Clinical Detection And Characterization Of Campylobacter Jejuni Derived From Animals And RacR Protein Expression

Campylobacter jejuni is an important zoonotic pathogen.Campylobacter disease is one of the common foodborne bacterial infections,which can cause a variety of clinical symptoms,including diarrhea,fever,vomiting and bloody stools.Since it can infect a variety of animals and humans and endanger public safety,it has now attracted attention at home and abroad.This subject aimed to establish a rapid molecular detection method,analyze the infection of Campylobacter jejuni in meat sheep and poultry in parts of Jiangsu,and detect some of its virulence genes.In order to establish an immunological detection method for Campylobacter jejuni,a prokaryotic expression system was used to express its RacR protein and polyclonal antibodies were prepared.1.Establishment of Duplex-PCR detection method for Campylobacter jejuniBased on the 16S rDNA and hipO gene of Campylobacter jejuni,two pairs of specific primers were designed to establish a Duplex-PCR detection method.This method can specifically amplify 16S rDNA partial gene fragments and hipO partial gene fragments of Campylobacter jejuni,the size of which is 756 bp and 305 bp,respectively,while the other eight quality control strains were amplified negative.The limit of detection of Campylobacter jejuni pure culture was 1 pg/?L DNA.The minimum detection limit of Campylobacter jejuni in sewage was 1.5 CFU/mL,which was 15 CFU/g in lamb stool samples.The establishment of this method provides technical support for the rapid detection of Campylobacter jejuni in clinic2.Analysis of infection status and drug resistance of Campylobacter jejuni in healthy meat sheep and diarrhea livestockIn the period from March 2018 to September 2019,a total of 540 clinically healthy lamb anal swab samples and 92 diarrhea livestock and poultry intestinal samples were collected in the main breeding areas of Jiangsu and were quickly detected using the established Duplex-PCR method.The results showed that a total of 10 samples were positive for Campylobacter jejuni and the positive detection rates of sheep,chicken and goose were 0.71%,10.5%and 12.5%respectively,in which the positive rates of healthy meat sheep was 0.74%and diarrhea livestock was 6.52%.Ten strains of Campylobacter jejuni were obtained through bacterial isolation,including 4 strains from sheep,4 strains from chicken and 2 strains from goose.Characteristic analysis of 10 strains revealed that the growth activity of sheep-derived isolates was significantly lower than that of chicken-derived isolates and goose-derived isolates.however,the ability to infect mice from sheep-derived isolates was higher than isolates of other species.The sensitive drug test found that each isolate was completely resistant to sulfonamides;the resistance to cephalosporins,tetracyclines,and quinolones was high.All sub-strains were multi-drug resistant strains and a total of 7 drug resistance profiles were generated,most of which are SXT-CRO-TE-CIP.3.Isolation of virulence-related genes and analysis of racR gene conservationIn order to understand the characteristics of the virulence factors of Campylobacter jejuni,PCR was performed on seven major virulence genes.The results showed that the tested strains all had temperature response regulator genes(racR)and the positive rates of adhesion-related genes(peb1A),chemotactic regulator genes(cheY),adhesion colonization genes(cadF)and invasion protein genes(iamA)were 90%,while which of the flagellin gene(flaA)was only 50%.In addition,none of the tested strains was detected plasmid gene(virBll).The carrier rate of virulence-related genes of Campylobacter jejuni isolates from sheep was higher than that avian-origin isolates.The detected strains carried more than 4 virulence genes,in which the dominant virulence factor type were racR-peb1A-cheY-cadF-iamAflaA and racR-peb1A-cheY-cadF-iamA.Through sequencing of racR,it was found that the gene only had a few base mutations between different strains,while no amino acid mutations were caused.This indicated that it was highly conserved and can be used as a candidate gene for rapid detection.4.Expression of Campylobacter jejuni RacR protein and preparation of polyclonal antibodySpecific primers for racR coding region were designed by referring to the gene sequence(NC002163.1)in Genbank.After PCR amplification using the isolate HC26 genome as a template,the target fragment was inserted into pJET1.2 plasmid and sequenced for identification.The recombinant plasmid pJET1.2-racR was double digested with EcoR I and Xho I and the target fragment was connected to the prokaryotic expression vector pET-28a(+)to construct the recombinant plasmid pET28a(+)-racR.E.coli BL21(DE3)containing the recombinant plasmid pET28a(+)-racR was able to solublely express the fusion protein His-RacR(29 KDa)after IPTG induction.After the His-RacR protein purified by affinity chromatography and PAGE gel recovery kit method was used to immunize mice,the titer of anti-His-RacR antibody in the mouse serum was proved to be 1:64000,which was detected by indirect ELISA method.Western Blot test indicated the immunoreactivity and antigenicity of the recombinant protein.The glass plate agglutination test showed that the serum can agglutinate the isolate HC26 and standard strain NCTC11168,which can be used for rapid identification of Campylobacter jejuni.