| The total number of yaks in China is about 14 million,accounting for 90% of the world’s total,which can be divided into two types: "Hengduan high mountain type" and "Qinghai-Tibet Plateau type".The living environment of yaks is in the high altitude,low oxygen and high temperature difference between day and night,which makes yaks have a strong adaptability to the ecological environment.In recent years,yak breeding industry has developed rapidly in the Qinghai-Tibet Plateau region,bringing huge economic benefits to local herdsmen and the whole breeding industry.However,the yak diarrhea caused by E.coli has greatly affected the yak industry in Tibet.At the same time,the problem of drug resistance caused by the irrational use of antibiotics;And the relationship between yak pathogenic E.coli and intestinal flora diversity is also the focus of this study.The variation of fecal microflora in yak diarrhea caused by E.coli was studied in this experiment.The pathogenic macrobacillus from yak was isolated,identified,virulence detected and drug resistance detected,and the results were analyzed by bioinformatics,which provided the basis for finding effective methods to treat yak diarrhea.Specific research work is as follows:1.Diversity analysis of microbial flora in yak diarrhea fecesThe diversity of fecal microflora of yak with diarrhea was analyzed by 16 s r RNA gene sequencing.We found that at the phylum classification level,Firmicutes,Actinobacteriota,Proteobacteria and Bacteroidia were the dominant bacterial flora in yak feces.At the family classification level,Micrococcaceae,Planococcaceae,Oscillospiraceae,Clostridiaceae,Lachnospiraceae and Enterobacteriaceae were the dominant bacterial flora in yak feces.At the genus classification level,Arthrobacter,Bacillus,Solibacillus and Escherichia-Shigella were the dominant bacteria in the faecal microflora of yak.At phylum level,Proteobacteria,Patescibacteria,Cyanobacteria and Fibrobacterota in faecal microbial community of yak diarrhea group increased significantly compared with that of healthy yak group.But Bacteroidia decreased significantly.Proteobacteri mainly includes E.coli,Helicobacter pylori,Salmonella and other pathogenic bacteria that often cause diarrhea in animals.This suggests that the diarrhoea of yaks may be mainly caused by the increase in the abundance of Proteobacteria.2.Isolation and identification of E.coli from yak diarrhea fecesThe sample bacteria were isolated and purified by conventional methods.At the same time,the samples were isolated and cultured by Gram staining.Then,the O-antigen serotype identification and 16 s r RNA identification of the isolated bacteria were carried out to determine the number and serotype of the isolated bacteria in this project.In this study,59 strains of E.coli were isolated and identified from 59 samples of diarrhea yak feces,and the isolation rate was 100%.The serotype identification results of E.coli O-antigen showed that 42(71.19%)of the59 isolates were identified as serotypes,belonging to 14 different serotypes,of which O1,O111 and O128 were the dominant serotypes,accounting for 11.86%(7/59)respectively.11.86%(7/59)and 8.47%(5/59).3.Detection of common virulence factors of pathogenic E.coli in yaks with diarrhea in TibetThe virulence genes of 59 strains of E.coli from Yak were detected by conventional PCR.The detection range of PCR was irp2 and Fyu A,which were commonly associated with Yersen’s virulence island.The genes F41,K88,K99,987 P and F18 controlling the expression of adhesin;Virulence genes Stx1 and Stx2 related to shiga toxin production.The results showed that only nine virulence genes,irp2,Fyu A,K99,987 P,Stx1,Stx2,STb,eae A and Hly A,were detected among 14 common pathogenic E.coli virulence genes from cattle.The detection rates were20.34%,23.73%,94.91%,71.19%,3.39%,5.08%,93.22%,5.08% and50.85%,respectively.Five virulence genes,F41,LT,STa,K88 and F18,were not detected.Virulence gene test showed that E.coli strains isolated from diarrhea yak feces in Tibet area carried more virulence genes,which may be related to the perennial stocking habit in Tibet.4.Drug resistance analysis of pathogenic E.coli from yakThe common drug resistance genes of 59 strains of E.coli from yak were detected by conventional PCR.The range of PCR detection were 19 kinds of drug resistance genes,including four classes of common sulfonamides,tetracycline,aminoglycosides and β-lactam.The results showed that 11 drug resistance genes were detected,including sul1,sul2,tet A,tet E,aac C2,aac C4,aad A1,aad A2,aac(3)-11 a,SHV and TEM.The detection rates were 23.73%,44.07%,27.12%,62.71%,6.78%,10.17%,25.42%,55.93%,81.36%,8.47% and 50.85%,respectively.The other resistance genes were not detected.It can be seen that aac(3)-11a(81.36%)had the highest carrying rate,followed by tet E(62.71%),aad A2(55.93%),TEM(50.85%)and sul2(44.07%).The detection rate ofβ-lactam resistance genes was the highest,so the use of such drugs should be reduced.The results of drug susceptibility test showed that 59 strains of E.coli from yak were 88.14% to erythromycin and 98.31% to clindamycin.It was also highly resistant,followed by tetracycline(50.85%)and ceftazidime(30.51%).5.Study on the pathogenicity of E.coli from yakWithin 6 h after the challenge,the mice all showed depression,loss of appetite and coarse hair,and most of the mice showed symptoms such as chills clustering and diarrhea.All mice died within 3 h after injection of F34 and F42,6 h after injection of F8 and F13,and 12 to 72 h after challenge of other groups.Necropsy of the dead mice showed that there was fluid in the abdominal cavity,spleen enlargement,lung congestion,enlarged heart congestion,brittle liver,thinning of the intestinal wall,mesenteric congestion,and yellow loose stool in the intestine.At the same time,pathological tissue sections were used to observe the organs of challenged mice.Under the microscope,it was found that different degrees of pathological injury appeared.Lung tissue showed diffuse bleeding,vascular congestion,part of alveolar sacs fused to form lumen,a small amount of cellulose exudation,alveolar cell necrosis,and necrotic alveolar structures fused with each other.Liver: There were no obvious abnormalities in the structure of liver cells,interlobular artery congestion in the portal area,interlobular vein congestion,blood filling in the duct,and central vein congestion.Spleen: There was no obvious abnormality in the distribution of red pulp and white pulp in the spleen,no obvious abnormality in the splenic cord structure,and the connective tissue thickened.Kidney: Some of the proximal convoluted tubules of the kidney are filled with blood,the tubular epithelial cells of the kidney are mildly vacuolar degeneration,the edge of the bristles is shed,the cells are flattened,and the lumen is dilated.Heart: The myocardium showed banded bleeding,some myocardial fibers were broken,and venous congestion.Intestine: The epithelial cells of the intestine are denaturated and necrotic,bleeding is severe,the villi become short and fall off,there are a large number of shed epithelial cells in the intestinal cavity,and the subbasement membrane is edema and bleeding.In summary,59 strains of E.coli were isolated and identified from80 stool samples of yak diarrhea in this study,and O1,O111 and O128 were found to be the dominant serotypes after O-antigen serotype identification.Through the diversity analysis of pathogenic E.coli and fecal microflora causing yak diarrhea,it was found that the increase in the abundance of Proteobacteria might be the main cause of yak diarrhea.At the same time,the 59 E.coli isolates generally carried multiple virulence genes,and the expression types of adhesion in yak E.coli and bovine E.coli were obviously different.In the pathogenicity experiment of E.coli,the results show that these pathogenic bacteria have obvious fatal infection to experimental mice. |
