| Colibacillosis was one of the epidemic diseases of piglets. It is caused by some certain serotypes of Escherichia coli and characterized by yellow scour of newborn piglets, white scour of piglets and edema disease of growing pigs. Its morbidity and mortality was 10%～30%, 80%～100%, respectively. It is one of the most important factors which impact the development of pig industry.In Shaanxi province, the disease distributed in many cities and counties such as Xi'an, Baoji, Hanzhong, which influenced development of pig industry seriously. Using high pathogenic Escherichia coli stains, O2, O8, O135, O138, O139, O141 etc, isolated from some pig farms in Shaanxi province, as productive strains, and multivalent inactivated vaccine of E.coli was produced by Yangling Green Bio-engineering Co. Ltd. But there still were two problems affected the normal productive process. One was storage and passage of bacterial stock; another was the unperfected fermentative technology which resulted in high cost, low production and poor economic benefits. So two aspects of studies were designed and carried out to optimize the technics. By observing the morphology, colony, livability and the pathogenicity to mice of various E.coli stains after storage.The stocks can be kept for 1 year in the optimal storage condition mixed with 15% glycerol in―20℃and for 2 years in 15% milk-sucrose lyophilized―20℃storage condition. The stocks can be kept for 90 days in 4℃in above two methods. The pathogenicity to mice inoculated with broth which cultured by various passages of bacteria was observed, and the bacteria from the infected animals were isolated and identified. The successive passage times were restricted within 10 generations. If beyond it, the bacteria were prone to mutant and would not be used as stocks.Several batchs of piglet E.coli multivalent antigens were propagated with routine powder medium in 50 liter fermentation vat. Through the means of plating count, basic culture conditions such as temperature, pH, ventilation quantity, glucose content and culture time were optimized . The results showed that piglet E. coli grew best in routine powder medium when the culture temperature was at 37±0.5℃, pH controlled in the range 7.0-7.2, ventilation volume increased progressively, adding 40% glucose solution according to the change of pH, the colony form unit (cfu) of E.coli can reach 15 billion per ml, which was 5-6 times of that before optimization (2×109 cfu-3×109 cfu). With the two technological innovation, the production costs of the piglet E.coli multivalent inactivated vaccine was decreased from 0.2 yuan RMB/bottle to 0.05 yuan RMB/bottle, creating additional net profits of 1.5 million yuan RMB every year for the company. |
