Construction And Immunogenicity Of An Attuanted Salmonella Typhimurium ΔcrpΔcya Mutant Strain

Salmonella typhimurium is a group of enteric pathogenic bacteria with a broadhost spectrum and pathogenic. A variety of livestock’s, poultry’s, birds’, rodents’ andmany other small animals’ and human’s intestinal could be infected, even lead todiseases like gastroenteritis, fever, paratyphoid fever and diarrhea. It can cause humanin food poisoning and some other desease, which is a huge threat to human health andbreeding industry. Therefore, it has important significance for veterinary, medical andpublic health. Due to drug resistance and food safety issues, vaccination was an idealchoice for the prevention and control of this disease. With the continuous deepening ofthe development of molecular biology and bacterial genetics, the use of geneticengineering method to prepare Salmonella typhimurium vaccine achieved a great deal,and it has been applied and shows a good prospect.In the current study, a safer attuanted Salmonella typhimurium vaccine wasdeveloped. So it lay the foundation for exploit a more safer and effective attenuatedSalmonella typhimurium vaccine and the further application in an oral live vectorvaccine for mucosal immunity. The attuanted strain, which was on the basis of thecyaSL1344, was developed through deleting the crp gene. The crp cyaSL1344mutant was constructed through the two-step method introduced by the transduction ofrecombinant suicide plasmid. Then compared the biological characteristics of thesestrains and made a preliminary study of the immunogenicity of crp cyaSL1344.1. Construction and identification of the Δcrp cyaSL1344The E. coil donor strain χ7213transformed with recombination suicide plasmidpREΔcrp was conjugated with the recipient S. typhimurium cyaSL1344strain. Thecrp mutant of S. typhimurium cyaSL1344was constructed by the allelic exchangeintroduced by the transduction of the recombination suicide plasmid. Then, theΔcrp cyaSL1344mutant was determined by PCR and sequencing analysis. 2. Comparison analysis for biological characteristics of theΔcrp cyaSL1344and other strainsThe phenotypes, growth properties, genetic stability and virulence in chick of theΔcrpΔcyaSL1344and the other3strains were characterized. The results of thephenotypes, growth properties and genetic stability showed that some significantlydifferences between the ΔcrpΔcyaSL1344and SL1344. ΔcrpΔcyaSL1344was not ableto ferment maltose, glycerol, sorbitol and H2S, but it could also ferment glucose, thatwas consistent with the parent strain ΔcyaSL1344and the ΔcrpSL1344exceptgalactose and rhamnose. The gelatin liquefaction test, urease test, catalase test,phenylalanine deaminase test results and so on and the serotype of the4strains wereconsistent. The ΔcrpΔcyaSL1344was stable with the recombinant crp gene and thevirulence was further reduced10~6than the SL1344strain.3. Oral immunization of chicken with Δcrp cyaSL1344A preliminary evaluation of the effects of humoral by attenuatedΔcrp cyaSL1344strain was made, then investigate the dynamic changes of serumlevels of IgG antibodies by using indirect ELISA. The results showed that: Comparedwith the negative control group,the serum levels of IgG antibodies was significantlyincreased, showed a increased tendency gradually and antibody concentration reachedthe maximum at the second or third week, then gradually decreased from21days afterimmunized. It showed that the Δcrp cyaSL1344was immunogenicity to chick.