Laboratory and Field Evaluation of a Locally Prepared Montanide® Adjuvanted Combined Hemorrhagic Septicemia-Mastitis Vaccine

Abstract

The present study represents a maiden attempt to develop and evaluate a combined hemorrhagic septicemia (HS) and mastitis vaccine in cows and buffaloes. The study was compartmentalized into two phases. In phase I (laboratory settings), isolates of Pasteurella multocida, Staphylococcus aureus and Streptococcus agalactiae isolated from field cases of HS and mastitis were scrutinized for virulence/pathogenicity and immunogenicity in laboratory animals. Bacterin- toxoids of S. aureus and Str. agalactiae were blended with prepared antigen of P. multocida, Montanide® ISA 201 VG, thimerosal and sodium azide to prepare combined HS- mastitis vaccine that was evaluated for sterility, safety and side effects under in vitro conditions and in vivo in cattle, buffaloes and mice. A challenge-protection assay conducted in immunized mice indicated 100% survival of challenged mice. The vaccine was physically stable in terms of pH, sedimentation, color, appearance, and syringibility for 6 months observation period at 37°C. In Phase II (field evaluation), the combined vaccine was evaluated in adult cattle and buffaloes and calves of cattle and buffaloes. To this end, a total of 70 S. aureus and Str. agalactiae free lactating buffaloes (n=45) and cows (n=25), 50 lactating cows (n=25) and buffaloes (n=25) positive for S. aureus/Str. agalactiae and dairy calves (buffalo calves n=70; cow calves n=50) aged up to 1 year were treated with 2 doses of combined HS-mastitis vaccine at 21 day interval and evaluated (where relevant) for 6 months in terms of ELISA based antibody titers against P. multocida, S. aureus and Str. agalactiae, incidence of HS, local and systemic reactions, incidence and prevalence of S. aureus and Str. agalactiae mastitis, severity of mastitis, milk somatic cell count, milk yield, cost effectiveness and vaccine efficacy. ELISA based antibody titers against P. multocida, S. aureus and Str. agalactiae were significantly (P˂0.05) higher in vaccinated groups than in unvaccinated groups. Two cases of HS were recorded in vaccinated animals vis-à-vis 7 cases in un-vaccinated animals. Incidence of S. aureus and Str. agalactiae over 180 days in vaccinated and un-vaccinated cows and buffaloes initially cultural –ve for these pathogens was 3 and 10, respectively; the corresponding figures in groups initially culture +ve for these pathogens being 2 and 12, respectively. Cumulative mean somatic cell counts in vaccinated groups were significantly lower (P<0.05) than those in respective unvaccinated controls. Milk yield was significantly higher (P<0.05) in vaccinated cows and buffaloes than in un-vaccinated controls. Mastitis severity scores were significantly lower (P<0.05) in vaccinated groups than in unvaccinated controls. The vaccine tested had a vaccine efficacy 84.78 and 90.25% against HS and mastitis, respectively with a financial benefit worth Rs 2,060,300. In sum, Montanide® adjuvanted combined HS-mastitis vaccine had preventative role against HS and both preventative and curative role against S. aureus and Str. agalactiae associated mastitis. In view of the preliminary nature of the study, additional work involving much larger number of cows, buffaloes and calves is clearly warranted.