Evaluation of the embryo transfer procedure proposed by the International Embryo Transfer Society as a method of controlling vertical transmission of Neospora caninum in cattle

OBJECTIVE: To evaluate efficacy of embryo transfer into seronegative recipients, using the procedure proposed by the International Embryo Transfer Society (IETS), for preventing vertical transmission of Neospora caninum in cattle. DESIGN: Prospective clinical trial. ANIMALS: 87 recipient cows and heifers and their embryo transfer calves from 22 donors originating from 9 dairy herds. PROCEDURE: Neospora caninum serologic status of donors and recipients was determined before collection and transfer of embryos. Viable embryos were washed and treated with trypsin. Recipients in experimental groups A (n = 50) and B (29) were seronegative and received embryos from seropositive and seronegative donors, respectively. Recipients in group C (n = 8) were seropositive and received embryos from seronegative or seropositive donors. Antibody titers against N caninum were determined monthly during pregnancy in recipients and in calf blood samples collected at birth. Tissues collected from stillborn calves and aborted fetuses were analyzed histologically and by immunohistochemical (IHC) methods. RESULTS: 76 calves and 11 fetuses and stillborn calves were examined. All calves from groups A and B were seronegative (n = 70) or lacked evidence of infection by use of tissue analysis (9). In group C, 5 of 6 calves were seropositive at birth, and IHC results were positive for 1 of 2 calves. Vertical transmission rate was significantly lower in groups A and B (0%) than in group C (75%). CONCLUSION AND CLINICAL RELEVANCE: Embryo transfer into seronegative recipients, using the procedure proposed by IETS, is an effective way to prevent vertical transmission of N caninum. Results provide support for pretransfer testing of all embryo transfer recipients.     

Rare detection of Neospora caninum in placentas from seropositive dams giving birth to full-term calves.

Neospora caninum is thought to be transmitted to cattle by dogs, the only known definitive host. Although aborted fetuses seem the most likely source of infective material for dogs, placentas from seropositive dams appear also as a potential source of infective material. The objective of the study was to evaluate the presence of N. caninum organisms in placentas of full-term calves born to seropositive cows. Sixteen placentas, 11 from Neospora-seropositive cows, were examined histologically and by immunohistochemistry and polymerase chain reaction (PCR) assay for the presence of N. caninum. Mild placentitis was observed in all placentas. Neospora caninum was not identified by immunohistochemistry, but placentas from 2 seropositive dams were positive for N. caninum by PCR. These results suggest that placentas of full-term calves from seropositive cows may be a potential source of N. caninum for dogs, but the incidence of this mode of transmission is likely to be low.     

In vitro isolation and characterization of bovine Neospora caninum in Korea

The brains of nine aborted bovine fetuses and two newborn calves born from dams suspected to be infected with Neospora caninum were homogenized and inoculated into Vero cells. All fetuses and calves were from cows determined as seropositive to N. caninum by an IFA test. Sera and thoracic fluids of all fetuses and calves also revealed high antibody titer to N. caninum by IFAT ranging from 1:800 to 1:3200. N. caninum was isolated from the brains of one aborted fetus and one newborn calf when the brain homogenates were grown continuously in Vero cell culture. N. caninum tachyzoites, giemsa-positive, were first observed on Days 45 and 56 postinoculation in the newborn calf and the aborted fetus, respectively. The isolates (KBA-1 and KBA-2) were morphologically and ultrastructurally similar to previously published Neospora isolates. The isolated parasites were confirmed as N. caninum by means of the antigenic reactivities, immunostaining, PCR and southern blotting, and electron microscopy.     

Congenital Neospora caninum infection in a calf with spinal cord anomaly

Neospora caninum was identified in a calf with spinal cord anomaly in Australia. The calf was full-term and born dead. The caudal cervical and cranial thoracic segments of the spinal cord of the calf were asymmetric because of marked unilateral reduction of ventral gray matter and focal cavitation. Mild focal disseminated nonsuppurative encephalomyelitis was associated with N caninum tissue cysts. Tissue cysts were 16 to 35 microns X 10 to 27 microns, and the cyst walls were 1 to 3 microns thick. In an immunohistochemical test, the parasite stained with N caninum serum but not T gondii serum.     

In vitro isolation of Neospora caninum from a stillborn calf in the UK.

Neospora caninum was isolated in Vero cell culture from the brain of a stillborn calf. This isolate (designated NC-LivB1) is the first to be obtained from cattle in the United Kingdom and was confirmed as N. caninum by immunofluorescence with specific antibodies and by internal transcribed spacer 1 (ITS1) sequence analysis. Differences were found between NC-LivB1, other bovine isolates and canine isolates of N. caninum and closely related protozoal parasites, using random amplified polymorphic DNA polymerase chain reaction (RAPD - PCR) techniques.     

Serological responses to Neospora caninum in experimentally and naturally infected water buffaloes (Bubalus bubalis)

The water buffalo (Bubalus bubalis) is an important natural host for Neospora caninum. Serologic responses to N. caninum were studied in experimentally and naturally infected water buffaloes in Brazil. Antibodies were assayed by the indirect fluorescent antibody test (IFAT) using a cut off value of 1:25. Six buffaloes were each inoculated subcutaneously with 5 x 10(6) live culture-derived tachyzoites of the cattle Illinois strain of N. caninum, and two calves were kept as uninoculated controls. Post-inoculation (p.i.) blood samples were collected weekly for 8 weeks and then monthly until 1 year p.i. All inoculated buffaloes developed IFAT titers of 1:100 or more between 7 and 11 days p.i. and the titers remained elevated until 7 weeks p.i. Antibody titers peaked to 1:1600 in 1, 1:800 in 3 and 1:400 in 2, usually by 3 weeks p.i. Antibody titers declined to 1:25 or 1:50 in all the six buffaloes by 12 months p.i. IFAT titers to N. caninum remained at an undetectable level (< 1:25) in both control uninoculated buffaloes. To follow the dynamics of N. caninum antibodies, sera from 29 buffaloes and their calves were collected for 1 year and assayed for N. caninum antibodies; 23 of 29 calves were seropositive (IFAT of 1:100 or more) at 1-2 day of age. Of these 23 calves, 17 remained seropositive during the study, while six became seronegative at four (two calves), six (one calf) seven (two calves) and eight (one calf) months of age. These findings suggest a high rate of neonatal transmission of N. caninum in buffaloes.     

Frequency of detectable serum IgG concentrations in precolostral calves

OBJECTIVE: To determine the prevalence of detectable serum IgG concentrations in calves prior to ingestion of colostrum and to assess whether a detectable IgG concentration was related to dam parity, calf birth weight, calf sex, season of calving, or infectious agents that can be transmitted transplacentally. ANIMALS: 170 Holstein dairy calves. PROCEDURES: Serum samples were obtained from calves prior to ingestion of colostrum, and serologic testing for bovine viral diarrhea virus (BVDV) and Neospora caninum was performed. Relative risk, attributable risk, population attributable risk, and population attributable fraction for calves with a detectable serum IgG concentration attributable to positive results for N caninum and BVDV serologic testing were calculated. Logistic regression analysis was used to determine whether dam parity, calf sex, season of calving, and calf weight were associated with precolostral IgG concentration. RESULTS: 90 (52.9%) calves had a detectable total serum IgG concentration (IgG >or= 16 mg/dL). Relative risk, attributable risk, population attributable risk, and population attributable fraction for calves with a detectable serum IgG concentration attributable to positive results for N caninum serologic testing were 1.66, 0.34, 0.014, and 0.03, respectively. Calf sex, calf birth weight, and season of calving were not significant predictors for detection of serum IgG in precolostral samples. CONCLUSIONS AND CLINICAL RELEVANCE: Prevalence of IgG concentrations in precolostral serum samples was higher than reported elsewhere. There was no apparent link between serum antibodies against common infectious agents that can be transmitted transplacentally and detection of measurable serum IgG concentrations.     

Association of serologic status for Neospora caninum with postweaning weight gain and carcass measurements in beef calves

OBJECTIVE: To determine the seroprevalence for Neospora caninum in a population of beef calves in a feedlot and the association of serologic status with postweaning weight gain and carcass measurements. DESIGN: Longitudinal observational study. ANIMALS: 1,009 weaned beef steers from 92 herds. PROCEDURE: Samples were obtained from all steers at time of arrival at a feedlot. Serologic status for Neospora spp was determined, using an agglutination test. Results of serologic testing were compared with calf growth and carcass data, using multivariate regression with generalized estimating equations. RESULTS: Of 1,009 calves, 131 (12.98%) were seropositive, and 54 of 92 (58.7%) consignments had > or = 1 seropositive calf. Median within-consignment prevalence for consignments in which there was > or = 1 seropositive calf was 20%. Seropositive status was associated with significant reductions in average daily gain, live body weight at slaughter, and hot carcass weight and an increase in ribeye area-to-hot carcass weight ratio. Seropositive status also was associated with significant increases in cost of treatment and significant reductions in income. Sick seropositive calves had the highest cost of treatment. An economic loss of $15.62/calf was projected for seropositive calves. CONCLUSIONS AND CLINICAL RELEVANCE: Significant reductions in postweaning weight gain, carcass weight, and economic return were associated with detection of antibodies to N caninum in beef calves in a feedlot.     

Ranch-management factors associated with antibody seropositivity for Neospora caninum in consignments of beef calves in Texas, USA

A study was conducted with a 1998 retained-ownership population of Texas (USA) beef calves to determine the ranch-management practices associated with calf seroprevalence to Neospora caninum. Management practices of 76 Texas ranches that consigned 760 calves to a retained-ownership feedlot program were reviewed from a mailed questionnaire. Ninety-nine of 760 (13%; 95% CI, 9.4%, 17.7%) calves were positive to N. caninum and 59% of the ranches consigned at least one positive calf. In the logistic multiple-regression model which controlled for overdispersion, increased odds of calf-level seropositivity was associated with seasonal calving patterns, with stocking>1cow/calfunit/2.2ha, using a round-bale feeder, allowing wildlife access to the weaning supplement, and self-reared replacement heifers. However, decreased odds of seropositivity was associated with using a cattle-working dog and with using a self-contained cattle feeder. There was substantial overdispersion due to ranch.     

Experimental studies on the transmission of Neospora caninum between cattle

Three studies were conducted to investigate the transmission of Neospora caninum between cattle by the oral route. In the first study, six calves were dosed with 10(7)N caninum tachyzoites (NC LivB1) in colostrum and/or milk replacer on four occasions. In the second study, two calves and two cows were fed placental tissues from N caninum -infected cows, and, in the third study, seven uninfected calves were fostered onto N caninum -infected dams. In the first study, all six calves developed antibody responses and five calves developed antigen-specific lymphoproliferation responses, including two calves initially challenged at 1 week of age. No evidence of N caninum infection was found in the brain or heart of these calves by histology or polymerase chain reaction (PCR). In the second and third studies, there was no evidence of N caninum infection in any of the calves and cows. The results confirm that calves up to 1 week of age can be experimentally infected via the oral route, but suggest that this is not an important natural route of transmission for N caninum between cattle.     

Vaccination of ewes for prevention of vertical transmission of Neospora caninum

OBJECTIVE: To evaluate the immunologic response of a killed tachyzoite vaccine against Neospora caninum and its effectiveness in preventing vertical transmission of N caninum in sheep. ANIMALS: 40 Dorset ewes seronegative for N caninum. PROCEDURE: Group-A ewes (n = 20) were vaccinated on days 1 and 126 with a killed N caninum tachyzoite preparation in a commercially available adjuvant. Group-B ewes (n = 20) were sham vaccinated. Blood samples were collected from ewes every 2 weeks and a recombinant ELISA (rELISA) was used to determine serum antibody titers against N caninum. During pregnancy, ewes were challenged with live N caninum tachyzoites. Precolostral serum was collected from lambs and tested for antibodies against N caninum by use of an indirect fluorescence antibody test and the rELISA. Tissue specimens from stillborn lambs or lambs that died within 2 weeks of birth were collected and examined for N caninum antigen and DNA by use of immunohistochemistry and polymerase chain reaction assay, respectively. RESULTS: Serum antibody titers against N caninum were significantly higher in group-A ewes, compared with group B ewes, following vaccination. Serum antibodies against N caninum were detected in 100% (33/33) of group-B lambs and 75% (18/24) of group-A lambs. In tissue specimens, N caninum DNA was detected in 9 of 11 group-B lambs and 0 of 10 group-A lambs. Histologically, N caninum tachyzoites were observed in 4 group-A lambs and 3 group-B lambs. CONCLUSIONS AND CLINICAL RELEVANCE: The killed tachyzoite vaccine against N caninum stimulated a humoral immune response in sheep and provided partial protection against vertical transmission.     

Mannosidosis in Galloway calves

Mannosidosis was diagnosed in four stillborn Galloway calves and an autolyzed full-term fetus from experimental matings of carrier animals. Gross lesions were moderate internal hydrocephalus, and pallor and enlargement of the liver and kidneys and arthrogryposis. Histologic changes in the central nervous system of each calf were marked foamy vacuolation of the cytoplasm of neurones in the cerebral cortex, thalamus and brainstem, and vacuolation of the Purkinje cells of the cerebellum. Spheroids were common throughout the brain and there was also consistent severe foamy cytoplasmic vacuolation of renal tubular epithelial cells and hepatocytes. The activities of alpha-mannosidase, the lysosomal enzyme whose activity is deficient in mannosidosis, and activities of five other lysosomal enzymes were compared in brain, liver, and kidney tissues of three mannosidosis-affected calves and normal calf tissues. Tissues from the affected calves had a marked deficiency of alpha-mannosidase activity compared with the normal tissues; the greatest deficiency was in the liver (99%) and brain (98%). Activities of the other lysosomal enzymes were elevated in the affected tissues compared with normal. Mannosidosis is a lysosomal storage disease that results from a defect in glycoprotein metabolism and affects man, Angus and Angus-related breeds of cattle, such as Murray greys, and the cat. The congenital disease is caused by an inherited deficiency of the lysosomal enzyme alpha-mannosidase, and is inherited in an autosomal recessive manner. Mannosidosis was recently reported in a number of aborted and stillborn Australian Galloway calves from an experimental breeding trial. This is more detailed account of the histological and biochemical results obtained during the trial.     

Determination of the seroprevalence of Neospora caninum in feedlot steers in Alberta

A study was conducted in Alberta to determine the seroprevalence of Neospora caninum in feedlot calves purchased from various auction markets throughout western Canada. Four feedlots (1 feedlot from each of the Airdrie and High River areas and 2 feedlots from the Strathmore area) were selected for sampling. At each feedlot, a random 10% sample of feedlot steer and bull calves entering the feedlot from September 2001 to December 2001 were enrolled in the study until there were a maximum of 500 animals enrolled per feedlot. Blood samples were collected from 1976 male animals at the time of entry to the 4 study feedlots. The animals represented 375 groups purchased from 70 sale points throughout British Columbia, Alberta, Saskatchewan, and Manitoba. Of the 1976 animals tested, 128 animals tested positive for antibodies to N. caninum. The prevalence and adjusted 95% confidence limits for N. caninum in beef calves on entrance to the feedlot in western Canada were 6.5% (95% CI, 5.1 to 8.2). There were no significant (P > or = 0.05) associations between the risk of treatment, the risk of designation as "chronic," and the risk of death and antibodies to N. caninum either before or after adjusting for feedlot, entry weight, entry date, and clustering of disease within lots at each feedlot. In addition, there was no significant (P > or = 0.05) association between serological status and feedlot entry weight or average daily gain. Note that there was no information available on feed conversion because the calves were mixed within existing commercial feedlot pens and the actual feed intake of each animal could not be determined. Adjustment for the concentration of antibodies to bovine viral diarrhea virus on arrival did not change any of the examined associations between N. caninum status and calf health or performance. The results of this study demonstrated that the prevalence of N. caninum in feedlot calves in western Canada was less than the prevalence reported in the United States. Additional studies are required to determine whether the inferior rate of gain and feed efficiency observed in the southern United States with animals testing positive for antibodies to N. caninum also occurs under the management conditions used in western Canada.     

Economic considerations for diagnostic and control options for Neospora caninum-induced abortions in endemically infected herds of beef cattle

OBJECTIVE: To compare economic outcome for herds not exposed to Neospora caninum with that for herds with various seroprevalences of N caninum infection and evaluate 3 control strategies. DESIGN: Economic simulation model. SAMPLE POPULATION: Beef herds with various seroprevalences of N caninum infection. PROCEDURE: A 5-year simulation model was used. Control strategies that were evaluated included culling females that fail to calve, selling seropositive females and purchasing seronegative replacements, and excluding the daughters of seropositive dams as potential replacements. RESULTS: For a 5-year period with low prices for feeder calves, endemic N caninum infection decreased mean return to fixed assets by 22.2% when true seroprevalence was 10% and by 29.9% when true seroprevalence was 70%. Percentage decrease in return to fixed assets was less dramatic when a 5-year period with high prices for feeder calves was evaluated. Analysis indicated that 2 control strategies (culling females that fail to give birth to a calf and selling seropositive female cattle and purchasing seronegative replacement female cattle) were not likely to be economically beneficial. The third control strategy (testing the entire herd for N caninum infection and excluding the female offspring of seropositive dams as replacements) appeared to be a reasonable control strategy. CONCLUSIONS AND CLINICAL RELEVANCE: For the assumptions in the model, endemic N caninum infection decreases return to fixed assets for cow-calf herds. Of the potential control strategies evaluated, testing the entire herd for N caninum infection and excluding the daughters of seropositive dams as potential replacements provided the best economic return.)     

Immune response to Neospora caninum native antigens formulated with immune stimulating complexes in calves

The aim of this study was to compare the immune responses to live Neospora caninum tachyzoites and N. caninum native antigens formulated with immune stimulating complexes matrix (ISCOM-matrix) in calves. Fifteen calves were used in this study: 3 were intravenously inoculated with 1 × 10(8) live tachyzoites (Group A), 3 were inoculated twice with N. caninum native antigens formulated with ISCOMs (Group B); 3 with N. caninum native antigens in phosphate-buffered saline (PBS) (Group C); 3 received ISCOM-matrix (ISCOMs without antigen) (Group D) and 3 were negative controls receiving PBS (Group E). The last four groups were inoculated subcutaneously. The specific total IgG and its subtypes were analyzed by an indirect enzyme-linked immunosorbent assays (ELISAs) and by Western blot. IFN-γ levels in plasma was quantified using a commercial kit. All calves were challenged intravenously with 1 × 10(8) live tachyzoites at week 11 after receiving the first dose. Parasitemia was assessed in plasma samples by semi-nested PCR. Neospora-specific antibodies were detected in animals from Groups A and B in the week 2 after inoculation. The ELISA OD values were higher in Group B compared with Group A from weeks 6 to 11 (P<0.05). Analysis of the subisotype specific antibodies in experimentally infected calves revealed a predominant IgG(2) response; however, a predominant IgG(1) response was observed in animals inoculated with N. caninum native antigens formulated with ISCOM-matrix. Control calves remained seronegative until challenge infection. The pattern of bands by Western blot was similar when testing sera from animals in Groups A and B. The levels of IFN-γ production after respective immunization schedules were similar between Groups A and B. Neospora-DNA was detected in plasma samples shortly after intravenous challenge in calves from all groups including those receiving the experimental vaccine formulation. The duration of the parasitemia was similar in all groups.     

Prevalence of Neospora caninum and persistent infection with bovine viral diarrhea virus in dairy-breed steers in a feedlot

OBJECTIVE: To determine the prevalence and effect of Neospora caninum infection and persistent infection (PI) with bovine viral diarrhea virus (BVDV) on weight gain, morbidity, and mortality rate in dairy-breed steer calves located on a feedlot in California. DESIGN: Prospective cohort observational study. ANIMALS: 900 dairy-breed steer calves in 2 pens. PROCEDURES: The 3- to 4-month-old calves were evaluated for serum antibodies against N caninum and infection with BVDV at entry to the feedlot. Five months later, sera were again analyzed for anti-N caninum antibodies; calves that were determined to have BVDV infection initially were retested to evaluate PI status. Average daily gain, morbidity, and deaths were recorded for all calves. RESULTS: Among 900 calves, prevalence of N caninum infection was 16.7% (95% confidence interval, 14.3% to 19.3%); prevalence of BVDV-associated PI was 0.2% (95% confidence interval, 0.03% to 0.9%). Morbidity rate and time to first illness were not significantly different between calves that were seropositive or seronegative for N caninum. At the second sample collection, weight and average daily gain of calves that were seropositive for N caninum was less than that of seronegative steers in 1 pen, whereas these measures did not differ between groups in the other pen. Statistical power was insufficient to evaluate the effect of BVDV PI on any outcome measurement. CONCLUSIONS AND CLINICAL RELEVANCE: Although N caninum serostatus had no significant effect on morbidity rate, some seropositive calves had reduced growth, compared with seronegative calves, 5 months after entry to the feedlot.     

Neospora caninum: oocyst challenge of pregnant cows

Three pregnant cows were each orally challenged at 10 weeks of gestation with 600 sporulated oocysts of Neospora caninum. The number of oocysts was limited by those available. In concurrent bioassays, one oocyst per os infected each of two gerbils. Challenged cattle developed Neospora-specific antibody, cell proliferation and gamma-interferon responses. N. caninum specific PCR demonstrated persisting infection in the brains of cows 4 months after calving. Abortion was not induced and there was no evidence of transplacental infection in the healthy calves born at full-term. This experiment suggests that the dose threshold for induction of abortion exceeds 600 oocysts.     

Epidemiology and impact of Neospora caninum infection in three Queensland tropical dairy herds

This study investigated the epidemiology of Neospora caninum in three tropical dairy herds in North Queensland, Australia. All animals in the herds were bled, and the sera were tested by ELISA for N. caninum antibodies. Herd records were examined, and the number of calves carried to term and the number of abortions which occurred over the lifetime of each animal were recorded to determine the abortion rate for each animal. Pedigrees were constructed for two of the herds to investigate whether vertical transmission was occurring. The seroprevalence of N. caninum ranged from 23% to 34%. The abortion rate in seropositive animals was significantly (p < 0.001) higher than in seronegative animals in all three herds (12-20.1% cf. 3.6-7%). Overall, the probability of a calf being seropositive was 3.5 times higher when the dam was also seropositive than when the dam was seronegative. Subsequent selective breeding employed by one herd reduced the N. caninum seroprevalence from 23% to 5% over a 9-year period. This study shows that N. caninum infection is prevalent in North Queensland dairy cattle, and both post-natal infection and vertical transmission are common.     

Abortion epidemic in a dairy herd associated with horizontally transmitted Neospora caninum infection

A dairy herd experienced an abortion epidemic during which 43 per cent of the cows at risk aborted. Neospora caninum infection was demonstrated in four of six fetuses suitable for examination and the group of at-risk cows that aborted had significantly higher N. caninum antibody concentrations than the at-risk cows that delivered a live calf at term (P<0.001). The antibody concentrations in the cow herd were significantly higher than in the youngstock (P<0.001), and the concentrations in the youngstock increased significantly (P<0.001) with age. When seven months to a year old, the calves born at term to the at-risk cows had significantly higher (P=0.007) antibody concentrations than age-matched calves born before the epidemic. At the time of the epidemic, there was a significant increase in the antibody levels of the herd that was not consistent with vertical infection alone, indicating that there appeared to have been a sudden large increase in the incidence of horizontal postnatal transmission of N. caninum to the cow herd, or to the surviving offspring of the at-risk cows, or to both of these groups.