

25

52

41

32

53

430

12AS

SN

CTC

1138

689

Stap

hylo

cocc

uslu

trae

−5

55

−−

−−

−−

55

−1

−−

−−

−1

264

Tena

ciba

culu

mm

ariti

mum

−5

13–

42–

55

55

55

55

−−

−0–

1−

−−

−18

7d2.

8560

5

cont

inue

d



216

Tena

ciba

culu

mm

ariti

mum

−5

34

15

53

12

55

−−

−−

−−

−−

2512

2.85

89

Tena

ciba

culu

mm

ariti

mum

−5

23

14

52

31

55

−−

−−

−−

−−

2212

0.85

ATC

C43

398

743

Tena

ciba

culu

mov

olyt

icum

−+

++

−+

+−

−−

++

−−

−−

−−

−−

324

Vago

cocc

usfe

ssus

−v

+w+w

−+

−v

−+

−−

−v

−−

v−

−−

369

Vago

cocc

uslu

trae

−−w

−+

−+

−−

−+

+−

+−w

−+

++

−−

477

Vibr

ioag

ariv

oran

s−

1w

1−

1−

−−

−−

w−

2−

−−

−−

−25

242.

85AH

LDA

1732

135

Vibr

ioal

gino

lytic

us−

5−

2w

41

−2

−5

1−

−−

−−

1−

−25

413

5

Vibr

iobr

asilie

nsis

−+

++

++

+−

−−

−−

−+

−−

−−

−25

241.

5LM

G20

546T

740

Vibr

ioca

lvie

nsis

−+

++

++

−−

−−

++

−+

−−

DSM

1434

7T21

6

Vibr

ioch

oler

aeno

n-01

−3

−3

12

1w

−−

11

−2

−1

−4

−−

2524

0.85

AHLD

A99

613

5

Vibr

iodi

abol

icus

−3

23

13

−−

22

3−

−−

−−

−−

−−

635

Vibr

iofis

cher

i−

+−

+−

+−

−−

−+

+−

−−

−−

+−

−22

24N

CM

B12

8150

6

Vibr

iofu

rnis

sii

−3

−2

14

22

−−

11

−1

−w

−4

−−

2524

0.85

ATC

C11

218

135

Vibr

ioha

liotic

oli

−1

w1

−1

w−

−−

−1

−−

−−

−3

−−

2524

2.85

AHLD

A17

3413

5

Vibr

ioha

rvey

i−

32

2−

1w

−−

15

1−

−−

−−

1−

−25

40.

85AT

CC

3508

413

5

Vibr

ioha

rvey

i−

42

2−

1w

−−

23

1−

−−

−−

2−

−25

42.

85AT

CC

3508

413

5

Vibr

ioha

rvey

i−

++

+−

−−

−−

−+

+−

−−

−−

−−

−AT

CC

3508

484

7

Vibr

iolo

gei

−+

++

−+

−−

−−

++

−−

−−

−−

−−

2224

ATC

C15

382

506

Vibr

iolo

gei

−+

++

−+

−−

−−

++

++

−−

−−

−−

2224

NC

MB

1143

506

Vibr

iolo

gei

−+

++

−+

−−

−−

++

−−

−−

−−

−−

2224

ATC

C29

985

506

Vibr

iom

edite

rrane

i−

52

22

1−

−−

−4

2−

−−

−−

2−

−25

242.

85AH

LDA

1733

135

Vibr

iom

imic

us−

5−

2−

41

−−

w5

1−

1−

−−

3−

−25

42.

85AH

LDA

1654

135

Vibr

iom

ytili

−−

23

13

−−

1−

2−

−−

−−

−2

−−

CEC

T63

263

5

Vibr

iona

trieg

ens

−+

++

−−

−−

−−

++

−−

−−

−−

−−

2224

NC

MB

1900

506

Vibr

ione

ptun

ius

−+

++

++

+−

+−

−−

−−

−−

−+

−22

241.

5LM

G20

536T

740

Vibr

ione

reis

−−

11

−2

−−

−−

−−

−−

−−

−−

−−

LMG

3895

635

Vibr

ioor

dalii

−+

++

−+

−−

−−

++

−−

−−

−+

−−

2224

NC

MB

2167

506

Wel

l1

23

45

67

89

1011

1213

1415

1617

1819

20Te

mp

Tim

eIn

ocSt

rain

Ref

Enzy

me

con

alk

C4

C8

C14

aryl

val

cys

trych

rac

pnp

a-g

alb-

gal

b-gl

ucr

a-g

lub-

glu

N-a

bglu

man

fuc

°CH

%N

aCl

Org

anis

mes

tes

tlip

lip

Tab

le4.

31.

Con

tinue

d.



217

Vibr

ioor

dalii

−5

−w

−4

w−

−−

ww

−−

−−

−−

−−

254

2.85

DF

3K42

8

Vibr

ioor

ient

alis

−+

++

++

+−

−−

++

−−

−−

−+

−−

2224

NC

MB

2195

506

Vibr

iopa

cini

i−

++

+−

++

v−

w+

+−

66−

+−

−−

−28

41.

5LM

G19

99T

306

Vibr

iopa

raha

emol

ytic

us−

5−

3w

53

−1

−5

1−

−−

−−

−−

−25

202.

85AT

CC

4399

613

5

Vibr

iopa

raha

emol

ytic

us−

+−

−−

−−

−+

−+

+−

−−

−−

+−

−AT

CC

1780

249

9

Vibr

iopa

raha

emol

ytic

us−

+−

+−

−−

−+

−+

+−

−−

−−

+−

−AT

CC

2796

949

9

Vibr

iopr

oteo

lytic

us−

21

2−

2−

−−

−−

w−

−−

−−

w−

−25

242.

85AH

LDA

1735

135

Vibr

ioro

tifer

ianu

s−

++

+−

++

−+

++

+−

−−

−−

−−

−25

242.

85LM

G21

460T

305

Vibr

iosa

lmon

icid

a−

++v

+−

−−

−−

−+

+−

−−

−−

−−

−22

24N

CM

B22

6250

6

Vibr

iosp

lend

idus

I−

++

++

+−

−+

−+

+−

−−

−−

+−

−22

24N

CM

B1T

506

Vibr

iosp

lend

idus

I−

++

++

+−v

−+

−+

v−

+v−

+−

+−

−28

1

Vibr

iosp

lend

idus

II−

++

+−

++

−+

−+

+−

−−

−−

−−

−22

24N

CM

B22

5150

6

Vibr

iota

petis

−+

++

−+

+−

−−

+−

−−

−−

−+

−−

2218

2.85

B109

0T58

7

Vibr

iotu

bias

hii

−2

13

12

−−

−−

−−

−−

−−

−−

−−

LMG

1093

663

5

Vibr

iotu

bias

hii

−+

++

−+

++

−−

−+

−−

−−

−+

−−

2224

NC

MB

1340

T50

6

Vibr

iotu

bias

hii

−+

++

−+

++

−−

−+

−−

−−

−+

−−

2224

NC

MB

1340

506

Vibr

iovu

lnifi

cus

biov

arI

−5

w1

−1

w−

−−

w1

−−

−−

−−

−−

254

0.85

AHLD

A17

1613

5

Vibr

iovu

lnifi

cus

biov

arI

−5

11

−1

ww

−−

−w

−−

−−

−−

−−

254

2.85

AHLD

A17

1613

5

Vibr

iow

odan

is−

+83

+−

+86

−−

−69

+−

−−

−−

77−

−22

24N

CIM

B13

582

506

Vibr

ioxu

ii−

++

++

+−

−−

−+

+−

−−

−−

−−

2524

1.5

LMG

2134

6T74

0

Yers

inia

ruck

eri

−1

−w

w4

1−

−−

21

−2

−1

−3

−−

2520

AHLD

A13

1313

5

Zobe

lliaga

lact

aniv

oran

s−

33

13

307d

2.5

61

Zobe

lliaul

igin

osa

−1

13

130

7d2.

561

*Is

olat

esN

CM

B13

82an

dN

CM

B14

55pr

evio

usly

iden

tifie

das

Flex

ibac

tera

uran

tiacu

sar

eno

wth

ough

tto

beFl

avob

acte

rium

john

soni

ae.

1=

cont

rolw

ell;

2=

2-na

phth

yl-p

hosp

hate

;3=

2-na

phth

yl-b

utyr

ate;

4=

2-na

phth

yl-c

apry

late

;5=

2-na

phth

yl-m

yris

tate

;6=

L-le

ucyl

-2-n

apht

hyla

mid

e;7

=L-

valy

l-2-n

apht

hyla

mid

e;8

=L-

cyst

yl-2

-nap

hthy

lam

ide;

9=

N-b

enzo

yl-D

L-ar

gini

ne-2

-nap

hthy

lam

ide;

10=

N-g

luta

ryl-p

heny

lala

nine

-2-n

apht

hyla

mid

e;11

=2 -

naph

thyl

-pho

spha

te;1

2=

naph

thol

-AS-

BI-p

hosp

hat e

;13

=6-

Br-2

-nap

hthy

l-a-D

-gal

acto

pyra

nosi

de;1

4=

2-na

phth

yl-b

-D-g

alac

topy

rano

side

;15

=nap

htho

l-AS-

BI- b

-D-g

lucu

roni

de;1

6=

2-na

phth

yl-a

-D-g

luco

pyra

nosi

de;1

7=

6-Br

-2-n

apht

yl-b

-D-g

luco

pyra

nosi

de;1

8=

1-na

phth

yl-N

-ace

tyl-b

-D-g

luco

sam

inid

e;19

=6-

Br-2

-nap

hthy

l-a-D

-man

nopy

rano

side

;20

=2-

naph

thyl

-a-L

-fuco

pyra

nosi

de.

Tem

p=

tem

pera

ture

ofin

cuba

tion;

Tim

e=

time

ofin

cuba

tion;

H=

hour

s;d

=da

ys;I

noc

=in

ocul

um;%

NaC

l ind

icat

esfin

alN

aClc

once

ntra

tion

inth

ein

ocul

um.M

I=M

anuf

actu

rer’s

Inst

ruct

ions

;nr=

notr

epor

ted;

Ref

=re

fere

nce

num

ber.

Ref

603

clas

sifie

da

reac

tion

of2

orle

ssas

nega

t ive.



218

Actinobacillus scotiae NCTC 12922 265

Actinomyces marimammalium CCUG 41710T = CIP 106509T 370

Aequorivita antarctica ACAM 640T, DSM 14231T 113

Aequorivita crocea ACAM 642T, DSM 14239T 113

Aequorivita lipolytica ACAM 641T, DSM 14236T 113

Aequorivita sublithincola ACAM 643T, DSM 14238T 113

Aeromonas allosaccharophilaHG15

CECT 4199 427

Aeromonas bestiarumHG2

CDC 9533-76 1,21,427

Aeromonas caviaeHG4

ATCC 15468 427,21

Aeromonas encheleiaHG16

CECT 4342 = LMG 16330 241,379,427

Aeromonas eucrenophilaHG6

ATCC 23309, CDC 0859-83 427,21

Aeromonas eucrenophila LMG 3774 = NCMB 74 379

Aeromonas eucrenophila LMG 13057 379

Aeromonas hydrophila dhakensis P21T = LMG 19562T = CCUG 45377T 383

Aeromonas hydrophila hydrophila ATCC 7966T = LMG 2844T 383

Aeromonas hydrophilaHG1

ATCC 7966T, CDC 9079-79 21,427,818

Aeromonas hydrophilaHG3

CDC 0434-84 427

A. hydrophila anaerogenes = A. caviae ATCC 15468 818

A. hydrophila anaerogenes ATCC 15467 Taxonomy 2000

Aeromonas janadaeiHG 9

CDC 0787-80, ATCC 49568 143,427

Aeromonas mediaHG 5B

CDC 9072-83, CDC 0435-84 427,21

Aeromonas mediaHG 5A

CDC 9072-83, CDC 0862-83 427,21

Aeromonas popoffii LMG 17541T 380

Aeromonas salmonicida achromogenes NCMB 1110 450,475

Aeromonas salmonicida pectinolytica DSM 12609T = 34 mel 615

A. salmonicida salmonicida ATCC 14174 450,818

A. salmonicida salmonicida SVLT -1, -2, -5, -6, Non-pigmented strains 450

A. salmonicida salmonicida NCMB 1102 475

Aeromonas salmonicidaHG3

CDC 0434-84 21

Aeromonas schubertiiHG12

CDC 2446-81, ATCC 43700, formerly Enteric group 501 348,427

Aeromonas sobria HG7 CIP 7433, CDC 9538-76 21,427,818

Aeromonas veronii sobriaHG8/10

CDC 0437-84 427

Aeromonas spp.HG11

CDC 1306-83 427

Aeromonas trota ATCC 49657T = LMG 12223T HG 14 142,427

Aeromonas veronii ATCC 35604T = CDC 1169-83 21,347

Aeromonas veronii HG 8, CDC-0437-84 21

Alteromonas aurantia ATCC 33046, NCMB 2052 286

Alteromonas citrea NCMB 1889 285

Alteromonas rubra NCMB 1890 283

Arcanobacterium phocae DSM 10002T, M1590/94/3T 636

Arcanobacterium pluranimalium CCUG 42575T = CIP 106442 480

Arthrobacter rhombi CCUG 38813T 600

Atopobacter phocae CCUG 42358T = CIP 106392 479

Bordetella bronchiseptica ATCC 19395, ATCC 4617, NCTC 8344 102

Brevundimonas diminuta ATCC 11568, LMG 2089 = CCUG 1427 685

Brevundimonas vesicularis ATCC 11426 = CCUG 2032 = LMG 2350 685

Organism Strain number Reference number

Table 4.32. Strains quoted in references.



219

Brucella maris biovar I NCTC 12890, 2/94 404

Brucella maris biovar II NCTC 12891, 1/94 404

Brucella maris biovar III 247,404

Carnobacterium inhibens CCUG 31728T, strain K1 411,412

Carnobacterium piscicola ATCC 35586 73,353,682

Chromobacterium violaceum 482

Corynebacterium aquaticum RB 968 BA 73,133

Edwardsiella ictaluri CDC 1976-78, ACC 33202 334

Edwardsiella tarda ATCC 15947T 374,640

Enterococcus faecalis ATCC 19433 638

Enterovibrio norvegicus LMG 19839T, CAIM 430T 741

Flavobacterium aquatile NCIB 8694T, LMG 4008T 89,92

Flavobacterium branchiophilum ATCC 35035, BGD-7721 802

Flavobacterium frigidarium ATCC 700810 = NCIMB 13737 376

Flavobacterium gillisiae ACAM 601T 533

Flavobacterium hibernum ATCC 51468 = ACAM 376T 532

Flavobacterium hydatis (C. aquatilis) ATCC 29551 720

Flavobacterium tegetincola ACAM 602T 533

Flexibacter polymorphus ATCC 27820 494

Granulicatella balaenopterae CCUG 37380T, M1975/96/1 478

Iodobacter fluviatilis NCTC 11159T 502

Janthinobacterium lividum NCIMB 9230, NCIMB 9414, DSM 1522 496

Lactococcus garvieae NCDO 2155 224,638

Lactococcus garvieae ATCC 49156T 236,464,682

Lactococcus lactis ATCC 19435 731

Lactococcus piscium NCFB 2778 835

Listonella anguillarum ATCC 14181 506

Listonella anguillarum NCMB 6 = ATCC 19264 (Bagge and Bagge strain) 341

Listonella anguillarum NCMB 407 = PL 1 341

Listonella anguillarum NCMB 571 (Hoshina) 341

Listonella anguillarum NCMB 828 = ATCC 14181 (strain 4063, Smith) 341

Listonella pelagia I (said to be V. natriegens) NCMB 1900T 506

Listonella pelagia II NCMB 2253 506

Mesophilobacter marinus IAM 13185 583

Moritella marina NCMB 1144T = ATCC 15381 82,506,766

Moritella viscosa NCIMB 13584T = NVI 88/478T 82,506

Mycoplasma crocodyli ATCC 51981 441

Mycoplasma mobile 163K 439

Myroides odoratimimus NCTC 11180, LMG 4029 565

Myroides odoratus NCTC 11036, LMG 1233 565

Nocardia seriolae JCM 3360 455

Nocardia spp., Australian strain 98/1655 117

Pantoea agglomerans ATCC 27155T, NCTC 9381 249,291

Pantoea agglomerans ATCC 27155T, ATCC 12287 325

Pantoea dispersa ATCC 14589T 291

Pasteurella skyensis NCTC 13204T, NCIMB 13593T 100

Phocoenobacter uteri NCTC 12872 266

Photobacterium angustum NCIMB 1895 745

Photobacterium damselae ssp. damselae ATCC 33539 = NCIMB 2184T 289,504,506,705,745

Photobacterium damselae ssp. damselae ATCC 35083 268,745

Photobacterium damselae ssp. piscicida ATCC 17911 & NCIMB 2058 289,745

Photobacterium damselae ssp. piscicida 751

Photobacterium damselae ssp. piscicida ATCC 29690, ATCC 17911 518

Photobacterium fischeri NCMB 1281T = ATCC 7744 506

Photobacterium fischeri ATCC 25918 745

Photobacterium fischeri ATCC 7744 340,818

Photobacterium iliopiscarium ATCC 51760 599,767



continued



220

Photobacterium leiognathi LMG 4228, NCIMB 1895 745

Photobacterium logei ATCC 15382 = NCMB 1143 = PS 207 506

Photobacterium logei NCMB 1143 506

Photobacterium logei ATCC 29985T 506

Photobacterium logei ATCC 15382 82

Photobacterium logei NCIMB 2252, ATCC 29985 506

Providencia friedericiana DSM 2620 559

Pseudoalteromonas antarctica CECT 4664T, NF3 115

Pseudoalteromonas citrea ATCC 29719T, NCMB 1889 285

Pseudoalteromonas elyakovii ATCC 700519T, KMM 162T 679

Raoultella planticola ATCC 33531 228

Raoultella terrigena ATCC 33257 228

Renibacterium salmoninarum ATCC 33209 671

Serratia fonticola ATCC 29844 290

Shewanella algae LMG 2265, IAM 14159 433,792,851

Shewanella baltica NCTC 10735, DSM 9439, CECT 323, IAM 1477, LMG 2250 851

Shewanella colwelliana ATCC 39565 815

Shewanella frigidimarina ACAM 591T 112

Shewanella gelidimarina ACAM 456T 112

Shewanella (Alteromonas) hanedai ATCC 33224 409

Shewanella japonica KMM 3299, LMG 19691 = CIP 106860 397

Shewanella oneidensis ATCC 700550T 782

Shewanella pealeana ANG-SQ1T 492

Shewanella putrefaciens ATCC 8071 433,792

Shewanella woodyi ATCC 51908T, MS32 112

Sphingobacterium spiritivorum NCTC 11386 365

Staphylococcus delphini DSM 20771T 778

Staphylococcus lutrae DSM 10244, M340/94/1 264

Streptococcus (difficile) agalactiae ND 2-22, CIP 103768 233

Streptococcus (difficile) agalactiae LMG 15977 776

Streptococcus iniae ND 2-16, CIP 103769 233

Streptococcus iniae ATCC 29177 626

Streptococcus parauberis NCDO 2020 224

Streptococcus phocae NCTC 12719, 8399 HI 700

Streptococcus uberis NCDO 2038 224

Tenacibaculum (Flexibacter) maritimum NCMB 2154T 801

Tenacibaculum (Flexibacter) ovolyticum NCIMB 13127 = EKD002 324

Vagococcus fessus CCUG 41755 369

Vagococcus fluvialis NCDO 2497, NCFB 2497 177,629

Vagococcus lutrae CCUG 39187 477

Vagococcus salmoninarum NCFB 2777 682,807

Vibrio aerogenes ATCC 700797 = CCRC 17041, FG1 692

Vibrio aestuarianus ATCC 35048, LMG 7909 149,747

Vibrio agarivorans CECT 5084, CECT 5085T = DSM 13756 514

Vibrio agarivorans AHLDA 1732 135

Vibrio brasiliensis LMG 20546T 740

Vibrio calviensis RE35/F12T = CIP 107077T = DSM 14347T 216

Vibrio (carchariae) harveyi ATCC 35084T 11,135

Vibrio cincinnatiensis ATCC 35912 120

Vibrio diabolicus CNCM I-1629 = HE800 635

Vibrio diazotrophicus ATCC 33466 319

Vibrio fluvialis ATCC 49515, NCDO 2497 = NCFB 2497 177,732

Vibrio fluvialis NCTC 11327 123,485

Vibrio furnissii ATCC 35016 = CDC B3215 123

Vibrio furnissii ATCC 11218 135

Vibrio furnissii Group F 687





221

Vibrio gazogenes ATCC 29988 818

Vibrio halioticoli IAM 14596T 678

Vibrio harveyi ATCC 14126T 11,818

Vibrio hollisae KUMA871, ATCC 33564T 346,580

Vibrio lentus CECT 5110T = DSM 13757 = 40MA 513

Vibrio mediterranei CECT 621T, LMG11258 149,631

Vibrio metschnikovii NCTC 8563 819

Vibrio metschnikovii NCTC 8443 818

Vibrio mimicus ATCC 33653T 210,507,818

Vibrio mytili CECT 632T 635

Vibrio natriegens ATCC 14048 44

Vibrio navarrensis CIP 1397-6 767,768

Vibrio neptunius LMG 20536T 740

Vibrio nereis LMG 3895T 635

Vibrio ordalii NCMB 2167T, ATCC 33509 = DF3K = Dom F3 506,680

Vibrio orientalis NCMB 2195T 506

Vibrio pacinii LMG 1999T 306

Vibrio pectenicida CIP 105190T, A365 470

Vibrio penaeicida JCM 9123, KH-1, IFO 15640 388

Vibrio proteolyticus AHLDA 1735 135

Vibrio proteolyticus CW8T2 788

Vibrio rotiferianus LMG 21460T 305

Vibrio salmonicida NCMB 2262T 232,506

Vibrio salmonicida NCMB 2245 506

Vibrio salmonicida 90/1667-10c 506

Vibrio scophthalmi A089, CECT 4638T 254,149

Vibrio shilonii (possibily V. mediterranei) ATCC BAA-91T = DSM 13774 = AK-1 59,458

Vibrio splendidus I NCMB 1T (= ATCC 33125T) 149,506

Vibrio splendidus I ATCC 33125 281,620

Vibrio splendidus II NCMB 2251 149,506

Vibrio tapetis B1090T, CECT 4600 108,587

Vibrio tubiashii NCMB 1340T 506

Vibrio tubiashii ATCC 19109T 321

Vibrio tubiashii LMG 10936T 635

Vibrio vulnificus 149

V. vulnificusbiovar I non-serovar E f

ATCC 27562 26,746

V. vulnificusbiovar 2, serovar E a

ATCC 33187 98

V. vulnificusbiovar 2, serovar E b

ATCC 33149 98

V. vulnificusbiovar 2, serovar 04 b

ATCC 33149 356

V. vulnificusbiovar 2, serovar E c

NCIMB 2138 98

V. vulnificusbiovar 2 serovar E d

NCIMB 2137 98

V. vulnificusbiovar 2, serovar E e

NCIMB 2136 98

V. vulnificusbiovar 2, serovar E g

Taiwanese strains 26

V. vulnificusbiovar 2

ATCC 33148 746

Vibrio wodanis NCIMB 13582 = NVI 88/441T 506

Vibrio xuii LMG 21346T

Yersinia rohdei ATCC 43380, CDC 3022-85 12

Yersinia aldovae ATCC 35236, CDC 669-83 85





5Technical Methods

5.1 Total Bacterial Count (TBC)

Diagnostic, research, or industry-based laborato-ries often monitor pond or tank water for bacte-rial load as an indicator of water quality, andhence as a disease indicator. This is done by atest known as the TBC.

There are a number of methods for TBCsand these may be found in most microbiologytextbooks. A basic method for TBCs is describedhere.

Generally, TBCs are performed using a goodgeneral-purpose medium that will support thegrowth of the majority of the organisms that areexpected to be cultured. A selective medium forVibrio species such as TCBS is often used as aculture medium for TBCs. However, this mediumhas a tendency to produce a lower count thanan equivalent sample inoculated to MSA-B plate(or BA in the case of freshwater samples) evenwhen the majority of bacteria cultured are Vibriospp. Therefore, it is not the best primary culturemedium.

The time between collection of the watersample and testing is important. Bacteria in thewater sample will multiply within a few hours atroom temperature. Thus, if there is a delay of morethan 1–2 h between collection and testing, aninaccurate assessment of the bacterial load of theoriginal water sample will be obtained. The watersample must be stored at 4°C either during trans-port or while waiting to be tested in the laboratory.There is no effect on bacterial count due to thesize of the collection container (Simon andOppenheimer, 1968).

There is no predetermined optimal bacterialload of pond or tank water, and this will varyanyway according to the number of fish in thewater, that is, the stocking density. It is suggestedthat laboratories regularly monitor a particulartank or set of tanks and keep a record of theirresults. The bacterial load or TBC is then relatedback to the health of the fish. If a daily log or graphis kept of the TBC against the health of the fish,then the laboratory can eventually determine theoptimal bacterial load for healthy fish, as opposedto the load seen when the fish appear diseased.

Suggested sites for monitoring bacterial loadare the intake water supply, the filtered water sup-ply, fish tanks and the feed supply. The sedimentat the bottom of the tank will have a greaterbacterial count than the water body, particularlywhen there are dead or diseased larvae or fish.

Research in an oyster hatchery indicated thatfour main genera were involved in the bacterialload; these were Vibrio species, Alteromonas spe-cies, Pseudomonas species and Flavobacteriumspecies. Vibrio species were the predominantorganism. Virulence testing showed that a third ofthese Vibrio species, which included Listonellaanguillarum, and two of the ten Alteromonasspecies were pathogenic. The isolates were notidentified to species level. Infection was not seen inthe larvae when they were dosed with the non-pathogenic bacteria. However, fatal infection wasproduced when the bacterial load of the patho-genic bacteria was 1 × 107–5 × 107. At a lowerdose of 1 × 105–5 × 105, infection still occurred,but took 2–3 days longer to develop and forclinical signs to be seen. Thus, infection rates are

©N.B. Buller 2004. Bacteria from Fish and Other Aquatic Animals:222 a Practical Identification Manual (N.B. Buller)



related to the bacterial load of pathogenic bacteria(Garland et al., 1983).

Bacteria are part of the diet of marine filterfeeders. In another study on healthy oysters(Crassostrea gigas) and horse mussel (Modiolusmodiolus), the total bacterial load of normal florain the haemolymph and soft tissue was 2.6 × 104

colonies/ml, and 2.9 × 104 colonies/g, respec-tively. The predominant flora and percentage ofthe total flora were Pseudomonas spp. (61.3%),Vibrio spp. (27%) and Aeromonas spp. (11.7%) inthe haemolymph, with the predominant bacteriain the soft tissues being Vibrio spp. (38.5%),Pseudomonas spp. (33%) and Aeromonas spp.(28.5%). When the oysters and horse musselwere challenged in virulence studies using Vibriosalmonicida, the TBC increased to 105 colonies/mlin the haemolymph and 6 × 107 colonies/g in thesoft tissue (Olafsen et al., 1993).

Table 5.1 indicates the bacterial countsobtained in both studies.

The microflora in the intestines of healthyjack mackerel (Trachurus japonicus) was found tobe 4.6 × 106 colonies/g and consisted of Vibriospecies. The total bacterial load in the stomachwas 2.6 × 105 colonies/g, with the predominantflora being Vibrio spp., Achromobacter spp.,with smaller counts of Pseudomonas spp.,Flavobacterium spp., Corynebacterium spp.,Bacillus spp. and Sarcina spp. (Aiso et al., 1968).

Equipment

A pipette or pipettes capable of measuring 100 mlto 1000 ml1.5 ml microfuge tubes

An agar medium that supports the growth ofthe target bacteria, such as BA for freshwater orMSA-B, or MA 2216 for salt waterInoculating loopBunsen burnerIncubator set to 25°C

Water

Collect a volume of waterDo dilutions at neat, 10−1, 10−2, 10−3

Dilutions

Neat = 100 ml to a plate and lawn inoculate(spread evenly over the plate)10−1 = 900 ml of sterile distilled water + 100 ml ofsample10−2 = 900 ml of sterile distilled water + 100 ml ofdilution 10−1

10−3 = 900 ml of sterile distilled water + 100 ml ofdilution 10−2

Method

Place 100 ml (or 10 µl) of each dilution into thecentre of an agar plate. Use a separate plate foreach dilution. Spread each inoculum evenly overeach plate using a sterile bent glass rod, or aflame-sterilized inoculating loop. It is important toensure that the inoculum is spread evenly overthe plate so that individual colonies are obtainedfor counting. Clumps of colonies will lead toerroneous results.

Technical Methods 223

Sample site Total bacterial count (cells/ml)

Seawater (filtered, no larvae)a 103–103 a

Tank watera 104–105 a

Sedimenta 107–108 cells/ga

Haemolymph healthy oysterb 2.6 × 104 b

Soft tissue healthy oysterb 2.9 × 104 b

Load of pathogenic bacteria at which mortality is seen. Rapid onset of infectiona 1 × 107–5 × 107 a

Load of pathogenic bacteria at which mortality is seen. Slower onset of infectiona 1 × 105–5 × 105 a

Haemolymph in diseased oysterb 105 b

Soft tissue in diseased oysterb 6 × 107 b

aData from Garland et al. (1983); bData from Olafsen et al. (1993).

Table 5.1. Total bacterial counts at sites in oyster hatcheries: indication of healthy and diseased states.



Incubation

Place plates in a sealed plastic container andincubate at room temperature or an incubator setto 25°C.

Counting the bacterial colonies

Examine the plates at 24 and 48 h.Count the number of colonies on a plate: using afelt pen, mark each colony from the back of thePetri dish to assist with countingCount = N

Calculation to obtain CFU/ml

If 100 ml is placed on to a plate and then lawninoculated, this is a plate dilution of 10−1. If 10 mlwere placed on a plate, this would be a platedilution of 10−2.

CFU/ml = N × dilution × plate dilution.

Thus if 268 colonies are counted fromthe 10−2 dilution and 100 ml was inoculatedto the plate, the count = 268 × 102 × 10 =2.68 × 105 CFU/ml.

5.2 Microscopy

Most laboratory staff will be familiar with the useof a light microscope. However, for those whoare new to the use of a microscope, some basicsare explained here.

Before use, a microscope should be set up forKoehler illumination. This ensures that the lightentering the microscope is focused so as to pro-duce an evenly illuminated field.

• Place a stained smear on to the stage. Focususing the ×10 objective.

• Close the field diaphragm – this is thediaphragm at the base of the microscope.

• Close the aperture of the iris diaphragm.This is the aperture just under the stage thatthe glass slide rests on.

• There will be a small circle of light. Centrethis circle of light using the condensercentring screws.

• Next, adjust the height of the condenserunder the stage so that the edge of the circleof light is in sharp focus.

• Now open the field diaphragm so that it justdisappears from view.

• Open the aperture diaphragm to suit thecontrast required.

Staff who are new to the use of a microscopeshould remember that examination of a Gram-stained smear of bacterial cells is best observedusing the oil immersion lens, which together withthe ×10 objective gives a magnification of 1000times. The aperture on the substage condensershould be open to half its setting with the lightintensity set so that it is comfortable for theeyes. For the examination of wet preparations thisaperture will need to be almost closed so that agood contrast is obtained and cellular structuresare defined.

Only use lens tissue paper for cleaning theobjective lens. Always remove the oil from thelens after use, as the oil can damage the structureswithin the objective. If oil does get on to any ofthe lenses, it can be removed by soaking somelens tissue with petroleum spirit and gentlywiping across the lens until the oil has beenremoved.

5.3 Storage of Isolates

Isolates may be stored at −80°C in LabLemco broth supplemented with 10% glycerol.For marine organisms use marine broth 2216supplemented with 30% glycerol (Bowman andNichols, 2002).

There are a number of different mediafor freeze-drying of bacteria. One method is tosuspend organisms in 1 ml of inositol horse serumin Wheaton serum bottles or appropriate con-tainer depending on freeze-drying equipment.Snap freeze in liquid nitrogen and followinstructions for freeze-drying equipment (seeChapter 7).

An analysis of different cryoprotectantstested for Flavobacterium columnare, F. psychro-philum and Tenacibaculum maritimum recom-mends the use of a medium containing two-thirds Brucella broth (Difco) and one-thirdhorse or fetal calf serum (Desolme andBernardet, 1996).

224 Chapter 5

240Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpWednesday, January 21, 2004 2:55:53 PM


6Techniques for the Molecular Identification of Bacteria

The polymerase chain reaction (Saiki et al.,1985; Mullis and Faloona, 1987) has wideapplications in both diagnostic and researchlaboratories. It is routinely being used in thediagnosis and identification of bacteria. Likewise,16S rDNA sequencing is being used more widelyand is a useful tool for the identification of bacte-ria that are difficult to identify by biochemicaltests. This chapter covers the basics of both thesetechniques.

6.1 Molecular Identification by PCRUsing Specific Primers

A number of specific primers for the detection ofaquatic organisms by PCR have been reported.However, most, if not all, of these primers willundergo a long period of validation before theyare routine in a diagnostic laboratory (Baderet al., 2003). It is suggested they be used intandem with biochemical identification methods.

The PCRs that have been reported for aquaticorganisms are summarized in Table 6.1. Each for-ward and reverse primer is listed together with therecommended annealing temperature, number ofcycles and expected product length in base pairs.The concentration of reagents is not given herebecause of the wide variety used for the individualPCRs. The reader is advised to either refer to thereference or optimize the particular PCR for theirlaboratory conditions and equipment.

DNA extraction techniques, basic PCR proto-cols and preparation of reagents are covered inthis chapter.

Aeromonas spp. PCR

The primers used for Aeromonas hydrophila(Nielsen et al., 2001) also produce a bandat 685 bp with an Australian strain of atypicalA. salmonicida (AHLDA 1334). Aeromonashydrophila strains that are positive usingthese primers are negative for the phenotypictests of LDC and cellobiose fermentation.This PCR may assist in determining that isolatesare A. hydrophila, as phenotypic tests are notalways reliable (Nielsen et al., 2001). However,phenotypic tests would need to be carried outto differentiate from atypical A. salmonicida.A. hydrophila, ATCC 7810, which is positivefor LDC, does not produce an amplified productby this PCR.

A PCR that detects the presence of theaerolysin gene, detected DNA from all haemolyticstrains of A. hydrophila that were cytotoxicto vero and CHO cells, and produced entero-toxin as detected by suckling-mouse assays.No aerolysin gene was detected in non-haemolytic strains of A. hydrophila, non-haemolytic strains of A. caviae, or strains ofA. sobria. Extraneous bands were found withsome strains of A. caviae and Plesiomonasshigelloides, but not at the correct molecularweight (Pollard et al., 1990). The aerolysingene is considered to be a useful virulencemarker for detecting virulent pathogenicAeromonas species (Kong et al., 2002). Perform-ing PCRs using the primers of Nielsen et al.(2001), and the primers of Pollard et al. (1990),may be useful in identifying virulent strains ofA. hydrophila.




226 Chapter 6

Aero

mon

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viae

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8C

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CTG

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GTT

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AGD

iges

twith

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uces

fragm

ents

180

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ers

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ific

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3038

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.,19

96

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iels

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al.,

2001

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mon

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5560

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uces

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mer

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sat

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ofge

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GG

7e5-

R78

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5535

242

Oak

eyet

al.,

1999

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mon

assa

lmon

icid

ass

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icid

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AGC

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GC

TCAC

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ASS-

RAA

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CC

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AGTG

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G60

3051

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iyat

aet

al.,

1996

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mon

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sein

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edPC

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nive

rsal

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imer

sAS

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CG

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GAT

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AS2

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CG

C55

3027

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.,19

97Ta

ylor

and

Win

ton,

2002

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mon

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Det

ects

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neSO

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CG

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GAC

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GG

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GG

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CG

Fille

reta

l.,20

00

Aero

mon

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CG

CC

CAG

GC

CG

GTG

CTG

AL2

ACC

ACTG

TGTG

GAC

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GG

TA66

3062

2Kh

anet

al.,

1999

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ards

iella

spp.

Ampl

ifies

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from

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dsie

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497R

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TAR

GC

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3545

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aan

dW

akab

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hi,

1999

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obac

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s ed

o n1 6

S rD

NA

Col

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GAA

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GC

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CTT

GC

GTA

GTG

6030

?Tr

iyan

toet

al.,

1999

Flav

obac

teriu

mco

lum

nare

with

in16

SrD

NA

FvpF

GC

CC

AGAG

AAAT

TTG

GAT

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5925

1192

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03

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ever

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Ref

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ific

prim

ers

avai

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rP

CR

dete

ctio

nof

aqua

ticor

gani

sms.



Techniques for the Molecular Identification of Bacteria 227

Flav

obac

teriu

mco

lum

nare

Gen

omov

ar1

Sequ

ence

with

in16

SrD

NA

Col

-Ta

TTC

AGAT

GG

CTT

CAT

TTG

Col

-Tb

CC

GTT

TAC

GG

GC

GTT

GG

AATA

CAG

5430

?Tr

iyan

toet

al.,

1999

Flav

obac

teriu

mco

lum

nare

Gen

omov

ar2

Sequ

ence

with

in16

SrD

NA

Col

-T1

ATTA

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GC

ATC

ATTT

AC

ol-T

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GTT

TAC

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CG

TGG

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A52

3062

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al.,

1999

Flav

obac

teriu

mco

lum

nare

Gen

omov

ar3

Sequ

ence

with

in16

SrD

NA

Col

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GAT

GTG

GC

CTC

ACAT

TGTG

Col

-Tb

CC

GTT

TAC

GG

GC

GTT

GG

AATA

CAG

5630

?Tr

iyan

toet

al.,

1999

Flav

obac

teriu

mps

ychr

ophi

lum

16S

rRN

APS

Y1G

TTG

GC

ATC

AAC

ACAC

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Y2C

GAT

CC

TAC

TTG

CG

TAG

5730

1089

Wik

lund

etal

.,20

00

Flav

obac

teriu

mps

ychr

ophi

lum

16S

rRN

AFP

1G

TTAG

TTG

GC

ATC

AAC

ACFP

2TC

GAT

CC

TAC

TTG

CG

TAG

5435

1088

Urd

acie

tal.,

1998

Flav

obac

teriu

mps

ychr

ophi

lum

16S

rRN

APs

y1C

GAT

CC

TAC

TTG

CG

TAG

Psy2

GTT

GG

CAT

CAA

CAC

ACT

45 5030 39

1100

Toya

ma

etal

.,19

94

Lact

ococ

cus

garv

ieae

Base

don

the

dihy

drop

tero

ate

synt

hase

gene

SA1B

10-1

-FC

ATTT

TAC

GAT

GG

CG

CAG

SA1B

10-1

-RC

GTC

GTG

TTG

CTG

CAA

CA

5830

709

Aoki

etal

.,20

00

Lact

ococ

cus

garv

ieae

B as e

do n

1 6S

rDN

ApL

G-1

CAT

AAC

AATG

AGAA

TCG

CpL

G-2

GC

ACC

CTC

GC

GG

GTT

G55

3511

00Zl

otki

net

al.,

1998

a

Lact

ococ

cus

garv

ieae

IRL

TTTG

AGAG

TTTG

ATC

CTG

GLg

RAA

GTA

ATTT

TCC

ACTC

TAC

TT45

3548

2Pu

etal

.,20

02

Lact

ococ

cus

pisc

ium

Doe

sno

tdiff

eren

tiate

from

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anta

rum

,L.

raffi

nola

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TTTG

ATC

CTG

GPi

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RC

GTC

ACTG

AGG

GC

TGG

AT45

3586

3Pu

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.,20

02

List

onel

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ysin

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VAH

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CAT

CG

CTC

AAG

AVA

H1-

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GAT

ATTG

ACC

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GAG

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5530

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Hiro

noet

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1996

List

onel

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CC

CG

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AVA

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CAG

TTC

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A62

2030

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.,20

01

Myc

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GC

GAA

CG

GG

TGAG

TAAC

ACG

T13

TGC

ACAC

AGG

CC

ACAA

GG

GA

5030

924

Tala

atet

al.,

1997

Myc

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teriu

mch

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give

fragm

ents

at81

2an

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trict

with

BanI

togi

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tsat

562

and

362

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laat

etal

.,19

97

cont

inue

d



228 Chapter 6

Myc

obac

teriu

mfo

rtuitu

mR

estri

ctw

ithAp

aIto

give

fragm

ents

at67

7,13

2,11

5bp

Res

trict

with

BanI

togi

vefra

gmen

tsat

562

and

362

bpTa

laat

etal

.,19

97

Myc

obac

teriu

mm

arin

umR

estri

ctw

ithAp

aIto

give

fragm

ents

at67

7,13

2,11

5bp

No

rest

rictio

nw

ithBa

nITa

laat

etal

.,19

97

Myc

obac

teriu

mtri

plex

-like

Nes

ted

PCR

For1

CG

AAAG

CG

TGG

GG

AGC

GAA

CA

For2

GG

TGTG

GG

TTTC

CTT

CC

TT

Rev

1AG

ACC

CC

GAT

CC

GAA

CTG

AGAC

CR

ev2

ACG

GG

CC

ATTG

TAG

CAT

55 55

38 5540

9

Her

bst e

tal.,

2001

Noc

ardi

age

nus

NG

1AC

CG

ACC

ACAA

GG

GG

NG

2G

GTT

GTA

ACC

TCTT

CG

A55

3059

6La

uren

teta

l.,19

99

Phot

obac

teriu

mda

mse

lae

ssp.

dam

sela

ean

dpi

scic

ida

Util

izes

pres

ence

orab

senc

eof

urea

sege

ne

Ure

-5TC

CG

GAA

TAG

GTA

AAG

CG

GG

Car

1G

CTT

GAA

GAG

ATTC

GAG

T

Ure

-3C

TTG

AATA

TCC

ATC

TCAT

CTG

CC

ar2

CAC

CTC

GC

GG

TCTT

GC

TG

60 60

30 30

448

267

Oso

rioet

al.,

2000

Pseu

dom

onas

angu

illise

ptic

aPA

FG

ACC

TCG

CG

CC

ATTA

PAR

CTC

AGC

AGTT

TTG

AAAG

4635

439

Blan

coet

al.,

2002

Pseu

dom

onas

plec

oglo

ssic

ida

Base

don

gyrB

codi

ngre

gion

Use

dTa

qMan

real

time

PCR

GBP

A-F

CC

TGC

TGAA

GG

ACG

AGC

GTT

CG

GBP

A-R

AAC

CAG

GTG

AGTA

CC

ACC

GTC

G68

50Su

kend

aan

dW

akab

ayas

hi,

2000

Ren

ibac

teriu

msa

lmon

inar

umD

etec

tsp5

7ge

neC

AAG

GTG

AAG

GG

AATT

CTT

CC

ACT

GAC

GG

CAA

TGTC

CG

TTC

CC

GG

TTT

Brow

net

al.,

1994

Ren

ibac

teriu

msa

lmon

inar

umD

etec

tsp5

7ge

neIn

tern

alpr

obe

FL7

CG

CAG

GAG

GAC

CAG

TTG

CAG

FL10

GG

TGTA

ACG

ATAA

TGC

GC

CA

RL1

1G

GAG

ACTT

GC

GAT

GC

GC

CG

A

RL1

1

60 60

35 35

349

149

Miri

amet

al.,

1997

Ren

ibac

teriu

msa

lmon

inar

umF G

ATC

GTG

AAAT

ACAT

CAA

GG

R GG

ATC

GTG

TTTT

ATC

CAC

CC

6030

149

Leon

etal

.,19

94

Salin

ivib

rio(V

ibrio

)cos

ticol

aIG

SGlu

(cos

B)-s

peci

ficpr

imer

VCO

S-F

CTG

ACG

CTA

TTC

TTG

CG

AVC

OS-

RG

TAAT

CAC

ATTC

GTA

AATG

C55

3518

6Le

eet

al.,

2002

Stre

ptoc

occu

sag

alac

tiae

(b-h

aem

olyt

ic,g

roup

B).

Sem

i-nes

ted

PCR

.Cro

ss-re

actio

nw

ithS.

porc

inus

DSF

1TG

CTA

GG

TGTT

AGG

CC

CTT

T

DSF

2G

GC

CTA

GAG

ATAG

GC

TTTC

T

DSR

1C

TTG

CG

ACTC

GTT

GTA

CC

AA

DSR

1

67 6730 30

450

265

Ahm

etet

al.,

1999

Org

anis

mFo

rwar

dpr

imer

5′–3

′R

ever

sepr

imer

5′–3

′AT

CBp

Ref

eren

ce

Tab

le6.

1.C

ontin

ued.




Stre

ptoc

occu

sin

iae

16S

rRN

ASi

n-1

(CTA

GAG

TAC

ACAT

GTA

CT(

AGC

T)AA

G)

Sin-

2G

GAT

TTTC

CAC

TCC

CAT

TAC

5535

300

Zlot

kin

etal

.,19

98b

Tena

ciba

culu

m(F

lexi

bact

er)m

ariti

mum

16S

rRN

AM

ar1

TGTA

GC

TTG

CTA

CAG

ATG

AM

ar2

AAAT

ACC

TAC

TCG

TAG

GTA

CG

5839

400

Bade

rand

Shot

ts,1

998

Cep

eda

etal

.,20

03Te

naci

bacu

lum

(Fle

xiba

cter

)mar

itim

um16

SrD

NA

MAR

1AA

TGG

CAT

CG

TTTT

AAA

MAR

2C

GC

TCTC

TGTT

GC

CAG

A45

30To

yam

aet

al.,

1996

Vibr

ioch

oler

ae01

ctxA

gene

CTX

2C

GG

GC

AGAT

TCTA

GAC

CTC

CTG

CTX

3C

GAT

GAT

CTT

GG

AGC

ATTC

CC

AC60

2556

4Fi

elds

etal

.,19

92

Vibr

ioch

oler

ae01

ElTo

rPo

sitiv

efo

r rtx

A,rtx

Can

dct

xBge

nes

Rtx

A-F

CTG

AATA

TGAG

TGG

GTG

ACTT

ACG

Rtx

A-R

GTG

TATT

GTT

CG

ATAT

CC

GC

TAC

G55

3041

7C

how

etal

.,20

01

Vibr

ioch

oler

aeno

n-01

Posi

tive

for r

txA

and

rtxC

gene

sR

txC

-FC

GAC

GAA

GAT

CAT

TGAC

GAC

rtxC

-RC

ATC

GTC

GTT

ATG

TGG

TTG

C55

3026

3C

how

etal

.,20

01

Vibr

ioch

oler

ae01

clas

sica

lPo

sitiv

efo

r ctx

Bge

neon

ly(n

egat

ive

for r

txA

and

rtxC

gene

s)

ctxB

2G

ATAC

ACAT

AATA

GAA

TTAA

GG

ATct

xB3

GG

TTG

CTT

CTC

ATC

ATC

GAA

CC

AC55

3046

0C

how

etal

.,20

01

Vibr

iodi

azot

roph

icus

IGSG

lu(d

iaA)

-spe

cific

prim

erVD

IA-F

AGAT

TCTC

TTG

ATG

AGTG

CC

VDIA

-RTA

CC

TAC

ATC

TCTA

AGAG

ACAT

AG55

3530

0Le

eet

al.,

2002

Vibr

iofis

cher

iLu

xAge

neLu

xA-F

GTT

CTT

AGTT

GG

ATTA

TTG

GLu

xA-R

TCAG

TTC

CAT

TAG

CTT

CAA

ATC

C40

4042

8Le

ean

dR

uby,

1995

Vibr

ioflu

vial

isIG

SGlu(fl

uA)-s

peci

ficpr

imer

VFLU

-FAT

AAAG

TGAA

GAG

ATTC

GTA

CC

VFLU

-RG

TATT

CC

TGAA

TGG

AATA

CAC

5535

278

Lee

etal

.,20

02

Vibr

ioho

llisae

gyrB

gene

HG

-F1

GC

TCTG

TCG

GAA

AAAC

TTG

AH

G-R

2AT

GC

TCAA

AATG

GAA

CAC

AG55

3036

3Vu

ddha

kule

tal.,

2000

Vibr

ioho

llisae

toxR

gene

HT-

F3C

TGC

CC

AGAC

ACTC

CC

TCTT

CH

T-R

2C

TCTT

TCC

TTAC

CAT

AGAA

ACC

G62

2430

6Vu

ddha

kule

tal.,

2000

Vibr

ioni

grip

ulch

ritud

oIG

SGlu

(nig

A)-s

peci

ficpr

imer

VNIG

-FC

ATTT

CTT

TGAA

ACAG

AAAG

TVN

IG-R

TAG

ATAA

GG

GG

ATTG

TTG

CTA

5535

114

Lee

etal

.,20

02

Vibr

iopa

raha

emol

ytic

usgy

rBge

ne.T

his

gene

may

also

bepr

esen

tin

V.al

gino

lytic

us

VP1

CG

GC

GTG

GG

TGTT

TCG

GTA

GT

VP2r

TCC

GC

TTC

GC

GC

TCAT

CAA

TA60

3028

5Ve

nkat

esw

aran

etal

.,19

98Ki

met

al.,

1999

Vibr

iopa

raha

emol

ytic

ustl

gene

.Thi

sge

nem

ayal

sobe

pres

enti

nV.

algi

noly

ticus

L-tl

AAAG

CG

GAT

TATG

CAG

AAG

CAC

TGR

-tlG

CTA

CTT

TCTA

GC

ATTT

TCTC

TGC

58.6

3045

0Be

jeta

l.,19

99

cont

inue

d



230 Chapter 6

Vibr

iopa

raha

emol

ytic

usto

xRge

neM

ayge

twea

kno

n-sp

ecifi

cba

nds

with

V.al

gino

lytic

us,

V.vu

lnifi

cus

ToxR

1G

TCTT

CTG

ACG

CAA

TCG

TTG

ToxR

2AT

ACG

AGTG

GTT

GC

TGTC

ATG

6320

368

Kim

etal

.,19

99

Vibr

iopa

raha

emol

ytic

usC

lone

dfra

gmen

tpR

72H

spec

ific

fort

his

spec

ies

VP33

TGC

GAA

TTC

GAT

AGG

GTG

TTAA

CC

VP32

CG

AATC

CTT

GAA

CAT

ACG

CAG

C60

3538

7or

320

Lee

etal

.,19

95R

ober

t-Pillo

teta

l.,20

02Vi

brio

pena

eici

da16

SrD

NA

VpF

GTG

TGAA

GTT

AATA

GC

TTC

ATAT

CVR C

GC

ATC

TGAG

TGTC

AGTA

TCT

6235

310

Saul

nier

etal

.,20

00

Vibr

iopr

oteo

lytic

usIG

SIA(p

roC

)-spe

cific

prim

erVP

RO

-CG

CAT

TCTT

ACG

AGTG

TGVP

RO

-RAT

TAG

TTG

TATT

CAA

ATA

5535

133

Lee

etal

.,20

02

Vibr

iosa

lmon

icid

aIG

S0(s

alA)

-spe

cific

prim

erVS

AM-F

TGC

GAT

TTAT

GAG

TGTT

CA

VSAM

-RAC

TCTT

CAT

TGAG

AGTT

CTG

5535

275

Lee

etal

.,20

02

Vibr

iosp

lend

idus

IGS0

(spn

A;sp

nD)-s

peci

ficpr

imer

VSPN

-FG

ATTT

AGTT

AAAG

CC

AGAG

CVS

PN-R

CC

TGAT

AAC

TGTT

TGC

CG

5535

240,

294

Lee

etal

.,20

02

Vibr

iotra

chur

i(ju

nior

syno

nym

ofV.

harv

eyi)

Pstl-

1aTG

CG

CTG

ACG

TGTC

TGAA

TTPs

tI-1b

AAG

CAG

CG

ATG

ACAA

GC

AGT

6035

417

Iwam

oto

etal

.,19

95b

Vibr

iotu

bias

hii

IGSIA

(tubA

)-spe

cific

prim

erVT

UB-

FTG

GG

TCTT

TCAG

GC

CC

GVT

UB-

RC

GAC

GAA

TGAC

CG

TTG

TC55

3539

4Le

eet

al.,

2002

Vibr

iovu

lnifi

cus

Nes

ted

PCR

P1 GAC

TATC

GC

ATC

AAC

AAC

CG

P3 GC

TATT

TCAC

CG

CC

GC

TCAC

P2 AGG

TAG

CG

AGTA

TTAC

TGC

CP4 C

CG

CAG

AGC

CG

TAAA

CC

GAA

57 59

50 50

704

222

Lee

etal

.,19

98

Vibr

iovu

lnifi

cus

Cyt

olys

in-h

aem

olys

inge

neF C

GC

CG

CTC

ACTG

GG

GC

AGTG

GC

TGR G

CG

GG

TGG

TTC

GG

TTAA

CG

GC

TGG

64.5

30–5

0C

olem

anet

al.,

1996

Yers

inia

ruck

eri

YER

8G

CG

AGG

AGG

AAG

GG

TTAA

GTG

YER

10G

AAG

GC

ACC

AAG

GC

ATC

TCTG

6025

575

Gib

ello

etal

.,19

99

Yers

inia

ruck

eri

16S

rDN

AR

uck1

CAG

CG

GAA

AGTA

GC

TTG

Ruc

k2TG

TTC

AGTG

CTA

TTAA

CAC

TTAA

5530

409

LeJe

une

and

Rur

angi

rwa,

2000

Yers

inia

ruck

eri

yruR

/yru

Iquo

rum

sens

ing

gene

IF2

GAG

CG

CTA

CG

ACAG

TCC

CAG

ATAT

IR2

CAT

ACC

TTTA

ACG

CTC

AGTT

CG

AC65

4010

00Te

mpr

ano

etal

.,20

01

Org

anis

mFo

rwar

dpr

imer

5′–3

′R

ever

sepr

imer

5′–3

′AT

CBp

Ref

eren

ce

Tab

le6.

1.C

ontin

ued.




Mul

tiple

xPC

RVi

rule

ntAe

rom

onas

spec

ies

Vibr

ioch

oler

ae

Vibr

iopa

raha

emol

ytic

us

Aero

-FTG

TCG

GSG

ATG

ACAT

GG

AYG

TGEs

p-F

GAA

TTAT

TGG

CTC

CTG

TGC

AGG

V par

a-F

GC

TGAC

AAAA

CAA

CAA

TTTA

TTG

TT

Aero

-RC

CAG

TTC

CAG

TCC

CAC

CAC

TTC

AEs

p-R

ATC

GC

TTG

GC

GC

ATC

ACTG

CC

CV p

ara-

RG

GAG

TTTC

GAG

TTG

ATG

AAC

6235

720

Kong

etal

.,20

02

Form

ultip

lex

reac

tion,

prim

ers

wer

eus

edat

0.2

mMfo

rAer

o,0.

1mM

forE

sp,a

nd1.

0mM

f or V

para

The

Aero

prim

erpa

irsde

tect

viru

lent

spec

ies

ofA.

hydr

ophi

la,A

.euc

reno

phila

,A.p

opof

fii,A

.sob

riaan

dA.

trota

Kong

etal

.,20

02

E.co

li(u

idA

gene

)

Vibr

ioch

oler

ae(c

txge

ne)

Vibr

opa

raha

emol

ytic

us(tl

gene

)(m

ayam

plify

V.al

gino

lytic

us)

Vibr

iovu

lnifi

cus

(cth

gene

)

Salm

onel

laty

phim

uriu

m(in

vA)

L-U

IDA

TGG

TAAT

TAC

CG

ACG

AAAA

CG

GC

L-C

TXC

TCAG

ACG

GG

ATTT

GTT

AGG

CAC

G

L-TL

AAAG

CG

GAT

TATG

CAG

AAG

CAC

TGL-

CTH

TTC

CAA

CTT

CAA

ACC

GAA

CTA

TGAC

L-IN

VAC

TCTA

CTT

AAC

AGTG

CTC

GTT

TAC

R-U

IDA

ACG

CG

TGG

TTAC

AGTC

TTG

CG

R-C

TXTC

TATC

TCTG

TAG

CC

GG

TATT

ACG

R-T

LG

CTA

CTT

TCTA

GC

ATTT

TCTC

TGC

R-C

THG

CTA

CTT

TCTA

GC

ATTT

TCTC

TGC

R-IN

VATT

GAT

AAAC

TTC

ATC

GC

ACC

GTC

A

55–6

030

147

302

450

205

273

Bras

her e

tal.,

1998

Univ

ersa

lpro

toco

lVi

brio

chol

erae

(toxi

nge

ne)

VC-1

GG

CAG

ATTC

TAG

ACC

TCC

TVC

-2TC

GAT

GAT

CTT

GG

AGC

ATTC

563

Wan

get

al.,

1997

(quo

tes

Fiel

dset

al.,

1992

)Vi

brio

vuln

ificu

s(c

ytol

ysin

gene

)VV

-1C

TCAC

TGG

GG

CAG

TGG

CT

VV-2

CC

AGC

CG

TTAA

CC

GAA

CC

A38

3W

ang

etal

.,19

97(q

uote

sBr

auns

etal

.,19

91)

Vibr

iopa

raha

emol

ytic

us(g

enom

icD

NA)

VIP-

1G

AATT

CG

ATAG

GG

TGTT

AAC

CVI

P-2

ATC

CTT

GAA

CAT

ACG

CAG

C38

1W

ang

etal

.,19

97(q

uote

sLe

eet

al.,

1995

)

AT=

anne

alin

gte

mpe

ratu

re;C

=nu

mbe

rofc

ycle

s;Bp

=pr

oduc

tsiz

ein

base

pairs

;IG

S=

inte

rgen

icsp

acer

s.



Edwardsiella spp. PCR

The sodB gene (coding for iron-cofactored super-oxide dismutase) from Edwardsiella spp. wasamplified (454 bp for all species) and sequenced.Differences in nucleotide sequence dividedthe species of Edwardsiella into a pathogenicand non-pathogenic cluster. Cluster I comprisespathogenic strains of E. tarda from Japaneseeel, Japanese flounder (Paralichthys olivaceus),Nile tilapia (Oreochromis niloticus) and ayu(Plecoglossus altivelis), atypical E. tarda from redsea bream, Edwardsiella species from Japaneseeel, and E. ictaluri. Non-pathogenic E. tarda andE. hoshinae were found in cluster II (Yamada andWakabayashi, 1999).

Flavobacterium columnare PCR

Primers FvpF and FvpR2, located within the 16SrDNA are said to be specific for F. columnare(Bader et al., 2003). Three primer sets, alsolocated within the 16S rDNA gene, differentiatethe three genomovars of F. columnare. Threeseparate forward primers are available, withgenomovars 1 and 3 having the same reverseprimer (see Table 6.1). Primers for amplifyinggenomovar 2 produce a product band at 621 bp,but also produce bands at 800 and 1000 bp in allFlavobacterium species (Triyanto et al., 1999).The sensitivity of the PCRs can be increased fromtissue samples by using universal primers (Baderet al., 2003) or universal primers followed by theFlavobacterium species-specific set of Col-72Fand Col-1260F (Table 6.1) (Triyanto et al.,1999).

Pseudomonas plecoglossicida PCR

This PCR was conducted using theTaqMan methodology for quantitative real-time PCR. The internal control and targetDNA probes were TGT-P 5′-(FAM)AGATGGCGTGGGCGTTGAAGTAGCGC(TAMRA)-3′, and ISD-P 5′-(VIC)CCTTCACCACCACGGCCGAGCGTGAG(TAMRA)-3′ (Sukenda and Wakabayashi, 2000).

Tenacibaculum (Flexibacter) maritimum PCR

A nested PCR using universal primers of 20F and1500R in the first PCR reaction, followed by anested PCR using T. maritimum-specific primersMar1 and Mar2 in the nested reaction allows

detection of this organism direct from fish tissue(Cepeda et al., 2003). Ready-to-Go PCR beads(Amersham Pharmacia Biotech) were used, with1 pmol of each primer in a 25 ml reaction. Cyclingconditions for the first PCR reaction used apreheating step of 95°C for 5 min, 30 cyclesof 95°C for 30 s, 57°C for 30 s, 72°C for 60 swith a final cycle that used an extension step at72°C for 5 min. The second PCR reaction used apreheating step of 94°C for 2 min, followed by 40cycles of 94°C for 2 s, 54°C for 2 s, 72°C for 10 swith a final cycle that used an extension step of4 min.

PCR for Vibrio spp.

Many of these PCRs will need to undergo aperiod of validation, and as more information isgathered and research done, the specificity ofthese PCRs will also be assessed. Such a case hasbeen found with PCRs for some of the Vibrio spe-cies. There is some doubt as to the specificity ofsome of the primers for Vibrio parahaemolyticusand V. alginolyticus. The gyrB gene has beensuggested to differentiate V. parahaemolyticusfrom V. alginolyticus (Venkateswaran et al.,1998); however, the primers also detectedthe gene in V. alginolyticus when the suggestedannealing temperature of 58°C was used. Speci-ficity for V. parahaemolyticus was improved at anannealing temperature of 60°C (Kim et al., 1999).The tl gene that encodes for a thermolabilehaemolysin and used in a PCR for detectingV. parahaemolyticus (Bej et al., 1999) was foundto occur not only in V. parahaemolyticus butwas also detected in species of V. alginolyticus(Robert-Pillot et al., 2002). The toxR gene, whichis involved in the regulation of many genes, isconserved amongst the Vibrio species; however,there is a low degree of homology that allows forselection of species-specific primers. A PCR thattargets the toxR gene in V. parahaemolyticus mayamplify non-specific amplicons from V. algino-lyticus and V. vulnificus (Kim et al., 1999). Stud-ies suggested that to date the PCR that appears tobe the most specific for V. parahaemolyticus is aPCR that detects a fragment termed R72H. Thissection of DNA is composed of a non-codingregion and a phosphatidylserine synthetase geneand the primers amplify an amplicon of either387 or 320 bp, both of which are consideredspecific for V. parahaemolyticus (Lee et al., 1995;Robert-Pillot et al., 2002).

232 Chapter 6



Multiplex PCR

A multiplex PCR enabled simultaneous detectionof the human pathogens E. coli, Salmonellatyphimurium, V. cholerae, V. parahaemolyticusand V. vulnificus from shellfish samples. Anoptimized PCR was used with 2.5 mM MgCl2,and an annealing temperature of 55°C (Brasheret al., 1998).

Nested PCR

Any of the above primers in Table 6.1 that aredesigned within the 16S rRNA can be used ina nested PCR reaction to improve detectionsensitivity. The first PCR reaction is done witheubacterial primers, which amplify the entire 16SrDNA, and the specific primers are then usedfor a nested PCR reaction. The detection ofFlavobacterium columnare was improved intissue samples when specific primers were used ina nested PCR with eubacterial universal primers(Triyanto et al., 1999; Bader et al., 2003). Fordetails on universal primers see section 6.3.

An optimized nested PCR was reportedfor Aeromonas salmonicida, Flavobacteriumpsychrophilum and Yersinia ruckeri. The firstround PCR used universal primers for the 16SrDNA gene and the second round of primers werespecies-specific for the three organisms tested.The PCR conditions were optimized so that thesame conditions could be used for all primers(Taylor and Winton, 2002). The universal primersof Weisburg et al. (1991) with forwardprimer fD2 5′-AGAGTTTGATCATGGCTCAG-3′ and reverse primer rP2 5′-GTTTACCTTGTTACGACTT-3′ were used togenerate a 1500 bp fragment of the 16S rDNAgene. This template was then used in secondround PCR with primers for Aeromonassalmonicida AS1 and AS2 (Høie et al., 1997),Flavobacterium psychrophilum PSY1 and PSY2(Toyama et al., 1994), and primers YER8 andYER10 for Yersinia ruckeri (Gibello et al., 1999).In the PCR reaction mix, the final concentrationof MgCl2 was 2.0 mM, dNTP was 200 mM, with100 pmol final concentration for each primer and1.25 U/50 ml of Taq. Thermocycling conditionswere also standardized with an initial denaturationstep of 95°C for 4 min, followed by 30 cycles of95°C for 45 s, annealing for 45°C for universalprimers and 55°C for specific primers, with anextension of 72°C for 90 s. A final cycle with an

extension step of 72°C for 4 min was used. A finalcycle of 4°C infinity was used as a holding cycle(Taylor and Winton, 2002).

Outline of steps for PCR using specific primers

Step Method1 Extract DNA from bacterial colonies2 Amplify DNA using specific primers3 Visualize DNA on agarose gel

DNA extraction from bacterial cells

There are many methods for the extraction ofDNA from bacterial cells and these include bothmanual methods and commercially availablekits. Such kits include Instagene (Bio-Rad),AquaPure genomic DNA kits (Bio-Rad),Chelex-based resin (Bio-Rad), Puregene (GentraSystems), PrepMan (Applied Biosystems),MasterPure (Astral Scientific – Epicentre),Wizard Genomic (Promega), and the DneasyTissue system and QiAamp system fromQiagen.

MANUAL METHOD 1. Suspend bacterial cells in100 ml of a solution of 1 mM EDTA-0.5% Triton-X-100. Boil in a microwave on HIGH setting for5 min (Lee et al., 1998).

MANUAL METHOD 2. Make a suspension ofbacterial cells to 0.5 McFarland density. Centri-fuge at 13,000 rpm for 5 min. Suspend pellet ofcells in a digestion buffer of 50 mM Tris-HCl(pH 8.5), 1 mM EDTA, 0.5% SDS and 200 mg/mlof proteinase K. Incubate for 3 h at 55°C withagitation. Heat inactivate the proteinase K for10 min at 95°C. Cool to 4°C, then centrifuge at13,000 rpm for 10 min. Use supernatant in thePCR reaction. However, the PCR master mix mustcontain a final concentration of Tween 20 toneutralize the effect of SDS, which is inhibitoryto Taq polymerase at concentrations as low as0.01% (Goldenberger et al., 1995).

This method is also suitable for tissue sampleswith an overnight incubation in digestion bufferfollowed by sonication (Goldenberger et al.,1995).

MANUAL METHOD 2 FOR GRAM-POSITIVE BACTERIA.

For Gram-positive bacteria treat cells withlysozyme (1 mg lysozyme/ml TE, pH 8.0).




Extraction of DNA from tissue

As for extraction of DNA from cultured bacterialcells, bacterial DNA can also be extracted fromtissue samples. There are many methods avail-able for both manual and commercially availablemethods. The following are some suggestions.

MANUAL METHOD 1. To 100 ml of homogenizedtissue add 100 ml of Chelex-100 resin (Bio-RadLaboratories or Sigma-Aldrich). Heat at 56°C for10 min. Add 200 ml of 0.1% Triton-X-100 andboil for 10 min. Cool on ice, then centrifuge at12,000 rpm for 3 min. Use 5 ml in PCR reaction(Khan and Cerniglia, 1997).

MANUAL METHOD 2. Culture 10 g of tissue into90 ml of TSBYE medium (30 g tryptic soy brothpowder with dextrose, (Difco); 6 g yeast extract;1 l of water). Incubate at 25°C overnight withshaking. Take 0.5 ml of the upper phase of thesample and mix with 1 ml of sterile phosphate buf-fered saline (PBS, 0.05 mol/l, pH 7.4). Centrifugeat 9000 g for 3 min. Wash the pellet three times inPBS and once with sterile water. Resuspend pelletin 50 ml of water, then dilute 1:10 with 1%Triton-X-100 and place in boiling waterbath for5 min. Place immediately on ice. Use 2 ml in PCRmixture (Wang et al., 1997).

6.2 PCR Protocols

Any PCR that is introduced into the laboratoryneeds to be optimized for the Taq enzyme that isused by that laboratory and the primers that areprepared. Differences may occur between differ-ent batches of primers and this may be seen withthe concentration of the dNTPs. For example,a pair of primers that may be optimal at a final

concentration of 100 mM dNTP mix in the PCRreaction; when re-ordered a concentration of200 mM may be optimal.

Method 1 – standard protocol

Table 6.2 suggests a standard PCR protocolthat may be used to amplify from most targetsequences. It is also a basic starting point fromwhich to do optimizations. When optimizing theconcentrations in the PCR mix, the suggestedranges for testing are: dNTP concentration(100–200 mM), primer concentration (0.1–1 mM),magnesium chloride concentration (1–4 mM)and Taq enzyme (1–2.5 Units). Too much ortoo little of any of these reagents will cause non-specific background, mispriming or insufficientproduct generated. In particular, high concentra-tions of dNTP, primer or MgCl2 will lead tonon-specific product (Saiki, 1989; Innis andGelfand, 1990). A negative and positive controlshould be included with each PCR run.

Sometimes bovine serum albumin, gelatin,Tween 20 or DMSO can be added to the PCRreaction mix to help stabilize the Taq enzyme.DMSO may assist in preventing the inhibition ofthe function of the Taq enzyme caused by inhibi-tors present in tissue or from contamination byagar and agar-containing media such as Stuartstransport medium. However, not more than 2%should be used, as greater than this concentrationis inhibitory to Taq.

It is suggested that the reagent volumesfor the PCR master mix be set up in an Excelspreadsheet with the appropriate formulae ineach cell. When a different number of samplesare tested then the master mix is easilycalculated.

Aliquot PCR master mix into 20 ml volumes.Add 30 ml of sterile paraffin oil to each tube if

234 Chapter 6

Reagent ×1 (ml) ×10 (ml) Final concentration

Water 11.8 = 25 minus the volume of reagentsPCR buffer (x10) 2.5 = 2.5 × 10 1dNTP mix (2 mM of each) 2.5 = 2.5 × 10 200 mM (0.2 mM)MgCl2 (25 mM) 2.2 = 2 × 10 2 mMPrimer forward (20 pmol = 5 mM) 0.5 = 0.5 × 10 0.1 mMPrimer reverse (20 pmol = 5 mM) 0.5 = 0.5 × 10 0.1 mMTaq enzyme (5 U/ml) 0.2 = 0.2 × 10 1 UnitTotal volume including volume of DNA to be added 25.2 = 25 × 10

Table 6.2. Standard PCR protocol.



thermocycler does not have a hot lid. Tubes canbe stored at −20°C until required.

Add 5 ml of template (DNA) and place tubeson thermocycler.

Method 2 – commercial PCR reaction mixture

Many companies offer master mix that containsbuffer, MgCl2, and dNTPs. The user addsthe DNA template, primers and water. Anexample is Ready-To-Go PCR beads fromAmersham Pharmacia Biotech. This is used as a25 ml reaction mix to which primers and 5 ml ofDNA are added. BIOMIX ready-to-go from AstralScientific (Bioline), PCR Master Mix (Promega),and IQ supermix (Bio-Rad laboratories) are otherexamples of commercially available PCR MasterMix.

Thermocycling conditions

The cycling conditions will depend on the typeof thermocycler, the annealing temperature ofthe primers and the type of Taq enzyme used,whether it is a hot-start enzyme or not.

A standard set of cycling conditions beginswith one cycle at 95°C for 1–5 min. Then, 25–35cycles of denaturation at 95°C for 30 s, annealingat 55–68°C 30 s, extension at 72°C for 30 s.A final cycle is suggested as for the last cyclebut with an extension time of 10 min. Thethermocycler can be set to do a hold cycle at 4°C.

The primer annealing temperature dependsupon the length of the primer, the GC content andconcentration. A recommended annealing tem-perature is 5°C below the true Tm of the primers.The Tm is usually provided on the data sheetsent with the primer. The Tm can be estimated bycalculating 2°C for A or T, and 4°C for G or C inthe primer sequence. The annealing temperatureaffects the specificity of the reaction. A lower thanoptimal annealing temperature may lead tomispriming of non-target sequence or the mis-extension of incorrect nucleotides at the 3′ end ofthe primers. A low extension temperature togetherwith high dNTPs also favours mispriming andnon-specificity (Saiki, 1989; Innis and Gelfand,1990).

The optimal number of cycles is between 25and 35. Increasing the number of cycles may leadto problems with the PCR, as a plateau effectis reached where product is no longer amplifiedand a result is that non-specific product may be

amplified preferentially. This plateau is reachedaccording to the number of target copies initiallypresent in the sample and the amount of DNAsynthesized. Also, reagent exhaustion occurs withan extended number of amplification cycles.The recommended number of cycles per startingmaterial is 25–30 (for 3 × 105 target molecules),30–35 (1.5 × 104 molecules), 35–40 (1 × 103

molecules), 40–45 (50 molecules) (Saiki, 1989;Innis and Gelfand, 1990).

Gel electrophoresis

A standard gel for the detection of amplifiedproduct is as follows. Prepare a 2% agarose gelusing chromosomal grade agarose (Bio-Rad).Load 5 ml of amplification product and 5 ml of2 × loading buffer into the wells. Run gel at5 v/cm for 2 h. On a Bio-Rad PowerPac 300,80 v for 90 min produces well-separated bands.Always include a molecular weight marker suchas a 100 bp marker.

The gel is stained for 1 h in 1 litre of distilledwater containing 50 ml of ethidium bromide.Ethidium bromide is a carcinogen and thereforeappropriate precautions need to be taken, such aswearing lab coats and gloves.

The gel is photographed over a UV trans-illuminator. Safety note: Do not expose the skinor eyes to UV light. Laboratory coats, gloves andface shields must be worn when operating UVtransilluminators that are not housed within aclosed instrument.

Reagent preparation of stock andworking solutions

dNTP concentration for PCR amplification

For dNTPs supplied as 100 mM. The finalconcentration in the PCR is required at either100 mM or 200 mM depending on results inoptimization tests.

Therefore, prepare each dNTP to 10 mM bydiluting 1:10. From this prepare a working mastermix of a dNTP solution by adding equal volumesof each dNTP plus an equal volume of water; thatis, a 1:5 dilution for each dNTP to give a finalconcentration of each dNTP of 2 mM. For a finalconcentration of 100 mM in the PCR master mix,use 1.25 ml. For 200 mM use 2.5 ml in a master mixof 25 ml total volume.




The nucleotides can also be purchased asa mixture containing all four dNTPs. An exampleis PCR nucleotide mix (Promega) with eachnucleotide at 10 mM concentration.

Preparation of primers

A data sheet is usually sent with the primer, whichdetails the concentration, melting temperatureetc of the primer. Prepare a stock solution of100 pmol in TE buffer (pH 7.0). Therefore, if thedata sheet states a primer is 3500 pmol, add350 ml of TE buffer to the dehydrated primer toobtain a stock solution of 100 pmol.

Prepare a working solution in distilledwater of the required concentration, usually20 pmol.

Primer quantification by spectrophotometry

An accurate quantification can be done byspectrophotometer. Dilute the stock solution ofprimer 1:20 (i.e. 50 ml + 950 ml of water).

The UV absorbance is read at 260 nm. Theconcentration of the primer or oligonucleotidein mg/ml = absorbance × dilution × weight perOD.

Single-stranded DNA (ssDNA) at a con-centration of 33 mg/ml has an absorbance of 1.Therefore 33 mg of ssDNA = 1 OD unit.

Therefore, in the above example, theconcentration of ssDNA mg/ml = absorbance ×20 × 33.

Double-stranded DNA (dsDNA): A260 =OD260 = 1 for a 50 mg/ml solution.

Oligomer – quantitation

For a 20-mer, a stock solution with A260 = 1,contains 5 nmol5 nmol = 33 mg/(20 × 325)For a 40-mer, a stock solution with A260 = 1contains 2.5 nmol2.5 nmol = 33 mg/(40 × 325)

Oligomer – conversion of pmol of primerto mg of primer

Multiply pmol by (length × 325)/1,000,000Example: 51809.88 pmol of a 20-mer (from datasheet)(51809.88 × 20 × 325)/1,000,000 = 336.7 mgprimer

Conversion of mg of primer to pmol of primer

Multiply by 1,000,000/(length × 325).Example: 365.73 mg of a 20-mer (from datasheet)(365.73 × 1,000,000)/(20 × 325) = 56,266.15pmol of primer

Primer concentration for PCR amplification

Micromolar concentrations of primer =pmol/mlThus 20 pmol of primer in 100 ml PCR mixture =20 micromolar (20 mM)

Equation for estimating volume required froma stock concentration

A basic formula for estimating the required vol-ume from a stock solution is as follows:(initial concentration) × (volume needed) =(final concentration) × (volume of sample)Therefore, if you have a stock solution of 5 mMof primer with a final concentration required of0.1 mM in a 25 ml reaction volume the equationis:5 mM × c = 0.1 mM × 25 mlc = (0.1 mM × 25)/5 mMc = 0.5 ml. Therefore add 0.5 ml of the 5 mMstock (or working solution) to a 25 ml reactionvolume.

Storage of oligonucleotides

Oligonucleotides should be stored at −20°C.Store in aliquots to prevent multiple freeze andthaw of oligonucleotides.

6.3 Molecular Identification by 16SrDNA Sequencing

Sequencing the 16S rRNA has been used exten-sively to study bacterial evolution and phylogeny.With a vast number of 16S rRNA sequencesavailable in the National Centre for Biotech-nology Information (NCBI) and the RibosomalDatabase Project (RDP) databases, sequencingthe 16S rDNA is an essential tool in bacterialsystematics and the identification of new species.

It is also a useful tool when used with bio-chemical tests for bacterial identification in thediagnostic laboratory. Ribosomal RNA containsvariable and highly conserved regions that evolve

236 Chapter 6



very slowly and, therefore, can be specific to agenus.

The 16S rRNA is found in the ribosomes,which consist of proteins and RNA. In prokaryotesthe ribosomes measure 70S, and consist of twosubunits, 50S and 30S. S is the sedimentationrate, or Svedberg unit, during centrifugation.The 16S rRNA is found in the 30 S subunit,whereas the 50S subunit contains the 5S and23S rRNA molecules. 16S is approximately 1600nucleotides, 23S, 3000 nucleotides and 5S isapproximately 120 nucleotides.

Within the gene encoding for the 16S rRNAare areas of sequence that are conserved acrossthe different genera, and it is these areas thathave been used for the design of the universaleubacterial primers. Not all primers will bind to theDNA of the bacterium of interest; however, if acombination of primers is used then a sectionof DNA will be amplified, which can then besequenced for identification.

Outline of steps for sequencing

Step Method1 Extract DNA from bacterial colonies2 Amplify 16S rDNA using universal

eubacterial primers3 Visualize DNA on agarose gel4 Clean up amplified product5 Quantify DNA concentration6 Perform sequencing PCR reaction7 Sequencing (send to a sequencing facility)8 Analysis of sequence information using

BIOEDIT

9 BLAST search for sequence identification

DNA extraction

DNA is extracted from bacterial colonies asdescribed previously.

Amplification of 16S rDNA using universaleubacterial primers

The rDNA contains a number of sites that areconserved across genera, and as such there are anumber of primers that can be used to amplify apart or all of the 16S rDNA for many genera. Theuniversal primers A, B and C amplify regions thatare universally conserved across prokaryotes andeukaryotes and which were initially proposed by

Lane et al. (1985). Since then, a number ofdifferent variations have been proposed. Someprimers are universal for bacteria only, someamplify the entire 16S rDNA, whereas othersamplify different regions. The convention foridentifying the primers is to name them accordingto the number of the E. coli position at which the3′ end of the primer anneals.

Some of the primers that are available toeither amplify all or part of the 16S rDNA aredetailed in Table 6.3. Not all primer combinationswill be appropriate for all bacteria. Table 6.4indicates primer combinations that have proveduseful. The primers may be ordered from acompany such as Qiagen or Invitrogen.

Primers A, B, and C are universal fororganisms from the three primary kingdoms,archaebacteria, eubacteria and eukaryotes (Laneet al., 1985). Primer EUBB (7–26) of Suzuki andGiovannoni (1996) is the same as primer 27f ofLane (1991), except for a degeneracy (M) thatpossibly makes the primer more universal.

Primer 27f was modified because comple-mentarity at the 3′ ends led to self-priming fol-lowed by primer depletion. The modified primerwas referred to as POmod (Wilson et al., 1990).Complementarity in primers and between primerpairs can lead to primer dimers, which is theproduct that is amplified in preference tothe desired sequence on the DNA template(Watson, 1989).

The suggested pairs of primers of 63f and1387r (Table 6.4) were found to improve uponthe primer pair of 27f and 1392r (Table 6.3)(Lane et al., 1991), particularly with difficult DNAtemplates (Marchesi et al., 1998).

The entire rDNA can be amplified with twosets of primer pairs. The primer pairs POmod andPC3mod amplify 789 base pairs of the rDNA andthe pairs P3mod and PC5 amplify the remainderof the rDNA of 721 base pairs (Wilson et al.,1990). Alternatively, the entire 16S rRNA can beamplified with 27f or EUBB as the forward primerand 1525 or EUBA as the reverse primer(Weisburg et al., 1991).

The universal primers 16/23S-F and16/23S-R were used to amplify the intergenicspacer (IGS) region between the 16S rRNA andthe 23S rRNA genes of the Vibrio species and thisregion was subsequently used to design specificprimers for the detection of eight Vibrio species. Astandard PCR reaction mix, and thermocyclingconditions with an annealing temperature of




238 Chapter 6

Primer(E. coli numbering) Primer sequence 5¢ to 3¢ Target Reference27f (EUBB) (7–26) AGAGTTTGATCMTGGCTCAG Most Eubacteria Lane, 1991; Weisburg et al.,

1991; Suzuki and Giovannoni,1996

20F AGAGTTTGATCATGGCTCAG Eubacterial Weisburg et al., 1991POmod (8–22)modified from 27f

AGAGTTTGATCMTGG Eubacterial kingdom only Wilson et al., 1990

63f (43–63) CAGGCCTAACACATGCAAGTC Marchesi et al., 1998357r (339–357) CTCCTACGGGAGGCAGCAG Most Eubacteria Lane, 1991; Weisburg et al., 1991530f (515–530) GTGCCAGCMGCCGCGG Most Eubacteria, Eukaryotes,

ArchaebacteriaLane, 1991

P3mod f (787–806) ATTAGATACCCTDTAGTCC Eubacterial kingdom only Wilson et al., 1990519r (519–536) GWATTACCGCGGCKGCTG Primer A. Universal for all kingdoms Lane et al., 1985; Lane, 1991PC3mod r (787–806) GGACTAHAGGGTATCTAAT Eubacterial kingdom only Wilson et al., 1990685r3 TCTRCGCATTYCACCGCTAC Most Gram-positives, cyanobacteria Lane, 1991907r (907–926) CCGTCAATTCMTTTRAGTTT Primer B. Universal for all kingdoms Lane et al., 1985; Lane, 1991926f AAACTYAAAKGAATTGACGG Most Eubacteria, Eukaryotes,


1100r (1100–1114) GGGTTGCGCTCGTTG Most Eubacteria Lane, 1991; Weisburg et al., 1991

1114f GCAACGAGCGCAACCC Most Eubacteria Lane, 19911387r (1387–1404) GGGCGGWGTGTACAAGGC Marchesi et al., 19981392r (1392–1406) ACGGGCGGTGTGTRC Primer C. Universal for all kingdoms Lane et al., 1985; Lane, 19911406f TGYACACACCTCCCGT Most Eubacteria, Eukaryotes,


PC5 r (1492–1507)Modified from 1492r

TACCTTGTTACGACTT Eubacterial kingdom only Wilson et al., 1990; Lane, 1991

1492r (1492–1512) TACGGYTACCTTGTTACGACTT Most Eubacteria, Archaebacteria Lane, 19911500R GGTTACCTTGTTACGACTT Eubacterial Weisburg et al., 19911525r (1525–1541) AAGGAGGTGWTCCARCC Universal for all kingdoms Lane, 1991; Weisburg et al., 19911525r EUB Amodified from 1525r

AAGGAGGTGATCCANCCRCA Eubacterial kingdom only Suzuki and Giovannoni, 1996

M = C:A; R = A:G; K = G:T; W = A:T (Lane et al., 1985). Other mixtures of nucleotides (known as degeneracies or wobbles) areS = C:G; Y = C:T; V = A:G:C; H = A:C:T; D = A:G:T; B = C:G:T; N = A:G:C:T. F = same sequence as rRNA. R = means thecomplement of rRNA sequence. The primer positions correspond to the nucleotide numbering system of E. coli 16S rRNA(e.g. 9–27). The C in the name of the primers of Wilson et al. (1990) refers to the complementary sequence.

Table 6.3. Universal eubacterial primers for 16S rDNA sequencing.

Forward primer 5′–3′ Reverse primer 5′–3′ Reference

27f GAGTTTGATCCTGGCTCAG 1392R ACGGGCGGTGTGTRC Lane, 199163f CAGGCCTAACACATGCAAGTC 1387R GGGCGGWGTGTACAAGGC Marchesi et al., 1998530F GTGCCAGCMGCCGCGG 1100R GGGTTGCGCTCGTTG Lane, 1991POmod. AGAGTTTGATCMTGG PC3mod. GGACTAHAGGGTATCTAAT Wilson et al., 1990P3mod. ATTAGATACCCTDTAGTCC PC5. TACCTTGTTACGACTT Wilson et al., 199016/23S-F. (1390–1407)TTGTACACACCGCCCGTC

16/23S-R. (474–456)CCTTTCCCTCACGGTACTG

Lee et al., 2002

Table 6.4. Suggested primer pairs.



55°C, was used. (Lee et al., 2002). See Table 6.1for specific primers.

Primers for sequencing the 16S rDNA gene forthe identification of Vibrio spp.

There is high sequence homology for the 16SrRNA of all Vibrio species and therefore sequenc-ing part of the 16S rDNA will not be of much usewhen trying to identify to species level. The entire16S rDNA needs to be sequenced for identifica-tion to species level. This can be achieved byamplifying the entire 16S rDNA with universalprimers (Table 6.5) and then using this amplifiedproduct as a template for eight sequencingprimers (Table 6.6) (Thompson et al., 2001a).

For the Universal PCR mix use a standardmaster mix. Use standard cycling parameters withan annealing temperature of 55°C. A productof approximately 1.5 kilo base pairs (kbp) isamplified.

Once the entire 16S rDNA is amplified andpurified, the sequencing primers are used inindividual sequencing PCR reactions to sequencethe 1.5 kbp product. For purification of theamplified product and for quantification of DNAsee page 240. Table 6.7 details the sequencingmaster mix.

PCR master mix for universal primers

Use the standard PCR protocol as a guide.DMSO at no more than a 2% final concentrationcan be added to improve the specificity ofhybridization of the primers (Wilson et al., 1990).Prepare PCR master mix in a single tube. Aliquot20 ml volumes to 0.6 ml or 0.2 ml microfugetubes depending upon the requirements ofthe thermocycler. Add 20–30 ml of paraffin oilto each tube (if hot lid is not available on thethermocycler). Tubes can be labelled and storedat −20°C until required for use.

For the PCR add 5 ml of template DNA to a20 ml reaction tube and place in thermocycler.

Thermocycling conditions for universal primers

The thermocycling conditions are much the sameas for the universal PCR, except that a relaxedannealing temperature is used. Therefore, thecycling conditions begin with one cycle at 95°Cfor 1–5 min. Then, 25–35 cycles of denaturationat 95°C for 30 s, annealing at 45–55°C 30 s,extension at 72°C for 30 s. A final cycle issuggested as for the last cycle but an extensiontime of 10 min. The thermocycler can be set to doa hold cycle at 4°C. The annealing temperaturecan be between 45 and 55°C.


Forward primer 5′–3′ Reverse primer 5′–3′ Reference

EUBB (7-26)AGAGTTTGATCMTGGCTCAG

EUB-AAAGGAGGTGATCCANCCRCA

Weisburg et al., 1991; Suzuki andGiovannoni, 1996; Thompson et al., 2001a

MH1AGTTTGATCMTGGCTCAG

MH2TACCTTGTTACGACTFCACCCCA

Thompson et al., 2001a

Table 6.5. Universal primers for amplification of 16S rDNA from Vibrio spp.

Primer name E. coli position Sequence 5′–3′ Reference

16F358 339–358 CTCCTACGGGAGGCAGT Modified from primer 357r from Lane, 199116F536 519–536 CAGCAGCCGCGGTAATAC Thompson et al., 2001a16F926 908–926 AACTCAAAGGAATTGACGG Modified from primer 926f from Lane, 199116F1112 1093–1112 AGTCCCGCAACGAGCGCAAC Thompson et al., 2001a16F1241 1222–1241 GCTACACACGTGCTACAATG Thompson et al., 2001a16R339 358–339 ACTGCTGCCTCCCGTAGGAG Modified from primer 342r from Lane, 199116R519 536–519 GTATTACCGCGGCTGCTG Modified from primer 519r from Lane, 199116R1093 1112–1093 GTTGCGCTCGTTGCGGGACT Thompson et al., 2001a. Similar to 1100r from Lane, 1991

Thompson et al., 2001a.

Table 6.6. Sequencing primers for the 16S rDNA from Vibrio spp.



Visualize DNA on agarose gel

To check that the DNA has been amplified,visualize the product on a 2% agarose gel.

Clean-up amplified product

Before sequencing, the amplified product needsto be purified from the other components in thePCR reaction. Commercial kits such as QIAquickPCR kit or QIAprep Spin kit (Qiagen), PCRKleen spin columns (Bio-Rad), Wizard

PCR Preps (Promega) can be used for post-PCRclean-up.

Quantify DNA concentration

Once the DNA is free of other contaminants itneeds to be quantified for the PCR sequencingreaction. This can be done spectrophoto-metrically by reading the absorbance at 260 nm.The concentration of double-stranded DNA inmg/ml = absorbance × dilution × weight per OD.Weight per OD for dsDNA is 50 mg/ml.

Alternatively, the concentration of DNAcan be estimated by gel electrophoresis in a 2%agarose gel and estimating the amount of DNAagainst a quantitative DNA molecular weightmarker, which has known molecular weightsin nanograms per band. Examples are Hyper-Ladder IV from Bioline, GeneRuler from MBIFermentas.

For the sequencing PCR, the concentrationof DNA needs to be 10–30 ng for a reactionvolume of 10 ml.

Concentrating DNA, ethanol precipitation

If the DNA needs concentrating to obtain thedesired concentration for sequencing, then anethanol precipitation is used. If oil has been usedas an overlay, remove as much as possible beforethe post-PCR clean-up or ethanol precipitation.This method is from Sambrook et al. (1989).

1. Add a 1/10 volume of 3 M sodium acetatepH 4.62. Add an equal volume of 95% ethanol (non-denatured alcohol)3. Centrifuge 13,000 rpm for 15 min at roomtemperature4. Carefully remove the supernatant5. Wash twice with 70% ethanol6. Wash once with 95% ethanol

7. Air dry, or vacuum dry8. Add the desired volume of water or TE bufferto the dried pellet.

3 M sodium acetate = 24.6 gm of sodiumacetate (molecular weight = 82.03) in 100 ml ofdistilled water.

Perform PCR sequencing reaction

The sequencing PCR is done using the BigDyeTerminator v3.0 kit from Applied Biosystems.

There is only one primer per sequencingreaction. Therefore, the universal forward primerused in the amplification PCR is used in onesequencing tube, and the reverse primer in asecond tube. It is advisable to do each primer induplicate or triplicate to check for errors due tomispriming.

The sequencing PCR master mix (Table 6.7)is the recommended protocol from Applied Bio-systems for use with their BigDye V3.0 CycleSequencing kit.

The full reaction volume is that recom-mended by the supplier. The volume of wateris used to adjust for the concentration of DNA.Sequencing results will not be obtained if the DNAis too dilute. For a thermocycler without a hot lid,overlay tubes with 30 ml mineral oil.

Thermocycling conditions for thesequencing PCR

Perform one cycle at 96°C for 2 min. Run 25cycles of 96°C for 30 s, 50°C annealing for 30 s,60°C for 4 min. Hold at 4°C. Times may beshorter, depending upon the thermocycler used.

Product purification before sequencing

The product from the sequencing PCR reactionneeds to be purified to remove salts and unincor-porated dye terminators otherwise the first 100

240 Chapter 6

Reagent Half reaction (ml) Full reaction (ml)

Water (ultra-pure) 0.5 0Terminator ready mix 4.5 8Primer F 0.5 1DNA (10-30 ng/ml) 5.5 11Volume ml (including DNA) 10.5 20

Table 6.7. Sequencing PCR master mix.



bases of the sequence will not be readable.Clean-up can be done by using Centrisepcolumns from Applied Biosystems, MicroBio-Spin 30 columns from Bio-Rad or DyeExDye-Terminator Removal system from Qiagen.

A manual protocol also gives good resultsand is based on an ethanol precipitation.

1. Into a 0.6 ml microfuge tube place 25 ml of95% ethanol (non-denatured, absolute alcohol)2. Add 1 µl of 3 M sodium acetate, pH 4.63. Add entire PCR product (10 ml if a halfreaction volume was used)4. Place on ice for 10–20 min. Some methodsrecommend a strict 10 min only5. Centrifuge 13,000 rpm at room temperaturein a table-top centrifuge for 30 min. Keep thehinges of the tube to the outside so that the DNApellet can be more easily located6. Carefully remove supernatant withoutdisturbing the pellet, which may not be visible7. Immediately add 125 ml of 70% ethanol andgently roll the tube8. Centrifuge 13,000 rpm for 5 min9. Carefully remove the supernatant withoutdisturbing the pellet10. Air dry or vacuum dry the pellet11. Send the dried product to a sequencingfacility.

Sequencing

The sequencing reaction is not covered in thismanual. Usually, this reaction is carried out by aspecialized laboratory.

Analysis of sequence information using BIOEDIT

The sequence information can be analysedby a number of different programs. Proprietarysoftware from Applied Biosystems is available forpurchase from the company.

However, there are a number of freewareprograms available and a very comprehensiveone is called BIOEDIT. It is available for downloadfrom http://www.mbio.ncsu.edu/Bioedit/bioedit

Citation: Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor andanalysis program for Windows 95/98/NT. NucleicAcids Symposium Series 41 95–98.

USING BIOEDIT. A brief explanation of theuse of BIOEDIT for analysing sequence data isexplained here, but the reader is referred to the

help notes under ‘General use of BIOEDIT’contained within the program.

The sequence information is usually e-mailedto the client laboratory. Open the e-mail and savethe attachments into a folder on the hard drive.

Open the BIOEDIT program. Select OPENSEQUENCE SET. This opens an ‘open file’ win-dow. The sequence files will have the ending .ab1.Select the sequence to be examined and doubleclick. This places the title of the sequence inthe left-hand side of the BIOEDIT window, and theDNA sequence in the right-hand side window. Achromatogram window is also seen. Position thetwo windows so that both can be seen and checkthe sequence for inaccuracies. In many cases wherea no base is called (N), checking the chromato-graph can interpret the correct base. Do thesame with the duplicate sequence. Once both arechecked a consensus sequence can be obtained.

First select the duplicate sequence by clickingin the left-hand window to highlight the sequencename. Go to the menu bar and EDIT, COPYSEQUENCE. Then click on the window con-taining the first sequence. Go to the menu barand EDIT, PASTE SEQUENCE. The primaryand duplicate sequences will now be in theone window.

While holding down the shift key, click onboth sequence names in the left-hand windowto highlight both sequences. Go to menu barSEQUENCE, PAIRWISE ALIGNMENT, ALIGNTWO SEQUENCES (optimal global alignment).A new window appears with the alignment resultfor the two sequences. From this window go tothe menu bar and select ALIGNMENT, CREATECONSENSUS SEQUENCE. A window nowappears with the consensus sequence. To savethe consensus sequence, highlight the consensussequence in the left-hand window, go to the menubar EDIT, COPY SEQUENCE. Open a new worddocument and select PASTE in the Word menubar. The consensus sequence will appear thus:

>ConsensusGGTACTGACC etc.

Re-name the sequence as appropriate. Thisformat is known as ‘fasta’ format, and is the formatrequired for a BLAST search.

ALIGNING TWO SEQUENCES USING CLUSTAL W.

BIOEDIT also contains the CLUSTAL program. Touse CLUSTAL within BIOEDIT to align two sequen-ces, select the NEW ALIGNMENT icon. Go to the




document with the sequence to be aligned. Makesure the sequence is in fasta format. Go back tothe BIOEDIT NEW ALIGNMENT window. UnderFILE, select IMPORT FROM CLIPBOARD. Thesequence name and sequence will appear in thewindow. Do this for the second sequence. UnderACCESSORY APPLICATION, select CLUSTAL.Run CLUSTAL. When it is finished, close theCLUSTAL window to see the alignment in theBIOEDIT window.

If the reverse complement is needed, underSEQUENCE go to NUCLEIC ACID, then toREVERSE COMPLEMENT.

CLUSTAL W citation: Thompson, J.D.,Higgins, D.G. and Gibson, T.J. (1994) Clustal W:improving the sensitivity of progressive multiplesequence alignment through sequence weighting,position-specific gap penalties and weight matrixchoice. Nucleic Acids Research 22, 4673–4680.

WEB SOFTWARE FOR MULTIPLE ALIGNMENTS.

http://dot.imgen.bcm.tmc.edu:9331/multi-align/multi-align.htmlhttp://www.ebi.ac.uk/clustalw/Clustal alignmentprogramhttp://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html Blast for alignment of two sequenceshttp://www.mbio.ncsu.edu/Bioedit/bioeditBioEdit program for sequence analysishttp://www.technelysium.com.au/chromas.htmlor http://bioinfo.weizmann.ac.il/pub/software/chromas (Chromas is a sequence editor)

BLAST SEARCH FOR SEQUENCE IDENTIFICATION.

The National Centre for Biotechnology Informa-tion is located at http://www. ncbi.nlm.nih.govSelect the BLAST icon. There is a tutorial there.However, BLAST is very easy to use. SelectNUCLEOTIDE BLAST. In the search windowpaste the sequence in fasta format. Press NOWBLAST IT. Press FORMAT to get the results. Theresults are placed in a queue and it may take 1 or 2minutes for the results to be downloaded to thedesktop.

Citation for BLAST: Altschul, S.F., Madden,T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller,W. and Lipman, D.J. (1997) ‘Gapped BLASTand PSI-BLAST: a new generation of proteindatabase search programs’. Nucleic AcidsResearch 25, 3389–3402.

The Ribosomal Database Project containsribosomal sequence information at http://rdp.cme.msu.edu/html/

6.4 Fluorescence in Situ Hybridization(FISH)

FISH is used increasingly in clinical laboratoriesfor the identification of bacteria from clinicalsamples (DeLong et al., 1989; Hogardt et al.,2000; Jansen et al., 2000).

Using the same principle, oligonucleotides orprimers that are species-specific as shown by aBLAST search of experimentation can be used todevelop sensitive FISH assays for aquatic bacte-ria. The primers listed in Table 6.1 would be agood starting point for developing specific FISHassays that could be applied to smears of coloniesgrown on agar plates. Although none of theseprimers has been tested as being suitable forspecific probes, it was suggested that eight prim-ers that were species-specific for V. costicola, V.diazotrophicus, V. fluvialis, V. nigripulchritudo, V.proteolyticus, V. salmonicida, V. splendidus andV. tubiashii would be suitable for use as specificprobes (Lee et al., 2002).

Outline of steps for FISH

Step Method1 Prepare smear from bacterial colonies2 Hybridization3 Washing4 Microscopic examination

Oligonucleotide probes

Primers should be selected from Table 6.1 and aBLAST search conducted to ascertain whether theprimers are species-specific. The single-strandedoligonucleotides are synthesized and covalentlylabelled with fluorescein isothiocyanate at the 5′end.

A universal positive probe is prepared usingEUB primer 5′-GCTGCCTCCCGTAGGAGT-3′.This sequence corresponds to positions 338–355on the E. coli numbering system. A universalnegative probe is prepared using primernon-EUB 5′-ACTCCTACGGGAGGCAGC-3′(Amann et al., 1990; Jansen et al., 2000).Fluorescein isothiocyanate is added to the 5′ endof each primer.

Prior to use, the probes are diluted to aconcentration of 10 ng/ml in hybridization buffer.

242 Chapter 6



Preparation of smears

Bacterial cells from a colony are emulsifiedin a drop (10–15 ml) of sterile saline or steriledistilled water within a marked area on a glassmicroscope slide. The suspension should not betoo thick, but should allow individual cells to beseen under the microscope. Prepare one dropper probe to be tested plus drops for the positiveand negative controls. Allow to air dry.

Once dry, the cells are fixed to the slide byusing a fixative solution of 4% formaldehyde in96% ethanol. Gram-positive organisms mustbe permeabilized before application of thehybridization buffer. The fixed slide is placed intopermeabilization buffer of 1 mg/ml of lysozymefor 5 min. Gram-negative organisms do notneed the permeabilization step (Jansen et al.,2000).

Hybridization

The fixed slides are hybridized in a hybridizationbuffer of 20 mM Tris-HCl, 0.9 M NaCl, 0.1%sodium dodecyl sulphate, pH 7.2, which containsthe probe at a concentration of 10 ng/ml. Thehybridization buffer (10–15 ml) can be placed ontop of the fixed cells. Gram-negative organismsare hybridized for 45 min, and Gram-positivesfor 2 h at a temperature of 50°C (Jansen et al.,2000).

One hybridization method pretreated theslides by placing them in ascending ethanol stepsof 50, 80 and 96% for 3 min each step. Thehybridization buffer then contained 30–40%formamide with 50 ng of probe. This concen-tration of formamide improved the specificity ofthe probe (Hogardt et al., 2000). However, aconcentration of 20% (vol/vol) of formamide didnot show an increase in probe specificity (Jansenet al., 2000).

Washing

The slides are washed in washing buffer of20 mM Tris-HCl, 0.9 M NaCl, pH 7.2, at atemperature of 50°C for 10 min (Jansen et al.,2000).

Examination of smears

The slides are mounted with VectaShield(Vector Laboratories, Burlingame, California), orCitifluor (Citifluor Ltd, London, UK).

The slides are examined for cells showingfluorescence (positive result), using a fluorescencemicroscope and filter set capable of detectingfluorescein. Fluorescence should be seen with thepositive control, and no fluorescence with thenegative control.




7Preparation of Media for Culture and Identification

7.1 General Isolation and Selective Media

Acetate agar. See Rogosa medium under ‘Test media’

Alkaline peptone water (APW) (May be used as an enrichment medium for Vibrio species; Furnisset al., 1978)

Reagent Amount Preparation of media Description of growth characteristicsPeptone 10.0 g Dissolve reagents in distilled water and

pH to 8.6. Dispense in 10 ml aliquotsto McCartney bottles and autoclave at121°C for 20 min.

May be used as an enrichment medium to isolateVibrio species from contaminated samples such asfaeces and polluted water. To be effective, the APWcultures should be subcultured at 6 h if incubated at37°C and overnight if incubated at 18–20°C.

Sodium chloride 10.0 gDistilled water 1000 ml

Amies transport medium (Oxoid)This transport medium can be purchased ready-made as swabs in tubes of media, or prepared from apowdered medium available from Oxoid.

Formulae: 10.0 g charcoal (pharmaceutical), 3.0 g sodium chloride, 1.15 g sodium hydrogenphosphate, 0.2 g potassium dihydrogen phosphate, 0.2 g potassium chloride, 1.0 g sodiumthioglycollate, 0.1 g calcium chloride, 0.1 g magnesium chloride, 4.0 g agar, 1000 ml distilled water,pH 7.2.

Anacker-Ordal agar (AO) (Used for growth of freshwater and marine Cytophaga, Flavobacteriumand Flexibacter spp.; Anacker and Ordal, 1955, 1959)

Reagent Amount Preparation of media Description of growth characteristicsBacto-tryptone (Difco) 0.5 g Add all reagents to

1000 ml of distilled water,and adjust pH to 7.2–7.4.Autoclave at 121°C for15 min (15 lb/20 min). Coolto 50°C and pour mediainto Petri dishes. Storeplates in sealed plasticbags at 4°C.

Using a cotton-tipped swab, collect samples from skinlesions and gills of fish and inoculate the plate. Colonies ofF. columnare appear at 2–5 days as yellow-pigmented,rhizoid colonies with a thin spreading growth. Seephotographic section.

Early colonies may be viewed with the aid of astereomicroscope. Subculture by cutting out a block of agarcontaining the colony, and invert on to new plate. A Pasteurpipette prepared in the shape of a ‘hockey stick’ with theend unsealed can be used to pick out a plug of agar.

Yeast extract (Difco) 0.5 gSodium acetate 0.2 gBeef extract (Difco) orLab-Lemco powder (Oxoid)

0.2 g

Agar (Difco or Oxoid AgarNo 1)

9 g/l

Distilled water 1000 ml

©N.B. Buller 2004. Bacteria from Fish and Other Aquatic Animals:244 a Practical Identification Manual (N.B. Buller)



Anacker-Ordal agar – marine (AO-M) (Used for growth of marine Cytophaga, Flavobacteriumand Flexibacter spp. and Tenacibaculum (Flexibacter) maritimum; Anacker and Ordal, 1955, 1959)Add artificial seawater salts (Sigma) at 38 g/l. If using seawater, use sterile at 50–100% final concentra-tion (Ostland et al., 1999b).

Anaerobe plates (ANA). For growth of anaerobic Gram-positive and Gram-negative organisms(Oxoid manual)

Reagent Amount Preparation of media Description of growth characteristicsOxoid Wilkins-ChalgrenAnaerobe agar

21.50 g Suspend powder in water in a 1000 ml Schottbottle with magnetic flea. Autoclave at 121°C for20 min then cool to 50°C in waterbath. Asepticallyadd blood. Pour into Petri dishes.

Inoculated plates are incubated atthe appropriate temperature in ananaerobic atmosphere.Distilled water 500 ml

Sterile equine blood 30 ml

Gram-negative anaerobe plates (ANA-GN). For growth of anaerobic Gram-negative organisms(Oxoid manual)

Reagent Amount Preparation of media Description of growth characteristicsOxoid Wilkins-ChalgrenAnaerobe agar

21.50 g Dissolve agar in water. Autoclave at 121°Cfor 20 min then cool to 50°C in a waterbath.Reconstitute 1 vial of supplement with10 ml sterile distilled water and add to base.Aseptically add blood. Pour into Petri dishes.

Inoculated plates are incubated atthe appropriate temperature in ananaerobic atmosphere.Distilled water 500 ml

Sterile equine blood 15 mlOxoid G-N selective supplement 10 ml

Artificial seawater – ASW: Sea salts (Sigma product number S 9883)

Reagent Amount Preparation of media Description of growth characteristicsSea salt (Sigma) 38 g Add salts to distilled water and

pH to 7.6. Autoclave at 121°C for15 min.

May be added to media for growth of marine organisms. Addto AO medium for isolation of marine Flavobacterium andTenacibaculum species.


Addition of 18.7 g/l makes a 50% seawater concentration of 17.5‰ salinity.

Artificial seawater medium (Lewin, 1974 – Used for isolation of marine Flexibacter/Flavobacterium group. Baumann et al., 1971, quoted in MacLeod, 1968, is the same medium butwithout the trace element mixture – Used for marine Vibrio species)

Reagent Amount Preparation of media Description of growth characteristicsNaCl 20.0 g Dissolve reagents in distilled water.

Add 1.0 ml of trace element solution.Adjust pH to 7.5 and autoclave at121°C for 20 min.

Can be used as an inoculatingmedium for biochemical identificationsets or as an initial isolation broth formarine organisms. Add agar at 15 g/land pour into Petri dishes if a solidmedium is required.

Lewin used this medium for marineFlavobacterium and Flexibacterspecies. The medium used byMacLeod and Baumann did notcontain the trace element solution.

MgSO4.7H2O 5.0 gKCl 1.0 gCaCl2.2H2O 1.0 gDistilled water 1000 mlB (soluble salt of element) 0.5 mg/ml Prepare a stock solution of trace

elements so that the final concentrationof each element is either 0.5 mg/l or0.01 mg/l.

Fe (soluble salt of element) 0.5 mg/mlMn (soluble salt of element) 0.5 mg/mlCo (soluble salt of element) 0.01 mg/mlCu (soluble salt of element) 0.01 mg/mlMo (soluble salt of element) 0.01 mg/mlZn (soluble salt of element) 0.01 mg/ml

Preparation of Media for Culture and Identification 245



Blood agar – BA

Reagent Amount Preparation of media Description of growth characteristicsOxoid ColumbiaBA base

19.5 g Suspend agar base in water. Autoclave at 121°C for15 min. Cool in waterbath to 50°C. Add blood to cooledagar, mix well and pour into Petri dishes to depth ofapproximately 3 mm. Store at 4°C in sealed plastic bags.(Plates can be left overnight on the bench and sealed inplastic bags the following day. This prevents too muchmoisture build-up once stored in the plastic bags.)

Plates are inoculated with a swab ofthe specimen, and incubated at theappropriate temperature andatmosphere. Plates are examineddaily for growth and haemolysis.

Distilled water 500 mlSterile equine blood 15 ml

Bordetella bronchiseptica selective agar – CFPA medium (Smith and Baskerville, 1979;Rutter, 1981; Hommez et al., 1983)

Reagent Amount Preparation of media Description of growth characteristicsColumbia agar base (Oxoid) 19.5 g Add agars to distilled water and autoclave at

121°C for 15 min. Cool to 50°C. Add sterileblood to cooled agar mixture. Reconstitute1 vial of Bordetella supplement with 2 ml ofdistilled water and add to mixture. Add 5 mlof antibiotic stock. Pour plates and store at4°C.

Colonies are 1 mm at 48 h and maybe haemolytic or non-haemolytic,opaque, smooth and pearl-like, orrough, translucent, raised in thecentre with an undulating margin,depending on phase variation.

Agar technical No. 3 (Oxoid) 10.0 gDistilled water 500 mlBordetella Pertussis supplement(Oxoid, Code SR082E)

2 ml

Sterile equine blood 15 ml

Penicillin 20 mg Antibiotic stock: Add 20 mg of each antibioticto 10 ml of normal saline. Store in fridge.Handle furaltadone with caution and usemask and gloves.

Furaltadone 20 mgNormal saline 10 ml

Brucella agar (Available from Difco or Oxoid media suppliers. See also under Farrell’s medium)

Burkholderia pseudomallei selective media – glycerol medium (Thomas et al., 1979)

Reagent Amount Preparation of media Description of growth characteristicsAgar No 3 (Oxoid) 2.4 g Add all reagents to distilled water (glycerol may

be warmed to aid pipetting). Autoclave at 121°Cfor 15 min. Cool to 50°C and pour into Petridishes. Store plates at 4°C.

Plates are a mauve colour.Colonies appear at 24 h, smooth,mauve-coloured with slight metallicsheen. As incubation increases,colonies become wrinkled andumbonate. Incubate for 4 days.

Glycerol 6.0 mlCrystal violet stock solution 0.5 mlDistilled water 194 mlCrystal violet – Stock(1/5000 dilution)

0.5 g Add 0.5 g of crystal violet to distilled water andstir until dissolved. Store at room temperature.


Burkholderia pseudomallei selective broth (Modified from Thomas et al., 1979)

Reagent Amount Preparation of media Description of growth characteristicsMacConkey broth (purple) (Oxoid) 100 ml Prepare MacConkey broth and add

reagents except antibiotics. Autoclave at121°C for 15 min. Cool to 50°C and addfilter-sterilized antibiotics. Asepticallydispense into sterile McCartney bottles.

Detection of Burkholderia can beimproved by incubating material inbroth for 24 and 48 h, followed bysubculture to plates.

Crystal violet 0.001 gGentamycin 0.8 mgStreptomycin sulphate 5000 units

246 Chapter 7



Burkholderia pseudomallei selective media – Ashdown’s medium (Ashdown, 1979a)

Reagent Amount Preparation of media Description of growth characteristicsTryptone soy agar 40 g Add all ingredients except gentamycin to distilled water.

Autoclave at 121°C for 15 min. Cool to 50°C. Add filtersterilized gentamycin. Mix well and pour into Petri dishes.

Colonies are flat, rough, wrinkledafter 3 days. Colonies ofBurkholderia pseudomallei absorbthe Neutral red after 3 days whereasPseudomonas species have no dyeuptake at 3 days.

Glycerol 40 mlCrystal violet 5 mgNeutral red 50 mgGentamycin 4 mgDistilled water 1000 ml

Carbon dioxide atmosphere. See under ‘Test media’

Cellobiose-Colistin agar – For selective isolation of Vibrio vulnificus (Massad and Oliver, 1987;Høi et al., 1998a)

Reagent Amount Preparation of media Description of growth characteristicsSolution 1 Adjust pH to 7.6. Autoclave at 121°C for

15 min and cool to 55°C.The medium is olive green to lightbrown. Incubate plates at 40°C for24–48 h. V. vulnificus appears as yellowcolonies surrounded by a yellow zonedue to fermentation of cellobiose.

V. cholerae appears as purple coloniessurrounded by a blue zone.

Bacto-peptone (Difco) 10 gBeef extract (Difco) 5 gNaCl 20 gBromothymol blue 40 mgCresol Red 40 mgAgar 15 gDistilled water 900 mlSolution 2 Filter sterilize. Add to cooled reagents of

solution 1 and mix. Dispense into Petri dishes.Cellobiose 15 gColistin 0.03 mg/ml

(4 × 105 U/l)Distilled water 100 ml

CFPA media. See under Bordetella bronchiseptica medium

Dermatophilus selective medium – polymyxin plates (Abu-Samra and Walton, 1977)

Reagent Amount Preparation of media Description of growth characteristicsColumbia agar base(Oxoid)

19.5 g Add agar base to distilledwater and autoclave at121°C for 15 min. Cool to50°C and aseptically addblood and polymixin.Polymixin B may bedissolved in sterile normalsaline first.

Grind scab material using a sterile pestle and mortar. Transferfinely ground material to a bijou bottle and add 2 parts ofdistilled water. Shake thoroughly then allow to settle for 15 min.Take a loopful of surface material and plate to BA and polymixinplates. Incubate at 25 or 37°C depending on the habitat of theinfected animal. Examine plates at 24 and 48 h for presence ofadherent pitted dry or mucoid colonies. This medium is notcompletely selective and some contaminating bacteria will grow.

Distilled water 500 mlSterile equine blood 50 mlPolymixin B (Use at aratio of 1000 IU/ml ofmedium)

62.5 mg

Dubos medium. For isolation of Cytophaga hutchinsonii (Bernardet and Grimont, 1989)

Reagent Amount Preparation of media Description of growth characteristicsNaNO3 0.5 g Add all reagents except cellobiose, to distilled water.

Adjust pH to 7.2 and autoclave at 121°C for 15 min.Cool to 50°C and add filter-sterilized cellobiose solution.Mix well and pour into Petri dishes.

K2HPO4 1 gMgSO4.7H2O 0.5 gKCl 0.5 gFeSO4.7H2O 0.01 gDistilled water 1000 mlAgar 15 gD-cellobiose 30% w/v Prepare cellobiose solution and filter-sterilize.

Note: For isolation of Cellulomonas species add 0.5 g yeast extract.




Edwardsiella ictaluri medium (EIM). For isolation of Edwardsiella ictaluri (Shotts and Waltman,1990)

Reagent Amount Preparation of media Description of growth characteristicsBacto-tryptone (Difco) 10 g Dissolve all reagents in distilled

water and adjust the pH to 7.0–7.2.Autoclave at 121°C for 15 min.Cool to 50°C and add the 10 mlfilter-sterilized solution 1 containingmannitol, colistin, bile salts andfungizone. Mix well and pour intoPetri dishes.

This medium is used for the isolation ofEdwardsiella ictaluri. Most Gram-negative bacteriaare inhibited with the exception of Proteus spp.,Serratia marcescens, Aeromonas hydrophila andYersinia ruckeri. Gram-positive bacteria areinhibited with the exception of Enterococci. 90%of E. tarda isolates grow on this medium.

E. ictaluri is seen as 0.5–1.0 mm green,translucent colonies at 48 h. Colonies of E. tardahave a similar size and appearance.Proteus species are 2–3 mm, brownish-green andmay swarm. S. marcescens colonies are 2–3 mmand reddish brown. Y. ruckeri are 1–2 mmyellowish green, A. hydrophila are 2–3 mmyellowish-green opaque colonies and Enterococciappear as 0.5 mm yellowish colonies.

Yeast extract (Difco) 10 gPhenylalanine 1.25 gFerric ammonium citrate 1.2 gSodium chloride 5.0 gBromothymol blue 0.03 gAgar (Difco) 17 gDistilled water 990 mlSolution 1Mannitol 3.5 g Add reagents to 10 ml of distilled

water and filter-sterilize.Colistin 10 mgBile salts 1 gFungizone 0.5 mgDistilled water 10 ml

Electrolyte supplement (Added to biochemical test media to improve growth of organism whenNa+ alone is insufficient; Lee et al., 1979)

Reagent Amount Preparation of media Description of growth characteristicsNaCl 100 g Add all reagents to distilled water and

autoclave at 121°C for 15 min.If addition of NaCl alone to biochemical identificationmedia does not improve the growth of an organism, thiselectrolyte supplement may improve growth. It is addedat a rate of 0.1 ml of supplement per 1.0 ml of medium.

MgCl2.6H2O 40 gKCl 10 gDistilled water 1000 ml

Erysipelothrix selective medium. See ‘Wood’s’ broth, and ‘Packer’s’ plates

Farrell’s medium. For the growth of Brucella spp. (Farrell, 1974)

Reagent Amount Preparation of media Description of growth characteristicsOxoid bloodagar base

20.0 g Add agar to distilled water and autoclave at 121°C for15 min. Cool to 50°C and leave at this temperature whilethe other reagents are prepared.

Incubate plates at 37°C in 10% CO2 for14 days.

Isolates from sea mammals appear after4 days incubation, whereas isolates fromseals do not appear until day 10 ofincubation, or do not grow. Incubation ofplates should continue for 14 days.

It has been suggested that the isolationof strains from seals may be improvedby decreasing the concentration orremoving bacitracin and/or nalidixic acidfrom the medium (Foster et al., 2002).

Always inoculate a non-selectivemedium such as blood agar or serumdextrose medium.

Colonies are 1–2 mm in diameter at 5days incubation, pale yellow,translucent, convex and round withentire edges.

Distilled water 500 mlNormal horseserum

25 ml Inactivate the horse serum at 50°C for 35 min.

25% Dextrose(D-Glucose)

20 ml Add 125 g of D-glucose to 375 ml distilled water anddissolve by gentle heating. Dispense into 20 ml aliquotsinto McCartney bottles and with lids loose, autoclave at121°C for 15 min. Store at 4°C.

Oxoid Brucellaselectivesupplement

10 ml Dissolve Brucella supplement in 5 ml sterile distilled waterand 5 ml methanol. Incubate for 10–15 min at 37°C andshake well to dissolve.

For complete Farrell’s medium aseptically combine allingredients while stirring.

Mix well and pour media into plates to an approximatedepth of 3 mm.

Final mediapreparation

Combine all prepared reagents (serum, dextrose,supplement) into agar. Mix well and pour into Petridishes to a depth of 3 mm.

The original medium uses the following antibiotic concentrations: bacitracin (25 units/ml), vancomycin (20 mg/ml), polymixin B(5 units/ml), nalidixic acid (5 mg/ml), nystatin (100 units/ml) and cycloheximide (100 mg/ml) (Farrell, 1974).

248 Chapter 7



Farrell’s medium is available from Oxoid as Brucella medium base, to which Brucella selective sup-plement (Oxoid code SR83) is added. The selective supplement is based on the Farrell formulation.

Flavobacterium maritimus media (FMM). May improve primary isolation for T. maritimum(Pazos et al., 1996)

Reagent Amount Preparation of media Description of growth characteristicsPeptone 5.0 g Add reagents and powders to sterile

seawater. Adjust pH to 7.2–7.4 and sterilizeby autoclaving at 121°C for 20 min.

Colonies pale-yellow, flat, irregular withuneven edges. Reduces growth of Vibriospecies and Aeromonas species that mayoutgrow Flexibacter on media such as MSA-Bor MA 2216.

Yeast extract 0.5 gSodium acetate 0.01 gAgar 15 gSterile seawater 1000 ml

Flavobacterium psychrophilum medium (FPM). May improve isolation rate and colony size forF. psychrophilum (Daskalov et al., 1999)

Reagent Amount Preparation of media Description of growth characteristicsTryptone T (Oxoid) 0.5 g Add all reagents (except sugars and

skimmed milk) to distilled water andautoclave at 121°C for 15 min. Cool ina 50°C waterbath.

Prepare supplements (galactose,glucose, rhamnose and skimmed milk)as separate 10% (w/v) solutions, andfilter by sterilization thorough a 0.22 mmpore size Millipore Millex porosity filter.Add filter-sterilized solutions to cooledagar medium. Pour into Petri dishes andstore at 4°C.

Colonies grow after 3–6 days andare an intense yellow colour with aspreading and irregular shape.

Yeast extract (Oxoid) 0.5 gBeef extract (Oxoid) 0.2 gSodium acetate trihydrate (Sigma) 0.2 gD(+) galactose (Sigma) 0.5 gD(+) glucose (BDH) 0.5 gL-rhamnose (Sigma) 0.5 gSkimmed milk (Oxoid) 0.5 gWater 1000 mlAgar – bacteriological agar no. 1(Oxoid)

9.0 g

This medium is based on Anacker Ordal (AO) agar (also known as Cytophaga agar) supplemented with galactose, glucose,rhamnose and skimmed milk. The size and number of colonies of F. psychrophilum isolated is increased compared to AO medium.Broth can be prepared without the addition of agar.

Flexibacter polymorphus media (Lewin, 1974)

Reagent Amount Preparation of media Description of growth characteristicsNaCl 20.0 g Dissolve reagents in distilled water.

Adjust pH to 7.5 and autoclave at121°C for 20 min.

The organism is maintained in 5 ml of the brothmedium and subcultured twice weekly. Filamentsseveral hundred microns in length and 1.5 mm wideare produced. They have rounded ends, areflexuous, unbranched and cylindrical. The growingfilaments may be peach coloured. Cultures in lategrowth phase may show a refractile granule at eachend, which is more easily seen after cell lysis.

Cobalamin is essential for growth.

Agar can be added to the medium for plate media.

The organism also grows on agar medium preparedwith seawater and yeast extract (10 mg/ml). Coloniesmay be peach coloured with a filamentous margin.

MgSO4.7H2O 5.0 gKCl 1.0 gCaCl2.2H2O 1.0 gFe (soluble salt) 0.5 mgB (soluble salt) 0.5 mgMn (soluble salt) 0.5 mgCo (soluble salt) 0.01 mgCu (soluble salt) 0.01 mgMo (soluble salt) 0.01 mgZn (soluble salt) 0.01 mgDistilled water 1000 ml


265Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpMonday, January 26, 2004 3:59:44 PM


Flexibacter maintenance medium (Lewin and Lounsbery, 1969; Lewin, 1974)

Reagent Amount Preparation of media Description of growth characteristicsTryptone (Difco) 1.0 g Add all reagents to filtered seawater

and adjust pH to 7.5. Dispensemedia into tubes. Autoclave at121°C for 20 min.

Optimum cobalamin is 0.3 mg/l.

The organisms are maintained in 5 ml of thebroth medium, incubated between 22 and32°C, and subcultured twice weekly.

The Flexibacteria may show a range ofpigmented filaments ranging from pink,orange or yellow colouration. On solidmedium they may produce fimbriate margins.

Agar can be added to the medium for platemedia.

A semi-solid medium can be produced using0.3% agar, and viable filaments can bemaintained for up to 1 month when grownin this medium at room temperature.

Casamino acids (Difco) 1.0 gMonosodium glutamate 5.0 gSodium glycerophosphate 0.1 gCobalamin 1.0 ugFe (soluble salt) 0.5 mgB (soluble salt) 0.5 mgMn (soluble salt) 0.5 mgCo (soluble salt) 0.01 mgCu (soluble salt) 0.01 mgMo (soluble salt) 0.01 mgZn (soluble salt) 0.01 mgFiltered seawater 1000 ml

Glycerol Lab Lemco broth (Used for storage of cultures at −80°C)

Reagent Amount Preparation of media Description of growth characteristicsLab Lemco broth(Oxoid)

0.64 g Add all reagents together. Pipettingof glycerol may be facilitated bypre-warming. Dispense into 2 mlvolumes into Bijou bottles.Autoclave at 121°C for 15 min.Store media at 4°C.

Used as −80°C storage medium. Using a sterilecotton-tipped swab, scrape bacterial growth from an agarplate and inoculate into 1 ml of Glycerol Lab Lemco mediumin a Nunc tube. Use an inoculum of approximately tube 5McFarland opacity. Place tube in cryobox in −80°C freezer.This medium is also suitable for liquid nitrogen storage.

Glycerol 20 mlDistilled water 80 ml

Helicobacter selective media. Use Skirrow’s medium (also known as VPT media; Skirrow, 1977)

Reagent Amount Preparation of media Description of growth characteristicsBlood agar baseNo. 2 (Oxoid)

20 g The agar is dissolved in waterand autoclaved at 121°C for15 min. After cooling to 50°Cadd 15 ml of sterile equineblood. Reconstitute 1 vial ofsupplement using 2 ml of steriledistilled water and add to cooledmedia. Mix thoroughly. Pourmedia into plates to anapproximate depth of 3 mm.Store plates at 4°C.

Other bases such as Columbia agar base (Oxoid), orBrucella medium base (Oxoid), or tryptone soy agar maybe used to prepare this medium. However, it is suggestedthat blood agar base No. 2 is more nutritionally rich andalso that trimethoprim is more active in this base.

To reduce contamination from other organisms, thesample can be filtered through a 0.65 mm filter and thefiltrate cultured to the plate (Butzler et al., 1973).

The plates are incubated at 37°C in a microaerophilicatmosphere of N2, H2, CO2 (80:10:10) for 2–4 weeks. Gasgenerating packs are available from commercial supplierssuch as MGC Anaero Pak Campylo from Mitsubishi GasChemical Company.

Helicobacter species will grow as pin-point colonies andalso appear as a thin spreading film across the plate.

Distilled water 500 mlSterile equine blood 15 mlOxoid Campylobactersupplement (Skirrow)

2 ml

This medium was developed by M.B. Skirrow (1977) for the isolation of Campylobacter species. It is also suitable for the isolation ofHelicobacter species and is widely quoted in many references. It is also available as ready-made media from a number of mediaproducers. The antibiotics are often supplied as a supplement and, depending on the originators of the medium, the supplementswill be named accordingly. For example, Oxoid produces ‘Campylobacter selective supplement (Skirrow) for additions ofvancomycin, polymyxin and trimethoprim, and Campylobacter selective supplement (Blaser-Wang) for additions of vancomycin,polymyxin, trimethoprim, amphotericin B, and cephalothin’. The additive containing the amphotericin B is recommended whenfungal contamination is suspected. The original concentrations described by Skirrow (1977) are vancomycin (10 mg/l), polymyxin B(2.5 IU/ml), and trimethoprim (5 mg/l). A series of papers describing the isolation and identification of Helicobacter cetorum fromdolphins and whales (Harper et al., 2000, 2002a,b) describes the use of TVP and CVA media from Remel. These are, in essence,Skirrow’s media and Blaser-Wang media, respectively.

250 Chapter 7



Hsu-Shotts agar – HS (For Flavobacterium columnare, F. psychrophilum; Bullock et al., 1986)

Reagent Amount Preparation of media Description of growth characteristicsTryptone 2 g Add all reagents to distilled water, except neomycin

sulphate. Autoclave at 121°C for 15 min. Cool to50°C. Add filter-sterilized neomycin sulphate, mixwell and pour into Petri dishes.

This is a semi-selective medium forthe isolation of Flavobacteriumcolumnare. At 48 h characteristicyellow, spreading colonies are seen.

Yeast extract 0.5 gGelatin 3 gAgar 15 gDistilled water 1000 mlNeomycin sulphate 4.0 mg/ml Prepare solution and filter-sterilize.

Note: HSM. Addition of 18.7 g/l sea salts (Sigma) to make a 50% seawater concentration (17.5%° salinity), and 200 IU/mlpolymyxin B is suitable for isolation of Tenacibaculum (Flexibacter) maritimum (Chen et al., 1995).

Inositol horse serum (Suspension medium for freeze-drying organisms)

Reagent Amount Preparation of media Description of growth characteristicsInositol 5 g Dissolve inositol in horse serum. Filter through

a 0.45 mm filter followed by further filtrationthrough a 0.22 mm filter for sterilization. Checksterility. Dispense into McCartney bottles andstore at 4°C.

Place 1 ml of sterile inositol horse serum into afreeze-dried vial (Wheaton serum bottle). Using asterile cotton swab or loop emulsify a heavysuspension of bacteria in the medium. Snap-freezein liquid nitrogen and follow instructions forfreeze-drying relevant to the equipment being used.

Horse serum 100 ml

KDM2. For growth and isolation of Renibacterium salmoninarum (Evelyn, 1977)

Reagent Amount Preparation of media Description of growth characteristicsTryptone 1 g Add all reagents to distilled water and

adjust pH to 6.5–6.8 (with NaOH).Dispense into tubes and autoclave at121°C for 20 min.

Store at 4°C for 1 month.

Incubate plates at 15°C for up to 2 months.Initial growth is visible between 2 to 8 weeks.Colonies are pin-point to 2 mm in size. Oldcolonies may have a granular or crystallineappearance. To assist in prevention ofovergrowth by fast growing colonies,examine plates every few days andaseptically remove these colonies.

Yeast extract 0.05 gL-cysteine (chlorhydrate) 0.1 gAgar 1.5 gDistilled water 100 mlFetal calf serum 5–10% For use, heat tubes. Cool to 50°C and

add calf serum. Pour into plates.

KDMC. For Renibacterium salmoninarum (Daly and Stevenson, 1985)

Reagent Amount Preparation of media Description of growth characteristicsKDM2 medium As above. Addition of charcoal acts as a

detoxifying agent.Activated charcoal (Difco) 0.1% For use heat tubes. Cool to 50°C and addcharcoal. Pour into plates.

Marine salt agar with blood (MSA-B) (Used as a general-purpose medium for isolation oforganisms from the marine environment)

Reagent Amount Preparation of media Description of growth characteristicsTryptone soy agar(Oxoid)

20.0 g Dissolve TSA and NaCl in waterand autoclave at 121°C for 15 min.Cool in waterbath to 50°C.

Aseptically add 15 ml blood tocooled agar, mix well and pourinto Petri dishes to depth ofapproximately 3 mm. Store at 4°C.

TSA with added blood has the advantageof providing differentiation betweenhaemolytic and non-haemolytic Vibrio spp.

Some Vibrio species will be haemolytic onblood agar and non-haemolytic on MSA-Bdespite the latter medium providing bettergrowth for most marine Vibrio species.

NaCl 7.5 g(= 2% w/v final conc)

Distilled water 500 mlSterile equine blood 15.0 ml




Marine 2216 agar (Difco) (ZoBell, 1941)

Reagent Amount Preparation of media Description of growth characteristicsBacto marine agar 2216(Difco – complete medium)

55.1 g Add powder to distilled water and boil to dissolve.Autoclave at 121°C for 15 min. pH 7.6 ± 0.2.

Plates are a light amber colour andslightly opalescent.


MA 2216 can be purchased from Difco as a broth or agar. Composition of MA 2216: 5.0 g Bacto peptone; 1.0 g Bacto yeast extract;0.1 g Fe(III) citrate; 19.45 g NaCl; 5.9 g MgCl2 (dried); 3.24 g NaSO4; 1.80 g CaCl2; 0.55 g KCl; 0.16 g Na2CO3; 0.08 g KBr; 34.0 mgSrCl2; 22.0 mg H3BO3; 4.0 mg Na-silicate; 2.4 mg NaF; 1.6 mg (NH4)NO3; 8.0 mg Na2HPO4; 15.0 g agar; 1000 ml distilled water.

Medium K (Used for isolation of Chryseobacterium (Flavobacterium) scophthalmum; Mudarris et al.,1994)

Reagent Amount Preparation of media Description of growth characteristicsYeast extract (Oxoid) 1 g Add all ingredients to seawater and

adjust to pH 7.2. Autoclave at 121°Cfor 15 minutes.

Orange-pigmented colonies are shiny,smooth, round, raised entire, 5–6 mm at48 h, 25°C.

On primary culture, isolates may showslight gliding motility, but lose this afterstorage.

Beef extract (Oxoid) 5 gCasein (Oxoid) 6 gTryptone (Oxoid) 2 gAnhydrous CaCl2 1 gAgar (Oxoid No. 1) 15 g750 ml seawater (aged 30 days)

Middlebrook 7H10-ADC medium (Used for isolation of Mycobacterium abscessus; Teska et al.,1997)

Reagent Amount Preparation of media Description of growth characteristicsNH4SO4 0.05 g Add chemical reagents to distilled water.

Autoclave at 121°C for 15 min. Cool to 50°C.Aseptically add sterile bovine albumin,nalidixic acid and cycloheximide, pyridoxineand biotin.

Media can be prepared as broths or add1.5 g of agar for preparation of plates.

Incubate at 25°C for 14–28 days.Growth occurs in 7 days. Youngcolonies may exhibit a lightblue-green tint. With age, coloniesare off-white to tan in colour.

KH2PO4 0.15 gNa2HPO4 0.15 gSodium citrate 0.04 gMgSO4 0.0025 gCaCl2 0.00005 gZnSO4 0.0001 gCuSO4 0.0001 gL-glutamic acid 0.05 gFerric ammonium citrate 0.004 gPyridoxine 0.0001 gBiotin 0.00005 gMalachite green 0.025 gGlycerol 0.5 mlNalidixic acid (35 mg/ml) 0.0035 gCycloheximide (400 mg/ml) 0.04 gBovine albumin V 0.5 mlD-glucose 0.2 gCatalase 0.0003%Distilled water 100 ml

This medium is modified from Middlebrook’s medium (Middlebrook et al., 1960) by the addition of ADC (albumin, catalase anddextrose).

252 Chapter 7



Mycoplasma medium – general purpose medium. Bacto Pleuropneumonia-like organism(PPLO) agar and broth with supplements (Difco). (Used for isolation of Mycoplasmas species. Thiscommercial medium, with the added supplements, is based on Hayflick’s medium)

Reagent Amount Preparation of media Description of growth characteristicsPPLO agar 35 g Agar plates

Add dehydrated media to distilledwater and autoclave at 121°C for15 min. Cool to 50–60°C.Aseptically add 300 ml Mycoplasmasupplement or 300 ml of Mycoplasmasupplement S. Mix well. Dispenseinto small (5 cm) Petri dishes. Storein plastic bags at 4°C.

Mince or grind tissue and place a few loopfulsinto a 3 ml broth. Incubate broth at 25 and37°C. A change in pH results in a colourchange from red to yellow, and may indicategrowth of Mycoplasma. Bacterial contaminationmay also change the pH of the medium. Thebacterial contamination can be filtered off bypassing the broth through a 0.22 mm filter.Place drops of the filtered broth to a fresh brothand to a plate.

Subculture 2 drops from a broth culture to aPPLO agar plate every 3 or 4 days. Incubateplates in air and 5–10% CO2 at 25 and 37°C.Subculturing on a Tuesday and a Friday maybe a convenient time frame for the laboratory.

Examine plates under a stereomicroscope forpresence of typical ‘fried-egg’ type colonies.

To differentiate Mycoplasma colonies fromcontaminating bacterial colonies, stain colonieswith Dienes stain. Mycoplasma colonies stainblue with a distinctly dense blue centre and alighter blue periphery. Mycoplasma coloniesretain the stain for 24 h, whereas bacterialcolonies decolorize after 30 min. See Dienesstain in ‘Tests’ section.


PPLO broth 21 g BrothsAdd dehydrated media to distilledwater and autoclave at 121°C for15 min. Cool to 50–60°C.Add either supplement as for agar.

Distilled water 700 mlPhenol red

Mycoplasma supplement 1 vial SupplementRehydrate vial with distilled waterand swirl to dissolve. Asepticallyadd 1 vial per 70 ml of PPLO agaror broth.

Sterile distilled water 30 ml

Mycoplasma supplement S 1 vial Supplement S

Rehydrate vial with distilled waterand swirl to dissolve. Aseptically add1 vial per 70 ml of PPLO agar orbroth.

Sterile distilled water 30 ml

DNA (optional) DNA solutionPrepare 0.2% solution by dissolving0.2 g DNA in 100 ml distilled water.Autoclave at 121°C for 15 min.Add 10 ml to 1000 ml of abovemedium to produce a finalconcentration of 1%.

DNA (optional) (Calbiochempolymerized calf thymusDNA) 0.2% solution

1%


Formulations

• PPLO agar = per litre contains 50 g Bacto beef heart infusion, 10 g Bacto peptone, 5 g sodiumchloride, Bacto agar 14 g. The broth does not contain the agar component.

• Mycoplasma supplement = per 30 ml vial is Bacto yeast extract 0.01 g, 1.6 g desiccated horseserum.

• Mycoplasma supplement S = Per 30 ml vial contains Bacto yeast extract 0.01 g, 1.6 g ofdesiccated horse serum, 55,000 units penicillin, 50 mg thallium acetate.

• Bacto heart infusion broth (Difco) contains 500 g beef heart infusion, 10 g bacto-tryptone, 5 gsodium chloride.

• The addition of 0.2% DNA can be added to the above medium. DNA is recommended forprimary isolation of the bovine mycoplasma M. bovigenitalium, and is thought to stimulate thegrowth of other Mycoplasma species as well. Therefore it is recommended as an optional mediacomponent (Freundt, 1983).




Mycoplasma medium – modified Hayflick medium (Chanock et al., 1962) (Modified and usedfor isolation of Mycoplasma mobile; Kirchhoff and Rosengarten, 1984. Also used for many animalmycoplasma species)

Reagent Amount Preparation of media Description of growth characteristicsBacto PPLO broth (Difco) 16.8 g Dissolve PPLO broth in distilled water and

autoclave at 121°C for 15 min.Cool to 50°C for agar containing media, orroom temperature for broths, and asepticallyadd other filter-sterilized ingedients.Dispense in 3 ml volumes to bijou tubes forbroths, or 5 ml volumes to small Petri dishesfor agar plates.

See above under Mycoplasmamedium – general-purpose medium.Horse or bovine serum 200 ml

Yeast extract (50% w/v) 10 mlDNA 0.02 gPenicillin 2000 IU/mlThallium acetate (1.25% w/v) 10 mlDistilled water 800 ml

For agar plates add 1.0% w/v purified agar (Oxoid) or use PPLO agar. Note: Handle thallous acetate with caution. Use gloves andmask.

Mycoplasma medium (Used for isolation of Mycoplasma from crocodiles; Kirchhoff et al., 1997)

Reagent Amount Preparation of media Description of growth characteristicsBrain heart infusion broth (Oxoid) 37 g Dissolve brain heart infusion, yeast extract

and glycerol in distilled water. Glycerol canbe warmed slightly to facilitate pipetting.Autoclave at 121°C for 15 min. Prepareother reagents and filter sterilize. Addeach reagent to cooled autoclaved media.Dispense into 5 cm Petri dishes. Store inplastic bags at 4°C.

Incubate plates in a candle jar orsimilar CO2 generating atmosphere,at 37°C.

Yeast extract (Oxoid) 2 gGlycerol 8 mlThallium acetate (10% w/v) 2.5 mlAmpicillin (5% w/v) 2 mlNAD (1% w/v) 10 mlDistilled water 700 ml

NAD, nicotinamide adenine dinucleotide; TTC, tetrazolium chloride.Kirchhoff et al. (1997) also used this medium as a base to study the metabolism of glucose, arginine hydrolysis, TTC andphosphatase activity.

Nutrient agar (Used as a general isolation medium)

Reagent Amount Preparation of media Description of growth characteristicsNutrient agar (Oxoid) 14.0 g Add agar powder to distilled water. Autoclave at 121°C

for 15 min. Cool to 50°C and dispense into Petri dishes.Store at 4°C.


Organic growth requirements

Some strains of Vibrio, e.g. Vibrio ordalii (previously V. anguillarum type II), Moritella (Vibrio) marinaand some strains of Vibro (Photobacterium) logei need 0.05% yeast extract for growth (Baumannet al., 1980). A 10× stock solution of yeast extract can be prepared and then 250 ml added to 5 ml ofbroth media.

254 Chapter 7



Packer’s plates (Selective medium for Erysipelothrix; Packer, 1943)

Reagent Amount Preparation of media Description of growth characteristicsColumbia blood agar base(Oxoid)

20.0 g Add agar base to water andautoclave at 121°C for 15 min.Cool to 50°C and asepticallyadd sodium azide, crystal violetand blood. Mix well and pourinto Petri dishes.

Colonies are 0.5–1 mm at 24–48 h. It is suggestedthis medium is used in conjunction with Wood’sbroth. Approximately 1 g of chopped or mincedtissue or sample material is placed into 10 ml ofWood’s broth. Incubate at 25°C and subculturefrom broth to BA and Packer’s plates at 24 and48 h. Examine plates at 24 and 48 h for typicalcolonies. Colonies of Erysipelothrix rhusiopathiaeare 0.5–1 mm, grey-green at 48 h, with slighta-haemolysis around the colony. They resemblea-haemolytic Streptococci.

See photographic section.


Crystal violet (stock = 0.25 gin 100 ml of distilled water)

2 ml Prepare stock solutions ofcrystal violet and sodium azide.Autoclave at 121°C for 15 min.Sodium azide (NaH3)

(stock = 1 g in 100 ml ofdistilled water)

12.5 ml

Pasteurella multocida selective media. NB plates (Rutter et al., 1984)

Reagent Amount Preparation of media Description of growth characteristicsColumbia agar base (Oxoid) 7.8 g Add agar to distilled water and autoclave

at 121°C for 15 min. Cool to 50°C andaseptically add blood and 1 ml of antibioticstock. Mix well and pour into Petri dishes.

This is a semi-selective medium.Colonies of Pasteurella multocidaappear as grey non-haemolyticcolonies 1–2 mm and may beslightly mucoid.


Neomycin sulphate (2.0 mg/ml) 4.0 mg Prepare antibiotic stock by addingneomycin and bacitracin to normal saline.Add 1 ml to cooled agar and blood mixtureas described above.

Bacitracin (3.5 mg/ml) 7.0 mgNormal saline 10 ml

Peptone yeast medium – PY (For growth of Cytophaga, Flavobacterium and Sphingobacteriumspecies; Takeuchi and Yokota, 1992)

Reagent Amount Preparation of media Description of growth characteristicsPeptone 1 g Add all reagents to distilled water and pH to 7.0. Autoclave

at 121°C for 15 min. Cool to 50°C and dispense into Petridishes.

Can be prepared as a broth by omitting the agar.

For growth of Pedobacter heparinus,Sphingobacterium spiritivorum, S.multivorum and Flavobacteriumspecies. Incubate at 28°C for 2 days.

Yeast extract 0.2 gGlucose 0.2 gNaCl 0.2 gAgar 1.5 gDistilled water 100 ml

Poly plates (See under Dermatophilus congolensis)

PYS-2 Medium (For growth of Vibrio rumoiensis; Yumoto et al., 1999)

Reagent Amount Preparation of media Description of growth characteristicsPolypeptone 8 g Add all reagents to distilled water and pH to 7.5.

Autoclave at 121°C for 15 min. Cool to 50°C andaseptically dispense into Petri dishes.

Colonies of Vibrio rumoiensis are circularand colourless. Growth can occur between2 and 34°C with an optimum at 27–30°C.

Yeast extract 3 gNaCl 5 gAgar 15 gDistilled water 1000 ml




R2A agar (Oxoid CM 906, or Difco) (Used for isolation of Antarctic Flavobacterium spp.;McCammon and Bowman, 2000)

Reagent Amount Preparation of media Description of growth characteristicsAgar CM906 18.1 g Add powder to distilled water and adjust

pH to 7.2. Boil to dissolve. Sterilize byautoclaving at 121°C for 15 min. Cool to50°C and pour into Petri dishes.

This is a nutritionally reduced medium that enhancesrecovery of heterotrophic bacteria from treatedwaters, and assists in recovery of bacteria that arestressed or chlorine-tolerant. The sodium pyruvateincreases the recovery of stressed cells.


Composition g/l of Oxoid CM 906: yeast extract (0.5), tryptone (0.25), peptone (0.75), dextrose (0.5), starch (0.50), di-potassiumphosphate (0.3), magnesium sulphate (0.024), sodium pyruvate (0.3), agar (15.0).

Rogosa acetate agar (Oxoid). See under ‘Tests’ section

Strontium chloride B enrichment broth (Use for isolation of Salmonella spp. and Edwardsiellatarda; Iveson, 1971)

Reagent Amount Preparation of media Description of growth characteristicsBacto tryptone (Difco) 0.5 g Add all reagents to the

distilled water. Dispenseinto 10 ml volumes inMcCartney bottles andautoclave at 121°C for20 min.

Final concentration ofstrontium chloride is3.4% and pH is 5.0–5.5.

Place macerated or chopped sample (0.5 ml) or swab ofsample into broth. Incubate at 37°C for 24 and 48 h.Subculture from broth on to selective media of MCA or DCA.

On MCA plates the colonies of Edwardsiella tarda andSalmonella spp. appear as non-lactose fermenting colonies.On DCA the colonies appear as pale pink to clear colonieswith or without a black centre (H2S). E. tarda colonies areslightly smaller in size than Salmonella and the black centretakes longer to develop.

Sodium chloride 0.8 gPotassium dihydrogenphosphate

0.1 g

Strontium chloride 60% 6.0 mlDistilled water 100 ml

Seawater – artificial. See under artificial seawater

Serum-dextrose agar (Non-selective medium for Brucella species; Alton and Jones, 1967)

Reagent Amount Preparation of media Description of growth characteristicsNutrient agar 95 ml Prepare nutrient agar and cool to 50°C. Add

5 ml of a stock solution of serum-dextrose per95 ml of nutrient agar. Mix and pour into Petridishes.

After 4 days growth, smoothcolonies of Brucella are small,round, glistening and blue orblue-green in colour whenexamined by obliquely reflectedlight. Rough colonies areyellow-white in colour and have adry granular appearance.

Serum-dextrose 5 ml to 95 ml ofnutrient agar

Stock solution ofserum-dextrose

1 g of dextrose per5 ml of serum

For a stock solution of serum-dextrose, dissolvepure dextrose (glucose) in inactivated serum,at a rate of 5 g of dextrose per 5 ml of serum.Filter-sterilize and store at 4°C or −20°C.

This medium is available from Oxoid as Brucella medium base (Code CM169). Inactivated horse serum (5%) is added to theautoclaved base medium.

256 Chapter 7



Shieh medium + Tobramycin (SM-T) (Selective medium for Flavobacterium columnare andF. psychrophilum; Decostere et al. 1997; Shieh, 1980)

Reagent Amount Preparation of media Description of growth characteristicsPeptone (Difco) 5 g Suspend all chemicals into

1000 ml of distilled water and pHto 7.2. Autoclave at 121°C for15 min. Cool to 50°C and addedfilter-sterilized solution oftobramycin. Mix and pour mediainto Petri dishes.

Ensure the moisture contentremains in the media by storingplates at 4°C in sealed plasticbags. Growth of F. columnare isoptimal when the plates are fresh,but reduces as the surfacemoisture decreases.

Using a cotton-tipped swab, collect samples fromskin lesions and gills of fish and inoculate theplate. Colonies of F. columnare appear at 2–5days as yellow-pigmented, rhizoid colonies with athin spreading growth. See photographic section.

Early colonies may be viewed with the aid of astereomicroscope and a plug of the colony cut outusing an unsealed Pasteur pipette prepared in theshape of a ‘hockey stick’. Subculture the colonyplug to a fresh plate.

Shieh medium plus tobramycin reduces thegrowth and colony size of Aeromonas hydrophilaand prevents growth of A. salmonicida, S.putrefaciens and Ps. fluorescens.

Yeast extract (Difco) 0.5 gSodium acetate 0.01 gBaCl2 (H2O) 2 0.01 gK2HPO4 0.1 gKH2PO4 0.05 gMgSO47H2O 0.3 gCaCl22H2O 0.0067 gFeSO47H2O 0.001 gNaHCO3 0.05 gTobramycin 0.5 mgNoble agar (Difco) 10 gDistilled water (pH 7.2) 1000 ml

Note: The original method states 1 mg/ml of tobramycin; however, 0.5 mg/ml is recommended for Australian strains of F. columnareas growth may be inhibited or reduced at 1 mg/ml of tobramycin (Dr Annette Thomas, Department of Primary Industries,Queensland, 2000, personal communication).

SKDM. (Selective medium for Renibacterium salmoninarum, Austin et al., 1983)

Reagent Amount Preparation of media Description of growth characteristicsTryptone 1.0 g Add all reagents to distilled water and adjust

pH to 6.8. Autoclave at 121°C for 15 min, thencool to 50°C.

Inoculate plates with infectiousmaterial and incubate in a humidatmosphere at 15°C for 12 weeks.Examine plates regularly for thepresence of pin-point colonies.Maximum size of colonies is 2 mm.Colonies are white or creamy, shiny,smooth, raised and round. Oldcolonies may have a granular orcrystalline appearance.

Yeast extract 0.05 gCycloheximide 0.005 gAgar 1.0 gDistilled water 100 mlFetal calf serum 10.0 g Filter-sterilize L-cysteine hydrochloride,

D-cycloserine, polymyxin B sulphate andoxolinic acid. Add these plus sterile fetal calfserum to autoclaved media. Pour into plates.

L-cysteine hydrochloride 0.1 gD-cycloserine 0.00125 gPolymyxin B sulphate 0.0025 gOxolinic acid 0.00025 g

Shewanella marine agar (SMA) (Used for isolation of Shewanella woodyi; Makemson et al., 1997)

Reagent Amount Preparation of media Description of growth characteristicsBacto peptone (Difco) 5.0 g Add all reagents to distilled water.

Autoclave at 121°C for 15 min.A medium suitable for growth of luminousmarine bacteria, especially Shewanella.Bacto yeast extract (Difco) 1.0 g

Bacto agar (Difco) 15.0 g1x sea salts (see below) 200 mlDistilled water 1000 ml

5× sea salts stock: 2.58 M NaCl, 0.125 M MgCl2, 0.125 M MgSO4, 0.1 M KCl, distilled water 1000 ml, pH to 7.5.




Siem selective medium (Selective medium for Aerococcus viridans; Stewart, 1972; Gjerde, 1984)

Reagent Amount Preparation of media Description of growth characteristicsGlucose 6.5 g Add powders to distilled water and

dissolve. Adjust pH to 7.4. Autoclave.Growth of Gram-positive cocci is promotedwith a change of medium from purple toyellow because of acid production.Incubate at 25°C for 5 days. Subcultureyellow-coloured broth to BA.

Yeast extract 4.5 gTryptone 15.0 gNaCl 6.4 gPhenyl ethyl alcohol 2.5 gBromocresol purple 0.008 gDistilled water 1000 ml

Skirrow’s medium See under Helicobacter selective medium.

SWT (A seawater-based complex medium for growth of marine organisms; Nealson, 1978; Boettcheret al., 1999)

Reagent Amount Preparation of media Description of growth characteristicsTryptone 0.5 g Add reagents to distilled water and autoclave at 121°C

for 15 min. Cool to 50°C and aseptically addfilter-sterilized seawater. Pour into Petri dishes.

Used for cultivation of Vibrio fischeri,Roseobacter spp., Stappia spp.Yeast extract 0.3 g

Glycerol 0.3 gSeawater 70 mlDistilled water 30 mlAgar 1.2 g

TCBS cholera medium (TCBS) (Semi-selective for growth of Vibrio spp.; Kobayashi et al., 1963;Nicholls et al., 1976)

Reagent Amount Preparation of media Description of growth characteristicsTCBS choleramedium (Oxoid)

44.0 g Add powder to distilled water.Autoclave at 121°C for 15 min.Cool to 50°C and dispenseinto Petri dishes. Store at 4°C.

This selective medium will grow the majority of Vibrio species.Some strains, however, such as Vibrio (Listonella) ordalii do notgrow on this medium. Aeromonas spp. and Pseudomonas willgrow weakly as small colonies approximately 1 mm at 24–48 h.

Vibrio species that ferment sucrose appear as yellow colonieswhereas sucrose-negative isolates appear as green colonies.


Notes:

• Some Vibrio species grow poorly on TCBS and a few strains do not grow at all on this medium.Some brands of TCBS are more inhibitory than others. Eiken and Oxoid brands supported thegrowth of a greater number of Vibrio species than BBL or Difco brands, in particular for V.cholerae and non-cholera Vibrio spp. Also, it is recommended that each batch of medium istested for growth of Vibrio spp. (Nicholls et al., 1976).

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• TCBS detects a lower number of Vibrio colonies than MSA-B when performing TBCs on water.Colonies that grow on MSA-B and not on TCBS on primary culture often grow on TCBS whensubcultured to that medium, and identify as Vibrio spp. biochemically.

• After prolonged incubation, sucrose-fermenting colonies that initially appeared yellow may turngreen once the sucrose in the medium has been used up or the pH of the medium changes.

• Formulation: yeast extract (5 g), peptone (10 g), sodium thiosulphate (10 g), sodium chloride(10 g), ox bile (8 g), sucrose (20 g), NaCl (10 g), ferric chloride (1 g), bromothymol blue (0.04 g),thymol blue (0.04 g), agar (1.4 g), water (1000 ml) to pH 8.6.

Tryptone soya agar (TSA) (Used as a general-purpose isolation medium)

Reagent Amount Preparation of media Description of growth characteristicsTSA (Oxoid) 20.0 g Dissolve TSA in water and autoclave at 121°C

for 15 min. Cool in waterbath to 50°C and pourinto Petri dishes to depth of approximately 3 mm.Store at 4°C.

Suitable as a general-purpose medium.Add NaCl (7.5 g) to give a finalconcentration of 2% to isolate bacteriafrom marine sources.


Tryptone yeast extract salt medium (TYES) (Used for the growth of Flavobacterium columnare;Triyanto and Wakabayashi, 1999)

Reagent Amount Preparation of media Description of growth characteristicsTryptone 0.4 g Dissolve reagents in water and autoclave at 121°C

for 15 min. Cool in waterbath to 50°C and pour intoPetri dishes to depth of approximately 3 mm. Storeat 4°C.

Used for the growth of F. columnare.Incubate plates at 25°C for 24 h.Colonies appear as yellow-pigmented,rhizoid, mucoid, or honeycomb-shapedcolonies.

Yeast extract 0.04 gMgSO4.7H2O 0.05 gCaCl2.10H2O 0.05 gDistilled water 100 ml

Tryptone yeast extract glucose agar (TYG) (Used for growth of F. psychrophilum, F. columnare,T. maritimum, Cipriano et al., 1996)

Reagent Amount Preparation of media Description of growth characteristicsTryptone 0.2 g Add all reagents, except

neomycin, to distilled water.Autoclave at 121°C for 15 min.Cool to 50°C, and asepticallyadd filter-sterilized neomycin.Pour into Petri dishes.

Colonies are yellow with a thin spreading margin, whichindicates the ability of the organism to exhibit glidingmotility. Optimal temperature for F. psychrophilum is14–20°C, for F. columnare 22–30°C, and 15–34°C forT. maritimum. Plates are examined daily for up to 7 days.Examination for colonies using a stereomicroscope mayassist in early detection of characteristic colonies.

Yeast extract 0.05 gGlucose 0.3 gAgar 1.5 gDistilled water 100 mlNeomycin sulphate 0.4 mg

Addition of 10 IU/ml of polymyxin B sulphate may assist as a selective medium for F. columnare (Shamsudin and Plumb, 1996).

TYG-M. Addition of 4 mg/ml neomycin sulphate, 200 IU/ml polymyxin B and 18.7 g/l ASW can beused for isolation of Tenacibaculum (Flexibacter) maritimum (Chen et al., 1995).




VVM Selective medium for Vibrio vulnificus (Cerdà-Cuéllar et al., 2001)

Reagent Amount Preparation of media Description of growth characteristicsD-cellobiose 15.0 g Dissolve all reagents in distilled water

and bring to boil. Cool to 50°C andadjust pH to 8.5 using 5 M NaOH. (Thismedia does not require autoclaving.)

VVM plates are violet-blue in colour.Vibrio vulnificus is seen as a brightyellow colony with a yellow halo ofdiffusion due to the fermentation ofcellobiose. Other cellobiose fermentingVibrio spp. such as V. campbellii,V. harveyi and V. navarrensis will growon VVM agar. Some strains ofV. aestuarianus, V. alginolyticus,L. anguillarum may also grow on thismedium.

NaCl 10.0 gYeast extract 4.0 gMgCl2.6H2O 4.0 gKCl 4.0 gCresol red 40.0 mgBromothymol blue 40.0 mgPolymyxin B 105 U/lColistin methanesulfonate 105 U/lAgar 15.0 gDistilled water 1000 ml

A modification of this medium, termed VVMc, has the same reagents except the polymyxin B, and recovery rates are virtually thesame (Cerdà-Cuéllar et al., 2001).

VAM (Presumptive differentiation medium for Listonella (Vibrio) anguillarum; Alsina et al., 1994)

Reagent Amount Preparation of media Description of growth characteristicsSorbitol 15 g Dissolve all reagents (except

ampicillin) in distilled water, andboil. Cool to 50°C. Adjust pH to 8.6with 5 M NaOH. Aseptically addampicillin. Pour into Petri dishes.

Store at 15°C. Plates are viablefor 3 weeks. After this time theampicillin begins to lose activity.

The uninoculated medium is a violet-blue colour. After48 h incubation at 25°C, L. anguillarum colonies areflat, round and bright yellow with a diffuse yellow halodue to the fermentation of sorbitol.

V. fluvialis, V. harveyi and V. metschnikovii are ableto grow as yellow colonies on VAM, and therefore mustbe differentiated biochemically from L. anguillarum.V. alginolyticus grows as a non-swarming blue colony.

Yeast extract 4 gBile salts 5 gNaCl 35 gAmpicillin 10 mgCresol red 40 mgBromothymol blue 40 mgAgar 15 gDistilled water 1000 ml

Wood’s broth (Selective broth for detection of Erysipelothrix rhusiopathiae; Wood, 1965)

Reagent Amount Preparation of media Description of growth characteristicsBacto tryptose (Difco) 7.5 g Add tryptose, Lab Lemco and NaCl

to distilled water, pH to 7.5 andautoclave at 121°C for 15 min.Cool to room temperature.

Place swab of material or choppedpieces of tissue into medium. Incubateat 37°C for 48 h. Subculture to BA at24 and 48 h.

Colonies of Erysipelothrix are 0.5 mmto 1 mm, grey-green at 48 h, with slighta-haemolysis around the colony. Theyresemble a-haemolytic Streptococci.


Lab Lemco powder (Oxoid) 1.5 gNaCl 2.5 gDistilled water 500 mlSterile horse serum (not inactivated) 25 ml Add antibiotics to 10 ml of sterile

distilled water. Aseptically add horseserum and antibiotic solution to cooledbase. Dispense 10 ml volumes toMcCartney bottles and store at 4°C.

Kanamycin 200 mgNeomycin 25 mgVancomycin 12.5 mg

Note: Wood and Packer (1972) modified this medium by replacing the distilled water with 0.1 M phosphate buffer (12.02 gNa2HPO4, 2.09 g of KH2PO4 and 1000 ml distilled water).

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Yersinia selective agar (Yersinia ruckeri does not grow on this medium)

Reagent Amount Preparation of media Description of growth characteristicsYersinia selectiveagar base (Oxoid)

29.0 g Add Yersinia selective base to distilled water andautoclave at 121°C for 15 min. Cool to 50°C.

Yersinia species grow as pinkcolonies with a darker ‘bulls eye’centre at 24–48 h.Distilled water 500 ml

Oxoid YersiniaSelective Supplement

2 ml Reconstitute 1 vial of supplement by aseptically adding1 ml distilled water and 1 ml ethanol. Mix to dissolveand aseptically add to base. Pour into Petri dishes.

7.2 Biochemical Test Media

Aesculin

Reagent Amount Preparation of media Description of testAesculin 0.2 g Dissolve all chemicals,

except aesculin, by boiling.Cool and add aesculin whilestirring. Dispense 5 ml ofmedia into tubes. Autoclaveat 121°C for 15 min.

Store tubes in the dark.

Bacterial cells are inoculated into aesculin broth and incubated atappropriate temperature for 24–48 h. Development of a black colouris positive. Because some bacteria, especially some Vibrio, causeblackening of the medium due to melanin production, true hydrolysisof aesculin must be tested for loss of fluorescence at 354 nmlongwave UV light (MacFaddin, 1980; Choopun et al., 2002).Fluorescence indicates a negative reaction.

Ferric citrate 0.1 gPeptone water(Oxoid)

3.0 g


Arginine dihydrolase (Møller). See under Decarboxylases

Arginine dihydrolase (Thornley). See under Decarboxylases

Brucella: Assay of metabolic activity (Alton and Jones, 1967; Jahans et al., 1997)

Reagent Amount Preparation of media Description of testL-alanine 1.25 g/l Dissolve various metabolic

substrates in sterile PBS,pH 7.2 at a concentrationof 1.25 g/l. Sterilize eachsolution by filtration through0.22 mm filter membrane.Store at 4°C.

Prepare MTT solution inPBS at 1.0 g/l. Sterilize byfiltration through a 0.22 mmfilter.

Substrate mediated metabolic activity is assayedby the stimulation of tetrazolium reduction.

Prepare a cell suspension in PBS and adjust theconcentration to 1010 organisms per ml. In aflat-bottomed microtitre plate, place 100 ml ofeach substrate into each well. Place 50 ml of cellsuspension in each well and incubate at 37°C in10% CO2 for 18 h.

After incubation, place 50 ml of MTT in each well.Incubate 1 h at room temperature and add 50 mlof formaldehyde to each well. After 2–4 h, readthe optical density at 630 nm. The metabolicindex for each substrate is expressed as a ratioof the OD with substrate against the OD withoutsubstrate.

L-asparagine 1.25 g/lL-glutamic acid 1.25 g/lL-arginine 1.25 g/lDL-ornithine 1.25 g/lL-lysine 1.25 g/lD-galactose 1.25 g/lD-ribose 1.25 g/lD-xylose 1.25 g/lMeso-erythritol 1.25 g/lUrocanic acid 1.25 g/l3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

1.0 g/l

Formaldehyde 40%




Brucella: growth on substrate media (Alton and Jones, 1967; Jahans et al., 1997)

Reagent Amount Preparation of media Description of testBasic fuchsin 20 mg/ml

(1/50,000)A 0.1% stock solution of each dye is made in distilledwater, and sterilized by being held in boiling water for1 h. Stock solution should be renewed after 3 months.

Each dye is then added to a base media such astryptone-soy agar or serum-dextrose agar. Alton andJones (1967) suggest that a laboratory shoulddetermine the concentration of each dye that givesoptimum results, the range being somewhere between1:25,000 and 1:100,000 (10–40 mg of dye per ml ofmedium), using the FAO/WHO reference strains.The concentrations in the ‘amount’ column aresuggested by Jahans et al. (1997).

The required amount of the dye stock solution is addedto the melted base medium, mixed and poured intoPetri dishes and allowed to set. Label each plate withappropriate dye identification.

Prepare a suspension of bacterial cellsin 0.5 ml of sterile normal saline.

The unknown culture should beinoculated on to the same plate alongwith the reference strains. Thereforemark off an appropriate number ofareas on a plate. Using a loop ofculture suspension, make five separatestreaks without recharging the loop onto each specifically marked area. Alsoinoculate a control plate that containsbasic medium only and no dye.Incubate plates at 37°C with andwithout CO2 for 4 days.

Record results of ‘growth’ or ‘nogrowth’ in the presence of each dye.

Safranin O 100 mg/ml(1/10,000)

Thionin 20 mg/ml(1/50,000)

Brucella selective media. See Farrell’s medium

Carbohydrate fermentation (General-purpose medium for detection of carbohydrate fermenta-tion; Vera, 1948, 1950)

Reagent Amount Preparation of media Description of testCystine tryptic medium (Gibco) 7.13 g Basal medium preparation

Boil distilled water and cysteine tryptic medium.Dispense 5 ml volumes into 10 ml tubes.

Carbohydrate preparationTo prepare the carbohydrates, make a 10% solution(3 g in 30 ml).For salicin use a 4% solution (1.2 g in 30 ml ofdistilled water).

For carbohydrates that can be autoclavedPipette 0.5 ml of carbohydrate solution per 5 ml ofbasal medium. Autoclave at 10 lb per 10 min.

For filter-sterilizationFilter-sterilize the carbohydrate solution through a0.22 mm filter.Autoclave basal medium 5 ml in 10 ml tubes. Cool to50°C and aseptically add the filter-sterilizedcarbohydrate solution, 0.5 ml to 5 ml of basal medium.

Fermentation isdetected by a colourchange from red toyellow.

References may statethat carbohydratesare added at 0.5% to1.0%. However,because reversion ofthe reaction mayoccur when 0.5% isused, using aconcentration of 1%ensures againstdepletion of thecarbohydrate by theorganism.

Distilled water 250 mlCarbohydrate (see below) Sterilization

methodCarbohydrateArabinoseAesculinGlucoseInositolLactoseMaltoseMannitolMannoseSalicinSorbitolSucroseTrehaloseXylose

SterilizationFilterAutoclaveAutoclaveAutoclaveAutoclaveFilterAutoclaveFilterAutoclaveAutoclaveFilterAutoclaveFilter

1% finalconcentration

Cystine tryptic medium contains Bacto tryptose, L-cystine, sodium chloride, sodium sulphite, agar and phenol red. With the additionof yeast extract at 0.01%, this medium is also suitable for testing carbohydrate fermentation of the Cytophaga, Flavobacterium,Flexibacter, Tenacibaculum group, plus certain Vibrio species that require yeast extract, such as V. ordalii, Moritella marina andsome strains of V. logei. Baumann et al. (1980) adds yeast extract at 0.05% for strains of Vibrio and Photobacterium spp. thatrequire organic growth factors.

Notes:

• In the literature, some original recipes used bromothymol blue as the pH indicator; however, itmay be toxic to a number of marine bacteria, and therefore phenol red is the recommended pHindicator in carbohydrate fermentation tests (Leifson, 1963).

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• Peptone-containing media may give misleading results for carbohydrate breakdown becauseweak acid production may be masked by peptone-breakdown products (Vera, 1950).

• Sucrose should be filter-sterilized, as it is thermolabile and will give false positive results if sterilizedby autoclaving (Stanier et al., 1966).

• To prevent colour change of the medium caused by CO2 build-up in screw-capped tubes wheninoculated with organism of interest, ensure that lids are loosened for about an hour before resultsare read. Preparing tubed media to only contain half their volume in media also helps to preventpH reversion caused by CO2 build-up.

• Most carbohydrates can be stored at room temperature for 2–3 weeks, with the main stocks storedat 4°C for longer term storage. However, some carbohydrates should always be stored at 4°C toprevent unwanted colour change in the medium. These are arabinose, mannitol, xylose and alsothe oxidative-fermentative media and ONPG media.

Alternative methods for carbohydrate fermentation for specific bacteria

Carbohydrate fermentation media for Flavobacterium spp.

• Broth medium containing 0.05% tryptone, 0.05% yeast extract, 0.0018% phenol red, and filter-sterilized carbohydrate at a final concentration of 0.2% (Wakabayashi et al., 1986).

• AO medium was used as a basal medium for carbohydrate fermentation; however, it wasunsuitable for this test, as colour changes were seen in the negative control tube (Bernardet andGrimont, 1989).

Carbohydrate fermentation media for marine bacteria (Leifson, 1963, used by Gauthier,1976b)

Reagent Amount Preparation of media Description of testCasitone (Difco) 0.1 g The ingredients are dissolved in half-strength

seawater and the pH is adjusted with HCl to 7.5.Autoclave and re-check the pH, which shouldbe about 8. Adjust with HCl if necessary.Filter-sterilize the carbohydrate and addaseptically to the base medium. Dispense 3 mlinto 13 × 100 mm tubes.

Fermentation is indicated by achange in colour from red to yellow.Yeast extract 0.01 g

Ammonium sulphate 0.05 gTris buffer 0.05 gAgar 0.3 gPhenol red 0.001 gArtificial seawater Half strengthCarbohydrate 1%

Notes: Einar Leifson (1963) modified this recipe from the original medium published in 1953 (Hugh and Leifson, 1953). The pHindicator in the 1953 medium (bromothymol blue) was found to be toxic for a number of marine bacteria and therefore phenol red isrecommended. This media is also used to test for oxidation/fermentation. Add individual carbohydrates to the base medium forfermentation tests. This method is similar to the general-purpose medium.

Carbohydrate fermentation media for Pseudomonas and Flavobacterium spp. (Gilardi,1983)

Reagent Amount Preparation of media Description of testOF basal medium(Difco)

9.4 g Add reagent in distilled water and dissolve by boiling.Dispense into 100 ml aliquots. Autoclave at 121°C for15 min. To each 100 ml aliquot aseptically add 10 ml ofa 10% carbohydrate solution. Aseptically dispense each100 ml of respective carbohydrate into 5 ml aliquots in10 ml tubes.

Colour change to yellow indicates apositive fermentation reaction.


Carbohydrate 10%solution

10 ml Filter-sterilize and add as above.

Composition of Difco OF medium: tryptone (2.0 g), sodium chloride (5.0 g), di-potassium phosphate (0.3 g), agar (2.0 g),bromothymol blue (0.08 g), in 1000 ml distilled water, pH 6.8.




Carbohydrate fermentation media using acid from ammonium salt sugar (ASS). Used forSphingobacterium, Flavobacterium, Shewanella putrefaciens, Pseudomonas spp. Used by Holmeset al. (1975) for testing Pseudomonas spp. Used by Bernardet and Grimont (1989) as an inoculationmedium for API 50CH when testing Flavobacterium. Quoted in Cowan and Steel (1970) from themethod of Smith et al. (1952).

Reagent Amount Preparation of media Description of test(NH4)2HPO4 1.0 g Add ingredients to distilled water and dissolve

by boiling or steaming.

Add indicator and autoclave at 115°C for20 min.

Allow medium to cool to 60°C and addfilter-sterilized carbohydrate solution.

Dispense into tubes and incline tube so thatmedium sets as a slope.

Inoculate and incubate at theappropriate temperature.Examine over 7 days. A colourchange from purple to yellow isindicative of fermentation ofcarbohydrate.

KCl 0.2 gMgSO4.7H2O 0.2 gYeast extract 0.2 gAgar 20 gDistilled water 1000 mlBromocresol purple 0.2% solution 4 mlCarbohydrate 10% solution 100 ml

Notes:

• Bacteria that do not give a reliable fermentation reaction with peptone-containing medium shouldbe tested in ASS. In general, peptone-containing media give the least positive reactions and ASSthe most positive reactions (Cowan and Steel, 1993 edition).

• Reactions using this medium are not reported in this book. Flavobacterium spp., reactionsreported in this book were the same by both the peptone-containing method (Carbohydrategeneral purpose medium) and ASS.

Carbohydrate fermentation media for Vibrio spp. (The following media were used by Baumannet al. (1971) for Vibrio spp. and Beneckea – adapted from methods of Stanier et al., 1966)

• Basal medium (BM): 50 mM tris (hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 7.5),190 mM NH4Cl, 0.33 mM K2HPO4.3H2O, 0.1 mM FeSO4.7H2O and half strength seawater.

• Basal medium agar (BMA): Mix equal volumes of double-strength BM with 20 g of Ionagar(Oxoid) per litre.

• Yeast extract broth (YEB): Add 5 g/l of yeast extract (Difco) to BM.• For Yeast extract agar (YEA) add 20 g of agar (Difco) per litre of YEB.• Carbohydrate fermentation medium (F-2): To YEB add 100 mM Tris-hydrochloride (pH 7.5),

1 g/l of Ionagar (Oxoid), 1 g/l of sodium thioglycollate, and 10 g/l of filter-sterilized glucose.• Carbohydrate fermentation medium (F-3): To BM add 25 mM Tris hydrochloride (pH 7.5),

0.5 g/l of yeast extract (Difco), 1 g/l of Ionagar (Oxoid), 2 ml/l of a 1.6% (w/v) alcoholic solution ofbromocresol purple, and 10 g/l of filter-sterilized carbohydrate.

Carbohydrate fermentation media for Nocardia spp. Basal inorganic nitrogen medium(Gordon et al., 1974, Quoted from Ayers et al., 1919)

Reagent Amount Preparation of media Description of test(NH4)2HPO4 1.0 g Add all reagents except

carbohydrate to distilled waterand adjust pH to 7.0. Dispense5 ml into tubes and autoclave at121°C for 15 min.

Aseptically add 0.5 ml of a 10%solution of each carbohydrate(autoclaved separately) to tubesand allow to set on a slant.

Production of acid due tofermentation of the carbohydrateis seen as a colour change frompurple to yellow.Incubate at 28°C and read from7–28 days.

With the addition of yeast extract,this medium is the same as ASS.

KCl 0.2 gMgSO4.7H2O 0.2 gAgar 15Distilled water 1000 mlBromocresol purple 0.04% solution 15 mlCarbohydrates to be tested (10% solution) 0.5 mlOptionalYeast extract (Difco)

Note: When testing N. seriolae, add 2% w/v yeast extract (Difco) to medium (Kudo et al., 1988).

264 Chapter 7



Carbon dioxide atmosphere (Used when incubation conditions require a carbon dioxideatmosphere)For laboratories that do not have access to a carbon-dioxide incubator, the following methods offer acheap alternative.

Method 1: Candle jar method. Place agar plates in an airtight container. Place a lighted candle in thejar and seal the lid. This method produces 2% CO2 (J. Lloyd, Department of Agriculture, Western Aus-tralia, 1985, personal communication) with other reports suggesting 6–8% CO2 (Cottral, 1978). If the lidis plastic, then protect from the heat produced by the candle by using an aluminium foil-covered device.

Method 2: ‘ENO salts’ method (Lloyd, 1985). 7.5 g of ENO salts per 10 l container generates 4%CO2. Use an appropriate container that has a well-sealed lid such as a household food container orMitsubishi Gas Chemical Company anaerobe box. Place 3 g of ENO salts (Sigma) into 20 ml of water fora container that measures 22 × 22 × 8 cm or 10 g of ENO salts into 40 ml of water for a container thatmeasures 30 × 30 × 14 cm. This technique needs to be performed quickly, therefore place salts into asmall piece of tissue paper. Place the water into a 50 ml urine container and place into the incubationcontainer. Quickly add the salts to the water and seal lid. Place box at appropriate temperature. ENO is asparkling antacid that contains per 5 g, sodium bicarbonate (2.32 g), sodium carbonate (0.5 g), citricacid anhydrous (2.18 g) and which can be purchased through Sigma chemicals.

Catalase test (from Cowan and Steel, 1970)Smear a colony on to a glass slide. Place a drop of 30% H2O2 on the smear. The presence of bubblesindicates a positive reaction. When picking growth from a blood agar plate, make sure no blood-containing agar is placed on the slide, as false-positive results may occur.

Cellulose digestion (Wakabayashi et al., 1989)To a broth culture medium appropriate to the growth requirements of the organism being tested, add apiece of cellulose filter paper. Observe for disintegration of the filter paper. A characteristic of theCytophaga species is that they digest cellulose, therefore when testing an organism suspected of beinga Cytophaga species use a broth of Anacker-Ordal medium.

Coomassie Brilliant Blue agar (CBBA). Used to detect the A+ protein layer of Aeromonassalmonicida (Udey, 1982; Evenberg et al., 1985; Cipriano and Bertolini, 1988; Markwardt et al.,1989)

Reagent Amount Preparation of media Description of growth characteristicsTSA 44.0 g Add Coomassie Blue and

TSA to distilled water.Autoclave at 121°C for15 min. Cool to 50°C andpour into Petri dishes.

Coomassie Brilliant Blue is a protein-specific dye that resultsin production of dark blue colonies of bacteria that have theA-layer protein. A-layer positive A. salmonicida stain deepblue. This medium is not selective and other bacteria willproduce blue-coloured colonies. However, it assists in theisolation and detection of A. salmonicida.

Coomassie BrilliantBlue R250 (Bio-Rad)

0.1 g


Citrate: Simmons method (Simmons, 1926)

Reagent Amount Preparation of media Description of testSimmons citrate (Difco) 3.63 g Suspend media in distilled water, and

bring to the boil.

Dispense 3 ml of media into tubes.Autoclave at 121°C for 15 min. Layon a slight incline to create slopes.

Development of a deep blue colour is positive.Simmons citrate (Difco) contains 1% NaCl. Citrateis tested as a sole carbon source. (Christensen’scitrate does not test for citrate as the sole carbonsource, as it contains other nutrients).





Congo Red. Tests for presence of extracellular galactosamine glycan in the Flavobacteriaceae(Johnson and Chilton (1966) who quote personal communication with E.J. Ordal for this test;McCurdy, 1969)

Reagent Amount Preparation of media Description of testCongo Red 10 mg Weigh 10 mg of Congo Red and add

to 100 ml of distilled water. Finalconcentration is 0.01% aqueous. Mix well,label bottle and store at room temperature.

Place 1–2 drops of Congo Red on a few isolatedcolonies growing on AO or Shieh’s medium. A positivereaction is red-colour development of colonies. Thiscolour lasts for a number of hours. This test detectsthe presence of extracellular glucans.


Congo Red agar (Used for detection of A protein layer of A. salmonicida; Ishiguro et al., 1985)Prepare tryptic soy agar (Difco) containing 30 g/ml of Congo Red. Incubate at 20°C and examine forred-coloured colonies after 48 h.

Dienes stain (Used to differentiate Mycoplasma from Bacteria; Dienes, 1939; Hayflick, 1965).

Reagent Amount Preparation of media Description of testMethylene blue 2.5 g Dissolve all reagents in

distilled water.

Store in a screw-capbottle.

Method I. Place some stain on to a coverslip and allow to dry. Agarblocks containing colonies are excised and placed on to a glassslide with colonies uppermost. Place coverslip on to agar blockcontaining colonies and press coverslip lightly so that there is goodcontact between the colonies and the stain.

Method II. Using a cotton wool swab directly apply the stain to theMycoplasma colonies.

Mycoplasma colonies stain blue with a distinctly dense blue centreand a lighter blue periphery. Mycoplasma colonies retain the stainfor 24 h, whereas bacterial colonies decolorize after 30 min.

Azure II 1.2 gMaltose 10 gSodium carbonate(anhydrous)

0.25 g


Some methods contain 0.2 g of benzoic acid.

Digitonin (Used to differentiate Mycoplasma species from Acholeplasma species)

Reagent Amount Preparation of media Description of testDigitonin(1.5% w/v)

1.5 g Dissolve digitonin in ethanol.Warm to 37°C to dissolve.

Place 20 ml of stock solutionon to a 6 mm sterile filterpaper disc (Oxoid). Dry at37°C for 1 h.

Plate suspect Mycoplasma colony to a 5 cm Mycoplasma agar plate. Thiswill either be from a broth or agar plate. When transferring colonies froman agar plate, use a sterile spatula or open-ended ‘hockey stick’ (madefrom a bent Pasteur pipette), to select a plug of agar containing a colony.Invert this agar plug, colony side down, on to a fresh agar plate and rubover the surface. If inoculating the plate from a broth culture, pipette thebroth culture on to the plate and then remove the excess. Allow the plateto dry for a few hours. Place the digitonin disc in the middle of the plate,and incubate at appropriate temperature under CO2 for 3–4 days.

Mycoplasmas are sensitive to digitonin and so a 4 mm zone of inhibitionof growth is seen from the edge of the disc (zone of 14 mm diameter).

Acholeplasma are resistant to digitonin and a zone of less than 1 mm isseen from the edge of the disc.

Ethanol 100 ml

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DNase (Used by West and Colwell (1984) and reported from all sources used in this manual)

Reagent Amount Preparation of edia Description of testDNase test agar(Oxoid)

19.50 g Suspend agar in water in 1000 ml.Autoclave at 121°C for 15 min thencool to 50°C in waterbath. Mix welland pour media into Petri dishes.

Spot-inoculate or streak as a single line, a loop of bacterialculture on to the plate. Incubate plates for 2–7 days at24°C. Flood plate with 1% HCl to precipitate the DNA.A positive reaction is seen as a clear zone around thebacterial streak. See photographic section.


See under HCl for preparation of 1M HCl.

Decarboxylases and arginine dihydrolase (Used in Cowan and Steel, 1970, West and Colwell,1984)

Reagent Amount Preparation of media Description of testDecarboxylase base Møller (Difco)(Møller, 1955)

2.1 g In separate bottles,dissolve each individualamino acid in the 200 mlof distilled water anddecarboxylase base.Dispense 5 ml into 10 mltubes. Autoclave at 121°Cfor 15 min.

A tube of basal medium without amino acid isalways inoculated in parallel with the test media.Inoculate with heavy inoculum. Overlay all tubeswith paraffin oil, and incubate for 7–14 days forsome strains. Most strains can be read at 48 h.

Check the bottom of tube and the cloudiness of thetube for signs of sufficient inoculum and growth.

For salt-requiring strains, add 0.5 ml of 20% NaClto tubes before inoculation with organism.

Distilled water 200 mlL-arginine HCl 2.0 gL-ornithine monohydrochloride 2.0 gL-lysine monohydrochloride 2.0 g

Control – use decarboxylase baseand distilled water only

See under Paraffin oil for sterilization proceduresfor the oil.

Arginine dihydrolase (ADH) (Method of Thornley, 1960. Recommended for marine bacteria)

Reagent Amount Preparation of media Description of testBacto-Peptone(Difco)

0.1 g Add reagents to distilled water and pHto 6.8. Dispense 5 ml into 10 ml tubes.Autoclave at 121°C for 15 min.

Note: the original pH described byThornley was 7.2, however pH 6.8 isrecommended by West and Colwell (1984).

A tube of basal medium without amino acid is alwaysinoculated in parallel with the test media. Inoculatewith heavy inoculum. Overlay all tubes with paraffinoil, and incubate for 7–14 days for some strains.Most strains can be read at 48 h.

Check the bottom of tube and the cloudiness of thetube for signs of sufficient inoculum and growth.

NaCl 0.5 gK2HPO4 0.03 gAgar 0.3 gPhenol red 0.001 gArginine HCl 1.0 gDistilled water

Note: Some Vibrio species are negative in Møller’s method for ADH, but positive by the method of Thornley. These are; Vibriomediterranei, V. mytili, V. orientalis, Vibrio splendidus biogroup I and some strains of V. tubiashii (Macián et al., 1996). The glucosein Møller’s medium appears to inhibit the reaction due to catabolite repression of the inducible ADH system (Macián et al., 1996).Baumann et al. (1971) found that Photobacterium spp. can produce alkaline products in Thornley’s medium, yet none possesses aconstitutive ADH system when tested with more sensitive analytical methods (West and Colwell, 1984). Different brands of peptonewere found to give different results and the Difco bacto-peptone was recommended (Thornley, 1960).

Thornley’s medium was modified by Baumann and Baumann (1981) by using basal medium with-out the Tris-HCl. See under Alternative media for carbohydrate fermentation, media for Vibrio species,for this recipe.




Furunculosis agar (For detection of pigment production from Aeromonas salmonicida; Bernoth andArtz, 1989)

Reagent Amount Preparation of media Description of testBacto-tryptone (Difco) 10 g Add all ingredients to distilled water.

Adjust pH to 7.3, and autoclave at121°C for 15 min. Cool to 50°C andpour into Petri dishes.

Pigment production is best detected by incubatingat 15–20°C for up to 7 days. At 25°C less pigmentproduction is seen.

Pigment is seen as a diffuse brown colorationaround each colony.

Yeast extract (Difco) 5 gL-tyrosine (Merck) 1 gNaCl (Merck) 2.5 gAgar (Oxoid L11) 15 gDistilled water 1000 ml

Note: Furunculosis agar is recommended as a superior medium for detection of pigment production, as a greater number of strainsshow pigment on FA than on TSA, NA or BHIA (Hirvelä-Koski et al., 1994, Hänninen and Hirvelä-Koski, 1997). However, FA is notthe best medium for primary isolation of Aeromonas salmonicida (Bernoth and Artz, 1989) and BA is recommended. (It may bepossible that by adding blood to FA, the medium would be a suitable primary isolation medium.)

Gelatinase (0% AND 3% NaCl) (Smith and Goodner, 1958. Used by West and Colwell, 1984)

ReagentAmount0% NaCl

Amount3% NaCl Preparation of media Description of test

Difco bacto Peptone 1.0 g 1.0 g Add reagents to distilledwater and autoclave at121°C for 15 min. Cool to50°C.

Use split Petri dishes, andlabel plates 0% and 3%per side of each plate.Pour appropriate mediainto each half of each plate.

Store at 4°C.

Spot inoculate the gelatin plate on both the 0%NaCl and 3% NaCl sides with approximately equalamounts of bacterial growth. Incubate at anappropriate temperature 24–48 h.

Production of gelatinase is seen as either a cloudyor clear zone around the area of bacterial growth.The plate should be held up to the light and readagainst a darkish background. Chilling the platesmay provide a sharper contrast between zonesand the unaffected portions of the media. Floodingthe plate with ammonium sulphate may assist withdefinition of zones of clearing.

Difco yeast extract 0.25 g 0.25 gOxoid gelatin 3.75 g 3.75 gNaCl – 7.50 gOxoid agar No 1 3.75 g 3.75 gDistilled water 250 ml 250 ml

This plate method of Smith and Goodner (1958) is used as it detects a change in the composition of gelatin rather than itsliquefaction and thus is a very sensitive method. Either use a split plate with one side 0% NaCl and the other side containing 3%NaCl. This allows the salt requirement of the organism to be detected at the same time. Alternatively, use small (5 cm) Petri dishesfor each NaCl concentration.

Glucose yeast extract agar (GYEA) (For colonial morphology and survival at 50°C of Nocardiaspecies; Gordon et al., 1974)

Reagent Amount Preparation of media Description of testYeast extract 10 g Add reagents to water and pH to 6.8.

Autoclave at 121°C for 15 min. May bepoured into plates, or prepared as tubedslants.

Plate media is used for observation of colonialmorphology.

Tubed slants are used for testing survival at 50°C.

Glucose 10 gAgar 15 gTap water 1000 ml

HaemolysisRecord from growth on BA or MSA-B within 7 days. For some Vibrio species, haemolysis may bemore pronounced on BA than on MSA-B even though their preferred growth medium is MSA-B.

268 Chapter 7



HCl (1N) (For detection of hydrolysis of DNA in DNase medium)

Reagent Amount Preparation of media Description of testConcentrated HCl, 32% 9.85 ml Add 9.85 ml of 32% acid to 80 ml

water, then make up to 100 ml.To a 24 or 48 h growth on a DNase plate flood with1 M HCl. Wait 1 min and observe over a black tilefor zone of clearing around bacterial growth.



Safety note: Always add acid to water, not vice versa. Other concentrations of HCl are available. To prepare from 35.4%, add 8.9 mlto 80 ml of water, and then make up to 100 ml. To prepare from 37%, add 8.5 ml of acid to 80 ml of water, and then make up to100 ml.

Hippurate hydrolysis (Hwang and Ederer, 1975)

Reagent Amount Preparation of media Description of testHippuric acidsodium salt

0.15 g Dissolve hippuric acid inwater. Dispense 0.4 mlper sterile, 5 mlyellow-capped plastictube. Store at −20°C.

Inoculate medium with a large loopful of bacterial growth from cultureplate. Incubate at appropriate temperature for 2.5 h. Add 200 ml ofninhydrin reagent and incubate a further 10 min. Development ofdeep purple colour is positive. (Original reference suggests a purplecolour as deep as the colour of Crystal violet used in the Gram stain.)


Reagent: Ninhydrin.

Ninhydrin reagent (Used for detection of hippurate hydrolysis; Hwang and Ederer, 1975)

Reagent Amount Preparation of media Description of testNinhydrin 0.35 g Dissolve ninhydrin in the acetone/butanol mixture.

Dispense into 5 ml tubes and store in the dark.

To a mixture of organisms incubated in hippuratesolution for 2.5 h, add 200 ml of ninhydrinreagent. Incubate 10 min and observe formationof purple layer.

Acetone 5 mlButanol 5 ml

Hydrogen sulphide productionMany of the references for the biochemical reactions use a method whereby a lead acetate strip (H2Sindicator) is suspended over a tube of growth medium containing cysteine (sulphur source). This is avery sensitive method. However, the preparation of the lead acetate strips is hazardous, (for methodsee Cowan and Steel, 1970). Alternative methods are available but may not have the same sensitivity.The test can be performed in an API 20E or a triple sugar iron tube. A hydrogen sulphide Biostrip(catalogue number TM343) is available from MedVet Science. When using these media be aware ofthe growth requirements of the test organism and the sensitivity of the test.

Indole (Cowan and Steel method 2, 1970; Colwell and West, 1984; MacFaddin, 1980)

Reagent Amount Preparation of media Description of testTryptone broth(Difco)

2.5 g Combine reagents and pHto 7.5. Dispense 5 ml pertube and autoclave at 121°Cfor 15 min.

Use a heavy inoculum and incubate 48 h at 25 or 37°C dependingon growth requirements of the bacterium. To read, add 6–7 dropsof Kovács reagent and shake tube. A positive result is developmentof a cherry red colour in the upper reagent layer on top of the brothmedium. No colour development indicates a negative result.

For organisms that require salt, add 0.5 ml of 20% NaCl to 5 ml ofmedium. Even though a salt-requiring organism may show growthin the tube at 48 h, if NaCl is not present a false-negative reactioncan still occur. See photographic section.

NaCl 1.25 gDistilled water 250 ml

Results between API 20E and tryptone broth may differ. Add NaCl to a final concentration of 2% for marine isolates (i.e. add 500 mlof 20% NaCl to 5 ml of test medium).




Indoxyl acetate hydrolysis (Mills and Gherna, 1987)Indoxyl acetate discs may be prepared by making a 10% (wt/vol) solution of indoxyl acetate inacetone. Add 50 ml to a blank disc, 0.64 cm in diameter. Discs are available from Oxoid. Allow discs todry and store at 4°C in an amber-coloured bottle. Shelf life of discs is approximately 6 months. Discsare also available commercially from companies such as Remel.

Test: Bacterial growth from an agar plate is scraped on to an indoxyl acetate disc and a drop ofdistilled water is added. A positive result is the development of a dark blue colour within 5–10 min.Alternatively, colonies from an agar plate can be emulsified in 0.3 ml of distilled water. Add an indoxylacetate disc. A positive result is the development of a dark blue colour within 10–15 min (Mills andGherna, 1987).

Kovács indole reagent (Kovács, 1928; Cowan and Steel, 1970; MacFaddin, 1980)

Reagent Amount Preparation of media Description of testp-dimethylaminobenzaldehyde 5 g Handle p-dimethylaminobenzaldehyde and HCl with caution.

Dissolve p-dimethylaminobenzaldehyde by warming in awaterbath at 50°C. Only leave long enough to dissolve,otherwise it turns pink then dark brown. Cool and slowly addthe HCl to the aldehyde–alcohol mixture. The reagent shouldbe light yellow to light brown in colour. Store reagent in abrown glass-stoppered bottle at 4°C.

See under ‘Indole’.Iso-amyl alcohol 75 mlConcentrated hydrochloric acid 25 ml

KOH (Tests for presence of Flexirubin pigments; Reichenbach et al., 1974, 1981)

Reagent Amount Preparation of media Description of testKOH 20% Place 1–2 drops of KOH on to a few isolated young colonies on AO medium.

Detection of a brown or red pigment is positive. Detects flexirubin pigment(Reichenbach et al., 1981). Some reports indicate a purple colour as positive(Mudarris and Austin 1989).

Note: Pigment production may be influenced by factors in the growth medium such as yeast extract, which promotes pigmentproduction through stimulated growth. Also, pH can affect pigment, where a lower pH may reduce pigment production(Reichenbach et al., 1974). (Reichenbach states 5% KOH in methanol.)

MacConkey agar

Reagent Amount Preparation of media Description of testMacConkey agar (Oxoid) 26.0 g Suspend the agar in the distilled

water and autoclave at 121°C for20 min. Cool to 50°C and pour intoPetri dishes. Store plates at 4°C.

Organisms that ferment lactose are seen as darkpink colonies. Non-lactose fermenting coloniesare seen as yellow-clear colonies. Salmonellaspp. and Edwardsiella tarda will appear asnon-lactose fermenting colonies.


Marine oxidative-fermentative medium (MOF) (Leifson, 1963). See under Oxidativefermentative marine.

McFarland (nephelometer) standards preparation

McFarlandTube No.

Sulfuric acid1% aqueous solution (ml)

Barium chloride,1% aqueous solution (ml)

Corresponding densityof bacteria – 106

International Units (IU)of opacity

1 9.9 0.1 300 35 9.5 0.5 1500 15

270 Chapter 7



Method: Mix the 1% sulphuric acid and the 1% barium chloride according to the table. Use clean, clear glass tubes. Plug or capthe tubes and seal with parafilm. Store tubes upright.Use: When comparing bacterial density against the tubes, ensure that the bacterial suspension is in a similar sized glass tube tothe standard. It may be best to prepare the standards in a tube or bottle that the bacterial suspensions will be normally prepared in.Sensitivity to vibriostatic agent (0/129 discs), prepare lawn inoculum at McFarland tube 1.API 20E, API 20NE, API 50CH. Inoculate with inoculum prepared to turbidity of McFarland tube 1.API ZYM. Inoculate with inoculum prepared to turbidity of McFarland tube 5.

(Taken from Difco manual, referenced in Gradwohl’s Clinical Laboratory Methods and Diagnosis. A.C. Sonnenwirth and L. Jarett(eds). C.V. Mosby Company, 1980, p.1363.)

Motility – Hanging drop method (Recommended in West and Colwell, 1984)Place a drop of suspension from an early stationary phase broth culture on to a coverslip. Place smalldollops of vaseline or plasticine at the corners. Invert coverslip and place on to a glass slide so that thedrop of culture hangs free of the glass slide. Examine under phase contrast, or bright-field with thesubstage condenser aperture closed down to give greater contrast. Weakly motile strains may notbe detected in semi-solid motility medium. For some organisms, motility is temperature dependent;therefore, ensure that growth medium is incubated at the appropriate temperature.

MRVP test medium (Clark and Lubs, 1915; Cowan and Steel, 1970; used by West and Colwell,1984)

Reagent Amount Preparation of media Description of testMRVP medium (Oxoid) 3.75 g Dissolve reagent in distilled water and

autoclave at 121°C for 15 min. Dispense5 ml per tube.

Add NaCl for marine organisms. Mustincubate for 2–3 days before testing.

VP reaction: Red coloration may develop upto 18 h after the addition of reagents.


For Voges-Proskauer test, see under VP.

Methyl Red reagent

Reagent Amount Preparation of media Description of testMethyl Red 40 mg Add methyl red reagent to ethanol and heat

to 50°C in a waterbath to assist dissolution.Make up to 100 ml total with distilled water.

More ethanol can be added if a precipitateforms.

After incubation at optimal temperature for at least 2days, add 3–4 drops of Methyl Red reagent.Persistence of a red colour indicates a positive result.


Ethanol 95% 40 ml

Mycoplasma characterization tests (Aluotto et al., 1970 with modifications)

Reagent Amount Preparation of media Description of testHeart infusion broth – stock Add dehydrated media to distilled water.

Using 5M NaOH adjust pH to 7.6.Inoculate a test and substrate controltube with 1 ml of a 24 h culture grownin broth.

Read tests daily for up to 2 weeks. A pHdrop of 0.5 unit or more in the glucose tubecompared with the appropriate substratecontrol tube is a positive reaction.

A rise of 0.5 pH unit in the arginine or ureatubes compared with the appropriatesubstrate tubes indicates a positivereaction.

The pH values are read by comparison witha set of standards ranging in pH from 5.6 to8.4.

Heart infusion broth 25 gDistilled water 1000 mlBasal medium Prepare yeast extract stock, and phenol

red stocks.

To 74 ml of HIB add horse serum, yeastextract, phenol red and test substrate.Using 5 M NaOH of 5 M HCl, adjust toappropriate pH depending on testsubstrate.

Prepare substrate control tubes, that is,basal medium without test substrate.

Filter sterilize and dispense in 5 mlamounts to sterile bijou or otherscrew-capped bottles.

Heart infusion broth (Difco)stock

74 ml

Horse serum (heated 56°C for30 min)

10 ml

Yeast extract (Oxoid) stocksolution of 10% w/v.Filter-sterilize.

5 ml

Phenol red (0.5% w/v)autoclaved

1 ml

Test substrate




Glucose (10% w/v) 10 ml pH 7.6Arginine (0.2%) 10 ml pH 7.0Urea (10% w/v) 10 ml pH 7.0OF test Dispense 1 ml of sterile medium into

15 × 45 mm tubes.Inoculate duplicate test and substratecontrol tubes with 0.5 ml of an overnightbroth culture. Overlay one test and onecontrol tube with sterile paraffin oil. Incubateat appropriate temperature and examinedaily for up to 2 weeks for pH changecompared with pH standards.

Fermentative organisms produce acid inboth the aerobic and anaerobic tubes,whereas oxidative organisms produce acidin the aerobic tube only.

Basal medium 90 mlGlucose (10% w/v) 10 ml,

pH 7.6

Tetrazolium (TTC) reduction Aseptically add reagents to HIA stock andpour into 5 cm Petri dishes.

TTC can also be tested in broth ratherthan on agar plates.

Inoculate duplicate plates with agar plugstaken from dense growth on culture plates.Agar plugs can be excised using a scalpelblade, alcohol flame-sterilized spatula, oropen-ended ‘hockey stick’ made from abent Pasteur pipette. Slide the inverted agarplug over the surface of the TTC plates.

Incubate one plate aerobically and the otheranaerobically for up to 2 weeks.

Development of a pink-red colour in 3–4days is positive.

HIA stock 74 mlHorse serum 20 mlYeast extract stock 5 mlTTC (2% w/v) 1 ml

Phosphatase Aseptically add reagents to HIA stock andpour into 5 cm Petri dishes.

Phosphatase can also be tested in brothrather than on agar plates.

Inoculate 3 plates with a drop from a 24 hbroth culture. Incubate plates at theappropriate temperature. On days 3, 7 and14, flood plate with 5 M NaOH. A positivereaction is the appearance of a red colour.

HIA 74 mlHorse serum 20 mlYeast extract stock 5 mlPhenolphthalein diphosphate(sodium salt) (1% w/v)

1 ml

Film and Spots 1 Aseptically remove egg yolks andhomogenize with an equal amount ofsterile distilled water. Add the homogenateto HIA at a final concentration of 10%.Pour into 5 mm Petri dishes.

Inoculate the egg yolk medium with testorganism, and incubate in CO2 at 37°C forup to 14 days. Examine macroscopicallyusing reflected light. An iridescent or pearlyfilm seen over areas of heavy growthindicates a positive reaction.

HIA 90 mlEgg yolk 10 ml

Film and Spots 2 An alternative method is to use 20%horse serum in the medium.

Inoculate plates with test organism andincubate in CO2 at 37°C for up to 14 days.The development of small spots in themedium indicates a positive reaction.

HIA 80 mlHorse serum 20 ml

Catalase Flood a plate containing 24–48 h growthwith 30% hydrogen peroxide. Production ofbubbles indicates a positive reaction.

HIA plateSee also Digitonin and Dienesstain

Note: This is the original medium proposed by Aluotto et al. (1970). Bacto heart infusion broth (Difco) contains 500 g beef heartinfusion, 10 g bacto-tryptone, 5 g sodium chloride. An alternative is to use Difco PPLO broth with Mycoplasma supplement, to whichthe phenol red and test substrate is added. Some Mycoplasmas may be inhibited by 1% arginine, as originally proposed by Aluottoet al. (1970), therefore 0.2% is recommended. TTC = 2, 3, 5-triphenyltetrazolium chloride.

pH standards: Add phenol red to basal medium and dispense 3-ml lots into 5 ml tubes. Adjust pHin tubes to give a pH range of 5.6–8.4.

272 Chapter 7



Control organisms:Glucose positive = M. bovirhinis ATCC 19884, and negative = M. arthritidis ATCC 19611.Arginine hydrolysis positive = M. arthritidis ATCC 19611, and negative is M. bovirhinis ATCC19884.Urea positive = T-strain mycoplasma, and negative = M. arthritidis ATCC 19611.OF test organisms: Oxidative = M. pneumoniae ATCC 15531, Fermentative = M. bovirhinisATCC 19884.TTC control organisms: Positive aerobic and anaerobic = M. bovirhinis ATCC 19884. Negativefor both is M. arthritidis ATCC 19611.Phosphatase control organisms: Positive = M. arthritidis ATCC 19611. Negative = M. bovirhinisATCC 19884.Film and spots control organisms: Positive = M. gallinarum ATCC 19708. Negative = M.arthritidis ATCC 19611.Sterol requirement: Inoculate into serum-free basal medium, and basal medium with 5 mg bovineserum albumin per ml, 0.01 mg of palmitic acid per ml, and different concentrations of cholesterol(1.0, 5.0, 10.0, 20 mg/ml). Growth is determined by sedimenting the mycoplasma and testing themedium for protein content by the biuret method. Paper disc inhibition method: Use a 1.5% w/vsolution of digitonin (Sigma), and 5, 10, 20% w/v solutions of sodium polyanetholesulphonate(Koch-Light Labs, UK). The widths of the zones of growth inhibition are measured. Zones ofinhibition are 5–10 mm wide.

Ninhydrin reagent Used for detection of hippurate hydrolysis (from Barrow and Feltham, 1993).See under Hippurate hydrolysis.

Nitrate broth (Quoted in Crosby, 1967; Cowan and Steel, 1970; West and Colwell, 1984)

Reagent Amount Preparation of media Description of testKNO3 0.25 g Add reagents to distilled water and

autoclave at 121°C for 15 min. Dispense5 ml into tubes.

Inoculate media with organisms and incubateat the appropriate temperature.

For organisms that require salt, add 0.5 ml of20% NaCl to 5 ml of test media.

Nutrient broth (Oxoid) 3.25 gDistilled water 250 ml

Reagent Amount Preparation of media Description of testNitrate reagent A Add sulphanilic acid to distilled water,

then dissolve in the acetic acid.After incubation for 24 or 48 h, add 5 dropsof nitrate reagent A, and 5 drops of nitratereagent B to nitrate broth. Appearance of ared colour is indicative of a positive reaction.

Tubes that do not show a red colour shouldbe tested with zinc dust (match headamount). Development of a red colourindicates a true negative result, whereasno colour indicates that nitrate is absent,indicating that it was reduced by theorganism to nitrite, which in turn wasreduced – therefore, a positive result.

Sulphanilic acid 1.28 gDistilled water 110 mlAcetic acid 50 ml

Nitrate reagent B Handle with caution and use mask andgloves.

Add dimethyl-a-naphthylamine todistilled water. Then add acetic acidand dissolve by heating to 50°C in awaterbath.

Dimethyl-a-naphthylamine 0.96 mlDistilled water 110 mlAcetic acid 50 ml

Nocardia spp.: growth medium. See Glucose yeast extract agar

0/129. See under Vibrio discs for description of test.0/129 = 2:4-diamino-6:7-diisopropyl pteridine (0/129) phosphate. Discs are available from Oxoidand other companies. Available at concentrations of 10 mg and 150 mg.




ONPG – o-nitrophenyl-b-D-galactosidase (Cowan and Steel (1970) using the method of Lowe,1962)

Reagent Amount Preparation of media Description of testOxoid peptone water 0.9 g Dissolve the peptone water in distilled water and

autoclave at 121°C for 20 min.Inoculate a tube of ONPG brothand incubate at the appropriatetemperature for 24–48 h.

A yellow colour indicates a positiveresult and indicates the presenceof the enzyme b-galactosidase.

Distilled water 60 mlONPG 0.15 g Dissolve the ONPG in the phosphate solution at pH

7.5 and filter-sterilize. Aseptically add to the peptonewater, and dispense 2.5 ml into sterile tubes.

Store at 4°C and keep protected from the light.

Na2HPO4 0.035 gDistilled water 25 ml

ONPG discs are available from Oxoid. Discs are recommended when testing Flavobacteriaceae.

Oxidase test (Kovács, 1956. Used in Cowan and Steel, 1970; West and Colwell, 1984)

Reagent Amount Preparation of media Description of testTetramethyl-p-phenylenediamine

1% aqueoussolution

Prepare a 1% solution ofoxidase reagent in water. Useimmediately.

The oxidase reagent must bestored in a stoppered darkglass bottle, protected from thelight and stored in the fridge.It should not be used if it hasbecome deep blue in colour.

Place a piece of filter paper in an empty Petri dish.Dampen with freshly prepared oxidase reagent. Usinga platinum loop or wooden orange stick or toothpick,smear a streak of bacterial growth across the paper.Appearance of a deep purple colour in 10–30 secindicates a positive reaction. A purple colour after 2 minmay be a false positive. Discard once the filter paperbecomes a blue colour.

Do not test for oxidase reaction from media that containscarbohydrate such as TCBS and MCA (Jones, 1981).

Note: Commercial oxidase strips are also available and are recommended, as they offer a standardized test method.

Oxidative-fermentative (O-F) media (Media of Hugh and Leifson, 1953. For marine organisms,use marine oxidative fermentative medium of Leifson, 1963)

Reagent Amount Preparation of media Description of testDifco bacto tryptone 0.8 g Mix all ingredients, except horse serum,

in distilled water and boil for 1 min todissolve. Cool slightly and pH to 7.1using 10 M NaOH. Dispense 5 ml totubes and autoclave at 121°C for 15 min.Cool to 50°C and aseptically add 2drops of sterile horse serum per tube.Store at 4°C.

Fermentative organisms produce an acidreaction (yellow colour) in both the sealed andopen tubes. Oxidative organisms produceacid at the surface of the open tube. Growth isseen at the surface of the medium with little orno growth at the bottom of the open tube, andno growth in the sealed tube. Organisms thatfail to either ferment or oxidize glucose mayproduce an alkaline reaction (purple) at thesurface of the open tube.

NaCl 2.0 gK2HPO4 0.12 gGlucose 4.0 gOxoid agar No 1 0.8 gIndicator (see below) 24 mlSterile horse serumnot inactivated

2 drops per5 ml tube


Notes from Hugh and Leifson (1953): Metabolism of carbohydrate involves two different mechanisms. One mechanism is calledfermentation and occurs without oxygen and is therefore an anaerobic process. The other mechanism is called oxidation andoccurs in the presence of oxygen and is therefore an aerobic process. The OF medium from Oxoid is based on Hugh and Leifson(1953).

OF indicator

Reagent Amount Preparation of media Description of testCresol red 0.15 g Dissolve the bromothymol blue in water, 0.3 ml of a

1% solution is added to each 100 ml of medium.Bromothymol blue 0.10 gNaOH 0.20 gDistilled water 500 ml

274 Chapter 7



Marine oxidative fermentative medium for marine organisms (MOF) (Used for carbohydratemetabolism for marine organisms; Leifson, 1963)

Reagent Amount Preparation of media Description of testCasitone (Difco) 0.1 g Dissolve ingredients in distilled water and adjust

pH to 7.5. Autoclave at 121°C for 15 min.Autoclave artificial seawater separately and addto ingredients in distilled water.

Aseptically add 10 ml filter-sterilized glucoseper 100 ml. Aseptically dispense 5 ml to 10 mltubes.

Inoculate 2 tubes of medium with organismand overlay the medium in one tube with alayer of sterile paraffin oil to approximately1 cm depth, or 0.5 ml. Incubate tubes atappropriate temperature.

Organisms that ferment carbohydrateacidify the medium in both tubes, whereasaerobic organisms acidify the medium inthe ‘open’ tube only.

For fermenting organisms record ‘F’ onresults sheet. For aerobes or oxidizingorganisms, record ‘O’ on results sheet.

Yeast extract 0.01 gAmmonium sulphate 0.05 gTris buffer 0.05 mlAgar 0.3 gDistilled water 50 mlArtificial seawater 50 ml

Phenol red(0.1% stock)

1 ml Phenol red: Used at a 0.001% finalconcentration. (Prepare a 0.1% solution anduse 1 ml of this per 100 ml of medium).

Glucose 1.0% Prepare a 10% solution and filter-sterilize.

Note: The indicator bromothymol blue, which is used in the conventional OF medium, may be toxic to some marine organisms.

Paraffin oil (Used for overlay in decarboxylase tests and OF test)

Reagent Amount Preparation of media Description of testParaffin oil As required Dispense into 100 ml bottles or any

appropriate-sized bottle. Hot-air sterilizeat 160°C for 60 min. Do not autoclave asthe oil goes cloudy.

Used as overlay in ADH, LDC, ODC and OF tests.

For ease of use, oil can be dispensed into a 1 l Schottbottle and capped with a Socorex 2 ml dispenser unit.Dispense 0.5 ml into 10 ml media tubes.

Physiological saline. See saline

Rogosa agar (RA) (Oxoid manual) (Selective medium for Lactobacilli; Rogosa et al., 1951)

Reagent Amount Preparation of media Description of testRogosa agar (Oxoid) 82 g Suspend Rogosa agar in distilled water and

bring to the boil to dissolve. Add glacial aceticacid and mix thoroughly. Heat to 90–100°C for2–3 min with frequent agitation. Distribute intosterile tubes or Petri dishes. Do not autoclave.

May assist to differentiate Lactobacillifrom other Gram-positive bacteria suchas Carnobacterium, Arcanobacteriumand Vagococcus species.

Distilled water 1000 mlGlacial acetic acid 1.32 ml

Ingredients (g/l) tryptone (10.0), yeast extract (5.0), glucose (20.0), sorbitan mono-oleate (1.0), potassium dihydrogen phosphate(6.0), ammonium citrate (2.0), sodium acetate (25.0), magnesium sulphate (0.575), manganese sulphate (0.12), ferric sulphate(0.034), agar (20.0).

Saline: physiological saline

Reagent Amount Preparation of media Description of testNaCl 0.85 g Add salt to distilled water. Dispense 10 ml into

McCartney bottles. Autoclave at 121°C for 15 min.May be used as an inoculum forcommercial kit tests and ‘Biochem Set’.Distilled water 100 ml




Salt – 20% stock solution

Reagent Amount Preparation of media Description of testNaCl 100 g Add NaCl to distilled water. Autoclave

at 121°C for 15 min.For ease of use, 20% NaCl can be dispensed into a 1 lSchott bottle and capped with a Socorex 2 ml dispenserunit with 0.5 ml graduations. Dispense 0.5 ml per 5 ml ofmedia to produce final NaCl concentration of 2% in test.


In most cases, the carbohydrate media tubes do not require NaCl in addition to the 2% NaCl inoculum fluid. However, liquid tubemedia such as aesculin, ADC, ODC, LDC, the decarboxylase control, MRVP, indole (TW) and nitrate do require that 500 ml of a20% NaCl solution is added to the 5 ml of media.

Salt tolerance0%, 3% NaCl. See Gelatin/ NaCl split plate. Using a wire loop, spot inoculate equal amounts of

the bacterium to the 0% NaCl side and the 3% NaCl side. After 1–2 days incubation at the appropriatetemperature, examine for areas of clearing or opacity. See photographic section.

10% NaCl. For tolerance to 10% salt, dispense equal volumes of TSB and 20% NaCl stocksolution into a sterile bijou bottle or 10 ml tube. Add bacteria to turbidity of 0.5 or tube 1 McFarlandstandard. Incubate at appropriate temperature for 24–48 h. Look for obvious increase in growth oforganism as seen by cloudiness in the medium.

Survival at 50∞C (Used for Nocardia species; Gordon et al., 1974)Inoculate slants of glucose yeast extract agar, and incubate in a 50°C waterbath for 8 h. Remove fromwaterbath, cool quickly and incubate at 28°C for 3 weeks. Examine tubes for growth.

Tryptophane deaminase (TDA) reagent (For use in the API 20E kit. Can be purchasedcommercially)

Reagent Amount Preparation of media Description of testFerric chloride 3.4 g Dissolve the ferric chloride in 90 ml of water,

and then make up to 100 ml.Used in the API 20E kit. A positive is a browncolour. Proteus strains tend to be positive.Distilled water 100 ml

Triple sugar iron agar (TSI)

Reagent Amount Preparation of media Description of testTriple sugar ironagar (Oxoid)

9.75 g Dissolve reagent indistilled water and pHto 7.4. Dispense in5 ml aliquots to 10 mltubes. Autoclave at121°C for 15 min. Allowto cool and solidify in aslanted position to givedeep butts.

Inoculate the tube using a straight wire. Stab into medium, and thenzig-zag the slope. H2S producers will turn the medium black.Fermentative organisms will acidify the TSI butt (yellow), whereasnon-fermentative organisms will grow on the slant only and show eitherno pH change or an alkaline reaction and, rarely, an acidic reaction.

For some organisms this test method may not be as sensitive as usinga lead acetate paper strip.

A hydrogen sulphide Biostrip (catalogue number TM343) is availablefrom MedVet Science.


Urea (Christensen, 1946)

Part A

Reagent Amount Preparation of media Description of testOxoid agar No 1 3.75 g Add agar to distilled water and autoclave at 121°C for

15 min. Cool to 50°C.Add 1% NaCl for marine organisms.


276 Chapter 7




Part B

Reagent Amount Preparation of media Description of testBBL brand ureaagar base

8.7 g Add reagents to distilled water and filter-sterilize througha 0.22 mm Millipore filter.

Add Part A to 25 ml of Part B. Aseptically dispense 5 mlvolumes per tube. Allow to solidify in a sloped position.

Inoculate the slant heavily over theentire surface with a loopful of bacteriaharvested from an agar medium. Brightpink indicates a positive reaction.


Note: Urea slopes should be prepared with a deep butt and a short slant (Gilardi, 1983).

Vibrio discs

0/129 = 2:4-diamino-6:7-diisopropyl pteridine (0/129) phosphate, vibriostatic reagent (mg). TheVibrio spp. (including Listonella spp., Moritella spp. and Photobacterium spp.) are sensitive tothis compound, commonly called vibriostatic agent (Shewan et al., 1954). This test assists in thedifferentiation of Vibrio species from other Gram-negative rods, particularly Aeromonas species, whichare resistant to vibriostatic agent. Almost all Vibrio species are sensitive to 0/129 at 150 mg and someare sensitive to 10 mg. However, Vibrio cholerae 0139 has developed resistance to 0129 at 150 mgconcentration (Albert et al., 1993; Islam et al., 1994).

Treat this test in the same manner as ‘sensitivity testing’ used to test an organism as sensitive orresistant to an antimicrobial reagent. Prepare an inoculum in normal saline to the opacity of McFarlandtube 1, a suspension that is just visible to the eye. Use a sterile cotton-tipped swab to lawn inoculate theplate. Use BA for freshwater organisms and MSA-B for marine organisms. Place the two discs on tothe agar surface so that they are at least 4 cm apart. Invert the plate, and incubate at the appropriatetemperature for 24 h. If there is insufficient growth incubate a further 24 h; however, normally the zonesare recorded at 24 h as further growth, especially when testing Vibrio species that swarm, may show afalse resistant result. Record zone sizes as ‘sensitive’ (S) or ‘resistant’ (R). For the 0/129 500 mg disc, azone of 9 mm is susceptible for Vibrio species (Bernardet and Grimont, 1989). A zone size of 22 mm isconsidered sensitive for Photobacterium damselae ssp. damselae (Love et al. 1981).

The discs are purchased from Oxoid or Rosco Diagnostics. The Oxoid codes for the twoconcentrations are DD14 for 0129 10 mg, and DD 15 for 150 mg.

Voges-Proskauer reaction See MRVP test (Clark and Lubs, 1915; Voges and Proskauer, 1898, asreported in Cowan and Steel, 1970. Used by West and Colwell, 1984)

Use for the detection of acetoin from glucose fermentation. The incubation time and temperaturerather than the method influence the production of acetoin.

MRVP test medium (The commercial medium is based on Clark and Lubs medium, 1915)

Reagent Amount Preparation of media Description of testMRVP medium(Oxoid)

3.75 g Dissolve reagent in distilled water andautoclave at 121°C for 15 min. Dispense5 ml per tube.

Add NaCl for marine organisms. Must incubate for2–3 days before testing.

Add VP reagent A, and VP reagent B. Red colorationmay develop up to 18 h after the addition of reagents.


MR = methyl red; VP = Voges-Proskauer.

VP test reagents (Barritt, 1936)

Reagent Amount Preparation of media Description of testReagent A Dissolve a-naphthol in ethanol.

Store at 4°C.After incubation of MRVP medium for 48 hat appropriate temperature, place 1 ml intoa test tube. Add 0.6 ml of reagent A, and0.2 ml of reagent B. Examine for pinkcolour up to 4 h at room temperature.

Tests for acetylmethylcarbinol (acetoin).

a-naphthol 5.0 gAbsolute ethanol 100 mlReagent B Weigh KOH. Make up to 100 ml

final volume with distilled water.Store at 4°C.

KOH 40.0 gDistilled water Add to final 100 ml

Note: VP reagent I and II in the API 20E kit are also suitable. Transfer 250 ml of medium to a microfuge tube. Add 150 ml and 50 mlof reagent I and II respectively, shaking after each addition. Read after 10–20 min.



Further Reading and Other Information Sources

Books on Fish Diseases

Austin, B. and Austin, D.A. (1999) Bacterial Fish Pathogens: Disease of Farmed and Wild Fish. 3rd revised edn. PraxisPublishing, Chichester, UK.

Woo, P.T.K. and Bruno, D.W. (eds) (1999) Fish Diseases and Disorders. Vol. 3: Viral, Bacterial and Fungal Infections.CAB International, Wallingford, UK.

Bergey’s Manual of Systematic Bacteriology, Vol I and Vol II. (1984) Holt, J.G. (ed.) Lippincott Williams and Wilkins,Baltimore, Maryland.

Diagnostic Manual for Aquatic Animal Diseases, 3rd edn (2000) Office International Des Epizooties (OIE), 12 rue deProny, F-75017, Paris, France.

Plumb, J.A. (1999) Health Maintenance and Principal Microbial Diseases of Cultured Fishes. Iowa State UniversityPress.

The fourth edition of the OIE Diagnostic Manual for Aquatic Animal Diseases will be available in July 2003. Thesemanuals are also available on line. http://www.oie.int/eng/normes/fmanual/A_summry.htm

Books on Biochemical Identification Tests

Cowan, S. and Steel, K. (1970) Manual for the Identification of Medical Bacteria. Cambridge University Press,Cambridge.

Barrow, G.I. and Feltham, R.K.A. (1993) Cowan and Steel’s Manual for the Identification of Medical Bacteria,3rd edn. Cambridge University Press, Cambridge.

MacFaddin, J.F. (1980) Biochemical Tests for Identification of Medical Bacteria, 2nd edn. Williams and Wilkins,Baltimore, Maryland.

MacFaddin, J.F. (2000) Biochemical Tests for Identification of Medical Bacteria, 3rd edn. Williams and Wilkins,Baltimore, Maryland.

Other Biochemical Identification Schemes

Alsina, M. and Blanch, A. (1994) A set of keys for biochemical identification of environmental Vibrio species. Journalof Applied Bacteriology, 76, 79–85.

Alsina, M. and Blanch, A. (1994) Improvement and update of a set of keys for biochemical identification of Vibriospecies. Journal of Applied Bacteriology, 77, 719–721.

Carson, J., Wagner, T., Wilson, T. and Donachie, L. (2001) Miniaturised tests for computer-assisted identification ofmotile Aeromonas species with an improved probability matrix. Journal of Applied Microbiology, 90, 190–200.

Schmidtke, L.M. and Carson, J. (1994) Characteristics of Vagococcus salmoninarum isolated from diseased salmonidfish. Journal of Applied Bacteriology, 77, 229–236.

278



Journals

Applied and Environmental Microbiology http://aem.asm.org/Aquaculture http://www.elsevier.com/locate/aquacultureBulletin of the European Association of

Fish PathologistsCurrent Microbiology http://link.springer.de/link/service/journals/00284/Diseases of Aquatic Organisms http://www.int-res.com/journals/dao/Fish PathologyInternational Journal of Systematic and

Evolutionary Microbiology http://ijs.sgmjournals.org/Journal of Applied Ichthyology http://www.blackwell-synergy.com/Journals/issuelist.asp?journal=jaiJournal of Applied Microbiology http://www.blackwell-synergy.com/Journals/issuelist.asp?journal=jamJournal of Aquatic Animal HealthJournal of Clinical Microbiology http://jcm.asm.org/Journal of Fish DiseasesVeterinary Microbiology http://www.elsevier.nl/locate/vetmic

Culture Collections

Ecole Nationale Vétérinaire de Toulouse, 23 chemin des Capelles, F-31076 Toulouse cedex 03, France. http://www.bacterio.cict.fr/collections.html

ACAM: Australian Collection of Antarctic Microorganisms, Antarctic CRC, University of Tasmania, Hobart,Australia.

AHLDA: Animal Health Laboratories, Department of Agriculture. 3 Baron-Hay Court, South Perth, WesternAustralia 6151.

ATCC: American Type Culture Collection. Corporate: ATCC, 10801 University Boulevard, Manassas, VA20110–2209, USA. Products & Services Orders: ATCC, PO Box 1549, Manassas, VA 20108–1549, USA.http://www.atcc.org/

CCUG: Culture Collection, University of Göteborg, Department of Clinical Bacteriology, Institute of ClinicalBacteriology, Immunology, and Virology, Guldhedsgatn 10A s-413, 46 Göteborg, Sweden.

CDC: Center for Disease Control, 1600 Clifton Rd, Atlanta, Georgia 30333, USA.CECT: Coleccion Espanola de Cultivos Tipo, Universidad de Valencia, Burjassot, Spain.CIP: Collection de l’Institut Pasteur, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris Cedex 15, France.CNCM: Collection Nationale de Culture de Microorganismes, Institut Pasteur, Paris, France.DSMZ (DSM): Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1B,

D-38124, Braunschweig, Germany. http://www.dsmz.de/dsmzhome.htmIAM: Institute of Molecular and Cellular Biosciences (formerly Institute of Applied Microbiology, Culture Collection –

IAMCC), The University of Tokyo, Yayoi, Bunkyo-Ku, Tokyo, Japan.KMM: Collection of Marine Microorganisms, Pacific Institute of Bioorganic Chemistry, Vladivostok, Russia.NCFB: National Collection of Food Bacteria (previously named NCDO). Transferred from the IFR (Institute of Food

Research), Reading, to National Collections of Industrial, Food and Marine Bacteria, 23 Machar Drive,Aberdeen AB24 3RY, UK.

NCIMB: National Collection of Industrial and Marine Bacteria, National Collections of Industrial, Food and MarineBacteria, 23 Machar Drive, Aberdeen AB24 3RY, UK. http://www.ncimb.co.uk/ncimb.htm

NCTC: National Collection of Type Cultures, Central Public Health Laboratory, Colindale Ave., London NW9 5HT,UK. www.phls.co.uk

RVAU: Royal Veterinary and Agricultural University, Copenhagen, Denmark.UB: University of Barcelona, Barcelona, Spain.

Bacterial Names/Taxonomy/Nomenclature

Bacterial nomenclature is continually being updated at the following websites:DSMZ (DSM): Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1B,

D-38124, Braunschweig, Germany. http://www.dsmz.de/dsmzhome.htm

Further Reading 279



List of Bacterial Names with Standing in Nomenclature: http://www.bacterio.cict.fr/NCBI: http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/

Fish Disease Web Sites

http://www.fishdisease.net This website is for aquatic animal health professionals and contains information onleaflets on different diseases, an image library on parasites, notification of conferences, jobs and contacts.

http://www.fishbase.org/home.htm or http://www.fishbase.org/search.html This website contains information onfishes that may be useful to fisheries managers, scientists and others. One of its features is the latest scientificname of the different fishes.

280 Further Reading



Appendix

Common Name and Scientific Name of Aquatic Animals

281

Common name Scientific nameAbalone Haliotis discus hannaiAdriatic sturgeon Acipenser naccariiAfrican cichlid Nimbochromis venustusAmazon freshwater dolphin Inia geoffrensisAmberjack Seriola dumeriliAmerican alligator Alligator mississippiensisAmerican crayfish Orconectes limosus, Pacifastacus leniusculus, Procambarus

clarkiiAmerican eel Anguilla rostrataAmerican plaice Hippoglossoides platessoidesAntarctic fur seal Arctocephalus gazellaArctic char Salvelinus alpinus L.Atlantic bottlenose dolphin Tursiops truncatusAtlantic cod Gadus morhua L.Atlantic croaker Micropogon undulatusAtlantic menhaden Brevoortia tyrannus LatrobeAtlantic salmon Salmo salar L.Atlantic walrus Odobenus rosmarus rosmarusAtlantic white-sided dolphin Lagenorhynchus acutusAustralian oyster Saccostrea commercialisAyu Plecoglossus altivelisBalloon molly Poecilia spp.Banana prawn Penaeus merguiensisBarramundi Lates calcarifer (Bloch)Beluga whale Delphinapterus leucasBighead carp Aristichthys nobilisBlack acara Cichlasoma bimaculatumBlack mullet Mugil cephalusBlack scraper Novodon modestusBlack skirted tetra Hyphessobrycon spp.Blenny Zoarces viviparusBlue fish Pomatomus saltatrixBlue mackerel Scomber australasicusBlue manna crab Portunus pelagicusBlue shrimp P. (Litopenaeus) stylirostris

Continued



282 Appendix

Common name Scientific nameBottlenose whale Hyperodoon ampullatusBoney bream Nematolosa come (Richardson)Borneo mullet Liza macrolepisBottle-nosed dolphin Tursiops truncatusBowhead whale Balaena mysticetusBream Abramis bramaBrine shrimp ArtemiaBrook salmon Salvelinus fontinalisBrook trout Salvelinus fontinalis (Mitchill)Brown bullhead Ictalurus nebulosus (Lesueur)Brown shark Carcharhinus plumbeusBrown-spotted grouper Epinephelus tauvina, E. coioidesBrown trout Salmo trutta m. fario, Salmo trutta m. lacustris L.Burnett salmon Polydactylus sheridani (Macleay)Californian sea lion Zalophus californianusCanadian shrimp Lismata amboiensCarp Cyprinus carpio L.Catfish Clarius batrachus L.Caucasian carp Carassius carassiusChanchito Chichlasoma facetum (Jenyns)Channel catfish Ictalurus punctatus (Rafinesque)Chinook salmon Oncorhynchus tschawytschaChub Leuciscus cephalisChum salmon Oncorhynchus keta (Walbaum)Cichlid Oreochromis mossambicusClam Tapes philippinarumCod Gadus morhuaCoho salmon Oncorhynchus kisutchCommon carp Cyprinus carpio L.Common dolphin Delphinus delphisCommon seal Phoca vitulinaCommon snook Centropomus undecimalisCommon wolf-fish Anarhichas lupusCoral prawn Metapenaeopsis spp.Crucian carp Carassius carassiusCultured flounder Paralichthys olivaceusCutthroat trout Salmo clarkiCuttle fish Sepia officinalisDab Limanda limandaDace Leuciscus leuciscus L.Damselfish Chromis punctipinnisDamselfish Pomacentridae, Amphiprion clarkii (Bennett),

Amblyglyphidodon curacao (Bloch)Danio Danio devarioDiscus fish Symphysodon discus, S. aequifasciatusDolphin Tursiops truncatus, T. gephyreusDolphin fish Coryphaena hippurus L.Eastern freshwater cod Maccullochella ikeiEastern mosquitofish Gambusia holbrookiEastern painted turtle Chrysemys picta pictaEel Anguilla japonica, A. reinhardtiiElephant seal Mirounga leoninaEuropean crayfish Astacus leptodactylus, A. pachypus, A. torrentium, A. astacus,

Austropotamobius pallipesEuropean eel Anguilla anguillaEuropean sea bass Dicentrarchus labrax Serranidae



Appendix 283

Common name Scientific nameFairy shrimp Branchipus schaefferi (Fisher),Chirocephalus diaphanus

(Prévost), Streptocephalus torvicornis (Waga)False killer whale Pseudorca crassidensFarmed mussel Perna pernaFathead minnow Pimephales promelasFighting fish Betta splendensFiremouth cichlid Cichlasoma meekiFlat-tailed mullet Liza dussumieri (Valenciennes)Flounder Paralichthys olivaceus, P. flesusFlounder Platichthys flesusFour bearded rockling Enchelyopus cimbrius L.Freshwater cod (Australian native) Maccullochella spp.Freshwater dolphin Inia geoffrensisFreshwater prawn Macrobranchium rosenbergiiFur seal Arctocephalus australisGilthead sea bream Sparus auratusGolden shiner Notemigonus crysoleucas (Mitchell)Goldfish Carassius auratus L.Goldsinny wrasse Ctenolabrus rupestrisGourami (three-spot) Trichogaster trichopterusGrass carp Ctenopharyngodon idellaGrayling Thymallus thymallus L.Greater weever Trachinus dracoGreen knife fish Eigemannia virescensGreen moray eel Gymnothorax funebrisGreen sturgeon Acipenser medirostrisGreenback flounder Rhombosolea tapirina GuntherGrey seal Halichoerus grypusGrouper Epinephelus guaz, E. coioidesGuppy Poecilia reticulata (Peters), Lebistes reticulatusHaddock Melanogrammus aeglefinus L.Halibut Hippoglossus hippoglossus L.Harbour porpoise Phocoena phocoenaHarbour seal Phoca vitulinaHarp seal Phoca groenlandicaHerring Arripis georgianusHooded seal Cystophora cristataHorse mackerel Trachurus trachurusIberian toothcarp Aphanius iberusJapanese abalone Sulculus diversicolor supratextaJapanese eel Anguilla japonicaJapanese flounder Paralichthys olivaceusJapanese medaka Oryzias latipesJewel tetra Hyphessobrycon callistus (Boulenger)Johnston crocodiles (freshwater) Crocodylus johnstoniKiller whale Orcinus orcaKing prawn Penaeus latisulcatusKnife fish Gymnotus carapoLake trout Salmo trutta m. lacustris, Salvelinus namaycush WalbaumLargemouth bass Micropterus salmoidesLemon shark Negaprion brevirostrisLittle penguin Eudyptula minorLiving dace Tribolodon hakonensis GuntherLoach Misgurnus anguillicaudatus CantorLobster Homarus gammarus L.

Continued



284 Appendix

Common name Scientific nameLocal mussel Mutilus edulisLong-tom Tylosurus macleayanus (Ogilby)Mackerel Scomber scombrusManila clam Tapes philippinarum, T. decussatus, Ruditapes philippinarumMasu salmon Oncorhynchus masouMenhaden Brevoortia patronusMinke whale Balaenoptera acutorostrataMinnow Phoxinus phoxinus L.Molly Poecilia velifera (Regan)Mud crab Scylla serrataMullet Mugil cephalusMurray cod Maccullochella peeliMussel (Far-eastern) Crenomytilus grayanus and Patinopecten yessoensisMussel Protothaca jedoensis, Mytilus edulis, Mytilus galloprovincialisNarwahl whale Monodon monocerusNeon tetra Paracheirodon innesi, Hyphessobrycon innesiNew Zealand fur seal Arctocephalus forsteriNew Zealand mussel Perna canaliculusNew Zealand sea lion Phocarctos hookeriNile tilapia Oreochromis niloticusNorth-east Atlantic mackerel Scomber scombrusNorthern elephant seal Mirounga angustirostrisNorthern fur seal Callorhinus ursinusNorthern pike Esox lucius L.Northern right whale dolphin Lissodelphis borealisNurse shark Orectolobus ornatusOctopus Octopus vulgaris, O. joubiniOne-spot bream Diplodus sargusOrnamental fish Pterophyllum scalareOscar Astronotus ocellatusOscar Apistogramma ocellatusOtter Lutra lutraOyster Ostrea edulisOyster (eastern) Crassostreae virginicaPacific herring Clupea harengus pallasiPacific oyster Crassostrea gigasPacific salmon Oncorhynchus spp.Pacific staghorn sculpin Leptocottus armatusPacific white shrimp Penaeus vannameiPacific white-sided dolphin Lagenorhynchus obliquidensParadise fish Macropodus opercularis (L.)Pejerrey Odonthestes bonariensisPenguins Aptenodytes patagonica, Eudyptes crestatus, Pyoscelis papua,

Spheniscus demersus, Spheniscus humboldtiPerch Perca fluviatilisPike Esox luciusPilchard Sardinops neopilchardusPilot whale Globicephala scammoniPink salmon Oncorhynchus gorbuschaPink snapper Chrysophrys unicolorPinkfish Lagodon rhomboidesPirarucu Arapaima gigas CuvierPlaice Pleuronectes platessaPompanos Trachinotus carolinus L.Rabbitfish Siganus rivulatus (Forsskål)Rainbow trout Oncorhynchus mykiss (Walbaum)



Appendix 285

Common name Scientific nameRainbow and steelhead trout Salmo gairdneriRed abalone Haliotis rufescensRed algae Jainia spp.Red claw crayfish Cherax quadricarinatusRed drum, Redfish Sciaenops ocellatusRed-eared slider turtle Chrysemys scripta elegansRed sea bream Pagrus majorRed swamp crawfish Procambarus clarkiiRedtail catfish Phractocephalus hemiliopterusRinged seal Phoca hispidaRisso dolphin Grampus griseusRoach Rutilus rutilus L.Rohu Labeo rohitaRosy barb Puntius conchoniusRudd Scardinius erythrophthalmusSablefish Anoplopoma fimbria (Pallas)Salmon Oncorhynchus kisutch (Walbaum)Saltwater crocodile Crocodylus porosusSand eel Ammodytes lancea (Cuvier), Hyperoplus lanceolatus

(Lesauvege)Sand lance Ammodytes personatus GirardSand whiting Sillago ciliata CuvierSaratoga Scleropages leichardiiSardine Sardinops melanostictus, Sardinops sagnaxScallop Pecten maximus, Argopecten purpuratusScaly mackerel fish Amblygaster posteraSea bass Dicentrarchus labraxSea bream Pagrus major, Evynnis japonicus, Sparus aurata,

Acanthopagrus latusSea catfish Arius felisSea horse Hippocampus angustus, H. barbouri, H. whitei, H. kudaSea lion Otaria flavescensSea mullet Mugil cephalus L.Sea trout Salmo trutta m. trutta L.Sea turtle Chelonia mydasSea-urchin Paracentrotus lividuSepiolid squid Euprymna scolopesShotted halibut Eopsetta grigorjewiShubunkin Carassius spp.Siamese fighting fish Betta splendens ReganSignal crayfish Pacifastacus leniusculusSilver black porgy Acanthopagrus cuvieriSilver bream Blicca bjoerknaSilver bream Acanthopagrus butcheri, A. australis (Owen)Silver carp Hypophthalmichthys molitrix ValenciennesSilver mullet Mugil curema (Valenciennes), Mugil cephalus (L.)Silver molly Poecilia spp.Silver perch Bidyanus bidyanus (Mitchell)Silver trout Cynoscion nothusSmall abalone Haliotis diversicolor supertextaSmallmouth bass Micropterus dolomieuiSnakehead fish Channa striatusSnakehead fish Ophicephalus punctatus, O. striatusSnub-nose garfish Arrhamphus sclerolepsis (Gunther)

Continued



286 Appendix

Common name Scientific nameSockeye salmon Oncorhynchus nerka (Walbaum)Softshell clam Mya arenariaSole Solea soleaSouth African abalone Haliotis midaeSouth American side-necked turtle Podocnemis unifelisSouthern elephant seal Mirounga leoninaSowerby’s beaked whale Mesoploden bidensSpanish mackerel Scomber japonicusSpanner crab Ranina raninaSpiny soft-shelled turtle Trionyx spiniferSpot Leiostomus xanthurusSpotted dolphin Stenella plagiodonSpotted moray eel Gymnothorax moringaSpotted wolf-fish Anarhichas minorSquid Loligo pealeiSquid SepiolaSquid Teuthoidea speciesStarfish Asterias rubensStingray Dasyatis pastinacaStriped bass Morone saxatilis (Walbaum), M. chrysopsStriped dolphin Stenella coeruleoalbaStriped mullet Mugil cephalusStriped-neck musk turtle Sternotherus minor peltiferSturgeon Acipenser naccariiSunfish Mola molaTasmanian lobster Jasus novaehollandiaeTilapia Oreochromis niloticus, O. aurusTilapia Tilapia nilotica, Tilapia aurea, Tilapia mosambicaTilapia Sarotherodon aureus (Steindachner)Tom cod Gadus microgadusTropical shrimp Stenopus hispidusTrout cod Maccullochella macquariensisTurbot Scophthalmus maximus L.Turtle Dermochelys coriaceaTurtle Pseudemis scriptaViviparous blenny Zoarces viviparusWeddell seal Leptonychotes weddelliiWestern rock lobster Panulirus cygnusWhite catfish Ictalurus catus L.White clawed crayfish Austropotamobius pallipesWhite leg shrimp P. (Litopenaeus) vannameiWhite perch Roccus americanus, Morone americanus (Gremlin)White whale Delphinapterus leucasWhitefish Coregonus spp.Whitespotted rabbitfish Siganus canaliculatusWhiting Merlangius merlangusWolf-fish Anarchichas lupus L.Wrasse Labrus berggyltaYellow bass Morone mississippiensisYellow perch Perca flavescensYellowtail Seriola quinqueradiata, S. lalandiYellowfin bream Acanthopagrus australis (Owen)Zebra danio Brachydanio rerio

This list was taken from the references used in this manual.



Glossary of Terms

287

a or aH (referring to greening of agar) Alpha haemolysisA Alkaline reactionAAHRL Australian Animal Health Reference LaboratoryACAM Australian Collection of Antarctic MicroorganismsADH Arginine dihydrolaseAes AesculinAFB Acid-fast bacteriaAHL Animal Health LaboratoriesAHLDA Animal Health Laboratories Department of Agriculture Culture CollectionAla L-alanineAmp Ampicillin disc 10 mgANA Plate media for anaerobic bacteriaAO Anacker Ordal agar for FlavobacteriaAO-M Anacker Ordal agar with added NaCl for growth of marine FlavobacteriaAPI 50CH API identification system from bioMérieux. Carbohydrate testsAPI 20E API identification system from bioMérieux. Fermentation and enzyme

testsAPI 20NE API identification system. Utilization testsAPI Rapid ID32 Strep API system for Streptococci and other Gram-positivesAPI 20 Strep API identification system for Streptococci and other Gram-positive

organismsAPI ZYM API identification system. Enzyme testsArab L-arabinoseArg ArginineAsp L-asparagineASW Artificial sea waterAT Annealing temperatureATCC American Type Culture Collection, Rockville, Maryland, USAb Beta (refers to clear zone or b haemolysis)BA Blood agarBGD Bacterial gill diseasebH b-haemolysisBHA Bacterial haemorrhagic ascitesBHIA Brain heart infusion agarBKD Bacterial kidney diseasebp Base pairs (in kilo-bases)BRD Brown ring diseaseBrucella agar Used for isolation of Brucella species



288 Glossary

C Number of cycles in the PCR reactionCa CalciumCat CatalaseCBBA Coomassie brilliant blue agarCCA Cellobiose Colistin agar for isolation of Vibrio vulnificusCCRC Culture Centre for Research and CollectionCCUG Culture Collection of the University of Göteborg, Department of Clinical

Bacteriology, Göteborg, SwedenCDC Centres for Disease Control and Prevention, Atlanta, Georgia, USACFPA Selective media for isolation of Bordetella bronchisepticaCFU colony forming unitsCit CitrateCNCM Collection Nationale de Culture de Microorganismes (Institut Pasteur,

Paris, France)CO2 Carbon dioxideCPC Cellobiose Polymyxin B Colistin agar for isolation of Vibrio vulnificusCr Coccoid rodsCR Congo RedCSF Cerebrospinal fluidcv Curved rodsd DaysDCA Desoxycholate-citrate agarDmso Dimethyl sulphoxideDNase Test for the detection of hydrolysis of DNADSM Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,

Braunschweig, GermanyEIM Edwardsiella ictaluri mediumEM Electron microscopyERM Enteric red mouthESC Enteric septicaemia of catfishEry Meso-erythritolF Fermentative (facultative anaerobe)FA Furunculosis agar for Aeromonas salmonicidaFAO Food and Agriculture Organization of the United NationsFINE Flounder necrotizing enteritisFM Farrell’s medium for Brucella speciesFPM Flavobacterium psychrophilum mediumG GelatinG Green-coloured colony on TCBS plateGlid Gliding motilityGal D-galactoseGlu GlucoseGlut Glutamic acidGm Gram stain reaction (blue colour = positive, red = negative)GUD Goldfish ulcer diseaseh HoursH2S Hydrogen sulphideHCl Hydrochloric acidHG (DNA) hybridization groupHip HippurateHS Hsu-Shotts agar for Flavobacterium columnareHSM For the isolation of Tenacibaculum maritimumI Inert reaction (in OF test)ID IdentificationIGS Intergenic spacerInd Indole



Glossary 289

Inos InositolISP No. 2 Yeast malt extract agar (Difco)JOD Juvenile oyster diseaseKCl Potassium chlorideKDM2 Medium for growth of Renibacterium salmoninarumKDMC Medium for growth of Renibacterium salmoninarumKf Cephalothin 30 mg discKOH Potassium hydroxide. 20%KUMA Kumamoto Prefectural Institute of Public Health (culture collection prefix)Lac LactoseLDC Lysine decarboxylaseLJM Lowenstein-Jensen medium for Mycobacteria speciesLMG Laboratorium Microbiologie Gent Culture Collection, Universiteit Gent,

BelgiumLPS LipopolysaccharideLys LysineMA 2216 Medium for growth of marine organisms. Commercially availableMAF Modified acid fast stainMalt MaltoseMan MannitolMan An Fermentation of mannitol under anaerobic conditionsMano MannoseMCA MacConkey agarMg MagnesiumMiddlebrook’s media For the isolation of Mycobacterium speciesmin MinuteMOF Marine oxidative-fermentative mediumMot MotilityMR Methyl RedMRVP Methyl Red Voges-ProskauerMSA-B Marine salt agar – blood medium for growth of marine organismsN NegativeNA Nutrient agarNaCl SaltNaCl 0/3 Plate media containing either 0% NaCl or 3% NaCl concentrationNB Nutrient brothNCFB National Collection of Food Bacteria, Agricultural and Food Research

Council (AFRC) Institute of Food Research, Reading Laboratory,Reading, Berkshire, UK (formerly National Collection of Dairy Organisms)

NCIM National Collection of Industrial Microorganisms, National ChemicalLaboratory, India

NCIMB National Collection of Industrial and Marine Bacteria, Aberdeen,Scotland, UK

NCTC National Collection of Type Cultures, Central Public Health Laboratory,London, UK

ND Not doneNeg NegativeNG No growthNH Non-haemolyticNit NitrateNK Not knownNLF Non-lactose fermentingnmol NanomolesNVI National Veterinary Institute, Oslo, Norwaynm Nanometrent Not tested



290 Glossary

O Oxidative (aerobic metabolism)OD Optical densityODC Ornithine decarboxylaseOF Oxidative-fermentative testONPG o-Nitrophenyl b-D-galactopyranosideOrn OrnithineOx Oxidase0%–3% Split plate containing gelatin, and concentration of salt at 0% and 3%0129 Vibriostatic agent – 2:4-diamino- 6:7-diisopropyl pteridine phosphatePacker’s plates Selective media for the isolation of Erysipelothrix speciesPBS Phosphate buffered salinePig Pigment productionpmol PicomolesPoly plates Media for the isolation of Dermatophilus speciesPos PositivePS Partially sensitive to vibriostatic discPY Peptone yeast medium for Pedobacter and Sphingobacterium speciesPYR L-Pyrrolidonyl-b-naphthylamidePYS-2 Peptone yeast medium 2R ResistantR2A Medium for isolation of marine Flavobacterium and other organismsRAA Rogosa acetate agarRBC Red blood cellsRib D-riboseS SensitiveSS Media for the isolation of Salmonella and ShigellaSAB Sabouraud’s medium for fungiSal SalicinShieh medium For the isolation of Flavobacterium columnareSiem agar Selective medium for isolation of Aerococcus viridansSKDM Selective medium for Renibacterium salmoninarumSkirrow’s medium For the isolation of Helicobacter speciesSor SorbitolSuc SucroseSW Swarming growth on agar plateSWT Seawater-based complex mediumTHA Todd-Hewitt agarTB Lab Tuberculosis laboratoryTCBS Thiosulphate–citrate–bile salts–sucrose agarTE Tris EDTA bufferTemp TemperatureTm Melting temperatureTre TrehaloseTSA Tryptone soy agarTSA-B Tryptone soy agar with added bloodTSA+NaCl Tryptone soy agar with added salt (2% final concentration)TSB Tryptic soy brothTSI Triple sugar ironTYG Tryptone yeast extract glucose agarTYG-M Tryptone yeast extract glucose agar with added NaCl for growth of

T. maritimumUro Urocanic acidV Variable reaction in literatureVAM Vibrio anguillarum medium for isolation of Listonella anguillarumVP Voges-Proskauer test



Glossary 291

VPT Vancomycin, Polymyxin, Trimethoprim – Skirrow’s medium for theisolation of Helicobacter species

Vs Variable or slow reactionUK United KingdomUSA United States of AmericaUV Ultraviolet lightVVM Vibrio vulnificus mediumW WeakWHO World Health OrganizationWood’s Broth Selective broth for the isolation of Erysipelothrix speciesXLD Xylose lysine desoxycholate agar for isolation of Salmonella speciesXyl D-xyloseY Yellow-coloured colony on TCBS plateYSA Yersinia selective agarZN Ziehl–Neilson strain for Mycobacteria− Negative reaction−cr Gram-negative curved rod−a Negative result, but may show a-haemolysis after a week+ Positive reaction+gb Gram positive cocco-bacilli+g+ Glucose fermentation positive, gas produced+g- Glucose fermentation positive, no gas produced+rt Positive at room temperature (25°C) but negative at 37°C+s Slow positive reaction. May take 2–4 days for reaction to occur+sr Gram-positive, short rod+w Weak positive reaction



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798 Waechter, M., Roux, F.L., Nicolas, J.-L., Marissal,E. and Berthe, F. (2002) Characterization of patho-genic bacteria of the cupped oyster Crassostreagigas. Comptes Rendus Biologies 325, 231–238.

799 Wakabayashi, H. and Egusa, S. (1972) Character-istics of a Pseudomonas sp. from an epizootic ofpond-cultured eels (Anguilla japonica). Bulletin ofthe Japanese Society of Scientific Fisheries 38,577–587.

800 Wakabayashi, H. and Egusa, S. (1973)Edwardsiella tarda (Paracolobactrum anguilli-mortiferum) associated with pond-cultured eeldisease. Bulletin of the Japanese Society ofScientific Fisheries 39, 931–936.

801 Wakabayashi, H., Hikida, M. and Masumura, K.(1986) Flexibacter maritimus sp. nov., a pathogenof marine fishes. International Journal of System-atic Bacteriology 36, 396–398.

802 Wakabayashi, H., Huh, G. and Kimura, N. (1989)Flavobacterium branchiophila sp. nov., a causativeagent of bacterial gill disease of freshwater fishes.International Journal of Systematic Bacteriology39, 213–216.

803 Wakabayashi, H., Sawada, K., Ninomiya, K. andNishimori, E. (1996) Bacterial hemorrhagic ascitesof Ayu caused by Pseudomonas sp. Fish Pathology31, 239–240.

804 Wallace, L.J., White, F.H. and Gore, H.L. (1966)Isolation of Edwardsiella tarda from a seal lionand two alligators. Journal of American VeterinaryMedical Association 149, 881–883.

805 Wallace, R.J., Brown, B., Tsukamura, M., Brown,J. and Onyi, G. (1991) Clinical and laboratoryfeatures of Nocardia nova. Journal of ClinicalMicrobiology 29, 2407–2411.

806 Wallach, J.D. (1977) Ulcerative shell disease inturtles: identification, prophylaxis and treatment.International Zoo Yearbook 17, 170–171.

807 Wallbanks, S., Martinez-Murcia, A.J., Fryer, J.L.,Phillips, B.A. and Collins, M.D. (1990) 16S rRNA

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808 Waltman, W.D., Shotts, E.B. and Hsu, T.C. (1986)Biochemical characteristics of Edwardsiellaictaluri. Applied and Environmental Microbiology51, 101–104.

809 Wang, G., Tyler, K., Munro, C. and Johnson, W.(1996) Characterization of cytotoxic, hemolyticAeromonas caviae clinical isolates and theiridentification by determining presence of a uniquehemolysin gene. Journal of Clinical Microbiology34, 3203–3205.

810 Wang, R.-F., Cao, W.-W. and Cerniglia, C. (1997)A universal protocol for PCR detection of 13species of foodborne pathogen in foods. Journalof Applied Microbiology 83, 727–736.

811 Watson, R. (1989) The formation of primer arti-facts in polymerase chain reactions. Amplifications1, 5–6.

812 Wauters, G., Janssens, M., Steigerwalt, A. andBrenner, D. (1988) Yersinia mollaretii sp. nov. andYersinia bercovieri sp. nov., formerly calledYersinia enterocolitica biogroups 3A and 3B.International Journal of Systematic Bacteriology38, 424–429.

813 Wayne, L.G., Brenner, D.J., Colwell, R.R.,Grimont, P.A.D., Kandler, O., Krichevsky, M.I.,Moore, L.H., Moore, W.E.C., Murray, R.G.E.,Stackebrandt, E., Starr, M.P. and Trüper, H.G.(1987) Report of the ad hoc committee on reconcil-iation of approaches to bacterial systematics. Inter-national Journal of Systematic Bacteriology 37,463–464.

814 Weiner, R., Segall, A. and Colwell, R. (1985)Characterization of a marine bacterium associatedwith Crassostrea virginica (the Eastern Oyster).Applied and Environmental Microbiology 49,83–90.

815 Weiner, R.M., Coyne, V.E., Brayton, P., West, P.and Raiken, S.F. (1988) Alteromonas colwellianasp. nov., an isolate from oyster habitats. Inter-national Journal of Systematic Bacteriology 38,240–244.

816 Weinstein, M., Litt, M., Kertesz, D., Wyper, P.,Rose, D., Coulter, M., McGreer, A., Facklam, R.,Ostach, C., Willey, B., Borczyk, A. and Low, D.(1997) Invasive infections due to a fish pathogen,Streptococcus iniae. New England Journal ofMedicine 9, 589–594.

817 Weisburg, W., Barns, S., Pelletier, D. and Lane, D.(1991) 16S ribosomal DNA amplification forphylogenetic study. Journal of Bacteriology 173,697–703.

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819 West, P., Lee, J.V. and Bryant, T.N. (1983)A numerical taxonomic study of species of Vibrioisolated from the aquatic environment and birds inKent, England. Journal of Applied Bacteriology 55,263–282.

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821 West, P.A., Brayton, P.R., Bryant, T.N. andColwell, R.R. (1986) Numerical taxonomy ofVibrios isolated from aquatic environments. Inter-national Journal of Systematic Bacteriology 36,531–543.

822 Westbrook, G., O’Hara, C.M., Roman, S. andMiller, J. M. (2000) Incidence and identification ofKlebsiella planticola in clinical isolates with empha-sis on newborns. Journal of Clinical Microbiology38, 1495–1497.

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824 Whittington, R. and Cullis, B. (1988) The suscepti-bility of salmonid fish to an atypical strain ofAeromonas salmonicida that infects goldfish,Carassius auratus (L.), in Australia. Journal of FishDiseases 11, 461–470.

825 Whittington, R., Gudkovs, N., Carrigan, M.,Ashburner, L. and Thurstan, S. (1987) Clinical,microbiological and epidemiological findings inrecent outbreaks of goldfish ulcer disease due toatypical Aeromonas salmonicida in south-easternAustralia. Journal of Fish Diseases 10, 353–362.

826 Whittington, R.J., Djordjevic, S., Carson, J. andCallinan, R. (1995) Restriction endonuclease anal-ysis of atypical Aeromonas salmonicida isolatesfrom goldfish Carassius auratus, silver perchBidyanus bidyanus, and greenback flounderRhombosolea tapirina in Australia. Diseases ofAquatic Organisms 22, 185–191.

827 Wiik, R., Torsvik, V. and Egidius, E. (1986)Phenotypic and genotypic comparisons amongstrains of the lobster pathogen Aerococcus viridansand other marine Aerococcus viridans-like cocci.International Journal of Systematic Bacteriology36, 431–434.

828 Wiklund, T. and Bylund, G. (1990) Pseudomonasanguilliseptica as a pathogen of salmonid fish inFinland. Diseases of Aquatic Organisms 8, 13–19.

829 Wiklund, T. and Bylund, G. (1993) Skin ulcer dis-ease of flounder Platichthys flesus in the northernBaltic Sea. Diseases of Aquatic Organisms 17,165–174.

830 Wiklund, T. and Dalsgaard, I. (1998) Occurrenceand significance of atypical Aeromonassalmonicida in non-salmonid and salmonid fishspecies: a review. Diseases of Aquatic Organisms32, 49–69.

831 Wiklund, T., Dalsgaard, I., Eerola, E. and Olivier,G. (1994) Characteristics of ‘atypical’, cytochromeoxidase-negative Aeromonas salmonicida isolatedfrom ulcerated flounders (Platichthys flesus (L.)).Journal of Applied Bacteriology 76, 511–520.

832 Wiklund, T., Tabolina, I. and Bezgachina, T.(1999) Recovery of atypical Aeromonassalmonicida from ulcerated fish from the Baltic Sea.ICES Journal of Marine Science 56, 175–179.

833 Wiklund, T., Madsen, L., Bruun, M. andDalsgaard, I. (2000) Detection of Flavobacteriumpsychrophilum from fish tissue and water samplesby PCR amplification. Journal of Applied Micro-biology 88, 299–307.

834 Willems, A., Busse, J., Goor, M., Pot, B., Falsen, E.,Jantzen, E., Hoste, B., Gillis, M., Kersters, K.,Auling, G. and Ley, J.D. (1989) Hydrogenophaga,a new genus of hydrogen-oxidising bacteria thatincludes Hydrogenophaga flava comb. nov.(formerly Pseudomonas flava), Hydrogenophagapalleronii (formerly Pseudomonas palleronii),Hydrogenophaga pseudoflava (formerly Pseudo-monas pseudoflava and ‘Pseudomonas carboxy-doflava’), and Hydrogenophaga taeniospiralis (for-merly Pseudomonas taeniospiralis). InternationalJournal of Systematic Bacteriology 39, 319–333.

835 Williams, A.M., Fryer, J.L. and Collins, M.D.(1990) Lactococcus piscium sp. nov. a newLactococcus species from salmonid fish. FEMSMicrobiology Letters 68, 109–114.

836 Willumsen, B. (1989) Birds and wild fish aspotential vectors of Yersinia ruckeri. Journal ofFish Diseases 12, 275–277.

837 Wilson, B. and Holliman, A. (1994) AtypicalAeromonas salmonicida isolated from ulceratedchub Leuciscus cephalis. Veterinary Record 135,185–186.

838 Wilson, K., Blitchington, R. and Greene, R. (1990)Amplification of bacterial 16S ribosomal DNA withpolymerase chain reaction. Journal of ClinicalMicrobiology 28, 1942–1946.

839 Wolters, W. and Johnson, M. (1994) Enteric septi-caemia resistance in blue catfish and three channelcatfish strains. Journal of Aquatic Animal Health 6,329–334.

840 Wong, F., Fowler, K. and Desmarchelier, P. (1995)Vibriosis due to Vibrio mimicus in Australian fresh-water crayfish. Journal of Aquatic Animal Health 7,284–291.

841 Woo, P.T.K. and Bruno, D.W. (1999) Viral,Bacterial and Fungal Infections. Fish Diseases andDisorders. Vol. 3. CAB International, Wallingford,UK.

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843 Wood, R. and Packer, R.A. (1972) Isolation ofErysipelothrix rhusiopathiae from soil and manureof swine-raising premises. American Journal ofVeterinary Research 33, 1611–1620.

844 Yabuuchi, E., Kaneko, T., Yano, I., Moss, C.W.and Miyoshi, N. (1983) Sphingobacterium gen.nov., Sphingobacterium spiritivorum comb. nov.,Sphingobacterium multivorum comb. nov.,Sphingobacterium mizutae sp. nov.: and Flavo-bacterium indologenes sp. nov.: glucose-nonfermenting Gram-negative rods in CDCGroups IIK-2 and IIb. International Journal ofSystematic Bacteriology 33, 580–598.

845 Yamada, Y. and Wakabayashi, H. (1999)Identification of fish-pathogenic strains belongingto the genus Edwardsiella by sequence analysis ofsodB. Fish Pathology 34, 145–150.

846 Yang, Y., Yeh, L., Cao, Y., Baumann, L.,Baumann, P., Tang, J.S. and Beaman, B. (1983)Characterization of marine luminous bacteriaisolated off the coast of China and description ofVibrio orientalis sp. nov. Current Microbiology 8,95–100.

847 Yii, K.-C., Yang, T. and Lee, K.-K. (1997) Isolationand characterization of Vibrio carchariae, acausative agent of gastroenteritis in the groupers,Epinephelus coioides. Current Microbiology 35,109–115.

848 Yuasa, K., Kitancharoen, N., Kataoka, Y. andAl-Murbaty, F.A. (1999) Streptococcus iniae, thecausative agent of mass mortality in Rabbitfish

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849 Yumoto, I., Kawasaki, K., Iwata, H., Matsuyama,H. and Okuyama, H. (1998) Assignment of Vibriosp. strain ABE-1 to Colwellia maris sp. nov., a newpsychrophilic bacterium. International Journal ofSystematic Bacteriology 48, 1357–1362.

850 Yumoto, I., Iwata, H., Sawabe, T., Ueno, K., Ichise,N., Matsuyama, H., Okuyama, H. and Kawasaki,K. (1999) Characterization of a facultativelypsychrophilic bacterium, Vibrio rumoiensis sp.nov., that exhibits high catalase activity. Appliedand Environmental Microbiology 65, 67–72.

851 Ziemke, F., Höfle, M.G., Lalucat, J. and Rosselló-Mora, R. (1998) Reclassification of Shewanellaputrefaciens Owen’s genomic group II asShewanella baltica sp. nov. International Journalof Systematic Bacteriology 48, 179–186.

852 Zlotkin, A., Eldar, A., Ghittino, C. and Bercovier,H. (1998a) Identification of Lactococcus garvieaeby PCR. Journal of Clinical Microbiology 36,983–985.

853 Zlotkin, A., Hershko, H. and Eldar, A. (1998b)Possible transmission of Streptococcus iniaefrom wild fish to cultured marine fish. Applied andEnvironmental Microbiology 64, 4065–4067.

854 ZoBell, C.E. (1941) Studies on marine bacteria. I.The cultural requirements of heterotrophicaerobes. Journal of Marine Research 4, 42–75.

855 Zorilla, I., Balebona, M.C., Moriñigo, M.A.,Sarasquete, C. and Borrego, J.J. (1999) Isolationand characterisation of the causative agent ofpasteurellosis, Photobacterium damselae spp.piscicida, from sole, Solea senegalensis (Kaup).Journal of Fish Diseases 22, 167–172.

328 References



Index

0/129culture appearance see photographic

sectionreagent 273test see Vibrio discs

Abalone 2, 67, 69, 281Haliotis discus hannai 2, 281Haliotis tuberculata 2Japanese abalone (Sulculus diversicolor

supratexta) 2Red abalone (Haliotis rufescens) 2, 67, 285Small abalone (Haliotis diversicolor

supertexta) 2, 71, 286South African abalone 67, 286

Abiotrophia 75A. adiacens see Granulicatella adiacensA. balaenopterae 38

see also Granulicatella balaenopteraeA. defectiva 75, 162, 210A. elegans 38, 75

see also Granulicatella elegansA. para-adiacens 162, 210

Acetate agar see Rogosa acetate agarAchromobacter 223

A. xylosoxidans ssp. denitrificans 38, 155Acinetobacter 32, 131

identification 91, 149, 178, 184, 190A. baumannii 38

identification 149A. calcoaceticus 20, 29, 38

identification 149, 197A. haemolyticus 38

identification 91, 149

Actinobacillus 29A. delphinicola 27, 28, 30, 38

identification 91, 154, 178, 184, 192,197, 212

A. scotiae 28, 38, 218identification 91, 154, 178, 184, 191

ActinomycesA. marimammalium 28, 29, 38, 218

identification 86, 91, 164, 208, 210A. viscosus 212

Aequorivita 75identification 86, 91, 129, 130A. antarctica 38, 218

identification 146, 212A. crocea 38, 218

identification 146A. lipolytica 38, 218

identification 146A. sublithincola 38, 218

identification 146Aerococcus

A. viridans 21, 38identification 160, 210, 258

A. viridans var. homari 13, 38identification 86, 91, 160, 200, 201,

212Aeromonas 75

DNA hybridization groups 75, 141identification 124, 139PCR primers 231A. allosaccharophila 10, 12, 39, 76, 218

identification 142, 178, 184, 196A. bestiarum 6, 39, 76, 218

identification 142, 178, 184, 196, 200,201

329



Aeromonas continuedA. caviae 218, 225

identification 142, 178, 184, 192, 193,200, 201

hosts and diseases 12, 32, 39, 76PCR primers 226

A. culicicolahosts and diseases 39identification 91, 142, 178, 200, 201

A. encheleia 218hosts and diseases 9, 39identification 86, 91, 142, 178, 184, 193,

200, 201taxonomy 76

A. enteropelogenes 40A. eucrenophila 218

hosts and diseases 6, 29, 39identification 86, 91, 142, 178, 200, 201PCR primers 231

A. hydrophila 218, 225culture appearance see photographic

sectionhosts and diseases 3, 4, 7, 15, 16, 20, 21,

29, 32identification 91, 178, 184, 192, 193,

196, 197, 200, 201, 212PCR primers 225, 226, 231taxonomy 75

A. hydrophila ssp. dhakensis 218hosts and diseases 12, 39, 76identification 76, 124, 142taxonomy 75

A. hydrophila ssp. hydrophila 218hosts and diseases 12, 39, 76identification 76, 124, 142taxonomy 75

A. ichthiosmia see Aeromonas veronii ssp.veronii

A. janadaei 12, 40, 218culture appearance see photographic

sectionhosts and diseases 16identification 142, 178, 184, 196, 212

A. media 218hosts and diseases 15, 40identification 92, 142, 200, 201

A. popoffii 40, 218identification 86, 124, 142, 178, 200,

201PCR primers 231

A. punctata 39A. salmonicida

identification 86, 124, 178, 184, 192, 212

A-protein layer 124detection with Coomassie brilliant blue

agar 265detection with Congo Red agar 266

luminescence 122PCR primers 233pigment detection 86, 124

Furunculosis agar 124, 268A. salmonicida ssp. salmonicida 218

antisera 136hosts and diseases 9, 11, 13, 22, 24, 31,

35, 37, 39, 77identification 92, 86, 140, 178, 184, 192,

196, 197, 200, 201PCR primers 226pigment 86non-pigmented strains 40taxonomy 77

A. shigelloides see Plesiomonasshigelloides

A. schubertii 39, 218hosts and diseases 12, 41identification 143

A. sobria 218, 225hosts and diseases 6, 12, 24, 41identification 142, 178, 184, 196, 197,

202, 203PCR primers 226, 231

Aeromonas species 15, 37, 223Aeromonas species PCR 225A. trota 218

hosts and diseases 12, 41identification 143, 179, 184, 196, 202,

203PCR primers 226, 231taxonomy 76

A. veronii ssp. sobria 218culture appearance see photographic

sectionhosts and diseases 12, 40identification 143, 202, 203, 212taxonomy 76

A. veronii ssp. veronii 218hosts and diseases 6, 12, 32, 41, 76identification 92, 142, 179, 184, 191,

195, 196, 202, 203taxonomy 76

Aeromonas group 501 39enteric group 41

Atypical A. salmonicidaculture appearance see photographic

sectiongrowth at 37°C strain 24, 41

330 Index



hosts and diseases 4, 6, 7, 9, 10, 11, 14,17, 18, 21, 22, 24, 34, 35, 37, 41, 77

identification 125, 178, 184oxidase-negative strains 10, 23, 34, 41,

140pigment 77, 125A. salmonicida ssp. achromogenes 22,

30, 32, 57, 77, 218identification 178, 184, 190, 200, 201hosts and diseases 5, 18, 21, 22, 40taxonomy 79

A. salmonicida ssp.achromogenes/masoucidaidentification 140, 197

A. salmonicida ssp. masoucida 40, 77hosts and diseases 40identification 140, 178, 184, 192

A. salmonicida ssp. novahosts and diseases 17, 40identification 92, 140

A. salmonicida ssp. pectinolytica 40, 218identification 92, 141

A. salmonicida ssp. smithia 40identification 140

Aesculin 130, 133media preparation 261, 262test interpretation 117, 118, 261

Agar digestion 129Agrobacterium stellulatum see Stappia

stellulata-likeAlbatross (grey headed albatross) 62AlcaligenesAlcaligenes denitrificans see Achromobacter

xylosoxidans subsp. denitrificansA. faecalis

hosts and diseases 29see also Halomonas aquamarina

A. faecalis var. homari 41identification 86, 98taxonomy 51

A. venustus see Halomonas venustaAlga 2

Marine alga 2, 57, 60Red alga (Delesseria sanguinea) 2, 63, 74Ulva lactuca 60

Alkaline peptone water 244Alligator 2, 47, 55, 56, 281Allomonas

A. enterica 42identification 92, 155, 179

Alteromonashosts and diseases 15, 31, 32, 42identification 86, 92

taxonomy 77A. antarctica see Pseudoalteromonas

antarcticaA. aurantia 218

see also Pseudoalteromonas flavipulchraA. citrea 218

see also Pseudoalteromonas citreaA. colwelliana see Shewanella colwellianaA. communis see Marinomonas communisA. denitrificans see Pseudoalteromonas

denitrificansA. distincta see Pseudoalteromonas

distinctaA. elyakovii see Pseudoalteromonas

elyakoviiA. espejiana see Pseudoalteromonas

espejianaA. hanedai see Shewanella hanedaiA. macleodii 77

identification 155, 212A. nigrifaciens see Pseudoalteromonas

nigrifaciensAPI 20NE see Alteromonas

nigrifaciens 197A. piscicida see Pseudoalteromonas

piscicidaA. rubra 218

see also Pseudoalteromonas rubraA. undina see Pseudoalteromonas undina

Alvinella pompejana see polychaete annelidAmblyglyphidodon curacao Bloch see

DamselfishAmeiurus species see CatfishAmies transport medium 84, 244Ammonium salt sugar (ASS) 139, 264Ammodytes lancea Cuvier see EelAmphiprion clarkii see DamselfishAmpicillin disc 114Anacker-Ordal agar (AO) 244, 245Anaerobe plates 245Anarhichas species see Wolf-fishAnchovy 2, 66Angel fish 45, 47Anguilla species see EelAO medium see Anacker-Ordal agarAphanomyces invadans 39, 42API identification systems 116

API 50CH 116, 200–207API Coryne 116, 208API 20E 116, 138, 178–196API 20NE 116, 197–199API 20 Strep 116, 209API rapid A 116

Index 331



API identification systems continuedAPI rapid ID 32 STREP 116, 210–211API ZYM 116, 139, 212–217

Apistogramma ocellatus see OscarAphanius iberus Valenciennes see CarpAptenodytes patagonica see PenguinAquaspirillum

hosts and diseases 42identification 86, 155

Arabinose see Carbohydrate fermentationArapaima gigas Cuvier see PirarucuArcanobacterium

culture requirements 86A. bernardiae

hosts and diseases 42identification 164, 202, 203, 208, 210

A. haemolyticumidentification 164, 208, 210

A. phocae 218hosts and diseases 28, 29, 42identification 92, 95, 164, 202, 203, 208,

210, 212A. pluranimalium 218

hosts and diseases 28, 42identification 92, 165, 208, 210

A. pyogenes 59hosts and diseases 42identification 92, 165, 202, 203, 210

Arctic Charr see SalmonidsArctocephalus species see SealsArdea herodias see HeronArginine dihydrolase

media preparation 267Møller’s method 117, 267test interpretation 117, 118

variable results in Vibrio species 117,120, 267

see also photographic sectionThornley’s method 117, 267

Argopecten purpuratus see ScallopAristichthys nobilis see CarpArtemia species see ShrimpArthrobacter

culture requirements 86A. agilis 42

identification 93, 165A. aurescens 212A. nasiphocae


A. rhombi 218hosts and diseases 11, 42identification 93, 165, 202, 203, 208

Artificial seawater medium 245Ascidians 59Ashdown’s medium see Burkholderia

pseudomallei selective mediaAtractoscion nobilis Ayres see BassASW see Artificial seawater mediumAtopobacter phocae 218


Atypical bacterial gill disease 60Atypical furunculosis 35Ayu 3, 39, 53, 60, 61, 65, 68, 281

BA see Blood agarBacillary necrosis 15, 72Bacillary necrosis of Pangasius 1, 7Bacillus 20, 29, 223

B. cereushosts and diseases 42identification 93, 164

B. mycoideshosts and diseases 7, 42identification 93, 164

B. subtilis 42Bacterial cold-water disease 49Bacterial gill disease 17, 23, 24, 31, 36, 49Bacterial haemorrhagic ascites (BHA) 3, 60Bacterial kidney disease (BKD) 3, 22, 23, 25,

36, 132Bacterial load 222Bacterial necrosis 14, 42, 71Bacterial stomatitis (mouth rot) 22, 36, 66Baitfish (Pimephales promelas, American

baitfish) 14Balaenoptera acutorostrata see WhaleBarramundi 4, 49, 57, 65, 70, 281Basal medium 264Basal medium agar 264Bass 3, 42, 47, 49, 57, 62, 285, 286

European seabass (Dicentrarchus labraxLinnaeus) 3, 36, 65, 282

Japanese seaperch 4Largemouth bass (Micropterus

salmoides) 3, 49, 56, 283Sea bass 39, 54, 56, 60, 66, 69, 285Sea bass (Lateolabrax japonicus Cuvier)

4Sea bass (Lates calcarifer Bloch) 4, 281Sea bass (Puntazzo puntazzo Cuvier) 4Striped bass (Morone saxatilis Walbaum,

Roccus saxatilis) 4, 44, 46, 53, 54, 57,65, 286

332 Index



White sea bass (weakfish, Atractoscionnobilis Ayres) 4, 55

Bearded dragon 47Benechea chitinovora


Bergeyella 78, 129, 130Betta splendens Regan see Siamese fighting fishBGD see Bacterial gill diseaseBidyanus species see PerchBHA see Bacterial haemorrhagic ascitesBiochemical identification tests 114

discs (Vibrio discs) 114growth at different temperatures 114inoculation of biochemical identification

sets 115freshwater isolates 115marine isolates 115

interpretation of biochemical identificationset (‘biochem set’) 117–123

recording results (Laboratory worksheet)177

using the biochemical identification tables123, 137, 140–176

flowchart 138BKD see Bacterial kidney diseaseBlack acara (Cichlasoma bimaculatum

Linnaeus) 16, 54, 55, 281Black disease 32, 39Black parch necrosis 32, 36, 66Black tiger prawn see ShrimpBlacksmith see DamselfishBLAST search 242Blaser-Wang media see Helicobacter selective

mediaBlenny 4, 41, 281, 286Blood agar (BA) 246Bordetella bronchiseptica 218

culture requirements 86Bordetella bronchiseptica selective agar

(CFPA) 246hosts and diseases 43identification 93, 153, 197, 212

Black scraper 52, 281Blicca bjoerkna Linnaeus see BreamBluefish 65, 281Brachionus plicatilis see RotiferBranchio-necrosis 42Branchipus schaefferi Fisher see ShrimpBRD see Brown ring diseaseBream 5, 53, 57, 282, 286

Black sea bream (Acanthopagrus latusHouttuyn) 5, 51, 66

Common bream 5Gilt-head sea bream (Sparus auratus

Linnaeus) 57, 65, 72, 283One-spot sea bream (Diplodus sargus

kotschyi Steindachner) 5, 284Red sea bream (Pagrus major) 5, 64, 66,

285Rock bream 66Sea bream (Sparus auratus Linnaeus) 60,

65, 67, 68, 69, 285Sliver bream (Blicca bjoerkna Linnaeus) 5,

40, 285Brevoortia patronus Goode see MenhadenBrevundimonas

B. diminuta 218hosts and diseases 43identification 93, 155, 197, 202, 203,

212B. vesicularis 218


Bromothymol blue indicator 262, 263, 275Brill see TurbotBrown ring disease (BRD) 14, 36, 72Brucella

culture requirements 86assay of metabolic activity 261Brucella agar 246Farrell’s medium 86, 248growth on substrate media 262serum-dextrose agar 256

hosts and diseases 12, 27, 29, 30, 43identification 93, 125, 144, 197taxonomy 77B. abortus

antisera 136hosts and diseases 12, 43identification 93, 144, 153

B. canishosts and diseases 43identification 144

B. cetaceae 219hosts and diseases 27, 28, 43, 77, 78identification 144, 124

B. maris see B. cetaceaeB. melitensis

hosts and diseases 43, 77identification 144

B. pinnipediaehosts and diseases 18, 28, 29, 43, 78identification 144

B. suis 43identification 144

Index 333



Brucella agar 246Brucella broth 224Budvicia aquatica

hosts and disease 44identification 94, 151

BurkholderiaB. cepacia 61

hosts and disease 44identification 155, 184, 190, 194, 195

B. pseudomalleiculture requirements 86

selective broth 246selective media (Ashdown’s medium)

247selective media (glycerol medium) 246

hosts and diseases 12, 27, 28, 29, 30, 44identification 94, 153, 179, 184, 190,

192

Callinectes sapidus see CrabCalloplesiops altivelis Steindachner see CometsCallorhinus ursinus see SealsCandida species 30

hosts and diseases 44identification 144

Capnocytophaga 78, 128, 129, 130Caranx hippos Linnaeus see Crevalle, Jack

crevalleCarassius species see Carp, GoldfishCarbohydrate fermentation

alternative method for Vibrio spp. 264carbohydrate fermentation media using

acid from ammonium salt sugar 264general purpose medium 262for Pseudomonas spp., Flavobacterium

spp., Nocardia spp., Shewanella spp.,Sphingobacterium spp. and marinespecies 263–264

test interpretation 118, 120, 262, 263see photographic section

Carbon dioxide atmosphere 247, 265Carcharhinus species see SharkCaretta caretta see TurtleCarnobacterium

hosts and diseases 7, 22, 35identification 78, 125–127taxonomy 78C. alterfunditum

hosts and diseases 44identification 165, 210, 212

C. divergenshosts and diseases 35, 44

identification 126, 165, 179, 202, 203,210, 212

C. divergens strain 6251hosts and diseases 44identification 165, 212

C. funditumhosts and diseases 44identification 165, 210, 212

C. gallinarumhosts and diseases 44identification 165, 202, 203, 210, 212

C. inhibens 219hosts and diseases 22identification 86, 94, 165, 209, 210, 212

C. inhibens strain K1hosts and diseases 44

C. mobilehosts and diseases 44identification 165, 202, 203, 210, 212

C. piscicola 219hosts and diseases 4, 7, 24, 44identification 86, 94, 125, 126, 127, 164,

202, 203, 209, 210, 212taxonomy 78

Carnobacterium speciesidentification 164

Carotenoid pigment detection 120, 129also see Congo Red

Carp 40, 41, 42, 45, 47, 49, 61, 65Bighead carp (Aristichthys nobilis) 5, 281Caucasian carp 6, 48, 282Common carp (Cyprinus carpio carpio

Linnaeus) 6, 76, 282Crucian carp (Carassius carassius Linnaeus)

6, 282Goldfish (Carassius auratus Linnaeus) 17Iberian toothcarp 6, 71, 283Koi carp 6, 49, 65Silver carp (Hypophthalmichthys molitrix

Valenciennes) 6, 58, 64, 285Spanish toothcarp (Aphanius iberus

Valenciennes) 6Carp erythrodermatitis 40, 41Catalase 130

method and test interpretation 120, 265for Mycoplasma spp. see Mycoplasma

characterization testsCatenibacterium species see Eubacterium

speciesCatfish 1, 6, 39, 40, 42, 48, 282, 285

African catfish 58Black bullhead (Ameiurus melas, Ictalurus

melas) 6, 49

334 Index



Blue catfish (Ictalurus furcatusValenciennes) 6, 36, 49

Brown bullhead (Ictalurus nebulosus) 7,44, 48, 282

Channel catfish 7, 36, 38, 39, 42, 44, 47,49, 74, 282

Freshwater catfish (Pangasiushypophthalmus Sauvage) 1, 7

Walking catfish (Clarias batrachusLinnaeus) 7, 39, 76

White catfish (Ameiurus catus Linnaeus) 7,47, 286

CBBA see Coomassie brilliant blue agarCellobiose-Colistin agar 247Cellulomonas culture media see Dubos mediaCellulophaga 129, 130Cellulophaga lytica


Cellulose digestion method 128, 265Centropomus undecimalis Bloch see SnookCetacea see Sea mammalsCFPA medium see Bordetella bronchiseptica

selective agarCFU see Colony forming unitsChanos chanos Forsskål see MilkfishChelonia mydas see TurtleCherax albidus see Crayfish, Australian

freshwater crayfishCherax quadricarinatus see Crayfish, red clawChirocephalus diaphanus Prévost see ShrimpCholera 12, 68Chorion 11, 66Chromis punctipinnis see DamselfishChromobacterium violaceum 219

hosts and diseases 12, 45identification 94, 155, 197

Chrysemys species see TurtleChryseobacterium 129, 130

C. balustinumhosts and diseases 45identification 94, 145, 197, 212

C. gleumhosts and diseases 45identification 94, 146, 197, 213

C. indologeneshosts and diseases 45identification 94, 146, 197, 213

C. indoltheticumhosts and diseases 45identification 146, 197, 213

C. meningosepticumculture requirements 86

hosts and diseases 45identification 94, 146, 197, 213

C. scophthalmumculture requirements 87

Medium K 252hosts and diseases 34, 45identification 94, 145, 197, 202, 203, 213

Chrysophrys unicolor see SnapperChub (Leuciscus cephalus Linnaeus) 7, 41, 282Cichlasoma bimaculatum Linnaeus see Black

acaraCichlasoma meeki Brind see Firemouth cichlidCichlid (Oreochromis mossambicus) 282

Firemouth cichlid (Thorichthys meeki,Cichlasoma meeki Brind) 16, 54, 283

CitrateChristensen’s citrate method 120Simmons method 120, 265test interpretation 118, 265

see photographic sectionCitrobacter diversus

identification 151C. freundii

hosts and diseases 6, 21, 24, 45identification 94, 125, 127, 149, 179

Clam 14, 67, 70, 73Manila clam (Ruditapes philippinarum) 14,

36, 72, 282, 284Mercenaria mercenaria 14Softshell clam (Mya arenaria) 286

Clarias species see CatfishCleaner fish see WrasseClostridial myositis 27, 28, 30Clostridium

C. botulinumculture requirements 87hosts and diseases 24, 46identification 95, 143toxin detection 87

C. perfringenshosts and diseases 27, 28, 30, 46identification 143

Clupea harengus membras Linnaeus seeHerring

Coalfish 7, 58Coastal fish 21Cockles 70Cod 58, 71, 282, 283, 284, 286

Atlantic cod (Gadus morhua Linnaeus) 7,40, 44, 52, 281

Coenonia 128, 129, 130Cold water disease 3, 23Colistium species see Turbot

Index 335



Colony forming units (CFU) 224Columnaris disease 6, 24, 31, 49

marine columnaris 66Colwellia maris


Comets (Calloplesiops altivelis Steindachner)16, 54

Black comets 49Conchiolin deposit 14, 62, 72Congo Red (CR) 120

media preparation 266for use see Carotenoid pigment detection

120Congo Red agar 265Coomassie brilliant blue agar (CBBA) 265Coral 8, 68

Oculina patagonica 8, 72Pocillopora damicornis 8, 68

Coral bleaching 70, 72Coregonus species see WhitefishCorynebacterium

C. aquaticum 219culture requirements 87hosts and diseases 4, 46identification 95, 164, 208

C. phocae see Arcanobacterium phocaeidentification 165

C. pseudodiphtheriticumidentification 165, 208

C. pseudotuberculosisidentification 165, 208

C. testudinorishosts and diseases 19, 46identification 95, 164, 208, 213

Corynebacterium species 29, 32, 223C. xerosis

identification 165, 208Coryphaena hippurus Linnaeus see Mahi-mahiCrab 8, 38, 48, 53, 67, 73, 281, 284, 286

Blue crab (Callinectes sapidus) 8Swimming crab (Portunus trituberculatus)

8Crane (Sandhill crane (Grus canadensis)) 25Crassostrea species see OysterCrawfish

American freshwater crayfish 8, 281Red swamp crawfish (Procambarus clarkii)

8, 281, 285Crayfish 8, 60, 281, 285

Australian freshwater crayfish 8, 70Marron 70, 71Red claw 8, 285

Yabby 8, 70Crevalle 8Crocodile 8, 47, 48, 54, 55, 283, 285

Caiman crocodile (Caiman crocodilus) 8Crocodilus acutus 8Crocodylus niloticus 8

Crumb-of-bread sponge see SpongeCryptococcosis 27Cryptococcus

culture appearance see photographicsection

differentiation from Candida species 127,144

C. lupihosts and disease 46identification 144

C. neoformans 12, 27C. neoformans var. gattii 46


CSF see Cerebrospinal fluidCulture and incubation 83, 85

culture medium 85freshwater animals 85incubation time and temperature 85, 115saltwater animals 85, 115specific culture requirements of organisms

86–90Culture collections 279Culture media preparation 244–261Cutaneous ulcerative disease 40Cyclobacterium marinum 128Cyprinus carpio carpio Linnaeus see CarpCystophara cristata see SealCytophaga

agar see Anacker-Ordal agarcarbohydrate fermentation 262cellulose digestion 265culture media

Anacker-Ordal agar 120, 121, 244peptone yeast medium 255

taxonomy 78, 128C. agarovorans see Marinilabilia

salmonicolor biovar agarovoransC. allerginae

identification 146, 213Cytophaga aquatilis see Flavobacterium

hydatisC. arvensicola


C. aurantiaca strain NCIMB 1382 78hosts and disease 46

336 Index



C. columnaris see Flavobacteriumcolumnare

C. fermentanshosts and disease 46identification 146, 213

C. flevensis see Flavobacterium flevenseC. heparina see Pedobacter heparinusC. heparinum see Pedobacter heparinusC. hutchinsonii 128

culture requirements 87Dubos medium 247

identification 146, 213C. johnsonae see Flavobacterium

johnsoniaeC. latercula

culture requirements 87hosts and disease 46identification 146, 213taxonomy 128

C. lytica see Cellulophaga lyticaC. marina see Tenacibaculum maritimumC. marinoflava

hosts and disease 47identification 146taxonomy 128

C. pectinovorum see Flavobacteriumpectinovorum

C. psychrophila see Flavobacteriumpsychrophilum

C. saccharophilum see Flavobacteriumsaccharophilum

C. salmonicolor see Marinilabiliasalmonicolor biovar agarovorans

C. succinicans see Flavobacteriumsuccinicans

C. uliginosum see Zobellia uliginosaC. xanthum see Flavobacterium xanthum

Dab 9, 41, 282Dace (Leuciscus leuciscus Linnaeus) 9, 282Damselfish (Pomacentrida) 9, 57, 60, 282

Blacksmith (Chromis punctipinnis) 9Staghorn damselfish 9Yellowtail clownfish (Amblyglyphidodon

curacao) 9Yellowtail clownfish (Amphiprion clarkii)

9Danio 16, 47, 282, 286

Danio devario Hamilton 16Sind danio 16

Dasyatis pastinaca see StingrayDelesseria sanguinea see Alga

Delphinapterus leucas see WhaleDelphinus delphis see DolphinDelya aquamarinus

identification 153see also Alcaligenes faecalis homari;

Halomonas aquamarinaDelya marina see Halomonas marinaDermatophilosis 47Dermatophilus

culture requirements 87D. chelonae


D. congolensishosts and diseases 47identification 95, 164selective medium (polymyxin plates)

247Dermatophilus selective medium (polymyxin

plates) 247Dermochelys coriacea see TurtleDicentrarchus labrax Linnaeus see BassDienes Stain 253, 266Dietzia maris (Rhodococcus maris)


Digitonin 266Diplodus sargus kotschyi Steindachner see

BreamDiscus fish 16, 282

Blue discus fish (Symphysodonaequifasciatus Pellegrin) 16, 54

Discs 114DNA extraction 233, 234DNase plate 130

media preparation 267HCl for detection of hydrolysis 269test interpretation 118

see also photographic sectionDolphin 46, 48, 51, 53, 56, 57, 64, 284, 285,

286Amazon freshwater dolphin (Inia

geoffrensis) 27, 65, 281, 282Atlantic bottlenose dolphin (Tursiops

truncatus) 27, 281, 282Atlantic white-sided dolphin

(Lagenorhynchus acutus) 27, 43, 281Bottlenose dolphin 43, 44Common dolphin (Delphinus delphis) 27,

43, 282Pacific white-sided dolphin

(Lagenorhynchus obliquidens) 27,284

Index 337



Dolphin continuedStriped dolphin (Stenella coeruleoalba) 27,

38, 43, 286Tursiops aduncas 27, 281Tursiops gephyreus 51White-sided dolphin 44

Dolphin fish see Mahi-mahiDorosoma cepedianum Lesueur see ShadDubos medium 247

Eagle (Bald eagle, Haliaeetus leucocephalus) 25Edwardsiella

E. anguillimortifera see Edwardsiella tardaE. hoshinae


hosts and disease 47identification 95, 127–128, 149, 184

E. ictaluri 219culture requirements 87

medium (EIM) 248hosts and diseases 1, 3, 5, 6, 7, 11, 16,

17, 28, 47identification 95, 128, 149, 179, 184,

193E. ictaluri anaerobic strains 7, 47, 87

identification 143, 149E. tarda 219

culture requirements 87, 256culture appearance see photographic

sectionhosts and diseases 2, 3, 5, 7, 8, 10, 11,

12, 15, 17, 19, 20, 23, 24, 25, 27, 33,34, 36, 47, 78

identification 96, 150, 179, 184, 185,194, 213, 270

taxonomy 78Edwardsiella species PCR primers 226,

232Eel 9–10, 39, 40, 41, 47, 49, 52, 53, 57, 58,

60, 67, 69, 73, 74, 281American eel 9, 281Anguilla reinhardtii 9, 282Elvers 10, 39European eel (Anguilla anguilla Linnaeus)

9Green moray eel (Gymnothorax funebris)

10, 55, 283Hyperoplus lanceolatus Lesauvege 10Japanese eel (Anguilla japonica) 10, 282Sand eel (Ammodytes lancea Cuvier) 10,

41, 285

Spotted moray eel (Gymnothorax moringa)10, 55, 286

Taiwanese eels 15Eigemannia virescens Valenciennes see Green

knifefishEIM see Edwardsiella ictaluri MediumElectric blue hap (Sciaenochromis ahli

Trewavas) 16Electrolyte supplement 248Elvers see EelEmpedobacter 129, 130Empedobacter brevis

culture requirements 87hosts and diseases 48identification 96, 147, 213taxonomy 78

Emphysematous putrefactive disease of catfish47

Enchelyopus cimbrius Linnaeus see RocklingEngraulis mordax see AnchovyENO salts 265Enteric redmouth (ERM) 23, 24, 25, 36, 74,

82Enteric septicaemia of catfish (ESC) 1, 6, 7, 36,

47Enterobacter 29, 48, 151

Enterobacter agglomerans see Pantoeaagglomerans

Enterococcus 209culture requirements 87identification notes 128taxonomy 78E. faecalis 219


E. faecalis var. liquefaciens 160E. faecium


E. seriolicida see Lactococcus garvieaeEnterovibrio norvegicus 219

culture requirements 87hosts and diseases 34, 47identification 96, 175, 179, 185, 192, 213luminescence 122taxonomy 78

Epalzeorhynchos kalopterus Bleeker see Flyingfox

Epinephelus coioides see GrouperEretmochelys imbricata see TurtleErysipelothrix rhusiopathiae


338 Index



culture requirements 87Packer’s plates 255Wood’s broth 260

hosts and diseases 8, 12, 27, 28, 47identification 96, 164, 210

Erysipelothrix selective medium 248Escherichia

E. colihosts and diseases 28, 29, 47identification 151PCR primers 231, 233

E. vulnerishosts and diseases 6, 17, 24, 47identification 150

Esox lucius Linnaeus see PikeEubacterium

hosts and diseases 19, 48identification 96, 143E. limosum

culture morphology 96E. tarantellae

culture requirements 87hosts and diseases 15, 48identification 96, 143

Eudyptes species see PenguinEudyptula minor see PenguinEuprymna scolopes see SquidExtracellular galactosamine glycan see

Carotenoid pigment detection; CongoRed

Facklamia miroungaeculture requirements 87hosts and diseases 30, 48identification 96, 162, 210, 213

False columnaris disease 49Farrell’s medium 248Fascaplysinopsis reticulata see SpongeFermentation see Carbohydrate fermentationFetal calf serum 224Film and spots see Mycoplasma characterization

testsFINE see Flounder necrotizing enteritisFin rot 39, 49, 60, 72FISH see Fluorescence in situ hybridizationFish bite 12, 51Fish gangrene 47Fish pasteurellosis 3, 4, 5, 11, 16, 18, 32, 33,

34, 35, 37, 57Flamingo 47Flatfish see Turbot

Flavobacteriaceae familycarbohydrate fermentation 262, 264gliding motility 121identification notes 128-130pigment detection 120using the API ZYM 139

Flavobacteriosis 45Flavobacterium species 32, 37, 223

culture mediaAnacker-Ordal agar 244, 245, 249artificial seawater medium 245carbohydrate fermentation 262

alternative methods for carbohydratefermentation 263

Flavobacterium maritimus media (FMM)249

Flavobacterium psychrophilum medium(FPM) 249

peptone yeast medium (PY) 255R2A agar 256

differentiation from Cytophaga species 128hosts and diseases 20PCR primers 226taxonomy 78F. aquatile 48, 219


F. balustinum see Chryseobacteriumbalustinum

F. branchiophilum 219culture requirements 87hosts and diseases 17, 23, 24, 31, 36, 49identification 96, 145, 179, 185, 190,

213F. breve see Empedobacter brevisF. cauliformans 48F. columnare 224

culture requirements 87culture appearance see photographic

sectionHsu-Shotts agar 251Shieh medium + tobramycin 257tryptone yeast extract salt medium 259tryptone yeast extract glucose agar 259

genomovars 129hosts and diseases 6, 10, 16, 17, 18, 19,

23, 24, 31, 37identification 97, 129, 145, 179, 185,

190, 191, 202, 203, 213PCR primers 226, 227, 232, 233media for freeze-drying 224taxonomy 79

Index 339



Flavobacterium species continuedF. flevense


F. frigidarium 219hosts and diseases 49identification 97, 147, 213

F. gillisiae 219culture requirements 88hosts and diseases 49identification 97, 147, 179, 185, 191

F. gleum see Chryseobacterium gleumF. gondwanensis see Psychroflexus

gondwanensisF. hibernum 219

hosts and diseases 49identification 97, 147, 179, 185, 191,

197F. hydatis 219


213F. indologenes see Chryseobacterium

indologenesF. indoltheticum see Chryseobacterium

indoltheticumF. johnsoniae


hosts and diseases 4, 49identification 97, 145, 179, 213, 214taxonomy 79

F. meningosepticum see Chryseobacteriummeningosepticum

F. mizutaiihosts and diseases 49identification 147

F. multivorum see Sphingobacteriummultivorum

F. okeanokoites see Planomicrobiumokeanokoites

F. pectinovorumhosts and diseases 49identification 97, 147, 214

F. piscicida see Photobacterium damselaessp. piscicida

F. psychrophilum 224culture requirements 87

Flavobacterium psychrophilum medium(FPM) 249

Hsu-Shotts agar 251Shieh medium + tobramycin 257tryptone yeast extract glucose agar 259

hosts and diseases 3, 22, 23, 24, 49identification 97, 145, 179, 185, 190,

202, 203, 214media for freeze-drying 224PCR primers 227, 233

F. saccharophilumhost and diseases 50identification 97, 147, 214

F. salegens see Salegentibacter salegensF. scophthalmum see Chryseobacterium

scophthalmumF. succinicans

host and diseases 50identification 97, 147, 214

F. tegetincola 219culture requirements 88host and diseases 50identification 97, 147, 179, 185, 190

F. uliginosum see Zobellia uliginosaF. xanthum

culture requirements 88host and diseases 50identification 98, 147, 179, 185, 190

F. yabuuchiae 64Flexibacter 37

culture mediaAnacker-Ordal agar 244artificial seawater medium 245Flexibacter maintenance medium 250Flexibacter polymorphus media 249

carbohydrate fermentation 262alternative methods for carbohydrate

fermentation 263culture requirements of marine species

88taxonomy 78, 79F. aggregans


F. aurantiacusAPI ZYM 214taxonomy 79

F. canadensishosts and diseases 50identification 147, 214

F. columnaris see Flavobacteriumcolumnare

F. eleganshosts and diseases 50identification 147

F. flexilis 128hosts and diseases 50identification 147, 214

340 Index



F. litoralishosts and diseases 50identification 147, 202, 203, 214

F. maritimus see Tenacibaculummaritimum

F. odoratimimus see Myroidesodoratimimus

F. odoratum see Myroides odoratusF. ovolyticus see Tenacibaculum

ovolyticumF. polymorphus 219

culture requirements 88media 249


F. psychrophilus see Flavobacteriumpsychrophilum

F. roseolusculture requirements 88hosts and diseases 50identification 148, 202, 203, 214

F. ruberculture requirements 88hosts and diseases 50identification 148, 202, 203, 214

F. sanctihosts and diseases 50identification 148, 214

F. tractuosushosts and diseases 50identification 148, 214

Flexirubin pigment 129KOH 121method and test interpretation 120

Flounder 10, 41, 47, 66, 73, 282, 283Greenback flounder (Rhombosolea tapirina

Günther) 10, 40, 283Japanese flounder (Paralichthys olivaceus)

11, 56, 57, 69Marine flounder 40Platichthys flesus Linnaeus 10Summer flounder (Paralichthys dentatus)

11, 69Winter flounder 53

Flounder necrotizing enteritis (FINE) 11, 69Fluorescence in situ hybridization (FISH)

242–243Flying fox (Epalzeorhynchos kalopterus Bleeker)

16, 65FPM see Flavobacterium psychrophilum

mediumFreeze-drying of bacteria 224, 251

Frogs 40, 47, 54, 65Bullfrogs 39, 45, 65Rana castesbeiana 65

Fundulus grandis Baird see MinnowFuruncles 10, 35, 36, 40, 41Furunculosis 5, 9, 11, 22, 23, 24, 35, 37, 40,

77Furunculosis agar (FA) 268

Gadus morhua Linnaeus see CodGaffkemia 13, 38Galactosamine glycan see Carotenoid pigment

detectionGaleocerdo curvieri see SharkGastric ulceration 27, 51Gastritis 27, 30Gavia immer see LoonGel electrophoresis 235Gelatin 130Gelatinase

gelatin/salt plate method 268test interpretation 118, 268

see also photographic sectionGelidibacter 78, 129, 130Giant freshwater prawn see ShrimpGill disease 34, 45, 49Ginglymostoma cirratum see SharkGliding motility 129

method and test interpretation 121Globicephala scammoni see WhaleGlucose 130

see also Carbohydrate fermentationGlucose yeast extract agar (GYEA) 268Glycerol Lab Lemco broth 250Glycerol medium see Burkholderia

pseudomallei, culture requirements,selective media

Golden Shiner (Notemigonus crysoleucasMitchill) 16, 283

Goldfish 17, 40, 41, 47, 49, 54, 60, 68, 74,283

Goldfish ulcer disease (GUD) 17, 40, 41Golf ball disease 27, 65Gourami 54, 58, 283

Three-spot gourami (Trichogastertrichopterus Pallas) 18

Gram-negative anaerobe plates 245Granulicatella

G. adiacenshosts and diseases 50, 75identification 98, 162, 209, 210

Index 341



Granulicatella continuedG. (Abiotrophia) balaenopterae 219

culture requirements (see Abiotrophiabalaenopterae) 86

hosts and diseases 30, 51identification 98, 160, 209, 210, 214

G. eleganshosts and diseases 12, 50identification 162, 209, 210

Grayling 11, 283Green knifefish 11, 47, 283Greenling 11Grouper 4, 11, 69, 282, 283

Orange-spotted grouper (Epinepheluscoioides) 4

Grus canadensis see CraneGUD see Goldfish ulcer diseaseGull, Ring-billed gull (Larus delewarensis) 25Guppy (Poecilia reticulata Peters) 17, 54, 283

Lebistes reticulatus 17GYEA see Glucose yeast extract agarGymnothorax species see EelGymnotus carapo see Knife fish

Haddock 11, 73, 283Haemolysis

Streptococcus iniae 121test description 121, 251, 268Vibrio species 121, 268

Haemophilus piscium 51, 79Hafnia alvei


hosts and diseases 21, 24, 51identification 98, 150, 179, 185, 194, 194,

195, 214differentiation from Yersinia ruckeri 129,

136taxonomy 79

Haliaeetus leucocephalus see EagleHalibut 11, 42, 53, 283, 285

Atlantic halibut (Hippoglossus hippoglossusLinnaeus) 11, 66

Greenland halibut (Reinhardtiushippoglossoides Walbaum) 11

Halichoerus grypus see SealHalichondria bowerbanki see SpongeHaliotis species see AbaloneHalomonas

H. aquamarinaculture requirements (see Alcaligenes

faecalis homari) 86


H. cupidahosts and diseases 51identification 150, 179, 185, 196, 197

H. elongataculture requirements 88hosts and diseases 51identification 98, 151

H. haloduranshosts and diseases 51identification 98, 151

H. marinahosts and diseases 51identification 151

H. venustahosts and diseases 12, 51identification 98, 155, 197

Halophilic 128Hayflick’s medium 253Helicobacter

culture requirements 88selective media 250

hosts and disease 27identification 159taxonomy 79H. cetorum

hosts and diseases 27, 30, 51identification 99, 159

Heron (Great blue heron, Ardea herodias) 25Hexagrammos otakii see GreenlingHerring 58, 60, 283, 284

Baltic herring 12Hippocampus species see SeahorseHippoglossus hippoglossus see HalibutHippurate hydrolysis 269Hitra disease 71Hole-in-the-head 36Holothuria 63Homarus species see LobsterHorse serum 224Hsu-Shotts agar 251Hsu-Shotts marine agar (HSM) 251Hugh and Leifson oxidative-fermentative media

274Human 12–13, 51Hydrogen sulphide production (H2S) 118,

269Hydrogenophaga

identification 155H. palleronii

host and diseases 51identification 155

342 Index



H. pseudoflavahost and diseases 52identification 99, 155

Hyperoplus lanceolatus Lesauvege see EelHyphessobrycon species see TetraHypophthalmichthys molitrix Valenciennes see

Carp

Ictalurus species see CatfishIndole 130

Kovács indole reagent 270media preparation 269test interpretation 118, 121, 269

see also photographic sectionIndoxyl acetate hydrolysis 270Inia geoffrensis see DolphinInositol see Carbohydrate fermentationInositol horse serum 224, 251Intestinal microflora 223Iodobacter fluviatilis 219

culture morphology 99hosts and diseases 52identification 155

Jack crevalle see CrevalleJanthinobacterium 37

J. lividum 219hosts and diseases 24, 52identification 153, 179, 185, 192, 197,

202, 203Japanese amberjack see YellowtailJapanese medaka 13, 54, 283Japanese sweetfish 49JOD see Juvenile oyster diseaseJuvenile oyster disease (JOD) 14, 62

KDM2 132, 251KDMC 251Klebsiella 29

identification 151K. oxytoca


K. planticola (now called Raoultellaplanticola)

identification 152, 202, 203K. pneumoniae


K. terrigena see Raoultella terrigena

Kluyvera ascorbata, K. cryocrescensidentification 151

Knife fish 283Koehler illumination 224KOH 121, 270

see also Flexirubin pigmentKoi carp see CarpKovács indole reagent see IndoleKuruma prawn see Shrimp

Labeo rohita see RohuLaboratory worksheet 177Labridae see WrasseLabrus berggylta see WrasseLactobacillosis 24, 44, 67Lactobacillus piscicola see Carnobacterium

piscicolaLactobacillus plantarum-like 52Lactobacillus

culture requirements 88Rogosa acetate agar 275

differentiation from similar genera 125–127hosts and diseases 22

Lactococcosis 52Lactococcus

culture requirements 88Rogosa acetate agar 244, 275

identification notes 130L. garvieae 219

hosts and diseases 10, 11, 13, 15, 24, 32,33, 34, 35, 52

identification 99, 126, 127, 130, 133,160, 204, 205, 209, 210differentiation from similar genera

126-127, 130using the API Rapid ID 32 Strep system

130taxonomy 78PCR primers 227

L. lactis 130, 162, 209, 219L. lactis ssp. cremoris 209, 211L. piscium 219


205differentiation from similar genera

126-127PCR primers 227

L. raffinolactis 209Lactose see Carbohydrate fermentationLagenorhynchus species see DolphinLagodon rhomboides see Pinkfish

Index 343



Laminaria 59Laminaria japonica see MakonbuLamprey 13Larus delewarensis see GullLates calcarifer Bloch see BarramundiLDC see Lysine decarboxylaseLebistes reticulatus see GuppyLeiognathus 58Leiostomus xanthurus see SpotLepidochelys olivacea see TurtleLeptocottus armatus Girard see Pacific Staghorn

sculpinLeuciscus cephalus Linnaeus see ChubLeuciscus leuciscus Linnaeus see DaceLight organ 33, 58, 68, 69Limanda limanda Linnaeus see DabListeria species 29Listonella

L. anguillarum 219antisera 136culture requirements 88

VAM differentiation medium 260culture appearance see photographic

sectionhosts and diseases 3, 5, 7, 9,13, 14, 15,

19, 22, 23, 24, 34, 35, 37, 53, 79identification 99, 130, 168, 179, 185,

192, 193, 198, 214luminescence 122PCR primers 227serotypes 79taxonomy 79, 81

L. ordalii see V. ordaliiL. pelagia 219

hosts and diseases 53identification 99, 168, 185, 191, 214luminescence 122taxonomy 79

L. ordalii see Vibrio ordaliiLiza species see MulletLizard 47Loach 283Lobster 13, 38, 48, 51, 53, 68, 70, 283, 286Loligo pealei see SquidLoon 25Luminescence 121–122Lumpsucker 53Lutra lutra see European otterLysine decarboxylase

media preparation 267method 120, 267test interpretation 118

see also photographic section

MA 2216 252MacConkey agar (MCA) 270Mackerel 13, 281, 281, 284

Horse mackerel 72, 283Japanese jack mackerel (Trachurus

japonicus) 13, 223Spanish mackerel 52, 286

Macrobranchium rosenbergii see ShrimpMacropodus opercularis see Paradise fishMahi-mahi (Dolphin fish) 14, 56Makonbu 13, 59Malaclemys species see TurtleMaltose see Carbohydrate fermentationMannheimia haemolytica


Mannitol see Carbohydrate fermentationMannose see Carbohydrate fermentationMarbled spinefoot see RabbitfishMarine 2216 agar 252Marine columnaris see Columnaris diseaseMarine oxidative fermentative medium (MOF)

275see also Oxidative fermentative media

Marine salt agar with blood (MSA-B) 251Marinilabilia

M. salmonicolor 128M. salmonicolor biovar agarovorans


Marinobacter hydrocarbonoclasticushosts and diseases 53identification 148

Marinomonas communis 156M. vaga 156

Mauremys caspica see TurtleMCA see MacConkey agarMcFarland (nephelometer) standards

preparation 270Medium K 252Melanochelys trijuga coronata see TurtleMelanogrammus aeglefinus Linnaeus see

HaddockMenhaden 14, 281, 284Mercenaria mercenaria see ClamMerlangius merlangus see WhitingMesophilobacter marinus 219

hosts and diseases 53identification notes 99, 131, 149

Methyl Red also see MRreagent preparation 271test interpretation 119, 122, 271

see also photographic section

344 Index



Micrococcus 20, 32M. luteus

hosts and diseases 24, 37, 53identification 160

Micropterus species see BassMicroscopy 224Middlebrook 7H10-ADC medium 252Milkfish (Chanos chanos Forsskål) 13, 69Minnow 14, 41, 284

Bullminnows (Fundulus grandis Baird) 14,65

Eurasian Minnow (Phoxinus phoxinusLinnaeus) 14

Fathead Minnow 49, 283Mirounga species see SealsMisgurnus anguillicaudatus see LoachModified Hayflick medium 254Modiolus modiolus see MusselMola mola see SunfishMolluscs 42, 53, 59, 68, 69, 71, 73

bivalve 14, 67, 70, 73Nodipecten nodosus 73

Molly 17, 49, 284Balloon molly 17, 48, 281Black molly (Poecilia sphenops

Valenciennes) 17Silver molly 17, 48, 285

Monitoring bacterial load see Total bacterialcount

Moraxella specieshost and diseases 20, 29, 31, 32, 53identification 149

MoritellaM. japonica

identification 169M. marina 219

culture morphology 99host and diseases 53identification 99, 169, 180, 185, 190,

215carbohydrate fermentation 262organic growth requirements 254

luminescence 122taxonomy 80

M. viscosa 219culture requirements 88luminescence 122hosts and diseases 22, 24, 53identification 100, 131, 169, 180, 185,

193, 204, 205, 215taxonomy 80

Morone americana, Gmelin see Perch

Motilitygliding motility 129hanging drop method 119, 271tube method 118

MRVP see Methyl red; Voges-ProskauerMSA-B see Marine salt agar with bloodMud skipper 15Mugil species see MulletMullet 71, 283, 284, 285

Black Mullet 15, 47, 281Borneo mullet 16, 282Flathead mullet (Mugil cephalus Linnaeus)

15, 285Grey mullet 15, 48Largescale mullet (Liza macrolepis Smith)

16Silver mullet (Mugil curema Valenciennes)

16, 285Striped mullet 16, 48, 286Wild mullet (Liza klunzingeri Day) 16, 65

Multiplex PCR 231, 233Mussel 14, 70, 73, 283, 284

Far-eastern mussel 59, 284Horse mussel (Modiolus modiolus) 223Protothaca jedoensis Lischke 63

Mustelus canis Mitchill see SharksMya arenaria see ClamMycobacterium

culture morphology 100culture requirements 88hosts and diseases 3, 4, 11, 13, 18, 20, 23,

35, 54, 55identification 100, 166, 167PCR primers 227, 228M. abscessus

hosts and diseases 13, 54Middlebrook 7H10-ADC 252

M. avium 166, 167M. chelonae

hosts and diseases 18, 22, 34, 54M. chelonae abscessus 16, 17M. fortuitum

hosts and diseases 16, 17, 18, 32, 54, 55M. marinum


hosts and diseases 3, 4, 11, 12, 19, 25,30, 34, 54

M. neoaurumhosts and diseases 23, 54, 61

M. peregrinumhosts and diseases 32, 54

Index 345



Mycobacterium continuedM. poriferae

hosts and diseases 32, 33, 55M. scrofulaceum

hosts and diseases 18M. simiae

hosts and diseases 16, 55Mycobacterium species new strain 55

identification 166M. triplex-like

hosts and diseases 10, 55M. ulcerans

hosts and diseases 11Mycoplasma 30

culture requirements 253characterization tests 271-273medium 253, 254modified Hayflick medium 254

differentiation from Acholeplasma speciessee Digitonin

differentiation from bacteria see Dienesstain

hosts and diseases 28, 55identification 100, 168taxonomy 80M. alligatoris

hosts and diseases 2, 55M. crocodyli 219

hosts and diseases 8, 55M. mobile 219

hosts and diseases 33, 55M. phocacerebrale see M. phocicerebraleM. phocarhinis see M. phocirhinisM. phocicerebrale

hosts and diseases 28, 55M. phocidae

hosts and diseases 28, 55M. phocirhinis

taxonomy 80M. testudinis


Myroidesculture requirements 88identification notes 129, 130taxonomy 78M. odoratimimus 219


M. odoratus 219culture requirements 88


NA see Nutrient agarNB plates see Pasteurella multocida selective

mediaNegaprion brevirostris Poey see SharksNeisseria cuniculi 29Neisseria species 31Nematolosa come see BreamNinhydrin 269, 273Nitrate 130

media preparation 273test interpretation 119, 273

Nocardiaculture requirements 89

carbohydrate fermentation media 264survival at 50°C 268, 276


microscopic appearance see photographicsection

PCR primers 228N. asteroides

hosts and diseases 25, 56N. brasiliensis

hosts and diseases 56N. crassostreae


N. flavoroseahosts and diseases 56

N. kampachi see N. seriolaeN. nova

hosts and diseases 56N. salmonicida


N. seriolae 219culture requirements 89

carbohydrate fermentation media264

hosts and diseases 4, 11, 22, 35, 56identification 101, 167

Nocardia species, Australian strain 219N. transvalensis

hosts and diseases 56Nodipecten nodosus see MolluscsNovodon modestus see Black scraper

346 Index



Notemigonus crysoleucas Mitchill see Goldenshiner

Nutrient agar 254Nutritionally variant Streptococci 38, 50, 75

OceanomonasO. baumannii


O. doudoroffiihosts and diseases 56identification 156, 198

Octopus 39, 53, 57, 61, 69, 71, 284Oculina patagonica see CoralODC see Ornithine decarboxylaseOdobenus rosmarus rosmarus see WalrusOdonthestes bonariensis see PejerreyOF see Oxidative-fermentative testOncorhynchus species see SalmonidsONPG 130

media preparation 274test interpretation 119

Orcinus orca see WhaleOrectolobus ornatus see SharksOreochromi species see TilapiaOrganic growth requirements 254, 262Ornamental fish 16-18, 36, 39, 41, 47, 54, 56,

60, 65, 74, 284Ornithine decarboxylase (ODC)

media preparation and method 267test interpretation 118

see also photographic sectionOrnithobacterium 78, 128, 129, 130Oryzias latipes Temminck and Schlegel see

Japanese medakaOscar 17, 54, 284

Apistogramma ocellatus 17Red oscar 60

Otter 66, 74, 284European otter 18, 43, 64

Oxidasemethod and test interpretation 122, 274

Oxidative-fermentative testmedia preparation 274, 275test interpretation 119for Mycoplasma spp. see Mycoplasma

characterization testsOyster 63, 64, 67, 68, 69, 72, 73, 281

Crassostrea gigas 72, 223Crassostrea virginica 62Eastern oyster 14, 63, 284Japanese oyster 15, 72

Mediterranean oysters 15, 69Pacific oyster 15, 56, 72, 284

Pacific staghorn sculpin 18, 55, 284Packers plates 255Pagrus major see BreamPangasius hypophthalmus Sauvage see CatfishPantoea

P. agglomerans 219hosts and diseases 13, 56identification 101, 150, 180, 185, 191

P. dispersa 219hosts and diseases 56identification 152, 180, 185, 191

Paracentrotus lividu see Sea urchinParacolobactrum anguillimortiferum see

Edwardsiella tardaParadise fish 284Paraffin oil 115, 275Paralichthys species see FlounderPasteurella

P. haemolytica see Mannheimiahaemolytica

P. multocidahosts and diseases 2, 27, 28, 56identification 101, 153, 180, 186, 190,

215Pasteurella multocida selective media 255P. piscicida see Photobacterium damselae

ssp. piscicidaP. skyensis 219

culture requirements 89hosts and diseases 22, 57identification 101, 153, 215

P. testudinisidentification 102, 153, 180

Pasteurellosis 27, 37, 57PCR (polymerase chain reaction) 225, 234, 235

multiplex PCR 231nested PCR 233primer preparation 236specific primers 225

Pecten maximus see ScallopPedobacter

P. heparinus 128culture requirements 89

peptone yeast medium 255hosts and disease 57identification 102, 148, 204, 205, 215

P. pisciumculture requirements 89identification 102, 148, 204, 205, 215

Index 347



Peled see WhitefishPelecannus occidentalis carolinensis see PelicanPenaeus species see Prawn; ShrimpPenguin 26, 44, 56, 58, 59

Aptenodytes patagonica 26, 59, 284Eudyptes crestatus 26, 59, 284Gentoo penguin 26, 62Little penguin (Eudyptula minor) 47Macaroni penguin (Eudyptes chrysolophus)

26, 62Pyoscelis papua 26, 59, 284Spheniscus demersus 26, 59, 284Spheniscus humboldti 26, 59, 284

Peptone 131, 132, 134, 244, 263, 264, 267Perca species see PerchPerch 18, 40

European perch (Perca fluviatilis Linnaeus)18, 284

Silver perch (Bidyanus bidyanus Mitchell)18, 40, 285

White perch (Morone americana Gmelin,Roccus americanus) 18, 57, 62, 286

Yellow perch (Perca flavescens Mitchill) 18,54, 286

Phenol red indicator 118, 125, 262, 263, 271,272, 275

Phoca species see SealPhocoena species see PorpoisePhocoenobacter uteri 219


198, 215Phosphatase see Mycoplasma characterization

testsPhotobacterium 31

taxonomy 80transport medium 84P. angustum 219

hosts and disease 57identification 102, 170, 180, 186, 192,

204, 205luminescence 122

P. damselae spp. damselae 219culture appearance see photographic

sectionculture requirements 89hosts and diseases 4, 5, 9, 10, 12, 15, 21,

22, 25, 27, 30, 31, 33, 34, 35, 57identification 102, 131, 169, 170, 131,

180, 186, 192, 196, 198, 204, 205, 215additional tests for biotypes 131

luminescence 122PCR primers 228

taxonomy 80P. damselae ssp. piscicida 219

antisera 136culture appearance see photographic

sectionculture requirements 89hosts and diseases 3, 4, 5, 11, 16, 18, 27,

31, 32, 33, 34, 35, 37, 57identification 102, 131, 170, 180, 186,

190, 191, 192, 198, 204, 205, 215PCR primers 228

P. fischeri see Vibrio fischeritaxonomy 80

P. histaminum see Photobacteriumdamselae ssp. damselae

taxonomy 80P. iliopiscarium 219

hosts and diseases 7, 12, 22, 58identification 102, 170, 180, 186, 196,

204, 205P. leiognathi 220



P. logei see Vibrio logeiP. phosphoreum

hosts and diseases 58identification 102, 170, 180, 186, 196luminescence 121

P. profundumhosts and diseases 58identification 170

Phoxinus phoxinus Linnaeus see MinnowPhycodurus equis see Sea dragonPhysiological saline see SalinePike 18

Northern pike 18, 284Pimephales promelas see BaitfishPinnipedia see SealsPirarucu 19, 62, 284 (also spelt Piarucu)Plaice 53, 69, 72, 281

European plaice 19Pleuronectes platessa Linnaeus 69, 284

Planococcushosts and diseases 37, 58identification 160P. citreus


P. kocuriihosts and diseases 58identification 102, 162

348 Index



P. okeanokoites see Planomicrobiumokeanokoites

Planomicrobium okeanokoiteshosts and diseases 58identification 102, 162

Platichthys flesus Linnaeus see FlounderPlaties 19, 49

Southern platyfish (Xiphophorus maculatusGünther) 19

Platyfish see PlatiesPlecoglossus altivelis see AyuPelican 26

Brown pelican (Pelecannus occidentaliscarolinensis) 26

Plesiomonas shigelloides 225culture appearance see photographic

sectionhosts and diseases 25, 31, 58identification 103, 153, 180, 186, 196,


Pleuronectes platessa Linnaeus see PlaicePocillopora damicornis see CoralPodocnemis unifelis see TurtlePoecilia species see MollyPolar bear 47Polaribacter 78, 129, 130Poly Plates see Dermatophilus selective mediumPolychaete annelid 68Polydactylus sheridani see SalmonidsPolymerase chain reaction 239Polymyxin plates see Dermatophilus selective

mediumPomacentrida see DamselfishPomadasys stridens Forsskål see Red Sea fishPomatomus saltatrix see Blue fishPorpoise 27, 38

Harbour porpoise (Phocoena phocoena)27, 38, 42, 43, 57, 65, 66, 283

Portunus species see CrabPost-stripping peritonitis 44Potassium hydroxide see Flexirubin pigmentPrawn see ShrimpPreservation of bacteria see storage of isolatesPrimers

degenerate primers 238sequencing primers 238for sequencing Vibrio species 239universal primers 233

Probiotic 11, 15, 23, 40, 44, 64, 70, 73Procambarus clarkii see CrawfishProteus

P. mirabilis 29

P. rettgeri see Providencia rettgeritaxonomy 80

Proteus species 20Protothaca jedoensis Lischke see MusselProvidencia

P. rettgerihosts and diseases 22, 58identification 103, 150, 180, 186, 190taxonomy 80

P. rustigianii 220culture morphology 103hosts and diseases 59identification 150, 180, 186, 190

Pseudemis scripta see TurtlePseudoalteromonas

culture morphology 103culture requirements 89hosts and diseases 2taxonomy 80P. antarctica 220

culture requirements 89hosts and diseases 59identification 103, 156, 198, 215

P. aurantia see Pseudoalteromonasflavipulchra

P. bacteriolyticahosts and diseases 13, 59identification 103, 153, 198

P. citrea 220hosts and diseases 59identification 103, 131, 156, 215

P. denitrificanshosts and diseases 59identification 103, 156

P. distinctahosts and diseases 59identification 156

P. elyakovii 220hosts and diseases 13, 59identification 103, 156

P. espejianahosts and diseases 59identification 103, 156

P. flavipulchrahosts and diseases 59identification 103, 104, 156

P. haloplanktis haloplanktisidentification 156, 198

P. haloplanktis tetraodonisidentification 156

P. luteoviolaceahosts and diseases 59identification 104, 156

Index 349



Pseudoalteromonas continuedP. maricaloris


P. nigrifacienshosts and diseases 59identification 157

P. piscicidahosts and diseases 9, 60identification 104, 153

P. rubrahosts and diseases 60identification 104, 157, 215

P. ulvaehosts and diseases 2, 60identification 104, 157, 180

P. undinahosts and diseases 2, 60identification 104, 157

Pseudocaranx dentex Bloch and Schneider seeWhite Trevally

Pseudomonascarbohydrate fermentation 264hosts and diseases 15, 20, 29, 31, 32,

37P. acidovorans 157P. aeruginosa

identification 157, 180, 186, 191, 192P. anguilliseptica

culture requirements 89hosts and diseases 3, 5, 9, 10, 12, 22, 24,

25, 33, 34, 35, 60identification 104, 132, 153, 180, 186,

190, 198, 215PCR primers 228

P. cepacia see Burkholderia cepaciaP. chlororaphis

hosts and diseases 25, 60identification 153, 198

P. diminuta see Brevundimonas diminutaP. doudoroffii see Oceanomonas

doudoroffiiP. fluorescens


culture requirements 89hosts and diseases 21, 29, 60identification 105, 153, 154, 180, 186,

190, 191, 192, 198P. fluorescens/putida 180P. marina see Halomonas marinaP. mendocina 157P. mesophilica 157

P. nautica see Marinobacterhydrocarbonoclasticus

P. palleronii see Hydrogenophagapalleronii

P. paucimobilis see Sphingomonaspaucimobilis

P. perfectomarina see P. stutzeriP. piscicida see Photobacterium damselae

ssp. piscicidaP. plecoglossicida

hosts and diseases 3, 60identification 105, 154, 180, 198, 215PCR primers 228, 232

P. pseudoalcaligeneshosts and diseases 60identification 105, 154

Pseudomonas pseudomallei seeBurkholderia pseudomallei

P. putidahosts and diseases 60identification 154, 180, 198

P. putrefaciens see Shewanella putrefaciensP. putrefaciens hosts and diseases 19, 21Pseudomonas species 223P. stanieri


P. stutzerihosts and diseases 21, 61identification 105, 154, 186, 190

Pseudorca crassidens see WhalePsychrobotes longicauda 63Psychroflexus 129, 130

taxonomy 78P. gondwanensis

hosts and diseases 61Psychrophilic 46, 128Psychroserpens 78, 129, 130Pterophyllum scalare 284

see also Ornamental fishPuffer fish 65Puffin 47Puntius conchonius Hamilton see Rosy barbsPY see Peptone yeast mediumPyoscelis papua see PenguinPYS-2 medium 255

R2A agar 246RAA see Rogosa acetate agarRabbitfish 19, 54, 63, 65, 284

Marbled spinefoot (Siganus rivulatusForsskål) 19

350 Index



White-spotted spinefoot (Siganuscanaliculatus Park) 19, 286

Rahnella aquatilishosts and diseases 12, 61identification 152, 180, 186, 191

Rainbow trout fry syndrome 37, 49Rams 17, 65Rana castesbeiana see FrogsRaoultella

R. ornithinolyticahosts and diseases 61

R. planticola 220API 50CH see Klebsiella planticola


R. terrigena 220hosts and diseases 61identification 152

Red disease 55Red Sea fish 19Red spot disease 13, 59, 60Red spot disease see Sekiten-byo diseaseRedfish 19, 48

Red drum 19, 285Red-leg frog disease 39Redpest 47Redsore disease 39Reef fish 65Reference strains 218–221Renibacterium salmoninarum 220

antisera 136culture requirements 89

KDM2 251KDMC 251SKDM 257culture appearance see photographic

sectionhosts and diseases 3, 22, 23, 25, 36, 61identification 105, 125, 126, 132, 164, 215PCR primers 228

Reinhardtius hippoglossoides Walbaum seeHalibut

Reptiles 19, 39, 45, 53, 54, 58Water monitor 47

Rhodococcushosts and diseases 61identification 105, 164R. equi 165R. fascians


R. maris see Dietzia marisRhombosolea tapirina Günther see Flounder

Rhynchopelates oxyrhynchus Temminck andSchlegel 21

Riemerella 78, 128, 129Roach 21, 40, 41, 285Roccus species see Bass and PerchRockfish (Schlegel’s black rockfish) 21, 41Rockling (Fourbeard rockling) 22, 283Rogosa acetate agar 256Roseobacter

culture mediumSWT 258

culture morphology 105R. gallaeciensis


Roseobacter strain CVSPculture requirements 89hosts and diseases 14identification 105, 154

Rosy barbs 17, 285Rotifer 22, 70, 71Rudd 22, 285Rudderfish see YellowtailRutilus rutilus Linnaeus see Roach

Saddleback disease 49Salegentibacter 129, 130

S. salegensculture requirements 89hosts and diseases 62identification 106, 148, 181

Salicin see Carbohydrate fermentationSaline, physiological saline 275Salinivibrio costicola ssp. costicola

hosts and diseases 62identification 106, 157PCR primers 228

Salmo species see SalmonidsSalmonella

antisera 136culture media

appearance on MCA 270strontium chloride B enrichment broth

256S. adelaide 29S. arizonae 19, 62

identification 150, 181, 186, 196S. durham 20, 62S. enteritidis 30, 62S. hartford 2, 25S. havana 30, 62S. heidelberg 28, 29, 62

Index 351



Salmonella continuedS. java 2, 25S. miami 2S. newport 28, 29, 30, 62S. oranienburg 28, 29, 62S. typhimurium 30, 62

PCR primers 233Salmonella group O type B 62

Salmonids 22–25, 36, 44, 49, 61, 66, 69, 74,77

Salmon 22–23, 40, 46, 50, 58, 60, 71,282, 284, 285

Atlantic salmon (Salmo salar Linnaeus)22, 36, 38, 39, 40, 44, 53, 54, 56, 57,60, 61, 62, 64, 66, 67, 69, 71, 73, 74,281

Blueback salmon 56Cherry salmon (Oncorhynchus masou

masou Brevoort, Yamame) 23, 51Chinook salmon (Oncorhynchus

tschawytscha Walbaum) 23, 44, 49, 54,56, 60, 61, 66, 282

Coho salmon (Oncorhynchus kisutchWalbaum) 23, 41, 46, 49, 61, 282

Pacific salmon 23, 284Sockeye salmon (Oncorhynchus nerka

Walbaum) 23, 286Trout 24–25, 39, 40, 46, 54, 57, 64, 283,

285Amago trout 25, 60Arctic charr (Salvelinus alpinus Linnaeus)

22, 44, 52, 62, 281Brook trout (Salvelinus fontinalis Mitchill)

24, 47, 61, 282Brown trout 24, 49, 51, 61, 67, 74, 282Cutthroat trout 44, 282Rainbow trout (Oncorhynchus mykiss

Walbaum) 24, 36, 41, 44, 45, 46, 47,48, 49, 51, 52, 53, 56, 57, 58, 60, 61,62, 64, 65, 67, 72, 73, 284

Sea trout 24, 65, 285Steelhead trout (Salmo trutta trutta

Linnaeus) 23, 284Salmon blood spot 74Salt

adding to biochemical sets 115artificial seawater salts 245marine salts mix 116salt requirement 11520% stock solution 276see also Saline

Salt tolerancemedia for detection 268, 276

Salvelinus species see SalmonidsSample collection 84Sample transport 84Sample preparation 84Sand lance 285Sand whiting 285Sardinops species see SardineSaratherodon species see TilapiaSardine 285

Pacific sardine 25Pilchard (Sardinops neopilchardus) 284South American pilchard (Sardinops sagax

Jenyns) 25Scallop 15, 39, 67, 71

Argopecten purpuratus 15, 285Pecten maximus 15, 61, 285

Sarcina species 223Scardinius erythrophthalmus Linnaeus see RuddSciaenochromis ahli Trewavas see Electric blue

hapSciaenops ocellatus Linnaeus see RedfishScleropages leichardii see SaratogaScomber species see MackerelScophthalmus maximus Linnaeus see TurbotScylla serrata see CrabSea bird 73, 74Sea dragon (Leafy sea dragon, Phycodurus

equis) 26Sea grass 26, 67Sea lion 28, 44, 47, 56, 284, 285

Californian sea lion (Zalophuscalifornianus) 62, 282

Sea mammals 26–31, 38, 47Sea salts see Artificial seawaterSea salts stock solution 257Sea urchin 30, 68, 285Seahorse 26, 57, 67, 69, 285Seals (Pinnipedia) 40, 42, 43, 46, 47, 55, 66,

283, 284Antarctic fur seal 62, 281Common seal (Phoca vitulina) 28, 42, 43,

282Grey seal (Halichoerus grypus) 29, 38, 39,

43, 44, 283Harbour seal 28, 55, 66, 283Harp seal (Phoca groenlandica) 29, 43, 44,

283Hooded seal (Cystophara cristata) 29, 38,

43, 283New Zealand fur seal (Arctocephalus

forsteri) 54, 67, 284Northern fur seal (Callorhinus ursinus) 29,

284

352 Index



Ringed seal (Phoca hispida) 29, 43, 44,285

South Georgian Antarctic fur seal 29Southern elephant seal (Mirounga leonina)

30, 48, 282, 286Weddell seals 43, 286

Seawater see Artificial seawaterSebastes schlegeli Hildendorf see RockfishSekiten-byo disease (red spot disease) 10Senegal 45Sepia officinalis see Cuttle fishSepiola species see SquidSequencing 236–242

BIOEDIT 241BLAST search 242

Seriola species see YellowtailSerratia

S. fonticola 220hosts and diseases 62identification 106, 152, 181, 186, 195

S. liquefaciensfor Arctic char isolates 150hosts and diseases 22, 34, 62identification 106, 150, 152, 181

S. marcescenshosts and diseases 4, 25, 62identification 150, 152

S. plymuthicahosts and diseases 62identification 106, 150, 181, 186, 191

S. rubidaeaidentification 152

Serum-dextrose agar 256Shad 26

Gizzard shad 26, 41Sharks 30–31, 44, 57, 69

Blacktip shark (Carcharhinus limbatus) 31Brown shark (Carcharhinus plumbeus) 30,

282Lemon shark (Negaprion brevirostris Poey)

31, 283Nurse shark (Ginglymostoma cirratum) 31,

284Sandbar shark 31Smooth dogfish (Mustelus canis Mitchill) 31Spiny dogfish 31Tiger shark (Galeocerdo curvieri) 31

Sheatfish 31Wels catfish 31

Shewanellacarbohydrate fermentation 264hosts and diseases 2S. algae 220


198, 215S. amazonensis

identification 157S. baltica 220


S. benthicaculture requirements 89hosts and diseases 63identification 158

S. colwelliana 220culture requirements 89hosts and diseases 14, 63identification 106, 158

S. frigidimarina 220hosts and diseases 63identification 106, 158, 181, 186, 190,

192S. gelidimarina 220

culture requirements 89hosts and diseases 63identification 106, 158, 181, 186, 190

S. hanedai 220culture requirements 89hosts and diseases 63identification 158

S. japonica 220culture morphology 106hosts and diseases 14, 63identification 158

S. oneidensis 220culture morphology 106hosts and diseases 63identification 158

S. pealeana 220culture requirements 89hosts and diseases 33, 63identification 106, 158, 198

S. putrefaciens 220hosts and diseases 19, 21, 31, 63identification 107, 154, 181, 186, 190,

191, 198, 215taxonomy 80

Shewanella sp. MR-1 see Shewanellaoneidensis

S. woodyi 220culture media

Shewanella marine agar 257culture requirements 89hosts and diseases 63identification 107, 158, 181, 186, 190

Index 353



Shewanella marine agar (SMA) 257Shieh medium + Tobramycin (SM-T) 257Shrimp 53, 57, 67, 69, 73, 281, 282, 286

Black tiger prawn (Penaeus monodon) 31,69

Brine shrimp (Artemia sp.) 31, 71, 282Chinese shrimp (Penaeus chinensis) 31, 71Fairy shrimp (Branchipus schaefferi Fisher)

32, 39, 283Fairy shrimp (Chirocephalus diaphanus

Prévost) 32, 283Fairy shrimp (Streptocephalus torvicornis

Waga) 32, 283Giant freshwater prawn (Macrobranchium

rosenbergii) 32, 39, 41, 283Kuruma prawn (Penaeus stylirostris,

P. japonicus) 32, 71Penaeid prawn 57, 69, 70Pacific white shrimp 54, 284Prawn 72White shrimp (Penaeus vannamei Boone)

32Shubunkin 285Siamese fighting fish (Betta splendens Regan)

17, 54, 283, 285Siberian sturgeon see SturgeonSIEM selective media 86, 258Siganus species see RabbitfishSillago ciliata see Sand whitingSilurus glanis Linnaeus see SheatfishSKDM 257Skin and tail rot 37Skin ulcers 37Skirrow’s medium see Helicobacter selective

mediaSMA see Shewanella marine agarSmooth dogfish see SharksSM-T see Shieh medium + TobramycinSnails 54, 61, 62Snakes 19, 47, 54Snakehead fish 32, 39, 42, 55, 56, 285, 286Snook 32, 282Sole (Solea senegalensis Kaup) 32, 53, 57,

286Dover sole (Solea solea) 32, 36, 66

Sorbitol see Carbohydrate fermentationSouth American pilchard see SardineSouthern platyfish see PlatiesSpadefish 32

Atlantic spadefish 32Sparus auratus Linnaeus see BreamSpecimen collection 84Spheniscus species see Penguin

Spiny dogfish see SharksSphingobacteriaceae 128Sphingobacterium

carbohydrate fermentation 264culture media

peptone yeast medium 255taxonomy 80S. heparinum see Pedobacter heparinusS. indologenes see Chryseobacterium

indologenesS. mizutae see Flavobacterium mizutaiiS. multivorum

culture requirements 89hosts and diseases 63identification 107, 148, 215

S. piscium see Pedobacter pisciumS. spiritivorum 128, 220

culture requirements 89hosts and diseases 64identification 107, 148, 215

Sphingolipids 128Sphingomonas paucimobilis

hosts and diseases 64identification 107, 158S. subarctica 198

Sponge 33, 55, 59Sporocytophaga 78Spot disease 13, 43, 59Squalus acanthias see SharksSquid 63, 69, 286

Hawaiian sepiolid squid (Euprymnascolopes) 33, 68, 285

Loligo pealei 33, 63, 286Sepiola affinis 33Sepiola robusta 33

St Peter’s fish see TilapiaStaphylococcus

hosts and diseases 2, 29, 32, 64identification 162, 163S. aureus 6


Staphylase test 136S. aureus anaerobius 162S. capitis 64, 162S. cohnii 64, 162S. delphini 220


S. epidermidishosts and diseases 29, 64identification 160

S. haemolyticus 64, 163

354 Index



S. hominis 64identification 107, 163

S. lutrae 220culture requirements 89hosts and diseases 18, 64identification 107, 160, 215

S. saprophyticus 64, 163S. simulans 64, 163S. warneri 25

culture requirements 89hosts and diseases 64identification 107, 161

S. xylosus 64, 163Stappia stellulata-like

culture requirements 90SWT 258


Stenella species see DolphinStenotrophomonas (Pseudomonas) maltophilia

158Sternotherus species see TurtleStingray 33, 57, 65, 286Storage of isolates 224Streptobacillus

S. moniliformisculture requirements 90hosts and diseases 64identification 107, 150

Streptobacillus moniliformis-like organism22

identification 108, 150Streptocephalus torvicornis Waga see ShrimpStreptococcosis 37Streptococcus

antiserum Groups 136S. agalactiae

hosts and diseases 5, 14, 16, 33, 37, 65identification 108, 161, 132, 211

Kuwait strains 132PCR primers 228serogroups 80

S. agalactiae (S. difficile) Group B Type Ib220


hosts and diseases 6, 17, 18, 25, 65identification 108, 132, 161, 209, 204,

205, 211PCR primers 228taxonomy 81

S. difficile see S. agalactiae (S. difficile)Group B Type Ib

S. dysgalactiae ssp. dysgalactiae LancefieldGroup L

hosts and diseases 28, 65identification 108, 161, 211taxonomy 81

S. dysgalactiae ssp. dysgalactiae, Group Chosts and diseases 65identification 161taxonomy 81

S. dysgalactiae ssp. equisimilishosts and diseases 65identification 163taxonomy 81

S. garvieae see Lactococcus garvieaeS. iniae 220


culture requirements 90hosts and diseases 3, 4, 5, 6, 11, 12, 16,

19, 25, 27, 33, 34, 35, 37, 65identification 108, 127, 132, 161, 204,

205, 209, 211differentiation from similar genera 125,

127haemolysis 121, 132

PCR primers 229S. milleri


S. parauberis 220culture requirements 90hosts and diseases 34, 66identification 108, 133, 161, 204, 205,

211S. phocae 220

hosts and diseases 28, 29, 66identification 108, 161, 204, 205, 209

S. porcinus 66identification 108, 163, 204, 205, 209PCR primers 228

S. shiloi see S. iniaeStreptococcus ssp. non-haemolytic 21Streptococcus ssp. non-haemolytic Group

B 14S. uberis 220

identification 133, 163, 204, 205, 211Streptomyces salmonis


Streptomycosis 66Striped jack 33, 60Striped piggy see Red Sea fishStriped trumpeter 66

Index 355



Strontium chloride B enrichment broth 256Stuarts medium 84Sturgeon 33, 58, 286

Adriatic sturgeon 33, 52, 281Siberian sturgeon 33

Sucrose 130see also carbohydrate fermentation

Sulculus diversicolor supratexta see AbaloneSurvival at 50°C see Nocardia spp.Sunfish 45, 286Swabs 84SWT 258Syndrome 93 32, 71Symphysodon aequifasciatus Pellegrin see

Discus fishSynodus variegatus Lacepède see Red Sea fish

TBC see Total bacterial countTCBS cholera medium 258

test interpretation 119, 122, 258, 259TDA reagent 276Tenacibaculum 129, 130

carbohydrate fermentation 262alternative methods for carbohydrate

fermentation 263T. maritimum 220, 224

culture requirements 90Flavobacterium maritimus media 249Hsu-Shotts marine agar 251tryptone yeast extract glucose agar 259

media for freeze-drying 224hosts and diseases 2, 3, 4, 5, 11, 22, 23,

25, 32, 36, 66identification 109, 129, 133, 145, 181,

186, 190, 206, 207, 215, 216PCR primers 229, 232

T. ovolyticum 220culture requirements 90hosts and diseases 11, 66identification 109, 145, 216

Tench 33, 55Terrapene carolina carolina see TurtleTetra 17

Black skirted tetra (Hyphessobrycon sp.)17, 281

Neon tetra (Hyphessobrycon innesi Myers)18, 49, 54, 56, 284

Serpae tetra 18Tetramethyl-p-phenylenediamine see Oxidase

testTetrazolium chloride reduction see Mycoplasma

characterization tests

Therapon oxyrhynchus see Coastal fishThorichthys meeki see CichlidThymallus thymallus Linnaeus see GraylingTilapia 33, 39, 60, 65, 286

Nile Tilapia (Oreochromi niloticus niloticusLinnaeus) 33, 284

St Peter’s fish 33Saratherodon (Tilapia) aureus 34, 286Sarotherodon niloticus 34

Tinca tinca see TenchTorticollis 45, 47Tortoise 19, 46, 47, 55, 57Total bacterial count (TBC) 222

Oyster hatcheries 223seawater 223tank water 223

Trachinotus carolinus (Pompanos) 284Trachinus draco (Greater weever) 283Trachurus japonicus see MackerelTransport medium 84

Amies transport medium 84Stuarts transport medium 84transport to the Laboratory 84

Trevally see CrevalleTrehalose see Carbohydrate fermentationTribolodon hakonensis (Living Dace) 283Trichogaster trichopterus Pallas see GouramiTrionyx spinifer see TurtleTriple sugar iron agar (TSI) 276Trout see SalmonidsTryptone soya agar (TSA) 259Tryptone yeast extract glucose agar (TYG)

259Tryptone yeast extract salt medium (TYES)

259TSI see Triple sugar iron agarTTC see Tetrazolium chloride reductionTurbot 34–35, 39, 41, 45, 48, 53, 54, 57, 60,

62, 66, 67, 68, 70, 71, 72, 78, 286Brill (Flatfish) 35, 67, 72Colistium guntheri 35Colistium nudipinnis Waite 35

Tursiops species see DolphinTurtles 19–21, 38, 39, 43, 47, 47, 54, 57, 67,

70, 286African mud turtle 62Caspian terrapin (Mauremys caspica) 20,

62Chelonia mydas 20, 62, 285Eastern box turtle (Terrapene carolina

carolina) 20, 62Eretmochelys imbricata 20Hawaiian green turtle 21

356 Index



Helmeted terrapin 62Leatherback turtle (Dermochelys coriacea)

21Loggerhead sea turtle (Caretta caretta)

21, 38Mississippi map turtle (Malaclemys kohni)

20, 62Northern diamondback terrapin

(Malaclemys terrapin terrapin) 20, 62Pacific green sea turtle 54Painted turtle (Chrysemys picta) 20, 43,

62Pseudemis scripta 21, 286Red-eared turtle (Chrysemys scripta

elegans) 20, 43, 285Ridley sea turtle (Lepidochelys olivacea)

21South American side-neck turtle

(Podocnemis unifelis) 43, 286Spiny soft-shelled turtles (Trionyx spinifer)

43, 286Stinkpot turtle (Sternotherus odoratus) 20,

62Striped-necked musk (Sternotherus minor

peltifer) 43, 286Travancore crowned turtle (Melanochelys

trijuga coronata) 20, 62Yellow-spotted Amazon turtle 62

TYES see Tryptone yeast extract salt medium(TYES)

TYG see Tryptone yeast extract glucose agar

Ulcer head disease of eels 41Ulva lactuca see AlgaeUrea 130

test method 276, 277Utilization, differentiation from fermentation see

Carbohydrate fermentation

Vagococcusidentification notes 124

differentiation from similar genera 124taxonomy 81V. fessus 220


V. fluvialis 220hosts and diseases 12, 66identification 109, 126, 163, 206, 207,

209, 211

V. lutrae 220hosts and diseases 18, 66identification 109, 126, 161, 211, 216

V. salmoninarum 220culture requirements 90hosts and diseases 22, 25, 67identification 109, 126, 126, 133, 161,

199, 206, 207, 209, 211differentiation from similar genera

126–127VAM 260Variegated lizardfish see Red Sea fishVarracalbmi


Vibrioalternative method for carbohydrate

fermentation 264differentiation from Aeromonas species

114haemolysis 121hosts and diseases 15, 31luminescence 121, 122PCR primers for sequencing 239taxonomy 81TCBS selective medium 122, 119transport medium 84Vibrio discs (0/129) 114, 119, 123, 277V. aerogenes 220


V. aestuarianus 220hosts and diseases 67identification 109, 175, 181

V. agarivorans 220culture appearance see photographic

sectionculture requirements 90hosts and diseases 2, 67identification 110, 133, 176, 181, 186,

191, 216V. alginolyticus


hosts and diseases 2, 5, 15, 20, 21, 31,67

identification 110, 133, 134, 171, 181,187, 193, 194, 199, 216, 260differentiation from V. harveyi 133

PCR primers 232V. anguillarum biovar II see V. ordalii

Index 357



Vibrio continuedV. brasiliensis 220


216V. calviensis 220


V. campbelliihosts and diseases 67identification 176

V. campbellii-like 35, 67V. carchariae see V. harveyiV. cholerae

antisera 136hosts and diseases 8identification 110, 133, 171, 187, 192,

193, 195, 196,199aesculin test 117, 133, 261

PCR primers 231, 233serotypes 81

V. cholerae 01 81hosts and diseases 12, 68identification 171, 181, 187, 195PCR primers 229

V. cholerae serotype 0139 Bengal 81hosts and diseases 12identification 110, 171resistance to Vibrio static agent 123

V. cholerae non-01 81culture appearance see photographic

sectionhosts and diseases 3, 4, 12, 17, 34, 68identification 134, 171, 181, 187, 195,

216PCR primers 229

V. cholerae-like 8, 68V. cincinnatiensis 220


V. coralliilyticusculture requirements 90hosts and diseases 8, 68identification 110, 134, 174, 199

V. costicola see Salinivibrio costicola ssp.costicola

V. cyclitrophicushosts and diseases 68identification 110, 176

V. damselae see Photobacterium damselaessp. damselae

V. diabolicus 220hosts and diseases 68

identification 110, 175, 181, 187, 194,199, 206, 207, 216

V. diazotrophicus 220hosts and diseases 30, 68identification 110, 175PCR primers 229

V. fischeri 219hosts and diseases 33, 68identification 110, 172, 173, 181, 187,

193, 206, 207, 216variable results for VP 134

PCR primers 229V. fluvialis 220


hosts and diseases 13, 21, 69identification 110, 134, 173, 181, 187,

193, 260PCR primers 229

V. furnissii 220culture appearance see photographic

sectionhosts and diseases 9, 13, 69identification 111, 134, 173, 181, 187,

192, 193, 216V. gazogenes 221


V. halioticoli 221culture requirements 90hosts and diseases 2, 69identification 111, 176, 181, 187, 190,

216V. harveyi 220, 221


hosts and diseases 2, 4, 5, 8, 11, 13, 16,31, 32, 69

identification 111, 133, 134, 171, 181,187, 194, 199, 206, 207, 216, 260differentiation from V. alginolyticus 133

taxonomy 81V. hollisae 221

culture requirements 90hosts and diseases 13, 21, 69identification 110, 176PCR primers 229

V. ichthyoenterihosts and diseases 11, 69identification 111, 174, 181, 187, 190,

199V. iliopiscarius see Photobacterium

iliopiscarium

358 Index



V. lentus 221hosts and diseases 15, 69identification 111, 175

V. logei 220carbohydrate fermentation 262hosts and diseases 23, 33, 69identification 111, 173, 216organic growth requirements 254

V. marina see Moritella marinaV. mediterranei 221

hosts and diseases 2, 8, 70identification 111, 134, 175, 182, 187,

196, 216arginine dihydrolase test differences 267

V. metschnikovii 221hosts and diseases 70identification 111, 175, 260

V. mimicus 221culture appearance see photographic

sectionculture requirements 90hosts and diseases 4, 8, 21, 34, 70identification 111, 134, 171, 182, 187,

195, 196, 199, 216V. mytili 221


199, 206, 207, 216arginine dihydrolase test differences 267

V. natriegens 219, 221hosts and diseases 70identification 176, 216taxonomy 79, 81

V. navarrensis 221hosts and diseases 70identification 111, 176, 182, 199

V. neptunius 221hosts and diseases 14, 22, 34, 70identification 111, 175, 182, 187, 192,

216ADH strain variation 134

V. nereis 221hosts and diseases 70identification 175, 182, 187, 190, 199,

206, 207, 216V. nigripulchritudo

hosts and diseases 71identification 176PCR primers 229

V. ordalii 221carbohydrate fermentation 262


culture requirements 90hosts and diseases 23, 25, 71identification 112, 174, 182, 187, 190,

216, 217organic growth requirements 254taxonomy 79, 80

V. orientalis 221hosts and diseases 71identification 112, 175, 217

arginine dihydrolase test differences267

V. pacinii 221identification 112, 175, 182, 217hosts and diseases 22, 31, 71

V. parahaemolyticusculture appearance see photographic

sectionculture requirements 90hosts and diseases 2, 6, 8, 9, 13, 31, 71identification 112, 134, 171, 182, 187,

194, 217PCR primers 229, 230, 231, 232, 233

V. pectenicida 221hosts and diseases 15, 71identification 112, 174, 182, 188, 190

V. pelagius see Listonella pelagiaV. penaeicida 221


199, 206, 207PCR primers 230

V. proteolyticus 221culture appearance see photographic

sectionhosts and diseases 31, 71identification 112, 135, 173, 182, 188,

196, 217PCR primers 230

V. rotiferianus 221hosts and diseases 22, 71identification 112, 175, 182, 188, 195,

217V. rumoiensis

culture mediumPYS-2 medium 255


V. salmonicida 221, 223culture requirements 90hosts and diseases 7, 23, 71identification 112, 174, 182, 188, 190,

195, 206, 207, 217PCR primers 230

Index 359



Vibrio continuedV. scophthalmi 221


191chitin hydrolysis 135differentiation from V. splendidus

biovar I 135V. shilonii 221


Vibrio species 223Vibrio species zoea 8

Vibrio species zoea see V. harveyiV. splendidus

hosts and diseases 5, 15, 35, 72identification 135, 173, 182, 188, 190,

192, 193, 206, 207test variations between strains 135

PCR primers 230V. splendidus-like 72V. splendidus I 34, 72, 135, 221


arginine dihydrolase test differences 267V. splendidus 221

hosts and diseases 15, 31, 72identification 112, 135, 173, 217

V. tapetis 221hosts and diseases 14, 36, 72identification 113, 174, 182, 188, 191,

199, 217V. trachuri

hosts and diseases 13, 72identification 172PCR primers 230taxonomy 81

V. tubiashii 221culture appearance see photographic

sectionhosts and diseases 14,15, 40, 72identification 113, 173, 174, 182, 188,

191, 192, 199, 206, 207, 217arginine dihydrolase test differences 267

PCR primers 230V. viscous see Moritella viscosaV. vulnificus 221

taxonomy, serotypes and biotypes 81culture requirements 90

cellobiose-colistin agar 247VVM 260


hosts and diseases 8, 9, 10, 13, 15, 31, 73identification 113, 135, 138, 171, 182,

183, 188, 189, 191, 193,194, 195, 196,217

PCR primers 230, 231, 232, 233V. wodanis 221

hosts and diseases 23, 73identification 113, 135, 176, 217salt requirement variation 134taxonomy 81

V. xuii 221hosts and diseases 32, 73identification 113, 175, 183, 189, 192,

217Vibrio discs (vibrio static agent 0/129)

test interpretation 114, 119, 123, 277Voges-Proskauer (VP)

media preparation 277test interpretation 119, 122, 277

VP see Voges-ProskauerVPT media see Skirrow’s mediumVVM selective medium 260VVMc 260

Weakfish see Sea bassWeeksella 78, 129, 130Weissella hellenica strain DS-12


Wels catfish see SheatfishWhale 43, 46, 282, 284

Beluga whale 30, 51, 281False killer whale (Pseudorca crassidens)

30, 44, 283Killer whale (Orcinus orca) 30, 283Minke whale (Balaenoptera

acutorostrata) 30, 44, 51, 284Pacific pilot whale (Globicephala

scammoni) 30, 284Sowerby’s beaked whale 30, 38, 286White whale (Delphinapterus leucas) 30,

54, 286White-spotted spinefoot see RabbitfishWhite Trevally 33Whitefish 35, 286

Cisco (Coregonus artedi Lesueur) 35Lake whitefish (Coregonus clupeaformis

Mitchill) 35Peled (Coregonus peled Gmelin) 35

Wild fish see Red Sea fishWinter disease 5, 53, 60Winter ulcers 22, 23

360 Index



Winter ulcer disease 53, 73Withering syndrome 2, 71Wolf-fish (Anarhichas lupus Linnaeus, A. minor

Olafsen) 35, 52, 282, 286Spotted wolf-fish 35, 285

Wood’s broth 260Worksheet see Laboratory worksheetWrasse (cleaner fish) 35, 40, 283, 286

Xiphophorus maculatus Günther see PlatiesXylose see Carbohydrate fermentation

Yamame see SalmonidsYeast extract 262Yeast extract broth or agar 264Yellowtail 35, 52, 56, 57, 60, 64, 66, 286

Seriola purpurascens 35Seriola quinqueradiata 35

Yellowtail clownfish see DamselfishYersinia

hosts and diseases 73, 74identification 152taxonomy 81Y. aldovae 221Y. frederiksenii

identification 135, 152Y. intermedia

identification 150, 183, 189, 191Y. kristensenii

identification 113, 152, 206, 207Y. pseudotuberculosis 152

Y. rohdei 221Y. ruckeri

antisera 136culture requirements 90PCR primers 230, 233hosts and diseases 6, 7, 9, 11, 14, 15, 18,

21, 22, 23, 24, 25, 33, 35identification 113, 136, 151, 183, 189,

191, 194, 195, 217differentiation from Hafnia alvei 129,

136motility at different temperatures 90,

113, 136see also photographic section

taxonomy and serotyping 82Yersinia selective agar (YSA) 260

Zalophus californianus see Sea lionZobellia 129, 130

Z. galactanovoranshosts and diseases 2, 74identification 113, 148, 183, 189, 191,

199, 206, 207, 217Z. uliginosa


199, 206, 207, 217Zoarces viviparus Linnaeus see BlennyZoBell’s agar see Marine 2216 agarZona radiata 11, 66Zoonotic 43, 44, 46, 48, 54, 55Zooxanthellae 72

Index 361



Bacteria from Fish and Other Aquatic Animals - N. Buller (2004) WW

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