Isolation, Identification, and Pathogenicity of Neospora caninum China Yanbian strain

BACKGROUND The aim of the study was to provide a point of reference to study the Neospora caninum infections in China. METHODS Genome DNA was extracted from the brains of aborted fetuses and specific PCR was performed with N. caninum Nc5-targeted specific primers. Fetal bovine brain tissues were homogenized and continuously cultured in Vero cells with double antibodies. The medium was replaced at 2-d intervals and the state of cells was observed. RESULTS A 608 bp Nc5 gene band was detected by PCR amplification. After sequencing, the sequence of the sample shared 99.5% homology with GenBank (AF061249). Brain homogenates were continuously cultured in Vero cells for 34 d and N. caninum was found. The results of IFAT and Nc5 gene-based PCR detection were N. caninum-positive, and the parasite was tentatively named N. caninum China Yanbian strain. BABL/c mice were inoculated with the separated parasites and showed clinical symptoms of ataxia and limb paralysis after 12 d. Only 3 mice survived. The blood of dying mice and the hearts, livers, spleens, lungs, kidneys, and brains of dead mice were collected aseptically. The Nc5 gene-based PCR showed that N. caninum may exist in brains, livers, and spleen. Based on immunohistochemical observations, we showed that N. caninum tachyzoites existed in the brains and livers. CONCLUSION We have successfully isolated bovine-specific N. caninum strain from brain tissues of aborted cattle in the China Yanbian region. This isolated strain has a strong infectious ability towards BABL/c mice.