Studies on various growth conditions for improved biomass of Clostridium perfringens vaccine production and immunogenicity

Enterotoxaemia has been reported worldwide in sheep and goats caused by bacterium Clostridium perfringens types B-11 and D-27. The bacteria multiply in intestine and produce toxin, which is absorbed in the body, resulting in death of animals. Prevention against enterotoxaemia requires proper vaccination to promote immunity to toxin (biomass) and to avoid the predisposing cause of the disease. Clostridium perfringens types B-11 and D-27 grown in different media included Nutrient Broth (NB), Rich Nutrient Broth (RNB), Brain Heart Infusion (BHI) and Reinforced Clostridial Medium (RCM) with 5% initial inoculum and incubated at 37% in anaerobiosis condition yielded moderate to dense growth. However, the better growth was obtained with higher biomass and maximum hemolytic unit (HU-480) production in Reinforced Clostridial Medium, moderate turbid growth with HU-256 in Brain Heart Infusion, while least-growth was observed in Rich Nutrient Broth (HU-184) whereas poor growth was obtained in Nutrient Broth (HU-140). Reinforced Clostridial Medium was found with better performance for biomass (Toxin) production of Clostridium perfringens species but the other media used during the study were not viable to use for biomass production. Different factors such as pH, shaking and time interval influenced the growth performance of biomass production of Clostridium perfringens in different media. All normal conditions were provided to record the biomass production in Reinforced Clostridial Medium. Clostridium perfringens types B-11 produced 40 HU/ml while type D-27 yielded 68 HU/ml at pH 6.3 without shaking at 3 hours interval and the total biomass was produced as 108 in normal conditions, when pH was changed and adjusted to 6.8 and 7.4 without shaking, generated 72 and 192 HU/ml by type B-11 respectively. Whereas type D-27 produced 144 and 320 HU/ml at pH 6.8 and 7.4 with same interval. The total biomass of Clostridium perfringens types B-11 and D-27 produced with pH 6.8 was 216 and 512 HU/ml at the pH of 7.4 but in case of pH 7.4 with shaking both the types B-11 and D-27 produced 1024 HU/ml which was quite double than the biomass produced without shaking. Internationally the dose of Clostridial perfringens vaccine was standardized as 250HU/dose. Based on the factors, Nutrient Broth had a production cost of paisa 28/dose, paisa 27/dose while Rich Nutrient Broth had a cost of Rs.1.62/dose. However, the Reinforced Clostridial Medium was used for vaccine production and had a production cost of paisa 14/dose. When simple modification in conditions was adopted, RCM was also observed as the best medium among other media used for propagation and also biomass (toxin) production for the vaccine purpose. The vaccine prepared was processed and checked free for bacterial and fungal contamination and tested for its quality by injecting subcutaneously in mice and all mice were survived with no local reaction. The vaccine also tested for its potency against Clostridium perfringens and found that the mice with booster dose of vaccine followed by a challenge dose of Clostridium perfringens were survived after 21 post-vaccination but in the case of mice, which did not receive vaccine were when challenge with the recommended dose of Clostridium perfringens all were dead. Further, it was also observed that all media used during the study, generated different biomass of vaccine, Reinforced Clostridial Medium produced 2000 while Nutrient Broth yielded 500 doses of vaccine. It is concluded that biomass production of Clostridium perfringens (enterotoxaemia) vaccine could be increased through modification in procedures during vaccine manufacturing process in the laboratories. It was also observed that RCM is the best medium among all four media used during investigation where Clostridial perfringens utilized properly and yielded more biomass as compared to other media. Further observed that pH, shaking and time interval for harvesting of bacterial growth (culture) had positive influence on biomass production for vaccine preparation. It was also recorded that Clostridium perfringens toxin is highly potent, protective and effective that generated high antibodies with 0.3ml close in mice against Clostridium perfringens challenge dose. However, by adaptation of the above conditions, a very cheap, safe and reduced in cost of vaccine production was also obtained.