Identification of anaerobic bacteria - AfriVIPApplied Veterinary Bacteriology and Mycology:...

Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria

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Author: Dr. J.A. Picard

Licensed under a Creative Commons Attribution license.

TABLE OF CONTENTS

INTRODUCTION ............................................................................................................................................................3

Methods of growing anaerobes ..................................................................................................................................3

The anaerobic jar ......................................................................................................................................................3

Anaerobic cabinets and glove boxes ........................................................................................................................4

Reducing agents .......................................................................................................................................................4

Media ........................................................................................................................................................................4

Preparation of media ................................................................................................................................................5

The Non-Clostridial Anaerobes ...................................................................................................................................5

Genus: Bacteroides ..................................................................................................................................................6

Bacteroides fragilis ........................................................................................................................................7

Prevotella melaninogenica (formerly Bacteroides) ........................................................................................7

Bacteroides corrodens ..................................................................................................................................8

Bacteroides oralis ..........................................................................................................................................8

Bacteroides capillosus ...................................................................................................................................8

Bacteroides praeacutus .................................................................................................................................9

Genus: Fusobacterium .............................................................................................................................................9

Fusobacterium necrophorum ........................................................................................................................9

Fusobacterium nucleatum ........................................................................................................................... 10

Fusobacterium varium ................................................................................................................................. 10

Fusobacterium mortiferum .......................................................................................................................... 11

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Genus: Dichelobacter ............................................................................................................................................. 11

Genus: Actinomyces ............................................................................................................................................... 12

Actinomyces bovis ....................................................................................................................................... 12

Genus: Eubacterium and Propionibacterium .......................................................................................................... 13

The clostridia .............................................................................................................................................................. 13

Habitat and pathogenicity ....................................................................................................................................... 14

Pathogenicity .......................................................................................................................................................... 15

Specimens .............................................................................................................................................................. 15

Direct microscopy ................................................................................................................................................... 15

Isolation procedures ............................................................................................................................................... 16

Biochemical identification ....................................................................................................................................... 16

Serological identification ......................................................................................................................................... 16

Individual clostridia ................................................................................................................................................. 19

Clostridium chauvoei ................................................................................................................................... 19

Clostridium novyi ......................................................................................................................................... 19

Clostridium septicum ................................................................................................................................... 19

C. sordellii ................................................................................................................................................... 20

Clostridium tetani ......................................................................................................................................... 21

Clostridium perfringens ............................................................................................................................... 22

Clostridium botulinum .................................................................................................................................. 22

REFERENCES ............................................................................................................................................................. 25

APPENDIX: MEDIA AND REAGENTS FOR THE ISOLATION OF ANAEROBIC BACTERIA .................................. 26


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INTRODUCTION

Anaerobic bacteria are those bacteria that grow only in the absence of free oxygen but fail to multiply in the

presence of oxygen on the surface of nutritionally adequate solid media incubated in air or an atmosphere

containing 5-10% CO2.

Anaerobes comprise a wide range of bacteria that are separated into Gram-positive and Gram-negative cocci

and bacilli, and two major additional groups, namely whether or not they produce spores.

METHODS OF GROWING ANAEROBES

A variety of methods are available for the culture of anaerobic bacteria. Exclusion of oxygen from part of the

medium is the simplest method, and it is affected by growing of the organism within the culture medium as a

shake or fluid culture. When an oxygen-free or anaerobic atmosphere is required for obtaining surface growth of

anaerobes, anaerobic jars provide the method of choice. More sophisticated methods for surface culture of

anaerobes are the pre-reduced anaerobically sterilized roll-tube technique, and use of the anaerobic cabinet or

glove box. These complex techniques provide the most meticulous anaerobic conditions and are appropriately

used for the isolation and study of anaerobic species that are highly sensitive to oxygen.

The anaerobic jar

Anaerobic jars are cylindrical vessels made of metal, glass or plastic, flanged at the top to carry an air-tight lid

which is held firmly in place by a clamp. The lid carries on it’s under surface the room-temperature catalyst

capsule. The difference between the BTL and the Gaspak jars is that in the former the lid is provided with two

valves through which air can be withdrawn and an anaerobic gas mixture introduced. The lid of the standard

Gaspak jar is not vented, because the jar is specifically designed for use with a disposable hydrogen-carbon

dioxide generator.

Anaerobic gas for use in the BTL jar is obtained from a cylinder of the compressed gas. As hydrogen is highly

explosive a mixture containing 5% hydrogen, 10% carbon dioxide and 85% nitrogen is used. Carbon dioxide in a

10% concentration improves the growth of many anaerobes and no anaerobes are adversely affected by this

CO2 concentration. Nitrogen is an inert gas without any risk of explosion, even with the addition of 5% hydrogen

which renders the mixture stable.

The use of a catalyst in the anaerobic jar speeds the development of anaerobiosis. Palladium (0,5%) is

contained in wire gauze attached to the underside of the lid of the jar. The sachet contains pellets of alumina

coated with finely divided palladium. Certain gases such as chlorine, sulphur dioxide, carbon monoxide and

hydrogen sulphide, oil, the vapour of some organic solvents and strong acids poisons the catalyst. Inactivation

by hydrogen sulphide is especially relevant since many anaerobes produce large amounts of this gas. The

catalyst is also inactivated by moisture, which is plentiful in the anaerobic jar, but it is readily reactivated in a hot

air oven at 160°C for 2 hours.

An anaerobic indicator is included to check on the development of anaerobiosis. As the jar becomes anaerobic,

the indicator dye (methylene blue) becomes colourless. The most commonly used chemical methods for


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indicating anaerobiosis depend on the fact that when methylene blue is placed in an anaerobic environment it is

reduced from its coloured oxidised form to a colourless reduced leuco-compound. The fact that indicators are

necessary in anaerobic work draws attention to the importance of examining the anaerobic apparatus before use

to ensure that it is in proper working order.

Anaerobic cabinets and glove boxes

An anaerobic cabinet is an air-tight cabinet in which conventional bacteriological techniques may be done in an

oxygen-free atmosphere. This technique has the advantage that all the operations of isolating and sub- culturing

anaerobes are conducted in the absence of oxygen. In addition to the roll tube method it enables the use of Petri

dish plate cultures.

An anaerobic cabinet consists of an air-tight chamber which is provided with glove ports. It is commonly fitted

with an air-lock through which materials are transferred into and out of the chamber. The anaerobic glove box is

constructed in such a way that it can be almost completely evacuated of air with a vacuum pump since it is

collapsible. Air in the interchange is removed by vacuum and replaced with a mixture of 85%N2, 5%H2 and 10%

CO2 (the same mixture that is present in the glove box.)

In some cases the temperature in the glove box is maintained at 37°C. Other models have built-in incubators

and in some instances the plates have to be removed from the glove box in an anaerobic jar and incubated

in a separate incubator.

Reducing agents

Although the addition of reducing agents to fluid and shake cultures is not necessary, it is essential for the

satisfactory culture of more exacting anaerobes. The following are some of the reducing agents used:

Thioglycollic acid; 0,01-0,2%

Glucose; 0,5-1%

Ascorbic acid; 0,1%

Sodium sulphide; 0,025%

Metallic iron (iron filings etc. in fluid media.)

Meat (as in cooked meat medium)

Media

The use of pre-reduced anaerobically sterilized media is not generally necessary in the routine laboratory, since

almost all of the anaerobes that are pathogenic can be isolated and identified by conventional anaerobic

techniques. It is however recommended for anaerobic blood culture.

Plate cultures are inoculated in the usual way. When dealing with very strict or otherwise demanding anaerobes,

it is desirable to use freshly prepared plates, since during storage the medium takes up oxygen from the

atmosphere in sufficient amounts to prevent the growth of these, even though complete anaerobiosis has

apparently been obtained in the jar. Alternatively, but less satisfactory, it may be convenient to store a set of

uninoculated plates under anaerobic conditions, the whole jar being kept in the refrigerator or the plates kept in

an anaerobic cabinet.


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Fluid media are inoculated after dissolved oxygen has been driven off by steaming or heating in a boiling water

bath. When inoculating with a Pasteur pipette, the inoculum should be pipetted gently into the depths of the

medium, care being taken not to introduce any air bubbles.

Preparation of media

The methods of preparation of media for the culture of anaerobes are the same as for aerobic bacteriological

media. Useful basic media are Columbia agar and Tryptose blood agar. Basic broth media may be solidified by

the addition of 1,5% Bacteriological agar.

To these basic media, whether fluid or solid, may be added any required enrichment, selective or indicator

substance that is compatible with bacterial growth (Table 1). It is convenient to select one or two basic media

from which most other media can then be prepared.

The following may be added to the medium for enhancement of growth:

Glucose 0,5 - 1% (for all anaerobes)

Sodium bicarbonate 0,1 %( for organisms whose growth is encouraged by carbon dioxide.)

Bile 20% (B. fragilis etc.)

Vitamin K and haemin (P. melaninogenica)

Table 1: Selective agents for anaerobic bacteria

Organisms Selected For Agents Amounts

(Per 100ml of medium)

Anaerobic cocci Neomycin 10 mg.

Bacteroides and Fusobacterium. Sodium azide/Brilliant green Oleandomycin

30,0 mg/1,8 mg 50 mg

Bacteroides Neomycin Vancomycin

10 mg

750 g

Some Bacteroides Sodium azide/Bile(ox gall) 20 mg./1,7 g.

Clostridium

Sodium azide Sorbic acid/Polymyxin B Phenyl ethyl alcohol Kanamycin

20 mg. 0,12 g/2,0 mg 0,25 g 10 mg

Cl. perfringens Sulphadiazine Neomycin

10 mg 10 mg

Fusobacterium Crystal violet / Streptomycin Kanamycin Neomycin

1,0 mg/1,0 mg 7,5 mg 10mg.

Fusobacterium and Veillonella. Polymyxin 1000 units

Gram-positive non-sporing anaerobes Vancomycin 750 g

THE NON-CLOSTRIDIAL ANAEROBES

The non-clostridial anaerobic bacteria may be divided into four groups for diagnostic identification purposes. An

anaerobe is classified as either Gram-positive or Gram-negative and either a coccus or a bacillus (Refer to Table

2). Diseases caused by the non-clostridial anaerobes are listed in Table 3.


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Table 2: Non-clostridial anaerobes

Gram’s stain Bacilli Cocci

Gram-Positive

Actinomyces Bifidobacterium Eubacterium Lactobacillus Propionibacterium

Peptococcus Peptostreptococcus

Gram-Negative

Bacteroides Fusobacterium Dichelobacter Prevotella

Veillonella

Genus: Bacteroides

Bacteroides are Gram-negative, non-sporing bacilli. They are strict anaerobes and many species are obligate

parasites, not occurring outside the body except perhaps in sewage. They differ from species of Fusobacterium

as they are commonly actively saccharolytic and do not produce indole or threonine deaminase.

Twenty-two species of Bacteroides have been described, of which five are of animal origin, two have been

isolated from termites, and the remaining 15 are associated with man. Bacteroides fragilis and Prevotella

melaninogenica are the species most commonly implicated as pathogens. Other commonly encountered

species are B. corrodens, B. oralis, B. capillosis and B. praeacutus.

Useful characteristics used for identification of certain species of Bacteroides are:

1. B. fragilis is by far the most commonly encountered pathogen in animal infections and unlike most other

species it is resistant to penicillin G.

2. Prevotella melaninogenica is also commonly encountered in infections. It is the only Bacteroides which is

proteolytic, which produces black pigmented colonies on horse blood agar, and whose colonies show red

fluorescence in ultraviolet light.

3. B. corrodens is the only species that cause pitting of agar media, and is the only oxidase-positive

Bacteroides spp. Table 3: Diseases caused by the non-spore-forming anaerobes

Organism Host(s) Disease

Actinomyces bovis A. suis A. viscosus A. hordeovulneris A. israelii Dichelobacter (Bacteroides) nodosus

Cattle Horses Pigs Dogs Dogs Humans and rarely pigs and cattle Sheep Goats, cattle and pigs. Calves, lambs, foals, piglets.

Bovine actinomycosis ("lumpy jaw") Fistulous withers Pyelonephritis Canine actinomycosis: Localised abscesses -pyothorax Localised abscesses, pleuritis, peritonitis, septic arthritis, visceral abscesses Human actinomycosis Bovine or porcine actinomycosis (rare) Contagious (virulent) foot rot. Occasional infections of interdigital skin.


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B. fragilis B. asaccharolyticus Prevotella melaninogenica Fusobacterium necrophorum F. russii F. nucleatum

Cattle Pigs Dogs, cats, horses, cattle. Cattle Cattle, sheep, dogs and cats. Cattle Sheep Pigs Horses Chickens Rabbits Cats Several species

Diarrhoeal disease Mastitis Abscesses Osteomyelitis Foot rot Osteomyelitis Calf diphtheria, liver abscesses, metritis, cellulitis, mastitis. Foot abscess, interdigital dermatitis, lip and leg ulcers 'Bull - nose', necrotic enteritis, liver abscess Thrush involving the frog, necrobacillosis of lower limbs Avian diphtheria Necrobacillosis of lips and mouth Soft-tissue infections Non-specific infections

Bacteroides fragilis

B. fragilis is a Gram-negative bacillus, about 0,4 x 3-5 μm in size, regularly shaped, with a straight or

slightly curved axis and rounded ends. Cells may contain one or more unstained vacuoles that distort the

bacillary body and may be mistaken for spores. It grows well on horse blood agar. After 24 - 48 hours of

incubation, colonies are low convex circular domes, 1-3 mm. in diameter, and semi-translucent or greyish-

white in colour. Most strains are non-haemolytic. B. fragilis is not inhibited by 20% bile, a feature which

distinguishes it from most other members of the genus. Growth of B fragilis is favoured by haemin, in the

absence of which atypical or negative biochemical reactions may be given.

Most strains of B. fragilis are resistant to benzyl penicillin (2 units), neomycin (1mg) and kanamycin (1mg).

Its resistance to penicillin distinguishes B. fragilis from most other Bacteroides spp. The organism is

sensitive to erythromycin (60g) and rifampicin (15g).

Prevotella melaninogenica (formerly Bacteroides)

Provetella melaninogenica is implicated in a variety of infections. Three subspecies are recognised on the

basis of saccharolytic and proteolytic activity- melaninogenica, intermedius and asaccharolyticus.

The cells from surface colonies are short Gram-negative rods, commonly more coccal than cocco-

bacillary in appearance. It grows well but slowly on horse blood agar. After 2-3 days incubation colonies

are 0,5 - 3mm. in diameter, circular, convex or umbonate, opaque and grey, brown or black in colour.

Blackening of the colony, which commences at its centre on about the third day of incubation, extends

towards the periphery to produce a shiny jet-black colony after 5 - 6 days. The development of

pigmentation is often associated with death of the culture. Prevotella melaninogenica is often difficult to

grow and maintain in pure culture although it usually grows well in the presence of other organisms such

as E. coli and B. fragilis. Many strains of P. melaninogenica require vitamin K in addition to haemin for

growth.

No strains of P. melaninogenica reduce nitrates and none grows in the presence of 20% bile.


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All strains of P. melaninogenica are sensitive to penicillin (2 units), erythromycin (60mg) and rifampicin

(15g). The organism is resistant to neomycin (1g) and sometimes to kanamycin (1g).

Bacteroides corrodens

B. corrodens is a Gram-negative bacillus, 0,5 x 1-2 μm in size with an occasional tendency for chain

formation.

It grows well, but slowly, on horse blood agar and produces small (1 mm. diameter), non-haemolytic

colonies after 4 - 5 days incubation. Young (48h) colonies are barely discernible with the naked eye and

present the appearance of pin-point depressions in the agar (pitting of agar). Mature colonies are circular

with entire or slightly undulating margins, low convex or umbonate, and semi translucent and greyish-

white in colour. Colonies of fresh isolates cause pitting of the agar which is best developed after 7 days of

incubation. The ability to pit agar media is commonly lost in stock laboratory strains and is not always

associated with fresh isolates.

Bacteroides corrodens is non-saccharolytic and non-proteolytic. Nitrate is reduced to nitrite and urease

and oxidase are produced. Neither indole nor hydrogen sulphide is formed.

Bacteroides corrodens is sensitive to penicillin (2 units), neomycin (1mg), kanamycin (1mg), erythromycin

(60g) and rifampicin (15g).

Bacteroides oralis

B. oralis forms part of the normal flora in animals. Although it is encountered in infections, its pathogenic

significance is not known.

Cells of B. oralis are 0,5 x 1-2 m in size with a marked tendency to chain formation. It grows well on

horse blood agar producing discreet circular colonies which are 0,5 - 2 mm. in diameter after 48 hours

incubation. Colonies are entire, shiny, translucent and non-haemolytic.

B. oralis ferments glucose, maltose, lactose and sucrose. It is also positive for aesculin and starch

hydrolysis. Unlike B. fragilis it does not ferment arabinose or xylose. The organism is non-proteolytic, does

not reduce nitrates and produces neither indole nor hydrogen sulphide. It does not grow in the presence

of 20% bile, but its growth is slightly enhanced by haemin and Tween 80.

B. oralis is resistant to kanamycin (1mg), but sensitive to penicillin (2 units), erythromycin (60g),

neomycin (1g) and rifampicin (15g).

Bacteroides capillosus

B. capillosus is part of the normal faecal flora of animals but has been encountered in a variety of soft

tissue infections.

Cells vary considerably in size, from 0,5 - 1,5 m wide and 1,5 - 8,0 m in length. Pleomorphism is often

prominent with bent filaments, distorted bacilli and irregular staining.


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Surface colonies on blood agar are 0,5 -2 m. in diameter, circular, entire, low convex, greyish,

translucent and non-haemolytic.

Bacteroides capillosus is non-proteolytic and weakly saccharolytic, fermenting only glucose with formation

of acid. Aesculin is hydrolysed, nitrate is not reduced and neither indole or hydrogen sulphide is produced.

It does grow in the presence of 20% bile.

Bacteroides capillosus is resistant to penicillin (2 units), kanamycin (1 mg) and neomycin (1 mg) but

sensitive to erythromycin (60 g) and rifampicin (15 g).

Bacteroides praeacutus

B. praeacutus forms part of the normal flora of animals but has been implicated in a variety of soft tissue

infections.

Cells of B. praeacutus show considerable variation in size, ranging from 0,5 - 1,5 m wide to 1,5 - 12 m

long. Pleomorphism is common with short chains, filaments and swollen forms.

Colonies on horse blood agar are small (0,5 mm. diameter) after 48 hours incubation, circular and flat with

scalloped or diffuse margins, greyish, translucent and non-haemolytic.

Bacteroides praeacutus is non-saccharolytic. Gelatinase is produced, but more complex proteins are not

attacked. Nitrate is reduced and hydrogen sulphide is produced but indole is not formed. It grows in the

presence of 20% bile.

Genus: Fusobacterium

The fusobacteria are Gram-negative, non-sporing mainly spindle-shaped bacteria and like the bacteroides, many

species are obligate parasites of animals. They are distinguished from the bacteroides by their weakly

saccharolytic activity and by their production of threonine deaminase and indole. The species most commonly

implicated as animal pathogens are F. necrophorum subsp. necrophorum, F. necrophorum subsp. fundiliforme,

F. russi, F. nucleatum, F. necrogenes, F. equinum and F. simiae.

Fusobacterium necrophorum

Fusobacterium necrophorum is a well recognised pathogen of man and a variety of animals. It occurs

normally in the upper respiratory and intestinal tracts. The most important virulence factor is a leukotoxin

that is produced by virulent strains of F. necrophorum. The organism is a cause of infections, especially in

relation to, or derived from the upper respiratory tract.

Two subspecies are recognised, namely, F. necrophorum subsp. necrophorum (formerly F. necrophorum

biotype A) and F. necrophorum subsp. fundiliforme (formerly F. necrophorum biotype B). Of the two F. n.

necrophorum is considered to be the most pathogenic causing calf diphtheria, interdigital phlegmon, and

liver and lung abscessation in ruminants. Occasionally F. n fundiliforme has been isolated from abscesses

in ruminants. A non-pathogenic and non-haemolytic strain that was classified as F. necrophorum biotype

C is now called F. varium.


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It is a Gram-negative bacillus with a tendency to pleomorphism and irregular staining. Cells are 0,5 x 5-10

m in size, the larger ones commonly being curved. Filament formation is common. Fusiform swelling of

bacilli is sometimes seen but free spheroids are rarely developed. In pathological material pleomorphism

may be the predominant feature - coccal, bacillary and filamentous forms all being represented.

Fusobacterium necrophorum grows well on horse blood agar. Colonies are 1 - 2 mm in diameter after 48

hours incubation, circular with scalloped or diffuse margins, high convex or umbonate, with a ridged

surface and semi translucent or opaque, and yellowish in colour. Most strains of the organism are beta-

haemolytic, and many are lipolytic on egg yolk agar.

It is non-saccharolytic although some strains ferment glucose weakly. Some strains produce gelatinase,

but more complex proteins are not attacked. Nitrate is not reduced but indole and hydrogen sulphide are

produced. It does not grow in the presence of 20% bile.

The organism is sensitive to penicillin (2 units), kanamycin (1mg/), erythromycin (60g) and rifampicin

(15g). It is resistant to vancomycin (5g).

F. n. necrophorum haemagglutinates a 15% solution of washed chicken blood cells suspended in saline,

whereas F. n. fundiliforme does not.

A RAPD-PCR using the WIL2 primers will distinguish the subspecies.

Fusobacterium nucleatum

Fusobacterium nucleatum is involved in soft tissue infections in man and animals, especially in relation to

the respiratory tract.

Cells of F. nucleatum are bacilli of classical fusiform appearance, being spindle-shaped, sharply pointed

and occasionally with central or eccentrically placed swellings. Cells are 1 x 5-10 m in size and free

spheroids are commonly seen.

It grows well on horse blood agar. Colonies are 1 - 2 mm. in diameter after 48 hours incubation, irregularly

circular, low convex, greyish, glistening, translucent and non-haemolytic.

Fusobacterium nucleatum is biochemically fairly inactive. Most strains weakly ferment fructose and some

weakly ferment glucose. The organism is otherwise non-saccharolytic, and is entirely non-proteolytic.

Indole is produced, nitrate is not reduced and hydrogen sulphide is not formed. It does not grow in the

presence of 20% bile.

The organism is sensitive to penicillin (2 units), kanamycin (1 mg.), rifampicin (15g), neomycin (1 mg.)

and erythromycin (60 g.) It is resistant to vancomycin (5 g.).

Fusobacterium varium

Fusobacterium varium has been implicated in a variety of soft tissue infections. It is a small Gram-

negative bacillus, 0,5 x 1-2 m in size. Coccobacillary forms are common.


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It grows satisfactorily on horse blood agar. Colonies may appear as mere pin-points or may measure up

to 1 mm. in diameter after 48 hours incubation. Colonies are flat circular disks, greyish, translucent and

non-haemolytic.

The organism grows well in 20% bile. Glucose, fructose and mannose are weakly fermented and some

strains weakly hydrolyse galactose. It is non-proteolytic, indole is produced, nitrate is not reduced and

hydrogen sulphide is not formed.

F. varium is sensitive to penicillin (2 units) and kanamycin (1 mg) but resistant to erythromycin (60 g),

rifampicin (15 g) and vancomycin (5 g).

Fusobacterium mortiferum

Fusobacterium mortiferum is implicated in a variety of soft tissue infections. It is a highly pleomorphic

bacillus. The cells are extremely variable in size, from 0,5 -2 m wide and 1 -10 m in length. Many cells

are grossly irregular in shape, and some are distorted with centrally placed swellings; large "ball" forms

are common.

It grows well on horse blood agar. Colonies are 1-2 mm. in diameter after 48 hours incubation, irregular,

low convex or slightly umbonate, greyish, semi translucent and non-haemolytic.

The organism is moderately saccharolytic, weakly fermenting glucose, lactose, mannose, salicin and

cellobiose. It is non-proteolytic, does not produce nitrate or produce indole or hydrogen sulphide. Aesculin

is hydrolysed and the organism grows well in the presence of 20% bile.

Fusobacterium mortiferum is sensitive to penicillin (2 units) and to kanamycin (1 mg), but it is resistant to

erythromycin (60 mg), rifampicin (15mg) and vancomycin (5 mg).

Genus: Dichelobacter

The only species of importance is Dichelobacter nodosus (formerly Bacteroides nodosus). Both virulent and

benign strains cause of foot rot in sheep where it is usually associated with other pathogens such as

Fusobacterium necrophorum, Trueperella (Arcanobacterium) pyogenes and Treponema penortha. Benign

strains are also known to cause occasional infections of the interdigital skin of goats, cattle and pigs.

Dichelobacter nodosus is a Gram-negative, fairly large (1,7 x 3-6mm), slightly curved, non-motile rod which is

often swollen at one or both ends. They occur singly or occasionally in pairs. Isolations of D. nodosus are most

likely to be made when Gram staining shows its presence in active foot rot lesions. The typical rods are in many

cases surrounded by radially disposed Gram-negative bacilli.

Samples for culture should be taken from necrotic material at the junction of healthy and separating tissue in the

feet of sheep, and transported in PRAS Cary-Blair or Stuart’s medium to the laboratory. Direct inoculation onto

hoof agar has proven to be a very sensitive method of isolation. Those portions of necrotic material showing

greatest numbers of D. nodosus on smears should be shaken vigorously for 30 seconds in 10ml. 0,25M sterile

sucrose and the suspension then seeded on anaerobic media, especially hoof agar.


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The ability to produce protease is associated with virulence. This is tested by the proteolytic index, gelatin-gel

protease thermostability test, the elastase test, the zymogram test and the protease ELISA.

Three basic colonial types are described for D. nodosus.

B-type: papillate or beaded (most pathogenic) from ovine footrot.

M-type: mucoid (less pathogenic) from non-invasive infections in sheep.

C-type: circular (non-pathogenic) and resulting from repeated passage in media.

The colonies, generally, are greyish-white and 0,5 - 3,0 mm. diameter in 3-7 days.

It is totally asaccharolytic and do not ferment any sugars. It is also negative for aesculin hydrolysis, catalase

production and urease and indole negative. It does not grow in 20% bile. It is however proteolytic and is

gelatinase and caseinase positive.

Genus: Actinomyces

Actinomyces occurs as part of the indigenous oral flora of mammals. A. israelii, A. eriksonii and A. odontolyticus

occur normally only in the human mouth while A. bovis appears to be normally confined to the bovine mouth.

Actinomyces species of veterinary importance A. bovis, A. suis, A. viscosus, A. hordeovulneris and A. israelii.

As a group the Actinomyces have been aptly described as "facultative but preferentially anaerobic organisms",

whose growth is usually enhanced by carbon dioxide.

Fluorescent antibody techniques provide the most rapid and specific diagnostic approach, on the basis of which

6 serological groups are recognised.

Actinomyces viscosus will not be dealt with in this section as they grow equally well in air + CO2 and are

therefore regarded as facultative anaerobes. Differential characteristics of the anaerobic animal pathogens and

other species encountered in clinical material are summarised in Table 4.

Actinomyces bovis

It is the cause of bovine actinomycosis (lumpy jaw), as well as poll evil and fistulous withers (supra-atlantal

and supraspinous bursitis, respectively) in horses.

“Sulphur” granules are the best specimens for direct examination in infections caused by A. bovis. If

smears are made from the granules and stained with the Gram stain, delicate, Gram-positive, branching

filaments can be observed. Occasionally short filaments or pleomorphic diphtheroidal forms may

predominate.

Actinomyces bovis is a carbon dioxide-dependent facultative organism. On brain-heart infusion agar micro

colonies are about 0,06 mm. in diameter after 24 hours incubation, circular, entire and flat, with a smooth

or granular appearance. Macro colonies are 0,5-1mm. in diameter after 7-14 days incubation. They are

circular, entire, low to high convex, opaque and white with a smooth or granular surface. The organism is

non-haemolytic on horse blood agar.


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Table 4: Actinomyces of animal importance and others isolated regularly from clinical specimens

TE

ST

S

Acti

no

myces b

ovis

A. is

raelii

A. o

do

nto

lyti

cu

s

A. h

ow

ellii

A. n

aeslu

nd

i

A. vis

co

su

s

Tru

ep

ere

lla

(Arc

an

ob

acte

riu

m)

pyo

gen

es

A. h

ord

eo

vu

lneri

s

Catalase - - - + - + - +

Urease - - - 0 + v - -

Gelatin hydrolysis - - - 0 - - + 0

Nitrate reduction - V + 0 + V - -

Mannitol fermentation - V - - - - - -

Salicin fermentation - + V - V V - 0

Raffinose fermentation - + - + + + - W

Xylose fermentation - + V + V + V +

Aero-tolerance M or An M or An M or An F F F F M An

V=variable W=weak positive M=carboxyphylic An=anaerobic F=grows aerobically and anaerobically

Genus: Eubacterium and Propionibacterium

The only species of veterinary importance occurring within the genera Eubacterium and Propionibacterium is

Eubacterium suis which causes pyelonephritis in sows. Eubacterium suis is a pleomorphic Gram-positive rod in

palisade and Chinese letter formation when viewed microscopically and stained with Gram’s stain. Colonies on

horse blood agar are non-haemolytic, 2-3 mm. in diameter, grey, smooth, circular with a shiny centre and a dull

edge. Although, with the exception of Eubacterium suis, it is adequate to identify these bacteria to only genus

level, care must be taken not to confuse them with non-sporing Clostridia or Actinomyces.

THE CLOSTRIDIA

The Clostridium species are large (0,3-1,3 x 3-10 m), Gram-positive, anaerobic, endospore producing rods

which usually bulge the cell. All the pathogenic species are straight rods except C. spiriforme that is curved or

spiral. Cells from older cultures have a tendency to decolourise and stain Gram-negative. Most species are

motile by peritrichate flagella except C. perfringens that is non-motile and also produces a capsule in animal

tissues.


14 | P a g e

The clostridia are fermentative, oxidase-negative and catalase-negative. The strictness of anaerobic

requirements varies among the species but they all prefer 10 per cent CO2. Most clostridia require enriched

media that include amino acids, carbohydrates, vitamins and blood or serum.

Optimum growth of most of the pathogenic species occurs at 37C. There are over 80 Clostridium species of

which about 11 are of veterinary importance. Most of the pathogenic species produce one or more exotoxins of

varying potency.

Habitat and pathogenicity

The clostridia have a wide distribution in soil, fresh water and in marine sediments throughout the world,

although some species or types are present only in localised geographical areas. Many of the pathogenic

clostridia are normal inhabitants of the intestinal tract of animals and man, and often cause endogenous

infections. Other clostridia are more commonly present in the soil and cause exogenous infections from wound

contamination or by ingestion.

Table 5: Diseases caused by the pathogenic clostridia

Clostridium species Hosts Diseases

NEUROTOXIC SPECIES Clostridium tetani C. botulinum (types A-F) C. argentinense

Horses, ruminants, humans etc. Man and other animals Humans (Argentina)

Tetanus Botulism Botulism

HISTOTOXIC SPECIES C. chauvoei C. septicum C. novyi type A type B type C C. haemolyticum (C .novyi type D) C. sordellii C. colinum

Cattle, sheep, pigs Cattle, sheep, pigs Sheep Chickens Sheep Cattle and sheep Sheep, (cattle, pigs) Water buffalo Cattle, (Sheep) Cattle, sheep, horses Game birds, young chickens, turkey poults

Blackleg Malignant oedema Braxy Necrotic dermatitis Big-head of rams Gas gangrene Black disease Osteomyelitis Bacillary haemoglobinuria Gas gangrene Quail disease (ulcerative enteritis)

ENTEROTOXAEMIAS C. perfringens type A type B type C type D

Humans Lambs Lambs (< 3 wks) Neonatal calves and foals Piglets, lambs, calves, foals Adult sheep Chickens Sheep (all ages except neonates) (goats, calves)

Food poisoning, gas gangrene Enterotoxaemic jaundice Lamb dysentery Enterotoxaemia Haemorrhagic enterotoxaemia Struck Necrotic enteritis Pulpy kidney disease, FSE


15 | P a g e

type E Calves and lambs (rare) Enterotoxaemia

SPECIES ACTIVATED BY ANTIMICROBIAL DRUGS C. spiriforme C. difficile

Rabbits Rabbits and guinea-pigs Foals and pigs Humans, hamsters, rabbits etc. Dogs, foals, pigs, lab. animals

Possible role in mucoid enteritis Spontaneous and antibiotic-induced diarrhoea Enterocolitis (natural) Antibiotic-induced enterocolitis Naturally occurring diarrhoea

Pathogenicity

Clostridia mainly cause disease through the production of potent exotoxins. These toxins vary in potency and

effect. Diseases caused by clostridia are listed in Table 5. Pathogenic Clostridium species are divided into the

following groups based on the type of toxin produced:

Neurotropic clostridia (C. tetani and C. botulinum) that produce potent neurotoxins but are non-invasive

and colonise the host to a very limited extent.

Histotoxic clostridia (C. chauvoei, C. septicum, C. novyi, C. haemolyticum, C. sordellii, C. perfringens

type A and C. colinum.) that produce less potent toxins than the first group but are invasive. This

includes the gas-gangrene producing clostridia.

Clostridia that produce enterotoxaemias (C. perfringens type A-E). Enterotoxins are formed in the

intestines and absorbed into the bloodstream producing a generalised toxaemia.

Clostridia (C. difficile and C. spiriforme) producing enteric diseases that can be antibiotic-induced.

Specimens

Specimens should be taken from recently dead animals as bacteria such as C. perfringens, C .septicum and

enteric facultative anaerobes are rapid post-mortem invaders. For isolation, blocks of affected tissue (20 mm x

20 mm x 20 mm) or fluids in air-free containers should be collected when possible rather than swab-taken

samples that expose the clostridia to the lethal action of atmospheric oxygen. Commercial systems are

satisfactory and the swab may be placed in Cary-Blair transport medium. In some clostridial diseases, such as

the enterotoxaemias, the toxin is required for diagnosis. The contents of the small intestine is collected from a

recently dead animal and submitted to the laboratory as soon as possible, as the toxins are labile.

Direct microscopy

Gram-stained smears from specimens are used to observe the morphological types of organisms present. The

fluorescent antibody (FA) stain is useful for specific identification.

1. Gram-stained smears from affected tissues may reveal large Gram-positive rods that tend to decolourise

easily when sporing C. spiriforme is an exception being curved or helical. The characteristic 'drumstick'

forms of C. tetani, due to the spherical forms being terminal and bulging the cell, may be seen in necrotic

material from wounds associated with tetanus. This is suggestive, but by no means conclusive, as other

clostridia, such as C. tetanoides and C. tetanomorphum have a similar morphology. In cases of suspected

enterotoxaemia, the presence of large numbers of fat Gram-positive rods in a smear of the small intestinal

mucosa, from a recently dead animal, is presumptive evidence of the condition.


16 | P a g e

2. The direct fluorescent antibody test is used routinely for disease associated with C. chauvoei, C. septicum,

C. novyi and C. sordellii as fluorescein-labelled antiserum can be obtained commercially. Affected tissue as

well as a piece of rib containing bone marrow (about 14cm long) is a useful specimen. A bacteraemia

usually occurs with these clostridial diseases so the bacteria would be expected to be present in the bone

marrow. This tissue has the added advantage of giving low background auto fluorescence and being one of

the last tissues to be invaded, post-mortem, by bacteria such as C. septicum.

Isolation procedures

Freshly prepared or pre-reduced blood agar is necessary for the isolation of clostridia, but special media are

required for isolation of more fastidious species such as C. chauvoei, C. haemolyticum and C. novyi types B and

C. A blood and MacConkey agar plate inoculated and incubated aerobically will detect any aerobic pathogens

that may be present and also indicate the degree of contamination of the specimen by facultative anaerobes.

Liquid and semi-solid media with a low REDOX potential such as cooked meat broth and thioglycollate medium

can be used to grow and maintain pure cultures of the clostridia. They are of limited use for primary inoculation

as any fast-growing anaerobes or facultative anaerobes will outgrow the Clostridium species of interest.

Immediately before inoculating broth media, boiling to expel absorbed oxygen should be undertaken, followed by

rapid cooling to 37C.

Most of the pathogenic species are strict anaerobes, the exception being C. perfringens that is relatively aero

tolerant. Isolation should however be performed under strict anaerobic conditions with the atmosphere

containing 2 - 10 % CO2 as this enhances the growth of clostridia.

Biochemical identification

Clostridia can be identified to species level by biochemical testing. Toxin types of, for example, C. perfringens or

C. botulinum can only be successfully identified by toxin-antitoxin neutralization tests (later in this section) or

PCR.

Lactose-egg-yolk-milk agar medium provides information not only on the production of lecithinase and lipase, but

also on lactose fermentation and proteolytic activity as evidenced by the digestion of milk. It also enables a

presumptive diagnosis of some anaerobes to be made from a single plate culture (Table 6).

Some differential characteristics of the most important species of Clostridium are shown in Table 7.

Serological identification

Neutralisation or protection tests are used to specifically identify the toxin(s) present and hence the clostridial

pathogen involved in the disease. These procedures are most commonly used in tetanus, botulism and in the

enterotoxaemias caused by C. perfringens. The methods used are described in the following section.


17 | P a g e

Table 6: Reactions of the most important clostridial species on lactose-egg-yolk-milk agar

Ag

en

t

Le

cit

hin

ase

Lip

ase

La

cto

se

Pro

teo

lysis

C. perfringens C. barati C. bifermentans C. sordellii C. botulinum A C. botulinum B C. botulinum C C. botulinum D C. botulinum E C. botulinum F C. botulinum G C. sporogenes C. novyi type A C. novyi type B C. novyi type C C. haemolyticum C. cadaveris C. cochlearium C. tetani C. tertium C. fallax C. chauvoei C. septicum C. sphenoides C. butyricum C. histolyticum

+ + + + - - - - - - - - + + - + - - - - - - - - - -

- - - - + + + + + + + + + - - - - - - - - - - - - -

+ + - - - - - - - - - - - - - - - - - + + + + + + -

- - + + + + or - - - - + or - + + - - - - - - - - - - - - - +


18 | P a g e

Table 7: Differential biochemical characteristics of the most important clostridia

T

ES

T

C.p

erf

rin

gen

s

C. sep

ticu

m

C .ch

au

vo

ei

C. n

ovyi A

C. n

ovyi B

C. h

aem

oly

ticu

m

C. so

rdellii

C. b

iferm

en

tan

s

C .b

otu

lin

um

C. te

tan

i

C. sp

oro

gen

es

C. co

lin

um

C .b

ara

ti

C. cad

averi

s

C .co

ch

leari

um

C. te

rtiu

m

C. fa

llax

C

. sp

hen

oid

es

C. b

uty

ricu

m

C. h

isto

lyti

cu

m

C. n

ovyi C

Nitrate + + + - + - - - - - - - v - - v v v - - -

Indole - - - - - + + + - + - - - + v - - v - - -

Glucose + + + + + + + + + - + + + + - + + + + - +

Maltose + + + + v - + + v - + + w - - + + + + - +

Lactose + + + - - - - - - - - - + - - + + + + - -

Salicin v + v - - - - + v - v + w - v + - w + - -

Sucrose + - + - - - - - v - v + + - - + + - + - -

Urease - - - - - - + - - - - - - - - - v - - - -

Gelatin + + + + + + + + + + + - - + - - - - - + +

+ = positive - = negative w = weak positive v = variable reactions


19 | P a g e

Individual clostridia

Clostridium chauvoei

C. chauvoei grows poorly on blood agar, but good growth may be obtained by supplementing the

medium with liver extract. Identification of the organism is greatly facilitated by incorporating sheep

blood into agar, resulting in colonies that are surrounded by a wide zone of haemolysis. It produces

oval subterminal spores together with numerous pleomorphic forms. Unlike C. septicum it ferments

sucrose but rarely salicin.

Clostridium novyi

C. novyi is divided into four types based on the production of the extracellular toxins. C. novyi type D

has recently been renamed C. haemolyticum. The distribution of the two main lethal toxins is shown in

Table 8. Unlike the other types of C. novyi, C. novyi type A can grow on normal blood agar and

produces large irregular colonies with a rhizoidal edge surrounded by a large zone of haemolysis. The

organisms are large Gram-positive rods with oval to cylindrical sub terminal spores not swelling the

sporangium.

Types B, C, and C. haemolyticum are extremely demanding, both in their nutritional requirements and

in their tolerance of oxygen and the medium of Moore should be used. The medium should be

prepared freshly and plates pre-incubated in an anaerobic gas mixture prior to inoculation. Provided

that such pre-incubated plates are quickly streaked and re-established under anaerobic conditions,

good growth ensues.

Table 8: Toxins produced by the different types of C. novyi

TYPE Alpha toxin Beta toxin

C. novyi A C. novyi B C. novyi C C. haemolyticum

+ + - -

- + -

+++

Colonies are smaller than those of C. novyi type A and usually rhizoidal in nature. The success of this

medium is largely attributable to the presence of cysteine and dithiothreitol. Under the usual conditions

of medium preparation, pouring and inoculation, oxidation of the cysteine in the medium proceeds

rapidly so that cysteine is likely to be largely inactivated by the time the cultures are set up.

Dithiothreitol protects the free thiol group of cysteine from oxidation, thus preventing its reducing activity

Clostridium septicum

Clostridium septicum grows readily on blood agar and, because of its tendency to spread, the agar

concentration is increased in order to obtain isolated colonies. Colonies of C. septicum are usually

irregular with a rhizoidal edge, but smooth, round colonies are produced by some strains.

Morphologically, the organism has characteristic filamentous forms and produces oval subterminal


20 | P a g e

spores. It can be differentiated from C. chauvoei on the basis of antiserum neutralization tests,

fluorescent staining, and fermentation of salicin but not sucrose.

C. sordellii

C. sordellii grows well on stiff agar and produces irregular colonies. Colonies lose their translucent

appearance and become white on aging due to the production of spores. The organisms are largely

Gram-positive rods with cylindrical spores not swelling the sporangium. The organism can be

differentiated from the closely related C. bifermentans by production of urease.

Identification of C. septicum, C. chauvoei, C. novyi and C. sordellii

1. Make 5 smears of the affected muscles and subcutaneous tissues. Liver impression smears may

also be made. Samples should be taken soon after death as false positives can be obtained with

decomposed specimens, particularly C. septicum and C. novyi.

2. Stain one of the smears with Gram’s stain and if no clostridia are observed, discontinue the test.

3. If clostridia are found, stain with Clostridium FA conjugates (see below).

4. Cultures can be undertaken in conjunction with the FA stains, particularly when it is important to

establish whether there has been a vaccine breakdown.

5. Two Columbia blood agar plates are streaked, one incubated aerobically and the other

anaerobically, both at 37°C for 48 hours.

Fluorescent antibody test

Specific fluorescein-labeled antisera are used for identification of C. chauvoei, C .septicum, C. novyi

and C. sordellii as follows:

1. Label two slides with a diamond pencil or wax pencil, pencil if frosted glass and circle two areas on

each slide with a diamond pencil (one circle per conjugate).

2. Smear a portion of the suspected lesion onto a microscope slide and leave until air dry.

3. Fix by immersing in cold 1:3 acetone-methanol solution (1 ml acetone plus 3 ml methanol) for 20

minutes.

4. Allow to air-dry by placing the slides in an incubator at 37°C for 10 minutes.

5. Place one drop of the appropriate conjugated antiserum on each circle, using a separate plastic

inoculation loop or pipette per conjugate, and spread gently.

6. Leave in a covered moist chamber, in an incubator at 37°C for 45 minutes. A large Petri dish

containing moistened filter paper is quite adequate.

7. Rinse off the excess of the reagents with 0,15M PBS, pH 9.

8. Place in a glass beaker or Petri dish and cover the slide/s with PBS and stir gently for 10 minutes,

using a magnetic stirrer.

9. Decant the PBS.

10. Allow the slides to air-dry. Cover the smear with a cover-slip using phosphate-buffered glycerol (9

parts glycerine to 1 part PBS, pH 8,4) as mounting fluid.


21 | P a g e

11. The slide is then examined with a conventional fluorescence microscope employing the filters

routinely used in fluorescence microscopy with FITC as conjugate. Switch on the microscope 10

minutes before use. Fluorescence is specific and the species is positively identified if fluorescence

(apple-green rods) occurs with the specific antiserum.

12. Positive control smears should be prepared with each batch of slides.

Clostridium tetani

C. tetani grows readily on blood agar, produces haemolysis and tends to have a spreading, swarming

growth. If 3% agar is used, separate colonies with a rhizoidal edge can be obtained. The organism

produces spherical terminal spores and the typical drumstick appearance is seen on smears. There are

10 serological types of C. tetani, based on flagella antigens, but the neurotoxin is antigenically uniform.

Injection of culture or culture filtrate into mice produces a characteristic spasm.

Tetanus toxin test

Suitable samples are tissue, serum or culture supernatant.

Preparation of samples

Macerate tissues and add an equal amount of Normal saline. Leave to stand for at least 3 hours or

overnight in the refrigerator. Serum is used as is.

Test

Centrifuge the sample or culture until the supernatant is clear. Collect the supernatant and mix 0,5 ml

with 0,5 ml of antitoxin diluted to 20 IU/ml. For the control, mix 0,5 ml supernatant with 0,5 ml normal

serum. Allow both solutions to stand for 30 minutes at room temperature.

Inject each of 2 mice intramuscularly in the back leg with 0,5 ml of test mixture and each of 2 mice

with 0,2 ml of the control. Observe for 4 days.

Results

Positive: Mice that have received antiserum stay alive, and those that did not die after showing

spasms and paralysis, especially at the injection site.

Negative: All 4 mice survive and do not show signs of tetanus.

Notes and precautions

Only use mice as they are the most susceptible of all the laboratory animals

Overnight cultures usually contain enough toxin. Cultures that are weak toxin producers can be

incubated for up to I week before testing.

Do not inject the mice subcutaneously or intravenously as delayed or irregular results could occur.


22 | P a g e

If the toxin levels in the samples are very high, all the mice may die but the mice receiving

antiserum will live longer. Dilute the supernatant 1:10 and 1:100 and retest.

Clostridium perfringens

This organism grows well on blood agar, producing smooth, round, glistening colonies. Colonies are

surrounded by a double-zoned haemolysis on blood agar because of theta toxin which produces a

clear zone of haemolysis which is surrounded by a zone of partial haemolysis produced by alpha toxin.

The organism is divided into five types, A - E, based on the extracellular production of alpha, beta,

epsilon and iota toxins. The toxins, and thus the type of C. perfringens can be identified both in culture

and in intestinal contents.

Clostridium perfringens toxin identification tests

The PCR may be used to establish whether alpha, beta or epsilon toxins are present (Table 9). It takes

one to two days to do, is accurate and can be done on cultures or samples.

Table 9: Toxin production by C. perfringens

C. perfringens type Toxin/s

A Alpha

B Alpha, beta, epsilon, iota

C Alpha, beta

D Alpha, epsilon

Should a PCR not be available, animal tests may be used.

Intestinal contents or culture supernatant are centrifuged to obtain a clear supernatant containing the toxin. This is mixed with varying amounts of antitoxins to neutralize the toxins and either injected into mice (intravenously) or into guinea pigs (intradermally). Some of the test mixtures contain trypsin to activate epsilon toxin. There are usually 8 mixtures in total.

The following day the mice are examined for deaths and/or the guinea pigs for skin lesions. From the

pattern of deaths or lesions, the toxin types can be determined.

Clostridium botulinum

C. botulinum is divided into types A - G, based on the production of specific lethal toxins. The name of

C. botulinum type G has been changed to C. argentinense. Intoxication results from the consumption of

foodstuffs containing toxin as a result of growth of the organism.

Types A, B, F and C. argentinense are responsible for classical botulism in humans, type E for human

botulism from marine sources, and type C and D for botulism in animals. Types A, B, F and C.

argetinense are all, except for a few strains of type B, strongly proteolytic. C. botulinum types C, D and

E resemble each other morphologically and culturally. They consist of large rods with oval to cylindrical


23 | P a g e

terminal spores not swelling the sporangium, and they produce irregular colonies with a granular

surface and rhizoidal margin. All are non-proteolytic.

Recovery of C. botulinum from food is not sufficient to establish a diagnosis of botulism. Food can be

contaminated without toxin being produced. A diagnosis is based upon the demonstration of a sufficient

amount of toxin.

Isolation

Great care should be taken when working with food or cultures that may contain botulinum toxin. To

culture C. botulinum from the suspected food, several samples are suspended in a small amount of

saline and heated at 65 - 80C for 30 minutes to eliminate non-sporing bacteria. Blood agar plates are

inoculated and incubated anaerobically. If the organism is identified biochemically as C. botulinum,

tests are conducted for toxigenicity. Filtrates are prepared from 5 - 10 day old cultures incubated at

30C in cooked meat medium.

Clostridium botulinum toxin test

Suitable samples are rumen and caecum contents and feed, but other samples such as intestinal

contents from any part of the intestine, liver or serum may be used. Toxin levels in the serum are

usually low.

Sample preparation

Organs such as liver should be minced or finely chopped. All samples such as intestinal contents are

placed in an equal amount of brain-heart infusion broth (BHI) or saline. Feed should be left in the broth

for at least 3 hours or preferably overnight to extract the toxin. 6 - 10ml of the supernatant is then used

for the test.

Serum is used without diluting it. At least 12 ml of serum is required for the complete test.

Screening test

1. Centrifuge the samples at 3 000 g for 15 minutes.

2. Collect the clear supernatant fluid.

3. Thoroughly mix 2 ml of supernatant fluid with 0,4 ml of penicillin/streptomycin and let stand at

room temperature for 30 minutes.

4. Store the rest of the supernatant fluid in the refrigerator.

5. Inject 0,5 ml of the supernatant/antibiotic mixture intraperitoneally into 2 adult mice. Store the rest

of the mixture in the refrigerator.

6. Serum is treated with pen/strep similar to supernatant and injected into mice twice a day at a dose

of 0,5 ml for a maximum of 4 days, until clinical signs or death occurs.

7. Examine the mice daily for 4 days for typical clinical signs. If the mice show a wasp waist or die

showing a hollow abdomen and enteritis, proceed to the definitive test. If the mice are negative on


24 | P a g e

the second day, they should again be injected with the mixture. If the mice remain negative for 4

days, the test is reported as negative.

Definitive test

Use the stored supernatant, and prepare the following suspensions:

1. 1 ml supernatant + 0,2 ml pen/strep + 0,2 ml D antiserum

2. 1 ml supernatant + 0,2 ml pen/strep + 0,2 ml C antiserum

3. 1 ml supernatant + 0,2 ml pen/strep + 0,2 ml normal serum

4. 1 ml supernatant heated at 100°C for 10 minutes.

5. Allow the suspensions to stand at room temperature for 30 minutes and inject mice in the same way

as above. If the mice remain negative they may be injected twice more on 2 consecutive days.

Serum samples are treated in the same fashion.

Possible results

Type C: Groups 1 and 3 are dead or show typical clinical signs and 2 and 4 are healthy.

Type D: Groups 2 and 3 are dead or show typical clinical signs and 1 and 4 are healthy.

If the results differ from this, the following may apply.

Group 3 is dead or shows typical clinical sings and groups 1, 2 and 4 are healthy. There may be

too little toxin present. Dilute the C and D antisera 1:10 and 1:100 with normal serum and repeat

the definitive test.

Groups 1, 2 and 3 are dead or show typical clinical signs and group 4 are healthy or dead. There

may be too much toxin or there may be a different toxin such as ionophore present. Dilute the

supernatant 1:10 and 1:100 and repeat the definitive test. Examine the mice more frequently so

that clinical signs as well as deaths are noted. If the results remain the same, there is probably

another toxin present.

If all the mice merely die without showing clinical signs, even if group 4 (botulinum toxin destroyed

by heat) dies, there is probably another toxin present.

Notes and precautions

The toxin may be present in the material in a protoxin form that needs activation by trypsin to become

toxic. This is more important if types B or E are suspected. It is not necessary to add trypsin to intestinal

contents. As farm animals usually are affected by toxins C or D and the toxin is usually present in large

amounts, veterinary samples are not usually treated with trypsin. Trypsin may be added to the

suspension at the same time as the broth or saline is added.


25 | P a g e

REFERENCES

1. Carter,G.R and Cole,J.R. Jr. Diagnostic Procedures in Veterinary Bacteriology and Mycology,

1990. Academic Press. ISBN 0-12-161775-0.

2. Donahue,J.M. Non-spore-forming Anaerobic Bacteria, In: Diagnostic Procedures in Veterinary

Bacteriology and Mycology (fifth ed.) 1990, edited by G.R.Carter and J.R.Cole, Jr. Academic Press.

ISBN 0-12-161775-0.

3. Quinn,P.J; Carter,M.E; Markey,B & Carter, G.R. Non-spore-forming Anaerobic Bacteria, In Clinical

Veterinary Microbiology,1994.Wolfe Publishing.ISBN 0 7234 1711 3.

4. Willis, A.T. Anaerobic Bacteriology: Clinical and Laboratory Practice (third edition),1977; Butterworths

and Co.; ISBN0-407-00081-X.

5. Palmer MA (1993) A gelatin test to detect activity and stability of proteases produced by

Dichelobacter (bacteroides) nodosus Veterinary Microbiology 36:113-122

6. Stewart DJ, Peterson JE, Vaughan JA, Clark BL, Emery DL, Caldwell JB and Kort AA (1986b)

The pathogenicity and cultural characteristics of virulent, intermediate and benign strains of

Bacteroides nodosus causing ovine footrot. Australian Veterinary Journal 63:317-326


26 | P a g e

APPENDIX: MEDIA AND REAGENTS FOR THE ISOLATION OF ANAEROBIC BACTERIA

1. VL (Viande-Levure) broth

Tryptone NaCl Beef extract Yeast extract Cysteine HCl Agar Distilled water.

10g. 5g. 2g. 5g. 0,4g 0,6g 1000ml

Heat to dissolve, adjust pH to 7,2-7,4, dispense,

sterilise by autoclaving at 115°C for 10 minutes.

This is a most useful basal broth from which an

almost complete range of fluid and solid media for

the identification of anaerobes can be prepared.

Sugars (filter sterilised 10% solutions of the

various sugars) are added after the broth has

been autoclaved.

Agar can be added at 15g /liter for plate media.

2. Egg yolk agar.

Prepare an egg yolk emulsion by mixing egg yolk

with an equal volume (about 20 ml. /egg yolk) of

sterile saline. Add the emulsion to sterile molten

VL agar base (at 50 - 55°C) in the proportion of

10%. Pour plates immediately.

3. Milk agar.

Melt the required amount of VL agar base and

cool to 55°C. Add skimmed milk to a concentration

of 25%. Pour plates immediately.

Skimmed milk is prepared from fresh cow's milk by

centrifugation to separate the cream. The milk is

pipetted and distributed in 20ml volumes in screw-

capped bottles and autoclaved at 121°C for 20

minutes.

This medium is used for the detection of

proteinase activity.

4. Gelatin agar.

Prepare the required amount of VL agar and add

0,4% gelatin.

Sterilise by autoclaving at 115°C for 10 minutes.

This medium is used for the detection of

gelatinase activity.

5. Bile broth.

This is VL broth containing 1% glucose and 20%

ox gall (Oxoid), dispensed in capped test tubes. It

is used to determine if growth is inhibited or

stimulated by bile.

6. Indole production.

Indole is detected in cultures growing in trypticase

nitrate broth. On the addition of Kovac's reagent to

an aliquot of the culture, a dark red colour in the

amyl alcohol surface layer constitutes a positive

indole test.

7. Nitrate reduction.

Nitrate reduction is tested for in cultures growing in

trypticase nitrate broth. On addition of the 2 nitrate

reagents to an aliquot of the culture, a pink colour

developing in the culture indicates the presence of

nitrite.


27 | P a g e

8. Urea medium.

To 100ml. of sterile VL broth base add aseptically

5 ml. of a 40% filter sterilised urea solution. Mix

well and distribute in sterile capped test tubes. On

the addition of phenol red (yellow at pH 6,8 and

red at pH 8,4) to the culture, a pink colour

indicates hydrolysis of urea.

9. Hydrogen sulphide production.

A lead acetate strip is wedged in the top of a

culture tube of VL broth, VL glucose broth or

cooked meat medium. Blackening of the strip

indicates a positive reaction.

10. Aesculin hydrolysis.

For the aesculin hydrolysis test either VL broth or

agar medium may be used with the addition of

0,01% aesculin and 1% ferric ammonium citrate.

A positive reaction in the broth is indicated by

blackening of the medium and on the agar

medium by blackening of the colonies.

11. Tryptose-sulfite-cycloserine (TSC) agar

For the selective isolation of Cl. perfringens

Tryptose

Yeast extract

Soytone

Ferric ammonium citrate

Sodium metabisulfite

Agar

Distilled water

15 g

5 g

5 g

1 g

1 g

20g

900ml

Heat with agitation to dissolve. Adjust pH to

7.6±0.2. Autoclave 15 minutes at 121°C.

Cool in a water bath to 50°C.

Add 80 ml of D-cycloserine solution (1 g in 200

ml distilled water).

Add 80 ml of 50% egg yolk emulsion.

Pour plates.

Positive: Black colonies surrounded by an

opaque white zone are indicative of lecithinase

activity.

Identification of anaerobic bacteria - AfriVIPApplied Veterinary Bacteriology and Mycology:...

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