title: Isolation and molecular characterisation of Neospora caninum in cattle in New Zealand.
abstract: OBJECTIVE
  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%.
  
  
  CONCLUSIONS
  This was the first successful isolation of N. caninum in New Zealand confirmed using molecular characterisation tests.
authors.affiliation: Institute of Veterinary Animal and Biomedical Sciences, Massey University, Private Bag 11222, Palmerston North, New Zealand.
authors.forename: C M
authors.initials: CM
authors.lastname: Okeoma
authors.forename: N B
authors.initials: NB
authors.lastname: Williamson
authors.forename: W E
authors.initials: WE
authors.lastname: Pomroy
authors.forename: K M
authors.initials: KM
authors.lastname: Stowell
authors.forename: L M
authors.initials: LM
authors.lastname: Gillespie