TABLE OF CONTENTS - African Veterinary Information … · Web viewActinomyces bovis It is the cause of bovine actinomycosis (lumpy jaw), as well as poll evil and fistulous withers

Applied Veterinary Bacteriology and Mycology: Identification of anaerobic bacteria

Applied Veterinary Bacteriology and Mycology:Identification of anaerobic bacteria

Author: Dr. J.A. PicardLicensed under a Creative Commons Attribution license.

TABLE OF CONTENTSINTRODUCTION............................................................................................................................................................. 2

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

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

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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INTRODUCTIONAnaerobic 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 ANAEROBESA 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 greenOleandomycin

30,0 mg/1,8 mg50 mg

Bacteroides NeomycinVancomycin

10 mg750 g

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

Clostridium

Sodium azideSorbic acid/Polymyxin BPhenyl ethyl alcoholKanamycin

20 mg.0,12 g/2,0 mg0,25 g10 mg

Cl. perfringens SulphadiazineNeomycin

10 mg10 mg

FusobacteriumCrystal violet / StreptomycinKanamycinNeomycin

1,0 mg/1,0 mg7,5 mg10mg.

Fusobacterium and Veillonella. Polymyxin 1000 unitsGram-positive non-sporing anaerobes Vancomycin 750 g

THE NON-CLOSTRIDIAL ANAEROBESThe 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

ActinomycesBifidobacteriumEubacteriumLactobacillusPropionibacterium

PeptococcusPeptostreptococcus

Gram-Negative

BacteroidesFusobacteriumDichelobacterPrevotella

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) DiseaseActinomyces bovis

A. suis

A. viscosus

A. hordeovulneris

A. israelii

Dichelobacter (Bacteroides) nodosus

CattleHorses

Pigs

Dogs

Dogs

Humans and rarely pigs and cattle

SheepGoats, 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 actinomycosisBovine 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

CattlePigs

Dogs, cats, horses, cattle.

Cattle

Cattle, sheep, dogs and cats.Cattle

Sheep

Pigs

Horses

ChickensRabbits

Cats

Several species

Diarrhoeal diseaseMastitisAbscesses

Osteomyelitis

Foot rot

OsteomyelitisCalf diphtheria, liver abscesses, metritis, cellulitis, mastitis.Foot abscess, interdigital dermatitis, lip and leg ulcers'Bull - nose', necrotic enteritis, liver abscessThrush involving the frog, necrobacillosis of lower limbsAvian diphtheriaNecrobacillosis 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

TEST

S

Actin

omyc

es b

ovis

A. is

rael

ii

A. o

dont

olyt

icus

A. h

owel

lii

A. n

aesl

undi

A. v

isco

sus

True

pere

lla

(Arc

anob

acte

rium

)

pyog

enes

A. h

orde

ovul

neris

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 CLOSTRIDIAThe 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.

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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 DiseasesNEUROTOXIC SPECIESClostridium tetani

C. botulinum (types A-F)

C. argentinense

Horses, ruminants, humans etc.

Man and other animals

Humans (Argentina)

Tetanus

Botulism

BotulismHISTOTOXIC SPECIESC. chauvoei

C. septicum

C. novyitype A

type B

type C

C. haemolyticum(C .novyi type D)C. sordellii

C. colinum

Cattle, sheep, pigs

Cattle, sheep, pigsSheepChickens

SheepCattle and sheep

Sheep, (cattle, pigs)

Water buffalo

Cattle, (Sheep)

Cattle, sheep, horses

Game birds, young chickens, turkey poults

Blackleg

Malignant oedemaBraxyNecrotic dermatitis

Big-head of ramsGas gangrene

Black disease

Osteomyelitis

Bacillary haemoglobinuria

Gas gangrene

Quail disease(ulcerative enteritis)

ENTEROTOXAEMIASC. perfringenstype A

type B

type C

type D

HumansLambs

Lambs (< 3 wks)Neonatal calves and foals

Piglets, lambs, calves, foalsAdult sheepChickens

Sheep (all ages except neonates)(goats, calves)

Food poisoning, gas gangreneEnterotoxaemic jaundice

Lamb dysenteryEnterotoxaemia

Haemorrhagic enterotoxaemiaStruckNecrotic enteritis

Pulpy kidney disease, FSE

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type E Calves and lambs (rare) Enterotoxaemia

SPECIES ACTIVATED BY ANTIMICROBIAL DRUGS

C. spiriforme

C. difficile

RabbitsRabbits and guinea-pigs

Foals and pigsHumans, hamsters, rabbits etc.Dogs, foals, pigs, lab. animals

Possible role in mucoid enteritisSpontaneous and antibiotic-induced diarrhoea

Enterocolitis (natural)Antibiotic-induced enterocolitisNaturally 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.

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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.

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

Age

nt

Leci

thin

ase

Lipa

se

Lact

ose

Prot

eoly

sis

C. perfringensC. baratiC. bifermentansC. sordelliiC. botulinum AC. botulinum BC. botulinum CC. botulinum DC. botulinum EC. botulinum FC. botulinum GC. sporogenesC. novyi type AC. novyi type BC. novyi type CC. haemolyticumC. cadaverisC. cochleariumC. tetaniC. tertiumC. fallaxC. chauvoeiC. septicumC. sphenoidesC. butyricumC. histolyticum

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

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

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

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

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Table 7: Differential biochemical characteristics of the most important clostridiaTE

ST

C.pe

rfrin

gens

C. s

eptic

um

C .c

hauv

oei

C. n

ovyi

A

C. n

ovyi

B

C. h

aem

olyt

icum

C. s

orde

llii

C. b

iferm

enta

ns

C .b

otul

inum

C. te

tani

C. s

poro

gene

s

C. c

olin

um

C .b

arat

i

C. c

adav

eris

C .c

ochl

eariu

m

C. te

rtium

C. fa

llax

C. s

phen

oide

s

C. b

utyr

icum

C. h

isto

lytic

um

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

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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 toxinC. novyi AC. novyi BC. novyi CC. 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

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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.20 | 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.

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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/sA AlphaB Alpha, beta, epsilon, iotaC Alpha, betaD 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

22 | 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

23 | 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 antiserum2. 1 ml supernatant + 0,2 ml pen/strep + 0,2 ml C antiserum3. 1 ml supernatant + 0,2 ml pen/strep + 0,2 ml normal serum4. 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.

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REFERENCES1. 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

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APPENDIX: MEDIA AND REAGENTS FOR THE ISOLATION OF ANAEROBIC BACTERIA1. VL (Viande-Levure) broth

TryptoneNaClBeef extractYeast extractCysteine HClAgarDistilled water.

10g.5g.2g.5g.0,4g0,6g1000ml

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.

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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.

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