Selecting the animal. Selecting tissues for submission

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1 Microbiology - Bacteriology Selecting the animal The best animal for diagnostic examination is an untreated, acutely ill one. In live animals, if the clinical signs are observed in more than one animal of a group, specimens should be collected from multiple clinically affected animals as well as some healthy animals which have been in contact with the affected individuals. If the case warrants it, and the owner permits it, an animal may be sacrificed in extremis in order to obtain high quality post mortem specimens. If this is not possible, the next best is a recently dead animal. Post mortem invasion of tissues by intestinal and environmental bacteria can be rapid, particularly in warm weather. This hampers identification of the causative agent. Selecting tissues for submission In live animals, samples should be collected from easily accessible affected sites as soon as possible following the onset of clinical signs. In dead animals, samples from tissues or organs with lesions are preferred. The following are a few guidelines: It is best to collect samples from the edge of lesions, including some healthy tissue. This is the site of active replication of organisms. In the above example of bastard strangles in a horse, tissue for culture is collected from the capsule of the retropharyngeal abscess (black circle). If there are no apparent gross lesions, select tissue from organ systems to which the clinical signs are referable e.g. transtracheal aspirate (live animal) or lung tissue (post mortem) from animals with clinical signs of pneumonia. Bone marrow can be sent from post mortal cases of suspected septicaemia, where autolytic change has rendered other organ samples unsuitable, as it is less susceptible to putrefactive organisms. Avoid taking samples from animals currently or recently on antibiotics. The best samples for bacteriology are from animals before they have received any antibiotics at all, but that is usually not possible. If uncertain as to what specimens to collect and/or how to collect them, contact the laboratory. 339

2 Sample collection Samples should be collected as aseptically as possible. This will prevent the aetiological agent from being overgrown by contaminating bacteria. Specimens should be handled gently as rough tissue handling exacerbates tissue artifacts and autolytic changes. Aerobic culture Swabs from different tissues should be collected individually or placed in separate sterile containers for microbial isolation. Containers suitable for sample collection and transport are microbiological swabs with transport medium, sterile plastic screw top jars or sterile serum tubes (without clot activator gel) for fluids. Avoid zipper seal bags as they tend to leak. Examination gloves are unsuitable as sample containers. Specimens for culture (biopsy material, exudates or faeces) must not come into contact with disinfectants or antiseptics and should be of adequate size, approximately 4cm 3. If the sample size is small, it is best to place it in an appropriate transport medium. Consider submitting a few air-dried smears of exudates, urine sediments and tissue impressions together with microbiological specimens, although cytological smears are prepared from all microbiological samples received at the laboratory. These smears may provide some important cytological diagnostic information. 340

3 Anaerobic culture Swabs from different tissues should be collected individually or placed in separate sterile oxygen free containers for microbial isolation. Specimens should be taken aseptically to prevent contamination. Containers suitable for anaerobic culture are charcoal swabs (first choice) and sealable plastic envelopes which have been folded to exclude oxygen. Fluid samples can be collected into sterile syringes and transferred to sterile screw top tubes which are sealed to prevent leakage. Syringes even with attached needles bent back and driven into the hub tend to autoexpress and leak with risk of exposure and no sample to culture. In addition, the bent over needles are a danger to lab staff. As strict anaerobes survive for only a short period of time in air, samples need to have oxygen excluded as soon as is practically possible. When using charcoal swabs ensure that the swab is inserted deep into the transport medium. Specimens for anaerobic culture must not come into contact with disinfectants or antiseptics and should be of adequate size, approximately 4cm 3. If the samples are sufficiently large and collected aseptically, the centre of the tissue often remains anaerobic without needing to take special precautions. Solid tissue samples should be transported at 4 C, while fluids should not be sent on ice as oxygen dissolves better in fluids at low temperatures. 341

4 Tuberculosis / Mycobacterial Culture To avoid confusion surrounding the term tuberculosis, convention limits it to diseases caused by Mycobacterium tuberculosis and Mycobacterium bovis. Other conditions are referred to as mycobacteriosis and atypical mycobacteriosis. Tuberculosis (M. bovis and M. tuberculosis) is a Controlled Disease in South Africa and further information regarding sample submission and testing will need to be obtained from the National Department of Agriculture, Directorate of Animal Health. Specimens from live animals for mycobacterial culture and PCR include: aspirates from cavities, lymph node biopsies, bronchoalveolar and gastric lavages, urine, faeces and the centrifuged deposit from about 50ml of milk in the case of a suspected mycobacterial / tuberculous mastitis. Lymph node and skin nodule biopsies can be submitted in 10% buffered formalin for histopathology, Ziehl-Neelsen staining and mycobacterial immunohistochemistry. Specimens from dead animals should be collected aseptically from any visible lesions or a selection of lymph nodes for mycobacterial culture and PCR. Duplicate samples are collected in 10% buffered formalin for histopathology, Ziehl-Neelsen staining and mycobacterial immunohistochemistry. Once the sample(s) have reached the laboratory, smears of sediment or tissue are prepared and stained with an acid-fast Ziehl-Neelsen stain to determine the presence of acid-fast organisms. Positive smear results should be confirmed on culture or PCR. 342

5 Blood culture Blood culture is considered in cases where a bacteraemia or septicaemia is suspected. It is best to collect the blood during a fever peak. Blood is collected under strict aseptic conditions directly into commercial blood culture bottles (aerobic and anaerobic). About 10ml of blood should be collected into each bottle. Since a bacteraemia can be intermittent, it is advisable to collect three to four blood samples within a 24 hour period. If blood culture bottles are not available then blood should be collected using heparin as an anti-coagulant and submitted to the laboratory within two to three hours of collection. 343

6 Bacteriological Serotyping Serotyping allows for identification of the organisms involved in disease outbreaks and has applications in epidemiological investigations. Streptococcal Isolates The latex agglutination test is performed on all streptococcal cultures in order to positively identify them. Specific antisera for groups A, B, C, D, F and G are commercially available Escherichia coli and Salmonella Escherichia coli are now typed by polymerase chain reaction (PCR) for virulence factors. The latex agglutination test is performed on all Salmonella cultures in order to positively identify them. Specific antisera for groups B, C, D, E and G are commercially available. Once grouping has been completed, the isolate may then be serotyped in order to determine the exact serovar of the isolate concerned e.g. Salmonella enterica subsp. enterica serovar Dublin 9, 12: g, p. Pasteurella multocida, Mannheimia haemolytica Actinobacillus pleuropneumoniae and Avibacterium paragallinarum Isolates These isolates may also be serotyped directly following their identification on primary culture. 344

7 Sample packaging and transport Samples are packed using the prescribed Triple Packaging System as per regulations SABS 0229:1996 (See Annexure 1 - Reference Ranges). Ensure there is adequate absorbent material to absorb any leakage. All packages should be watertight and correctly labeled. Fresh and formalin-fixed specimens should be packaged separately, as formalin leakage will render fresh tissues unsuitable for culture. Submission forms should contain as complete a history as possible and include animal species, age, sex, numbers affected and any other relevant clinical information. Recent treatment with antimicrobials as well as vaccination history should also be included as these can affect interpretation of results. Submission forms containing this information should be placed in separate zipper plastic envelopes to the samples, to avoid any smudging of ink. All specimens (except fluids for anaerobic culture) should be transported at 4 C in sealed insulated containers. 345

8 Specimen collection for specific syndromes Skin conditions Abscesses: Recently formed abscesses should be selected. If the abscess is intact, an attempt should be made to draw up pus into a syringe. A swab of the pus can be taken from the edge of the abscess, where the likelihood of obtaining viable organisms is greatest. The centre of an abscess is often sterile. Air-dried smears can be made from the pus and edge scrapings. Aerobic and anaerobic cultures should be performed. Lumps and vesicles: Remove the lump and send half in formalin for histopathology and the other half on ice for bacterial, fungal or viral isolation. Samples taken with biopsy needles or punches can also be used. Open lesions: A biopsy is best, half for culture and half for histopathology. Important to remember is that secondary infections are common in these types of lesions which can mask the original aetiology. Crusts can be sent in a sterile container for bacterial isolation. Otitis externa: Prior to collection of samples for culture, it is recommended that heatfixed smears of the exudate be prepared and examined/submitted to the laboratory. Both ears should be sampled by inserting a sterile swab deep into the ear canal. The swabs are then placed in bacterial transport medium, labeled left and right and submitted to the laboratory for aerobic culture. Musculoskeletal infections Gangrenous myositis: Six impression smears are made from the cut surfaces of incisions into affected muscle groups for Gram s staining and fluorescent antibody tests. Fresh tissue sample blocks (4cm 3 ) collected from the same area as the smears should be submitted in a sterile, wrapped, sealed plastic envelope (to exclude oxygen) for anaerobic culture targeting Clostridium spp. Osteomyelitis: Blood cultures (aerobic and anaerobic) provide the best chance of isolating the causative bacteria. As only about 75% of cases produce positive cultures, a negative culture result does not necessarily exclude a bacterial cause. If there has been surgical intervention at the site, bone samples for culture can be obtained. If Brucella canis or other Brucella infections are suspected, serum should also be submitted for serology. Septic arthritis: A note should be made of whether an individual joint or a polyarthritis is present. If an infectious agent is suspected, a joint fluid aspirate or, preferably, a synovial biopsy, should be taken and sent for bacterial, mycoplasmal and fungal culture. Blood cultures should be performed if systemic infections are suspected. If Borrelia burgdorferi is suspected, serum samples should also be submitted for serology. Diarrhoea Live animal: For microbiological examination, faecal samples collected from the rectum are best. Fresh faecal smears should be examined for the presence of motile protozoa and bacteria. 346

9 Dead animal: Portions of affected intestine (open-ended) and mesenteric lymph nodes should be submitted for aerobic culture. In addition, a 3cm portion of ileum/affected intestine should be tied-off at both ends and submitted for anaerobic culture. Respiratory disease Upper respiratory tract infections: A laboratory diagnosis is usually only requested when the infection is unresponsive to therapy. Nasal discharges or swabs for bacterial isolation usually yield very poor results as many potential pathogens normally reside in the nasal cavity. Lower respiratory tract disease: In live animals, transtracheal aspirates or bronchoalveolar lavages should be submitted for cytology and culture. If the pneumonia is long standing, both aerobic and anaerobic culture should be carried out. If there is a pleural effusion, fluid should be withdrawn and submitted for aerobic, anaerobic and mycoplasma isolation. Consider submitting a few air-dried smears, although cytological smears are prepared from all samples received at the laboratory. In dead animals, affected lung tissues and mediastinal lymph nodes should be collected. These should be submitted for aerobic culture and, if indicated, anaerobic, fungal or mycoplasma culture. Pleural fluid should also be collected and handled in the same fashion as for the living animal. Nervous signs Live animal: In the live animal, cerebrospinal fluid (CSF) can be used for cytology, microbiological examination and antibody detection. Dead animal: Brain tissue is collected aseptically as per the standard guidelines for aerobic and anaerobic culture above. Urogenital tract Cystitis: Collection of urine via cystocentesis is best as the sample is less likely to contain contaminating bacteria. A catheterised sample is also acceptable. Bacteriological counts should be performed on mid-stream urine. A count of 10 5 CFU/ml or more is considered significant. Endometritis: Two guarded swabs should be taken of the endometrial discharge one for cytology and the other for microbiological culture. Infections of the male genital tract: See Reproduction Bovine Venereal Disease. 347

10 Further reading 1. Hirsh DC, MacLachlan N J and Walker RL Veterinary Microbiology, 2 nd edn. Blackwell Publishing. 2. Quinn PJ, Carter ME, Mosby B and Carter GE Clinical Veterinary Microbiology. Wolfe Publishing. 3. Songer JG and Post KW Veterinary Microbiology Bacterial and Fungal Agents of Animal Disease. Elsevier-Saunders. 4. University of Pretoria Applied Veterinary Bacteriology and Mycology: Identification of Anaerobic Bacteria (AVB812). Course Notes. 348