Production and Evaluation of Immunopotentiator Oil Adjuvantcd Haemorrhagic septicaemia vaccine.

Abstract

Haemorrhagic septicaemia (HS), septicaemic pasteurellosis or septicaemic fever is an acute infectious disease of cattle and buffalo. Pastcurclla multocida is the causative agent of H.S. in cattle and buffa1o. The disease is sporadic throughout the year but it may occur in epizootic form during the rainy season. The organism is of special significance in the tropical countries including Pakistan where it causes heavy economic losses. The orgamsm is heterogeneous in characteristics. Vaccine is the only accepted method to control the disease. Up till now, only available vaccine was alum precipitated formalin killed bacterin, which gives protection for only 12 - 14 weeks. Therefore, animal needs vaccination after every 3 months for complete protection. This project was designed to provide such a vaccine, which would give protection for full one year. To solve this problem, it was planned to isolate the causative organism from typical cases of H.S. and to serotype this organism along with vaccinal strains and this was decided to prepare the immunopotcntiator oil adjuvanted vaccine from this field isolate. A nwnber of samples were collected from various areas of Pakistan and serotyping was done and it was classified as Carter type - 2. This field isoht? was cultured in different media to get the maximum bacteril growth i. c. nutrient broth, casein sucrose yeast broth and tryptose yeast extract broth. For dense cultures, a new media containing peptic digest of blood was also tried, which gave 1 mg/ml of dry weight of bacteria. For vaccine production, these bacteria were centrifuged at 7,000 rpm for 30 minutes. The supernatant was discarded and sediment was resuspended in 0.85% normal saline having 0.3% fonnaline and concentration of the orgamsm was adjusted hv spectrophotometer. Pasteurella multocida was also grown in TSY broth in a fennentor and centrifuged at 7000 rpm for 30 minutes at 4 °C and reconstituted in 0.85% saline containing 0.3% fonnaline. Concentration of the organism was adjusted by spectrophotometer at 640 nm. Vaccine was prepared with paraffin oil, sunflower oil and peanut oil by using Tween 80 and Span 80 as emulsifiers and bacterin and water in on type emulsion \Y?L3 prepared. Viscosity of vaccine was measured with 1 ml graduated pipette. Stability (shelf life) of vaccine was recorded by storing the vaccine at room temperature, 4°C and 37°C. Different batches of vaccine showed different behavior. Batch VII and XII of vaccine showed all the desirable characters. HLB value of this batch was determined as 6.2. The vaccine was injected to rabbits and goats as experimental animals in the first phase. Scrum samples of selected animals were coJlected to determine the antibody titer of these animals. Antibody titers were determined using lHA and ELISA. Encouraging results were found in these animals. Later on buffaloes and cows at different farms were vaccinated with oil based vaccine and alum precipitated vaccine at different livestock farms throughout Pakistan. Blood samples were collected with an interval of 15 days, to determine the immune response, antibody titers and duration of protection was determined using IHA and ELISA. For this purpose hyper immune sera was raised in rabbits and goats by injecting bacterin with 0.3% fonnalinized normal saline. This h_yμ?r immune sera was used in indirect hacmagg1utination test (lHA). For lHA, antigen was prepared by heating the organism at 100°C for 1 hour. Serums of experimental animals were used to determine the duration of protection from the disease. The details of antibody level in experimental animals have been presented graphically in results. However GMT showed that antibody titer is 0 above the protective level and NlAB HS VACClNE can be used safely to protect the animals from haemorrhagic septicemia for longer period of time (more than one year) as compared to conventionally available alum precipitated vaccine. Keeping in view the results given by this vaccine <.i;,1..l demand from the livestock farmers, it has been planned to prepare the vaccine on commercial scale. Now this vaccine is available in the market with the brand name of NIAB HS VACCINE.