Isolation and molecular characterisation of Neospora caninumin cattle in New Zealand

AbstractAIM: To isolate Neospora caninumfrom the brains of naturally infected cattle and use molecular techniques to characterise the isolates.METHODS: Neospora caninumtachyzoites 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 anti-body 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 caninumtachyzoites were isolated from all three cases and first observed in vitrobetween 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. caninumin 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. caninumin New Zealand confirmed using molecular characterisation tests.