Serum antibody profile and reproductive performance during two consecutive pregnancies of cows naturally infected with Neospora caninum.

The objective of this study was to record how the antibody levels change over time during pregnancy in dairy cows naturally infected with the protozoan parasite Neospora caninum, and relate this to the reproductive performance. Eighteen cows with antibodies to N. caninum were serum sampled monthly during their first pregnancy and 13 of them were also followed for a second pregnancy. In all, five pregnancies ended in abortion and two in stillbirth. Antibodies to N. caninum in serum were demonstrated by immune stimulating complex enzyme-linked immunosorbent assay (iscom ELISA). The N. caninum antibody titres remained well above the 1:100 cut-off limit for the test used during 2 years in all cows. In the non-aborting cows, mean values of antibody titres to N. caninum rose 1.5-2.5 dilution steps to reach a plateau 4-5 months before parturition, and thereafter decreased from 2 months before parturition. These changes were statistically significant (p < 0.001). The same pattern was seen in the aborting cows. The consistent pattern of rise in antibody titres observed during both pregnancies in all cows indicated a reactivation rather than a reinfection of the parasite at mid-gestation.

An IgG avidity ELISA to discriminate between recent and chronic Neospora caninum infection.

The avidity of IgG antibodies directed to Neospora caninum was measured using an IgG avidity enzyme-linked immunosorbent assay (ELISA) employing N. caninum proteins incorporated into immunostimulating complexes as antigen. In this ELISA, low-affinity antibodies were eluted by adding an incubation step with urea after the serum incubation. The antibody titers obtained with and without incubation with urea were then used to calculate the IgG avidity values. Analysis of sequential sera collected from experimentally infected calves revealed that the avidity increased during the course of infection. Three weeks after infection, the IgG avidity was 9-18%, and 24 weeks later it had increased to 58-76%. Cattle naturally infected for more than 6 months all had avidities >50%. The results in this study, however preliminary, indicate that the IgG avidity ELISA can be used to discriminate between recent and chronic N. caninum infections and may therefore be a valuable complement to IgG assays in epidemiologic studies of N. caninum infection in cattle.

Oral Neospora caninum inoculation of neonatal calves.

Four calves born to cows seronegative for Neospora caninum were dosed orally within 6 h after birth with tachyzoites of the bovine N. caninum Nc-SweB1 isolate added to colostrum. Two of the calves were dosed via stomach tube and two by feeding bottle. The latter two calves showed transient fever and passed blood-stained diarrhoea 1-2 weeks after inoculation. From 5 weeks after inoculation they developed a significant antibody response which remained high until the calves were euthanised and necropsied at 15 and 19 weeks after inoculation, respectively. The two calves inoculated by stomach tube showed no clinical signs and they remained seronegative throughout the study. At necropsy of the seropositive calves, no pathological lesions were seen, and parasites were not detected by immunohistochemistry. Neospora caninum was not re-isolated in cell culture from the brains of the seropositive calves; however, N. caninum DNA was detected in brain from both of them by PCR. The data suggest that oral infection of N. caninum via colostrum might be a possible route of vertical transmission in newborn calves, in addition to transplacental infection.

Characterization of a Swedish bovine isolate of Neospora caninum.

The brain of a stillborn calf, seropositive to Neospora caninum and born to a seropositive cow, was homogenized and cultured on Vero cells, where growth of Neospora-like tachyzoites was detected after 8 weeks. The ultrastructural features of the new isolate (Nc-SweB1) corresponded to those of previously published Neospora isolates. In indirect immunofluorescence tests, antigens on Nc-SweB1 tachyzoites were recognized by antibodies raised to a canine N. caninum isolate (Nc-1) but not by antibodies to Toxoplasma gondii, Sarcocystis cruzi, S. tenella, Eimeria alabamensis, Babesia divergens, or B. motasi. Immunoblot analyses revealed no major antigenic difference between Nc-SweB1 and Nc-1, whereas several differences were seen between Nc-SweB1 and protozoa related to N. caninum. The sequences of 16S-like rRNA and the internal transcribed spacer 1 of Nc-SweB1 revealed complete homology with corresponding sequences of two canine N. caninum isolates. Thus, no dissimilarity between Nc-SweB1 and the canine isolates was found, confirming that Nc-SweB1 is N. caninum and suggesting that Neospora-like organisms isolated from cattle are indeed N. caninum.

Neospora species infection in a herd of dairy cattle.

OBJECTIVE: To investigate the route of transmission of Neospora sp in a herd of dairy cattle in which sporadic abortions had been observed since the establishment of the farm in 1980. DESIGN: Serum samples were screened for antibodies to Neospora sp, and records from an artificial insemination program were analyzed. ANIMALS: 58 female cattle. PROCEDURE: An ELISA was used to screen serum samples of antibodies to Neospora sp. Fertility, calf mortality, and relationships between specific cattle were investigated. Statistical analysis was performed on the fertility data. RESULTS: Antibodies were detected in 17 of 58 (29%) tested cattle. All seropositive cattle were descendants of 2 cows purchased in 1980. Cattle that were descendants of those 2 cows were compared with their herdmates, but significant differences were not detected in the number of inseminations per confirmed pregnancy or in the number of cattle that required more than 1 insemination/ pregnancy. Since 1980, there were 323 confirmed pregnancies in the herd, and calf mortality (prenatal and perinatal mortality) was 24 of 323 (7%). CLINICAL IMPLICATIONS: Congenital transmission of Neospora organisms together with the apparent lack of horizontal transmission observed in the herd reported here indicated that Neospora sp has the ability to be transmitted from dam to offspring for several generations. This mode of transmission would explain the maintenance of infection in a population of cattle despite the lack of a definitive host for the parasite.