Isolation and molecular characterisation of Neospora caninum in cattle in New Zealand

TitleIsolation and molecular characterisation of Neospora caninum in cattle in New Zealand
Publication TypeJournal Article
Year of Publication2004
AuthorsOkeoma, C.M., Williamson N.B., Pomroy W.E., Stowell K.M., and Gillespie L.M.
JournalNew Zealand Veterinary Journal
Pagination364 - 370
Date Published2004
ISBN Number00480169 (ISSN)
Keywordsanimal cell, animal health, Animalia, article, Bos taurus, Brain, brain slice, Calf, cattle, cell culture, cell ultrastructure, controlled study, correlation analysis, cow, DNA sequence, DNA sequencing, histopathology, immunofluorescence test, Immunofluorescent antibody test, immunohistochemistry, Isolation, molecular biology, mortality, Neospora caninum, nonhuman, parasite isolation, parasitosis, pathogenicity, polymerase chain reaction, primer DNA, sequence analysis, statistical analysis, technique, Transmission electron microscopy, Vero cell, Vero cells, veterinary medicine

AIM: To isolate Neospora caninum from the brains of naturally infected cattle and use molecular techniques to characterise the isolates. METHODS: Neospora caninum tachyzoites were isolated in Vero cell culture from the brains of a cow and two calves. The isolates were characterised using polymerase chain reaction (PCR) methods, DNA sequencing, an immunofluorescent antibody test (IFAT), transmission electron microscopy (TEM), and immunohistochemistry (IHC). The brains of the three cattle were subjected to histopathological examination. A pathogenicity study was conducted in 120 BALB/c mice. RESULTS: Neospora caninum tachyzoites were isolated from all three cases and first observed in vitro between 14 and 17 days post-inoculation. Parasites were sub-cultured and maintained in Vero cell culture for more than 6 months. PCR products were generated for all three isolates, using two different primers. Sequencing of the PCR products and a subsequent BLAST search identified the isolates as N. caninum. In addition, the isolates tested positive using IFAT and IHC, and ultrastructure revealed by TEM was characteristic of N. caninum. Histopathological examination revealed lesions characteristic of N. caninum in 1/3 brains. In the pathogenicity study using BALB/c mice, the mortality rate was 3-7%. CONCLUSION: This was the first successful isolation of N. caninum in New Zealand confirmed using molecular characterisation tests.

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