| Clostridium perfringens（C.perfringens）is one of the important zoonotic food-borne pathogens,it can exist widely in poultry farms and cause necrotizing enteritis（NE）in poultry,bringing huge economic losses to farms;it can also contaminate poultry products,endangering food safety and causing food poisoning in human.C.perfringens contamination of poultry products comes from slaughtering and product processing,and there is a risk of transmission to human along the food chain.China is a large duck farming country and a large consumer of duck products.To investigate the prevalence characteristics and spread laws of C.perfringens along the duck breeding chain,as well as the prevalence distribution and evolution laws of C.perfringens from breeding stage to slaughtering stage under different breeding modes,not only can provide a reference for preventing and controlling the prevalence and spread of C.perfringens in duck farms,but also can provide a scientific basis for controlling the contamination of C.perfringens in slaughterhouses and duck products,reducing the potential risk of foodborne diseases caused by C.perfringens.During June to November 2020,this study investigated the prevalence,toxin type,antibiotic resistance and genetic diversity of C.perfringens from the breeding duck farm to commercial meat duck farm along its breeding chain in a large duck breeding enterprise in Shandong Province.On this basis,whole genome sequencing（WGS）was used to study the genomic characteristics of epidemic strains in breeding duck farms.In order to explore the genetic diversity and genetic relationship of C.perfringens strains from the breeding and slaughtering chain under different breeding modes,according to the antibiotic resistance,host source,breeding and slaughtering stages,79 strains were selected for multilocus sequence typing（MLST）from the breeding and slaughtering chain under the fermentation bed breeding mode,and compared with strains from the breeding and slaughtering chain under the ground free-range mode in the same duck farm with the same breeding background（Provenance,feed,and immunization were same.）,to explore the impact of different breeding modes on C.perfringens prevalence,and the pollution situation and evolution laws of C.perfringens along the breeding and slaughtering chain.The results of the breeding chain showed that 45.26% of the 855 samples were identified as positive for C.perfringens,and the positive rates were 84.52%,26.51% and 59.79% in the breeding duck farm,hatchery and commercial meat duck farm,respectively.In the hatchery,the positive rates of gut,yolk sac,albumen,eggshell membranes and allantoic fluid samples from healthy duck embryos were 30.77%,1.61%,3.77%,9.68% and 6.25%,respectively;the positive rate of cloacal samples from newborn ducklings was 60.00%;the positive rate of dead embryo gut samples was 27.40%,and the number of contaminated C.perfringens was high.Not only were C.perfringens isolates obtained from the same batch of duck embryos at different incubation stages,but also the number of C.perfringens in the gut of duck embryos increased gradually with embryonic development.A total of 617 C.perfringens strains were isolated,94.81% of isolates were type A,4.05% of isolates were type F and the remaining strains were type G.The results of drug sensitivity tests showed that 62.50% of the strains were multidrug resistant.The MLST results showed that 66 representative strains from the breeding duck farm to commercial meat duck farm were divided into 60 sequence types（STs）,and 69.70% of the strains formed 10 clonal complexes（CCs）.Among them,CC2 strains were the most prevalent（15.15%,10/66）and the dominant strains in the breeding duck farm,accounting for 58.82%（10/17）of the strains analyzed in the breeding duck farm,while strains of the commercial meat duck farm had abundant genetic diversity（Simpson’s diversity index of STs was 0.9565）.Some strains of the breeding duck farm and hatchery were distributed in the same ST or CC,suggesting that the infected strains of duck embryos may come from the hatchery environment or breeding duck farm,with the possibility of vertical transmission.The phylogenetic tree results showed that the evolutionary relationships of strains from the same farm were relatively close.The MLST results of strains from different breeding duck farms showed that most strains from the same breeding duck farm were distributed in the same ST or CC,and the strains were closely related;The WGS results of the epidemic strains from breeding duck farms in Zibo and Liaocheng showed that epidemic strains carried multiple virulence genes and different antibiotic resistance genes.The genetic relationships of the breeding duck farm isolates based on the MLST were mostly consistent with those based on the core-genome single nucleotide polymorphism（SNP）phylogenetic tree.The MLST results of strains from the breeding and slaughtering chain under fermented bed breeding mode showed that 79 representative strains were identified as 71 STs,and37.97% of the strains formed 8 CCs.Some isolates from the breeding stage and slaughtering stage were distributed in the same ST or CC,indicating that C.perfringens of the breeding stage may contaminate the carcasses and their products in the slaughterhouse with the production process;some isolates of cloaca and environment were closely related in the duck farm;some isolates from processing equipment,carcasses and products were closely related in the slaughterhouse.The MLST results of strains from the breeding and slaughtering chain under different breeding modes showed that the STs of C.perfringens isolates from duck flocks in different breeding modes differed significantly under the same provenance and feed condition,indicating that different breeding modes could change the genetic diversity of C.perfringens.The front and back batches of ducks were slaughtered sequentially in the slaughterhouse,and the strains from their carcasses and products were distributed in the same CC,indicating that cross-contamination of C.perfringens may exist among them.In summary,the prevalence of C.perfringens in duck breeding and slaughtering chains was mainly type A,and type G strains were isolated from the duck farm for the first time.62.50% of the strains were multidrug resistant.Some duck embryos were infected with C.perfringens during incubation period,and infected strains should originate from the hatchery or breeding duck farm,with the possibility of vertical transmission.C.perfringens strains from the breeding duck farm formed dominant genotypes after long-term competition and evolution.The genetic relationships of the breeding duck farm isolates based on the MLST were mostly consistent with those based on the core-genome SNP phylogenetic tree.In addition to provenance and feed,breeding modes significantly affected the genetic diversity of C.perfringens.Cross-contamination of C.perfringens was observed between pre-slaughter and post-slaughter batches of duck carcasses and products at the slaughterhouse.Contamination and spread of C.perfringens along the breeding chain,cross-contamination of C.perfringens among breeding,slaughtering and product processing,widespread multidrug resistance of strains and presence of netB or cpe positive strains posed a potential threat to farms,slaughterhouses and public health. |
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