Flourescent antibody test (FAT) was applied to determine the cross-reactivities of monoclonal (mAb), polyclonal (pAb) antibodies to Neospora, Toxoplasma and Cryptosporidium and antisera from cattle naturally infected with Neospora canium against antigens from a number of sources. Both mAb and pAb to Neospora reacted strongly (FAT titre up to 2560) with the homologous antigens and demonstrated weak titre (80) or no reaction with both Toxoplasma and Cryptosporidium antigens. Also mAb and pAb to Toxoplasma gondii reacted at titres of 80 - 640 with homologous antigens and at titres of 10-40 with N. caninum. No cross-reactions with either mAb or pAb antibodies to N. caninum and T. gondii were observed with Cryptosporidium parvum. The same results were observed with C. parvum mAb when tested with both N. caninum and T. gondii antigens. Sera from cattle naturally infected with N. caninum had titres ranging from 80- 640 with N. caninum antigens, and 10- 40 with T. gondii and C. parvum antigens. At low dilutions, the complete surfaces of Neospora and Toxoplasma parasites were fluorescent, while in higher dilutions only dotted fluorescence appeared on the apical complex. These results indicated the presence of cross-reactivity between Neospora and Toxoplasma but not with Cryptosporidium. Accordingly the recommended cut-off antibody titre for diagnosis of neosporosis is 80.
In order to attempt isolate the protozoan parasite Neospora caninum, an N. caninum seropositive pregnant Sahiwal Friesian cross heifer from a large-scale dairy farm in Malaysia was kept for observation until parturition at the Veterinary Research Institute, Ipoh. The heifer gave birth to a female calf that was weak, underweight and unable to rise. Precolostral serum from the calf had an N. caninum indirect fluorescent antibody test titre of 1:3200. It died 12 h after birth and necropsy was performed. Brain homogenate from the calf was inoculated into 10 BALB/c mice that were kept for 3 months after which brain tissue from the mice was inoculated onto 24 h fresh monolayer Vero cell lines. The cell cultures were examined daily until growth of intracellular protozoa was observed. DNA of the organisms from the cell cultures was analyzed by PCR and DNA sequencing. DNA fragments of the expected size were amplified from the isolate using N. caninum-specific primers, and sequence analysis of ITS1 clearly identified the isolate as N. caninum. This is the first successful isolation of N. caninum from a bovine in Malaysia, and the isolate is designated Nc-MalB1.