Construction And Characterization Of A ApxⅣ △orf1 Mutant Of Actinobacillus Pleuropneumoniae

Porcine contagious pleuropneumonia(PCP) caused by Actinobacillus pleuropneumoniae (APP) is a severe,contagious pulmonary disease of pigs that causes important economic losses in industrialized pigs production worldwide.Immunization is the main method to prevent it.The commercial vaccines mainly are killed whole cell bacterins and subunit vaccines,which generally reduce the mortality of APP infection.However those vaccines frequently failed to offer protection against heterogenous serovar.By contrast,the live attenuated vaccines could re-present protective antigens and stimulate a lasting immune response that may be more efficacious in preventing the disease.Therefore,studies of A.pleuropneumoniae vaccines tend to focus on live attenuated vaccines constructed by inactivating virulence-associated genes.Based on above considerations,by deleting the orf1 gene of the SLW03 strain(APP-1,Δapxâ… C/Δapxâ…¡C) which constructed by Dr Lin,we developed an attenuated A.pleuropneumoniae serovar 1 strain,SLW05,which was a potential vaccine candidate to prevent and control porcine contagious pleuropneumonia.The main research was described as follows:1.Construction of recombination suicide plasmid(pEMORF)According to the GenBank(No:AF021919) sequences,The upstream and downstream of orf1 gene were amplified and cloned from SLW01 strain genome.Those sequences were highly conserved compared with the sequences published.The upstream and downstream of orf1 gene were respectively subcloned into suicide plasmid pEMOC2, which contained a sucrose sensitive(sacB) gene.The recombination suicide plasmids were designated as pEMORF.2.Selection and identification of the mutantsThe E.coil donor strainβ2155 transformed with plasmid pEMORF,was conjugated with the recipient SLW01 and SLW03,respectively.After transconjugation,Chlorampheni col-resistant(Cm^R) transconjugants were analyzed for the presence of a first crossover event by PCR.This first step selected for clones in which the whole plasmid had been incorporated into the recipient chromosome.Colonies with the correct PCR profile were incubated in TSB(supplemented with NAD and sterile bovine serum).After incubation for 24h,sucrose-resistant(Suc^R) colonies exhibiting a non-mucoid phenotype were tested for the chloramphenicol sensitivity(Cm^S) phenotype,which was indicative of loss of plasmid vector sequences.Following this,Suc^R and Cm^S colonies were identified using PCR to determine the presence of the second crossover,and mutants were constructed. 3.Characterization of the mutantsFor investigated the genetic stabily of the mutants,the mutants were grown on TSA plates for more than ten generations and determinde by PCR.The results indicating that the genetic of mutants were stabily.For growth of mutants,cultures of the parent strains and mutant strains were grown with shaking at 37℃in TSB(supplemented with NAD and sterile bovine serum) overnight.The cultures were subinoculated into fresh supplemented TSB at a 1:1000 dilution.The OD₆₀₀ of the bacterial cultures was determined at intervals of 1h,no obvious difference was observed in the in vitro growth curves of parent strains and mutant strains,indicating that deletion of the orf1 gene had no significant influence on the growth of App-1.By test the LD₅₀ of mutants and parent strains in mice,the virulence of mutants is lower compare to the parent strains.4.The virulence of the mutant strain SLW05 in pigsPigs were randomly divided into two experimental groups of four.Groups 1 were challenged intratracheally with 3×10⁹CFU of SLW05 in 1ml TSB.Groups 2 were challenged intratracheally with 3×10⁹CFU of SLW03 in 1ml TSB.The pigs were examined clinically at least once a day or as needed.Body temperatures were recorded for each pig along with clinical symptoms like depression,dyspnea,and coughing.The results indicating that the virulence of SLW05 is lower than the SLW03,and Apxâ…£plays an important role in pathogenicity.5.The immune and protective assay of the mutant strain SLW05 in pigsThirty-six 6-week-old pigs were randomly divided into six experimental groups of six.Groups 1 and 2 were vaccinated twice intratracheally with 8×10⁹CFU of SLW05 in 1ml TSB.Groups 3 and 4 were vaccinated twice intratracheally with 8×10⁹CFU of SLW03.Groups 5 and 6 served as unvaccinated control groups inoculated intratracheally with 1ml TSB.Two weeks after the booster immunization,groups 1,3 and 5 were challenged intratracheally with SLW01(serovar 1).Groups 2,4 and 6 were challenged intratracheally with JL03(serovar 3).Before changlleng,tested the antibody of Apxâ… ,Apxâ…¡and Apxâ…£,the difference of antibody level is not obivious.Postmortem examination showed severe lung lesions,pleural effusion and adhesive pleuritis in pigs.The difference of lung lesions scores is obivious(P＜0.05).