Establishment Of Triple PCR Detection Method For E.coli In Sheep/Goat And Biological Characterization Of Clinical Isolates

As one of the common meats on people’s tables,mutton is favored by consumers,and in recent years,the level of mutton consumption has steadily increased,and the scale of mutton and sheep/goat breeding is also expanding.However,the intensive and off-site fattening breeding model in East China provides favorable conditions for the spread of various pathogens.Among the Animal-derived pathogenic Escherichia coli（E.coli）,Shiga toxin-producing E.coli（STEC）and extraintestinal pathogenic E.coli（Ex PEC）are two important zoonotic pathogens with important public health significance.Several studies have shown that STEC and Ex PEC can be transmitted to humans through the food chain,posing a huge threat to public health security.However,compared with the studies on E.coli from poultry and pigs,there are few studies on E.coli from sheep/goat,and most of them only focus on the identification of E.coli in diarrhea cases,and for food-borne E.coli,STEC such as O157:H7 is usually concerned,but there are only a handful of studies on Ex PEC.In addition,there are many serotypes of E.coli,and the predominant serotypes prevalent in different regions are different.Therefore,in order to clarify the epidemic characteristics of pathogenic E.coli in sheep/goat breeding,slaughtering and processing progress in East China,this study established a triple PCR for the detection of O8,O9,O89 serotypes E.coli as dominant serotypes based on previous laboratory investigations.The detection method is used to conveniently and quickly identify the three serotypes in clinical and food testing.In this study,samples of diarrhea and respiratory diseases from sheep/goat farms in some provinces in East China,as well as mutton and environmental samples from slaughterhouses,were collected for the isolation and identification of E.coli,and the serotypes,phylogenetic groups,virulence genes of the isolated strains and drug resistance were detected and analyzed,and then the pathogenicity of some representative strains,especially STEC and Ex PEC,was analyzed in mice infection model.This study yielded the following results:（1）Through primer design and condition optimization,a triple PCR method with good repeatability,specificity and sensitivity for the detection of E.coli serotypes O8,O9 and O89 was successfully established.The obtained 217 E.coli strains were identified by serotype,and a total of 32 E.coli strains of serotype O8（14.75%）,22 strains of E.coli serotype O9（10.14%）and 16strains of E.coli serotype O89（7.37%）were detected..It is proved that the method can be used for the identification of clinical samples and food-derived E.coli.（2）217 strains of E.coli isolated from stool、intestine and lung samples of diarrhea and respiratory diseases in different farms,as well as mutton and environmental samples from slaughterhouses.The results showed that the A group and B1 group accounted for the majority of E.coli from each source,and the detection rates of omp A,crl A,yijp,fim H,mat and ibe B were at a high level in each source of E.coli.The eae and hly A genes,which are closely related to STEC,and the ibe B and yijp genes,which play a key role in the pathogenic process of E.coli meningitis in Ex PEC,have high detection rates in slaughterhouse-derived E.coli.Through virulence gene analysis,63 strains of STEC and 18 strains of Ex PEC were defined in this study,and both types of E.coli with a high risk of zoonotic disease were also detected in slaughterhouse samples,proving that they were widely popular in the breeding process and had a high risk of contamination during slaughtering.（3）The drug susceptibility characteristics of the isolated strains are detected by the K-B paper method.A total of 17 antibacterial drugs in 9 categories were detected.The results showed that the isolates were generally resistant to clindamycin,ampicillin,doxycycline,enrofloxacin,amikacin,kanamycin,amoxicillin and co-trimoxazole,with a resistance rate higher than 70%.In addition,these strains have a broad spectrum of resistance,with 98.62%of the strains defined as multi-drug resistant（MDR）.（4）According to the detection results of the molecular biology characteristics of each strain,18 representative strains were selected,and their pathogenicity was detected by intraperitoneal injection in the mice model,and its virulence was assessed.Through screening,half of the more pathogenic strains were further measured and histopathological observation was carried out.The results showed that the two slaughterhouses had the lowest LD₅₀,5×10^6.38CFU and 5×10^6.50CFU,respectively.The LD₅₀ of other strains were 5×10^7.38 CFU,5×10^7.50CFU and 5×10^7.83 CFU from low to high.The histopathological observation showed that the lung lesions of the mice in each challenge group were obvious,and the liver also had different degrees of lesions.Histopathological observations showed that the lung lesions in mice were obvious.In summary,this study established a triple PCR method that can quickly detect O8,O9 and O89 serotypes of E.coli,and applied it to the detection of pathogenic E.coli in the process of mutton and sheep/goat breeding and slaughtering and processing in East China.The study of the phylogenetic group,virulence gene and drug resistance of isolated strains from different sources,as well as the pathogenicity detection of representative strains,especially the identification and analysis of STEC and Ex PEC,enriched the epidemiological data of sheep/goat-derived pathogenic E.coli in East China and proved its high risk of contamination in the slaughtering process.This result provides a basis and guidance for the prevention and control of E.coli in the process of sheep/goat breeding,and warns that the monitoring and control of the risk of E.coli contamination in sheep/goat breeding and mutton processing should be strengthened.