Assessment of the Use of PCR as an Early Diagnostic Indicator of Bovine Tuberculosis in Dairy Farms

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Abstract
Background: Zoonotic tuberculosis has been mainly associated with the consumption of bovine dairy products and its control has been prioritized by the Food and Agriculture Organisation. Objective: As such, the aim of this study is to assess whether the polymerase chain reaction (PCR) can be used as an early diagnostic indicator of Mycobacterium bovis infection in bovine dairy farms. Methods: Milk samples (n=78) were collected from all the animals older than 8 years of age, in all (n=4) bovine dairy farms located in a specific agricultural region of Greece, with high prevalence in bovine tuberculosis. Detection of DNA belonging to slow growing members of the genus Mycobacterium was conducted in pooled samples using two PCR assays targeting 16S-rRNA and 65-kDa heat shock protein (hsp65). Randomly selected PCR products were submitted to sequence analysis for confirmation of the specificity of the amplification process. DNA isolation and PCR testing were conducted in compliance with ISO17025 accreditation requirements. Results: The overall percentage of positivity was 47.7%, and ranged among farms from 0% to 76.9%. PCRpositive animals were detected in both farms that were at the time of investigation positive with the tuberculin skin test (TST), whereas the only farm with a negative TST record tested also negatively with PCR. Interestingly, one farm that was negative with TST since 2012 but had a long prior record of high level TST-positivity, tested positively with PCR. Conclusion: In conclusion it can be stated that PCR can be used for the detection of mycobacteria in pooled samples of milk collected from the older animals of a dairy farm, as an early and sensitive diagnostic indicator. This can support TST monitoring for the control of bovine tuberculosis, and improve detection of farms, in which routine monitoring should be intensified. The specific approach offers significant practical benefits that compensate for the additional cost of PCR.

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