| Clostridium perfringens(C.perfringens)is a common pathogenic microorganism in nature.It is a Gram-positive spore-forming,anaerobic bacterium that can cause many diseases in animals and humans,for example,human food poisoning and poultry necrotizing enteritis(NE).China is a big producer and consumer of poultry products,studies on the prevalence characteristics and genetic relationships of C.perfringens in chicken rearing and slaughtering stages can not only provide references for the effective prevention and control of NE in chicken farms,but also have important significance for reducing C.perfringens contamination and reducing food safety risks in the production of chicken products.From December 2018 to August 2019,this study investigated the prevalence,toxin type distribution and antibiotic resistance of C.perfringens from four chicken farms indifferent regions(Guangdong,Pingyin,Tai’an),and the integrated rearing and slaughtering company in Weifang.In addition,feces samples of patients suffering from diarrhea and food poisoning in Tai ’an area were collected,and the genetic relationship between poultry strains and strains causing human food poisoning and bacterial diarrhea was evaluated.Total of 1,445 samples were collected and cultured for C.perfringens.Eight toxin genes including cpa,cpb,cpb2,etx,iota,cpe,net B and tpe L were detected by PCR.The antibiotic resistance of isolates was detected by the K-B disk diffusion method,and representative strains were selected for multilocus sequence typing(MLST)according to antibiotic resistance phenotype and host source,and genotypes and genetic relationships of representative isolates were detected.The results showed that C.perfringens was detected in 35.99% of the 1445 samples,and the positive rates were 27.48 %(285/1037)and 54.50 %(206/378)at the farm stage and the slaughter stage,the positive rate of feces samples from patients with food poisoning and bacterial diarrhea was 96.67 %(29/30).The positive rates of C.perfringens in the 5 farms are different,and the highest positive rate is Farm 3(Pingyin)(62.90 %),and the lowest positive rate is Farm 5(Guangdong)(6.00 %).The contamination of samples in each slaughter stage is different,the positive rate of samples in the evisceration stage is the highest(90.00%),which is the risk point of carcass contamination during the slaughter process.After chilling,the positive rate of chicken carcass decreased to 39.73 %,which indicated that this stage could effectively reduce the spread of C.perfringens in the slaughter chain.In this study,a total of758 strains of C.perfringens were isolated,of which 97.23% of the strains were type A,2.51 %(19/758)of the strains were type F,and the rest were type G.The toxin gene detected results showed that 37.59 %(285/758)of the strains carried the cpb2 gene.The results of the antibiotics susceptibility test showed that 61.63 % of the isolates at the rearing stage were resistant to at least five antibiotics,and 68.66 % of the isolates at the slaughter stage were resistant to at least five commonly used antibiotics.MLST results showed that 69 representative strains(chicken strains and environmental strains)isolated from the farm stage were divided into 61 STs,and the Simpson’s diversity index of STs(sequences types)for the strains from five farms was 0.9979.37.84% of the isolates were divided into seven CCs(CC1-CC7),of which the most popular CC is CC3,which is the popular subtype of Farm 5,accounting for 63.64 % of the strains in Farm 5(7/11).Although the genetic diversity of C.perfringens MLST was rich,the isolates from the same rearing farm were relatively close in genetic relationship.And some cloacal isolates and feed isolates were distributed in the same ST or CC,which indicated that feed was closely related to C.perfringens from chicken.The 91 representative isolates of chicken rearing and slaughter chain in Weifang area were divided into 74 STs,and 40.66 %(37/91)strains could be divided into seven CCs.Some strains isolated from rearing stage and from slaughter stage are distributed in the same ST or CC,which indicates that C.perfringens from chicken may spread from farms to slaughterhouses and contaminate chicken products.In addition,some environmental strains from slaughterhouses belong to the same STs or CC as those from chicken carcasses or chicken products,indicating that there may be cross-contamination between slaughterhouse environment and chicken products.The MLST results of human strains and poultry strains in Tai ’an showed that some human strains and poultry strains belonged to the same ST or CC,which indicated that some poultry strains had close genetic relationship with human strains,which indicated that poultry strains had the risk of spreading to human beings through food chain.In summary,C.perfringens is common in rearing and slaughtering links,and it has a wide range of drug resistance.And the most popular toxin type is type A.Although MLST genetic diversity is rich,the isolates from the same farm are relatively close relatives.The contamination rate of C.perfringens is the highest in the evisceration stage,which is the critical control point of risk.The strains from environment of slaughterhouse and the strains of chicken products,the strains of rearing stage and slaughtering stage have the same ST and CC,this results indicated that slaughter environment and rearing stages were closely related to C.perfringens contamination in chicken products,and corresponding measures should be taken to reduce food public health risks. |
PDF Full Text Request