Hemorrhagic septicemia is a contagious bacterial disease of large ruminants principally in cattle and buffalo with high morbidity and mortality. The disease is endemic in nature and outbreaks are common during hot, humid and wet season. The acute and fatal nature and brief duration of the disease limit the antimicrobial therapy. In Pakistan, the disease causes heavy economic losses to dairy industry. Vaccination therefore, is an option for controlling the disease. For a quality vaccine, biomass production of P. multocida along with well developed capsule (immunogen) is necessary. The problem associated with the production of a quality vaccine is poor biomass production of P. multocida when grown in ordinary or routine media.
Present study was designed to isolate P. multocida from sick animals and its molecular characterization in the laboratory and study factors (temperature, media composition, pH incubation time and agitation or shaking) affecting its immunogen production and "in process quality control" factors (biological titer, dry mass, adjuvant and storage time) that affect antibody response. Finally, biomass production of the organism using biofermentor and monitoring of the antibody response of buffaloes to inactivated Montanide ISA-70 based P. multocida vaccine.
Each of the field isolates showed grey, viscous, mucoid, translucent and non hemolytic colonies on blood agar. There was no growth on MacConkey's agar. It was Gram negative coccobacilli or thin rods and bipolar when stained with Leishman's stain. The isolates were positive for Catalase, Oxidase, Hydrogen sulphide and Indole production along with nitrate reduction while it was negative for urease production, citrate utilization and gelatin liquefaction. The bacteria fermented glucose, sucrose, mannitol, mannose, but failed to ferment arabinose, maltose, salicin, lactose, dulcito and inositol.
Polymerase chain reaction (PCR) was performed on isolated colonies by using P. multocida specific and HS causing serotype B specific primers. P. multocida specific PCR gave product of 465 bp while HS causing serotype B specific primers amplified a product of approximately 590 bp.
Growth of the bacteria in casein yeast sucrose broth was optimized under different conditions. CSY broth showed dense growth of P. multocida during incubation for 18 hours. A temperature in between 35°C and 40°C showed its optimum growth. Poor growth was observed below 30°C and no growth was detected at 50°C and above. No growth occurred at pH 0.5 and 10.0 but best growth was obtained at pH 7.0 and 8.0. There was positive correlation between shaking in terms of rpm and growth. There was optimum growth at 500 rpm for 24 hours.
Inactivated HS Vaccine was prepared from dense growth in biofermentor on the basis of dry mass and bacterial count. The effect of biomass, adjuvant, storage of the vaccine, priming alone or with boosting on its potency was also studied along with boosting effect of montanoid ISA 70 oil based vaccine. Dry mass 1.7 mg/dose produced protective antibody titer while bacterial count 10-14/ml was sufficient to produce the protective antibody titer. Montanoid ISA 70 based vaccine provided immunity to buffalo calves better than aluminium hydroxide gel and bacterins. Boosting with oil based vaccine can help to keep the animal immunized for whole year. For better results of vaccine, it can be stored at 4oC for six months.
It is concluded that the proposed study improved quality of the vaccine and reduced volume of the vaccine dose, cost of its production and frequency of vaccination.