The Cultivation Of Attenuated Salmonella From Duck And Its Biological Characteristics

This study attempted to cultivate one attenuate salmonella as the candidate vaccine strain for the control and prevention of duck salmonellosis. The salmonellae were separated from the duck farm in the sichuan and chongqing of china, chose one with strong virulent and high separation rate as the original strain, through UV-MNNG compound mutation process, the mutant which attenuated was confirmed by the LD50 detection to the duckings, then the biological character of attenuate strain was studied. The result show as following:1. selecting result of original strain:30 strains of salmonellae which the glass plate agglutination obviously were chose, according to the O antigen classification, there were 16 strains of O7,8 strains of Os and 6 strains of O4, chose one with strong virulent from O7 as the original strain, its serotype is 6,7:1,V:enx, which called S.bonn.2. The optimum induced mutation condition of UV and MNNG:the original broth with 1×108 CFU/ml concentration processed by stiring under 254 nm wavelengh UV radiation with 20 cm vertical height for 15 seconds and spinning in 37℃、100 r/min table concentrator by adding MNNG to the final concentration of 0.5 mg/ml, both treatment lead to about 80～90% mortality of original broth, so the two induced mutation conditions were combined as the optimum compound mutation condition.3. Virulent variation of mutant strain:the LD50 of original strain to one week old duckings is 7.4×107 CFU, while the mutant strain’s is 3.9×108 CFU, is 5.2 times to the original strain, indicating that the mutant strain was attenuated.4. The biological characteristic of mutant strain:the serotype of mutant is identical with the original strain after culture in vitro for 35 generations, no matter the colony or under the microscope, the morphology of the mutant stain didn’t change, indicating that the mutant strain could abdicate steadily, after culture in vitro for 35 generation, the virulent of mutant strain was more attenuated, but in the 95% confidence interval, indicating that the virulence of the mutant strain is stable. The biochemical testing result make no different between the two strains, what different is the colony of mutant strain is smaller than the original strain, and its flagella could express in liquid medium but not in solid medium, while the flagella of the original strain could express in both solid and liquid medium. By extracting the genome of the mutant strain and original strain, and amplifying the fliC which encode the I phase flagella through the specific primers, both of them could amplify the fliC, and it may be relate to the mutant in the gene which control the expression of the I phase flagella. Duckings were immune by mutant strain through oral and inject administration, high level of antibody were detected in all administration after the second immunization, but the antibody level of the injection group is higher than the oral group, and the antibody level of the higher dose group is higher than the lower dose group. Those result show that the mutant strain had fine immunogenicity, if the immunization does and delivery route are appropriate, it can induce high level of antibody, the toxic attack test also show that the duck immune with mutant strain can resist the attack of wild strains of the same serotype.