Department of Food and Environmental HygieneFaculty of Veterinary Medicine

University of HelsinkiFinland

TAXONOMIC STUDIES ON CANINE ANDFELINE GASTRIC HELICOBACTER

SPECIES

KATRI JALAVA

HELSINKI 1999

Department of Food and Environmental HygieneFaculty of Veterinary Medicine

University of HelsinkiFinland

TAXONOMIC STUDIES ON CANINE ANDFELINE GASTRIC HELICOBACTER

SPECIES

KATRI JALAVA

ACADEMIC DISSERTATION

To be presented with the permission of the Faculty of Veterinary Medicine,University of Helsinki for public criticism in Auditorium Maximum, Hämeentie

57, Helsinki, on 11th June, 1999 at 12 noon.

HELSINKI 1999

ISBN 951-45-8701-4 (PDF version)Page-numbering differs from the printed version

Helsinki 1999-09-27

Helsingin yliopiston verkkojulkaisut

ACKNOWLEDGEMENTS........................................................................................................6

LIST OF ORIGINAL PUBLICATIONS ...................................................................................9

1. ABSTRACT .........................................................................................................................10

2. REVIEW OF THE LITERATURE ......................................................................................12

2.1. Helicobacter pylori ........................................................................................................12

2.2. History of gastric Helicobacter spp ...............................................................................12

2.3. Taxonomy of the genus Helicobacter ............................................................................16

2.4. Isolation and detection of gastric helicobacters.............................................................18

2.4.1. Primary isolation.....................................................................................................18

2.4.2. Rapid urease test .....................................................................................................19

2.4.4. Visualisation of "gastrospirilla" by light microscopy.............................................20

2.4.5. Visualisation of "gastrospirilla" by electron microscopy .......................................20

2.4.3. PCR.........................................................................................................................21

2.5. Identification of culturable "gastrospirilla" ...................................................................21

2.5.1. Classical phenotypic tests.......................................................................................22

2.5.2. Protein profile analysis ...........................................................................................22

2.5.3. DNA-DNA hybridisation .......................................................................................23

2.5.4. 16S rDNA sequencing ............................................................................................23

2.5.5. Restriction fragment length polymorphism (RFLP) of 23S rRNA gene................25

2.6. Polyphasic taxonomy.....................................................................................................25

2.7. Molecular typing...........................................................................................................27

2.7.1. Typing methods ......................................................................................................27

2.7.2. Plasmid profiling ....................................................................................................28

2.7.3. RFLP.......................................................................................................................28

2.7.4. PFGE ......................................................................................................................29

3. PURPOSES OF THE STUDY .............................................................................................31

4. MATERIALS AND METHODS .........................................................................................32

4.1. Gastric biopsy samples of dogs and cats .......................................................................32

4.2. Primary microbial isolation ...........................................................................................32

4.3. Primary identification of field isolates from Finnish dogs and cats ..............................32

4.4. Extended phenotypic characterisation of cultured "gastrospirilla" ...............................33

4.5. Electron microscopy......................................................................................................33

4.5.1. Analysis of in vivo electron micrographs ...............................................................33

4.5.2. Ultrastructural analysis of cultured organisms .......................................................33

4.6. 16S rDNA sequencing ...................................................................................................33

4.6.1. Amplification of 16S rDNA by PCR......................................................................34

4.6.2. Sequencing..............................................................................................................34

4.6.3. Phylogenetic analysis .............................................................................................34

4.7. DNA-DNA hybridisations.............................................................................................35

4.7.1. DNA isolation.........................................................................................................35

4.7.2. Dot blot DNA-DNA hybridisations........................................................................35

4.7.3. Optical DNA-DNA hybridisation ...........................................................................35

4.8. Numerical analysis of SDS-PAGE whole-cell protein profiles.....................................36

4.9. Molecular typing of H. felis ..........................................................................................36

4.9.1. In situ DNA isolation and pulsed-field gel electrophoresis (PFGE) ......................36

4.9.2. Plasmid DNA extraction.........................................................................................37

4.9.3. 16 + 23S ribotyping ................................................................................................37

4.10. 23 rRNA gene polymorphism analysis....................................................................37

5. RESULTS.............................................................................................................................39

5.1. Primary isolation (I, II, III) ............................................................................................39

5. 2. Phenotypic analysis (I, II, III).......................................................................................44

5.2.1. Primary identification of Finnish field isolates (I, II, III).......................................44

5.2.2. Extended phenotypic characterisation (III) ............................................................44

5.3. ELECTRON MICROSCOPY (I, II, III)........................................................................45

5.3.1. In vivo electron micrographs (I, III) .......................................................................45

5.3.2. Ultrastructure of cultured strains (I, II, III).............................................................45

5.4. 16S rDNA sequencing (II).............................................................................................46

5.5. DNA-DNA hybridisation (I, II, III)...............................................................................47

5.6. Numerical analysis of protein profiles (III) ...................................................................48

5.7. 23S rRNA gene polymorphism (V)..............................................................................50

5.8. Molecular subtyping of H. felis (IV) .............................................................................50

5.8.1. PFGE typing and genome size determination ........................................................50

5.8.2. Plasmid profiling ....................................................................................................51

5.8.3. 16 + 23S ribotyping ................................................................................................51

6. DISCUSSION.......................................................................................................................52

7. CONCLUSIONS ..................................................................................................................57

8. REFERENCES .....................................................................................................................59

6

ACKNOWLEDGEMENTS

This study was carried out at the Department of Food and Environmental Hygiene, Faculty of

Veterinary Medicine, University of Helsinki during the years 1995-1998. The Foundation of

Emil Aaltonen, the Finnish Academy of Science, the Faculty of Veterinary Medicine, the

Foundation of Finnish Veterinary Science and the University of Helsinki have supported this

work. I give special thanks to the anonymous members of the Foundation of Emil Aaltonen:

thank you for trusting our group while we still were very small. Without your 3-year funding

this would not have been possible. I hope I have fulfilled your expectations.

I am deeply grateful to my supervisor Prof. Marja-Liisa Hänninen for her kind guidance

and support. You were never too busy to talk or give practical advice. Your talent for

knowing that there is science outside veterinary field and your skills for ideas of scientific

work were invaluable.

I also wish to thank the heads of the department, Prof. Timo Pekkanen and Prof. Hannu

Korkeala for creating a relaxed and enjoyable atmosphere in our team. Also the tea, almond

cookies, smoke of cigars and ever lasting diets will be unforgettable, Timi! To all the people

of the department; especially my long-time room mate Johanna Björkroth (what will happen

to “background noise”?), Sebastian Hielm, Maria Fredriksson-Ahomaa and the other

scientists. A very special thank you to the technical people Mirja Eerikäinen, Urszula Hirvi,

Raija Miettinen, Johanna Seppälä, and all the people in the library and computer services for

all the help.

It has been a privilege to work with Matti Kaartinen, Eila Pelkonen, Irmeli Happonen,

Antti Sukura and Pirkko Niemelä, in this faculty and also a very informative time in the

Hartman Institute to learn the secrets of sequencing. Also our foreign collaborators; Peter

Vandamme (Belgium), Stephen On (Denmark), Jani O’Rourke (Australia), Adrian Lee

(Australia), Cora de Ungria (Australia), Enevold Falsen (Sweden), Kate Eaton (Ohio, USA)

and Floyd Dewhirst (Massachusetts, USA) are gratefully thanked.

To Peter and Stephen, for making taxonomy both fun and serious business. You taught me

with such sophisticated methods how to write Scientific English! (“Where did you plan to

publish this??? In the Journal of Bad English? Journal of Incoherent Science?”) Thank you for

7

being my guardian angels and big brothers. You know and I know that I would have not

reached this point without you. I will probably hate you and love you for the rest of my life…

I wish to express my sincere gratitude to Prof. Sinikka Pelkonen and Prof. Hannele

Jousimies-Somer for the reviewing the thesis manuscript and to Stephen On for the revision

of the English text and useful discussions of this thesis and the papers II, III and V.

To all the members of the University Senate of 1995-1998: it has been a privilege to get to

know you, to learn from you and to know what really is the University of Helsinki. Especially

to Prof. Arto Mustajoki and Prof. Eero Puolanne; how much I learned from you and how

much I admire your passion to work for the good of our University.

A very special thank you to Pia Bäcklund, Vesa Kanninen, Sirkku Paajanen, Jade Erkko,

Anna-Maija Teppo and Kirsti Tervaportti for being such wonderful “dog sitters”. And of

course, Jaffa and Emil, for all the tail wagging while mummy has been upset and tired.

I am deeply grateful to Rev. Manuel Prado, Teresa Areia and everybody in Opus Dei for

spiritual guidance and continuous support.

Finally, to my family (especially Leevi Oskari!) to Marko, Minna, Anu and all my other

friends. Your love and support made this all come true.

8

.

To my parents:

To my charming and loveable mother

and

To my “silly” father who has always been so proud of us

9

LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following papers as listed in the text with Roman numericals (I-V)

I Hänninen, M.-L., Happonen, I., Saari, S. and Jalava, K. (1996) Culture and characteristics of

Helicobacter bizzozeronii, a new canine gastric Helicobacter sp. Int. J. Syst. Bacteriol. 46,

160-166.

II Jalava, K., Kaartinen, M., Utriainen, M., Happonen, I. and Hänninen, M.-L. (1997)

Helicobacter salomonis sp. nov., a canine gastric Helicobacter sp. related to Helicobacter

felis and Helicobacter bizzozeronii. Int. J. Syst. Bacteriol. 48, 975-982.

III Jalava, K., On, S.L.W., Vandamme, P.A.R., Happonen, I., Sukura, A. and Hänninen, M.-L.

(1998) Isolation and identification of Helicobacter spp. from canine and feline gastric

mucosa. Appl. Environ. Microbiol. 64, 3998-4006.

IV Jalava, K., De Ungria, M.C., O'Rourke, J., Lee, A., Hirvi, U. and Hänninen, M.-L. (1999)

Characterization of Helicobacter felis by pulsed-field gel electrophoresis, plasmid

profiling and ribotyping. Helicobacter. In press.

V Jalava, K., Hielm, S., Hirvi, U. and Hänninen, M.-L. (1999) An identification scheme based

on 23S rRNA gene polymorphism does not differentiate canine and feline gastric

Helicobacter spp. Lett. Appl. Microbiol. In press.

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1. ABSTRACT

The first culture of Helicobacter pylori in Australia in 1982 by Warren and Marshall rapidly

revolutionised the science of gastroenterology in human medicine. H. pylori is the most

widespread infection in man, occurring world-wide with approximately half of the human

population estimated as being infected with this pathogen. Previous studies during the last 100

years have shown the presence of Helicobacter-like organisms (so-called "gastrospirilla") in

the gastric mucosa of virtually all adult cats and dogs. The purpose of this study was to

develop and improve culture methods for these "gastrospirilla" and to characterise the strains

isolated.

We demonstrated that a variety of different Helicobacter species can be cultured from

gastric biopsy samples of dogs and cats. Two new species; namely H. bizzozeronii and H.

salomonis were isolated and described in the present study. In addition, we isolated two

strains, which resemble ”Flexispira rappini” in morphology from the same specimen, yet the

taxonomy of these ”Flexispira"-like organisms remains unsettled. The overall isolation rate of

Helicobacter-like spp. from canine and feline gastric specimen was 51% and 14%,

respectively.

The main difficulties in isolating these organisms are to support growth on their initial

culture, subsequent recognition and isolation from mixed cultures and finally the species

identification. The procedure of isolation of canine and feline "gastrospirilla" is very

challenging, yet possible, as was shown in the present study. Whilst the methods described

here for isolation of these bacteria present considerable progress over those used previously,

further improvements in culture conditions for these bacteria are still required, especially with

samples derived from cats.

Due to the inertia of Helicobacter spp. in most classical tests used for microbial

identification, a polyphasic approach must be applied for species characterisation of these

species. Morphological features as detected in electron micrographs were found to be

important, but by no means conclusive for species identification. It was observed that no less

than the accepted gold standard of DNA-DNA hybridisation has to be used to delimit species

boundaries. Dot blot DNA-DNA hybridisation method was proved to be a good screening

method which could guide the selection of strains for quantitative DNA-DNA reassociation

study. We strongly recommend the use of highly standardised SDS-PAGE as a means of

11

species identification for culturable Helicobacter spp. as both the dot blot and quantitative

DNA-DNA hybridisation results are in excellent agreement with the results of the protein

profile analysis.

Results of the 16S rDNA sequence comparisons between culturable and unculturable

Helicobacter spp. within the "gastrospirilla" group indicated all strains to be highly related by

this method, essentially representing a phylogenetic complex. Therefore, the true genetic

relationship between the culturable and unculturable "gastrospirilla" remains unknown, and

their complex taxonomy waits to be resolved. It is however clear that the widely used 16S

rDNA is not applicable for the definitive species-level identification.

The genetic diversity of different H. felis strains isolated from cats and dogs

geographically representing three different continents was shown to be very high with three

different molecular typing methods, namely ribotyping, plasmid profiling and pulsed-field gel

electrophoresis.

Accumulating data strongly indicate the need for further work concerning the specificity

and need to improve sensitivity of PCR assays for detecting the various Helicobacter spp.

known to inhabit the gastric mucosa of domestic pets and humans. Nonetheless, the

development of non-cultural methods for direct detection of the bacteria from the biopsy

samples, such as species-specific PCR tests would also be of benefit.

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2. REVIEW OF THE LITERATURE

2.1. Helicobacter pylori

The successful culture of Helicobacter pylori in Australia in 1982 by Warren and Marshall

rapidly revolutionised the discipline of gastroenterology in human medicine (Warren and

Marshall, 1983). H. pylori is now regarded as the most widespread infection in man: it has a

world-wide distribution and it is estimated that approximately half of the human population is

infected with this pathogen (Dunn et al., 1997). Most infections are believed to be acquired

during childhood and appear to persist for decades (Blaser, 1995). H. pylori is known to be

the most important causal agent of human gastritis, gastric and duodenal ulcers, and has

recently been classified as a first class human carcinogen by IARC (International Agency for

Research on Cancer )(Anonymous, 1994).

H. pylori is an unusual organism. It has developed several characteristics to survive in an

unquestionably hostile ecological niche. Acidity of the stomach is deleterious for most micro-

organisms, but this pathogen has developed strategies to overcome this problem. Copious

urease production helps to neutralise acidity, whilst the spiral morphology and flagella help

the bacterium to move rapidly within the viscous gastric mucosa. It has mechanisms to evade

the host immune system and it closely colonises the mucosa, resulting in a lifelong infection.

H. pylori is a pathogen highly adapted for human infection; the only recognised natural

animal reservoirs are primates and experimental animal models to study the mechanism and

treatment of the disease few (Blaser, 1995).

2.2. History of gastric Helicobacter spp

Before the isolation of H. pylori from human gastric mucosa, man and animals were known to

harbour gastric ”spirillum” or ”spirochete-like organisms. The first observation was in 1881

from dogs; ”a spiral curve or helix with a straight axis, not bended, moves quickly or slowly

depending of the rotation” (Rappin, 1881). More studies of gastric spiral organisms of cats

and dogs were performed around 1900. These morphological studies are still amazingly

accurate and sophisticated taken into account the limitations of the technology. Three

morphologically distinct bacteria were observed in canine gastric mucosa; spiral organisms in

cats and dogs were morphologically different from each other. The motility of the bacteria

13

was attributed to the flagella that were seen. By application of available staining techniques,

the organisms were defined as gram negative, they were found exclusively in the stomach,

most heavily colonising the fundus. They were very common in adult dogs and cats, but

virtually absent in young puppies or kittens (Bizzozero, 1893; Salomon, 1896; Regaud,

1909a; Regaud, 1909b; Lucet, 1910, Ball and Roquett, 1911; Dubosque and Lebailly, 1912;

Kolmer and Wagner, 1916; Kasai and Kobayashi, 1919; Edkins, 1920; Lim, 1920). An

especially noteworthy study was performed by Salomon (1896), who was the first to describe

three morphologically different spiral bacteria within the gastric mucosa of dogs and cats; he

was even able to propagate these bacteria in vivo in the stomachs of mice (Salomon, 1896).

The nomenclature of these bacteria was inconsistent, however. These organisms were first

named Spirillum rappini, in honour of the first scientist to describe them (De Toni and

Trevisan, 1889). Names such as Spirillum stomachii (Salomon, 1896; Lehmann and

Neumann, 1899), Spirochaete regaudi (Ball and Roquett, 1911; Kolmer and Wagner, 1916;

Kasai and Kobayashi, 1919) Spirella canis (Dubosque and Lebailly, 1912), Spirella regaudi

(Edkins, 1920) were also used to depict the gastric spiral canine and feline organisms.

Notably, the first description of humans infected with large, tightly spiral gastric bacteria

occurred in 1939 (Doenges, 1939), this study went largely unnoticed for many years. The

sixth edition of Bergey's manual (published in 1948) lists three separate gastric Spirillum

species in the genus VI Spirillum of the family Pseudomonacae (Breed et al., 1948);

significantly, none reappeared in the next edition (Breed et al., 1957). After the development

of electron microscopic techniques, three different morphological groups of bacteria were

described in detail within the canine gastric mucosa (Weber et. al, 1958, Weber and

Schmittdiel, 1962, Lockard and Boler, 1970). Again, feline organisms were morphologically

thinner and more tightly curved than those of canine organisms (Weber et al, 1958, Weber

and Schmittdiel, 1962). It was suggested that these distinct morphological types seen in dogs

were merely variable forms of the cell body which occurred as a consequence of moving

(Lockard and Boler, 1970). These organisms were suggested to be more closely related to

bacteria assigned to the order Spirochaete on the basis of their morphology, resembling thus

Treponema microdentium and Borrelia vincentii (Lockard and Boler, 1970).

Interest in spiral organisms associated with the gastric mucosa of animals increased

dramatically following the discovery of H. pylori in human disease; mainly due to lack of

animal models for H. pylori; and the possible zoonotic threat of gastric helicobacters. Since

three morphologically distinct bacterial types had been detected in vivo within the canine

14

gastric mucosa, there were attempts to culture these bacteria in vitro (Henry et al., 1987,

McNulty et al., 1989). Their classification, however, remained obscure; in 1987 these

organisms were suggested to be spirilla-like, showing features of both Spirillum and

Spirochaeta (Henry et. al., 1987). The first successful isolation of microaerophilic, large,

tightly helical bacteria with periplasmic fibrils around the cell body was reported from cats

and dogs in Australia in 1988 (Lee et al., 1988; Paster et. al., 1991). These strains

ultrastructurally resembled the so-called “type 2 organisms” of Lockard and Boler (1970), and

the success in their culture facilitated more detailed taxonomic studies. These isolates were

subsequently determined as members of the genus Helicobacter and designated H. felis

(Paster et al., 1991). Efforts by Lee et al. (1988) to isolate a second, morphologically similar

organism lacking surface periplasmic fibrils (resembling the "type 3" organisms of Lockard

and Boler, 1970) were unsuccessful. These organisms were numerous, compared to bacteria

resembling H. felis in cats and dogs. Finally, a third, highly distinctive bacterium with a

complex fusiform cell body had also been detected in morphological sections ("type 1" of

Lockard and Boler, 1970). Organisms with this morphology were originally isolated from

ovine abortions in 1985 (Kirkbride et al., 1985) and more recently have been cultured in vitro

from canine gastric mucosa (Eaton et al., 1996).

Figure 1. Three different morphological types of organisms detected with electron microscopyin canine gastric mucosa as described by Lockard and Boler in 1970. Ref: Lockard and Boler(1970) HUOM. Kuvan käyttö verkkoversiossa on kielletty.

15

Towards the end of the 1980’s, large, tightly spiral gastric bacteria distinct from H. pylori

but resembling the unculturable bacteria observed in domestic pets were observed in the

human gastric mucosa (Dent et al., 1987). The human organisms were initially named

”Gastrospirillum hominis” (McNulty et al., 1989). Following the proposal of the genus

Helicobacter (Goodwin et al., 1989) these organisms were considered members of this genus

by comparison of 16S rDNA sequences and subsequently named ”H. heilmannii” (O’Rourke

et al., 1992, Solnick et al., 1993). The incidence of these bacteria in human gastritis was

notably lower (ca. 0.2-1.0 %) compared to that associated with H. pylori (Heilmann and

Borchard, 1991). Several efforts were made to culture these organisms in vitro (Dent et al.,

1987, McNulty et al., 1989, Lee et al., 1988), but all failed until 1996 when one such strain

was isolated in Denmark (Andersen et al., 1996, Andersen et al., 1999). Unexpectedly,

Fawcett et al. reported recently that H. pylori strain ATCC 43504 when grown in blood agar

plates, resembled morphologically H. pylori and whilst grown in broth media, the

morphological appearance resembled that of "H. heilmannii" (Fawcett et al., 1999). Both

these "morphological forms" were shown to represent H. pylori with urease and cytotoxin-

associated gene PCR. This phenomenon needs considerable work to be settled.

In the following text, gastric, morphologically similar, large, tightly helical Helicobacter

spp. will be referred to as "gastrospirilla". Although there is no formal description,

"gastrospirilla" cells are generally described as large spiral rods, 0.4 to 0.9µm in diameter and

4 to 10µm in length. They are motile by means of polar bundles of sheathed flagella. Cells

have 3 to 8 spiral turns and some strains have periplasmic fibrils, but these are not detectable

when using common light microscopy. The presence of "gastrospirilla" cells is always

associated with urease activity in gastric biopsy samples. The named organisms within this

group (whether validly published or not) so far include ”G. hominis”1, ”G. hominis”2, ”G.

suis”, ”G. lemur”, ”H. heilmannii” and H. felis. To date, "gastrospirilla" have been detected in

a wide variety of animals, including dogs (Salomon, 1896), cats (Salomon, 1896), rats

(Salomon, 1896), monkeys (Kasai and Kobayashi, 1919, Cowdry and Scott, 1936), baboons

(Curry et al., 1987), pigs (Queiroz, 1990), lemurs (O'Rourke et al., 1992) captive exotic

carnivores (Jakob et al., 1997) and humans (Doenges, 1939).

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2.3. Taxonomy of the genus Helicobacter

The taxonomy of the genus Helicobacter has rapidly evolved since many new Helicobacter

spp. have been described since 1989. H. pylori (formerly Campylobacter pyloridis, later

corrected to C. pylori), the type species of the genus, and H. mustelae (formerly C. pylori

subsp. mustelae, later elevated to species status as C. mustelae), were the first two taxa

assigned to Helicobacter (Goodwin et al.,1989). Since 1991, at least one species has been

described each year and there are presently 18 validly published species assigned to the genus.

A distinct phylum within the class Proteobacteria of the eubacteria was proposed by

Vandamme et al. (1991): rRNA superfamily VI. This superfamily encompassed the genera

Campylobacter, Helicobacter, Arcobacter and Wolinella, and both Helicobacter and

Wolinella formed rRNA cluster III within the superfamily. Bacteria referred to previously as

”Flexispira” were found to be closely related to the genus Helicobacter. Similarly, two former

enteric Campylobacter species (C. fennelliae and C. cinaedi) were reclassified as H.

fennelliae and H. cinaedi and a revised genus description was given (Vandamme et al., 1991).

A typical characteristic of the entire Helicobacter genus is the fastidious nutritional

requirements of the individual species that warrant special skills to isolate and culture these

organisms (Vandamme et al., 1996). Furthermore, the whole superfamily VI shows

exceptional inertia in common biochemical reactions, thus decreasing the number of tests

which can be used for classification (On and Holmes, 1995, On, 1996, On et al., 1996). Also,

some species descriptions are based on a limited number of strains and therefore the

intraspecies variation cannot be properly evaluated (Bronsdon et al., 1991, Eaton et al., 1993,

Franklin et al., 1996). In addition, many names commonly used have not been properly

validated and thus causing confusion as to the taxonomic status of these bacteria. These

problems have risen either due to the availability of only one, or a few isolates for study, or

the inability to isolate the organisms (Table 1). As a consequence, there are several problems

to be resolved in the taxonomy of Helicobacter spp.

The importance of phenotypic analyses must not be underestimated in modern taxonomic

studies. In order to describe a new species, a clear phenotypic differential to existing taxa at

the appropriate taxonomic level must be presented (Wayne et al., 1987). Moreover, the

determination of intraspecies variation is important for routine diagnostic purposes and

therefore, the species descriptions should be performed with a representative number of

unrelated strains (On, 1996; Vandamme et al., 1996). Furthermore, any phylogenetically

based scheme should show phenotypic consistency (Wayne et al., 1987). This provision is

17

particularly difficult to fulfil within the superfamily VI as all the members exhibit marked

inertia phenotypically (On, 1996).

Taxon Year Source Country Isolated/detected/described by

“Flexispira rappini”, “H. rappini” 1985 Sheep placenta USA Kirkbride et al., 1985

Helicobacter sp., CLO-3 1985 Human proctitis USA Totten et al., 1985

“H. heilmannii”, “G. hominis” 1987 Human gastric mucosa GB Dent et al., 1987

“G. suis” 1990 Porcine gastric mucosa Brazil Mendes et al., 1990

“G. lemur” 1992 Lemur gastric mucosa USA Dewhirst et al., 1992

Helicobacter sp. Bird-B 1994 Wild birds USA Dewhirst et al., 1994

Helicobacter sp. Bird-C 1994 Wild birds USA Dewhirst et al., 1994

Helicobacter sp. strain Mainz 1994 Human arthritis Germany Husmann et al., 1994

“H. westmaedii” 1997 Human blood Australia Trivett-Moore et al., 1997

“H. colifelis” 1998 Feline colon mucosa USA Foley et al., 1998

“H. suncus” 1998 Mouse gastric mucosa Japan Goto et al., 1998

Table 1. Provisional Helicobacter spp. and related groups, requiring validation (as of April

1999)

DNA-DNA hybridisations have been performed to validate the distinct taxonomic status

of certain Helicobacter spp., including H. cinaedi and H. fennelliae (Totten et al., 1985), H.

nemestrinae (Bronsdon et al., 1991), H. canis (Stanley et al., 1993), H. pullorum (Stanley et

al., 1994) and H. pametensis (Dewhirst et al., 1994). Many Helicobacter spp. have been

described without any results of DNA pairing experiments, whereby the principal justification

for species-status has been based on numerical comparison of 16S rDNA sequence similarity

data. These include H. felis (Paster et al., 1991), H. muridarum (Lee et al., 1992), H.

acinonychis (Eaton et al., 1993), H. hepaticus (Fox et al., 1994), H. bilis (Fox et al., 1995), H.

cholecystus (Franklin et al., 1996), H. trogontum (Mendes et al., 1996) and H. rodentium

(Shen et al., 1997). Remarkably, in all these species (except H. felis and H. muridarum), the

similarity of the 16S rDNA sequence to one, or even several examples of existing species has

been estimated to exceed 97%. Therefore, according to recommendations for the indications

to use of 16S rDNA sequence analyses in bacterial taxonomy (Stackebrandt and Goebel,

1994), DNA-DNA hybridisation experiments should have been performed in all the

aforementioned cases to confirm their taxonomic position.

18

The taxonomy of the "gastrospirilla" is especially complex. These organisms were first

classified on the basis of their morphology to a new genus ”Gastrospirillum” gen. nov. and

the organisms of the human source were designated ”Gastrospirillum hominis” sp. nov. (Dent

et al., 1987, McNulty et al., 1989). As a consequence, the "gastrospirilla" later observed in

pigs were designated ”G. suis” (Mendes et al., 1990), and in lemurs as ”G. lemur” (Dewhirst

et al., 1992). After the PCR technique was applied to determine the phylogenetic position

these unculturable bacteria, they were found to be members of the genus Helicobacter both on

the basis of the 16S rDNA and urease gene sequence data (Solnick et al., 1993, Solnick et al.,

1994). The 16S rDNA sequences of these taxa generally exhibited more than 97% sequence

similarity to each other. However, two 16S rDNA sequences were identified from two

different patients with less than 97% similarity, and therefore these two different entities were

named as ”G. hominis”1 and ”G. hominis”2 (Solnick et al., 1993). Nonetheless, these bacterial

strains were suggested to represent a new species, ”H. heilmannii”, despite the clear

differences in 16S rDNA sequences (Solnick et al., 1993). It has been inferred recently on the

basis of multiple clones of a 580 bp fragment of the urease gene, that humans can be infected

with at least three different variants of ”H. heilmannii” (Dieterich et al., 1998). Furthermore,

the "gastrospirilla" seen in pigs were found to closely resemble ”G. hominis”1 by 16S rDNA

sequence similarity, whilst "gastrospirilla" in dogs and, to some extent, in cats appeared more

closely affiliated to ”G. hominis”2 (Mendes et al., 1995). At present, the taxonomy of the

"gastrospirilla" is perplexing and in a state of considerable flux.

2.4. Isolation and detection of gastric helicobacters

2.4.1. Primary isolation

Culture of helicobacters in vitro is very demanding and special culture conditions are

necessary to succeed. These include a microaerobic atmosphere (oxygen level of 5-7%) with

high humidity, an incubation temperature of 37oC, and a rich growth medium. Additional

atmospheric hydrogen (H2 %) is either required, or stimulates growth of certain helicobacter

species (Goodwin and Armstrong, 1990, Vandamme et al., 1991, Dunn et al., 1997). The

primary growth may take up to 7 days to appear (Bronsdon et al., 1991, Dunn et al., 1997)

and mixed cultures may occur with campylobacters (Lee et al., 1988).

19

Gastric biopsies are taken either via an endoscope from humans (Dunn et al., 1997) or

from recently euthanised animals (Lee et al., 1988). The biopsies should be cultured as soon

as possible. If storage prior to culture is necessary, the biopsies should ideally be stored in a

transport medium at room temperature. Several media have been tested for this purpose and

the most widely used are Stuart’s medium (Kjoller et al., 1991), Brucella broth with glycerol

or cysteine-albumin media with glycerol (Alpert et al., 1989), or skimmed milk with glycerol

(Han et al., 1995). Short-term storage (<6 hours) in saline is also appropriate (Han et al.,

1995).

A variety of culture media have been used for isolation of gastric helicobacters. Both

selective and non-selective media have been used. The most commonly used are Brucella-,

Brain Heart Infusion-, Trypticase Soy- or Blood Agar Base agar media, each supplemented

with added 5-10% blood. Media supplementation with charcoal or cornstarch instead of blood

has been used to support the growth of H. pylori (Buck and Smith, 1987). One of the most

common selective media used for primary isolation is Skirrow’s medium containing Brucella

blood agar supplemented with trimethoprim, vancomycin, polymyxin B and amphotericin B

(Warren and Marshall, 1983, Fox et al., 1988, Lee et al., 1988, Eaton et al., 1991). The

biopsies may be ground first with a glass grinder in liquid media, or directly rubbed against

the agar media (Goodwin et al., 1985). Biopsies are cultured on freshly prepared media and

agar plates are incubated in microaerobic conditions (6% of O2) , with the lids uppermost, in a

humid atmosphere (Warren and Marshall, 1983, Goodwin and Armstrong, 1990).

Bacterial growth usually appears after three to five days of incubation, but may take up to

7 days (Lee et al., 1988). Subculture is often difficult as the primary area of isolation may be

small; any growth should be inoculated onto a small area of growth medium (Goodwin and

Armstrong, 1990). The growth appears either as translucent, small, pinpoint colonies (Warren

and Marshall, 1983, Fox et al., 1988, Eaton et al., 1991, Bronsdon, 1991) or as swarming

film-like growth without distinct colonies as was described for H. felis (Lee et al., 1988) and

”F. rappini” (Kirkbride et al., 1985).

2.4.2. Rapid urease test

It was noted shortly after its initial isolation that H. pylori (as well as the other gastric

helicobacters) produced copious quantities of urease (Warren and Marshall, 1983, Fox et al.,

1988, Lee et al., 1988, Eaton et al., 1991, Bronsdon, 1991). This led to the development of

rapid (60 minute) tests that detected this enzyme directly in biopsy samples colonised with

20

helicobacters. The tests usually contain 10% urea (either in agar, solution or tablet form) and

phenol red as an indicator. As the urease enzyme of the gastric helicobacter hydrolyses urea,

the pH rises and a colour change occurs; any positive result can be recorded in a few minutes

or several hours. The density of the bacteria in the biopsy has a direct correlation with the

sensitivity of the test, both with H. pylori (Laine et al., 1996) and canine gastric helicobacters

(Happonen et al., 1996a).

2.4.4. Visualisation of "gastrospirilla" by light microscopy

In animals or humans infected with "gastrospirilla", the bacteria are distributed relatively

freely in the gastric mucosa (McNulty et al., 1989, Happonen et al., 1996b), in contrast to H.

pylori infection where bacteria are usually tightly associated with the mucosal cells (Dunn et

al., 1997). Consequently, the touch cytology- (Debongie et al., 1995) and brush cytology

(Happonen et al., 1996) techniques have proved to be excellent methods for primary detection

of "gastrospirilla" by light microscopy. Moreover, the brush cytology technique has proved

superior to both the urease test, and conventional histological examinations, when examining

for "gastrospirilla" in canine or feline gastric samples (Happonen et al., 1996a). In this

procedure, a small round bottle-brush is used to collect the mucus from the gastric mucosa

during endoscopy by rolling the brush over the test site and subsequently depositing the

sample on the slide. The slide is air dried, stained with May-Grünwald-Giemsa and any

bacteria are visualised with light microscopy (Happonen et al., 1996a). Only the presence of

"gastrospirilla" can be recorded with this method; no certain species distinction can be made

(Happonen et al., 1996a).

2.4.5. Visualisation of "gastrospirilla" by electron microscopy

Three different morphological types of "gastrospirilla" in canine and feline gastric mucosa

were observed when tissue samples were examined by electron microscopy in various studies

in the 1950’s and 1970’s (Weber et al., 1958, Weber et al., 1962 and Lockard and Boler,

1970). These remain among the most delicate studies performed on the morphology of these

bacteria to date. More recently, it has been noted that spiral bacteria without periplasmic

fibrils are most commonly found in the gastric mucosa of cats and dogs, whilst cells

resembling H. felis (i.e. tightly helical rods entwined by surface periplasmic fibrils) appear to

be comparatively infrequent (Henry et al., 1987, Lee et al., 1988, Otto et al., 1994, Eaton et

al., 1996, Happonen et al., 1996a and Happonen et al., 1996b). The third, fusiform organism

21

(resembling ”F. rappini” in morphology) was detected in an early morphological study

(Lockard and Boler, 1970), but was not detected in electron micrographs in a more recent

study, despite the culture of these organisms in vitro (Eaton et al., 1996). The sensitivity of

electron microscopic techniques to detect "gastrospirilla" is somewhat lower than that of the

urease test or histology (Happonen et al., 1996a and Happonen et al., 1996b). This may be due

to the small surface area investigated by electron microscopy (McNulty et al., 1989,

Happonen et al., 1996a and Happonen et al., 1996b), since in human biopsies "H. heilmannii"

is less prolific, and more sporadically distributed in the gastric mucosa than H. pylori (Holck

et al., 1997).

2.4.3. PCR

PCR (polymerase chain reaction) is a powerful method for detecting low numbers of bacteria

from clinical material and does not require viable bacteria (Persing et al., 1993, Vaneechoutte

et al., 1997). It has also been applied to the diagnosis of H. pylori by direct amplification of

H. pylori DNA from biopsy samples. The accuracy varies greatly due to inhibitory factors in

the gastric biopsy, the choice of primers, and the PCR reaction itself (Dunn et al., 1997,

Persing et al., 1993). Nonetheless, well-designed PCR methods are superior to other methods

in terms of sensitivity when detecting gastric helicobacters from saliva, dental plaque, gastric

juice or faeces (Madinier et al., 1997, Mapstone et al., 1993, Wahlfors et al., 1995, Westblom

et al., 1997, Yoshida et al., 1998) and are also non-invasive. Quantitative PCR assays have

also been developed to estimate the severity of infection with H. pylori and H. felis (Kong et

al., 1996, Monteiro et al., 1997). However, the use of PCR-based methods carries a risk of

false positive results (Persing et al., 1993)

2.5. Identification of culturable "gastrospirilla"

Accurate identification of strains belonging to the genera Campylobacter, Arcobacter and

Helicobacter is difficult, particularly for the latter (On, 1996). This is due to the fastidious

growth requirements, slow growth rate, and remarkable inertia in common biochemical tests

exhibited by these bacteria (On and Holmes, 1995, On, 1996). Furthermore, new Helicobacter

species have been described each year since 1991, so it is difficult for a routine

microbiological laboratory to keep up with current taxonomic developments. In any case,

there remain many unresolved problems in Helicobacter taxonomy. Therefore, species

identification requires considerable effort. The following list of methods is not

22

comprehensive, but presents some of the most widely used identification tests used within this

group of bacteria.

2.5.1. Classical phenotypic tests

Minimal standards for describing new Campylobacter spp. have been proposed (Ursing et al.,

1994), but no comparable recommended scheme exists for Helicobacter spp. This, combined

with the difficulties in growing the organisms has led to minimal and probably insufficient

testing of new isolates; even when proposing new species, the biochemical testing of the

isolates is usually minimal. As a consequence, there are often too few clear phenotypic

differences between species (On, 1996). Lack of standardisation of the test conditions has

further led to inconsistency, and contradictory results have been obtained from different

studies (On, 1996). A large panel of standardised tests for the identification of all

Campylobacter spp., Helicobacter spp. and related organisms has been developed, but has not

established wide acceptance (On, 1996).

2.5.2. Protein profile analysis

The suitability of one-dimensional sodium dodecyl sulphate- polyacrylamide gel

electrophoresis (SDS-PAGE)-based whole-cell protein profile analysis for speciation of

bacteria has long been established (Owen and Jackman, 1982; Kersters, 1985). It was noted

that the results obtained from protein profile analysis were in good agreement with results of

whole genome DNA-DNA hybridisations. Since DNA codes for proteins via RNA, it is

logical that the results of whole cell protein profile analysis correlate with results obtained

from DNA-DNA hybridisation studies. Therefore, the more laborious and time-consuming

DNA-DNA hybridisation method has been replaced to some extent, or at least is used in

conjunction with, protein profile analysis. However, the interpretation of the protein profile

patterns requires considerable expertise (On, 1996, Vandamme et al., 1996). The observation

that whole-cell protein patterns of campylobacters are relatively insensitive to growth

conditions and age of the bacterial cells, makes it a very suitable method for this fastidious

group of bacteria (Vandamme et al., 1996). Therefore, this method has been applied to a

number of Campylobacter spp. (Vandamme et al., 1990) and Helicobacter spp. (Costas et al.,

1993).

23

2.5.3. DNA-DNA hybridisation

The DNA-DNA hybridisation method to study the genetic relationship of different bacterial

strains was developed in the 1960’s. The method uses single-stranded whole cell DNA

(derived from native double stranded DNA by denaturation) from two bacterial strains and

compares their capacity to hybridise with each other to form double stranded DNA under

different temperatures and ionic concentrations (stringency). Thus this technique gives

information about the whole genome DNA similarity between two bacterial strains expressed

as hybridisation values (Owen and Pitcher, 1985). Yet, it should be noted that it is not

possible to convert a percentage DNA-DNA hybridisation value into a percentage of whole-

genome sequence similarity (Vandamme et al., 1996). The DNA-DNA hybridisation remains

the classical gold standard method for species delineation (Wayne et al., 1987; Stackebrandt

and Goebel, 1994). Several techniques have been employed, including the agar gel method

(McCarthy and Bolton, 1963), membrane filter method (Gillespie and Spiegelman, 1965),

hydroxyapatite method (Brenner et al., 1969), S1 endonuclease method (Sutton, 1971, Crosa

et al., 1973) and spectrophotometric method (De Ley et al., 1970). This method requires large

amounts of pure DNA, and therefore, is relatively laborious and time-consuming. More recent

modifications include the use of non-radioactive labels in the membrane filter method

(Jahnke, 1994), reduction of the DNA concentration (Huss et al., 1983) and optimisation of

the DNA-DNA hybridisation conditions (Grimont et al., 1980, Hartford and Sneath, 1993).

2.5.4. 16S rDNA sequencing

The development of molecular-genetic methods during the 1980’s and 1990's led to the

recognition of the suitability of ribosomal RNA molecules to be good indicators of

phylogenetic relationships (Woese, 1987). The 5S, 23S and the more widely used 16S rDNA

gene sequences were proposed to reflect best phylogenetic relationships among bacteria due

to their stability, relatively slow evolutionary change rate, occurrence in all organisms and

functional constancy of these molecules (Woese, 1987). The 16S rRNA gene is approximately

1500 bp long and it is usually present as several copies in a bacterial cell (Woese, 1987). It is

known that there are both conserved and variable regions within the gene, and genus or

species specific primers or probes may be designed if sequences are known. H. pylori carries

two copies of 16S rRNA genes (Taylor et al., 1992, Tomb et al., 1996), while the copy

number of any other Helicobacter spp. is not known. In recent years, the extensive use of

sequence-based analyses of ribosomal genes has led to the establishment, and rapid expansion

24

of large international sequence databases such as EMBL (European Molecular Biological

Laboratory) and GenBank. It should be noted that this rapid development has led to a

situation where 16S rDNA sequencing presently dominates, or has even replaced the use of

many classical taxonomic methods. However, restrictions apply to this method (Vandamme et

al., 1996)

It has become clear that phylogenetic analyses cannot replace polyphasic taxonomic

studies. It was noted in 1992 that 16S rDNA sequence identity is not sufficient to guarantee

species identity (Fox et al., 1992). Two clearly separate Bacillus spp. were shown to be

distinct species by means of DNA-DNA pairing experiments and phenotypic properties, yet

they shared 99.5% 16S rDNA sequence similarity (Fox et al., 1992). In 1994 a literature

survey was performed to compare the results from a number of 16S rDNA sequence analyses

and DNA-DNA pairing experiments (Stackebrandt and Goebel, 1994). The authors

considered that, if the 16S rDNA sequence homology of a given group of bacteria was less

than 97% to existing species, they were not likely to demonstrate more than 60% relatedness

in DNA-DNA hybridisation experiments. However, if the sequence homology exceeded 97%,

DNA-DNA hybridisation experiments were necessary to define species boundaries as it was

noted that above this value of sequence similarity, the DNA-DNA hybridisation value may be

either low or as high as 100% (Stackebrandt and Goebel, 1994). It was also concluded that

16S rDNA analysis is a valuable addition to polyphasic taxonomy and could be used as a

screening method to determine if more time-consuming DNA-DNA hybridisation

experiments should be performed (Stackebrandt and Goebel, 1994, Vandamme et al., 1996).

A more complex situation is evident when molecular sequencing data is used as a sole

criterion to define species boundaries. This occurs when DNA from unculturable bacteria is

subjected to direct PCR amplification with primers designed to amplify the 16S rDNA region

and the product is cloned and sequenced. As a consequence, descriptions of several new, less

than adequately described, bacterial species and genera have been published (Murray and

Schleifer, 1994). A category ”Candidatus” has been proposed for the descriptions based on

sequences of unculturable organisms to solve this matter (Murray and Schleifer, 1994), and as

a result 19 ”Candidatus” descriptions have been proposed up to date (Medline, November,

1998).

25

2.5.5. Restriction fragment length polymorphism (RFLP) of 23S rRNA gene

Despite the current popularity of sequence-based analyses, they are not rapid, cheap or easy

species identification methods for routine clinical laboratories. Therefore, methods using

PCR-amplified-rDNA which is then digested with restriction endonucleases have been

developed for species identification (Gurtler et al., 1991, Jayaro et al., 1991). These methods

amplify parts of the 16S or 23S rRNA genes and may omit or include the ribosomal spacer

region. Universal primers of conserved regions are used. The amplicons are then digested by

restriction enzymes and the patterns visualised by electrophoresis in agarose gels. These PCR-

rDNA restriction fragment length polymorphism (RFLP) patterns are usually species-specific

and can be used as identification tools in contrast to other PCR typing methods which are

usually more discriminatory (Gurtler et al., 1991; Jayaro et al., 1991). One such method has

been developed for Helicobacter spp. This identification scheme is based on a 23S rRNA

gene amplicon, which is digested with HaeIII, HpaII HhaI and HinfI. These RFLP profiles

could be used to differentiate l2 Helicobacter spp. to species level (the study included one H.

felis strain of the "gastrospirilla" group) (Hurtado and Owen, 1997). The method was able to

differentiate the phylogenetically highly related group of H. pylori, H. acinonychis (formerly

H. acinonyx) and H. nemestrinae from each other.

2.6. Polyphasic taxonomy

In simple terms, taxonomy is an attempt to organise the constantly expanding information

base concerning natural diversity into a meaningful hierarchical system. Classically it has

been divided into three parts; 1) classification of organisms into groups on the basis of

similarity, 2) nomenclature of these groups according to international rules and 3)

identification of unknown organisms (Krieg and Holt, 1984, Vandamme et al., 1996).

However, studies using just one or few methods to classify bacteria often give confusing

results. An approach, which utilises a large amount of data from phenotypic and genotypic

analyses, is called polyphasic taxonomy. Here, a taxon (whether it be a species, genus, family,

superfamily, subdivision, division or domain of any group of living organisms) is defined

with all necessary information from classical phenotypic analysis to highly sophisticated

nucleic acid sequence data (Vandamme et al., 1996). Colwell first used the term “polyphasic

taxonomy” in 1970 (Colwell, 1970). It should be remembered that this approach contains no

strict or defined guidelines describing its application, but involves a proper definition of the

problem to be resolved and the subsequent application of the most suitable methods for this

26

purpose (Vandamme et al., 1996). As a consequence, the end result should serve to the best

possible extent all practical users of taxonomy; clinicians, veterinarians, epidemiologists, and

other scientists (Vandamme et al., 1996).

The most important unit of taxonomy is the species, since it is the lowest category that

cannot be subdivided. It must be noted that bacterial species are not like animal species,

which may be defined by specific criteria, namely the possibility of two individuals of

opposite gender to produce offspring with the capacity to reproduce. The definition of

bacterial species requires considerable flexibility. At present, consensus guidelines define a

bacterial species as a group of strains (including a type strain) that are characterised by a

certain degree of phenotypic consistency, a significant degree of DNA-DNA homology

(preferably over 70%) and over 97% of 16S rDNA homology (Wayne et al., 1987;

Stackebrandt and Goebel, 1994; Vandamme et al., 1996). In addition to these requirements, it

is important to note that at least two phenotypical differences should exist to other defined

species (Vandamme et al., 1996) Thus, these guidelines give precedence of the phenotype

over the genotype (Wayne et al., 1987, Vandamme et al., 1996).

Much confusion has been caused by introducing more taxonomic groups. A category of

subspecies or biotype has been referred to organisms that differ phenotypically, but exhibit

hybridisation value of approximately 40-60%. Also the opposite occurs, if two genetically

distinct groups can be identified, yet not differentiated from each other by clear phenotypic

means, they should not be considered as separate species, but have been designated as

genospecies, genomic species, genomovars or genomic groups (Wayne et al., 1987, Ursing et

al., 1995). Much of these other categories have caused more problems than they have solved

(Vandamme et al., 1995).

Two groups of strains can be considered as two separate species, if they exhibit clear

phenotypic differences and share insignificant degree of binding in DNA-DNA hybridisation

experiments (less than 30%) or are show less than 97% similarity in their 16S rDNA

sequences (Stackebrandt and Goebel, 1994; Vandamme et al., 1996). If two such groups of

strains can be differentiated phenotypically, but the 16S rDNA sequence similarity is higher

than 97%, DNA-DNA pairing experiments should be performed to delineate species

boundaries (Stackebrandt and Goebel, 1994). In many cases these rules cannot be applied, but

then the polyphasic taxonomic approach is the method of choice (Vandamme et al., 1996).

27

2.7. Molecular typing

2.7.1. Typing methods

Bacteria can be differentiated below the species level. Modern molecular subtyping methods

may be based upon interstrain differences in lipopolysaccharide (LPS), fatty acids, proteins or

nucleic acid sequence (Persing et al., 1993). Nucleic acid-based methods have developed

significantly during the past 20 years and the change from conventional biotyping, serotyping,

bacteriophage typing and bacteriocin typing scheme to genotyping methods has been rapid.

The molecular typing methods were first applied to problems of epidemiology and hospital

infections, but their potential for studies of genetic diversity and genomic structure has been

recognised recently. Therefore it is possible to classify strains of the same species in terms of

genetic similarity (Römling et al., 1994).

Nucleic acid-based typing techniques detect differences in the nucleic acid sequence of

chromosomal DNA and thus study the genetic structure of microbes. Most of them apply

restriction enzymes that recognise and cleave the DNA in a highly specific manner, thereby

revealing restriction fragment length polymorphism (RFLP) between the strains. A simple

point mutation or larger mutational event can cause loss or acquisition of a restriction site.

Restricted fragments can be analysed directly (restriction endonuclease analysis, REA; and

pulsed-field gel electrophoresis, PFGE) or by detecting only selected DNA fragments by

hybridisation, e.g. with a ribosomal gene probe (ribotyping). Also plasmids can be analysed

with these methods. None of these methods can be considered as “gold standard”, but

differences in typeability, reproducibility and discriminatory power do occur and has to be

taken into account when a particular method is chosen (Maslow et al., 1993a).

H. pylori has been studied with several different typing methods. Various molecular

typing studies show this species to be highly variable at the genetic level. These data include

RFLP analysis of whole-genomic DNA (Langenberg et al., 1986), RAPD (randomly

amplified polymorphic DNA) analysis (Akopyants et al., 1992), MLEE (multilocus enzyme

electrophoresis) analysis (Go et al., 1996), plasmid profiling (Simor et al., 1990), ribotyping

(Owen et al., 1992, Tee et al., 1992) and PFGE profiling (Taylor et al., 1992).

The following overview is not comprehensive, but gives examples of some of the most

widely used bacterial typing methods (Swaminathan et al., 1993, Maslow et al., 1993a).

28

2.7.2. Plasmid profiling

The first nucleic acid-based method to be described was plasmid profiling. It was used for the

first time in 1979 to study a nosocomial infection of Klebsiella pneumoniae (Sadowski et al.,

1979). The advantage of this method is its simplicity and low cost, but there are several major

disadvantages. Not all bacterial strains carry plasmids; they can lose the plasmids upon

subculturing; and the bands obtained are relatively few and therefore the discriminatory

power is low. Furthermore, a particular plasmid may appear after isolation in several bands as

supercoiled, relaxed circular or linear DNA with different electrophoretic mobilities (Maslow

et al., 1993a). Of all the Helicobacter spp., only H. pylori has been studied with this method

and approximately 35% of strains examined have harboured plasmids (Simor et al., 1990).

2.7.3. RFLP

First, whole-genomic DNA is isolated from bacterial cells, which is then digested with

restriction enzymes that cut the DNA into fragments approximately 1000 to 20,000 base pairs

(bp) in length. These are separated electrophoretically according to their size in an agarose gel

to give a complex genomic "fingerprint". These patterns can be analysed and stored as

computer files and compared with other profiles. The advantages of this method is its wide

applicability and universal use, whilst the disadvantages include the need for pure, good

quality DNA for analysis and the exceptionally complex DNA fragment patterns that are

difficult to interpret (Sambrook et al., 1989). As the number of bands generated by restriction

enzymes are so numerous, reducing the number of component bands can facilitate

interpretation of the data. One such approach is to transfer DNA from agarose gels to a

membrane (Southern blotting) and to subsequently apply a labelled probe, which hybridises

only to a subset of DNA fragments that can be specifically recognised by the probe.

Variations in position and number of these fragments are called as restriction fragment length

polymorphism, RFLP.

The most widely used probe involves the use of ribosomal DNA, usually 16S, 23S or

16+23S rRNA genes, that are present in several copies in most bacterial species. This method

is called ribotyping (Grimont and Grimont, 1991). It is a widely adopted typing method,

because the same universal probe (usually derived from E. coli rDNA) can be used for

different species and most species harbour multiple copies of the rRNA operon. Thus banding

patterns composed of several bands are obtained, depending on the cleavage sites of the

29

restriction enzyme within the probe. The discriminatory power is good with bacterial species

carrying multiple copies of rRNA operons. The main benefits of ribotyping are the relatively

simple banding patterns, good reproducibility and possibility for automatisation. The

disadvantages include the laborious procedure, taking several days using standard methods,

and low discriminatory potential when applied to some bacterial species. Nonetheless, it is a

widely accepted taxonomic tool as a species identification method with some difficult groups

of bacteria due to its benefits (Swaminathan et al., 1993). Furthermore, recently an automated

ribotyping system has been developed (Riboprinter®, Qualicon Inc., DE, USA). It is highly

automated, needs only cultured bacterial cells, and gives an answer within 8 hours. Yet, the

equipment is still relatively costly and not at hand to the of most research institutes.

2.7.4. PFGE

One of the most discriminatory typing methods is PFGE because it detects mutations in any

of the cleavage sites of the restriction enzyme used. In this method, bacterial cells are

embedded in agarose plugs in order to obtain intact DNA. Slices of the plugs are subjected to

digestion of rare-cutting enzymes (most commonly used recognise 6 or 8 bp site). The

resulting DNA fragments are relatively large (between 10-1000 kb) and will be separated

under specific electrophoretic conditions (Chu et al., 1986). The gel loaded with slices is

placed into a running chamber and is surrounded by hexagonal array of electrodes. The

electric current is changed between the opposite pair of electrodes, thus forming a pulse time.

This change in electrophoretic conditions causes the DNA to move along the gel with back

and forward fashion, thus resulting a high level of resolution facilitated with this method

(Tenover et al., 1997). The choice of the enzyme is related to the purpose, if the genome size

determination is desired, relatively few bands are eligible, whereas for typing purposes a sum

of 20-40 bands is preferred ((Römling et al., 1994)

The main advantage of PFGE is its discriminatory power and relatively simple banding

patterns, but long and laborious DNA isolation procedures, and digestion of the samples mean

that results may take from few days up to a week to obtain (Maslow et al., 1993b; Matushek

et al., 1996; Gautom, 1997). Furthermore it represents the entire bacterial genome in a single

gel and the result is highly reproducible (Maslow et al., 1993a). Some consensus concerning

the interpretation of the PFGE patterns has been obtained and this interpretation is quite

straightforward. When compared epidemiologically related strains, one can determine that

30

indistinguishable patterns represent the clones of the same outbreak or source. Also patterns

with 2-3 band differences (i.e. one genetic change in enzyme recognition site) are being

considered as probably related. It has been noted that some bacterial species exhibit this

phenomenon when subcultured for a period of time or strains isolated multiple times from a

patient. Two strains are considered possibly related if a difference of 4-6 fragments is present

and an isolate is not of the same ancestor if ≥ 7 band difference occurs (Tenover et al., 1995).

Furthermore, PFGE can be used for the determination of the genome size by summing up the

resulting fragments.

Large computerised databases hold a promise for the future. At present, PFGE patterns of

selected food borne pathogens (certain Escherichia coli and salmonella) can be compared

rapidly in real time through the internet, such as PulseNet. This network permits rapid

comparison of the PFGE patterns through an electronic database established at the Centers for

Disease Control and Prevention (CDC) in Atlanta, USA

(http://www.cdc.gov/ncidod/dbmd/pulsenet/pulsenet.htm).

As mentioned above, the genetic diversity of H. pylori has been studied by PFGE (Taylor

et al., 1992). In addition, the genetic diversity of two animal helicobacters, H. mustelae and H.

hepaticus has been determined with PFGE and is markedly less than that of H. pylori

(Saunders et al., 1997, Taylor et al., 1994). The genome sizes of H. pylori, H. mustelae and H.

hepaticus have also been determined with the PFGE technique and range between 1.3 to 1.8

Mb. Just recently, the whole genome of one H. pylori strain has been sequenced (Tomb et al.,

1997). The genome sizes of their closest relatives, Campylobacter spp. varies also between

1.1 and 1.7 Mb (Chang and Taylor, 1990).

31

3. PURPOSES OF THE STUDY

The main aim of this thesis was to investigate and clarify the taxonomic relationships of the

so-called "gastrospirilla" group within the genus Helicobacter. As mentioned earlier,

taxonomic studies of this genus as a whole is complicated due to two main reasons,

difficulties in culturing helicobacters in vitro and inertia in common biochemical tests,

resulting in difficulties in correct species identification. Furthermore, the comparison of

culturable and unculturable "gastrospirilla" by means of cell morphology and PCR-amplified

16S rDNA sequences has led to a situation where the nomenclature of these organisms has

been more or less arbitrary. These problems can only be solved with improved in vitro

culturing methods and applying a polyphasic approach for species delineation. Therefore, the

aims of this study were:

1. To develop and improve isolation methods for large, spiral gastric Helicobacter spp. (i.e.

"gastrospirilla") of dogs and cats

2. To establish a large reference collection of "gastrospirilla" and to characterise new isolates

3. To characterise and classify any novel "gastrospirilla"-like organisms

4. To apply a polyphasic approach to species delineation of H. felis and the isolated new

species

5. To study the genetic and geographical diversity of H. felis (a model for H. pylori

infection) with several typing methods.

6. To study the efficacy of RFLP analysis of the 23S rRNA gene for the differentiation

"gastrospirilla"-like organisms

32

4. MATERIALS AND METHODS

Katri Jalava performed all the studies in the Department of Food and Environmental Hygiene,

Faculty of Veterinary Medicine, except were stated. Manufacturers of the reagents etc. are

presented in respective papers (I-V).

4.1. Gastric biopsy samples of dogs and cats

During the period 1990-1997 we examined 95 canine and 22 feline gastric mucosal biopsies

as described in I, II, III. Shortly, biopsies were taken from 37 clinically healthy pet dogs, 23

patients with upper gastrointestinal signs (vomiting, nausea or abdominal discomfort), 18

euthanised pet dogs (health status unknown) and 17 healthy experimental dogs. Feline gastric

biopsies were taken from 22 cats, of which five were clinically healthy, two suffering from

upper gastrointestinal problems and 15 were euthanised pets (health status unknown). The

biopsy samples were taken from the corpus (body) area under light anaesthesia via endoscope

from the live animals or immediately post mortem from the euthanised pets. The animals for

endoscopic studies were withheld from food for 16 hours prior to examination

4.2. Primary microbial isolation

The handling of biopsy samples, transport media used and culture techniques are as described

in I, II, III. Briefly, the biopsy samples were either cultured directly or stored in transport

medium for 2-12 hours before culturing. The biopsies were either first crushed with BHI

(Brain Heart Infusion) broth and then spread onto agar plates or rubbed directly against the

agar plates. Both freshly-prepared BHI agar and Brucella agar, with cattle or horse blood and

Skirrow’s antibiotic supplement were used for isolation. Plates were incubated

microaerobilically and regularly checked for bacterial growth and a few drops of BHI broth

added to the plates during the incubation period to avoid drying of the media.

4.3. Primary identification of field isolates from Finnish dogs and cats

All strains were initially characterised by determining their type of growth on blood agar

plates and cellular morphology in gram stain, type of motility in wet preparations, reactions in

common biochemical tests, and whole-cell protein content as resolved by sodium dodecyl

33

sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE), and the profiles were prepared

and electrophoresed with a minigel system (running length 4 cm) and the patterns were

visually compared with those of type strains of relevant Helicobacter species as described in

I, II and III.

4.4. Extended phenotypic characterisation of cultured "gastrospirilla"

Thirty-four representative strains of H. felis (n=16, including three atypical H. felis strains),

H. bizzozeronii (n=10), H. salomonis (n=6) and ”F. rappini” (n=2) as listed in Table 3 were

characterised with 65 phenotypic tests listed in a probability matrix for identifying

campylobacters, helicobacters and related taxa as described in III. Dr. Stephen On performed

this study at the Danish Veterinary Laboratory, Copenhagen, Denmark.

4.5. Electron microscopy

4.5.1. Analysis of in vivo electron micrographs

The 52 biopsies used for in vivo electron micrograph analyses are listed in Table 3. The

biopsies were handled as described in I and III. A more detailed study of in vivo electron

microscopy will be presented elsewhere.

4.5.2. Ultrastructural analysis of cultured organisms

Electron microscopic examination of bacterial cell morphology was performed on 26 selected

isolates as listed in Table 3. The strains were handled as described in I, II and III. Shortly,

bacterial cells were resuspended in physiological NaCl and a turbid suspension was stained with

1% phosphotungstic acid and examined in a JEOL 1200 EX electron microscope.

4.6. 16S rDNA sequencing

Katri Jalava carried out this study in the Haartman Institute, Faculty of Medicine, University

of Helsinki, under the supervision of docent Matti Kaartinen.

34

4.6.1. Amplification of 16S rDNA by PCR

The 16S rDNA from selected canine helicobacter strains (H. bizzozeronii, CCUG 35545 T, H.

salomonis CCUG 37845 T and H. salomonis CCUG 37848) was amplified by PCR as described

in II. Briefly, the 16S rRNA gene sequence from genomic bacterial DNA was obtained by PCR

amplification with two primers. Biotinylated HY-2B primer (anneals to the positions 45-67 of E.

coli 16S rRNA) and A-2 primer (positions 1524-1541). The PCR conditions and purification of

the single stranded DNA from biotinylated PCR product were performed.

4.6.2. Sequencing

16S rDNA sequences were determined as described in II. Dr. Floyd Dewhirst performed the

sequencing of strain CCUG 37845T at the Forsyth Dental Centre, Boston, Massachusetts,

USA. In short, nucleotide sequences were determined using Sanger's dideoxy-method and

Sequenase 2.0R polymerase. For sequencing the partial 16S rRNA gene sequences directly from

PCR templates, the primer A-2 together with five additional primers were used. Ten ml of avidin

beads with attached single stranded PCR product were used as template in the

dideoxy-sequencing reactions.

4.6.3. Phylogenetic analysis

The phylogenetic analysis, similarity matrices and phylogenetic trees were constructed as

described in II. The strains examined in this study are listed in Table 3. Dr. Floyd Dewhirst

performed the final phylogenetic analysis at the Forsyth Dental Centre, Boston,

Massachusetts, USA. Shortly, the 16S rRNA gene sequences were entered into RNA, a program

for analysis of 16S rRNA data in Microsoft Quickbasic for use on IBM-PC compatible

computers, and aligned. The database contains approximately 100 Helicobacter, Wolinella,

Arcobacter and Campylobacter sequences. Similarity matrices were constructed from aligned

sequences by using only those base positions for which 90% of the strains had data, and were

corrected for multiple base changes. Phylogenetic trees were constructed by the

neighbour-joining method.

35

4.7. DNA-DNA hybridisations

4.7.1 DNA isolation

Whole-genome DNA for DNA-DNA hybridisation assays, ribotyping and 23S rRNA gene

polymorphism studies was performed as described in I and II. Briefly, DNA was isolated by

the method of Pitcher et al. (1989), with modifications designed to remove the RNA and

proteins. RNase and proteinase K treatments were performed after the dissolution to TE and the

DNA was reprecipitated as in the first phase.

4.7.2. Dot blot DNA-DNA hybridisations

Dot blot DNA-DNA hybridisations were performed as described in I, II and III. The probes used

were whole-genome DNA samples from H. bizzozeronii CCUG 35545T, H. salomonis Elviira,

H. felis CCUG 28539T and H. felis DS 3. The strains from which target DNA samples were

derived are listed in Table 3. In short, dot blot DNA-DNA hybridisations were performed by dot

blot assay on nylon membranes using a vacuum-blotting apparatus. Aliquots of 50 ng, 5 ng and

0.5 ng aliquots of diluted chromosomal DNA were added to the nylon membranes. The probes

were labelled by random priming with digoxigenin-dUTP. The membranes were incubated with

the probes overnight at 68ºC. The results were determined visually on the basis of the intensity of

alkaline phosphatase colour that was developed. The tests were repeated three times.

4.7.3. Optical DNA-DNA hybridisation

Optical DNA-DNA hybridisation experiments were performed as described in II. The strains

used are listed in Table 3 and hybridisations were performed on at least two occasions, except

where stated otherwise. In brief, the initial reassociation method of de Ley was applied with

reduced DNA concentration of 35 µg/ml. The DNA samples were adjusted to a concentration of

approximately 300 µg/ml. DNA was subsequently fragmented by sonication and dialysed

against 2x SSC overnight at 4oC. The samples were stored at -20oC before the measurement.

Hybridization was performed with a Lambda 11 spectrophotometer with a PTP 6 heating

element and Winlab and Templab software. The renaturation of the DNA was performed and

the optimal reassociation temperature (69oC) was used as recommended. The degree of binding

was calculated using the formula recommended by de Ley (1970). The hybridisations were

performed at least twice except where stated.

36

4.8. Numerical analysis of SDS-PAGE whole-cell protein profiles

The strains included in the highly standardised SDS-PAGE analysis are shown in Table 3.

The growth conditions, protein sample preparation, electrophoresis and numerical analysis

were as described in III. Dr Peter Vandamme performed the protein electrophoresis and

numerical analysis at the University of Ghent, Ghent, Belgium. Shortly, the strains were

grown on Mueller-Hinton agar with 5% horse blood at 37oC in a microaerobic atmosphere for

72 hours. Protein samples were prepared, separated by PAGE (running length 9 cm), digitised

and subjected to comparative numerical analysis. Numerical analysis of the protein profiles

was performed by using the GelCompar system version 4.0. The profiles were recorded and

stored on a PC computer. The similarity between all pairs of traces was expressed by the

Pearson product moment correlation coefficient converted for convenience to a percent value.

4.9. Molecular typing of H. felis

The genetic variation of H. felis strains isolated in several parts of the world (Sydney,

Australia; Bologna, Italy; Ohio, USA and Helsinki; Finland) was studied with PFGE, plasmid

profiling and ribotyping (see Table 3 for strain selection for each analysis). Dr. Cora de

Ungria, Dr. Jani O’Rourke and Prof. Adrian Lee performed the plasmid profiling at the

University of New South Wales, Sydney, Australia.

4.9.1. In situ DNA isolation and pulsed-field gel electrophoresis (PFGE)

In situ DNA isolation, digestion of the samples, electrophoretic conditions and genome size

determination was performed as described in IV. Briefly, the cells were harvested in 0.9%

PBS To inhibit DNases the cells were suspended in PBS containing 3.5 to 4.0%

formaldehyde solution for approximately 1 h. After three washes with PBS, the cells were

suspended in PIV adjusted to an approximate concentration of 1.0 x 109 as determined by

hemocytometer and were mixed with an equal volume of 2% low melting point agarose. The

plugs were lysed overnight in lysis solution at 37°C. Further lysis was performed by treating

the plugs with ESP for two 2 h periods and one overnight period at 50°C. Inactivation of

proteinase K was performed by two 1 h incubations at 37°C in 0.1 phenylmethylsulfonyl

fluoride in TE buffer. The plugs containing H. felis DNA were washed three times with TE

buffer and 1 mm cut slices were digested with 11 rare-cutting restriction enzymes (ApaI, AscI,

AvrII, KspI, NotI, NruI, PacI, RsrII, SfiI, SpeI, and XbaI) according to the manufactures

37

instructions. Samples were electrophoresed in a 1% (weight/vol) agarose gel in 0.5x TBE at

9°C and 200 V for 14 to 22 hours with different sets of pulse time. The diversity of the strains

digested with SpeI, ApaI, and AscI and the enzymes SpeI and NotI were used for the

determination of the genome size of H. felis. Low-Range, Mid-Range I, Lambda Ladder, and

Yeast Chromosome PFG markers (New England Biolabs) were used as molecular

weight/standard markers. The gels were stained with ethidium bromide in 0.5x TBE. The

genome size was determined by calculating the size of the fragments in relation to molecular

size markers. All DNA extractions and digestions were repeated at least twice.

4.9.2. Plasmid DNA extraction

Plasmid DNA was extracted from H. felis strains as described in IV by Dr. Cora de Ungria at

the University of New South Wales, Sydney, Australia. Shortly, plasmid DNA was extracted

from H. felis using the Wizard mini-prep kit. Profiles were obtained by electrophoresis of the

plasmid DNA on 0.7% agarose gels run in TAE at 4oC for 2 hours. Lambda HindIII EcoRI

marker and a supercoiled marker were used as the molecular size standards.

4.9.3. 16 + 23S ribotyping

Ribotyping was performed as described in IV. Shortly, a cDNA probe was prepared from E.

coli 16+23S rRNA by reverse transcription. The probe was labelled with digoxigenin. DNA

from selected bacterial strains (Table 3) was isolated as stated above. Four restriction

endonucleases, HaeIII, PvuII, ClaI and PstI were tested for cleaving 3 µg DNA of selected

strains. The digested samples were run in a 0.8% agarose gel overnight (25 V). DNA was

transferred from gels to membranes, fixed with UV irradiation. Hybridisations were

performed using procedures as described in IV.

4.10. 23S rRNA gene polymorphism analysis

The strains used for the analysis of 23S rRNA gene polymorphism are listed in Table 3. The

methods employed for these experiments are described in V. Shortly, bacterial DNA was

isolated as stated above. The PCR reaction conditions and primers were as outlined earlier

(Hurtado and Owen, 1997). The PCR products were analysed electrophoretically, stained with

ethidium bromide and visualised with UV light. Initially, PCR products of all strains were

digested with four restriction endonucleases HaeIII, HpaII, HhaI and HinfI The fragments

38

were run electrophoretically in 3 mm thick 2.5 % MetaPhor agarose at 170 V (6V/cm) at 14oC

with circulating 1 x TBE (Tris Borate EDTA) buffer for 3 hours. A standard molecular

marker was used to estimate the fragment sizes. Analysis of the fragments and calculation of

their respective fragment sizes was performed using the GelCompar computer system version

4.0 program.

39

5. RESULTS

5.1. Primary isolation (I, II, III)

Fifty-one gastric Helicobacter isolates were obtained from the 95 canine and 22 feline biopsy

samples examined. Forty-eight of these were of canine origin; three were isolated from cats.

Three of the dog strains were lost during subcultivation and thus could not be examined in

detail. The isolation rates from the canine and feline biopsies (as indexed to urease-positive

biopsy samples) were 51% (48/95) and 14% (3/22), respectively. Primary bacterial growth

appeared after 3-12 days of incubation as a thin spreading film on Brucella or BHI blood agar

plates. No single colonies were produced and the primary area of growth was often very

small. However, if air-dried plates were used, colonies could be observed although the

bacterial growth was poor or scanty. The isolation rate stayed approximately the same

throughout the study period (I, II, III).

Four different species were isolated from the samples, as shown below. Two new species,

H. bizzozeronii (I) and H. salomonis (II) were described and are presented here with all other

validly described Helicobacter spp.(Table 2). Several strains identified as H. felis were also

isolated (III, Table 3). Two isolates were defined as ”F. rappini”-like organisms, and three

mixed cultures were obtained (III).

Of the taxa encountered in this study, H. felis proved the easiest to isolate. Relatively

profuse growth was obtained after 4-7 days of incubation and strains were readily

subculturable. Similarly, H. salomonis also grew comparatively quickly and profusely on the

primary plates, although examination of gram-stained bacterial films indicated that cells

transformed rapidly to coccoid forms and maintenance by subculture proved difficult. Most

H. bizzozeronii isolates grew slowly, with primary growth observed within 4-12 days of

incubation. Subculture of H. felis, H. bizzozeronii and H. salomonis was expedited by the

addition of a few drops of BHI broth to plate cultures. All commercial microaerobic

atmospheres tested (gas generating kit model BR 56 with palladium catalyst, and gas

generating kit model BR 38 without catalyst [both Oxoid]), as well as evacuation/gas

replacement systems supported growth of these bacteria when the aforementioned

subculturing procedures were employed. Additional hydrogen was not essential in order to

obtain growth (I, II, III)

40

Helicobacter spp. Year Original source Country Originally described by

H. pylori 1982 Human gastric mucosa Australia Warren and Marshall, 1983

H. cinaedi 1985 Human intestine USA Totten et al., 1985

H. fennelliae 1985 Human intestine USA Totten et al., 1985

H. mustelae 1988 Ferret gastric mucosa USA Fox et al., 1988

H. nemestrinae 1991 Macaque gastric mucosa USA Bronsdon et al., 1991

H. felis 1988 Feline and canine gastric mucosa Australia Paster et al., 1991

H. muridarum 1992 Rodent intestines Australia Lee et al., 1992

H. acinonychis 1993 Cheetah gastric mucosa USA Eaton et al., 1993

H. canis 1993 Canine faeces Switzerland Stanley et al., 1993

H. pametensis 1994 Wild bird and pig faeces USA Dewhirst et al., 1994

H. pullorum 1994 Poultry and human faeces Switzerland Stanley et al., 1994

H. hepaticus 1994 Mice liver and intestines USA Fox et al., 1994

H. bilis 1995 Mice liver and intestines USA Fox et al., 1995

H. trogontum 1996 Rat colon mucosa Brazil Mendes et al., 1996

H. cholecystus 1996 Hamster gallbladder USA Franklin et al., 1996

H. bizzozeronii (a) 1996 Canine gastric mucosa Finland Hänninen et al., 1996

H. salomonis (a) 1997 Canine gastric mucosa Finland Jalava et al., 1997

H. rodentium 1997 Rat intestine USA Shen et al., 1997

described in the present study

Table 2. Validly described Helicobacter spp. as of December 1998

Four mixed cultures with two different Helicobacter spp., and one mixed culture

containing Helicobacter sp. and Campylobacter sp. were obtained. Two of these mixed

cultures were recognised by examination of gram-stained slide preparations and were

subsequently purified. The first purified Helicobacter strain was contaminated with a

Campylobacter-like organism and the pure culture (H. bizzozeronii, strain 13) was obtained

after several subcultures on plates containing 100 IU of polymyxin B per ml (Burnens and

Nicolet, 1993). The second mixed culture contained ”F. rappini” and H. bizzozeronii strains.

As ”F. rappini” strains grow more quickly, the pure ”F. rappini” strain (strain 19) was

obtained by carefully taking bacterial growth from the edge of the culture and subculturing

onto fresh media. After these procedures, strains 13 and 19 were handled as pure cultures in

the present study. The remaining three mixed cultures were revealed by the comparison of the

protein pattern of the early culture with that of the later culture and were not successfully

separated into pure cultures of the respective taxa (strain Jutta, H. bizzozeronii/H. felis; strain

Tuohimetsa, H. bizzozeronii/H. felis; and strain Loko 18, H. bizzozeronii/H. salomonis).

41

Table 3. The strains isolated in this study from Finnish dogs and cats, reference strains and reference sequences used for phylogenetical analysis

Species Designationa Origina,b Source Analysis performed Sequence accession number PFGE c,d,e,f,g,h,i,j Other notes type

Helicobacter acinonychis LMG 12684T, CCUG 29263T, CCUG Cheetah gastric mucosa c,e,h M88148H. bilis ATCC 51630 T GenBank Murine liver h U18766H. bizzozeronii CCUG 35545T, Storkis This study Canine gastric mucosa c,c*,d,e,f,h,k Y09404 H. bizzozeronii CCUG 35546, 14 This study Canine gastric mucosa c,c*,d,e,f,kH. bizzozeronii Wiberg This study Canine gastric mucosa c,d,e,gH. bizzozeronii 5F This study Canine gastric mucosa c,d,e,f,kH. bizzozeronii 10 This study Canine gastric mucosa c,d,e,fH. bizzozeronii 12A This study Canine gastric mucosa c,c*,d,e,kH. bizzozeronii Heydar This study Canine gastric mucosa c,d,e,gH. bizzozeronii Yrjala This study Canine gastric mucosa c,d,e,f,kH. bizzozeronii Emo This study Canine gastric mucosa d,e,fH. bizzozeronii Loko 21 This study Canine gastric mucosa c,d,eH. bizzozeronii 11 AM This study Canine gastric mucosa d,eH. bizzozeronii Moppi This study Canine gastric mucosa e,gH. bizzozeronii Pinky This study Canine gastric mucosa e,gH. bizzozeronii Renny This study Canine gastric mucosa e,g,kH. bizzozeronii Bertta This study Canine gastric mucosa e,gH. bizzozeronii Roope This study Canine gastric mucosa e,kH. bizzozeronii Nummijarvi This study Canine gastric mucosa c,gH. bizzozeronii 9F This study Canine gastric mucosa c,fH. bizzozeronii 13 This study Canine gastric mucosa cH. bizzozeronii Loko 20 This study Canine gastric mucosa c,f,gH. bizzozeronii Rinne This study Canine gastric mucosa c,gH. canis CCUG 12739 T GenBank Canine faeces h L13464H. cinaedi ATCC 35683 T GenBank Human faeces h M88150H. felis CS 1, CCUG 28539T CCUG Feline gastric mucosa c,c*,e,f,i,j,k M57398 1H. felis CS 2 O’Rourke Feline gastric mucosa i,j 2H. felis CS 5 O’Rourke Feline gastric mucosa d,e,i,j 1H. felis CS 6 O’Rourke Feline gastric mucosa d,e,i,j 3H. felis CS 7 O’Rourke Feline gastric mucosa d,e,i,j 4H. felis CS 8 O’Rourke Feline gastric mucosa d,e,i,j 3H. felis DS 1 O’Rourke Canine gastric mucosa d,e,i,j,k 5

42

H. felis DS 2/c O’Rourke Canine gastric mucosa i,j 6H. felis DS 3 O’Rourke Canine gastric mucosa c,c*,d,e,i,j M37643 7H. felis DS 4, CCUG 28540 CCUG Canine gastric mucosa c,c*,d,e,f.i,j 7H. felis DS 5 O’Rourke Canine gastric mucosa d,e,i,j 8H. felis Dog 1, 1602 Eaton Canine gastric mucosa d,e,f,h,i,j U51870 9bH. felis Dog 2, 2301 Eaton Canine gastric mucosa d,e,h,i,k U51871 18H. felis Dog 3, Dog 7 Eaton Canine gastric mucosa c,c*,d,e,f,h,i U51872 19H. felis Into This study Canine gastric mucosa c,d,e,f,i,j,k 9aH. felis Hellu This study Canine gastric mucosa c,d,e,i 10H. felis Kukka This study Canine gastric mucosa e,gH. felis Teppo This study Canine gastric mucosa eH. felis Loko 14 This study Canine gastric mucosa c,gH. felis Solanti This study Canine gastric mucosa c,i 13H. felis Tellu This study Canine gastric mucosa cH. felis Vilma This study Canine gastric mucosa c,i 11H. felis Cintti This study Feline gastric mucosa c,d,e,g,I,k 12H. felis Loki 13 This study Feline gastric mucosa c,f,g,I,k 15H. felis Fievel This study Feline gastric mucosa c,f,g,i 14H. felis 15/144 Cattoli Canine gastric mucosa i,j 16H. felis 15/390 Cattoli Canine gastric mucosa i,j 16H. felis 16/937 Cattoli Canine gastric mucosa i 17H. felis/H. bizzozeronii Tuohimetsa This study Canine gastric mucosa c,d,e,f,g mixed cultureH. felis/H. bizzozeronii Jutta This study Canine gastric mucosa c,f,g mixed cultureH. fennelliae ATCC 35684 T GenBank Human faeces h M88154H. hepaticus ATCC 51448 T GenBank murine liver h U07574H. salomonis InkinenT, CCUG 37845T This study Canine gastric mucosa c,c*,d,e,f,g,h,k Y89351H. salomonis 06A, CCUG 37848 This study Canine gastric mucosa c,e,f,h,k Y09405H. salomonis Alma This study Canine gastric mucosa c,fH. salomonis Elviira This study Canine gastric mucosa c,c*,fH. salomonis Vilho This study Canine gastric mucosa c,d,e,f,kH. salomonis Mini This study Canine gastric mucosa c,c*,d,e,f,gH. salomonis Ko.K. III This study Canine gastric mucosa c,d,e,f,kH. salomonis Remu This study Canine gastric mucosa c,d,e,f,g,kH. salomonis Mikkola This study Canine gastric mucosa kH. salomonis/bizzozeronii Loko 18 This study Canine gastric mucosa c,d,e,f,g mixed cultureH. mustelae CCUG 25715T CCUG Ferret gastric mucosa c,e,h M35048

43

H. muridarum CCUG 29262T, ATCC 49282T CCUG Murine intestinal mucosa e,h M80205H. nemestrinae CCUG 32350T, ATCC 49396T CCUG Macaque gastric mucosa e,h X67854 H. pametensis CCUG 29255 T GenBank Tern faeces h M88147H. pullorum NCTC 12824 T GenBank Broiler chicken caecum h L36141H. trogontum ATCC 700114 T GenBank Rat colon mucosa h U65103H. pylori CCUG 17874T, ATCC 43504T CCUG Human gastric mucosa e,h,k M88157”Flexispira rappini” CCUG 23435, ATCC 43879 ATCC Human faeces e”F. rappini” ATCC 43966 ATCC Aborted sheep fetes h”F. rappini” Hilli This study Canine gastric mucosa c,d,e,f”F. rappini” 19 This study Canine gastric mucosa c,d,e,f”Gastrospirillum hominis”1 unculturable GenBank Human gastric mucosa h L10079, unculturable”G. hominis”2 unculturable GenBank Human gastric mucosa h L10080, unculturablea ATCC, American Type Culture Collection, Rockville, MD.; Cattoli, G. Cattoli, University of Bologna, Bologna, Italy; CCUG, Culture Collection, Department of ClinicalBacteriology, University of Göteborg, Göteborg, Sweden; Eaton, K. A. Eaton, Department of Veterinary Pathobiology, Ohio State University, Columbus; LMG, CultureCollection, Laboratorium voor Microbiologie, University of Ghent, Ghent, Belgium; O'Rourke, J. L. O'Rourke, School of Microbiology and Immunology, University of NewSouth Wales, Sydney, Australia.b Own isolate unless indicated otherwise.c Strain used in dot blot DNA-DNA hybridisation studyc* Strain used in quantitative DNA-DNA hybridisation studyd Strain used in phenotypic characterisatione Strain used in highly standardised protein profile analysisf Strain used in electron micrographic analysisg In vivo electron micrographic analysis of the biopsy performedh 16S rDNA sequence used for phylogenetical analysisi Strain used for PFGE and ribotypingj Strain used for plasmid profilingk Strain used for 23S rRNA gene polymorphism study

44

5. 2. Phenotypic analysis (I, II, III)

5.2.1. Primary identification of Finnish field isolates (I, II, III)

All primary gastric helicobacter isolates were urease positive when first tested. Light

microscopic examination of gram-stained bacterial films revealed that isolates belonged

to one of three morphological categories, corresponding to the taxa H. felis and H.

bizzozeronii (tightly helical cells), H. salomonis (plump, less tightly coiled cells) and ”F.

rappini” (straight, cigar-shaped cells with tapering ends). Furthermore, two forms of

motility were observed; a rapid, screw-like motion proved typical for H. felis, H.

bizzozeronii, and ”F. rappini” (with the unique cellular morphology of the latter clearly

distinct), whilst the movement of H. salomonis strains was relatively slow and wavy. The

species distribution of canine isolates is shown in III, Table 2, p. 4001. Three strains were

isolated from cats (2 from healthy pets and one from an euthanised pet); these were all

shown to be H. felis by the minigel SDS-PAGE system. All strains isolated in this study

were characterised with standard phenotypic tests, which revealed only minor differences

between the taxa. H. salomonis strains differed from H. felis and H. bizzozeronii by their

ability to reduce triphenyl-tetrazolium chloride (TTC) and their inability to grow at

temperatures higher than 40°C. H. bizzozeronii strains differed from H. felis only by their

ability to hydrolyse indoxyl acetate, with H. salomonis strains also positive in this test (I,

II). The visual examination of SDS-PAGE protein profiles with the mini-gel system

clearly differentiated H. felis, H. bizzozeronii and ”F. rappini” (I, II, III). The

differentiation of H. salomonis from H. bizzozeronii was more difficult with this method

(II, III)

5.2.2. Extended phenotypic characterisation (III)

Considerable difficulties in culturing bacteria for phenotypic testing were encountered

with the methods used; consequently, two strains (strain 10, H. bizzozeronii and strain

CCUG 37848, H. salomonis) could not be tested. Table 3 (III) summarises the results of

those tests with discriminatory value in species differentiation. Tests clearly

distinguishing H. bizzozeronii, H. felis and H. salomonis were not identified in the scheme

applied, although certain traits (distinctions in cell morphology, growth at 37 oC on

unsupplemented blood agar and resistance to 5-flurouracil [100 mg/l]) provided a broad

distinction between H. bizzozeronii/felis and H. salomonis. "Flexispira rappini"-like

45

strains were readily distinguished from the other taxa in being tolerant to 1.0%, 1.5% and

2.0% bile, with other traits (elongated loosely-spiral cell morphology, alkaline

phosphatase production, reduction and growth of triphenyl-tetrazolium chloride medium,

growth on potato starch medium) providing some additional discrimination from the other

Helicobacter species isolated (III, Table 3, p. 4003). In contrast with results obtained from

freshly isolated strains (I, II) no urease activity was detected in four strains ("F. rappini"-

like Hilli; H. bizzozeronii 11AM, Loko 21 and 12A), despite repeated examination and

overnight incubation in the urease test medium (III). Furthermore some of the results of

the phenotypic analyses (III) were contradictory to results obtained previously (I, II).

5.3. ELECTRON MICROSCOPY (I, II, III)

5.3.1. In vivo electron micrographs (I, III)

Relatively few bacterial cells were observed in electron micrographs prepared from tissue

samples. Furthermore, these results did not always correlate with corresponding

ultrastructural examinations of cultured bacteria. Moreover, only two types of organisms

could be seen; either tightly coiled organisms without the periplasmic fibrils, resembling

H. bizzozeronii or atypical H. felis; or similar bacteria with the periplasmic fibrils, thus

resembling typical H. felis strains. Thus, organisms resembling H. salomonis or ”F.

rappini” organisms were not detected. In seven animals (13%) where organisms

resembling H. felis were seen, no yield in culture was obtained. Furthermore, in two

animals H. felis cells were seen in tissue samples, yet H. bizzozeronii growth was

obtained. H. felis-like cells were observed only in 12 samples (23%), constituting only a

small proportion of spiral organisms, most of which resembled H. bizzozeronii. In 36

animals (64%) only H. bizzozeronii-like organisms were seen. Conversely, although H.

bizzozeronii-like strains were observed as the sole gastric colonisers in these animals,

typical H. felis strains were isolated from four (7%) of these samples, of which two were

from cats.

5.3.2. Ultrastructure of cultured strains (I, II, III)

Typical examples of the ultrastructural cell morphology of each species, and of the

atypical H. felis strains, are reproduced in Figure 2 (and in III, Figure 1, p. 4002). Typical

H. felis cells were corkscrew-like, possessing 1-2 periplasmic fibrils, whilst H.

46

bizzozeronii strains were similar but slightly more helical and lacking periplasmic fibrils.

H. salomonis was thicker and only slightly curved, whilst the ”F. rappini”-like organisms

were cigar-shaped with tapering ends and a remarkable net-like arrangement of fibrils on

the cell surface. As expected, the atypical H. felis strains closely resembled H.

bizzozeronii.

Figure 2. Electron micrographs of canine and feline gastric helicobacters. (A) H. felis(CCUG 28539T) Note the periplasmic fibrils around the cell body. This is the only speciesthat has been cultured in vitro from cats. (B) H. bizzozeronii (CCUG 35545T). (C) H.salomonis (06A). (D) ”F. rappini” (Hilli). Note the net-like surface and cigar-shape ofthe cell body. Magnification 7.000x.

5.4. 16S rDNA sequencing (II)

Approximately 95% of the 16S rDNA sequence was determined for the Helicobacter strains

examined by this method. Only those base positions (1404) for which 90% of the strains

possessed data were included in the calculation of similarity. A phylogenetic tree based on

this analysis is shown in Figure 3 (and in II, Figure 3, p. 980). This analysis showed that H.

felis, H. bizzozeronii, H. salomonis and “G. hominis”2 were highly related and formed a

47

distinct cluster within rRNA homology group III (i.e. the Helicobacter phylogenetic branch)

of rRNA superfamily VI.

Figure 3. Phylogenetic tree for 20 strains of Helicobacter species on the basis of 16S rDNAsequence similarity. The scale bar represents a 4% difference in nucleotide sequence asdetermined by measuring the length of horizontal lines connecting any two species.

5.5. DNA-DNA hybridisation (I, II, III)

Dot blot DNA-DNA hybridisation assays performed at 74oC revealed that the H. felis, H.

bizzozeronii and H. salomonis probes all reacted strongly when tested against other strains of

their respective species (I, II, III). With quantitative DNA -DNA hybridisation experiments,

all species gave inter-strain DNA reassociation values exceeding 65% (II, Table 4, p. 980)

and between-species values below 40%. In particular, inter-strain hybridisation values of H.

salomonis strains exceeded 90%, while comparable results obtained between the type strain

of H. felis and an atypical H. felis strain were 66% (II, Table 4, p. 980).

48

5.6. Numerical analysis of protein profiles (III)

The whole-cell protein patterns of H. felis, H. bizzozeronii and H. salomonis strains

isolated in the present study were compared with those of reference strains of other gastric

helicobacters (see for strain selection in Table 3). A dendrogramme illustrating the results

of the numerical comparison of the whole-cell protein patterns of these strains is shown in

Figure 4 (and in III, Figure 2, p. 4003), and comprises four major clusters at the 77%

similarity level. Clusters I and II comprise H. bizzozeronii and H. salomonis respectively,

grouping above a similarity level of 79.8% and 84.8%, respectively. Cluster III comprises

the Australian reference strains of H. felis (Lee et al., 1988; Paster et al., 1991), our own

isolates, and the atypical H. felis strains without the periplasmic fibrils described by Eaton

et al. (1996). Cluster IV comprises the two isolates morphologically resembling ”F.

rappini”. The similarity level between the whole-cell protein patterns of the latter isolates

is 91.6% and this cluster is linked to the ”F. rappini” reference strain LMG 8738 at a

similarity level of 77.9%. The type strains of H. acinonychis, H. mustelae, H.

nemestrinae, and H. pylori each occupy distinct positions in the dendrogramme.

Representative whole-cell protein patterns of the different gastric helicobacter isolates

examined are shown in III, Figure 3, p. 4004.

The whole-cell protein patterns of the H. bizzozeronii strains are fairly homogeneous

with some variability primarily localised in the high molecular weight region (molecular

weight > 60,000). The majority of the H. felis strains have very similar whole-cell protein

patterns (III, Figure 3, p. 4004). However, strains lacking the periplasmic fibrils are

characterised by the absence of two prominent bands (estimated molecular weight, ca.

54,000 and 80,000 Da) which are present in all other strains (III, Figure 3, p. 4004). Two

additional strains have slightly aberrant patterns, characterised by an additional low

molecular weight protein band of approximately 33,000 (Into), or by a slightly different

size of the prominent protein band with an approximate molecular weight of 80,000 (DS

3). The patterns of the H. salomonis strains are very homogeneous and are typically

characterised by an unusual prominent high molecular weight protein band (approximate

size: 95,000, in III, Figure 3, p. 4004). Finally, the whole-cell protein patterns of the two

”F. rappini”-like strains are similar to, but clearly different from that of the ”F. rappini”

reference strain (LMG 8738) included in the analysis.

Two subcultures of the mixed culture Loko 18 were included in the analysis. As

illustrated in Fig. 4, the 1995 subculture (listed as Loko 18-95) was identified as H.

49

bizzozeronii; while the 1996 subculture (listed as Loko 18-96) was identified as H.

salomonis. The subculture of strain Tuohimetsa included in the present analysis was

identified as H. felis. It seems logical that the initial mixed cultures which were dominated

by the slow-growing H. bizzozeronii strains, were later dominated (after a considerable

number of subcultivations in vitro) by the faster growing H. salomonis and H. felis in the

mixed populations.

Figure 4. Dendrogramme derived from the numerical analysis of the whole-cell proteinpatterns of representatives of the strains examined. Strains marked Loko 18-95, and Loko18-96, represent the 1995 and 1996 subcultures of strain Loko 18. H. felis strains markedby an asterisk lack the periplasmic fibrils. Roman numbers I through IV refer to clusterdesignations discussed in the text.

50

5.7. 23S rRNA gene polymorphism (V)

The 23S rRNA-gene based PCR identification scheme described by Hurtado and Owen

(1997) and evaluated in this study did not produce species-specific patterns for H. felis, H.

bizzozeronii and H. salomonis. These species could be differentiated, as a group, from

other Helicobacter spp. using PCR-RFLP profiles derived from HaeIII, HpaII HhaI and

HinfI analyses. However, species-specific PCR-RFLP profiles with these enzymes were

not obtained. Furthermore, strains of a given species did not yield identical profiles. With

the aforementioned enzymes, variation was observed between the profiles of individual

strains although one profile type predominated a given species (V).

5.8. Molecular subtyping of H. felis (IV)

5.8.1. PFGE typing and genome size determination

Most H. felis strains were distinguished from each other with PFGE by the use of

restriction enzymes NotI, SpeI, ApaI and AscI. However, the patterns of the two Italian

strains, 15/144 and 15/390 were indistinguishable with all enzymes tested, as were the

type strain of H. felis (CS1) compared to CS5; DS3 compared to DS4; and CS6 compared

to CS8. One or two single band shifts were seen in DS3 and DS4. No association to host

species or country of origin was seen among the profiles. One of the Finnish isolates with

periplasmic fibrils (Into) showed great similarity to an American isolate without the

periplasmic fibrils (1602). None of these strains originated from related animals and they

were isolated at different time points (data not shown). The different PFGE types are

listed in Table 3 (and in IV, Table 1). Representatives of SpeI digestion patterns are

presented in Figure 5. All in situ DNA extractions produced reproducible patterns when

experiments were repeated on different occasions.

The genome size of H. felis was found to be approximately 1.6 Mb as calculated from

the fragment sizes of bands produced with NotI and SpeI-based PFGE analyses of five

strains in IV, Table 2. This estimate is the mean value from the five strains examined.

51

Figure 5. SpeI macrorestriction patterns of 21 H. felis isolates. Fragments are between9.4 and 146 kb in size. A low range PFGE marker is included, with molecular sizes (topto bottom) of 146, 97, 48.5, 23.1 and 9.4 kb.

5.8.2. Plasmid profiling

All strains showed multiple plasmid bands with sizes ranging from 2 to >16kb with no

common plasmid detected amongst the strains (IV, Figures 3A and 3B). In agreement with

the results obtained from the PFGE studies, similar plasmid profiles were seen in the

following pairs of strains; CS1 and CS5, CS6 and CS8, DS3 and DS4, 15/144 and 15/390. In

addition, the plasmid profiles of Into and 1602 were similar except for the presence of two

additional plasmid bands (estimated sizes 3 kb and 5.1 kb) in isolate Into (IV, Figure 3B).

5.8.3. 16 + 23S ribotyping

ClaI and HaeIII proved to be the most suitable enzymes for ribotyping H. felis with the 16

+ 23S rDNA probe. The level of species diversity demonstrated was almost as high as

observed using PFGE analysis. A few common bands were seen in some strains, but

neither species specific bands, nor bands with an association to host species or

geographical region could be detected among the strains. HaeIII ribotypes are shown in

IV, Figure 5. The pairs of strains giving similar PFGE profiles produced identical

ribotypes with all enzymes tested, including the two morphologically different strains,

Into and 1602.

52

6. DISCUSSION

Previous studies have demonstrated the presence of Helicobacter-like organisms in the

gastric mucosa of virtually all adult cats and dogs (Otto et al., 1994; Hermann et al., 1995;

Eaton et al., 1996; Happonen et al., 1996b; Neiger et al., 1998). We demonstrated that a

variety of different Helicobacter species can be cultured from gastric biopsy samples of

animals, particularly dogs, provided adequate isolation procedures are used. Two new

species, namely H. bizzozeronii (I) and H. salomonis (II) were isolated and described in

the present study. The overall isolation rate of Helicobacter-like spp. from canine and

feline gastric specimen was 51% and 14%, respectively (III), which exceeds those

reported previously (Eaton et al., 1996, Neiger et al., 1998). We were also successful in

isolating a diverse range of taxa (I, II, III), in contrast with other studies (Haziroglu et al.,

1995; Cattoli et al., 1996; Eaton et al., 1996).

Clearly, the method used for sampling and primary isolation is critical. Fresh biopsy

samples were essential, since no growth was obtained with samples stored for more than

12 hours or frozen at -70°C (unpublished observations). In addition, the prerequisites in

obtaining bacterial growth were the long incubation period, high atmospheric humidity,

and use of moist, freshly prepared media, each of which contributed to the high level of

contaminants encountered. Fasting the animals prior to biopsy was found to be important

in order to decrease the number of contaminating organisms, particularly since

helicobacter growth was often first seen on the plate in a relatively small area, making

subculture difficult. In addition, contamination of the primary plates of the cat biopsies

was more severe than comparable investigations of canine samples. This may help to

explain the high proportion of culture failures in cats. However, H. pylori is often

distributed unevenly within the human gastric mucosa (Bayerdorffer et al., 1989) and it is

conceivable that some of the culture failures encountered here were also due to similar

phenomena, since only one biopsy sample was used for culture. Nonetheless, no

substantial methodological differences between the present investigation and those of

other workers were evident, although it is surmised that careful handling of samples, and

a close attention to methodological detail played an essential role in the results obtained

here.

53

Although the methods used in this study for canine biopsies yielded a considerably

higher isolation rate (44% overall) compared with other studies (10%) (Eaton et al.,

1996), 56% of biopsies in which helicobacters were observed failed to give positive

results for culture. Only three strains were isolated from cats and all of these were proved

to be H. felis strains. It has been clearly observed in early morphological studies

comparing canine and feline "gastrospirilla" that the majority of feline mucosal samples

show a more tightly coiled, thinner and longer organism than those in comparable dog

samples (Weber et al., 1958; Weber and Schmittdiel, 1962). It may be that these tightly

coiled organisms lacking periplasmic fibrils in cats represent a different species from H.

bizzozeronii, and our method is not adequate for isolating it.

The identification of strains to species level was challenging in the present study. In

primary identification tests, ”F. rappini”-like isolates were readily distinguished from the

other taxa encountered by virtue of their distinctive morphology, which was evident from

both light and electron microscopy. Furthermore, H. salomonis isolates could be

presumptively identified by their less helical cell morphology and unusually slow and

sporadic motility (in contrast with H. bizzozeronii and H. felis), although the visual

examination of protein patterns using the minigel system proved a less useful means of

identification. The unequivocal differentiation of H. bizzozeronii from H. felis was

especially problematic. No clear differences were noted in primary identification tests, yet

the visual examination of SDS-PAGE mini gels separated H. felis with periplasmic fibrils

from H. bizzozeronii and H. salomonis. However, the description of atypical H. felis

strains lacking cellular periplasmic fibrils (Eaton et al., 1996) invalidated this

characteristic as an unequivocal means of distinguishing H. felis and H. bizzozeronii,

although all Finnish H. felis isolates proved typical in that respect. Whilst useful,

ultrastructural differences could not therefore be relied upon as a wholly accurate means

of speciation. Furthermore, the complex nature of the technique is not easily applied to

routine use. Moreover, the differences noted here between the cell morphology of the

bacteria observed in vivo and those obtained in vitro may suggest that mixed populations

of different taxa are much more common than the available data allow us to conclude.

However, it is equally possible that the cellular morphology of some species is different

on artificial media and in the host environment as has been described for H. felis (Lee et

al., 1988) and was noted with H. salomonis (II).

54

Even with the extended phenotypic identification scheme of 65 tests, it proved

impossible to clearly differentiate the closely related species H. bizzozeronii, H. felis and

H. salomonis, although some useful traits for broadly distinguishing the latter species

from the former two taxa were noted (III, Table 3, p. 4003). Interestingly, urease was not

detected in one "F. rappini"-like strain and three H. bizzozeronii isolates, suggesting a

spontaneous loss of enzyme activity. This phenomenon has been described before for H.

pylori (McNulty et al., 1984) and H. mustelae (Costas et al., 1991).

The prospect of strain misidentification due to mixed cultures is evident from the

examples described in this study, although this problem is not easily solved especially as

discrete colonies are not observed in primary cultures of the "gastrospirilla" species

studied here. This must be taken into account when examining suspect helicobacter

cultures from a canine or feline gastric biopsy. Multiple species may coexist in the gastric

tract of domestic pets (I, II, III, Lee et al., 1988; Happonen et al., 1996b) and the bacterial

growth of many of these helicobacters have a tendency to swarm. This is strikingly

illustrated by the differences in morphology between the organisms seen in in vivo

electron micrographs (III) and that of the isolated cultures (II,III). Furthermore, no tests

based on inhibitory substance tolerance were found in the present study to facilitate

differentiation between Helicobacter spp. studied, and thus no recommendations for

species-selective media can be made at present. Logically, repeated subcultivation of

these mixed cultures and transfer of the strains after a relatively short incubation period

will favour growth of the less fastidious strains.

16S rDNA sequences for strains representing the species H. felis, H. bizzozeronii, and H.

salomonis were 98.2%-100% similar to one another, clearly indicating a close phylogenetic

relationship and revealing that these organisms belong to the genus Helicobacter. H.

salomonis CCUG 37845T differed from H. bizzozeronii, H. felis and "G. hominis”2 only by

1.0%, 0.8% and 1.4% sequence difference, respectively. Furthermore, the two sequenced H.

salomonis strains differed by 1.7% from each other. Recently, Eaton et al. (1996) described

three H. felis strains without periplasmic fibrils. The Dog 3 strain of these authors showed a

sequence difference of 0.7% and 0.6% compared to those derived from H. felis strains CS1T

and H. felis DS3, respectively. Because of this high degree of sequence similarity between

these culturable and unculturable Helicobacter species and sequence variability within the

species, 16S rRNA gene sequence information cannot be used to establish species

boundaries (Majewski et al., 1988). Therefore, DNA-DNA hybridisation was necessary to

delimit species boundaries for the strains in this cluster.

55

The dot-blot DNA-DNA hybridisation method used in the present study was found to be

a good screening method for the selection of strains for quantitative DNA-DNA

reassociation analyses. DNA homology of representative strains from each group designated

H. felis, H. bizzozeronii and H. salomonis were confirmed using quantitative DNA-DNA

reassociation to represent distinct species. Moreover, the atypical H. felis strain (Dog 3) was

confirmed to belong to this species as this strain showed levels of DNA relatedness of 66%

with H. felis CS1T. This strain displayed hybridisation values of only 12% and 26% with H.

bizzozeronii CCUG 35545T and H. salomonis CCUG 37845T, respectively, confirming that it

belongs to neither of these species. Isolation of the DNA for the quantitative DNA-DNA

hybridisation studies was extremely laborious.

The efficacy of highly standardised whole-cell protein analysis for identifying

helicobacters and related organisms is well established (Costas et al., 1993, On, 1996,

Vandamme et al., 1991) and was confirmed in the present study. All H. felis, H.

bizzozeronii, and H. salomonis strains identified by quantitative- or dot-blot DNA-DNA

hybridisation methods to the relevant species (I, II) formed discrete clusters after

numerical analysis of protein patterns. H. salomonis, H. bizzozeronii, and H. felis were all

readily recognisable. The three American H. felis strains lacking periplasmic fibrils

clustered among the other H. felis strains, although two prominent protein bands were

absent. The whole-cell protein analysis confirmed the identification of these strains as H.

felis (Eaton et al., 1996, Lee et al., 1988), but emphasises their aberrant nature.

The development of non-cultural methods for direct detection of bacteria from the

biopsy samples, such as species-specific PCR tests would also be of benefit. Such

methods were employed by Neiger et al. to infer that 78% of cats harboured ”H.

heilmannii”, and none H. felis (Neiger et al., 1998). The applicability of genes such as the

urease genes used by Neiger et al. for the differentiation of gastric helicobacters has not

been studied in detail and therefore the sensitivity and specificity of such approaches is

unknown at present. Thus, it is possible that the ”H. heilmannii” strains detected by

Neiger et al. (1998) represent misidentified strains of H. felis, H. bizzozeronii or H.

salomonis. As shown in the present study, 16S rDNA sequence similarities between H.

felis, H. bizzozeronii, H. salomonis and ”H. heilmannii” is extremely high (II);

consequently, this widely used gene does not seem suitable as a target for species-specific

PCR. Furthermore, the scheme based on 23S rRNA gene polymorphism developed by

Hurtado and Owen (1997) for Helicobacter spp. could not be used for species

56

identification of the phylogenetically highly related species H. felis, H. bizzozeronii and

H. salomonis (see V). These species were, however, differentiated as a group from the

other culturable Helicobacter spp.

The zoonotic potential of gastric helicobacters has been the subject of considerable

interest, especially since the isolation of H. pylori from cats (Handt et al., 1994). Although

subsequent studies have indicated that cats do not represent a significant reservoir for

human H. pylori infection (Eaton et al., 1996; El-Zataari et al., 1997), the issue

concerning ”H. heilmannii” is far less certain. Several reports suggest ”H. heilmannii” as

a possible zoonosis (McNulty et al., 1989; Solnick et al., 1993). However, the latter

organism is ultrastructurally indistinguishable from H. bizzozeronii and the atypical H.

felis strains of Eaton et al. (1996) in vivo and all these taxa show a complex relationship

by 16S rDNA sequence analysis (II; Eaton et al., 1996). Clearly, it is crucial to accurately

determine the taxonomic position of ”H. heilmannii” to properly evaluate its zoonotic

potential, but such studies have been hindered by the failure of most workers to culture

the organism (McNulty et al., 1989; Solnick et al., 1993). It is feasible that the methods

described in this report for the culture of canine and feline helicobacters could be applied

to isolate human ”H. heilmannii” strains, facilitating the necessary investigations. In this

respect, it is encouraging to note that one such strain has been described (Andersen et al.,

1996; Andersen et al., 1999) but a detailed polyphasic study is necessary to ascertain the

relationship of this human isolate to H. bizzozeronii, H. felis, H. salomonis, "H. heilmannii",

"G. hominis"1, "G. hominis"2, "G. suis" and "G. lemur".

The genetic diversity of different H. felis strains isolated from cats and dogs and

originating from three different continents (Sydney, Australia; Columbus, Ohio, USA;

Bologna, Italy; Helsinki, Finland) was very high. This was demonstrated by each of the

three techniques employed: PFGE-, and plasmid profiling, and ribotyping. Both PFGE-

and plasmid profiling are suitable methods for small-scale epidemiological studies, whilst

ribotyping was not able to discriminate all the isolates. The molecular patterns of strains

passaged through animals should be examined to evaluate the genomic stability of H.

felis. Furthermore, isolates from the same colony of animals or known relatives should be

examined in order to resolve the routes of transmission of the infection. We believe that

both PFGE technique and plasmid profiling will be of value for typing H. felis and other

Helicobacter spp.

57

7. CONCLUSIONS

Isolation of canine and feline "gastrospirilla" is very demanding, yet possible, as was

shown in the present study. We have described and successfully used an isolation method

to detect two new Helicobacter spp., H. bizzozeronii and H. salomonis, from the gastric

biopsies of dogs and cats. In addition, we have isolated two strains that resemble ”F.

rappini” in morphology, although the taxonomic position of these ”Flexispira"-like

organisms requires further investigation. Whilst the methods described here for isolation

of these bacteria represent a considerable advance over those used previously, further

improvements in culture conditions for these bacteria are still required, especially when

examining cats.

The culture and characterisation of H. bizzozeronii and H. salomonis have extended

knowledge of the ecology, prevalence, and culture characteristics of "gastrospirilla". The

methods employed in this study can be applied to determine the clinical significance of

gastric Helicobacter spp. in dogs and cats with gastrointestinal disease, as well as for

animal model studies of H. pylori. Furthermore, potential virulence genes can be studied

from the culturable gastric organisms.

Successfully cultured and taxonomically identified species are needed and can be used

for the development of new selective culture media for the differentiation of H. felis, H.

bizzozeronii and H. salomonis. Such studies may resolve the problem of mixed cultures of

these species obtained from gastric biopsies. Similarly, organisms growing in culture can

be used for the development of species-specific PCR-based methods of direct detection of

these species in gastric biopsy samples.

Results of the 16S rRNA gene sequence comparisons between culturable and

unculturable Helicobacter spp. within the "gastrospirilla" group reveal highly similar

sequences for H. felis, H. bizzozeronii, H. salomonis, "G. hominis"1 and 2 and "G. suis".

Therefore, the true relationship between these "strains" remains unknown. It is evident

that this widely used gene cannot be used as a species identification tool for these

bacteria. Its application to the taxonomy of these bacteria has been confusing and the true

genetic relationship between cultured and uncultured animal and human "gastrospirilla"

remains to be solved by more discriminative methods such as DNA-DNA hybridisation.

58

Similarly, the inertia of "gastrospirilla" in commonly used biochemical tests, and

those comprising an extended (65 tests) phenotypic identification scheme, indicate that a

polyphasic approach must be used for definition of these species. Certain biochemical

features, detailed morphological determination by light- and electron microscopy,

standardised SDS-PAGE protein profile analysis and DNA-DNA hybridisation should be

used.

The widely accepted "gold standard" for delimiting species boundaries is DNA-DNA

hybridisation. This technique must be applied in taxonomic studies of strains in this group of

culturable Helicobacter spp. Dot-blot DNA-DNA hybridisation was found to be a good

screening method that could be used for the selection of strains for quantitative DNA-DNA

reassociation studies. For routine purposes we strongly recommend the use of highly

standardised SDS-PAGE protein profile analysis for identification of culturable

"gastrospirilla", since the results obtained were in excellent agreement with both the dot-

blot, and quantitative DNA-DNA hybridisation analyses.

The genetic diversity of different H. felis strains isolated from cats and dogs and

originating from three different continents was very high and comparable to that observed

in its close taxonomic relative, H. pylori. The genome size of H. felis was determined to

be 1.6 Mb.

Available data strongly indicate the need for further work concerning the specificity

and application of PCR assays for detecting the various Helicobacter spp. known to

inhabit the gastric mucosa of domestic pets and humans.

59

8. REFERENCES

Alpert, L.C., Graham, D.Y., Evans, D.J., Yoshimura, H.H., Hazell, S.L., Evans, D.G. andKlein, P.D. (1989) Diagnostic possibilities for Campylobacter pylori infection. Eur. J.Gastroenterol. Hepatol. 1, 17-26.

Andersen, L.P., Norgaard A., Holck, S., Blom, J. and Elsborg, L. (1996) Isolation of"Helicobacter heilmannii" from human tissue. Eur. J. Clin. Microbiol. Infect. Dis. 15, 95-96. (Letter.)

Andersen, L.P., Boye, K., Blom, J., Holck, S., Norgaard A. and Elsborg, L. (1999)Characterization of a culturable "Gastrospirillum hominis" (Helicobacter heilmannii)strain isolated from human gastric mucosa. J. Clin. Microbiol. 37, 1069-1076.

Anonymous. (1994) Schistosomes, liver flukes and Helicobacter pylori. IARC Monogr.Eval. Carcinog. Risks Hum. 61, 1-241.

Ball, V. and Roquet, M. (1911) Spirochètes et affections hémorragiques gastro-intestinales du chien. Journal de Médecine vétérinaire et de zootechnie. 14, 257-260.

Bayerdorffer, E., Oertel, H., Lehn, N., Kasper, G., Mannes, G.A., Sauerbruch, T. andStolte, M. (1989) Topographic association between active gastritis and Campylobacterpylori colonisation. J. Clin. Pathol. 42, 834-839.

Bizzozero, G. (1893) Ueber die schlauchförmigen Drüsen des Magendarmkanals und dieBeziehungen ihres Epitheles zu dem oberflächenepithel der Schleimhaut. Archiv füfMicroskopiche Anatomie Entwickungs mechanik. 42, 82-152., Tafel VII

Breed, R.S., Murray, E.G.D. and Hitchens, A.P. (eds.) (1948) Bergey's manual ofdeterminative bacteriology. 6th Edition. The Williams & Wilkins company, 217-218.

Breed, R.S., Murray, E.G.D. and Hitchens, A.P. (eds.) (1957) Bergey's manual ofdeterminative bacteriology. 6th Edition. The Williams & Wilkins company.

Brenner, D.J., Fanning, G.R., Johnson, K.E., Citarella, R.V. and Falkow, S. (1969)Polynucleotide sequence relationships among members of Enterobacteriacae. J. Bact. 98,637-650.

Bronsdon, M.A., Goodwin, C.S., Sly, L.I., Chilvers, T. and Schoenknecht, F.D. (1991)Helicobacter nemestrinae sp. nov., a spiral bacterium found in the stomach of a pigtailedmacaque (Macaca nemestrina). Int. J. Syst. Bacteriol. 41, 148-153.

Buck, G.E. and Smith, J.S. (1987) Medium supplementation for growth of Helicobacterpyloridis. J. Clin. Microbiol. 25, 597-599.

Burnens, A.P. and Nicolet, J. (1993) Three supplementary diagnostic tests forCampylobacter species and related organisms. J. Clin. Microbiol. 31, 708-710.

60

Cattoli, G., Zanoni, R., Benazzi, C., Della Salda, L., Serraino, A. and Sanguinetti, V.(1996) Isolation of Helicobacter felis from dogs in Italy, p. 341-343. In Newell D. et al.(eds.), Proceedings of Campylobacters, Helicobacters and related organisms. PlenumPress, New York.

Chang, N. and Taylor, D.E. (1990) Use of pulsed-field agarose gel electrophoresis to sizegenomes of Campylobacter species and to construct a SalI map of Campylobacter jejuniUA 580. J. Bacteriol. 172, 5211-5217.

Chu, G., Vollrath, D. and Davis, R.V. (1986) Separation of large DNA molecules bycontour-clamped homogenous electric fields. Science, 234, 1582-1585.

Colwell, R.R. (1970) Polyphasic taxonomy of the genus Vibrio: numerical taxonomy ofVibrio cholerae, Vibrio parahaemolyticus, and related Vibrio species. J. Bacteriol. 104,410-433.

Costas, M., On, S.L.W., Owen, R.J., Lopez-Urquijo, B. and Lastovica, A.J. (1993)Differentiation of Helicobacter species by numerical analysis of their one-dimensionalelectrophoretic protein patterns. System. Appl. Microbiol. 16, 396-404.

Costas, M., Owen, R.J., Morgan, D.D. and Goodwin, C.S. (1991) Loss of urease activityin Helicobacter mustelae. Lett. Appl. Microbiol. 14, 260-264.

Crosa, J.H., Brenner, D.J. and Falkow, S. (1973) Use of a single-strand specific nucleasefor analysis of bacterial and plasmid deoxyribonucleic acid homo- and hetero-duplexes. J.Bact. 115, 904-911.

Curry, A., Jones, D.M. and Eldridge, J. (1987) Spiral organisms in the baboon stomach.Lancet ii, 634-635 (letter).

De Ley, J., Cattois, H. and Reynaerts, A. (1970) The quantitative measurements of DNAhybridisation from renaturation rates. Eur. J. Biochem. 12, 133-142.

Debongie, J.C., Donnay, M. and Mairesse, J. (1995) Gastrospirillum hominis(”Helicobacter heilmannii”): a cause of gastritis, somewhat transient, better diagnosed bytouch cytology? Am. J. Gastroenterol. 90, 411-416.

Dent, J.C., McNulty, C.A.M., Uff, J.C., Wilkinson, S.P. and Gear, M.W.L. (1987) Spiralorganisms in the gastric antrum. Lancet i, 96.

Dewhirst, F.E., Paster, B.J., Fraser, G.J. and Olsen, I. (1992) Phylogeny of 9 major groupsof eubacteria based on 16S rRNA sequence comparisons, p. 6 and separate handout. InAbstracts on the Conference on Taxonomy and Automatic Identification of Bacteria.Czechoslovak Society For Microbiology, Prague, Czech Republic.

Dewhirst, F.E., Seymor, C., Fraser, G.J., Paster, B.J., and Fox, J.G.. (1994) Phylogeny ofHelicobacter isoaltes from bird and swine feces and description of Helicobacterpametensis sp. nov. Int. J. Syst. Bacteriol. 44, 553-560.

61

Dieterich, C., Wiesel, P., Neiger, R., Blum, A. and Corthésy-Theulaz, I. (1998) Presenseof multiple ”Helicobacter heilmannii” strains in an individual suffering from ulcers andhis two cats. J. Clin. Microbiol. 36, 1366-1370.

Doenges, J.L. (1939) Spirochetes in the gastric glands of macacus rhesus and of manwithout related disease. Arch. Pathol. 25, 469-477.

Duboscq, O. and Lebailly, C. (1912) Spirella canis, n. g., n. sp., spirille de l'estomac duchien. Compt. Rend. Acad. Sci. 154, 835-837.

Dunn, B.E., Cohen, H. and Blaser, M.J. (1997) H. pylori. Clin. Microbiol. Rev. 10, 720-741.

Eaton, K.A., Radin, M.J., Kramer, L., Wack, R., Sherding, R., Krakowka, S. and Morgan,D.R. (1991) Gastric spiral bacilli in captive cheetahs. Scand. J. Gastroenterol. 26(suppl.181), 38-42.

Eaton, K.A., Dewhirst, F.E., Radin, M.J., Fox, J.G., Paster, B.J., Krakowka, S. andMorgan, D.R. (1993) Helicobacter acinonyx sp. nov., isolated from cheetahs withgastritis. Int. J. Syst. Bacteriol. 43, 99-106.

Eaton, K.A., Dewhirst, F.E., Paster, B.J., Tzellas, N., Coleman, B.E., Paola, J. andSherding, R. (1996) Prevalence and varieties of Helicobacter species in dogs fromrandom sources and pet dogs: Animal and public health implications. J. Clin. Microbiol.34, 3165-3170.

Edkins, J. S. (1920) Spirella regaudi in the cats. Parasitology 15, 296-307, plates VIII-X.

El-Zaatari, F.A.,Woo, J.S., Badr, A., Osato, M.S., Serna, H., Lichtenberger, L.M., Genta,R.M. and Graham, D.Y. (1997) Failure to isolate Helicobacter pylori from stray catsindicates that H. pylori in cats may be an anthroponosis - an animal infection with ahuman pathogen. J. Med. Microbiol. 46, 372-376.

Fawcett, P.T., Gibney, K.M. and Vinette, K.M.B. (1999) Helicobacter pylori can beinduced to assume the morphology of Helicobacter heilmannnii. J. Clin. Microbiol. 37,1045-1048.

Foley, J.E., Solnick, J.V., Lapointe, J.-M., Jang, S. and Pedersen, N.C. (1998) Isolation ofa novel enteric Helicobacter species in a kitten with severe diarrhea. J. Clin. Microbiol.36, 908-912

Fox, G.E., Wisotzkey, J.D. and Jurtshuk, P. Jr. (1992) How close is close:16S rRNAsequence identity may not be sufficient to guarantee species identity. Int. J. Syst.Bacteriol. 42, 166-170.

Fox, J.G., Taylor, N.S., Edmonds, P. and Brenner, D.J. (1988) Campylobacter pylorisubsp. mustelae subsp. nov. isolated from the gastric mucosa of ferrets (Mustela putoriusfuro), and an emended description of Campylobacter pylori. Int. J. Syst. Bacteriol. 38,367-370.

62

Fox, J.G., Yan, L.L., Dewhirst, F.E., Paster, B.J., Shames, B., Murphy, J.C., Hayward, A.,Belcher, J.C. and Mendes, E.N. (1995) Helicobacter bilis sp. nov., a novel Helicobacterspecies isolated from bile, livers, and intestines of aged, inbred mice. J. Clin. Microbiol.33, 445-454.

Fox, J.G., Dewhirst, F.E., Tully, J.G., Paster, B.J., Yan, L., Taylor, N.S., Collins, M.J.,Gorelick, p.R. and Ward, J.M. (1994) Helicobacter hepaticus sp. nov., a microaerophilicbacterium isolated from livers and intestinal mucosal scrapings from mice. J. Clin.Microbiol. 32, 1238-1245.

Franklin, C.L., Beckwith, C.S., Livingston, R.S., Riley, L.K., Gibson, S.V., Besch-Williford, C.L. and Hook, R.R. (1996) Isolation of a novel Helicobacter species,Helicobacter cholecystus sp. nov., from the gall bladders of syrian hamsters withcholangiofibrosis and centrilobular pancreatitis. J. Clin. Microbiol. 34, 2952-2958.

Gautom, R.K. (1997) Rapid pulsed-field gel electrophoresis protocol for typing ofEscherichia coli O157:H7 and other gram negative organisms in one day. J. Clin.Microbiol. 35, 2977-2980.

Gillespie, D. and Spiegelman, S. (1965) A quantitative assay for DNA-DNA hybrids withDNA immobilized on a membrane. J. Mol. Biol. 12, 829-842.

Goodwin, C.S., Blinkow, E.D., Warren, J.R., Waters, T.E., Sanderson, C.R. and Easton,L. (1985) Evaluation of cultural techniques for isolating Campylobacter pyloridis fromendoscopic biopsies of gastric mucosa. J. Clin. Pathol. 38, 1127-1131.

Goodwin, C.S. and Armstrong, J.A. (1990) Microbiological aspects of Helicobacterpylori (Campylobacter pylori). Eur. J. Clin. Microbiol. Infect. Dis. 9, 1-13.

Goodwin, C.S., Armstrong, J.A., Chilvers, T., Peters, M., Collins, M.D., Sly, L.,McDonnell, W. and Sharper, W.E.S. (1989) Transfer of Campylobacter pylori andCampylobacter mustelae to Helicobacter gen. nov. as Helicobacter pylori comb. nov. andHelicobacter mustelae comb. nov., respectively. Int. J. Syst. Bacteriol. 39, 397-405.

Goto, K., Osashi, H., Ebukuro, S., Itoh, K., Tohma, Y., Takakura, A., Wakana, S., Ito, M.and Itoh, T. (1998) Isolation and characterization of Helicobacter species from thestomach of the house musk shrew (Suncus murinus) with chronic gastritis. Curr.Microbiol. 37, 44-51.

Grimont, P.A.D., Popoff, M.Y., Grimont, F., Coynalt, C. and Lemelin, M. (1980)Reproducibility and correlation study of three deoxyribonucleic acid hybridisationprocedures. Curr. Microbiol. 4, 325-330.

Grimont, F., and Grimont, P.A.D. (1991) DNA fingerprinting, p. 249-280. InStackebrandt, E. and Goodfellow, M. (eds.), Nucleic acid techniques in bacterialsystematics: Modern microbiological methods. John Wiley & Sons, Chichester.

Gurtler, V., Wilson, V.A. and Mayall, B.C. (1991) Classification of medically importantclostridia using restriction endonuclease site differences of PCR-amplified 16S rDNA. J.Gen. Microbiol., 137, 2673-2679.

63

Han, S.W., Flamm, R., Hachem, C.Y., Kim, H.Y., Clarridge, J.E., Evans, D.G., Beyer, J.,Drnec, J. and Graham, D.Y. (1995) Transport and storage of Helicobacter pylori fromgastric mucosal biopsies and clinical isolates. Eur. J. Clin. Microbiol. Inf. Dis. 14, 349-352.

Handt, L.K., Fox, J.G., Dewhirst, F.E., Fraser, G.J., Paster, B.J., Yan, L.L., Rozimiarek,H., Rufo, R. and Stalis, I.H.. (1994) Helicobacter pylori isolated from the domestic cat:public health implications. Inf. Immunol. 62, 2367-2374.

Happonen, I., Saari, S., Castren, L., Tyni, O., Hänninen, M.-L. and Westermarck, E.(1996a) Comparison of diagnostic methods for detecting gastric Helicobacter-likeorganisms in dogs and cats. J. Comp. Pathol. 115, 117-127.

Happonen, I., Saari, S., Castrén, L., Tyni, O., Hänninen, M.-L. and Westermarck, E.(1996b) Occurence and topographical mapping of Helicobacter-like organisms and theirassociation with histological changes in apparently healthy dogs and cats. J. Vet. Med.Assoc. 43, 305-315.

Hartford, T. and Sneath, P.H.A. (1993) Optical DNA-DNA homology in the GenusListeria. Int. J. Syst. Bacteriol. 43, 26-31.

Haziroglu, R., Diker, S., Guvenc, T. and Kul, O. (1995) Canine gastritis associated withHelicobacter felis. Dtsch. Tierärztl. Wschr. 102, 474-476.

Heilmann, K.L. and Borchard, F. (1991) Gastritis due to spiral shaped bacteria other thanH. pylori: clinical, histological, and ultrastructural findings. Gut. 32, 137-140.

Henry, G.A., Burns, J.L. and Charbonneau, D.L. (1987) Gastric spirillosis in beagles. 31,831-836.

Hermanns, W., Kregel, K., Breuer, W. and Lechner, J. (1995) Helicobacter-likeorganisms: Histopathological examination of gastric biopsies from dogs and cats. J.Comp. Pathol. 112, 307-318.

Holck, S., Ingeholm, P., Blom, J., Nørgaard, A., Elsborg, L., Adamsen, S. and Andersen,L.P. (1997) The histopathology of human gastric mucosa inhabitated by Helicobacterheilmannii-like (Gastrospirillum hominis) organisms, including the first culturable case.APMIS 105, 746-756

Hurtado, A. and Owen, R.J. (1997) A rapid identification scheme for H. pylori and otherspecies of Helicobacter based on 23S rRNA gene polymorphism. System. Appl.Microbiol. 20, 222-231.

Huss, V.A.R., Festl, H. and Schleifer, K.H. (1983) Studies on the spectrophotometricdetermination of DNA hybridisation from renaturation rates. Syst. Appl. Microbiol. 4,184-192.

Hussmann, M., Gries, C., Jechnichen, p., Woelfel, T., Gerken, G., Ludwig, W. andBhakdi, S. (1994) Helicobacter sp. strain Mainz isolated from an AIDS patient with septic

64

arthritis: case report and non-radioactive analysis of 16S rRNA sequence. J. Clin.Microbiol. 32, 3037-3039.

Jahnke, K.-D. (1994) A modified method of quantitative colorimetric DNA-DNAhybridisation on membrane filters for bacterial identification. J. Microbiol. Methods 273-288.

Jakob, W., Stolte, M., Valentin, A. and Schröder, H.-D. (1997) Demonstration ofHelicobacter pylori-like organisms in the gastric mucosa of captive exotic carnivores. J.Comp. Pathol., 116, 21-33.

Jayaro, B.M., Doré, J.J.E., Baumbach, G.A., Matthews, K.R. and Oliver, S.P. (1991)Differentiation of Streptococcus uberis from Streptococcus parauberis by polymerasechain reaction and restriction length polymorphism analysis of 16S ribosomal DNA. J.Clin. Microbiol. 29, 2774-2778.

Kasai, K. and Kobayashi, R. (1919). The stomach spirochete occurring in mammals. J.Parasitol. 6, 1-11.

Kersters K. (1985) Numercal methods in the classification of bacteria by proteinelectrophoresis. In Computer Assisted Bacterial Systematics (M. Goodfellow, D. Jonesand F.G. Priest, eds.), Academic Press, London, UK, pp. 337-368.

Kirkbride, C.A., Gates, C.E., Collins, J.E. and Ritchie, A.E. (1985) Ovine abortionassociated with an anaerobic bacterium. J. Am. Vet. Med. Assoc. 186, 789-791.

Kjoller, M., Fischer, A. and Justensen T. (1991) Transport conditions and number ofbiopsies necessary for culture of Helicobacter pylori. Eur. J. Clin. Microbiol. Inf. Dis. 10,166-167.

Kolmer, W. and Wagner, R.J. (1916). Ueber eine im Magenfundus des Hundesvorkommende saprophytiche Spirochäte. Zbl. Bakt. Parasitenkd. Infektionskr. Hyg. Abt.1 78, 383-384.

Kong, L., Smith, J.G., Bramhill, D., Abruzzo, G.K., Bonfiglio, C., Cioffe, C., Flattery,A.M., Gill, C.J., Lynch, L., Scott, P.M., Silver, L., Thompson, C., Kropp, H. and Bartizal,K. (1996) A sensitive and specific PCR method to detect Helicobacter felis in aconventional mouse model. Clin. Diagn. Lab. Immunol. 3, 73-78.

Krieg, N.R. and Holt, J.G. (eds.) (1984) Bergey's manual of systematic bacteriology. TheWilliams & Wilkins company, Baltimore, MD .

Laine, L., Chung, D., Stein, C., El-Beblawi, I., Sharma, V. and Chandrasoma P. (1996)The influence of size or number of biopsies on rapid urease test results: a prospectiveevaluation. Gastrointest. Endosc. 43, 49-53.

Langenberg, W., Rauws, E.A.J., Widjojokusumo, A., Tytgat, G.N.J. and Zanen, H.C.(1986) Identification of Campylobacter pyloridis isolates by restriction endonucleaseDNA analysis. J. Clin. Microbiol. 24, 414-417.

65

Lee, A., Hazell, S.L., O`Rourke, J. and Kouprach, S. (1988) Isolation of a spiral-shapedbacterium from the cat stomach. Infect. Immunol. 56, 2843-2850.

Lee, A., Phillips, M.W., O’Rourke, J.L., Paster, B.J., Dewhirst, F.E., Fraser, G.J., Fox,J.G., Sly, L.I., Romaniuk, P.J., Trust, T.J. and Kouprach, S. (1992) Helicobactermuridarum sp. nov., a microaerophilic helical bacterium with a novel ultrastructureisolated from the intestinal mucosa of rodents. Int. J. Syst. Bacteriol. 42, 27-36.

Lim, R.K.S. (1920) A parasitic spiral organism in the stomach of the cat. Parasitology 12,108-112, plate VII.

Lockard V.G. and Boler, R.K. (1970) Ultrastructure of a spiral microorganism in thegastric mucosa of dogs. Am. J. Vet. Res. 31, 1453-1462.

Lucet, M. (1910) Sur la presence de Spirochètes dans un cas de gastro-entèritehémorragique chez le chien. Compt. Rend. Acad. Sci. 151, 260-262.

Madinier, I.M., Fosse, T.M. and Monteil, R.A. (1997) Oral carriage of Helicobacterpylori: a review. J. Periontodol. 68, 2-6.

Majewski, S.I.H. and Goodwin, C.S. (1988) Restriction endonuclease analysis of thegenome of Campylobacter pylori with a rapid extraction method: evidence forconsiderable genomic variation. J. Infect. Dis. 157, 465-471.

Mapstone, N.P., Lewis, F.A., Tompkins, D.S., Lynch, D.A.F., Axon, A.T.R., Dixon, M.F.and Quirke, P. (1993) PCR identification of Helicobacter pylori in faeces from gastritispatients. Lancet 341, 447. (letter).

Maslow, J.N., Mulligan, M.E. and Arbeit, R.D. (1993a) Molecular epidemiology:application of contemporary techniques to the typing of microorganisms. Clin. Infect. Dis.17, 153-164.

Maslow, J.N., Slutsky, A.M. and Arbeit, R.D. (1993b) Application of pulsed-fieldelectrophoresis to molecular epidemiology, p. 563-572. In Persing,,D.H., Smith, T.F.,Tenover, F.C. and White, T.J. (eds.). Diagnostic molecular microbiology, principles andapplications. ASM press, Washington, DC, pp. 563-572.

Matushek, M.G., Bonten, M.J.M and Hayden, M.K. (1996) Rapid preparation of bacterialDNA for pulsed-field gel electrophoresis. J. Clin. Microbiol. 34, 2598-2600.

McCarthy, B.J., Bolton, E.T. (1963) An approach to the measurement of geneticrelatedness among organisms. Proc. Nat. Acad. Sci. 50, 156-164.

McNulty, C.A.M., Dent, J.C., Curry, A., Uff, J.S., Ford, G.A., Gear, M.W.L. andWilkinson, S.P. (1989) New spiral bacterium in the gastric mucosa. J. Clin. Pathol. 42,585-591.

Mendes, E.N., Queiroz, D.M.M., Rocha, G.A., Moura, S.B., Leite, V.H.R. and Fonseca,M.E.F. (1990b) Ultrastructure of a spiral micro-organism from pig gastric mucosa(Gastrospirillum suis). J. Med. Microbiol. 39, 64-68.

66

Mendes, E.N., Queiroz, D.M., Dewhirst, F.E., Paster, B.J., Carvalho, S.D., Magnago,A.G.P., Moura, S.B., Rocha, G.A. and Oliveira, A.M.R. (1996) Reservoir hosts for human”Gastrospirillum” infection. VIIIth International Workshop on gastroduodenal pathologyand Helicobacter pylori. Edinburgh, Scotland 7.-9.7.1995. Abstract 104.

Mendes, E.N., Queiroz, D.M., Dewhirst, F.E., Paster, B.J., Moura, S.B. and Fox, J.G.(1996) Helicobacter trogontum sp. nov., isolated from the rat intestine. Int. J. Syst.Bacteriol. 46, 916-921.

Monteiro, L., Hua, J., Birac, C., Lamouliatte, H. and Megraud F. (1997) Quantitativepolymerase chain reaction for the detection of Helicobacter pylori in gastric biopsyspecimens. Eur. J. Clin. Microbiol. Inf. Dis. 16, 143-149.

Murray, R.G.E. and Schleifer, K.M. (1994) Taxonomic notes: A proposal for recordingthe properties of putative taxa of procaryotes. Int. J. Syst. Bacteriol. 44, 174-176.

Neiger, R., Dieterich, C., Burnens, A., Waldvogel, A., Corthésy-Theulaz, I., Halter, F.,Lauterburg, B. and Schmassmann, A. (1998) Detection and prevalence of Helicobacterinfection in pet cats. J. Clin. Microbiol. 36, 634-637.

On, S.L.W. (1996) Identification methods for campylobacters, helicobacters, and relatedorganisms. Clin. Microbiol. Rev. 9, 405-422.

On, S.L.W. and Holmes, B. (1995) Classification and identification of campylobacters,helicobacters and allied taxa by numerical analysis of phenotypic tests. System. Appl.Microbiol. 18, 374-390.

On, S.L.W., Holmes, B. and Sackin, M.J. (1996) A probability matrix for theidentification of campylobacters, helicobacters and allied taxa. J. Appl. Bacteriol. 81,425-432.

O'Rourke, J., Solnick, J., Lee, A. and Tompkins, L. (1992) Helicobacter heilmannii(previously Gastrospirillum), a new species of Helicobacter in humans and animals. VWorkshop in gastroduodenal pathology and Helicobacter pylori. In Irish J. Med. Science161(suppl.10), 31.

Otto, G., Hazell, S.H., Fox, J.G., Howlett, C.R., Murphy, J.C., O'Rourke, J.L. and Lee, A.(1994) Animal and public health implications of gastric colonization of cats byHelicobacter-like organisms. J. Clin. Microbiol. 32, 1043-1049.

Owen, R.J. and Jackman, P.J.H. (1982) The similarities between Pseudomonaspaucimobilis and allied bacteria derived from analysis of deoxyribonucleic acid andelectrophoretic protein patterns. J. Gen. Microbiol. 128, 2945-2954.

Owen, R.J. and Pitcher, D. (1985) Current methods for estimating DNA base compositionand levels of DNA-DNA hybridisation. In Chemical Methods In Bacterial Systematics,Society for Applied Bacteriology, p. 67-93.

67

Owen, R.J., Hunton, C., Bickley, J., Moreno, M. and Linton, D. (1992) Ribosomal RNAgene restriction patterns of Helicobacter pylori: analysis and appraisal of Hae III digestsas a molecular typing system. Epidemiol. Infect. 109, 35-47.

Paster, B.J., Lee, A., Fox, J.G., Dewhirst, F.E., Tordoff, L.A., Fraser, G.J., O'Rourke,J.L., Taylor, N.S. and Ferrero, R. (1991) Phylogeny of Helicobacter felis sp. nov.,Helicobacter mustelae, and related bacteria. Int. J. Syst. Bacteriol. 41, 31-38.

Persing, D.H., Smith, T.F., Tenover, F.C. and White, T.J.(eds.) (1993) Diagnosticmolecular microbiology - principles and applications. 1. ed. American Society forMicrobiology, Washington, D.C. 641 p.

Pitcher, D.G., Saunders, N.A. and Owen, R.J. (1989). Rapid extraction of bacterialgenomic DNA with guanidium thiocyanate. Lett. Appl. Microbiol. 8, 151-156.

Queiroz, D.M.M., Rocha, G.A., Mendes, E.N., Lage, A.P., Carvalho, A.C.T. and Barbosa,A.J.A. (1990a) A spiral micro-organism in the stomach of pigs Vet. Microbiol. 24, 199-204.

Rappin (1881) Contribution à l'étude des bactéries de la bouche à l'état normal et dans lafièvre typhoïde. PhD thesis. Collège de France, Nantes.

Regaud, C.L. (1909a) Sur une curieuse localisation de spirilles parasites dans lescanalisations glandulaires de la muqueuse gastrique normale, chez le chien et le chat.Compt. Rend. Soc. Biol. 66, 229-231.

Regaud, C.L. (1909b) Sur les spirilles parasites des glandes gastriques du chien et du chat.Compt. Rend. Soc. Biol. 66, 617-618.

Römling, U., Heuer, T. and Tummler, B. (1994) Bacerial genome analysis by pulsed fieldgel electrophoresis techniques. In Advances in Electrophoresis Ed. Chambrach A. et al,VCH, Weilheim, vol. 7, 353-406.

Salomon, H. (1898) Über das Spirillum des Säugetiermagens und sein Verhalten zu denBelegzellen. Zbl. Bakt. Parasitenkd. Infektionskr. Hyg. Abt. 1 19, 433-442 and Taf. VIIIand Taf IX.

Sambrook, J., Fritsch, E.F and Maniatis, T. (1989) Molecular cloning, a laboratorymanual. Cold Spring Harbor Laboratory, Cold Spring Harbor.

Saunders, K.E., McGovern, K.J. and Fox, J.G. (1997) Use of pulsed-field gelelectrophoresis to determine genomic diversity in strains of Helicobacter hepaticus fromgeographically distant locations. J. Clin. Microbiol. 35, 2859-2863.

Swaminathan, B. and Matar, G.M. (1993) Molecular typing methods. In Persing,,D.H.,Smith, T.F., Tenover, F.C. and White, T.J. (eds.). Diagnostic molecular microbiology,principles and applications. ASM press, Washington, DC, pp. 26-50

68

Shen, Z., Fox, J. G., Dewhirst, D.E., Paster, B.J., Foltz, C.J., Yan, L., Shames, B. andPerry, L. (1997) Helicobacter rodentium sp. nov., a urease-negative Helicobacter speciesisolated from laboratory mice. Int. J. Syst. Bacteriol. 47, 627-634.

Simor, A.E., Shames, B., Drumm, B., Sherman, P., Low, D.E. and Penner, J.L. (1990)Typing of Campylobacter pylori by bacterial DNA restriction endonuclease analysis anddetermination of plasmid profile. J. Clin. Microbiol. 28, 83-86.

Solnick, J.V., Rourke, J.O., Lee, A. and Tompkins, L.S. (1992) Gastrospirillum is a newspecies of helicobacter in humans. Gastroenterology 102, A699.

Solnick, J.V., O'Rourke, J., Lee, A., Paster, B.J., Dewhirst, F.E. and Tompkins, L.S.(1993) An uncultured gastric spiral organism is a newly identified Helicobacter inhumans. J. Inf. Dis. 168, 379-385.

Solnick, J.V., O'Rourke, J., Lee, A. and Tompkins, L.S. (1994). Molecular analysis ofurease genes from a newly identified uncultured species of Helicobacter. Inf. Immun. 62,1631-1638.

Stackebrandt, E. and Goebel, B.M. (1994) Taxonomic note: A place for DNA-DNAreassociation ans 16S rRNA sequence analysis in the present species definition inbacteriology. Int. J. Syst. Bacteriol. 44, 846-849.

Stanley, J., Linton, D., Burnens, A.P., Dewhirst, F.E., Owen, R.J., Porter, A., On, S.L.W.and Costas, M. (1993) Helicobacter canis sp. nov., a new species from dogs: an integratedstudy of phenotype and genotype. J. Gen. Microbiol. 139, 2495-2504.

Stanley, J., Linton, D., Burnens, A.P., Dewhirst, F.E., On, S.L.W., Porter, A., Owen, R.J.and Costas, M. (1994) Helicobacter pullorum sp. nov., - genotype and phenotype of anew species isolated from poultry and human patients with gastroenteritis. Microbiology140, 3441-3449.

Sutton, W.D. (1971) A crude nuclease preparation suitable for use in DNA reassociationexperiments. Biochim. Biophys. Acta 240, 522-531.

Taylor, D.E., Chang, N., Taylor, N.S. and Fox, J.G. (1994) Genome conservation inHelicobacter mustelae as determined by pulse-field gel electrophoresis. FEMS Microbiol.Lett. 118, 31-36.

Taylor, D.E., Eaton, M., Chang, N. and Salama, S.M..(1992) Construction of aHelicobacter pylori genome map and demonstration of diversity at the genome level. J.Bacteriol. 174, 6800-6806.

Tee, W., Lambert, J., Smallwood, R., Schembri, M., Ross, B.C. and Dwyer, B.Ribotyping of Helicobacter pylori from clinical specimens. J. Clin. Microbiol. 30, 1562-1567.

Tenover, F.C., Arbeit, R.D., Goering, R.V., Mickelsen, P.A., Murray, B.E., Persing, D.H.and Swaminathan, b. (1995) Interpreting chromosomal DNA restriction patterns produced

69

by pulsed-field gel electrophoresis: Criteria for bacterial strains typing. J. Clin. Microbiol.33, 2233-2239.

Tenover, F.C., Arbeit, R.D. and Goering, R.V. (1997) How to select and interpretmolecular strain typing methods for epidemiological studies on bacterial infections: Areview for healthcare epidemiologists. Infect. Control. Hosp. Epidemiol. 18, 426-439.

Tomb, J.F., White, O., Kerlavage, A.R., Clayton, R.A., Sutton, G.G., Fleichmann, R.D.,Ketchum, K.A., Klenk, H.P., Gill, S., Dougherty, B.A., Nelson, K., Quackenbush, J.,Zhou, L., Kirkness, E.F., Peterson, S., Loftus, B., Richardson, D., Dodson, R., Khalak,H.G., Glodek, A., McKenney, K., Fizegerald, L.M., Lee, N., Adams, M.D., Venter, J.C.et. al. The complete genome sequence of the gastric pathogen H. pylori. Nature. 388, 539-547.

Totten, P.A., Fennell, C.L.,Tenover, F.C., Wezenberg, J.M., Perine, P.L., Stamm, W.E.and Holmes, K.K. (1985) Campylobacter cinaedi (sp. nov.) and Helicobacter fennelliae(sp. nov.): Two new campylobacter species associated with enteric disease in homosexualmen. J. Inf. Dis. 151, 131-139.

Trivett-Moore, N.L., Rawlinson, W.D., Yuen, M. and Gilbert, G.L. (1997) Helicobacterwestmaedii sp. nov., a new species isolated from blood cultures of two AIDS patients. J.Clin. Microbiol. 35, 1144-1150.

Ursing, J.B., Lior, H. and Owen, R.J. (1994) Proposal of minimal standards for describingnew species of the family Campylobacteraceae. Int. J. Syst. Bacteriol. 44, 842-845.

Ursing, J.B., Rosello-mora, R.A., Garcia-Valdes, E. and Lalucat J. (1995) Taxonomicnote: a pragmatic approach to the nomenclature of phenotypically similar genomicgroups. Int. J. Syst. Bacteriol. 45, 604.

Vandamme, P., Pot, B., Falsen, E., Kersters, K. and De Ley, J. (1990) Intra- andinterspecific relationships of veterinary campylobacters revealed by numerical analysis ofelectrophoretic protein profiles and DNA:DNA hybridisations. Syst. Appl. Microbiol. 14,295-303.

Vandamme, P., Falsen, E., Hoste, R., Segers, B., Tytgat, P. and DeLey, J. (1991) Revisionof Campylobacter, Helicobacter and Wolinella taxonomy: emendation of genericdescriptions and proposal of Arcobacter gen. nov. Int. J. Syst. Bacteriol. 41, 88-103.

Vandamme, P., Pot, B., Gillis, M., De Vos, P., Kersters, K. and Swings, J. (1996)Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol. Rev. 60,407-438.

Vaneechoutte, M. and Van Eldere, J. (1997) The possibilities and limitations of nucleicacid amplification technology in diagnostic microbiology. J. Med. Microbiol. 46, 188-194.

Wahlfors, J., Meurman, J.H., Toskala, J., Korhonen, A., Alakuijala, P., Janatuinen, E.,Kärkkäinen, U.M. and Nuutinen, P.J.P. (1995) Development of a rapid PCR method for

70

identification of Helicobacter pylori in dental plaque and gastric biopsy specimens. Eur. J.Clin. Microbiol. Inf. Dis. 14, 780-786.

Warren, B.J. and Marshall, J.R. (1983) Unidentified curved bacilli on gastric epitheliumin active chronic gastritis. Lancet. i, 1273-1275.

Wayne, L.G., Brenner, D.J., Colwel, 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. andTrüper, H.G. (1987) Report of the ad hoc committee on reconciliation of approaches tobacterial systematics. Int. J. Syst. Bacteriol. 37, 463-464.

Weber, A.F., Hasa, O. and Sautter, J.H. (1958) Some observations concerning thepresence of spirilla in the fundic glands of dogs and cats. Am. J. Vet. Res. 19, 677-680.

Weber, A.F. and Schmittdiel, E.F. (1962) Electron microscopic and bacteriologic studiesof spirilla isolated from the fundic stomachs of cats and dogs. Am. J. Vet. Res. 23, 422-427.

Woese, C.R. (1987) Bacterial evolution. Microbiol. Rev. 51, 221-271.

Yoshida, H., Hirota, K., Shiratori, Y., Nihei, T., Amano, S., Yoshida, A., Kawamata, O.and Omata M. (1998) Use of a gastric juice-based PCR assay to detect Helicobacterpylori infection in culture-negative patients. J. Clin. Microbiol. 36, 317-320.