10.3 Gamma interferon assays

The gamma interferon assay (IFNγ) is an in vitro form of the CITT. It is based on detection of γ interferon produced by specific circulating lymphocytes upon stimulation of heparinized whole blood in vitro with PPD-B and PPD-A. Detection of IFNγ is carried by a sandwich ELISA, using two monoclonal antibodies to bovine gamma-interferon, after incubation of the blood for about 16–24 hours with PPD-B and PPD-A. The IFNγ test is reportedly more sensitive than the tuberculin test and can detect infected animals that are negative to the later. The sensitivity and specificity are estimated at 81.8 and 99.1% [65]. It has been observed that more infected cattle can be identified by using both the tuberculin and the IFNγ tests and it is recommended that both tests be conducted in parallel [66]. The advantage of the IFNγ is that infected animals are detected early and only one visit to the farm is required. It is particularly convenient for animals that are difficult to capture or handle, such as cattle reared in ranches or under nomadic pastoralism, or wildlife, as they need only to be captured once rather than twice. It however requires more technical expertise and facilities and is costly (approximately 10 USD, for consumable materials per test).

the media. Thereafter, the caps are tightened and the tubes incubated vertically for six 12 weeks. Liquid media include BACTEC 460, Mycobacterial Growth Indicator Tube (MGIT), which have enriched Middlebrook 7Ha with antibiotics and growth promoters are added. BACTEC 460 MGIT media is fully automated and can monitor the growth of mycobacteria by the use of oxygen quenching or fluorescent sensor. Mycobacteria may not be recovered in the cultures for a number of reasons:

Direct smear of a tuberculous lymph node lesion from a cow, showing presence of acid-fast bacilli (arrowed).

1. Extended period between sample collection and analysis, leading to death of

2. Nonviability of the bacilli due to necrosis and calcification of granulomas.

Cultures suspected to be mycobacteria are then stained by acid fast method for confirmation. All laboratory procedures must be conducted in a class II biosafety cabinet in a laboratory environment that has been found safe and secure following

Molecular tools for differentiating Mycobacterium species have been developed [70]. Polymerase chain reaction (PCR) technique involves detection of the genetic material that is unique and specific to a species. Convenient commercial kits are available. Genotype Mycobacterium (Hain, Nehren, and Germany) are line probe assays available in three different formats: Genotype MTBC differentiate species in MTBC; GenoType Mycobacterium common mycobacteria (CM) detects most frequently encountered Mycobacteria species and Genotype Myco-

Mycobacteria species. This kit uses reverse hybridization technology on a solid membrane matrix consisting of nitrocellulose strips. The DNA probes are immobilized on parallel lines on the strips. Biotinylated DNA apricon fragments of the 16S-23SrRNA spacer region are incubated with the labeled strips and hybridization detected colorimetrically by addition of an enzyme, Streptavidinalkaline phosphatase, and a chromogenic substrate. A precipitate is formed on

3. Organisms may be inactivated by the decontamination process.

4.Samples may contain microorganisms other than mycobacteria.

bacterium additional species (AS) detects less frequently encountered

the organism.

ZN 1000. ©2018. JKN Kuria.

Diseases Caused by Bacteria in Cattle: Tuberculosis DOI: http://dx.doi.org/10.5772/intechopen.82051

Figure 5.

risk assessment.

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10.6 Molecular diagnosis
