ABSTRACT
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.
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.