Study On Gene Engineering Subunit Bacterin Against Oedema Disease Of Swine And Etiology Of F18～+E. Coli

Porcine postweaning diarrhea (PWD) and edema disease(ED) associated with F18fimbriae Escherichia coli (E. coli) strains which adhere to and colonize the microvilli ofsmall intestinal epithelial cells and secrete shiga-like toxin, are the most widespreadcauses of death in weaned pigs and acount for substantial economical losses in swinefactory. However, commercial vaccines and effective drug therapies are still lacking so far,most probably due to a large number of different F18~+ E. coli serogroups implicated inporcine cases of PWD and ED, which makes it difficult to prevent carriers fromintroducing F18~+ E. coli to a naive herd. So in this study, a recombinant G S T taggedFedF protein in genetic engineering strains of E. coli was produced and a FedF-basedenzyme-linked immunosorbent assay (ELISA) was established for detection of FedFspecific antibodies in porcine sera. Nevertheless, we constructed a recombinantimmunogen, SF, formed by fusion of FedF to SLT-Ⅱe B subunit, its in vivoimmunogenieity and as an addictive to a univalent formalin-inactivated vaccine. Theprincipal results describe as the following.1. Isolation, detection and identification of F18~+ Escherichia coli and sequenceanalysis of fragments encoding major subunit of Ee strain.12 strains of F18~+ Escherichia coli were detected and identified among the 284 strainswhich were isolated from the year 2002 to 2004 and 24 strains were isolated from the 130healthy fattern pigs by using fectal swabs from the year 2004 to 2006. Among the 36strains of F18~+ Escherichia coli, 32 strains were serotyped and belong to 17 kinds ofserotype. Predominant sero types are O8, O9, O138, O139, O101 and O78, whichoccupied 53.7％among these strains. These strains were tested with resistance to 14 kindsof antibiotics. As a result, a total of 36 stains were all resistant to 15 antibiotics to agreater or less degree. Ee strain, one F18~+ VTEC field strain from Wuhan, belong to O139serotype and further proved by conventional methods to harbor F18 fimbriae andShiga-like toxin variantⅡ. F18 fimbriae of Ee strain belong to F18ab confirmed by fedAsequence analysis. The nucleotide sequence of FedF, SLT-ⅡeB, fedA and SLT-ⅡeA of Eestrain is 100％, 100％,99％,98％homology with that of 107/86 strain, respectively.2. Development of a FedF based ELISA for the detection of antibodies against F18~+Escherichia coil and its evaluation of serological prevalence of F18~+ Escherichia coilin ChinaPostweaning diarrhea or edema in piglets caused by F18~+ E. coli is a disease spreading worldwide, but little data is available about its epidemiology and effectivemonitoring and prevention measures are lacking. In this study, a FedF-based indirectenzyme-linked immunosorbent assay (ELISA) was developed which employed afragment of the FedF protein of F18~+ E. coil expressed through genetic engineering anddetects antibodies against F18 fimbfiae. This FedF-ELISA and bacterial isolation andidentification were applied to assay 55 sera from the fattern pigs and the two methodsshowed an agreement ratio of 92.8％. The FedF-ELISA was used to conduct a survey ofFedF seroprevalence in China in which 2593 pig serum samples from fifteen pig farms (3each from five provinces) were tested. In the farms FedF seropositive prevalence rangedfrom 8.4％to 45.1％with a medium number being 31.8％, Sows and piglets had higherFedF seropositive rates (medium 50％and 42％, respectively) than curling and fatteningpigs (medium 16％and 26％, respectively). In conclusion, the FedF-based ELISA wasfound to have adequate specificity and sensitive for detection porcine antibodies producedduring active infection of F18~+ E. coli, as well as antibodies passively acquired from thesows. The serological epidemiology data provides valuable information for the preventionand control of F18~+ E. coli-induced diseases.3. Fusion of FedF and SLT-Ⅱe B subunit resulting in effective protection againstF18~+VTEC infectionPostweaning diarrhea or edema in piglets caused by F18~+ Escherichia coli (E. coli) isa disease spreading worldwide, but commercial vaccines and effective drug therapies arestill lacking so far. Herein, a recombinant protein, named SF, was constructed by fusion ofFedF fragement of F 18 fimbriae to the carboxyl terminus of truncated SLT-Ⅱe B subunit.The antiserum obtained from SF vaccinated rabbits could protect the Vero-E6 cell from50％cytotoxic effect of SLT-Ⅱe at a dilution of 1:512 and inhibit adhesion of 10~6 Cfu ofEe E. coli to intestinal epithelial cell at a dilution of 1:100. Different groups of BALB/cmice were immunized with SLT-Ⅱe B subunit, FedF, SF, respectively. The antibody titerfor SLT-Ⅱe B subunit and FedF elicted by SF was significantly higher than that raised byby SLT-Ⅱe B subunit or FedF alone. The protection efficacy against lethal Ee E. coli forSF immunized mice was augmented from 20％to 70％compared with the miceimmunized with SLT-Ⅱe B subunit or FedF alone and pathological changes challenged byEe strain was also relieved. In conclusion, we demonstrated that fusion of FedF andSLT-Ⅱe B subunit improved respective immunogenicity in mice and this SF fusionprotein has great potential as suitable antigens in developing a promising vaccine againstF18~+VTEC. 4. Protective Efficacy of the Genetic toxoid Bacterin against F18~+VTEC in MiceThe fusion of the exotoxin SLT-IIeB and FedF of Eecherichia coli Ee strain wasexpressed in E. coli, the recombinant protein was purified and mixed with Ee strain, thenemulsified with oil - emulsion adjuvants to get a sort of mixed subunit bacterin. Group sof BALB/ c mice were immunized twice at week 0, 3, and then challengedintraperitoneally with Ee strain at week 6. Antibodies were detected with ELISA andsomatic agglutination test. After second immunization, antibody level increased obviouslyand the immune protection rate against Ee strain was 70％and 91.7％in subunit bacteringroup and bacterin group, respectively. It was concluded that the mixed subunit bacterinhad good protection against Ee strain, which built a good foundation for the furtherresearch of high efficacy vaccine against porcine edema disease.5. Protective efficacy of the genetic toxoid bacterin against F18~+VTEC in pigletsThe efficacy of a new genetic toxoid bacterin containing recombinant SF fusionprotein and Ee strain was determined. Weaned piglets were vaccinated twiceintramuscularly with the new genetic toxoid bacterin or univalent formalin-inactivatedvaccine at 3-week internals. The antibody for SLT-IIe B subunit, FedF and Somaticantigen was detected respectively by ELISA and agglutination test. The biochemicalindicators for GST, TBIL, Crea and NO were detected by commercial Kits. The Pigletswere challenged by intravenous injection through precaval vein with 160×10~9CFU of Eestrain. The antibody titer for SLT-IIe B subunit and FedF elicted by Subunit bacterin wassignificantly higher than that raised by univalent formalin-inactivated vaccine alone. Theprotection efficacy against lethal Ee E. coli for Subunit bacterin immunized pigs wasaugmented from 60％to 100％compared with the pigs immunized with univalentformalin-inactivated vaccine alone and pathological changes challenged by Ee strain wasalso relieved. In conclusion, we demonstrated that the addition of fusion of FedF andSLT-IIe B subunit to vaccine improved protection efficacy in pigs and this subunitbacterin hold great potential as suitable antigens in developing an effective vaccineagainst F18~+VTEC.