Epidemiological Investigation And Inactivated Vaccine Preliminary Study Of Enterotoxigenic Escherichia Coli(ETEC)in Piglets From Northern Jiangsu

Enterotoxigenic Escherichia coli(ETEC)is a major pathogenic bacterium which causing diarrhea in piglets,and causing yellow-white scour of newborn piglets from newborn to nursing after infection.It is also an intestinal pathogen that difficult to eradicate on scale pig farm.The main clinical manifestations of sick piglets are diarrhea,dehydration,emaciation,yellow dysentery or white dysentery,also death of seriously piglets.It is a significant infections disease of swine colibacillosis.The clinical investigation showed that yellow-white scour of newborn piglets is the most frequently colibacillosis,its pathogenic ETEC has high drug resistance and high pathogenicity,which greatly threatened the healthy development of swine industry.In this study,we investigated the prevalence of ETEC in large-scale pig farms from northern Jiangsu.Otherwise,its biological characteristics such as serotype,antibiotic resistance and pathogenicity has been analyzed,then developed an o antigen inactivated vaccine which has broad-spectrum immune protection effect.On this basis,effective monitoring and control piglet diarrhea of large-scale pig farms in northern Jiangsu.1 Isolation and identification of Enterotoxigenic Escherichia coli in piglets from northern JiangsuIn this study,562 samples of fresh feces,rectal cotton swabs and small intestine samples were collected from 21 large-scale pig farms in northern Jiangsu,and 792 strains of suspected Escherichia coli were isolated from 528 samples.The results showed that there were 141 strains of ETEC has been isolation,which accounted for 25.1%of the total number of diseased strains,which identified by plate marking,colony morphology observation and microscopic morphology observation.The suspected strain was identified by PCR and 16s rRNA sequencing.Then the O antigen serum type of 141 strains were identified by glassy plate agglutination test combined with PCR method.The antibiotics resistance and sensitivity of the isolated strains were analyzed with 14 antimicrobial standard drug sensitive tablets,moreover the overall antibiotics resistance was analyzed.By means of mouse attack test analyse the virulence of isolated strain,and the immune protection effect of inactivated vaccine was analyzed by mice immune protection test.The glassy plate agglutination test and PCR molecular biology were used to identify O antigen serum type of isolated strain.Seven kinds of polyvalent O antigen serum and 37 kinds of single factor O antigen serum were used.The serum type of 141 ETEC strains was identified,the results showed that there were many O antigen serotypes with 85 strains were identified,accounting for 60.3%of the total number of ETEC strains.In addition,one strain showed self-solidifying and two strains showed polyvalent agglutination but univalent agglutination.Among which 08,0101 and 0128 were the dominant serotype,accounted for 29.4%,20%,11.8%of the stereotyped strain respectly,and accounted for 17.7%,12.1%,7.1%of the total strain respectly.The proportion of three dominant serotype in type ETEC strains is 61.2%,accounted for 36.9%of the total ETEC strains.Other serotypes include 09,03,020,0149,0111,0138,0139,0141,025,0115,0119,0153,045 and 089,etc 15 species serotype.2 Biological characteristics analysis of Enterotoxigenic Escherichia coli in piglets from northern JiangsuThe virulence factors such as enterotoxin and pathogenicity islands were identified of 141 ETEC strains.The results showed that all ETEC isolates strains contained ST virulence gene,25%strains contained LT and Ler virulence gene.In addition,10%of the strains containing Irp2,fyuA and eaeA virulence gene.Antibiotic resistance analysis fourteen antibiotic sensitive tablets of 141 ETEC strains by antibiotic susceptibility test.The results showed that ETEC isolates strains was highly resistant to neomycin,erythromycin,tetracycline,gentamicin,doxycycline,amoxicillin,trimethoprim/sulfamethoxazole,and the proportion of resistant strains was more than 80%.The percentage and number of resistant strains were 95%(134/141),100%(141/141),80.9%(114/141),100%(141/141),92.2%(130/141),96.5%(136/141),99.3%(140/141),83%(117/141)respectly.Besides,50.4%(71/141)of ETEC were sensitive to enrofloxacin,66%(93/141)and 51.8%(73/141)of ETEC were medium sensitivity to polymyxin B and cefotaxime respectly.Multidrug resistance(MDR)was mainly concentrated in 8 to 11 species drug resistance,which accounted for 68%(96/141)of the total ETEC,and accounted for 17%(24/141),16.3%(23/141),19.1%(27/141),15.6%(22/141)respectly.The proportion of strains with 10 species resistant strains was the largest.Routine ICR mice were selected in animal experiments.Nine highly virulent strains were picked out by screening the pathogenicity of 141 ETEC strains.All of them could kill mice with 1×107CFU.Then the pathogenicity of dominant serotypes 08 and 0101 were screened respectively,and the related literature was consulted to attack the mice with 5×107CFU.The results showed that 8 strains of 08 serotype were virulent strain,accounted for 38.1%of total 08 serotype strains,other 5 strains were virulent strain of 0101 serotype,accounted for 27.8%of total 0101 serotype strains.Based on the results of virulent strain,an 08 virulent strain was selected to measure its median lethal dose(LD50)and minimum lethal dose(MLD).The LD50 of the 08 serotype virulent strain was 1.4×107CFU and the MLD was 3×107CFU.3 Preliminary study on inactivated vaccine of Enterotoxigenic Escherichia coli in piglets from northern JiangsuThe inactivated vaccine immune effect in mice results showed that the monovalent inactivated vaccine could protect the same serotype virulent strain completely,and the protective rate was 100%.Two virulent strains of dominant serotype were inactivated to make bivalent inactivated ETEC vaccine,then mice were immunized could able to form effective protective.The bivalent inactivated vaccine protective rate over 83%of two species dominant serotypes virulent strains.There is a high protective rate against high concentration of infection and no accidental death.Moreover it’s also has partial protective effect on other serotype virulent strains.Comparison the immune serum antibody titer of before and after,the results showed that after two free serum antibody titers increased rapidly,but first immunization in mice can form effective protection.Comparing the immune effects of different immunization doses,it was found that the best effect was to immunize mice with 2×108CFU.The effect of inactivated vaccine against different pathogenic strains showed that the inactivated vaccine prepared by three different pathogenic strains could effectively protect the mice.