Isolation And Identification Of Bacterial Pathogens Of Porcine Respiratory Disease Syndrome And Development Of Monoclonal Antibodies Against Pasteurella Mutocida Toxin

Porcine respiratory disease complex has caused severe economic losses to modern intensive swine industry in the world.Due to the high proportion of cases caused by secondary bacteria infection,the isolation and identification of swine respiratory bacteria with antibiotics resistance analysis are of great significance for the diseases diagnosis and clinical antibiotics using.At the same time,it can also provide experimental support for further research.To date,the most hazardous bacteria to pigs are Streptococcus suis and Haemophilus parasuis.Especially,Streptococcus suis is an important zoonosis,which can cause some diseases in swine and human.At present,Streptococcus suis can be divided into 33 serotypes according to the specificity of capsule antigen.The disease caused by Haemophilus parasuis secondary infeciton is the most common bacterial disease in pigs.Swine atrophic rhinitis(AR)is a worldwide distribution disease.It is mainly caused by toxigenic Pasteurella multocida(T+Pm),which can secrete Pasteurella muluocida toxin.Inoculation with PMT alone can reproduce all the major symptoms of AR in the experiment pigs.The toxin is a protein with a molecular weight of about 146 kDa.The detection of PMT protein and its antibody is an important method for the diagnosis of AR.The specific contents of this study are as following:1.Isolation and identification of bacterial pathogens in swine respiratory bacteriaBacteria were isolated and cultured from 84 lung and spleen samples from several provinces of Jiangsu,Shandong,Zhejiang,Shandong and Guangxi.The bacteria isolates were initially identified by morphology and Gram staining.The suspected isolates were further identified by PCR with species-specific primers or amplified by 16s rRNA gene universal primer PCR and identified using sequencing.The following bacteria strains were isolated:35 strains of Streptococcus suis,13 strains of Haemophilus parasuis,10 strains of Pasteurella multocida and 3 strains of Actinobacillus pleuropneumoniae.Among of them,Streptococcus suis is one of the most important pathogens.The serotypes of Streptococcus suis isolated in this experiment were classified by multiple PCR.The distribution of serotypes of SS is as follows:except for 2 strains which could not be identified,the others were type 2,9,16,8,3,29,25,31,4,and 18,respectively.The corresponding number of bacteria were 5 strains,3 strains,1 strain,2 strains,1 strain,1 strain,1 strain,1 strain,1 strain,1 strain,1 strain,and 1 strain,respectively.According to the above results,type 2 and type 9 were the dominant serotypes.2.Antimicrobial susceptibility of swine respiratory bacterial pathogensThe antimicrobial susceptibility of Streptococcus suis,Haemophilus parasuis and Pasteurella multocida was conducted by K-Bauer method.And the result of SS is as follows:isolates were sensitive to cefotaxime and penicillin at a rate of 97%and 94.3%,respectively,followed by amoxicillin,and the sensitivity rate was 86%.The isolates showed a higher antimicrobial resistance for lincomycin,kanamycin and tetracycline at a rate of 86%,77.2%and 78%respectively,followed by trimethoprim at a rate of 62.8%.The results showed that only 1 SS was sensitive to 11 antibiotics,11 and 10 of these isolates were resistant to 4 and 3 kinds of antibiotics respectively.All of the HPS isolates were fully sensitive to florfenicol and enrofloxacin at a rate of 100%,followed by amoxicillin and doxycycline at a rate of 92.3%respectively.The results showed that 3 HPS were sensitive to 5 antibiotics,1 and 2 of these isolates were resistant to 4 and 3 kinds of antibiotics,respectively.Antimicrobial susceptibility of 10 P.multocida strains indicates that most of isolates were sensitive to cefotaxime,penicillin and amoxicillin,but several strains are resistent to lincomycin and sulfonamides.Surprisingly,a plasmid,pLH4,was extracted from one strain with sulfonamides resistence.Plasmid sequencing shows that pLH4 contains two resistant genes,sulll and strA,to resist sulfonamides and streptomycin.3.Development of monoclonal antibodies against Pasteurella multocida PMT toxinIn order to prepare biological material to establish immunological method to detect PMT,BALB/c mice were immunized with prokaryotic expressed and purified recombinant PMT protein.Hybridoma cells were screened using indirect ELISA screening and subcloned.Three hybridoma cell clones,secreting antibodies stably,were screened and identified and named 3E3,5G6 and 2H4,respectively.The results of western blot showed that the antibodies secreted by three hybridoma cell clones can specifically react with the T+Pm and recombinant PMT.The cell supernatant titers of McAb 3E3,5G6 and 2H4 were 1:3200,1:3200 and 1:6400,respectively.Identification of monoclonal subclasses suggests that MAbs 3E3,5G6 and 2H4 belong to IgG1/?,IgG1/? and IgG2a/? respectively.When cultured in-vitro for 20 generations,the antibodiy titers were basically consistent,which indicated that the hybrydoma clones can be subcultured and secrete antibody stably.Additon test showed that 3 MAbs reacted with the same antigen determinant.