Screening Of Neospora Caninum Specific Genes、Establishment Of Detection Methods And Their Applications

Neospora caninum is an intracellular protozoan that infects domestic and wildcanids as well as many warm-blood animals as shown by the isolation of viableparasites. N. caninum and T. gondii are structurally, genetically and immunologicallyclosely related protozoan parasites in the phylum Apicomplexa. These twoorganisms have nearly identical morphology and can cause similar pathology anddisease, which has frequently led to N. caninum being incorrectly identified as T.gondii. A diagnostic method specific for N. caninum should have no cross-reactionwith T. gondii. The specific aim of the present study is to screen and identify specificgenes of N. caninum and to establish a diagnostic method.1. N.caninum tachyzoites were cultured in vero cells in RPMI1640medium and T.gondii were cultured in BALB/c mice. The parasites and infected host cell wereharvested and passed through20and27-gauge needles, and tachyzoites werepurified by a40%Percoll gradient. RNA was extracted by TRIZOL from N.canimumand T. gomdii ticks according to the manufacturer’s protocol. A SSH cDNA librarywas established by the PCR-SelectTMcDNA Subtraction Kit from Clotech anddscDNA was prepared using the Super SMARTTMPCR cDNA Synthesis Kit as permanufacturer’s instructions. To isolate N. caninum specific sequences from theN.canimum ‘tester’ cDNA, T. gondii cDNA was used in the present study as the‘driver’cDNA. Through two suppressive subtractive hybridizations and nested PCR,the N. caninum specific sequences were cloned into the T vector, sequenced, andanalyzed by Bioinformatics.2．Nestd-PCR primers have been designed using Primers version5.0based on the N.caninum specific gene identified and numbered in the present study asNCLIV₀04830. All reaction conditions of nested-PCR were optimized for detecting N.caninum specific DNA sequences with the optimal annealing temperature at55℃a ndoptimal Mg2+concentration at1.5mmol/L. This optimal PCR condition candetect the genomic DNA level from one parasite with no cross-reaction with T.gondii, H.heydomi and canine Cystosospora spp DNA. The results indicated thatnested-PCR detection method of N.caninum was of good both specificity andsensitivity.3. An epidemiologic study of N. caninum was carried out in Shen Yan by nestedPCR. The results showed that infection rate was38.9%in police dog training facilityand35.2%for farmer’s free range dogs respectively. The Infection in juvenile dogwas significantly higher than that in adult dog.. Sequence analysis of ITS1gene from37isolates from hosts infected only with N. caninum, but not H.heydomi, showedthat there were four genotypes with26isolates belonged to NC-PR and no isolatebelonged to NC-1. From Genetic Mutations Network it seemed that N. caninum andH. heydomi may be originated from a common ancestor, and other N. caninumisolates may be mutated forms of NC-PR.4. Two genes suitable for antigens were identified from SSH library, the N. caninum40KD surface antigen（P40） and the putative protein similar to T. gondii MIC6named N. caninum MIC6. P40contains two surface antigen domains （SRS） andbelongs to surface antigen super family. For further study, P40and MIC6wereexpressed in E. coli and purified by a commercial kit. The final concentrations ofP40and MIC6were3.07mg/ml and0.96mg/ml, respectively. Dot-blot andWestern-blot showed that P40and MIC6had no cross-reactions with serum fromhost infected with T.gondii, which suggested that these two antigens could be usedfor specific sero-diagnosis of N.caninum.5. N. caninum specific Indirect-ELISA was base on purified P40and MIC6wasestablished and all reaction conditions were optimized as followings：the optimalprotein concentration for antigen coating of P40-ELISA was0.25g/well; theoptimal serum titer were1:20; the optimal antigen coating concentration ofMIC6-ELISA iwas0.1g/well; the optimal serum titer were1:40; the optimal blocking buffer were5%MILK; the optimal blocking time were1.5h and2h,respectively; the optimal time of reaction with serum were2h and1.5h, respectively;and the optimal time of reaction with substrate were25min and20min, respectively.The ELISA index value iwere N×2.172and N×2.158, respectively, where N is theOD value obtained for the negative serum. The intra-assay and inter-assay variancesof the P40-ELISA were4.7%-9.6%and1.5%-6.7%，respectively and the intra-assayand inter-assay variances of the M-ELISA were4.3%-7.9%and2.6-5.8%，respectively. The specificity and sensitivity of P40-ELISA were94%and97%,respectively and the specificity and sensitivity of MIC6-ELISA were100%and88%,respectively. All results showed that P40-ELISA and MIC6-ELISA developedestablished in the present study could provide a specific and sensitive assay fordetecting neosporasis.6. Seroprevalence of antibodies of N.caninum in cattle and human from Chang Chuncity was investigated. A total of113（16.5%） serum samples were positive forantibodies against N. caninum. There was an apparent association of N. caninuminfection with age of cattle. In addition,10suspected positive human serum sampleswere later confirmed negative by Western-blot.In summary, the studies successfully established N.caninum and T.gondii SSHcDNA library and screened candidate gene to N.caninum diagnosis. N.caninumspecific nested-PCR、NcP40-ELISAand NcMIC6-ELISA diagnostic methods wereestablished and applied in clinical diagnosis of neosporosis.