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

Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Author: Mnr J.J. Gouws Licensed under a Creative Commons Attribution license. TABLE OF CONTENTS TABLE OF CONTENTS...1 INTRODUCTION...2 Table 7.1: Summary of diseases caused by Brucella serovars...2 Bacteriological Methods...3 Safety precautions...3 Preferred specimens for the isolation of Brucella...3 Processing of specimens for the isolation of Brucella...4 Identification of Brucella serovars...5 Table 7.2: Differential characteristics of Brucella and selected Gram-negative organisms...5 Typing of Brucella isolates...7 Table 7.3: Distinguishing characteristics of Brucella serovars and biovars...8 REFERENCES...9 APPENDIX 7... 10 1 P age

INTRODUCTION The members of the genus Brucella are best known for their ability to cause infertility and abortion in livestock and undulant fever in man. Bovine brucellosis is an important cause of abortions in cattle in many countries of the world and the laboratory diagnostician can therefore expect to encounter Brucella abortus when culturing material from foetuses. Brucella ovis, a cause of epididymitis in rams and sporadic abortions in ewes, also occurs in Africa. In Africa, Brucella melitensis has been reported from Northern Kwazulu-Natal province in South Africa, Swaziland, Mozambique, Zimbabwe, Kenya, Somalia, Nigeria, Egypt, Sudan, Niger, Libya and Tunisia. It is a cause of abortions in goats and sheep, and sometimes in cattle. Brucella canis has been detected in dogs in the Western Cape province of South Africa. The remaining biovars in the genus are Brucella suis, Brucella ceti, Brucella pinnipedialis and Brucella neotomae. Brucella melitensis can also persist in certain antelope (Saiga tatarica), but these do not occur in South Africa. Brucella abortus has been isolated from yaks, camels, Asian buffalo and various species of game, including African buffalo, bison and elk. Five biovars of Brucella suis exist and biovars 1 and 3 infect pigs. The other biovars are responsible for infections in European wild hares, reindeer and caribou, and certain rodents. A summary of the disease caused by members of the genus Brucella are included in Table 7.1. Table 7.1: Summary of diseases caused by Brucella serovars Brucella abortus Brucella melitensis Species Host Disease Cattle Sheep, goats and pigs Horses Man Goats and sheep Cattle Man Abortion and orchitis Sporadic abortion Bursitis (fistulous withers) Undulant fever Abortion Occasional abortion Malta fever Brucella ovis Sheep Epididymitis and sporadic abortion Brucella suis Pigs Man Abortion, orchitis and herd infertility. Arthritis. Undulant fever Brucella canis Dogs Abortion, epididymitis and infertility in males. Disco spondylitis. Man Undulant fever. Brucella neotomae Desert wood rat Non pathogenic Brucella ceti Cetaceans and dolphins Abortion and neurological disease in dolphins Brucella pinnipedialis Pinnipeds Uncertain 2 P age

BACTERIOLOGICAL METHODS Safety precautions Brucellosis in humans can be caused by several of the Brucella species, with Brucella melitensis and Brucella suis considered the most pathogenic. The incubation period is usually four weeks, but may vary greatly. The acute form is often preceded for up to three months by symptoms such as fatigue, fever and headaches. Brucellosis may resemble influenza in the early stages. The term undulant fever comes from the cyclical nature of the fever in brucellosis. Affected persons often suffer from night sweats and extreme tiredness, especially in the afternoon. In chronic cases, ocular disturbances and joint pains occur and patients may eventually suffer from anxiety and depression. Treatment is based on a prolonged course of antibiotic therapy. Material which is potentially infected with Brucella must be handled with extreme caution. A Biosafety level 3 laboratory is preferred, but at the very least a separate room with only one entrance and a prominent biohazard notice is required. The room should contain a Class II or Class III biohazard cabinet. Persons handling infected material or cultures should work in the biohazard cabinet and wear disposable gloves and an impermeable gown. If it is necessary to handle potentially infectious material outside the cabinet, an eye shield and face mask, or preferably a respirator, should also be worn. Mouth pipetting is strictly prohibited. The prevention of aerosols is important and all items should be placed in disinfectant after use. These items should be autoclaved before further handling. A suitable disinfectant, such as 70% alcohol should always be available, both for routine cleaning and in case of breakages or spills. Preferred specimens for the isolation of Brucella Specimens should be placed on ice immediately after collection and frozen if they are to spend more than twelve hours in transit. All specimens must be packed in leak proof containers and clearly labelled. It must be remembered that a poorly packaged specimen may pose a risk to both the public and the laboratory staff. Samples of milk for the isolation of Brucella should include milk from each quarter or each side as infection of the udder may be confined to one quarter or one side. Approximately 20ml should be collected from each teat at the end of milking and the samples are better kept separate than pooled. Brucella may be isolated from vaginal swabs taken within six weeks after abortion or parturition. Blood culture may be performed on blood samples collected aseptically into a citrated tube. Foetal membranes contain large numbers of Brucella organisms in cases of Brucella-induced abortion or even infected full-term births. Infected cotyledons are the preferred specimens and are a dirty greyish yellow colour. Stomach contents, spleen and lung are the best specimens from aborted foetuses. In mature cows during necropsy, mammary lymph nodes should be taken and samples from the mandibular and medial iliac lymph nodes together with uterine caruncles included if possible. In heifers and bulls, additional tissues are necessary and these include medial, retropharyngeal, parotid, superficial cervical (prescapular) and mesenteric lymph nodes, and spleen. 3 P age

Samples from female sheep and goats are the same as for cattle. In pigs, mandibular, gastro-hepatic, internal iliac, mammary and retropharyngeal lymph nodes should be collected, as well as any tissues showing inflammatory lesions. If uncertainty exists as to which samples are best, all major lymph nodes can be collected in any species. In male animals, in addition to the samples already mentioned, male reproductive organs should be collected. These include testes, prostate, epididymes, seminal vesicles and associated lymph nodes. Processing of specimens for the isolation of Brucella As the number of viable organisms in a sample may be very small, careful processing of samples will increase the likelihood of isolation. Recipes for some of the media used will be found at the end of this chapter. Farrell's medium is the solid medium of choice. Blood should be cultured in liquid medium, as the number of organisms in blood is usually very small and liquid medium allows culture of a larger volume of sample than solid medium. Nutrient broth containing serum is suitable. Approximately 10 ml of blood is inoculated into 50 to 100 ml of broth and incubated. The sample can be subcultured periodically onto solid media. A biphasic method for the culture of blood is also described (Alton et al, 1988). Milk is centrifuged at 6 000 to 7 000 g for 15 minutes to separate the cream and the sediment from the skim milk. The cream and sediment are inoculated onto a solid selective medium. Separation of the milk can also be achieved by allowing the sample to stand overnight. Other liquids, such as foetal stomach content, may be spread directly onto solid selective media. In cases where the number of organisms is likely to be small, liquid media can also be used. Vaginal swabs are streaked onto a solid selective medium and then transferred to a liquid medium. Foetal membranes are often grossly contaminated and samples may be washed in sterile normal saline prior to culture. Not less than three different lots of saline should be used. The sample can then be cut with a sterile instrument and the cut surface streaked onto a solid selective medium. A sterile swab may be used to inoculate a second plate from the surface of the first, if necessary. Solid tissues may be cut with a sterile instrument and the cut surface streaked onto a solid selective medium. When available, a stomacher may be used to macerate organs in diluent. The macerated tissue is then inoculated onto solid selective medium or/and liquid medium. Use of the stomacher increases the likelihood of isolation of Brucella. All cultures for diagnostic purposes must be incubated at 37ºC in an atmosphere of 5 to 10% CO2. Anaerobic or polycarbonate jars together with disposable CO2 generator envelopes (GasPaks) can be used. Gas may also be supplied from a cylinder containing pure CO2 or an appropriate mixture of CO2 and air. A tube containing a CO2 indicator solution should always be included in the container. The reagent consists of a 0.1 % solution of sodium bicarbonate in water to which a few drops of a 0.5 % solution of bromothymol blue has been added. The indicator is blue in a normal atmosphere and green in 10 % CO2. Yellow or greenish-yellow indicates too much CO2. A colour change occurs after approximately one hour. 4 P age

Identification of Brucella serovars Brucella colonies are visible on solid media after 48 to 72 hours. On blood agar, the colonies are small (approximately 1 mm diameter), round, grey and non-haemolytic. On Farrell's medium, the colonies are small (approximately 1 to 2 mm diameter), round, convex, translucent, have smooth margins, and are a pale honey colour. On a Gram s smear from a culture, the organisms are Gram-negative cocco-bacilli, usually arranged singly, but may occur in pairs or small groups. Individual bacteria measure approximately 0.6 to 1.5 µm by 0.5 to 0.7 µm. The bacteria are partially acid-fast and stain red against a blue background with the modified Ziehl Neelsen (Stamp's) stain. As the organisms are intracellular, they tend to be seen in small clumps on smears from tissues. Brucella does not grow well in liquid media and up to 7 days incubation may be necessary before turbidity is visible. Liquid media should be subcultured on to solid media at frequent intervals, starting on the third day. Cultures on solid media should be examined every second day for up to ten days before being discarded as negative. Brucella can sometimes grow on MacConkey agar, producing non-lactose fermenting colonies. Brucella spp. are non-motile. They do not grow anaerobically. Their optimum growth temperature is 37ºC, but growth can occur between 20 and 40ºC. They are catalase-positive and usually oxidasepositive, but negative strains do occur. Acid production does not occur from carbohydrates, except for Brucella neotomae. Brucella species are indole negative and do not liquefy gelatin. Several Gramnegative organisms which may be confused with Brucella exist (Table 7.2). The smooth Brucella species will react with antisera prepared against smooth Brucella cultures. Rough Brucella species and rough variants of smooth species will not react with antisera prepared against smooth cultures, but will show agglutination with a 1 in 1 000 solution of neutral acriflavine in distilled water. Brucella ovis and Brucella canis are always rough. Antisera can be prepared against rough Brucella species and these will distinguish between rough Brucella and other bacteria. Table 7.2: Differential characteristics of Brucella and selected Gram-negative organisms Tests Brucella Bordetella bronchiseptica Campylobacter foetus Moraxella Psychrobacter phenylpyruvicus Acinetobacter Yersinia enterocolitica Morphology Small coccobacilli Small coccobacilli Comma shaped Diplococci Broad cocco-bacilli Diplococci Rods Motility at 37ºC - + + - - - - 5 P age

Motility at 20ºC - - - - - - + Lactose fermentation on - - - - - - - MacConkey agar Acid production from glucose Haemolysis - (except B. neotomae) - - - - Variable + on blood agar - + - Variable - Variable - Catalase + + + (except B. ovis, B. neotomae + Variable + + Oxidase and occasional + + + + - - B. abortus strains) + (except B. ovis and Urease Nitrate reduction occasional B. abortus strains) + (except B. ovis) + - Variable + Variable + + + Variable V - + Citrate reduction - + - - - Variable - Suspected isolates of Brucella may be tested against antisera using a slide agglutination method. A drop of antiserum is mixed with a suspension of bacteria and observed for agglutination. Lack of agglutination is considered a negative reaction. A drop of normal serum can be used as a control. Specific antisera against Brucella abortus and Brucella melitensis are used for typing. Details of the methods for preparation of antisera in rabbits can be found in Alton et al, 1988. Freshly isolated strains of Brucella are usually in the smooth form, except for Brucella ovis and Brucella canis. However, the isolation of rough variants from samples has been described. Rough variants cannot be typed with mono-specific antisera and do not show lysis with smooth Brucella phages. In cases where changes 6 P age

have occurred during culture, it is necessary to select a smooth colony for typing. Agglutination with acriflavine can be used to distinguish rough from smooth colonies. Typing of Brucella isolates The different serovars of the genus Brucella are very closely related and can only be distinguished through the use of specific tests. These tests also help to distinguish the different biovars from one another as well as to identify the vaccine strains (Table 9.3). The method below is most commonly used by diagnosticians. A pure culture which has grown at 37ºC in 10% CO2 for 48 hours is used for typing. The culture may be on blood agar or Farrell's. Tests for catalase and oxidase are performed directly from this culture, as is slide agglutination with mono-specific Brucella abortus and Brucella melitensis antisera. Urease activity is determined by inoculation onto Christensen's medium and is often visible within ten to fifteen minutes, but plates should be kept for up to 24 hours before being considered negative. Smears prepared from the culture may be stained with Gram's and Stamp's stains. A very dilute suspension is used for the tests: one colony in 10 ml of saline or nutrient broth. Blood agar is a suitable solid medium for all typing tests. Inoculate a lawn of bacteria onto three blood agar plates. They are to be used as follows: Plate 1 Phages can be applied to this lawn. Many different phages have been described for Brucella, but for diagnostic purposes the use of the Tb (Tbilisi) phage only is sufficient. Lysis is visible as a clear zone after 48 hours incubation in 10 % CO2 at 37ºC. Plates 2 and 3 Two sets of impregnated filter paper discs are applied to the surface of the plates. One consists of antibiogram discs and one of dye discs. Sensitivity is visible as a clear zone around a disc after 48 hours incubation in 10 % CO2 at 37ºC. The use of discs is a variation on the use of dye and antibiotic media. Details on the preparation of these media can be found in Alton et al, 1988. Brucella abortus biovar 1 is the most common biovar in South Africa and accounts for 90 % of field strains of abortus. Biovar 2 is the second most common and accounts for almost all of the remaining 10 %. Other biovars are extremely rare in South Africa. Some characteristics of Brucella are unstable and all cultures should be typed as soon as possible after initial isolation. Sub-culturing should be kept to a minimum. 7 P age

Table 7.3: Distinguishing characteristics of Brucella serovars and biovars Tests Brucella melitensis Brucella abortus biovars Brucella Brucella Brucella Brucella suis biovars biovars canis neotomae ovis 1 2 3 4 5 6 9 S19 1 2 3 Rev 1 1 2 3 4 5 Urease (+) (+) (+) (+) (+) (+) (+) (+) + + + + + + - + + + + + Oxidase + + (+) + + + + + + + + + + - - + + + + - Lysis by phage (Tb) + + + + + + + + - - - - - partial - - - - - - lysis CO2 requirement (+) (+) (+) (+) - - - - - - - - - - + - - - - - Growth on media containing thionine 1:50 000 - - + - + + + - + + + + - - + + + + + + Growth on media containing basic fuchsin 1:50 000 Agglutination: abortus antiserum Agglutination: melitensis antiserum Agglutination: rough antiserum Growth on media containing erythritol 1mg/ml Growth on media containing penicillin 5 iu/ml Growth on media containing streptomycin 2.5µg/ml + - + + + + + (-) + + + - - (-) (-) - + (-) - + + + - - + - - - + + + - + + + + - - - - + + - + - + - + - - - - - + + - - - - - - - + - - - - + - - - - - + - + - + - - + 8 P age

REFERENCES These notes cover only the essentials necessary for the isolation and typing of Brucella species. It is strongly recommended that any laboratory which intends doing Brucella diagnostics purchase the following book: 1. ALTON, G.G., JONES, L.M., ANGUS, R.D. & VERGER, J.M. 1988. Techniques for the brucellosis laboratory. Paris: Institut National de la Recherche Agronomique. Other useful references 1. ELBERG, S.S. 1981. A guide to the diagnosis, treatment and prevention of human brucellosis. Geneva: World Health Organisation. 2. HOLT, J.G., KRIEG, N.R., SNEATH, P.H.A., STALEY, J.T. & WILLIAMS, S.T. 1994. Bergey's Manual of Determinative Bacteriology. 9 th ed. Baltimore: Williams & Wilkins. 3. QUINN, P.J., CARTER, M.E., MARKEY, B. & CARTER, G.R. 1994. Clinical Veterinary Microbiology. London: Wolfe. 4. Quin, P.J., Markey, B.K., Leonard, F.C., FitzPatrick, E.S., Fanning, S., Hartigan, P.J. Veterinary Microbiology and Microbial Disease. Second Edition, Wiley-Blackwell, 2011. ISBN 78-1-4051-5823-7. 9 P age

APPENDIX 7 Media for use in the Brucella laboratory Nutrient agar agar peptone sodium chloride meat extract distilled water 20g 10g 5g 5g 1litre Place the ingredients in a container and expose to flowing steam for approximately one hour. Adjust the ph to 7.8. Place the container in the autoclave. Take up to 126ºC and then cool immediately to precipitate phosphates. Filter the medium through paper pulp and adjust the ph to 7.4. Distribute in measured quantities into flasks. Sterilize at 120ºC for 15 minutes. Addition of serum Equine or bovine serum can be used. Serum should be free of Brucella antibodies. Pass through a sterilizing filter, autoclave, cool to 56ºC and add to medium at a rate of 2 to 5%. Serum dextrose agar Nutrient agar is prepared, autoclaved, cooled to 56ºC and 5 ml of filter sterilized stock solution of serum containing 20% dextrose added per 95 ml of medium. Farrell's medium The following quantities of antibiotics are added to 1 litre of melted and cooled serum dextrose agar: cyclohexamide 100 mg bacitracin 25000 units polymyxinb 5 000 units vancomycin 20 mg nalidixic acid 5 mg nystatin 100000 units Modified Ziehl Neelsen (Stamp) stain The modified Ziehl Neelsen or Stamp's stain can be performed on smears from cultures as well as on impression smears from organs. Impression smears are made from a clean cut-surface of an organ. The cut surface is first blotted on clean absorbent paper and then pressed gently onto a slide. Smears are air dried before staining. Heat fix the smear by passing through a flame. Stain for 10 minutes with a 1:10 dilution of stock solution of Ziehl Neelsen carbol fuchsin. Stock solution is made up of 1 g of basic fuchsin dissolved in 10 ml of absolute ethanol and added to 90 ml of a 5 % phenol solution. Rinse in tap water. Differentiate with 0.5 % acetic acid for a maximum of 30 seconds. Rinse thoroughly in tap water. 10 P age

Counter stain with 1 % methylene blue for 20 seconds. Brucella organisms stain red against a blue background. They may easily be confused with Chlamydophila and Coxiella as these organisms have an almost identical appearance. An impression smear alone is therefore not sufficient basis for a diagnosis of brucellosis and should be supported by culture. A note on vaccine strains Brucella melitensis Rev 1 is the strain used in the vaccine against Brucella ovis and melitensis in sheep and goats. Brucella abortus S19 is the strain used in the vaccine against Brucella abortus in cattle. Both strains may infect humans. 11 P age