Production and Evaluation of Immunopotentiator Oil Adjuvanted Haemorrhagic Septicaemia Vaccine

Abstract

Hemorrhagic septicemic (HS), septicemic pasteurellosis or septicemic fever is an acute infectious disease of cattle and buffalo. Pasteurella Multocida is the causative against H.S. in cattle and buffalo. 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 organism is heterogenous 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 to full one year. To solve this problem, it was planned o isolate the causative organism from typical cases of H.S. and to serotype this organism along with the vaccinal strains and this was decided to prepare the immunopotentiator oil adjuvanted vaccine from this field isolate. A number of samples were collected from various areas of Pakistan and serotyping was done and it was classified as CARTER TYPE-2. This field isolate was cultured in different media to get the maximum bacterial growth i.e., nutrient broth, casein sucrose yeast broth and tryptase yeast extract broth. For dense cultures, a new media containing peptic digest of blood was tried, which gave 1 mg/ml of dry weight of bacteria. For vaccine production, these bacteria were centrifuged at 7000 rpm for 30 minutes. The supernatant was discarded and sediment was resuspended in 0.85% normal saline having 0.3% formalin and concentration of the organism was adjusted by spectrophotometer. Pasteurella multocida was also grown in TSY broth in a fermenter and centrifuged at 7000rpm for 30 minutes a 4oC and reconstituted in 0.85% saline containing 0.3% formalin. Concentration of the organism was adjusted by spectrophotometer at 640nm. 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 il type emulsion was prepared. Viscosity of vaccine was measured with 1ml graduated pipette. Stability (shelf life) of vaccine was recorded by storing the vaccine at room temperature 4oC and 37oC. different baches of vaccine showed different behavior. Batch V11 and X11 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. Serum samples of selected animals were collected to determine the antibody titer of theses animals. Antibody titers were determined using LHA and ELISA. Encouraging results were found in these animals. Later on, buffaloes and cows at different farms are vaccinated with oil-based vaccine and alum precipitated vaccine at different livestock farms throughout Pakistan. Blood samples were collected with a n 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 were raised in rabbits and goats by injecting bacterin with 0.3% formalinized normal saline. This hyper immune serum was used in indirect hemagglutination test (IHA). For IHA, antigen was prepared by heating the organism at 100oC for 1 hour. Serums of experimental animals were used to determine the duration of protection from the disease. The detail of antibody level in experimental animals have been presented graphically in results. However, GMT showed that antibody titer is above the protection level and NIAB HS vaccine can be used safely to protect the animals from hemorrhagic 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 vaccine and demanded from the livestock farmers, it has been planned to prepare the vaccine on commercial sale. Now this vaccine is available in market with the brand name of NIAB HS VACCINE.