Comparative evaluation of pncA gene, IS6110 and 12.7-Kb fragment based PCR assays for simultaneous detection of Mycobacterium tuberculosis complex (M. tuberculosis and M. bovis) in cultured strains and clinical specimens

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.

Standardization and development of Pasteurella multocida inactivated adjuvanted vaccine against septic pasteurellosis in pigs

In piggery, septic pasteurellosis caused by Pasteurella multocida (B:2) is an issue of concern, which needs an effective vaccine. Here, we prepared a double emulsified (DE) vaccine containing 2.5 mg inactivated antigenic mass of pig field strain (B:2) (named as soron) isolated from an outbreak of septicaemic death in pigs and P. multocida P52 cattle strain (B:2) and studied their efficiency in terms of immunity to direct challenge, duration of immunity and the role of humoral and cell-mediated immunity. Both of these strains showed presence of hgbB, pfhA, nanH, ptfA, and tbPA virulence genes. The sequence analysis of bands of 760 bp product using capsular primers were obtained for soron and P52 revealed 99.2% homology between these two strains, indicating differences at genetic level. nanH and pfhA genes of soron shared 99.2% and 92.7% homology with P52, respectively suggesting differences between these two strains at genetic level. SDS-PAGE analysis of cell wall of both strains showed presence of about 15 major protein bands whereas Western blot analysis with 21 day soron immunized pig serum showed 16, 33, 47, 63 and 83 kDa polypeptides in both strains. The duration of immune responses were monitored at 3, 6 and 9 months post immunization in pigs. By direct challenge, pigs showed that the vaccines were protective at 21 days and up to 270th day post immunization. Vaccines induced a serum ELISA IgG response that peaked on 60 DPI which declined gradually up to 270th DPI in both vaccines. Stimulation index measured by lymphocyte proliferation test (LTT) indicated that the vaccine, induced cell-mediated immune response and in general percent stimulation index (SI) was higher in pigs immunized with soron vaccine at 15 days post challenge infection. The results showed that pig strain (soron) would be a potential homologous strain of P. multocida for the vaccine against pasteurellosis in place of use of cattle P. multocida P52 strain.

Recombinant CFP-10 as antigen for diagnosis and quantification of IFN-γ expression by real-time PCR in guinea pigs sensitized with Mycobacterium bovis

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.

Tuberculin response in guinea pigs with recombinant proteins cocktail prepared from Indian strain of Mycobacterium bovis (3/86 Rv)

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.

Detection of Mycobacterium avium & M. tuberculosis from human sputum cultures by PCR-RFLP analysis of hsp65 gene & pncA PCR.

BACKGROUND & OBJECTIVE: Identification of mycobacteria by conventional methods is slow, labour intensive and may at times fail to produce precise results. Molecular techniques developed in the recent past, overcome these disadvantages facilitating rapid identification of most species. We undertook this study to characterize mycobacteria isolated from sputa of human patients suspected to have tuberculosis by conventional methods and later, by polymerase chain reaction-restriction fragment length polymorphism analysis (PRA) of hsp65 gene and pncA PCR. METHODS: Twenty two mycobacteria isolated from 30 sputum samples were identified based on growth rate, pigmentation, cultural and biochemical properties and subjected to PRA of hsp65 gene involving amplification of hsp65 gene and digestion of the product with BstEII and HaeIII in separate reactions and analysis of digests by 3 per cent agarose gel electrophoresis. The mycobacteria were simultaneously evaluated by M. tuberculosis-specific and M. bovis-specific pncA PCR assays in separate reactions. RESULTS: With the conventional biochemical tests, the 22 sputum culture isolates were identified as M. tuberculosis (19) and M. avium complex (MAC) (3). PCR of hsp65 gene yielded 439 bp product in all the mycobacteria tested. The RFLP patterns of three MAC isolates with BstEII and HaeIII were identical to reference M. avium strain with two fragments in each of the digest. M. intracellulare reference strain showed a distinct pattern with 3 fragments each in both enzyme digests. The PRA of hsp65 confirmed MAC isolates as M. avium. M. tuberculosis isolates including H37Rv and M. bovis strains could not be discriminated by PRA of hsp65. The two pncA PCR assays (M. bovis-specific and M. tuberculosis-specific) detected specifically the respective organisms with an amplification product of 185 bp. The MAC strains yielded no amplification product in both the pncA PCR assays. INTERPRETATION & CONCLUSION: PRA profiles of hsp65 could differentiate MAC isolates into M. avium and M. intracellulare but could not distinguish between M. tuberculosis and M. bovis. pncA PCR assays were found specific in detecting the respective mycobacterial species. The study confirms utility of pncA PCR assays in differential identification of M. tuberculosis and M. bovis and that of PRA of hsp65 in the identification of M. avium.