Recent reports from New Zealand indicate Neospora caninum has a possible role in causing abortions in sheep. Transmission of N. caninum via semen has been documented in cattle. This study aimed to investigate if horizontal transmission through semen was also possible in sheep. Initially, 6-month old crossbred ram lambs (n=32), seronegative to N. caninum, were divided into 4 equal groups. Group 1 remained uninoculated whilst the remainder were inoculated with N. caninum tachyzoites intravenously as follows: Group 2 - 50 tachyzoites; Group 3 - 10(3) tachyzoites; Group 4 - 10(7) tachyzoites. Semen samples were collected weekly for 8 weeks for the detection of N. caninum DNA and quantified using quantitative PCR (qPCR). Plasma collected 1 month post-inoculation was subjected to ELISA (IDEXX Chekit) and Western blot. At 2 weeks post-infection, three rams from Group 1 (uninoculated) and three rams from Group 4 (10(7)tachyzoites/ml) were mated with two groups of 16 ewes over two oestrus cycles. Ewe sera collected 1 and 2 months post-mating were tested for seroconversion by ELISA and Western blot. All experimentally infected rams seroconverted by 1 month with ELISA S/P% values ranging from 11% to 36.5% in Group 2, 12-39.5% in Group 3 and 40-81% in Group 4. However, none of the ewes mated with the experimentally infected rams seroconverted. For the Western blot, responses towards immunodominant antigens (IDAs) were observed in ram sera directed against proteins at 10, 17, 21, 25-29, 30, 31, 33 and 37 kDa. Rams in Group 2, 3 and 4 were noted to have at least 3 IDAs present. None of the ewes showed any of the 8 prominent IDAs except for the one at 21 kDa which was seen in 30 out of 32 ewes in both groups. N. caninum DNA was detected intermittently in the ram's semen up to 5 weeks post-inoculation with the concentrations ranging from that equivalent to 1-889 tachyzoites per ml of semen. Low concentrations of N. caninum DNA were also detected in the brain tissue of two rams (Groups 1 and 4). These results suggest that although N. caninum DNA can be found in the semen of experimentally infected rams, the transmission of N. caninum via natural mating is an unlikely event.
To determine if toltrazuril was effective in eliminating Neospora caninum infection from congenitally infected lambs. Twenty-eight ewes were allocated to 3 groups where animals in Groups A and B were inoculated with 1 × 10(7)N. caninum tachyzoites on Day 120 of gestation and Group C was maintained as a negative control group. Lambs born from ewes in Group A were treated with toltrazuril (20mg/kg) on Days 0, 7, 14 and 21 after birth. Lambs in Groups B and C were untreated. All lambs in Groups A and B were seropositive at 12 weeks of age. At 12 weeks of age, no differences between lambs in Group A and Group B were observed in serological results (ELISA and western blot), presence of N. caninum-related brain histopathological lesions or the number of organisms detected by qPCR. Group C remained negative for serology, detection of N. caninum DNA as well as histopathology throughout the study. Results indicate that N. caninum congenitally-infected lambs had a continuing infection with N. caninum despite being treated with toltrazuril.
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.