ABSTRACT
PCR based molecular techniques help in discrimination of two closely related Mycobacterium tuberculosis and M.bovis. Here, we analyzed 24 M. bovis, 39 M. tuberculosis, 21 fresh acid-fast positive sputum samples and standardmycobacterial strains with pncA, 12.7 Kb and IS6100 based PCR assays. DNA from cultures and sputum yielded a positiveamplification of 185 bp with M. tuberculosis specific reverse primer pncAMT-2 but not with M. bovis specific reverseprimer pncAMB-2 and all M. bovis strains showed a positive amplification of 185 bp with M. bovis specific reverse primerpncAMB-2 but not with M. tuberculosis specific reverse primer pncAMT-2. The 12.7 Kb fragment based PCR performed onDNA extracted from cultures of M. tuberculosis and sputum yielded product of 168 bp while M. bovis showed 262 bpproducts. M. tuberculosis complex specific IS6110 PCR assay performed on DNA extracted from M. tuberculosis, M. boviscultures and sputum samples yielded M. tuberculosis complex specific 123-bp amplified products. The sequence analysis ofrepresentative PCR products of IS6110 and 12.7 Kb fragment showed 99-100% and 100% identity in amplicon products,respectively. To test reliability of primers, M. tuberculosis and M. bovis cultures were mixed and subjected to IS6110, pncAand 12.7 Kb PCR assay. pncA primers could not successfully and reliably discriminate the mixed culture, however, 12.7 Kbfragment primers successfully discriminated the mixed culture of M. tuberculosis and M. Bovis.
ABSTRACT
Culture filtrate protein (CFP-10) is a low molecular weight protein and is an early secretory protein in Mycobacterium tuberculosis culture filtrate and plays key roles in tuberculosis pathogenesis and in the stimulation of immunity. Keeping in view the important role of CFP10, we investigated the CFP10 gene in Indian Mycobacterium bovis (M. bovis) (3/86Rv) and its expression in suitable prokaryotic host for its diagnostics potential by single intradermal test (SID). Real-time PCR was used to quantify mRNA interferon-γ expression levels. The study concluded that the expression of IFN-γ mRNA in blood was found up to 19.962, 20.795, 9.633, 34.511 and 1.688 times in rCFP10, bovine PPD (purified protein derivative), avian PPD, conA and PBS stimulated groups, respectively as compared to healthy control group. The mRNA expression level of bovine PPD and avian PPD stimulated group was found statistically significant (at P <0.05) and among conA and rCFP10 stimulated group, it was highly significant (at P <0.01) in comparison to ‘without antigen stimulated’ group.
ABSTRACT
The bovine tuberculosis caused by Mycobacterium bovis is a serious disease among cattle worldwide resulting in considerable economic loss. There is a need for a diagnostic test that can discriminate M. bovis infection from BCG vaccination and NTM sensitization in animals. In this study, we intended to find out the potential use of recombinant antigens from Indian strain of Mycobacterium bovis (3/86Rv) for the intradermal tuberculin test of cattle. Immunodominant proteins MPB64, MPB83 and ESAT6 from M. bovis (3/86 Rv) Indian strain were recombinantly overexpressed, purified and immunologically characterized (rMPB64, rMPB83 and rESAT6). Four different cocktail combinations viz., cocktail I of protein antigens contained rMPB64, rMPB83, rESAT6, rCFP10 with protein concentration of 0.5 µg each; cocktail II contained 0.5 µg of each of rMPB64, rMPB83, rESAT6; cocktail III with 1 µg of each rESAT6, rCFP10; and cocktail IV contained rMPB64 and rMPB83 with 1 µg concentration of each protein, were administered at a dose of 0.1 mL. The DTH response was measured in heat killed M. bovis and non-tuberculous mycobacteria (NTM) sensitized, bacille Calmette-Guerin (BCG) vaccinated and control guinea pigs.The first cocktail of rMPB64, rMPB83 and rESAT6 containing 1.5 µg showed almost similar to cocktails II and III but stronger DTH response even at lower individual protein concentrations (each 0.5 µg) than the rESAT6 and rCFP10 protein of third cocktail with higher individual protein concentration (each 1 µg). The fourth cocktail with rMPB64 and rMPB83 elicited less DTH response as compared to the all other formulated cocktails. Cocktail I of four protein antigens elicited highest response at 24 h. Guinea pig model sensitized with heat killed M. bovis was found to be an efficient model for evaluating DTH response elicited by recombinant proteins cocktails. None of the cocktails elicited positive erythematous reaction in NTM sensitized and BCG vaccinated guinea pigs. A diagnostic test based on above cocktails could discriminate M. bovis infection from BCG vaccinated and NTM sensitizatized cattle.