Establishment And Application Of Test Method For Neospora Canium Infection In Dogs

Objective:The Neospora caninum is an obligate intracellular parasite belongs to phylum Apicomplexa,parasitise in the body of a variety of animals,such as cattle,sheep,horses,etc.It is considered to be one of the main reasons of abortion in livestock around the world.The result is neosporosis when livestock carry N.caninum.Canine（domestic dogs,coyotes,wolves,grey wolves and wild dogs）are both the intermediate and definitive host for N.Caninum.Neosporosis may result in neuromuscular injury and even death in dogs when it is serious.N.caninum oocysts in dog feces can pollute soil and warter in farm,leading to Neosporosis and economic loss.Thus it plays an important role in the prevention and control of neosporosis,which is to establish an indirect ELISA test method in dogs.Indirect ELISA is a common method in serological detection.When we establish an indirect ELISA method,we need to do as follows:firstly coat the specific antigen on ELISA plates,then the antigen binds with antibodies which we want to test in the serum,enzyme labeled antibody is also needed for the second binding with antibodies in the serum.In this way,we can achieve the purpose of detecting pathogens.Indirect ELISA has many advantages,such as easy operating,short reaction time and high detection flux.It has been uesed widely in disease screening in animals which has large populations.The NcP40 protein is one of the N.caninum specific surface antigens.It had been confirmed NcP40 protein was a diagnostic marker for neosporosis.Our research expressed the NcP40 protein,and established an indirect ELISA detecting method.It provided technical supports for the prevention and control of neosporosis in dogs.Methods:Our research designed primers to amplify the sequence encoding the NcP40protein by PCR.We achieved a DNA fragment.The PCR fragment was inserted into an Xho I and EcoRI digested pET-30a expression vector.Recombinant plasmid was transformed into receptive cells of BL21,following by induction with IPTG.Finally,rNcP40 protein contained His tag was expressed.SDS-PAGE and Western blotting were used to identify the rNcP40 protein we expressed.We established the indirect ELISA method after coating the ELISA plates with the purified rNcP40 protein.After optimizing the conditions of coating the rNcP40 protein,serum and enzyme labeled antibody,the established indirect ELISA reaction conditions were determined.Comparing the established ELISA with IFAT kit,the ROC curve was made.Then we used this established method to dectect samples of dog serum from Changchun,Siping,Changbai,Yanji and Baichen.Results:1.We achieved a DNA fragment of 777bp.The rNcP40 protein contained His tag was expressed,which weights 35 KD.SDS-PAGE and Western blotting were used to identify the r NcP40 protein expressed.It was confirmed that the rNcP40 protein had good immunogenicity.2.The established indirect ELISA reaction conditions were as follows:the optimum package of polypeptide was determined by the concentration of 0.104μg each reaction hole,the 50 mmol/L Tris-HCl（pH 8.4）buffer solution was optimal for package,the package time was 37℃2 h,the best sealing solution was 1%BSA plus5%skim milk,the optimum sealing time was 4℃overnight,the serum optimum dilution muliples was 1:40,the best of the second antibody was 1:40000,the serum and the second antibody best incubation conditions ware 37℃1 h and 37℃30min,moreover 2%skim milk was the best buffer solution,the TMB chromogenic solution was 50μL each reaction hole,it needed to be at RT for 5 min avoiding lighting,at last 2 mol/L H₂SO₄ was needed to terminate the reaction,OD_450nm was needed to be tested within 10 min.Comparing the established ELISA with IFAT kit,the ROC curve was made with detecting 180 samples of dog serum at the same time.After analysing,we got the following imformations:cut-off value which was calculated at an S/P of 0.32,a Youden index of 0.701,the assay yielded a sensitivity of 69.4%,a specomcificity of 87.0%and a coincidence rate of 82.2%3.Then we used this established method to dectect 1415 samples of dog serum from Changchun,Siping,Changbai,Yanji and Baichen.Of the samples from these five cities’dogs,13.8%tested positive for N.caninum antibodies.At the same time,comparing the established ELISA with commercial ELISA kit from Bio X,the serological positive rate was 13.8%.The coincidence rate of the two method about these 1415 samples was 96.8%.Compared with the previous studies,seropositivity of N.caninum antibodies in dogs was increasing.The establishment of this indirect ELISA method provided technical supports for the prevention and control of neosporosis in dogs.Conclusions:1.We achieved a DNA fragment of 777bp.The rNcP40 protein expressed had good immunogenicity.2.Established an indirect ELISA method.3.We used the established method to dectect samples of dog serum from Jilin province.The total serological positive rate was 13.8%.The establishment of this indirect ELISA method provided technical supports for the prevention and control of neosporosis in dogs.