| Avian Salmonellosis is an acute or chronic disease caused by one or more members of Salmonella, pathogenic salmonella in chickens including pullorum, fowl typhoid and paratyphoid. Septicemia and enteritis are the most common clinical manifestation. Salmonellosis generally exists in all parts of the world, and it not only can seriously harm the development of the poultry industry but also can pass the poultry products into the food chain and severely threaten the health of human beings, and it is a zoonotic infectious diseases. For a long time, the disease was prevented and treated by using antimicrobial drugs in most of the chicken farms in China. At present, due to overuse and misuse of antimicrobial drugs, drug-resistance of Salmonella has been significantly enhanced accompanying with continuously widened spectrum of multiple drug resistance, which has caused great difficulties on prevention and treatment of the disease. In recent years, Salmonella vaccine has been used to immune the poultry and good results have been achieved in many foreign countries. In this study, we have done research on the isolation and identification of salmonella from 108 samples of 26 chicken farms in 7 districts of Sichuan Province, and the epidemic strains have been done research on development of attenuated strain and preparation of attenuated vaccine. The purpose is to provide important reference data for the epidemiological investigation, vaccine immunity and drug therapy of salmonella infection in Sichuan.The bacteria from 108 samples of 26 chicken farms were isolated by selective medium of salmonella and identified by following test:morphological observation, PCR detection, biochemical identification, serotype identification, drug sensitive experiment and animal pathogenicity experiment. The results showed that 28 salmonella strains were obtained from 108 samples, and the separation rate was 25.9%, and 9 strains isolates were in line with the biochemical characteristics of Salmonella pullorum and 19 strains were in line with the biochemical characteristics of Salmonella gallinarum. The serotypes of most isolates were O2, O9, O9,12. The 28 Salmonella strains were pathogenic to chickens, and were sensitive to Amikacin, Neomycin, Cefoperazone, Ofloxacin, Gentamicin, and resistant to most of antimicrobial drugs.The 10 strains Salmonella gallinarum from 9 chicken farms were selected to detect its virulence and immunogenicity, and one strain was obtained and named TQ-1-1, which had better immunogenicity and weaker virulence. TQ-1-1 was taken as starting strain and mutated by UV and HNO2, and the strains obtained by mutagenesis were preliminarily screened according to the virulence. The obtained attenuated strains were continuously subcultured and detected for biological characteristics. The results showed that attenuated Salmonella gallinarum strain was successfully screened and named CN505-120, which had stabile hereditary and good immunogenicity, and the median lethal dose to mice was 1.85×109 CFU, the virulence of CN505-120 was 14 times lower than TQ-1-1.Attenuated CN505-120 strain was greatly cultured in the nutrient agar medium, and then the cultured bacteria was preliminarily screened according to bacterial concentration, reaction time of protective agent and bacteria, dispensing volume of the vaccine, and the optimal conditions for the preparation of the heat-resisting lyophilized protective agent live vaccine were optimized. The finished vaccine was developed and detected by following test:physical character, viable count, safety inspection, optimal immunization dose, immune efficiency, the period of immune protection, storage and validity, etc. The results indicated that attenuated Salmonella gallinarum vaccine was obtained, and which had good security and immunogenicity, and was conducive to storage and transport. The finished attenuated vaccine can be used in chicken production. |
