| Escherichia coli is a ubiquitous bacteria in nature,generally does not cause disease,but some serotypes and virulence factors with pathogenicity,can cause human and animal co-infection,is a zoonotic pathogenic bacteria.Piglet yellow dysentery,piglet white dysentery and piglet edema caused by pathogenic Escherichia coli have high morbidity and mortality,which bring huge economic loss to the pig industry.The problem of drug resistance caused by the abuse of antibiotics makes the prevention and control of live pig Escherichia coli disease more and more difficult.From February to August 2018,diarrhea rate of piglets caused by unknown reasons increased significantly in several large-scale pig farms in Western Fujian province,causing serious economic losses to farmers.In order to determine the pathogenic bacteria of this epidemic,this study collected tissue samples from diseased piglets for the isolation and identification of Escherichia coli.Then the isolates were subject to examination of phylogenetic classification,virulence gene,antimicrobial resistance and so on.This study provided scientific basis for the prevention and treatment of live pig Escherichia coli in this area.The specific research work is as follows:1.Isolation and identification of Escherichia coli from pigs and phylogenetic clustering: Through the methods of bacterial culture andstaining,gram's staining and 16 S rRNA sequence analysis,44 strains of Escherichia coli were isolated and identified from 62 tissue samples collected by piglets with diarrhea in this study,with a separation rate of70.97 %.Phylogenetic analysis showed that 26 of the 44 isolates were A group,12 strains were B1 group,4 strains were B2 group and 2 strains were D group.The results indicated that 44 strains of Escherichia coli isolated were mainly symbiotic group,accounting for 86.36 %(38/44)of the total number of isolates.2.Porcine Escherichia coli related virulence gene test: To investigate the virulence genes of 44 strains of Escherichia coli,the common swine and the common avian pathogenic Escherichia coli related virulence genes were tested by PCR.The virulence gene detection suggested that among the 12 virulence genes related to swine pathogenic Escherichia coli,only 5 virulence genes of EAST1,LT,STb,STa and F4 were detected,and the detection rates were 43.18 %,25.00 %,22.72 %,13.63 %,11.36 % respectively.The virulence gene test results of avian pathogenic Escherichia coli showed that luxs,ompA,irp2,iroC,iucD,cvaC,fyuA,tsh and iss were detected among the 13 related virulence genes,and the detection rates were 90.91 %,75.00 %,34.10 %,27.27 %,25 %,13.63 %,9.09 %,9.09 %,4.55 % respectively.3.Analysis of drug resistance of porcine Escherichia coli: To understand the drug resistance of 44 isolated strains,12 commonly usedclinical antimicrobial agents were selected for drug sensitivity test.The results showed that 44 strains resistance to florfenicolr,ceftiofur,sulfamethoxine,ampicillin,sulfamethoxazole/trimethoprim,doxycycline,enrofloxacin,ciprofloxacin,levofloxacin,gentamycin,cefotaximed,amikacinand,and the resistance rates were 93.12 %,93.12 %,93.12 %,90.91 %,84.09 %,75.00 %,75.00 %,68.18 %,61.36 %,59.09 %,40.91 %,20.45 %,the results showed that the resistance of 44 strains of escherichia coli to the selected animicrobial agents was serious.Four related resistance genes TEM,CTX,OXA and SHV were detected for chloramphenicol related resistance genes floR and beta-lactamase,the reults showed that the detection rates of TEM,CTX,OXA and SHV were81.82 %,70.45 %,45.45 %,45.45 % and 0 %,respectively.Further analysis showed that the coincidence rates of drug resistance phenotype and drug resistance gene detection of chloramphenicol and lactamase of44 isolates were 97.22 % and 93.18 %,respectively.It was suggested that the resistance mechanism of isolated bacteria might be relevant to the existence of related resistance genes.4.Pathogenicity study of Escherichia coli in pigs: In order to understand the pathogenicity of the isolates of A group and B1 group,then this work combined with the virulence gene detection and drug resistance analysis results of the above isolates.The 3 isolates in the A group(MX-15,MX-19,MX-42)and the 3 isolates in the B1 group(MX-6,MX-10,MX-30)were tested for mouse pathogenicity.Compared with the control group,the results of animal pathogenicity test showed that the mice were all died within 26 hours after inoculation with the A group of isolates.The clinical symptoms were mainly rough hair,mental depression,and a large amount of secretions outside the eyes.Pathological examination and histopathological observation showed that the liver,spleen,lung and intestine of mice in the treatment group showed severe pathological damage.Compared with the mice inoculated with the A group isolates,the mice all died within 34 hours after inoculation of the B1 group isolates,and showed similar Clinical symptoms and pathological changes.The above results indicate that the isolated strains of group A and group B1 have obvious pathogenicity to test mice and can cause fatal infection,suggesting that we should pay attention to in clinical practice.To sum up,A total of 44 strains of escherichia coli were isolated and identified in this study,and the main strains were symbiotic types(group A and group B1)of escherichia coli.Further study found that the 38 isolated strains of symbiotic e.coli generally carried multiple virulence related genes,and multiple drug resistance was serious.The pathogenic test results of symbiotic type(group A,group B1)escherichia coli showed that they were obviously pathogenic to mice and could cause fatal infection.Therefore,Attention should be paid to symbiotic Escherichiacoli in clinical practice. |
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