Establishment And Preliminary Application Of ELISA Test Method For Neospora Caninum Antibodies At Rabbits' Sera

Neospora caninum is an apicomplexan parasite that is the etiologic agent of neosporosis.Neosporosis has emerged as a serious disease of cattle and dogs worldwide.Additionally,clinical neosporosis has been reported in sheep,goats,deer,a rhinocerus,and horses and antibodiesto N.caninum have been found in the sera of water buffaloes,red and gray foxes,coyotes,camels and felids.Even the N.caninum antibodies were detected in the human serum was reported.In conclusion,there is no evidence that N.caninum infection is zoonotic.But it is has risks for infecting human beings.Neospora could infect rabbits,in order to reduce neosporiasis infecting supplies economic losses,make diagnosis to prevent the disease is very important.At present,there is no diagnostic method for the neosporiasis infecting at rabbits.We expressed the Nc-p43 gene from N.caninum in bacteria BL21 and demonstrated that the antigenic domain of Nc-p43 is localized in the C-terminal 2/3 parts of the molecule.The DNA fragments Both DNA fragments inserted into pET32 a vector.The plasmids were then used to transform the BL21 strain.Recombinant proteins were purified by passing through a Ni-NTA column.The result that we successful made the recombinant plasmid pET-Nc-p43,and have a high level expressed.When the purified the protein fused C-terminal 2/3 parts(P fragment)of Nc-p43 was used as an antigen in ELISA and used in ELISA.Polystyrene 96-well microtiter plates were coated overnight at 4 �C with 5 ?g/ml of recombinant protein Ncp43 P.The plates were then blocked using 0.01 M PBST with 10% mouse serum at 37 �C for 1h.After three washes with PBS-T,The positive and negative control sera and serum samples were diluted at 1:100 in 0.01 M PBS-T and added in duplicate to the plate.The secondary peroxidase-conjugated IgG was diluted at1:1000.The CV of the difference between batches was 1.6%-5.0%,and within batches was 0.4%-2.3%.The CV result was reported that the method has a good stability for detecting.Detecting the T.gndii antibodies positive serum show that no cross reaction.According to the Cut off=Negative OD value +3SD for calculating.The cut-off value for positive reactions was calculated as 0.36.We determine the OD>0.4 was the N.caninum antibodies positive and OD<0.3 was negative.Between them was suspected.We observed that the meted coincidence rate with the VMRD ELISA Kit was 97.5%.The results from this study suggest that the recombinant protein expressed in BL21 is a suitable antigen for use in immunodiagnosis to detect N.caninum in rabbit exposed to this parasitosis.Among 468 rabbits sera collected from JiLin province,149 sera(31.8%)were found to react with Ncp43 P positively.