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

Abstract

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 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 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.

KEY WORDS:

• Neospora caninum
• cow
• calf
• brain
• isolation
• Vero cells
• immunofluorescent antibody test
• immunohistochemistry
• polymerase chain reaction
• DNA sequencing
• transmission electron microscopy

Acknowledgements

The authors are grateful for the cooperation of all technicians involved in these studies. The corresponding author is the recipient of a New Zealand Overseas Development Assistance (NZODA) scholarship. Professor John Ellis of the University of Technology Sydney provided the Nc-Liverpool culture (courtesy of Professor AJ Trees, University of Liverpool), used as a control organism in these studies. The Global Fund of the New Zealand Dairy Board funded this work.