Screening And Evaluation Of Novel Molecules For Diagnosis Of Contagious Caprine Pleuropneumonia

Contagious Caprine Pleuropneumonia is a severe respiratory disease caused by Mycoplasma capricolum subsp.capripneumoniae(Mccp),which mainly affects goats and some wild ruminants,listed as one of the notifiable animal diseases by the World Organization for Animal Health(OIE).The epidemic area of CCPP covers more than 40 countries in Asia,the Middle East and Africa.The main symptoms of CCPP are respiratory manifestations,which are similar to a variety of diseases such as Peste des Petits Ruminants(PPR),Pasteurellosis,goats pneumoniae caused by M.ovipneumoniae,etc.The clinical signs of CCPP are diverse and complex,so the disease can only be confirmed by laboratory diagnosis.The accurate diagnosis of CCPP has always been one of the key technical difficulties in the prevention and control of this disease.The specific and sensitive diagnostic method is one main challenge of research,due to the close relationship between Mccp and other members of Mycoplasma mycoides cluster.Exploring the specific diagnostic molecules of Mccp as well as developing accurate and effective diagnostic methods further,is the necessary technical support for effective prevention and control of CCPP.In this study,new diagnostic molecules of CCPP were investigated mainly through two approaches.Firstly,the gene sequences theoretically specific to Mccp were screened by comparative genomic analysis.Then,the gene 87.1 which is potentially specific antigen gene was selected,and the recombinant protein of this gene was obtained by using the prokaryotic expression technique.The specificity and reactivity of recombinant protein 87.1 were assessed by western blotting(WB)and indirect ELISA.On the other hand,the specific gene sequence 92.1 was selected and used to design the primers 92.1F/R,and the specificity of the primers was assessed by the PCR system.Secondly,Mccp and its close mycoplasmas were used as the capture antigens to screen specific monoclonal antibody(MAb)to Mccp.The preparation of MAb included steps of mice immunization,preparation of hybridoma,cell clone screening,and purification of antibody,etc.A blocking ELISA(bELISA)based on the selected MAb 7H11 as the indicator antibody was established and evaluated subsequently.The results showed that the recombinant protein 87.1 reacted with Mccp positive sera in WB and iELISA,and it could also indicate Mccp antibody levels in serum by iELSIA.There was no cross reaction with positive sera to M.mycoides subsp.capri(Mmc)and M.capricolum subsp.capricolum(Mcc),however,the background value of negative serum was high.In summary,the recombinant protein 87.1 can be used as a new diagnostic target of CCPP,but reaction conditions still need to be optimized.In PCR,the primer set of 92.1F/R was able to amplify a 509 bp of targeted fragment from the genome DNAs of Mccp strains M1801,zly-14,M1601 and zly-1309 F,but there is no amplification from M.agalactiae(Ma)strain PG2,Mmc strain PG3,Mmc strain YG(formerly known as MmmLC),M.ovipneumoniae(Mo)strain Y98,Mcc strain CKid,M.bovis(Mb)strain 08 M,and M.leachi(Ml)strain PG50.It indicates that the primers 92.1F/R can be used as a new set of primers for CCPP molecular diagnosis.In the bELISA system,Mccp positive sera were detected,but positive sera to Mmc,Mcc,Mo,Ma,Ml,and Mb were not detected.Meanwhile,false negative results were found in the goat sera from the late phase of experimental infection with Mccp which means that the MAb 7H11 may be a candidate for CCPP diagnosis but remains further evaluation.