Establishment And Application Of Indirect ELISA Assay For Detection Of Antibodies Against Neospora Caninum

Neosporosis is a common parasitic disease caused by Neospora caninum inhabiting the nucleated cells of various animals.Neosporiasis can cause abortion and stillbirth of pregnant animals,and neurological dysfunction and dyskinesia of newborn animals.It is recognized as one of the main causes of bovine abortion worldwide,seriously endangering the sustainable development of animal husbandry.However,there have been no effective drugs or vaccines to control neosporidiosis,and the transmission of neosporidiosis is prevented mainly by eliminating cattle with neosporosis and N.caninum infection.Based on this situation,firstly,the present study investigated the antibody levels against N.caninum in cattle from some areas of China by using the commercial kit.Secondly,the srs67 gene of N.caninum was cloned,expressed and purified.Finally,an indirect ELISA method for detection of N.caninum antibody based on SRS67 recombinant protein was established,and preliminary used in clinic.The following results were obtained.The antibody levels against N.caninum in cattle from some parts of China were investigated.Blood samples were collected from 279 Holstein cows and 69 Simmental in partial regions of Ningxia Hui Autonomous Region,Henan Province,Shaanxi Province,Gansu Province and Inner Mongolia Autonomous Region.The antibody levels against N.caninum in serum samples were determined by using a commercial detection kit.The total positive rate was 20.40%（71/348）.The positive rates antibody levels among cattle of different breeds and ages was significantly different（P<0.01）.In the term of regional distribution,though there were no significant differences in the positive rates between Gansu and Henan,significant differences were found among other regions（P<0.05）.The srs67 gene of N.caninum was cloned,prokaryotic expressed and purified.Molecular biology softwares were used to analyze sequences of the srs67 gene and the predicted SRS67 protein.The open reading frame of N.caninum srs67 gene was 1056 bp.The SRS67 protein was predicted to have one transmembrane structure but with no signal peptides.N.caninum srs67 gene（903 bp）without the transmembrane structure was cloned into the prokaryotic expression vector p ET-32a,and the recombinant expression plasmid PET-32a-srs67 was constructed and successfully expressed.The size of the recombinant protein of N.caninum SRS67 was found to be 53 k Da by using Western blot.The recombinant protein reacted with positive serum samples against N.caninum.The indirect ELISA method for detection of antibody against N.caninum was established.After optimizing the conditions,the protein coating concentration for established indirect ELISA method based on the recombinant protein of N.caninum SRS67 was 1.25μg/μL coated at 37℃for 2 h.The blocking was did using 5%skim milk powder+1%BSA overnight at 4℃.The tested serum sample was diluted into 1:200 times with PBS-T and incubated at 37℃for 1 h.The concentration of HRP rabbit anti-bovis Ig G antibody was1:1000 and incubated at 37℃for 1.5 h.The TMB chromogenic solution was kept in dark place at 37℃for 15 min.OD_{450 nm}-OD_{630 nm}in the serum samples above 0.303 was identified to be positive.The sensitivity of the method established in this study was 1:1600,and no cross-reaction was found with bovine serum samples against Toxoplasma gondii and Cryptosporidium.The intra-and inter-assay coefficients of variations were within 10%.The compliance rate was 87.5%compared with the commercial kit.Using this established method,a total of 175 blood samples of cattle from Henan,Inner Mongolia and Ningxia were detected.The total positive rate of N.caninum antibody was 14.28%,with significantly different（P<0.05）between Henan and Inner Mongolia.In conclusion,the positive rates of antibody against N.caninum in cattle in the investigated regions were high.The established indirect ELISA method for detection of N.caninum antibody has strong specificity,high sensitivity,good repeatability and high coincidence rate.The results of the study provide basic data for the epidemiological studies of bovine neosporosis in China and a technical mean for the diagnosis of bovine neosporosis.