Development of a molecular diagnostic test applied to experimental abattoir surveillance on bovine tuberculosis

Abstract

One of the most essential systems applied to the eradication of bovine tuberculosis by Mycobacterium bovis is the epidemiologic surveillance of animals slaughtered in abattoir by means of inspection and sample taking of lesions compatible with tuberculosis, confirming the existence of the disease through culture and molecular detection, which takes weeks before a result can be obtained. An interesting alternative is to develop high-throughput molecular systems for the direct detection of M. bovis on biological samples. In this sense, our research has developed a molecular detection system by means of a real time based PCR process which is applied directly to bovine biological samples and it allows to differentiate between Mycobacterium tuberculosis Complex, Mycobacterium avium Complex and other atypical mycobacteria that are interesting from the veterinary point of view. The sensitivity was analyzed by applying a conventional extraction system based on guanidine thiocyanate and a robotized system based on the selective magnetic capture of mycobacterial DNA. The molecular detection system showed a high specificity and a detection threshold of only 2-3 genomes. The sensitivity depended on the DNA extraction system being used and on the kind of lesions on which it was used; the sensitivity ranged from 61.11% for samples with non visible lesions to 80.64% for chronic lesions, with an average sensitivity of 73.87% when using the manual extraction system and between 27.77 and 74.19% (average sensitivity 47.74%) when using the automated robotic system. In conclusion, our multiplex real time PCR assay represents a fully controlled, highthroughput diagnostic tool for the rapid detection of Myobacterium presence directly in animal clinical specimens, which could be a practical tool in the context of bovine tuberculosis abattoir surveillance programs and granuloma submission programs.

Assessing the histopathology to depict the different stages of bovine tuberculosis infection in a naturally infected herd

“…Although only three samples were positive by bacterial culturing, eight of them were positive by PCR. Molecular evidence of Mycobacterium bovis has been described as more sensitive than bacteriology since it requires a low bacterial load (Parra et al 2008). Therefore, all the histopathological, bacteriological, molecular and immunological ϐindings support the hypothesis that those animals were recently infected.…”

Culture and Molecular Method for Detection of Mycobacterium tuberculosis Complex and Mycobacterium avium subsp. paratuberculosis in Milk and Dairy Products

“…Therefore, the need for reliable detection and risk-based investigations of different matrices is obvious in the framework of the official food control. Present reports about the development of molecular methods for the detection and differentiation of MTBC deal with clinical human and veterinary samples (3,9,11). They cannot be transmitted for the investigation of food, as low contamination levels per gram of sample material are to be expected in these matrices.…”

Diagnóstico histopatológico e molecular de lesões sugestivas de tuberculose em búfalos abatidos nos municípios de Macapá e Santana, estado do Amapá

“…Parra et al (2008), desenvolvendo sistema de PCR múltipla em tempo real, diretamente de amostras biológicas, obtiveram taxas de sensibilidade variando de 61,11% (lesões não visíveis) até 80,64% para lesões tuberculosas crônicas, permitindo a diferenciação entre membros do CMA e outras micobactérias não-tuberculosas. Furlanetto et al (2012), empregando PCR-múltipla diretamente em fragmentos de tecido, detectaram presença de M. bovis em 7% (14/182) das lesões, mostrando maior sensibilidade do teste molecular comparado a análise microbiológica (1,5%).…”