Isolation,Identification,Analysis Of Antimicrobial Resistance,MLST Molecular Typing Of Salmonella From Wild Birds In East China And Preliminary Study On Its Pathogenicity

Salmonella is an important zoonoses pathogen worldwide.The animal-derived food contamined by Salmonella is the main source of Salmonellosis in human.The circulating infection by salmonella between humans and animals is commonly named as the salmonella cycle,which has complicated relationship,and the wild birds is also an important part of the cycle.Therefore,the distribution of salmonella in wild birds and the correlation research from salmonella isolates between wild birds and poultry is of great significance for the poultry dustry and food safety.In this study,the carrying situation,antimicrobial resistance and MLST molecular typing of salmonella isolates from wild birds in eastern China,and pathogenicity of S.Choleraesuis and S.Choleraesuis biotype Kunzendorf were investigated.The study will provide epidemiological data and theoretical basis for the prevention and control of salmonellosis.1.Isolation,identification and antimicrobial resistance analysis of Salmonella from wild birds in east China from 2015 to 2018To investigate the prevalence and antimicrobial resistance of Salmonella in wild birds,5889 fecal samples of wild birds were collected in eastern China during 2015-2018,Salmonella were isolated and identified by mPCR and glass plate agglutination.Their resistance to 21 commonly used antibiotics was determined by the method of Kirby-Bauer,and their 11 main carring resistance genes,which locate on the gene island-1(SGI),were detected by PCR.As the results,a total of 56 isolates were identified as Salmonella with the isolation rate of 0.95%.The isolates included 30 strains(53.57%)of S.Typhimurium,10 strains(17.86%)of S.Choleraesuis biotype Kunzendorf,7 strains(12.50%)of S.Enteritidis,5 strains(8.93%)of S.Potsdam,and 4 strains(7.14%)of S.Choleraesuis.The results of the antibiotic sensitivity test showed that more than 50%of isolates were resistant to penicillin,oxacillin,erythromycin and rifampicin,and all isolates were resistant to oxacillin and rifampicin.The multi-drug resistance rate of these isolates was 10.71%.The determination results of carring resistance genes indicated that the detection rates of blaCMY-2?blaTEM-1-like?blaCTX-M?blaOXA-1-like?sull?aacC4?Aac(6')-1b?floR and tet(G)were 42.9%,67.9%,26.8%,5.4%,60.7%,10.7%,48.2%,23.2%and 41.1%,respectively,blaPSE and blaSHV were not detected.The detection rate of earring resistant gene was higher than that of resistant phenotype in all isolates The above results indicated that S.Typhimurium was the predominant serotype in wild birds in eastern China,and it is the first report for isolation of S.Choleraesuis biotype Kunzendorf and S.Choleraesuis in wild birds.2.MLST molecular typing of salmonella isolates from wild birds in east China7 housekeeping genes of salmonella,named as aroC,thrA,purE,dnaN,sucA,hisD and hemD were amplified by PCR,respectively.With MLST,the 56 isolstes could be divided into 7 type ST,ST808,ST99,ST19,ST1498,ST11,ST314 and ST413.Among them,S.typhimurium isolstes belonged to multiple types ST,including ST99,ST19 and ST1498.However,all S.enteritidis,S.Choleraesuis,S.Choleraesuis biotype Kunzendorf and S.Potsdam isolstes belonged to single type ST,which were ST11,ST808,ST314 and ST413,respectively.The attenuated live vaccine strain C500 of S.Choleraesuis was ST1753 as a reference,which is different from S.Choleraesuis isolates.3.Preliminary study on the pathogenicity of of S.Choleraesuis and S.Choleraesuis biotype Kunzendorf from wild birdsThe growth curve was deternined through cultured single colony,and the results showed that there was no significantly difference between isolates and attenuated vaccine strain C500.2 strains of S.Choleraesuis and 2 strains of S.Choleraesuis biotype Kunzendorf were selected for SPF chichens experiment,and vaccine strain C500,was used as the control.3-day-old SPF chickens were challenged intraperitoneally with 107,108 and 109 CFU of the selected strains respectively,the results showed that 5 strains were high virulent for chicken,chickens from group PI and K1,K2 all died within three days,while one chicken of each group P2 or C500 survived at the dosage of 107 CFU,indicated that P1,K1 and K2 strains were more virulent than P2 or C500 strains in SPF chickens.The pathogenicity of PI and K2 was evaluated by the median dose(LD50)in SPF chickens or in mice.In SPF chickens,the LD50 of K2,P1 and C500 were 9.0×106.6 CFU,3.0×105.8 CFU and 8×107.5 CFU,respectively,indicated that the isolates were more virulent than C500.While the LD50 of them were 3.0×107.5 CFU,9.0×107.6 CFU and 8×107.5 CFU,respectively,indicated that the virulence of the isolates was similar as that of C500 in mice,and the pathogenicity of the two isolates was no significantly difference in mice.The results of pathological lesions indicated that the dead chickens and mice were characterized by acute inflammation.In conclusion,the predominant serotype and resistance of salmonella from wild birds are consistent with those from poultry.For the first time,S.Choleraesuis and S.Choleraesuis biotype Kunzendorf were isolated from wild birds in China.Preliminary study on the pathogenicity of S.Choleraesuis and S.Choleraesuis biotype Kunzendorf from wild birds showed that the pathogenicity of isolate strains were stronger than that of vaccine strain C500 in SPF chickens.However,the pathogenicity of all isolate strains and vaccine strain were similar in mice,and its pathogenesis still needs further study.