Isolation,Identification And Genotyping Of Clostridium Perfringens Isolated In Duck Breeding To Slaughtering Chains In Some Areas Of Shandong Province

Clostridium perfringens?C.perfringens?,as an important zoonotic microbes,can not only lead to poultry necrotizing enteritis,cause huge economic losses to breeding industry,but cause humans food poisoning and other digestive tract diseases,bring certain threat to public health.With the widespread use of antibiotics,the resistance of C.perfringens to antibiotics is increasing,and multi-drug resistance has developed obviously,which seriously affects the effect of clinical treatment.Poultry is a reservoir of C.perfringens,and serious C.perfringens contamination often occurs during poultry slaughtering and processing.There were many reports of C.perfringens in different hosts and foods,but few publications have evaluated prevalence and molecular characteristics of C.perfringens isolated from duck production chains and duck farms.China is the largest producer of meat ducks in the world,and Shandong province is a major province of duck breeding.Therefore,studying on the prevalence,serotype distribution,antibiotic resistance and genetic evolution of C.perfringens during duck breeding and slaughtering process in Shandong,not only can fill in the gaps in research of the prevalence of C.perfringens in duck source and provide theoretical basis for rational use of antibiotics in breeding farms,but also have important significance for public health and food safety.From December 2018 to June 2019,a total of 939 samples were collected from the breeding and slaughtering chain of two duck groups from an integrated breeding and slaughtering company in Weifang and from other four duck farms in Weifang,Liaocheng,Tai'an.After isolation and identification of samples,eight toxin genes of C.perfringens were identified by PCR amplification,antibiotic resistance of isolates against 12 kinds?10 classes?common used antibiotics was detected by Kirby-Bauer disk diffusion method.And multilocus sequence typing?MLST?was used to determine the genotypes of the selected isolates and the genetic relation of C.perfringens from different sources.In this study,498 of 939 samples?53.04%?were determined to be positive for C.perfringens,with a positive rate of 50.16%in farm samples and 58.86%in slaughterhouse samples.The positive rate of cloaca samples in different duck farms varied,among which the antibiotic-free farm?Farm 2?had the highest positive rate?72.5%?,which was significantly different from other farms?P<0.05?.In addition,the positive rate of C.perfringens in early stage of duck breeding was relatively low?22.00%?.The contamination of samples in each slaughtering stage was different,the carcass of the dehairing stage was observed to be the highest pollution rate?85%?,which was an important risk point for C.perfringens contamination in the slaughtering stages of this study;the positive rate of carcass samples after disinfecting?28.33%?was significantly lower than that in other stages?P<0.05?,indicating disinfection before precooling stage was the key to cut off the transmission of C.perfringens in the slaughter chain.A total of 859 strains of C.perfringens were obtained and all strains were identified as C.perfringens type A by PCR amplification,in which 34.69%of the isolates carried cpb₂ gene and 0.93%isolates carried cpe gene.Antimicrobial susceptibility testing showed that 78.79%of the isolates showed multi-drug resistance,43.27%of isolates were resistant to at least 5 commonly used antibiotics.The drug resistance of isolates in different farms varied,the multidrug resistance of C.perfringens from the antibiotic-free meat duck farm?64.58%?of Liaocheng?Farm 2?and fermentation bed duck farm?67.44%?of Weifang?Farm 4-2?were significantly lower than that of other farms?P<0.05?,indicating raising without using antibiotics and raising ducks in fermentation bed had certain effects on reducing the resistance of isolates.Multilocus sequence typing?MLST?results showed that 117 representative isolates can be divided into 74 sequences types?STs?,among which ST3?17.09%?was the most common?17.09%?and ST3 was a prevalent ST in Farm 3,Farm 4 and the slaughterhouse.30.59%of85 isolates from farms were classified into five clonal complexes?CC?,the most prevalent CC?CC1?,which was popular subtype of Farm 2,accounting for 15.29%of all isolates.Although all the isolates were type A,the genetic diversity of the isolates varied widely in different regions,the Simpson diversity index of isolates in Liaocheng,Tai'an and Weifang farms was0.5941,0.9198 and 0.9627,respectively,and the Simpson diversity index of isolates from slaughterhouse was 0.9023.A portion of cloacal isolates and environmental isolates were distributed in the same ST or CC,indicating close genetic relationship between cloacal isolates and environmental isolates.The isolates of the same genotype existed in each stage in breeding and slaughtering chains,indicating that C.perfringens from duck source could be contaminated from breeding farm to slaughterhouse and from upstream to downstream along slaughtering chain to contaminate products.A part of isolates from duck carcasses or products were the same ST type or CC with the isolates from slaughterhouse environment,indicating contamination might existed.A portion of human and duck-derived strains were found to phylogenetically close,suggesting that C.perfringens may pose a potential threat to humans through the food chain.