Etiology And Pathogenicity Of Some Pig Farms Intestinal Pathogenic Escherichia Coli In Pig In HuBei Province

Escherichia coli is the main enteric pathogen causing bacterial diarrhea in newborn piglets.After infecting piglets,it shows symptoms such as diarrhea,dehydration,yellow diarrhea or pullorum,and even death,causing serious harm to the pig industry.In this study,the etiology of bacterial diarrhea in piglets in Hubei Province was investigated,and the bacterial isolation,identification,drug resistance analysis and virulence research of suspected samples were carried out to analyze the epidemic situation of bacterial diarrhea in Hubei Province and study the prevention and control technology.And scientific medicine to provide reference.1.Isolation,identification and multi-locus sequence typing of Escherichia coliIn order to explore the prevalence of Escherichia coli in pig farms,214 piglet fecal swabs,rectum and small intestine samples from pig farms in Suizhou,Xiangyang,Wuhan and Yichang,Hubei Province from 2021 to 2022 were collected.After bacterial culture,colony morphology,Gram staining,PCR,sequencing analysis and sequence typing,153 strains of Escherichia coli were identified,with an isolation rate of 71.5%,including 31 strains of enterotoxigenic Escherichia coli.The results of sequence typing showed that there were 14 ST types in the 153 strains,and ST46 and ST165 accounted for 22.88%and 20.92%,respectively.Among the 8clone complexes,CC-46 and CC-165 contained relatively more strains.There is a diversity of ST-type isolates.2.Study on the biological characteristics of Escherichia coli（1）Drug resistance analysisIn order to understand the drug resistance of the isolated strains,17 common antibiotics were selected for drug susceptibility tests.The results showed that the isolated strains were all resistant to 17 drugs to a certain extent,and showed multidrug resistance to more than 6 drugs.The drug resistance rate of Escherichia coli to ampicillin,lincomycin,tetracycline and sulfisoxazole was 100%.The drug resistance genes related toβ-lactamases and aminoglycoside-modifying enzymes were detected,and the carrier rates of bla _TEMand bla _CTX-M-9Gwere 58.82%and35.29%,respectively;The carrier rates of aad A1,aad A2,aac（3’）-IIc were 76.47%,41.18%,and 41.18%,respectively.The carrier rate of bla _TEM+bla _CTX-M-1Gwas23.53%.The carrier rate of aad A1+aac（3’）-Ⅱc was 29.41%.（2）Virulence gene detectionIn order to understand the carrying status of virulence genes,the virulence genes LT-I,STa,STb,EASTI,irp2,fyu A,etc.of the isolated strains were amplified by PCR.The results showed that the detection rates of irp2,EASTI,and fyu A single genes were 58.82%,52.94%,47.06%,and the detection rates of STa,STb;and LT-I were 35.29%,35.29%,and 23.53%,respectively.（3）Cell adhesion testCombined with the results of virulence gene detection and drug resistance analysis,five ST-type E.coli,including ST165,ST515,ST46,ST602 and ST32,were selected for adhesion test.The results showed that the adhesion ability of ST165,ST515 and ST602 to IPEC-J2 cells was significantly higher than that of the control group（P<0.05）;the adhesion ability of ST46 to cells was relatively low.（4）Determination of cellular inflammatory factorsIL-6,IL-8 and TNF-αwere detected by fluorescence quantitative PCR on 5ST-type Escherichia coli respectively.The results showed that compared with the blank control group,the IL-6,The expressions of IL-8 and TNF-αwere significantly up-regulated（P<0.001）.3.Pathogenicity study of Escherichia coli（1）Clinical symptoms and anatomical lesionsIn order to understand the pathogenicity of the isolated strains,combined with the above-mentioned virulence gene detection and drug resistance analysis results,five ST-type E.coli.The mice in the experimental group were inoculated with 0.2m L/mice（2×10⁹CFU）of bacteria by gavage respectively,and the mice in the blank group were administered an equal dose of PBS.The results showed that the mice in the experimental group had various symptoms such as diarrhea,messy coat,and lethargy.Seven days after inoculation,the mortality rates of mice in ST165 group and ST32 group were 83.33%and 66.7%,respectively;the other three groups were all 0.After necropsy,the mice in the infection group were observed to have edema,congestive and translucent intestinal tract,a small amount of yellow content mixed in the interior,a large number of bleeding spots on the surface of the intestinal mucosa,mild congestion in the liver,a large number of bleeding spots in the lung,and swelling of the tissue around the spleen.The mice in the blank group had no obvious abnormality.（2）Observation of histopathological sectionsHistopathological observation was performed on the mice in the infected group and the blank control group,respectively.The results showed that the liver,spleen,lung,and intestinal tract of the mice in the infected group showed different degrees of pathological damage.Among them,the ST165 infection group had the highest mortality rate in the intestinal tract.A large area of autolysis can be seen in the tissue;there is congestion in the liver sinus with a small amount of inflammatory cell infiltration;the lymphocytes in the spleen nodules are reduced and loosely arranged;In the ST32 infection group,the intestinal villi were slightly swollen,congested and necrotic,the mucosal epithelial cells were necrotic and exfoliated,and the nuclei of necrotic cells were pyknotic and fragmented;hepatocytes were balloon-like degeneration,with centered nuclei,vacuolated cytoplasm,and focal infiltration of inflammatory cells;There was mild congestion in the red pulp with more granulocyte infiltration;the alveolar wall was thickened with a small amount of inflammatory cell infiltration.To sum up,Escherichia coli isolates in some areas of Hubei are generally multi-drug resistant and carry a variety of virulence genes,of which 153 isolates have ST-type diversity.This study provides a theoretical basis for the prevention and control of colibacillosis.