Isolation And Identification Of Pathogenic Escherichia Coli Causing Diarrhoea Of Donkey Foals And Development Of A Compound Vaccine

The donkey industry is an important regional animal husbandry in China.Diarrhoea is one of the most common diseases in the large-scale donkey field,also one of the main factors affecting the survival rate of foals.1.Investigation of diarrhoea in large scale donkey fieldIn 2015 ~ 2017,the survey of donkeys in the 49 large scale donkey farms showed that the incidence rate of diarrhoea in 0 ~ 6 month old foal was about 67.3 %(33/49),and the incidence of individual foal was 23.1 %(1099/4755),with the highest incidence of 15 ~ 45 d.About 15 % of the cases died during the onset of 48 h ~ 72 h.The development and performance of the foals were seriously affected after the onset of the disease,resulting in serious economic losses.The factors that cause diarrhea in donkeys are more complicated,which are related to long-distance transportation stress,drinking water uncleanness,poor hygiene,infection of parasites(Ascaris)and invasion of pathogenic microorganisms.The infection of pathogenic Escherichia coli(E.coli,serotype O78,O101 and O8)is the most important pathogenicity factor.The pathological changes of the foal of the diarrhoea include the abscission of mucous membrane of the intestinal tissue,the fracture of villi,the edema and bleeding of the mucous membrane,which conforms to the histologic characteristics of the infectious enteritis.2.Isolation and identification of pathogenic E.coli in diarrhoea cases of donkey foal2015 ~ 2017,416 strains of Escherichia coli were isolated from diarrhea materials in Liaocheng donkey farm.After purification,electron microscopy,automatic biochemical identification,animal attack test and 16 S rRNA sequencing,85 strains of pathogenic E.coli were identified.The electron microscope analysis showed that the average size of the pure culture was 329.4 nm×223.5 nm.Bacteriological identification can be quickly completed by automatic bacterial identification instrument through biochemical identification.The pathogenic Escherichia coli sensitive drugs including florfenicol,Amikacin,tobramycin,non sensitive drugs including piperacillin,ampicillin,cephalothin,cefotaxime and Cefoxitin etc.The analysis of 16 S rRNA sequence by DNAStar Megalign showed that the homology of 7 strains of E.coli donkey source was 98.2% ~ 99.7%.The homology with cattle,horses,pigs,birds and human strains were 97.0% ~ 99.9%,88.6% ~ 91.0%,97.7%~ 99.6%,95.8% ~ 97.3 and 98.8% ~ 99.9%,respectively.3.Genome sequence analysis of pathogenic E.coli in donkey's foalThe original data of two strains of pathogenic Escherichia coli isolates(E.coli La18,E.coli Lb73)were filtered and the original data were 2.55 G,and the original data were SRP148724 in the NCBI database.The sequences were compared with the reference genome sequences of the horse origin Escherichia coli(E.coli 3.2608 Contig0022)and bovine Escherichia coli(E.coli O157:H7 strain FRIK2455)registered in NCBI,respectively.Comparison of the evaluation results,the quality control result of mutation of a total of 35586 SNP,20805 SNP,a total of 4157 InDel and 23821 InDel;the mutation statement and notes,obtained by comparing genomic mutation density differences,chromosome discovery and NZ-AEZS000010.1 chromosome,NZ-CP015843.2,mutation,mutation frequency in this experiment;the types of mutations in the histogram analysis,13 types of mutations found in 3(downsteam-gene-variant,synonymous-variant,upstream-gene-variant)mutation type 175222,counting the highest,respectively 27751,178117 reach;through mutation position distribution statistics found: 3 genomic regions(DOWNSTREAM,EXON,UPSTREAM)the highest number of mutations.4.Preparation of a new vaccine for the pathogenic Escherichia coli of foalsE.coli La18 isolates were stored in CGMCC for preservation of bacteria(registration number was CGMCC No.14058).The compound vaccine was prepared by using its K99 subunit components.The clinical protective rate was determined after stability test,toxin protection test,safety test and comparative test.The optimum proportion of the bacteria to the vaccine subunit was 7 billion 500 million: 2.375 mg.The rate of clinical protection was reached 95.7%.In conclusion,the results of isolation,identification,complete genome sequencing and composite vaccine development of Pathogenic E.coli in donkey's diarrhea cases will provide important theoretical basis and technical support for the prevention and treatment of diarrhoeal diseases.