| Neospora caninum is an intracellular obligate protozoa recognized in1984and firstlyreported in Europe. Dogs, cows, sheep, goats, cattle, horses, deer and other animals were mainlyinfected. Neospora caninum is mainly found in central nervous system, muscle, liver, brain andother internal organs, causes abortion in pregnant animals and neurological diseases in calves.Neosporosis leads to serious economic losses for livestock husbandry. There is no effective drugand vaccine to control this disease currently, so it is particularly important to develop a highsensitive and specific method for detecting neosporosis.Studies showed that Neospora caninum can infected special economic animal—sika deer,and affect its health and related products, yet there was no method for diagnosis of neosporosisin sika deer. Therefore, this study aimed at developing a sandwich Dot-ELISA for detectingNeospora caninum infection in sika deer, by utilizing anti-Neospora caninum MIC6monoclonalantibody. And this effort can be helpful to control neosporosis in sika deer. This study mainlyincludes，Preparation of MIC6monoclonal antibodies of Neospora caninum The BALB/c mice wereimmunized with recombinant MIC6protein and than the spleen cells were fused with SP2/0cells. Two hybridoma cell lines1A1and1H11were obtained by screening, which was IgG1andIgG2b subtype respectively. And the number of chromosome was90and94respectively. It wasbetter to combine caprylid acid-ammonium sulfate method and protein affinity chromatographyto purified antibody in ascites. The antibody titers in ascites were5.12×10（41A1）and1.024×105（1H11）and the concentration was0.974mg/mL and2.01mg/mL respectively. The twoMcAbs could react specifically with Neospora caninum and had no cross-reaction withToxoplasma gondii.Localization of MIC6protein antigen of Neospora caninum Immunofluorescence techniqueswas used for the localization of the MIC6protein antigen in Neospora caninum. Result observedby confocal laser scanning fluorescence microscopy showed that the protein was located in theapical region of Neospora caninum tachyzoites. Experimental infection with Neospora caninum in sika deer The main purpose of the currentstudy was to experimentally infect sika deer with Neospora caninum，and to observe its clinicalsymptoms, conduct histopathological examination and immunohistochemistry. Infected sikadeer were characterized with fever, depression anorexia and weight loss after inoculation. Atautopsy, white spots in the surface of liver and bleeding plaques in lung were observed, meninxwere characterized with congestion and edema. With HE staining and immunohistochemistrytests, haemorrhage in spleen, congestion in kidney, necrosis in tubular epithelial cells, acuteglomerulonephritis, vascular congestion in the muscle fibers, and cysts in the brain were found,while no cyst was observed in other tissues. Circulating antigen in serum was found in theamount of circulating antigen decline after the first rise.Establishment and application of double-antibody sandwich Dot-ELISA for detection ofNeospora caninum infection in sika deer Based on anti-Neospora caninum MIC6monoclonalantibodies, a double-antibody sandwich Dot-enzyme-linked immunosorbent assay（sandwichDot-ELISA）were established for detecting Neospora caninum infection in sika deer, and themethod had a new criteria to conclude result by comparing double-antibody sandwich ELISAwith double-antibody sandwich Dot-ELISA. The checkerboard assay results showed that the1H11MAbs labeled by HRP was diluted1:100as the detection antibody and the concentrationof packet capture antibody (1A1MAbs) was0.3μg each well. The serum dilution was1:8. Thesandwich Dot-ELISA had good specificity, sensitivity and reproducibility and nocross-reactivity with Toxoplasma gondii in tests. Serum samples coming from a sika deer farmin Changchun were tested positive rate was15.2%(12/79) by the method, and when tested bydouble-antibody sandwich ELISA, the positive rate is also15.2%(12/79). The effort in thisstudy can provide new method in diagnosing Neospora caninum infection in sika deer. |
