Evaluate comparative efficacy of tuberculin test, gross and histopathology, Ziehl-Neelsen staining and polymerase chain reaction technique for the detection of tuberculosis in dairy cattle

Abstract

Tuberculosis (TB) is an archaic infectious disease of human and animals caused by Mycobacterium spp and responsible for considerable morbidity and mortality. TB in man and animals is mostly caused by Mycobacterium bovis (M. bovis, bovine TB) and Mycobacterium tuberculosis (M. tuberculosis, human TB). This study, was, aimed at rapid and specific identification of bovine TB and human TB in dairy cattle by using intradermal tuberculin test, gross and microscopic pathology, Ziehl-Neelson staining of tissue sections and PCR detection of the specific causes of TB. Out of 100 dairy cattle tested using intradermal tuberculin test in the Bangladesh Agricultural University dairy farm during the period between 2013 and 2014, 05 yielded hypersensitivity reactions to intradermal tuberculin test. The tuberculin test positive cattle (N=5) were sacrificed and investigated systematically. Gross lesions observed were recorded during necropsy and lungs, liver, spleen, kidney and lymphnodes were collected in 10% buffered neutral formalin for histopathological examination. A small portion of lungs were snap frozen and preserved at-20 0 C for the extraction of genomic DNA and PCR detection of TB. Grossly nodular masses in lungs and enlarged mesenteric lymphnodes were observed in four cases. Accumulation of macrophages, lymphocytes and fibrous connective tissue was seen in microscopic section of lungs, spleen and lymphnodes. Tissue sections stained with Ziehl Neelsen staining, showed rod shaped bright red color acid fast bacilli in three cattle. The multiplex PCR used successfully amplified fragment of 16srRNA gene specific for Mycobacterial infections (1030bp) and infection due to Mycobacterium tuberculosis complex (372bp) in four cases. Results of uniplex PCR showed that three cattle were infected with M. bovis (MPB83 gene specific 600bp amplicon) and one cattle with M. tuberculosis (H37Rv Rv3479HP gene specific 667bp amplicon). Out of five tuberculin test positive cattle sacrificed in this study, four were found to infect with TB; one cattle could have reacted due to infection with saprophytic mycobacterium or other infections that cross reacted to tuberculin test. The multiplex and uniplex PCR adapted and designed in this study found sensitive, accurate and conclusive. Dairy cattle in this farm were infected with M. bovis and M. tuberculosis. M. bovis and M. tuberculosis are extremely zoonotic pathogens. It needs to examine all of the dairy cattle with tuberculin test and PCR test twice in a year, sacrifice the test positive cattle at early onset to prevent dissemination in farm animals and human. People working in the farms also need to sit for tuberculin test, Ziehl Neelsen staining and PCR test of cough to detect infected and carrier state of infection.