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ThesisItem Open Access INVESTIGATIONS ON ACIDIC SERINE PROTEASE V2 (APRV2) OF DICHELOBACTER NODOSUS AS A POTENTIAL VACCINE CANDIDATE AGAINST VIRULENT FOOTROT(Department of Veterinary Microbiology, 2015-12-05) WANI, AASIM HABIB; Sharma, MandeepABSTRACT Dichelobacter nodosus, a Gram negative anaerobe secretes extracellular proteases involved in digestion of the hoof and underlying soft tissue of ruminants causing footrot. Various whole cell and fimbriae based, monovalent and multivalent vaccines have been used, but they fail to provide cross protective immunity against different serogroups. Among the proteases, AprV2 has been considered most important virulence factor and is conserved across different D. nodosus serogroups. Considering the role of AprV2 in footrot pathogenesis, this study measured the immunogenic potential of AprV2 in sheep. AprV2 gene was amplified from a virulent D. nodosus that was characterized using genotypic and phenotypic methods. AprV2 was expressed in E. coli and purified through affinity chromatography. Safety and potency tests were carried out using rAprV2 in combination with FCA and IFA in sheep. Immune response comparison of rAprV2 vaccinated sheep (group I) was made with whole cell vaccinated (group II) and control sheep (group III). Indirect ELISA revealed a significant increase in rAprV2 specific (P < 0.05) IgG immune response in group I and group II sheep. IgG titres of 320 were measured at day 15 and 45 in group I sheep while group II sheep revealed titres of 5120 and >10240 at 15 and 45 days respectively. PBMCs (2×106 cells/well/ml) from each sheep on each time point were stimulated with rAprV2 (25 µg), heat killed D. nodosus (10 µg) and Concanavalin A (5 µg) as a positive control for 4 hours and monitored for measuring early Th1 (IFN-Ɣ and TNF-α) and Th2 (IL-4 and IL-6) response. It was observed that in group I sheep, significant (P ˂ 0.05) elevation was seen in IL-4 and IL-6 on 15th day when stimulated with rAprV2 and only IL-6 on 15th and 45th day when stimulated with heat killed D. nodosus in comparison to control sheep. In group II sheep IL-4, TNF-α and IFN-Ɣ were significantly (P ˂ 0.05) elevated on 15th day and only IFN-Ɣ on 45th day when stimulated with rAprV2 while on stimulation with heat killed D. nodosus, IL-6 and TNF-α were elevated significantly (P ˂ 0.05) on 15th day while IL-6, TNF-α and IFN-Ɣ were significantly (P ˂ 0.05) low on day 45 when compared to control animals. Intra-group comparison of group I sheep to day 0 revealed that only TNF-α was significantly (P ˂ 0.05) elevated at 45th day when stimulated with heat killed D. nodosus. Intra-group comparison of group II sheep revealed significant (P ˂ 0.05) elevation in all four cytokines on 15th and 45th day when stimulated with rAprV2, when stimulated with heat killed D. nodosus, TNF-α and IFN-Ɣ were significantly (P ˂ 0.05) elevated on day 15 and IL-6 and TNF-α on 45th day. In general, only IL4 levels were elevated on 45th day than observed on 15th day in rAprV2 vaccinated sheep, however, insignificant. In conclusion, vaccination with rAprV2 induced superior humoral antibody response (IgG) and Th2 type cell mediated immune response (IL-4