| Diagnostics |
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| Written by BTB Website Administrator | |
| Monday, 17 April 2006 | |
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Several tests can be used to determine the TB status of animals. Not all tests are validated for use in all species; therefore culture of the Mycobacterium bovis organism remains the Gold Standard for confirmation of infection with bovine tuberculosis. The following tests and procedures are all used in South Africa to determine the disease status in both domestic and wild animals. Intradermal tuberculin test The intradermal tuberculin test is most probably the test used in the widest range of species suspected to be infected with M. bovis. There are slight differences in the application of the test and interpretation of results between species, but the general method remains the same. Method: The skin is examined for the presence of any lesions that would interfere with the test. Rectangular areas (30mm x 20mm) are shaved on either side of the neck and the skin thickness of each of the sites measured and recorded. A volume of 0,1ml (0,1mg/ml) of bovine purified protein derivative (PPD) was injected intradermally in the centre of the clipped area on the left side and 0,1ml (0,05mg/ml) avian PPD on the right side of the neck using a McClintock syringe. After a period of 72 hours had elapsed, the skin at each of the two injection sites is examined and measured. The increase in skin thickness of the avian PPD injection site was subtracted from that of the increase in skin thickness of the bovine PPD site to get a final result. Different interpretations are used in different species, for example in lions only the bovine injection site is measured after 72 hours and in buffalo the difference between bovine and avian must be more than 4mm for a positive result compared to the 2mm difference for cattle (refer to publications for detail on cattle/ buffalo skin test data) Pros:
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Interferon-gamma(γ) assay The IFN-g assay is a rapid assay on blood to measure cell-mediated immunity (CMI) for the diagnosis of BTB in cattle. The assay is based on the principle that lymphocytes in the blood of the M. bovis infected animal detect specific mycobacterial antigens present in the bovine purified protein derivative (PPD). This recognition involves the generation and secretion of the cytokine, interferon-gamma (IFN-g). Tuberculin PPD antigens are presented to lymphocytes in whole blood cultures and the production of IFN-g from the stimulated T cells is detected using a monoclonal antibody-based sandwich enzyme immunoassay (EIA). Lymphocytes from uninfected cattle do not produce IFN-g and hence the increase IFN-g levels correlates with infection. The test was developed and patented by CSIRO, Australia. Method: PPD antigens are added to the whole blood in 24 well plates and then incubated for 20 – 24 hours. The supernatant is removed after incubation and used in the monoclonal antibody-based sandwich enzyme immunoassay. Several steps of adding conjugate and substrate as well as washing the test plates are incorporated before the stopping solution is added as the final step. The optical density of the reaction was measured at 450nm using an EL312e spectrophotometer. The amount of IFN-g in each plasma sample is estimated and this indicates whether an animal is infected with M. bovis. This assay has been modified for use in buffalo (Michel et al 2000). Different outcomes such as positive, equal reactor, multiple reactor, avian reactor, invalid reaction and negative are all possible when the IFN-g assay is run. The standard interpretation of the IFN-g assay is used as described by the Onderstepoort Veterinary Institute (OVI). Although the test has not been validated for any other species in SA, it might be possible in future. Pros:
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Necropsy The animal is either euthanased for a survey, killed at the abattoir or died of disease and then the carcass is submitted for necropsy. Method: When submitted to an abattoir the standard necropsy is combined with routine meat inspection. Heads are moved from the carcasses and placed on hooks together with the pluck from the same carcass onto a semi-automated system. Lungs are thoroughly palpated for the presence of tuberculous granulomas and lymph nodes from the head and thorax are removed for inspection. All the different parts of one carcass are clearly identified with cards carrying the slaughter number of each individual animal. The carcasses are eviscerated and the abdominal viscera placed on a stainless steel working bench, where a thorough examination of the reticulum, rumen, omasum, abomasum, small and large intestine as well as the kidneys can be carried out. During the processing of the carcass, all the lymph nodes are removed for slicing and detailed examination. A field necropsy is similar to that of the abattoir procedure except that the carcasses are not hung and most of the organs can be evaluated in situ rather than being removed from the carcass. Lymph nodes are still removed from the carcass to be sliced, examined and specimens taken for mycobacterial culture. Pros:
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Mycobacterial culture Methods as described by Nel, Kleeberg and Gatner (1980) are now in use for isolation and identification of mycobacteria. Method: Lesions are excised and homogenized. The homogenized tissue is divided in two parts, one is decontaminated with 2% HCl, and the other with 4% NaOH. The samples are left at room temperature for 15 minutes, then centrifuged at 1 650g. The supernatant fluid is discarded, and the sediments washed by centrifugation and then inoculated onto each of two tubes of:
The tubes are incubated at 37C and observed for growth of colonies of acid-fast bacteria at 1 and 2 weeks, and then at 2-weekly intervals for 10 weeks. As soon as colonies of acid-fast bacteria are observed, they are sub-cultured onto the same medium that supported primary growth. The isolates are then identified by:
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Histopatology When a carcass is submitted for necropsy and specimens are collected for mycobacterial culture, a separate set of specimens may be collected in 10% buffered formalin for histopathology as an ancillary diagnostic test. Method: Specimens are collected and preserved in 10% buffered formalin, and later prepared routinely for light microscopy by embedding them in paraffin wax. Sections cut to a thickness of 4 - 6mm are routinely stained with haematoxylin and eosin, and selected sections with the Ziehl-Neelsen acid-fast stain. Pros:
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ELISA’s and Lymphocyte stimulation assays The ELISA is an antibody based assay where sera from test animals are used to detect animals infected with BTB. Generally only cases with advanced disease where cellular immunity has changed over to humeral immunity test positive. Infected animals with no visible lesions or only early tuberculous lesions may be missed when this diagnostic method is applied, therefore false negative test results will be a problem. The Lymphocyte stimulation tests measures the activity of the lymphocytes within the white blood cell component to give an indication of the cellular immune status. If the reaction to the bovine stimulant is higher than that of the control stimulant an animal is regarded as being positive for BTB. Unfortunately due to the long incubation period (5 days) the test is fairly cumbersome and not regarded as useful in a free range system as is found in most of the wild animals in South Africa. These assays have been tried in buffaloes and lions but resulted in very poor sensitivity and specificity and therefore were not pursued any further. However, these assays have been implemented with great success in the diagnosis of BTB in red deer in new Zealand (Griffin & Cross 1989; Griffin, Cross & Buchan 1991). |
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| Last Updated ( Tuesday, 03 April 2007 ) |
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