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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria

Chapter 10: Regular and coryneform non-sporing Gram-positive rods

Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 10: Regular and coryneform non-sporing Gram-positive rods

Author: Dr. J.A. Picard

Licensed under a Creative Commons Attribution license.

TABLE OF CONTENTS

Table 10.1: Differences between Listeria monocytogenes and Erysipelothrix rhusiopathiae......3

Listeria........................................................................................................................................................ 3

Table 10.2: Differentiation of Listeria species..............................................................................4

Erysipelothrix Rhusiopathiae................................................................................................................... 6

Corynebacterium species......................................................................................................................... 8

Table 10.3: Distinguishing features of corynebacteria of veterinary importance..........................9

Table 10.4: Identification characteristics of regular, non-sporing, Gram-positive rods................12

Table 10.5: Biochemical and morphological characteristics of genera of Gram-positive rods isolated from animals.................................................................................................................. 13

Table 10.5 continued: Biochemical and morphological characteristics of genera of Gram-positive rods isolated from animals.............................................................................................15

Table 10.6: Diseases associated with pathogenic coryneforms and Rhodococcus equi............................................................................................................17Use of laboratory animals................................................................................................................................................... 17

Rhodococcus equi................................................................................................................................... 17

Trueperella (previously Arcanobacterium) pyogenes..........................................................................18

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Brochothrix thermasphactum................................................................................................................. 19

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INTRODUCTIONGram-positive rods are divided into two groups based on morphology and the guanine-cytosine (G+C) content of the DNA. Those with a high G+C content and irregular cell shape are included in the actinomyces group and are discussed in a different chapter, with Trueperella (Arcanobacterium) pyogenes, Corynebacterium and Rhodococcus equi being dealt with in this chapter. Those with a low G+C content and with irregular staining include the genera Bacillus, Clostridium, Listeria, Erysipelothrix and Lactobacillus. The latter three will be discussed in this chapter.

Listeria monocytogenes and Erysipelothrix rhusiopathiae are important animal pathogens and Lactobacillus spp. are common commensals of the gastrointestinal tract and mucous membranes of animals. All these agents have a similar morphology and must be distinguished by culture morphology and various identification tests. Refer to Table 10.1

Table 10.1: Differences between Listeria monocytogenes and Erysipelothrix rhusiopathiae

Characteristic Erysipelothrix rhusiopathiae Listeria monocytogenes

Disease

Septicaemia, diamond skin disease, arthritis and endocarditis in pigs. In other animals and humans skin and joint lesions

Septicaemia, abortions and meningoencephalitis in most animals (rare disease in South Africa)

Morphology Smooth colony: short rodsRough colony: filaments

Thick rods

Gelatine stab after two weeks at 22°C Brush growth Umbrella-like growthMotility Non-motile MotileBlood agar α-haemolysis β-haemolysisGrowth at 4°C - +Aesculin hydrolysis - +Catalase production - +MR and VP reactions - +Pathogenicity Kills pigeons but not guinea pigs Kills guinea pigs but not pigeons

LISTERIA Listeria species are medium-sized (0.5-2μm in length), Gram-positive rods that are non-acid-fast and are motile at 22°C. Although they are facultative anaerobes, their growth is enhanced by 5 – 10% CO 2. They are catalase-positive, oxidase-negative, hydrolyse aesculin, tolerate 10% salt (NaCl) and will not grow on MacConkey agar.

They are widely spread in nature and have been isolated from both sick and healthy animals as well as from the soil, water, silage and sewage.

Only some strains of L. monocytogens (more common) and L. ivanovii have been associated with disease. Pathogenic strains of L. monocytogenes can cause meningoencephalitis, septicaemia and abortions in primarily domestic ruminants, but also other animals and humans (zoonosis). Sheep and goats may sometimes harbour the bacteria without showing clinical signs. When such animals are stressed they may then begin to shed the bacteria and may become sick. The main source of infection in ruminants has been the feeding of poor quality (alkaline) silage.

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Occasionally L. monocytogenes is shed in the milk of infected goats, cows and ewes. In humans disease is often associated with the intake of contaminated dairy products, such as improperly pasteurised milk, poultry meat and coleslaw.

LABORATORY DIAGNOSIS

Specimens

Septicaemia: material from lesions in the liver, kidney and spleen.

Meningoencephalitis: spinal fluid, brainstem, and tissue from several sites on the medulla oblongata.

Abortion: Placenta (cotyledons), foetal abomasal contents and/or uterine discharges.

Direct microscopy

Histopathology examination (of formalin fixed specimens) is more useful as Gram-positive rods will be seen in association with lesions characteristic of listeriosis.

Isolation

Suspected material is inoculated on blood and MacConkey agar to rule out any contaminants. Selective media containing antibiotics or blood agar containing 0.5% potassium tellurite to inhibit many Gram-negative bacteria can be used. Examples of selective media are PALCAM (polymixin-acriflavin-lithium chloride-ceftazidime-esculin-mannitol) agar where grey-green colonies form and Oxford (lithium chloride-phenylethanol-moxalactam) agar where black colonies surrounded by a black halo form.

In the visceral form of the disease sample material can be directly inoculated onto agar, but in the neural form of the disease nervous tissue should be placed in a broth and the cold enrichment technique followed. The cold enrichment technique is a more sensitive method for recovering Listeria species. Small pieces of brain tissue are homogenized and a 10% suspension is made in nutrient broth. The suspension is placed in a refrigerator at 4°C and sub-cultured weekly onto blood agar for up to 12 weeks before being considered negative.

Table 10.2: Differentiation of Listeria species

Cha

ract

eris

tics

L. m

onoc

ytog

enes

L. in

nocu

a

L. s

eelig

eri

L. w

elsh

imer

i

L. iv

anov

ii

L. g

rayi

L. m

urra

yi

L. d

enitr

ifica

ns

Irregular rods with uneven staining - - - - - - - -

β-haemolysis +/- - (+) - + (wide zone)

- - -

CAMP-test (S. aureus) +/- - (+) - - - - -CAMP-test (R. equi) (+) - - - + - - -Acid production from:L-Arabinose - - - - - +Dextrin d - - + + +Galactose d - d + + +Glycogen - - - - - +Lactose d + + + + +

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D-Lyxose - - + + -Mannitol - - - - - + + -Melezitose d d d - - -Melibiose - - - - - -α-Methyl-D-glucoside + + + + + -α-Methyl-D-mannoside + + -/+ + -L-Rhamnose + d - d - - d -Sorbitol - d - - - -Soluble starch - - - + +Sucrose - d d - - +D-xylose -/+ - + + + - - +Voges-Proskauer + + + + + + + -Hydrolysis ofCellulose - - - - - +Hippurate + + + - - -Starch d d - - - +Lecithinase d d + - - -Phosphatase + + + + + -Reduction of NO3 to NO2 - - - - - - + +Pathogenicity for mice + - - - + - - +?

Serotypes

1/2a,1/2b, 1/2c, 3a, 3b, 3c, 4a,

4ab, 4b, 4c, 4d, 4e, 7

1/2a,1/2b,1/2c,US,4b,4d,

6b

1/2b,4c,6a,6b,U

S

5 Specific

L. ivanovii subsp. londoniensis is ribose negative, it is also positive to N-acetyl-β-D-mannosamine, whereas L. ivanovii is negative.

Cultural and staining characteristics

Small round transparent colonies appear on the blood agar after 24 hours of incubation. They become greyish-white with a diameter of 0.5-2 mm after 48 hours of incubation. Listeria monocytogenes and the non-pathogenic L. seelegeri produce a narrow zone whereas L. ivanovii produces a wide zone of β-haemolysis. Sometimes haemolysis can only be noted under the colonies. Smooth and rough colony forms may be seen.

Gram-stain reveals the presence of Gram-positive rods. Primary cultures are often pleomorphic and may be confused with Corynebacterium and Streptococcus species. The organisms easily decolourize and may be mistaken for Gram-negative rods.

Identification

Listeria species can be differentiated from other related genera e.g. from motile Corynebacterium spp. by aerobic acid production from glucose, inability to hydrolyse urea and positive VP reaction; from Streptococcus spp. by its catalase positive reaction; from Lactobacillus spp. by it being motile and catalase positive; from Kurthia spp. by being a facultative anaerobe and its aesculin hydrolysis.

All the Listeria species hydrolyse aesculin. L. monocytogenes has a typical tumbling motility in a 2 to 4 hour broth culture incubated at 25°C when examined by the hanging drop method. When grown on semi-solid motility medium or gelatine they have a characteristic umbrella-shaped growth on the subsurface.

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Modified CAMP tests using Staphylocccus aureus and Rhodococcus equi help distinguish the β-haemolytic Listeria species. (Table 10.2)

Other tests that can be used to distinguish the different Listeria species include acid production from carbohydrates (Table 10.2), API-Listeria test, phage typing and DNA fingerprinting.

Determination of pathogenicity

As many strains of L. monocytogenes and L. ivanovii can be non-pathogenic, laboratory animals can be used to assess the virulence of this agent.

1. Anton test: inoculation into the conjunctiva of a rabbit or guinea pig. L. monocytogenes will cause a purulent keratoconjunctivitis within 24 to 36 hours after inoculation.

2. Intraperitoneal inoculation of mice with a broth culture of either L. monocytogenes or L. ivanovii. If virulent, death will occur within 5 days with necrotic lesions present in the liver.

3. Inoculation of the chorioallantoic membrane of embryonated hen’s eggs. 4. Cell culture cytotoxicity using the human intestinal cell line Caco-2.

Serotyping

Strains of Listeria species can be divided into serotypes on the basis of somatic (O) and flagellar (H) antigens.

Antimicrobial susceptibility

In vitro the organism is sensitive to penicillin, ampicillin, gentamycin, potentiated sulphonamides, erythromycin, tetracycline and chloramphenicol. Since aminoglycosides enhance the activity of penicillin, this combination is recommended in the therapy of disease. In human isolates resistance has been found to develop to chloramphenicol, erythromycin and tetracycline. Cephalosporins are ineffective.

ERYSIPELOTHRIX RHUSIOPATHIAERhusos= red; Pathos = pain; Thrix = hair (describes the disease in pigs)

Organisms in this group of bacteria are slender, Gram-positive, non-motile bacteria. They generally do not grow on MacConkey agar. There are both smooth and rough variants. The smooth colonies are often present in peracute and acute disease, and the rough forms predominate in chronic disease. The organisms grow with a test tube brush appearance in a gelatine stab.

Pigs carry these organisms on the tonsils and in the intestines. They are present in the soil and pig manure and will survive for prolonged periods in an alkaline environment. It has also been isolated from the slime layer on the skin of fish and from free-living avian species that died from septicaemia.

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It causes swine erysipelas and can manifest as a septicaemia, a skin form known as “diamond skin disease”, chronic arthritis and valvular endocarditis (heart valve disease). In sheep, cattle and horses the disease may manifest as a polyarthritis. A severe septicaemia disease often occurs in turkeys. There have been a few reports of valvular endocarditis in dogs. Humans mainly develop a localized skin infection known as erysipeloid that usually involves the hands and fingers. Rarely systemic infection occurs.

Direct microscopy

Gram stained smears made from the blood of the heart will reveal the presence of large numbers of slender Gram-positive rods in acute disease and long filaments in chronic disease. The filaments stain poorly with Gram’s stain.


Specimens:

Liver, spleen, kidney, heart and synovial tissue/fluid can be used. Recovery of the organism from skin lesions and chronic forms of the disease can be difficult.

Isolation Procedures:

Suspected erysipelas material is inoculated onto blood agar. Blood agar containing sodium azide (1:1000) or crystal violet (0,01%) may be added to suppress contaminants. Inoculated plates are put in a candle jar or 10% CO2 in order to accelerate growth and incubated at 37°C for 24 – 48 hours.

Cultural and staining characteristics

Non-haemolytic pin-point colonies are seen after 24 hours of incubation. After 48 hours a zone of greenish haemolysis appears just under the colonies. On further incubation a zone of clear (β) haemolysis may develop. Smooth colony types (S) are often small (0,1-1,5 mm in diameter) and round, whereas rough (R) colonies are larger, flatter, and more opaque with irregular borders. This colony variation is best seen after 48 hours of incubation.

Gram stain of S-colonies reveals the presence of slender Gram-positive rods indistinguishable from L. monocytogenes, whereas R-colonies reveal filaments of varying length.

Identification

Biochemically, they are catalase-negative, aesculin-negative and H2S positive (Table 10.1).

After stab inoculation of TSI agar and an incubation period of 24 hours at 37°C, a thin black line indicating H2S production is produced.

Rough colony forms give a “bottle-brush” type of growth in nutrient gelatine after 5 days of incubation at room temperature.

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Carbohydrate tests should be carried out in peptone water with added sterile serum (0.5 – 1%) or in nutrient broth plus the test carbohydrate with phenol red as an acid indicator. Although the fermentation pattern is variable, they usually ferment lactose, glucose, levulose and dextrin.

Serotyping

This is usually only done in reference laboratories. Twenty-two serovars have been identified on the basis of somatic antigens. Serovars 1 and 2 account for 70 – 80% of all isolates. Serovar 7 is now known as E. tonsillarum, an avirulent commensal in the oropharynx of pigs.

CORYNEBACTERIUM SPECIESCoryne = club shaped.

Members of this genus are Gram-positive, small pleomorphic rods (about 0.5 μm in diameter). They are non-spore forming, not acid-fast, catalase positive and oxidase negative. The majority are non-motile except for the plant pathogens. These bacteria are frequently arranged in parallel “palisades” or at sharp right-angles to each other “Chinese letters”. Corynemycolic and corynemycolinic acids are present in the cell walls. Many have metachromic granules (high-energy phosphate stores) in their protoplasm, which is particularly noticeable in the type species Corynebacterium diphtheriae, the cause of diptheria in humans.

Members of this genus and related genera belong to the actinomycetes group, which includes Corynebacterium, Actinomyces, Arcanobacterium, Brevibacterium, Mycobacterium, Rhodococcus, Nocardia, Bifidobacterium and many others.

Many occur widely in nature and those isolated from animals are often found as commensals on the skin and mucous membranes. More than 30 Corynebacterium species have been identified, of which about a third are parasitic on humans and animals and are often found in clinical samples. Only a few, however, cause disease.

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Table 10.3: Distinguishing features of corynebacteria of veterinary importanceC

hara

cter

istic

Cor

yneb

acte

rium

ca

pito

vis

C. c

ampo

real

ensi

s

C. s

tria

tum

C. j

eike

ium

C. c

oyle

ae

C. a

uris

C. a

rgen

tora

tens

e

C. a

myc

olat

um

C. b

ovis

C. p

seud

otub

ercu

losi

s

C. m

astit

idis

C. c

ystit

idis

C. r

enal

e

C. p

ilosu

m

C. l

ipop

hilo

flavu

m

Pigment lemon - - - lemon yellowHaemolysis - - β - - -Catalase + + + + + +O/F F F F O F O F F O F O F F F OLipophilic - - - + - - - - + - + - - - +Gelatin hydrolysis -Urease - - - - - - - - + + V + + + +Bile aesculin -Nitrate - - + - - - - - - V - - - + -ALP + + + + + + v + + v + + - + +Acid phosphatase + - + +Acid productionGlucose + v + + + - + + + + - + + + -Lactose -Maltose - - - v - - - v - + v - - - -Mannitol -Ribose - - +Sucrose - - v - - - - v - v v - - - -D-xylose -

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Specimens

Pus or exudates from recent abscesses or suppurative lesions, or swabs from the edge of abscesses. Mid-stream urine for the isolation of members of the “C. renale” group.

Direct microscopy

Pleomorphic rods are usually observed within the cytoplasm of neutrophils or free in pus. They may be confused with streptococci, staphylococci, enterococci and Listeria species.

Isolation

For routine isolation, blood and MacConkey agars can be used. The inoculated plates are incubated at 37°C for 24 – 48 hours. If no contaminants are suspected, the specimen can also be inoculated into a serum-containing or brain heart infusion broth. A selective medium containing fosfomycin, nalidixic acid and the culture supernatant of Rhodococcus equi has been developed for the culture of Corynebacterium pseudotuberculosis and is known as FNR medium.

Identification

Colony morphology

A rapid visual identification of certain similar Gram-positive rods is available in Table 10.3.

C. bovis: small, white, dry, non-haemolytic colonies that tend to appear in the wells of plates inoculated with milk, as it is a lipophilic organism.

C. pseudotuberculosis: small, white and dry colonies, which after 48-72 hours of incubation will be surrounded by a narrow zone of β-haemolysis. After several days of incubation the colonies can reach 3 mm in diameter and appear dry, crumbly and cream in colour.

C. kutscheri: resembles the colonies of C. pseudotuberculosis. Occasional strains may be β-haemolytic.

C. renale, C. pilosum and C. cystitidis: very small, non-haemolytic. With time colonies of C. renale become cream to light yellow, C. pilosum yellow and C. cystitidis remains translucent to white in appearance.

CAMP test

The CAMP tests are quick presumptive tests for C. pseudotuberculosis, C. renale and R. equi. Bovine or sheep blood agar should be used for the test. The results are as follows:

Staphylococcal β-haemolysinC. pseudotuberculosis InhibitionC. renale EnhancementR. equi Enhancement

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A. pyogenes Enhancement

There is also enhancement (synergism) between the R. equi “equi factors” and the haemolysin of C. pseudotuberculosis.

Biochemical tests

The definitive identification of the corynebacteria is based upon biochemical tests (See tables 10.4 and 10.5). APICoryne can also be used to identify these bacteria.

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Table 10.4: Identification characteristics of regular, non-sporing, Gram-positive rods

CharacteristicsCatalase-negative

Facultative anaerobeCatalase-positive

Facultative anaerobe Strict aerobic

Lactobacillus Erysipelothrix Brochothrix Listeria Kurthia Caryophanon Renibacterium

Cell morphologyUsually straight rods, sometimes coccobacilli

Sender rods to filaments

Sender rods to filaments

Short rods often short chains and filaments

Regular rods in chains, cocci in old cultures

Short rods in chains Short rods often in pairs

Multi cellular rods (trichomes) - - - - - + -

Diameter of rods 0.5-1.6 0.2-0.5 0.6-0.8 0.4-0.5 0.7-0.9 1.4-3.2 0.3-1.0Motile -/(+) - - + (25°C) + + -Catalase reaction -/ (+) - + (media dependent) + + + +Growth at 35°C + + - + +H2S production - + - - -/ (+)Acid from glucose + + + + +

Major fermentation products

Mainly lactate (some may also give acetate, ethanol and CO2)

Lactate Mainly lactate Lactate No acid No acid No acid

Normal habitatWidespread in fermentable materials, rarely pathogenic

Widespread pathogen in vertebrates

Meat products, non-pathogenic

Widespread in decaying material, may be a vertebrate pathogen

Faeces of farm animals, meat products, non-pathogenic

Cow dung, non-pathogenic

Pathogen in salmonid fish

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Table 10.5: Biochemical and morphological characteristics of genera of Gram-positive rods isolated from animals.

Cha

ract

eris

tic

Act

inom

yces

Agr

omyc

es

Ara

chni

a (P

. pro

pion

icus

)

Arc

anob

acte

rium

Art

hrob

acte

r

Aur

eoba

cter

ium

Bre

viba

cter

ium

Bro

chot

hrix

Car

yoph

anon

Cas

eoba

cter

Cel

lulo

mas

Cor

yneb

acte

rium

Cor

yneb

acte

rium

(pla

nt

path

ogen

s)C

urto

bact

eriu

m

C. a

quat

icum

Der

mab

acte

r

Der

mat

ophi

lus

Erys

ipel

othr

ix

Exgu

obac

teriu

m

Kur

thia

Lact

obac

illus

List

eria

Mic

roba

cter

ium

Myc

obac

teriu

m

Noc

ardi

a

Oer

skov

ia (C

alla

tom

onas

tu

rbat

a)Pr

oprio

niba

cter

ium

Prop

ioni

fera

x

Ren

ibac

teriu

m

Rho

doco

ccus

Rot

hia

Sang

uiba

cter

Stre

ptom

yces

Turic

ella

Source*All G H H G M

G

Ch

D

M F Ch G A P PG H S PC

S

H FM A A MF A A A M

S

H F A H H A H

Shape§PB BF PB PC RC P RC R@ M

R

PR

C

P P P P BF FR RC R R P P PB BF P R R P FB

Aerobic growthD +C

O

(+) + + + + + + + + + + + + + + D + + + + + (D) + + + + +

Anaerobic growth + (+) + + - - - + - + + +d - - + + - + + - - - + + - - + -

37°C + + + + D D -d - + (+) + + D + + + + + + + + + + + - D + +

Adherence

(mycelium)

D + + - - - - - - - - - - - + - - - - - D- + + - - D+ - +

Aerial hyphae - - - - - - - - - - - - - - + - - - - - D- + - - - D- - +

Microcols filament D + + - - - - - - - - d - - +# - - - - - - - + - - D A +

Pigment¥ DR (Y) - - D Y DY - -/P Y DY YB DY Y - Y - G D/ - - DY + YR Y YR W YR - Y + W

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Y (+)

Haemolysis D D + - + - - D D D - D - D

Oxidase D - - -/+ - - - d - - - - - D - -

Catalase D - - - +- + + + + + + + + + + - + - + +- + + + D + + + +

Acid fast (milk)- - - - D - - - - - -

(+)

- - - - - - - D - (+) - - (+) - -

Motility- - - - - D - - + - D - - D + - +S - + +/- -

(+)

+2

5

+ D - D - - - - - + - -

Glucose acid+ 3w + + - (+) +/- + - (+) + +

(-)

+ (+) + + + (+) + - + + - - + + + + - - + + - -

Glucose gas - - (+) - - - - - - - - - - - - - D - D D - - D - - - +

Nitrate +/- - + D D - - + D - V - - - V - - D - D+ + + +/- + - D + V D -

Urea D - - D - - - D -/+ - - - + - - - - - D D+ D +/- + D - D -

Gelatine-/+ PG

§

D - + D + - - - (+) D D + - + + - V - - - D + D + - D V + -

Aesculin D + - - D D +/- - V + - + - D + + + - - + + -

TSI D D - - + - - -

H2S (LA) D + D D D - - D D -/+ - - D D +

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Hippurate - - - D D - D - + D + - D

Indole - + - - - - - - D - - - - - - -

Table 10.5 continued: Biochemical and morphological characteristics of genera of Gram-positive rods isolated from animals

Cha

ract

eris

tic

Starch hydrolysis D -/+ D D D - - - - + D + + + - + - D D D - + - V D -

DNase D - + + D + - - + +/- + + - D (+) + D + - + - + -

Casein +/- - - - + D + - - D - + + - + - - - D + - D

Phosphatase - - - - - D D D - + D D + -

Arginine D - - D + - - - D - D

Sabouraud’s agar - - + +

ONPG - D + - + - D + D + + - - - +

Penicillin S S S S S S S S R R S S S

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Filter paper trypsin

- - - - - - - + - - - - - - - - D - - -

Volatile fatty acids - - + - - - - - - - - - - - - - - - - - - + - - - -

A: all; C: poultry, P: pigs, S: sheep; F: faeces; M: meat; Ch: cheese; M: milk; G: soil; P: plants; D: skin, H: humans §: PG: poor growth,

R: rods, FR: filamentous rods, BF: branching filaments, P: pleomorphic; RC: cocco-bacilli, R@: long rods in loops, MR: multi celled rods, Y: yellow; P: pink; DY: dull yellow, #: only in co2,

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Table 10.6: Diseases associated with pathogenic coryneforms and Rhodococcus equi.

Bacterial species Main host(s) Disease(s) Natural habitatC. bovis Cattle Uncertain Teat canal of cowsC. kutscheri Mice, rats (guinea

pigs)Caseopurulent foci in the liver, kidneys, lungs and lymph nodes

Mucous membranes of carrier rodents

C. pseudotuberculosis Sheep, goats

Horses, cattle

Caseous lymphadenitis(Bacterial icterus)

Ulcerative lymphangitis,Contagious acne

Skin, mucous membranes and gastrointestinal tract of carrier sheep and the soil of sheep pens

C. renale, C. pilosum, C. cystidis

Cattle Pyelonephritis, cystitis Bovine genital tract, especially in bulls

C. renale Sheep

Pigs

Balanoposthitis “Pizzle rot”

Kidney abscesses

Unknown

Unknown“C. ulcerans” Cattle Mastitis (rare)R. equi Foals (2–4 months)

Older horses and other animals

Suppurative bronchopneumonia, ulcerative colitis

Abscessation

Soil rich in organic material and faeces of foals

Soil

Gemella morbillorum Any Opportunistic infections (rare) Genital tract of humansThese genera are subject to taxonomic changes.

Use of laboratory animals

Should one be uncertain as to the identification of C. pseudotuberculosis, guinea pigs can be used as they develop characteristic lesions.

0,2 ml of a 1:200 dilution of a broth culture is inoculated intramuscularly into the thigh. A swelling develops in 1 to 3 days, with later enlargement of the inguinal lymph node and suppuration of the local lesion. Due to spread of the bacteria, it often dies or is euthanized after 3 to 4 days. At necropsy thick-walled, abscesses containing whitish pus are found in or on the liver, spleen and other organs. The presence of the organism is confirmed by culture. Rabbits and mice can also be used, but develop less characteristic lesions.

Since the infection can spread between laboratory animals, the inoculated animals must be kept in isolation for the duration of the test.

RHODOCOCCUS EQUIThese are Gram-positive, aerobic, non-motile, pleomorphic coccobacilli. They grow well on blood agar forming non-haemolytic, smooth, pinkish, shiny, mucoid colonies. The bacteria commonly appear coccoid on direct examination of clinical material, but may be rod shaped and rarely have limited branching.

It is a common cause of bronchopneumonia and ulcerative colitis in 2 to 4 month old foals and rarely in immunosuppressed adult horses. In other species it causes opportunistic suppurative lesions e.g. cervical lymphadenitis in pigs, pyaemia in dogs, ulcerative lymphangitis, abscesses and arthritis in adult horses. Tuberculosis-like lesions in the lymph nodes of pigs have also been reported.17 | P a g e



LABORATORY IDENTIFICATION

Direct microscopy

Gram-stained smears of clinical material reveal the presence of coccoid sometimes rod-shaped and rarely branched Gram-positive organisms. They may occasionally stain positive (pink) with acid-fast stains.

Isolation procedures

Clinical material should be streaked onto blood agar and incubated aerobically at 30 to 37°C for 24-48 hours. To isolate it from soil a selective medium is available – refer to Appendix 1.

Cultural characteristics

Within 48 hours, the organisms appear as smooth, mucoid, translucent tear-drop colonies, 3 to 5 mm in diameter. With age they become larger, mucoid and salmon-pink in colour. Glucose-yeast extract medium enhances pigment formation.

Identification

They are generally non-reactive in standard tests, are catalase- and urease-positive and do not ferment carbohydrates.

TRUEPERELLA (PREVIOUSLY ARCANOBACTERIUM) PYOGENESThese are pleomorphic and commonly observed as clusters of Gram-positive, club-shaped, short rods but may also appear cocco-bacillary or as short, branching rods. They are non-motile and catalase-negative. The organisms are commonly isolated from purulent conditions in sheep, cattle, swine and occasionally horses. It is often recovered as opportunistic infections from wounds, pneumonia, genital tract and udder infections.

LABORATORY IDENTIFICATION

Direct Microscopy

Gram-stained smears of pus or mastitis milk samples in T. pyogenes infections usually reveal large numbers of small, highly pleomorphic, Gram-positive forms. They tend to be a mixture of cocci, rods and pear-shaped cells. Occasionally short branching forms may be seen.

Isolation procedures

It grows well on blood agar incubated in 10 percent C02 at 37°C and also grows well anaerobically.

Cultural characteristics18 | P a g e



Aerobic cultures are dry, white, fine (1mm) colonies at 48 hours, growth not very visible at 24 – 48 hours. At times colonies may appear smooth or mixed rough and smooth. Small zones of fuzzy β-haemolysis are seen even before colony growth is observed. It becomes clearer with time

Identification

Typically Gram-positive, plump pleomorphic rods that are catalase negative. A rapid presumptive test for T. pyogenes is its ability to pit a Loeffler’s serum slope in 24 – 48 hours (Appendix 1). A. pyogenes will also give a positive CAMP test with the -haemolysin of Staphylococcus aureus. So too will A. neuii and some Corynbebacterium species.

BROCHOTHRIX THERMASPHACTUMIt is a spoilage bacterium of meat. It will grow on almost all media, but has a strict temperature requirement.

1. Suspend meat in 0.1% peptone water and shake vigorously2. Inoculate the fluid onto blood agar and the selective agar (see Appendix 1) and incubate at 22°C

for up to 5 days. Colonies are usually visible in 48 hours.

Identification

Non-haemolytic small white to yellowish colonies Does not grow at 37 °C (grows best at 22 °C) Gram-positive regular small rods that often forms chains or clumps Catalase positive, Oxidase and Indole negative Thiogel: non-motile, grows throughout the media, gelatine negative OF: Fermentative, glucose positive Casein, DNAse, nitrate, H2S and gas negative

REFERENCES1. Van Netten, P, Perales I, van de Moosdijk, A., Curtis, GWD, & Mossel, DAA, 1989. Liquid and

solid differential media for the detection and enumeration of Listeria monocytogenes and other Listeria spp. International Journal of Food Microbiology, 8, 299-316.

2. Zhao, H. K., Hiramune, T., Kikuchi, N., Yanagawa, R., Ito, S., Hatta, T., Serikawa, S. & Oe, Y. 1991. Selective medium containing fosfomycin, nalidixic acid, and culture supernatant of Rhodococcus equi for isolation of Corynebacterium pseudotuberculosis. Journal of Veterinary Medicine. Series B, 38, 10, 743-748.

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APPENDIX 1Glucose-yeast extract medium

Yeast extract 10g

Glucose 10g

Agar 15g

Tap water 1000ml

Loeffler’s serum slant

This medium is used to isolate Corynebacterium diptheriae, but also can be used to demonstrate proteolytic activity of T. pyogenes.

Bovine serum 70ml

Infusion dextrose broth (dried)

2,5g

Whole egg dried 7.5g

Distilled water 1000ml

Final pH: 7.6

Medium preparation

Mix, and dispense in glass heat resistant bottle. Arrange so that they are in a slant position and autoclave at 120°C for 15 minutes. Properly prepared slants are smooth and greyish-white in colour.

Test procedure

A loopful of the bacterial colony is made in the centre of the slope, taking care not to break the surface of the medium. Incubate the medium at 37°C for 24 – 48 hours. Pitting of the slope gives a positive result for T. pyogenes.

NANAT Selective medium for the isolation of Rhodococcus equi.

Base mediumPeptoneSodium chlorideYeast extractGlucoseSodium hydrogen sulphiteSodium thiosulfateDipotassium hydrogen phosphateSodium hydrogen carbonateNutrient broth (horse meat broth)AgaroseWater

20g5g5g5g0.2g1.2g2g2g300g15g1000ml

Dissolve by heating and adjust to pH 7.2, bottle and sterilize at 121°C for 15 min.

Cool to 55°C and add the following 4 antibiotics to reach the following concentrations

Novobiocin

actidone [cycloheximide]

potassium tellurite

nalidixic acid

25g/ml

40g/ml

50g/ml

20g/ml

Brochothrix selective agar

PeptoneYeast extractGlycerolK2HPO4

MgSO4

Agar

20g2g15ml1 g1 g3g

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Weigh and add to Schott bottle and make up to 1000 ml using deionised water. The pH should be 7.0.

Autoclave at 121 °C for 15 minutes

Then add:

Streptomycin sulphateCycloheximideThallium acetate

500 µg/ml50 µg/ml50 µg/ml

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