Development And Preliminary Application Of Indirect ELISA Diagnostic Method For Neosporosis In Cattle

Neosporosis is a protozoosis, which was caused by Neospora caninum of cells in cattle, sheep, dogs and other animals. It can cause motor disorder and nervous system disease for the neonatus and abortion, stillborn for pregnant animals, especially, abortion of cow cattle, which has brought about enormous economic loss for the livestock farming. It is important that early, accurate diagnosis for controlling neosporosis. Therefore, NcSRS2 was chosen as the surface protein for development of indirect ELISA in neosporosis in cattle for diagnosis.Specific primers were designed and synthesized according to the reported NcSRS2 gene from N. caninum in GenBankTM. NcSRS2 gene was amplified by PCR using DNA as template, then cloned into pMD18-T vector. The recon was transformed into the competent E. coli JM109 for obtaining the positive clone, then was sequenced. In order to get high expression, the gene encoding truncated NcSRS2 (NcSRS2t) lacking a signal peptide and hydrophobic regions was subcloned. The positive plasmid pMD18-T-NcSRS2t and the expression vector pET-28a(+)were digested by restriction endonuclease digestion and purified, then transformed into the competent E. coli JM109. The plasmid was extracted and identified, then the positive plasmid was transformed into the optimal competent E. coli BL21. The recombinant strain E. coli BL21 (pET-28a-NcSRS2t) expressing fusion protein NcSRS2t was constructed. It was proved that the recombinant plasmid pET-28a-NcSRS2t contained the fusion gene NcSRS2t, which had correct sequence and ORF by identification of restriction endonuclease digestion and sequence analysis. The recombinant strain E. coli BL21 (pET-28a-NcSRS2t) was innoculated into LB medium (included Kan 50μg/mL) at the ratio of 1%,and cultured for 2.5 hours at 37℃, then induced by 1mmol/L IPTG for 4h. The fusion protein NcSRS2t was expressed at high level. The expressed protein could be reacted with antibodies of N. caninum by western blot.The ELISA plate was coated by the purified recombinant NcSRS2t antigen, the optimal reaction conditions of indirect ELISA were determined by experiments. The optimal coating buffer was carbonate buffer solution (0.05M pH9.6), and the optimal coating concentration of antigen for microplate was 5.0μg/mL. Five percent skim milk was added as the optimal blocking agent and incubated for 1 hour at 37℃. The best serum dilution buffer was 5% skim milk, and the optimal dilution concentration of serum samples was 1:100. The working concentration of HRP-labeled rabbit anti-bovine IgG was 1:5 000. The cut off value for indirect ELISA was determined from 218 negative bovine serum samples by statistics analysis. If the OD value is more than or equal to 0.32, the test sample will be positive, or negative. Fifty positive and 128 negative serum samples from the cow cattle (confirmed by the IDEXX Neospora caninum antibody test kit) were detected for the antibodies against N. caninum using the developed indirect ELISA. It was found that the specificity and sensitivity of the developed indirect ELISA was 93.0% and 90.0%, respectively.A Total of 540 sera of dairy cattle, which obtained from Keshan, Hailin, Shuangcheng Gannan, Kedong, Fuyu, Mudanjiang, Daqing in Heilongjiang province were sampled and used to evaluate serological screening for neosporosis infection by developed indirect ELISA. The result indicated that the overall seroprevalence of neosporosis in dairy cattle in some regions above in Heilongjiang provienc was 13.33%, and the seroprevalence of neosporosis in different regions was from 5.56% to 23.81%. The seroprevalence of neosporosis in aborting cows (14.88%) was higher than that in non-aborting cows (10.26%), with most Neospora-induced abortions occurring at 5-6 months of gestation. There was no apparent association of neosporosis seropositivity with age or number of pregnancies (P > 0.05), but the difference was statistically significant in breed and raising methods(P﹤0.05). It is very important that development of indirect ELISA and preliminary application in neosporosis in cattle for the preparation of ELISA kit and prevention of neosporosis.