DETECTION OF SPECIFIC CAUSES OF TUBERCULOSIS IN THE DAIRY CATTLE OF BANGLADESH AGRICULTURAL UNIVERSITY BY SEQUENCING AND SEQUENCE ANALYSIS

Abstract

Tuberculosis (TB) is a chronic disease of man and animal. In this study intradermal tuberculin test, necropsy, histopathology, polymerase chain reaction (PCR) and sequencing techniques were used to diagnose specific cause of TB in dairy cattle of Bangladesh Agricultural University, Mymensingh. Intradermal tuberculin tests were carried out on randomly selected 100 dairy cattle. Tuberculin test positive cattle (N=05) were examined at necropsy and granulomas in lungs was seen in three cattle. Caseous necrosis and swelling of lymphnodes was seen in prescapular (N=01) and mesenteric (N=02) lymphnodes. In a case nodular lesion was seen in lungs and mesenteric lymphnodes. Portion of infected lungs and lymphnodes were snap frozen, extracted genomic DNA and PCR protocols was adapted targeting MPB83 gene. Result of PCR showed amplification of 600bp fragments in five cases. The MPB83 gene although specific for M. Bovis, the gene is less abundantly expressed by M. tuberculosis. To differentiate infectivity due to M. Bovis and M. Tuberculosis, two more PCR were adapted targeting pncA and oxyR genes. Out of five cattle tested in PCR all samples generated pncA specific 185bp and oxyR gene specific 270bp amplicons. The sequencing of MPB83, pncA and oxyR genes were carried out. Results of sequencing did not show mutation in MPB83 gene. Sequencing of pncA gene showed replacement of nucleic acid base (guanine to cytosin) in position 169 in cattle no. 5. Similarly, sequence analysis of oxyR gene (n=05) showed replacement of nucleic acid base (adenine to guanine) in position 285 in cattle no. 5. The cattle no. 5 was confirmedly infected with M. Tuberculosis and rest of the cattle were infected with M. Bovis. The tuberculin tests, necropsy, histopathology and PCR amplification of MPB83 gene may not contribute species specific detection of Mycobacterial infectivity in cattle. Sequencing and sequence analysis of pncA and oxyR genes found to differentiate infectivity in cattle due to M. Bovis and M. Tuberculosis.  Both of the bacterial species are extremely zoonotic and dairy cattle of Bangladesh Agricultural University were infected with both M. Bovis and M. Tuberculosis. It needs to tests all the dairy cattle twice in a year with tuberculin tests and dispose test reactors in order to minimize zoonotic risk.