Construction For Goose Source Salmonella Typhimurium Crp?hfq Genes Deleted Strains And Characteristics Evaluation Of Biological And Immunological

Salmonella typhimurium is an enteric pathogenic bacteria,which is more common in waterfowl culture.Clinical symptons such like gastroenteritis,fever,diarrhea,and even death may be caused after infection which is posing a serious threat to the development of waterfowl breeding industry.In order to explore ways to control the spread of Salmonella typhimurium and to reduce the harm of Salmonella typhimurium to the goose industry in Guangdong,according to the results of PFGE molecular typing on156 Salmonella typhimurium strains isolated from goose in Guangdong,our researh group picked one of the main circulating strain,termed as A29 as the base for further crp and/or hfq genes deleting.Moreover,the biological characteristics,drug sensitivity,mice pathogenicity and immunogenicity of these gene deleted strains were evaluated.The main research contents are briefly described as below:Taking crp gene encoding cyclic adenosine monophosphate receptor protein and hfq gene encoding RNA chaperone as the aims of gene deleting,we constructed crp gene knockout strain A29?crp,hfq gene knockout strain A29?hfq and crp/hfq double gene knockout strain A29?crp?hfq by using?-Red homologous recombination technology.Furthermore,the plasmid p BR322 was used as a vector to construct a related gene recovery plasmid,and three plasmid-recovery strains of the deletion strain were successfully obtained.The biological characteristics of A29,A29?crp,A29?hfq,A29?crp?hfq and their plasmid-recovery strains were determined and compared.The results showed that the growth and biochemical characteristics of the gene-deleted strains were differed from wild type strain,and no significant differences found between the plasmid-recovery strains and A29.Compared with A29,A29?crp and A29?crp?hfq had shorter cell length(P<0.001)and smaller colony diameter(P<0.001);Rhamnose,xylose and maltose could not be decomposed,salicin could be decomposed,urease and hydrogen sulfide test showed negative;The growth rate of both A29 and A29?crp was consistent,but OD₆₀₀ of A29?crp decreased before mid-log phase,increased after mid-log phase.Compared with A29,the length of A29?hfq was significantly longer(P<0.001),it could not decompose xylose,and the urease test showed negative;the expression of flagellar gene was affected,and the mobility of flagellar gene decreased in semi-solid medium;the growth rate slowed down and OD₆₀₀decreased in growth period.The results of genetic stability analysis showed that the deletion sites of the three gene-deleted strains could be inherited stably after passage 60 generations.Drug sensitivity tests were carried out on A29,A29?crp,A29?hfq and A29?crp?hfq.The results showed that the sensitivity of A29?crp to aminoglycosides,carbapenems,ciprofloxacin,trimethoprim and azithromycin decreased(P<0.001),while the sensitivity to tetracyclines increased(P<0.05);the sensitivity of A29?hfq to12 drugs except penicillins and cefazolin was all increased(P<0.001).The sensitivity of A29?crp?hfq to aminoglycosides,carbapenems and ciprofloxacin decreased(P<0.001),while the sensitivity to tetracyclines,trimethoprim,chloramphenicol and azithromycin increased(P<0.001).Experimental results of artificial infection in mice showed that compared with A29,the virulence of A29?crp decreased 21.7 times,while 202.2 times decresing for A29?hfq and 1256.1 times for A29?crp?hfq.Pathological observation showed that A29?hfq and A29?crp?hfq were less sensitive to liver damage in mice.Furthermore,the immunized mice could be completely protected by inoculated gene-deleted strains at about 10^-1 LD₅₀ doses following challenged by lethal dosage of wild type strain.A29?hfq had the least effect on weight loss in mice,and the mice immunized with A29?crp?hfq were more protective against wild-type strains.Serum tested by EILSA indicated that IFN-?content increased about 8 times after immnization.In this study,A29?crp,A29?hfq and A29?crp?hfq were successfully constructed and may serve as the vaccine candidates for the prevention and control of salmonellosis in goose,and lay a foundation for the functional linkage between crp and hfq genes of Salmonella typhimurium.