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Research on the Bivalent Vaccines Against Procine Edema Disease and Paratyphoid of Swine

Author: ManXiaoYing
Tutor: WuBin
School: Huazhong Agricultural University
Course: Preventive Veterinary Medicine
Keywords: procine edema disease Shiga-like toxin Escherichia coli(SLTEC) recombinant bacteria bio-characteristics genetically engineered vaccine
CLC: S858.28
Type: Master's thesis
Year: 2009
Downloads: 83
Quote: 1
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Abstract


Porcine Edema disease（EDS） is a peracute toxemia caused by specific pathotypes of Shiga-like toxin Eseherichia coli thought its morbility is sloe,mortality reaches high. Even the survivor may grow slowly,hence this disease induces enormous lost in stockbreeding.S.Choleraesuis is the main pathogen of Piglet’s Paratyphoid for 2-4 -month-old piglets and has an important status in swine industry.Edema disease is caused by hemolytic E coli that produce F18ab pili and Shiga toxin 2e（also known as verotoxin 2e）.FedA,fedB,fedC,fedD,fedE and fedF encoding F18ab were cloned.Smeds A etal（2001） focused on purifing FedF for adhesion studies and show that anti-FedF antibodies were able to inhibit E.coli adhesion to porcine enterocyteS。SLT-IIe is responsible for edema disease of pigs.SLT-IIe is a bipartite molecule composed of a single enzymatic intracellularly active A-subunit（SLT-IIeA） and a pentamer of B-subunits（SLT-IIeB）.The receptor binding capacity of SLT-IIe is associated with the B subunit.SLT-IIeB has immunological activity to protecte Vero cells from native SLT-IIe.Salmonella as the vector of vaccine has received wide attention in veterinary medicine.The major advantage of Salmonella is that it can be administered by oral or intranasal route,easily genetically manipulated and cause little stimuli to animals.In addition,as an intracellular invasive bacterium,Salmonella can effectively present antigen,stimulate specific humoral and cellular immune response against both Salmonella and heterologous antigen,stimulate mucosal and systemic immune at the same time.Based on above considerations,this study was aimed to develop a safer vaccine through fusion proteins expressing in Salmonella expression system which against Porcine Edema disease and Piglet’s Paratyphoid.The main research was described as follows:1.Construction of recombination plasmid pYA-SFThe SLT-IIeB and FedF gene which amplified fragment size of were 204bp and 840bp,respectively were amplified and cloned from WH3 genome according to the GenBank sequences of E.coli strain EC0412A.The former carried the restriction endonuclease EcoRI and SalI,the latter with restriction endonuclease HindⅢand SalI. Plasmid pMD 18-SLT-IIeB-FedF was got by conventional biological methods,then tandem gene SLT-IIeB-FedF were cloned into the pYA3493 vector,resulting in pYA-SLT-IIeB-FedF（abbreviated as pYA-SF）.2.Construction and identification of recombinant strain C501（pYA-SF） Recombinant expression plasmid pYA-SF were transformed to△asdC500 strain by electroporation and recombinant strains were selected by LB plate without DAP and confirmed by PCR.The recombinant strains could amplificate the fragement of 2,229 bp,while the patent strain C500 4,000bp with the prime pa7 and pa6.meanwhile the recombinant strain also amplificated the specific fragement of the gene of SLT-IieB and FedF.All of the facts proved that construction of recombinant strain was succeed and correct.3.Biological characterization of recombinant strain C501（pYA-SF）Phenotypic and biochemical characteristics of study showed that the recombinant strain C501（pYA-SF） was the same with parental strain C500.The growth curve of recombinant strain gave the evidences that it was similar to parental strain C500,while grew slightly faster than△asdC500（pYA3493）did.Genetic characteristics of study showed that the recombinant strains had the ability of a stable genetic.The results of SDS-PAGE showed that the expression of recombinant strains can be secreted,bacause the recombinant strain C501（pYA-SF） had a clear expression at about 37 kDa band and△asdC500（pYA3493） did not.4.The immune and protective assay of recombinate bivalent vaccine in miceELISA was used to assay serum antibodies after being inocubated subcutaneously in mice.The results showed that the IgG titers of sera were highest at week 2 after oral immunization and upgraded after being inocubated again.The oral vaccines conferred complete protection against fatal infection with the virulent parent S.choleraesuis strain （C78-1） with the dose of 107.1 CFU and none of 9 phosphate-buffered saline （PBS）-treated controls that survived.The other mice vaccinated s.c.survived intranasal challenge with 2.5 times the 50%lethal dose of virulent SLTEC（WH3） compared with 4 of 9 trivalent inactivated vaccine controls and 8 of 9 recombinant strain controls that survived.5.The immune and protective assay of recombinate bivalent vaccine in swineThe strains were safe to piglets after being inocubated subcutaneously with different dose.Piglets immunized s.c.with 6×109 CFU C501（pYA-SF） produced robust elicited Salmonella- or rSF-specific serum IgG antibodies and survived the oral challenge with 5 times of lethal doses（LD） of wild-type Salmonella choleraesuis C78-1.Simultaneously, piglets survived the intravenous challenge with dose of 1.5mL edema toxin compared with 1 of 4 vector-treated controls and 0 of 4 phosphate-buffered saline（PBS）-treated controls that survived.It indicatated that the recombinant C501（pYA-SF） vaccine showed the potential as a new recombinant recombinate bivalent live vaccine against both edema disease and paratyphoid.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Pig
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