| Campylobacter is recognized as one of the most important food-bornes and zoonosis pathogen,which could cause human bacterial gastroenteritis.In this study,220 Campylobacter spp.strains were isolated and identified from the duck slaughter chain in a duck-processed company at Chengdu,Sichuan Province,followed by antimicrobial resistance analysis,virulence-associated genes detection and pulsed-field electrophoresis(PFGE)typing.The results were as follows:1 Isolation and identification of the Campylobacter spp.strains from the duck slaughter chainFrom 2015 to 2016,656 samples were collected from the duck slaughter chain,among them 227 samples were collected from the cecum(pre-slaughter),138 samples were collected during the defeathering process(cotton swab and water sample),155 samples were collected during the eviscerated process(cotton swab and water sample),136 samples were collected from the segmented duck feet,wing and whole duck(post-slaughter).According to GB 4789.9-2014 and the references,three pairs of primers were selected and synthesized to amplify the target genes:16S rRNA(Campylobacter spp.),ceuE(C.coli)and hipO(C.jejuni).Triplex PCR assay was then applied to identify the species of Campylobacter spp.The results revealed that 220 Campylobacter strains were identified,109 strains were C.jejuni,103 strains were C.coli,and 8 strains belonged to the other species of Campylobacter spp..The total isolation rate of Campylobacter spp.reached 33.54%,while C..jejuni and C.coli shared 96.36%of all the isolated Campylobacter spp.strains.The isolation rate of the cecum,defeathering process,eviscerated process and segmented duck was 79.3%,6.52%,18.71%,1.47%respectively.2 Antimicrobial susceptibility assay of the isolated Campylobacter spp.from the duck slaughter chainThe recovered Campylobacter spp.strains were tested for the antimicrobial susceptibility against 8 kinds of antimicrobial agents using a broth microdilution method.The results indicated that Campylobacter isolates were most frequently resistant to tetracycline(96.36%),followed by resistance to clindamycin(92.27%),the resistance rate of azithromycin(66.82%)was in the middle,the resistance rates of ciprofloxacin(37.27%),gentamicin(41.36%),nalidixic acid(44.45%),erythromycin(47.27%),chloramphenicol(42.73%)were relatively low.47 kinds of antibiotic resistant profiles in total were obtained and the multiple resistance rate reached 75.91%.33,27 and 4 kinds of antibiotic resistant profiles were obtained for C.jejuni,C.coli,and other Campylobacter species,meanwhile the multiple resistance rate was 71.56%,79.61%,87.50%,respectively.When it comes to the different slaughter process,the isolated Campylobacter presented 40(pre-slaughter),6(defeathering process),14(eviscerated process)and 2(post-slaughter)kinds of antibiotic resistant profiles,meanwhile the multiple resistance rate was 78.33%,77.78%,58.62%and 100%,respectively.3 Virulence-associated genes detection of the isolated Campylobacter spp.from the duck slaughter chain(1)Virulence-associated genes detectionThe prevalence of cadF(adhesion-associated gene),iamA and ciaB(invasion associated protein gene),flaA(flagellin gene),cheY(chemotaxis gene),cdtA,cdtB and cdtC(toxin regulation gene),virBll(plasmid gene)was 100%,71.82%,42.73%,77.27%,92.73%,60%,92.27%,54.09%,7.73%,respectively.Among the 220 isolated Campylobacter spp.,183 strains carried with 5?9 virulence-associated genes(83.18%).More specifically,1 strain carried with 9 virulence-associated genes,37 strains carried with 8 virulence-associated genes,and 59 strains carried with 7 virulence-associated genes.104 C.jejuni carried with 5?9 virulence-associated genes(95.41%),76 C.coli carried with 5?9 virulence-associated genes(73.79%).The proportion of the strains carried with 5?9 virulence-associated genes in the Campylobacter of cecum,defeathering process,eviscerated process and intersected duck was 81.67%,88.89%,93.10%,50.00%,respectively.(2)Virulence-associated genes patterns48 kinds of virulence-associated genes patterns were obtained among all the 220 isolated Campylobacter spp.,and most of them were typical C.jejuni or C.coli patterns.20 kinds of virulence-associated genes patterns were obtained among 109 C.jejuni,in which flaA,cadF,cdtB,cheY,iamA,ciaB,cdtC were the dominant patterns(detected in 56.59%C.jejuni).34 kinds of virulence-associated genes patterns were obtained among 103 C.coli,in which flaA,cadF,cdtA,cdtB,cheY were the dominant patterns(detected in 19.79%).45,5,6,and 2 kinds of virulence-associated genes patterns were obtained in the Campylobacter of cecum,defeathering process,eviscerated process and intersected duck,respectively.4 PFGE typing of the isolated Campylobacter spp.from the duck slaughter chain68 randomly selected duck-origin Campylobacter spp.were enzyme-digested by using Sma I as restriction enzyme.Analyzed from the results of PFGE typing,the PFGE band similarities of 35 C.jejuni were between 36.00%?99.80%,and 15 kinds of patterns were obtained;the PFGE band similarities of 33 C.coli were between 28.93%?100.00%,and 11 kinds of patterns were obtained.In conclusion,the total isolation rate of Campylobacter spp.was 33.54%,the main species recovered were C.jejuni and C.coli,while the dominant species was C.jejuni,a reduction amount of Campylobacter was observed from pre-slaughter to post-slaughter process.The antibiotic resistance of duck origin Campylobacter was rather serious and multi-drug resistance was common in these strains,higher antibiotic resistance was found in C.coli compared to C.jejuni,the strains from the duck products also revealed a highly antibiotic resistance.The virulence-associated genes widely existed among the Campylobacter isolates,while virulence-associated genes were more commonly detected in C.jejuni,its indicated that the pathogenicity of Campylobacter is affected by several factors.The results indicated that a cross-contamination existed in the duck slaughter chain,meanwhile a trend of Campylobacter spread was observed from pre-slaughter to post-slaughter process. |
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