Study Of Diagnosis Technique And Molecular Epidemiology Of Contagious Bovine Pleuropneumonia

Contagious bovine pleuropneumonia (CBPP) is a subacute or chronic contagious disease of cattle caused by Mycoplasrna mycoides subsp mycoides SC (MmmSC) in the A-list of communicable animal diseases of the Office International des Epizooties (OIE).This disease was transmitted to China in 1920's which had caused great economic loss to China's cattle industry. By means of large-area vaccination from 1950's, CBPP had been controlled effectually in China. China proclaimed the eradication of CBPP in 1996, but we did not get Disease-free Recognition from World Organization for Animal Health, because we did not give the related technical data of the eradication of CBPP to the related international organize.From 2002, The Veterinary Bureau of Agricultural Ministry of China has carried out a program of nation-wide epizootiological survey of CBPP and makes technical prepare for Disease-free Recognition.The data of seroepidemiology is very important in the epizootiological survey. We are using complement fixation test (CFT) recommended by OIE, which it is difficult to extend in field. We do not have better diagnosis method using with CFT, so developing sensitive, specific and convenient method of serodiagnosis is important to seroepidemiology survey.The most effective method to diagnose seropositivity of CBPP still is pathogeny isolation, but quick and specific PCR assay has already been one of more important technologies to detect pathogen. Sensitive and specific PCR assay can diagnose the pathogen in shortest time which can help pathogen isolation.Though CBPP spreaded in China more than 50 years ,we did not have the data of molecular epidemiology of CBPP.According to the epizootiological data ,There are two ways of saying "how CBPP was transmitted to China". One route of infection into China is through trade of cattle between Russia and China and the other is from Australia to Shanghai China to introduce well-bred cattle. Based on the differences of MmmSC genome and antigenicity, MmmSC can be divided into European and African-Australian groups. These two groups present different pathogenicity and epidemiological characteristics. But we do not know which strains MmmSC spreading in china belongs to and from where it was introduced. In the study of serological diagnosis, the evidences show the N-terminal of lipoprotein LppQ is a much ideal CBPP diagnosis antigen. The lipoprotein LppQ encoded by lppQ gene is specific to MmmSC and found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. No serological cross-reactions were observed with the related mycoplasmas of the Mycoplasma mycoides cluster.The N-terminal domain of the mature lipoprotein LppQ is hydrophilic, and it induces a strong, specific, early and persistent immune response in naturally and experimentally infected animals.Mycoplasma-specific TGA (Trp) codon are utilized as stop codon in most other organisms. The lppQ N-terminal fragment from MmmSC HVRI X strain, the Chinese strain for CF antigen production, was mutated with one-step overlapping extension PCR. Sequence analysis confirmed the successful mutation from A to G in codon 198 in the lppQ gene. The fragment containing the mutation site was subcloned into the pET32a expression vector. The recombinant protein with molecular weight of 42kDa was purified by Ni-NTA His·Bind purification kit, with purity up to 95%. Western blot indicated that the standard positive serum of CBPP could react with the recombinant protein. The purified protein was diluted to 0.33μg/ml, and coated to microtiter ELISA plates. Indirect ELISA reaction conditions were optimized.After the statistical analysis, assessment is that the S/P values above 0.5 were considered as positive, below 0.4 as negative and between 0.5 and 0.4 as suspicious. The result of test in which 100 experimentally infected and 150 non-CBPP serum samples is detected shows the sensitivity of ELISA is 95%(95/100), and the specificity is 98%(147/150) .Comparison detection of 3,817 bovine from 3 different provinces was performed using international standard CFT and indirect ELISA method. The Kappa value is 0.701, which is middle or high agreement between the two methods.In research of diagnosing pathogen, we designed and synthesized two pairs of primers, MC1, MC2 and SC1, SC2 in polymerase chain reaction (PCR) assay. The MC primers, be specific for mycoides cluster, allowed amplification of a 462 bp fragment from 6 members of the mycoides cluster assayed. The SC1, SC2 primers, be specific for MmmSC, selectively amplified a 277 bp fragment from MmmSC (?), with three specific internal restriction sites for the endonucleases VspⅠ, DraⅠand Bsp143Ⅰallowing confirmation of the identification. No amplification occurred with MmmLC (Y-Goat strain) assayed. The sensitivity assessed by direct agarose gel analysis for the PCR assay with SC primer was 100CFU. This assay was used to detect pathogen in Xinjiang. The PCR result of 83 suspicious lung sample collected from Xinjiang is negative which was match to the result of CFT and pathogen isolation.In the study of molecular epidemiology, we collected 6 MmmSC strains from different endemic areas in China and compared their molecular characteristics to those of PG1 and Y-goat. PCR amplification of long fragments showed that there was no 8.84-kb deletion in all of 6 MmmSC Chinese strains, PG1 and Y-goat. lppB gene and its encoding protein presented in all African strains, but not European strains. By comparison we found that the LppB complete gene sequence of 6 MmmSC Chinese strains was 99% homologous to that of PG1, while was less than 93% homologous to that of Y-goat. The antiserum prepared by the LppB protein C terminus of recombinantly expressed PG1 strains could have hybridization reaction with PG1, Y-goat and 6 Chinese strains at 66kDa sites in Western-blot, indicating that LppB gene and its encoding protein existed in 6 Chinese strains. Multilocus sequence analysis (MLSA) of the strains from various regions including 6 Chinese strains showed that 6 Chinese strains were identical to African. This result was also supported by the outcome of selective primer amplification. Based on the above results, we propose that Chinese MmmSC strains are identical to African and Australian at molecular characteristics.Based on the above studying, we develop sensitive and specific indirect ELISA method which receives satisfactory evaluation in Neimenggu and Xinjiang .specific PCR diagnosis method by which can know result of suspicious samples in 8 hours .we use the PCR to detect suspicious lung samples in Xinjiang ,which result is same as pathogen isolation and serology detection. By Analyses the molecular characteristics of 6 MmmSC strains from different endemic areas in China, we propose that Chinese MmmSC strains are identical to African and Australian first time, suggesting that CBPP spread in China has come from Australia.