DIAGNOSIS
Field and laboratory tests
Tuberculin Skin TestThe Tuberculin skin test is an OIE-recommended international standard test for ante-mortem diagnosis of bTB. This test induces a T-cell mediated delayed-type hypersensitivity (DTH) reaction in case of exposure to MTC or other mycobacteria containing cross-reactive antigens. In case of the single intradermal test (SIT), purified protein derivatives (PPD) of tuberculin, which is a crude antigen preparation obtained from heat-killed cultures of M. bovis (PPDb) is used. Upon injection with PPDb, a DTH reaction occurs resulting in an inflammatory reaction (erythema, swelling, and induration) at the area of injection in case of test-positive animal. Regions with high exposure to environmental mycobacteria, employ a single intradermal comparative cervical tuberculin test (SICCT) using PPDb and an avian PPD (PPDa) to help improve test specificity.
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SIT SOP
SICCT SOP |
Microbiological AssaysMicroscopic Examination
Microscopic examination is performed for identification of acid-fast bacilli in clinical samples. Direct smear of clinical samples such as tissues presenting tuberculous lesions is prepared followed by Ziehl-Neelsen staining. The organism appear as pink or red coloured rods under 100X oil immersion objective. Bacterial culture Bacterial culture is used for isolation of the bacteria and is the recommended gold standard for the diagnosis of bTB. The process is time-consuming and may take up to 8-12 weeks. Nasal swabs, milk sample, lesions found in the lungs, lymph nodes of head and thorax, and other abdominal organs can be collected for bacterial isolation. Obtaining decontaminated and good quality sample is critical for successfully culturing the agent. Typically solid media using Lowenstein-Jensen media or Middlebrook 7H10 or liquid media using the Mycobacteria Growth Indicator Tube (MGIT) tubes are used for culture purposes. M. bovis produces smooth and off-white (bluff) colonies in the pyruvate-based solid media, which can further be examined by using Ziehel –Neelsen staining technique. |
Ziehl-Neelsen Staining SOP
Bacterial culture SOP
MGIT SOP |
Interferon-Gamma Release Assay (IGRA)
This in vitro blood-based assay is an ancillary test for diagnosis of bTB. This is based on the principle that sensitized lymphocytes, in the blood of infected animals, release interferon-gamma on re-exposure to mycobacterial antigens in vitro. In this test, heparinized whole blood of the suspected animal is stimulated for 16-24 hours with tuberculin PPD antigens (PPDb and PPDa), the plasma supernatant is then harvested and analyzed for IFN-γ using the ELISA kit. Enzyme-linked Immunosorbent Assay (ELISA) An ELISA kit is used for detection of antibodies specific to M. bovis antigens in cattle serum and plasma samples. Antibody-based detection of bTB is known to be less sensitive, and hence not regularly used for diagnosis. |
IGRA SOP
ELISA SOP
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Polymerase Chain Reaction (PCR)
A number of PCR-based assays have been developed for the detection of MTC-specific genetic material. Insertion sequence, IS1081, is present in all MTC species and hence is a widely used PCR target. More recently, single nucleotide polymorphism (SNP)-based Mycobacterium species-specific real-time PCRs have been developed and are in use. The PCR protocol provided below speciates MTC, and also helps differentiate the human Mycobacterial lineages. Whole Genome Sequencing (WGS) Whole genome sequencing is rapidly progressing from a research tool to being used for clinical applications. WGS also provides constructive details regarding evolution and genetic diversity of the organism. The USDA has adopted a routine WGS analysis of clinical bovine tuberculosis samples and has provided detail information on interpreting WGS results. |
MTBC PCR SOP
WGS information
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LFA is an immunochromatographic strip test that is usually used for the diagnosis of bTB in wild animals and now approved for domestic animals as well. It is a two module system that uses PPD-B and PPD-A in test line 1 of module 1 and 2 respectively (that helps to differentiate between pathogenic and environmental mycobacteria) and recombinant fusion protein (ESAT-6::CFP-10) in test line 2 of both the modules. In this test serum sample is collected from the suspected animal and added on the LFA pad with the diluent. Development of colour band indicates the presence of antibodies against the Mycobacteria.
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LFA SOP
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About usKapur lab
204 Wartik lab Pennsylvania State University, UP |
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