Development of maternal and foetal immune responses in cattle following experimental challenge with Neospora caninum at day 210 of gestation

This study examined the immunological responses of pregnant cattle and their foetuses following an experimental challenge with live Neospora caninum tachyzoites at day 210 of gestation. Animals were bled prior to and weekly throughout the experiment and sacrificed at 14, 28, 42 and 56 days post inoculation (dpi). At post mortem examination, samples of lymph nodes and spleen were collected from both dam and foetus for immunological analysis. Subcutaneous (sc) inoculation over the left prefemoral (LPF) lymph node of pregnant cattle at day 210 of gestation, led to the vertical transmission of parasites by 14 dpi, however no foetal deaths were observed in the infected animals. Foetuses from infected dams mounted Neospora-specific humoral and cell-mediated immune (CMI) responses by 14 dpi. These responses involved anti-Neospora IgG, antigen-specific lymphocyte proliferation, and the production of the cytokines IFN–γ, interleukin (IL)-4 and IL-10. There was also evidence of innate immunity during the response against Neospora from infected dams, with statistically significant (p < 0.05) increases in mean expression of toll like receptors (TLR)-2 on 56 dpi in maternal spleen, LPF, right prefemoral (RPF), left uterine (LUL) and right uterine (RUL) lymph nodes and TLR-9 in retropharyngeal (RLN), LPF and RPF lymph nodes from 28 dpi. Statistically significant (p < 0.05) increases in mean TLR-9 were detected in spleen samples from foetuses of infected dams, compared to the foetuses from control animals. Our results show that vertical transmission of the parasite occurred in all infected dams, with their foetuses showing effective Neospora-specific cell mediated, humoral and innate immune responses.     

Maternal and foetal immune responses of cattle following an experimental challenge with Neospora caninum at day 70 of gestation

The immune responses of pregnant cattle and their foetuses were examined following inoculation on day 70 of gestation either intravenously (iv) (group 1) or subcutaneously (sc) (group 2) with live NC1 strain tachyzoites or with Vero cells (control) (group 3). Peripheral blood mononuclear cell (PBMC) responses to Neospora antigen and foetal viability were assessed throughout the experiment. Two animals from each group were sacrificed at 14, 28, 42 and 56 days post inoculation (pi). At post mortem, maternal lymph nodes, spleen and PBMC and when possible foetal spleen, thymus and PBMC samples were collected for analysis. Inoculation with NC1 (iv and sc) lead to foetal deaths in all group 1 dams (6/6) and in 3/6 group 2 dams from day 28pi; statistically significant (p ≤ 0.05) increases in cell-mediated immune (CMI) responses including antigen-specific cell proliferation and IFN-γ production as well as increased levels of IL-4, IL-10 and IL-12 were observed in challenged dams compared to the group 3 animals. Lymph node samples from the group 2 animals carrying live foetuses showed greater levels of cellular proliferation as well as significantly (p ≤ 0.05) higher levels of IFN-γ compared to the dams in group 2 carrying dead foetuses. Foetal spleen, thymus and PBMC samples demonstrated cellular proliferation as well as IFN-γ, IL-4, IL-10 and IL-12 production following mitogenic stimulation with Con A from day 14pi (day 84 gestation) onwards. This study shows that the generation of robust peripheral and local maternal CMI responses (lymphoproliferation, IFN-γ) may inhibit the vertical transmission of the parasite.     

Induced transplacental transmission of Neospora caninum in cattle.

Three Jersey cows were inoculated SC and IM with 26 million Neospora caninum tachyzoites at 129 (cow 1), 126 (cow 2), and 81 (cow 3) days after mating. Cows remained clinically normal for at least 1 month after inoculation of N caninum. Cow 1 was euthanatized 32 days after inoculation because of gangrenous mastitis. Cow 1 had a live fetus with no gross lesions; however, microscopic lesions were seen in the fetus and consisted of severe nonsuppurative necrotizing encephalitis of the cerebral white matter. Neospora caninum was identified in lesions by staining with anti-N caninum serum in an immunohistochemical test, by bioassays in mice, and by inoculation of bovine monocyte cultures with fetal tissue homogenate. Neither N caninum nor lesions were associated with infection with the protozoon identified in tissues of cow 1. Cows 2 and 3 aborted small autolysed fetuses 101 and 74 days, respectively, after inoculation with N caninum; the fetuses and attached placenta were unsuitable for laboratory investigations. Cows 2 and 3 remained clinically normal 4 months after abortion. Results of this study indicated that N caninum can be transmitted transplacentally in cattle.     

Repeated transplacental transmission of Neospora caninum in dogs

Four litters of German Shorthaired Pointers from one owner developed a toxoplasmosis-like illness. According to the records, 29 of 39 dogs had hind limb paralysis. Six dogs from 2 litters were necropsied and had generalized encephalomyelitis. Tachyzoites and tissue cysts of Neospora caninum were found in the brain and spinal cord of each dog. Lesions were found in the eyes, extraocular muscles, or both in all of the dogs, and N caninum was detected microscopically in the eyes (retina and choroid in 1 dog), extraocular muscles, or both in 5 of the 6 dogs. Ocular lesions consisted of focal retinitis, choroiditis, mild nonspecific iridocyclitis, and myositis of extraocular muscles. Organisms stained with anti-N caninum serum, but not with anti-Toxoplasma gondii serum in an immunohistochemical test, except in 1 dog. In one dog, aged thick-walled N caninum tissue cysts reacted mildly with anti-T gondii serum.     

Natural postnatal Neospora caninum infection in cattle can persist and lead to endogenous transplacental infection

A serological follow-up study of 3.5 years duration was done of a dairy herd that had experienced a mass seroconversion to Neospora caninum following a point source exposure shortly before the 17th of January 2000. A total of 913 blood samples of 244 animals at seven sampling dates were used to investigate the seroprevalence dynamics in the herd. Most postnatally infected cattle remained seropositive during the period of investigation but 11 animals became seronegative after 6-27 months indicating transient infection. Six animals seroconverted later than the main group of 45 animals and 5 animals became seronegative after at least two seropositive records possibly due to a low infection dose or difference in the haplotypes of the infected animals. In total 58% (14/24) of the offspring of postnatally infected dams was seropositive. Nine of 16 (56%) daughters originating from inseminations after the postnatal infection of their dams were seropositive indicating endogenous transplacental infection.     

Phenotypic characterisation of the cellular immune infiltrate in placentas of cattle following experimental inoculation with Neospora caninum in late gestation

Despite Neospora caninum being a major cause of bovine abortion worldwide, its pathogenesis is not completely understood. Neospora infection stimulates host cell-mediated immune responses, which may be responsible for the placental damage leading to abortion. The aim of the current study was to characterize the placental immune response following an experimental inoculation of pregnant cattle with N. caninum tachyzoites at day 210 of gestation. Cows were culled at 14, 28, 42 and 56 days post inoculation (dpi). Placentomes were examined by immunohistochemistry using antibodies against macrophages, T-cell subsets (CD4, CD8 and γδ), NK cells and B cells. Macrophages were detected mainly at 14 days post inoculation. Inflammation was generally mild and mainly characterized by CD3⁺, CD4⁺ and γδ T-cells; whereas CD8⁺ and NK cells were less numerous. The immune cell repertoire observed in this study was similar to those seen in pregnant cattle challenged with N. caninum at early gestation. However, cellular infiltrates were less severe than those seen during first trimester Neospora infections. This may explain the milder clinical outcome observed when animals are infected late in gestation.     

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.     

Natural transmission routes of Neospora caninum between farm dogs and cattle

Twelve dairy herds with evidence of post-natal infection with Neospora caninum were compared with 21 control herds with no evidence of post-natal infection. On the former farms, dogs consumed placenta or licked uterine discharge in 75 and 67% of the farms, respectively, while on control farms these activities occurred in 38 and 24% of the farms, respectively. On all control farms and all but three post-natally infected farms the dogs were fed colostrum or milk. Defecation of dogs on the feeding alley was observed in 92% of the post-natally infected farms and in 24% of the control farms. The same trend was observed for defecation of dogs in grass silage, in 75% of the post-natally infected farms and in 19% of the control farms; and in corn silage, in 50% of the post-natally infected farms and in 10% of the control farms. Consumption of placenta, material of aborted foetuses or uterine discharge in combination with defecation on the feeding alley, storage of grass or corn silage was observed in 19% of the control farms and in 75% of the post-natally infected farms. This study supports the hypothesis that farm dogs may become infected by foetal fluids or placental material of infected cattle, and may subsequently cause a post-natal infection of cattle in the herd by shedding oocysts.     

Seroprevalence of Neospora caninum infection following an abortion outbreak in a dairy cattle herd

OBJECTIVE: To investigate the seroprevalence of Neospora caninum infection in a commercial dairy cattle herd, 15 months after detection of an abortion outbreak. PROCEDURE: Sera from the whole herd (n = 266) were examined for N caninum antibodies by indirect fluorescent antibody test (IFAT) and immunoblot analysis. Herd records were reviewed to collate serological results with abortion history, proximity to calving, and pedigree data. RESULTS: The seroprevalence of N caninum infection was 24% (63/266) for IFAT titre > or = 160, 29% (78/266) for immunoblot positive (+ve), and 31% (82/266) for IFAT > or = 160 and/or immunoblot +ve; 94% (59/63) of animals with IFAT > or = 160 were immunoblot +ve. The association between seropositivity (IFAT > or = 160 and/or immunoblot +ve) and history of abortion was highly significant (P < 0.001); the seroprevalence was 86% (18/21) in aborting cows, compared with 30% (50/164) in non-aborting animals. The abortion rate for seropositive cows was 26% (18/68) compared with 3% (3/117) for seronegative animals. IFAT titres of infected cows were higher within 2 months of calving than at other times (P < 0.001). The association between seropositivity in dams and daughters was highly significant (P = 0.009). CONCLUSIONS: The abortions were associated with N caninum infection and there was evidence of reactivation of latent infection close to calving and congenital transmission of infection. Immunodominant antigens identified by immunoblots may prove useful for improved diagnostic tests.     

Incidence and vertical transmission rate of Neospora caninum in sheep

The infection by Neospora caninum in sheep can lead to abortion and the birth of weak and debilitated lambs. The aim of this study was to assess the incidence of natural infection by Neospora caninum and the vertical transmission rate among sheep. A flock of 50 sheep was monitored for serum antibody titres against N. caninum and seroconversion over a period of six months using an indirect ELISA technique. The offspring of the herd was also investigated regarding anti-N. caninum antibodies to determine the vertical transmission rate through the indirect immunofluorescence technique. The initial and final prevalences of infection by N. caninum were 26.0% (13/50) and 72.0% (36/50), respectively, and the incidence of infection by N. caninum in the present study was 62.2% (23/37). The vertical transmission rate found was 15.4% (2/13). A high incidence of infection by N. caninum in sheep was observed, and this is the first report assessing the incidence of N. caninum among naturally infected sheep.     

Neonatal Neospora caninum infection in dogs: isolation of the causative agent and experimental transmission

Neospora caninum infection was diagnosed in 5 young dogs from 2 litters with a common parentage. The pups were born healthy, but developed hind limb paresis 5 to 8 weeks after birth. The predominant lesions were polyradiculoneuritis and granulomatous polymyositis. Neospora caninum was seen microscopically in sections of naturally infected pups, and was isolated in cell cultures, mice, and dogs inoculated with infected canine tissues. Antibodies to N caninum were detected in sera of infected dogs by indirect fluorescent antibody test.     

Neospora caninum infection and congenital transmission: serological and parasitological study of cows up to the fourth gestation

In this paper, a parasitological and serological study performed in three cows up to the fourth gestation is reported in order to clarify the extent of vertical propagation and, secondly, in which period of gestation the recrudescence of previous infection occurs. The cows selected for the study delivered healthy but congenitally infected calves in first pregnancy. The parasite was found, by biological tests in Swiss mice, in all the placentas of the three cows examined, during the three subsequent gestations, at calving. The parasite was found, at slaughtering, in the brains of all nine calves born clinically healthy from the three cows as well. The serological profile, performed at monthly intervals on serum of the cows, showed that IgG and IgM increased in the third trimester of gestation; this rise of antibodies was constantly observed during the three gestations and in all three cows. In the calves, the IgG titres increase after colostrum consumption and an IgM peak at birth, were indicative of a late infection. These findings, along with negative results obtained by a serological study conducted simultaneously on 38 cows housed in the same stable as the experimental animals and the negative results obtained in isolating parasite or antibodies from farm dogs, suggest that N. caninum infection can be maintained over several bovine generations and that recrudescing persistent infection, rather than a new infection, explains the Neospora infection of calves.     

Experimental infection with a low virulence isolate of Neospora caninum at 70 days gestation in cattle did not result in foetopathy

The Nc-Spain 1H isolate of Neospora caninum, which was newly obtained from the brain of a congenitally asymptomatic infected calf, demonstrated a reduced in vitro tachyzoite yield and viability rate, as well as low virulence in mouse models. The objective of the present study was to determine the ability of this isolate to induce foetal death in a pregnant bovine model. For this purpose, 13 naïve pregnant heifers were divided into three groups and were experimentally challenged with either 10⁷ tachyzoites of Nc-1 (group 1, n = 5), Nc-Spain 1H (group 2, n = 5) isolates or phosphate-buffered saline (group 3, n = 3) intravenously at 70 days of gestation. After inoculation, pregnancy was monitored and dams were sacrificed when foetal death was detected. The remaining animals were slaughtered at 45 days post-infection. Maternal and foetal samples were collected for examination by histology and parasite DNA detection. Parasitaemia, specific anti-N. caninum IgG and interferon γ responses were also studied. At 3–4 weeks after infection, foetal death was detected in 3 out of 5 Nc-1-infected dams. However, no evidence of foetal death was observed in either Nc-Spain 1H-infected or control groups during the period studied. The most severe histopathological lesions were observed in the placenta and foetal organs from Nc-1-infected cattle that exhibited foetal death. It was in these animals that N. caninum DNA was more frequently detected. Parasitaemia was observed in all Nc-1-infected dams and in only 3 out of 5 Nc-Spain 1H-infected animals. The magnitude of the immune response was significantly higher in the Nc-1-inoculated group than in the group inoculated with the Nc-Spain 1H isolate. These data reveal the reduced virulence of the Nc-Spain 1H isolate in cattle.     

Seroprevalence of Neospora caninum infection in dairy and beef cattle in Paraguay

Seroprevalence of Neospora caninum in 879 beef and dairy cattle in different locations of Paraguay was determined by an ELISA. In the survey, 262 (29.8%) cattle were positive to N. caninum, and animals with anti-Neospora antibody titre were observed in all the locations tested in the country. Serum samples taken from a herd that exhibited persistent abortion had the highest percentage of animals being positive to the parasite (17/30, 56.7%). In the same herd, abortion was significantly more likely in animals with high anti-Neospora antibody titre. Immunoblot analysis demonstrated that the banding pattern from positive Paraguayan cattle was similar to that seen with the positive control sample. In conclusion, N. caninum infection is present among Paraguayan beef and dairy cattle, and it may be an important cause of bovine abortion in Paraguay.     

Congenital Neospora caninum infection in dairy cattle and associated calfhood mortality.

A prospective cohort study was undertaken on two central California dairies, A and B, to estimate prevalence of congenital infection with Neospora caninum, to characterize temporal variation in prevalence, to determine if occurrence of congenital infection was associated with specific dam and calf attributes, and to estimate the effect of congenital infection on calfhood mortality. Of the 405 calves enrolled over a period of 2 1/2 y on dairy A and dairy B, 30.6% (85/278) and 53.5% (68/127), respectively, were seropositive precolostrally to N. caninum, as determined by an ELISA test. Adult cow seroprevalence at calving was 36.0% (82/228) for dairy A, and 57.9% (33/57) for dairy B. No evidence was found for a significant increasing or decreasing trend in adult and precolostral seroprevalence through the study period (P > or = 0.26). For both herds combined, 81% of seropositive cows (93/115) and 5% of seronegative cows (8/170) had congenitally infected calves. Seroprevalence did not increase with cow age on either dairy (P > or = 0.47). The probability of a calf being congenitally infected was not associated with dam age, dam lactation number, dam history of abortion, calf gender, or length of gestation (P > or = 0.11). High dam ELISA values at calving were significantly associated (P < or = 0.001) with an increased probability of congenital infection in her calf. Results of survival analyses of female calves available for follow-up indicated a consistently greater survivorship to 90 d in congenitally infected calves than in noninfected calves on both dairies, which was significant for dairy A (P = 0.07, n = 186) but not for dairy B (P = 0.69, n = 72), thus indicating that congenital infection does not necessarily have a detrimental effect on calf health. The findings of a similar magnitude in congenital infection rate and adult cow prevalence, the lack of increasing seroprevalence with cow age, the lack of an effect of dam age on precolostral seropositivity, and the constant seroprevalences during the study period, suggest that, in the two dairies studied, congenital transmission constituted a substantial amount of infection and was likely the major mode of transmission of N. caninum.     

Neospora caninum serostatus and culling of Holstein cattle

OBJECTIVE: To determine whether time until culling or risk of culling was associated with Neospora caninum serostatus among Holstein cattle in dairy herds in Ontario. DESIGN: Retrospective cohort study. ANIMALS: 3,412 cows in 56 herds. PROCEDURE: Blood samples were collected, and serum was tested for antibodies against N. caninum. Information on cows that were culled was collected during the 1- to 2-year period that producers were unaware of serostatus of individual cows in their herds. RESULTS: Herd prevalence of N. caninum-seropositive cows ranged from 0 to 68.3% (median, 7.0%). During the time of the study, 184 of 359 (51.3%) N. caninum-seropositive cows were culled, compared with 1,388 of 3,053 (45.5%) seronegative cows. Mean time from blood sample collection to culling for seronegative cows (289 days; 95% confidence interval, 280 to 299 days) was not significantly different from mean time for seropositive cows (296 days; 95% confidence interval, 269 to 323 days). Survival analysis indicated that N. caninum serostatus was not associated with time to culling or risk of culling. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that N. caninum serostatus of Holstein cows in Ontario was not significantly associated with either time to culling or risk of culling. Thus, N. caninum serostatus alone should not be used to determine whether cows should be culled.     

Comparison of serological methods for the diagnosis of Neospora caninum infection in cattle

The aims of this study were to evaluate the performance and agreement of various commercial and in-house Neospora caninum antibody assays used in dairy cattle in North America, and to investigate reproducibility of two assays performed in different laboratories. From 1998 to 2005, three enzyme linked immunosorbent assays (ELISAs, a competitive ELISA-VMRD Inc., an indirect ELISA-Biovet Inc., and another indirect ELISA-Herdchek IDEXX Corp.), two indirect fluorescent antibody tests (IFATs, VMRD Inc., and in-house USDA) and one N. caninum agglutination test (NAT, in-house USDA) were utilized to test 397 randomly selected dairy cattle serum samples from 34 herds in eastern Canada for antibodies to N. caninum. The manufacturers' recommended cut-off values were used to evaluate test performance and agreement between tests. One IFAT (VMRD Inc.) performed well (sensitivity and specificity: 0.97 and 0.97, respectively) using reference sera (n = 452), therefore, results from this IFAT on the 397 samples could subsequently be used as the reference standard to calculate test characteristics for the other assays. Only 11% of the 397 sera were found to be N. caninum-positive with the IFAT. Prevalence-adjusted bias-adjusted kappa (PABAK) ranged from 0.06 to 0.99. Positive agreement was moderate to very good (P(pos) = 0.25-0.96). Negative agreement was very good for all assays (P(neg) > 0.94) except NAT (P(neg) = 0.66). Sensitivity was > or =0.89 for all assays except the NAT, which had a significantly lower sensitivity (0.66). Specificity was high (>0.94) for all assays except for one indirect ELISA (specificity = 0.52). This indirect ELISA did not perform satisfactorily when used in 1998, but an improved version of the ELISA performed as one of the best assays in 2004. Reproducibility of the competitive ELISA was excellent, but the reproducibility of the indirect ELISA that was improved was low (concordance correlation coefficient = 0.90 and 0.36, respectively). The performance characteristics observed for most assays in this study make them useful for screening antibodies to N. caninum in cattle.     

Models for experimental infection of dogs fed with tissue from fetuses and neonatal cattle naturally infected with Neospora caninum

Three models were designed to investigate the development and enteroepithelial phase of Neospora caninum in dogs, and to induce oocyst production by the parasite. In the first model, three dogs were fed raw fetal bovine tissue on two occasions. The bovine fetal tissue had been stored at 4 °C for 14 days and 16 days, respectively, and had tested positive for neosporosis using histopathology and immunohistochemistry. In the second model, nine dogs from the beginning of the experiment until euthanasia were fed portions of tissue from bovine fetuses that been stored at 4 °C for between 1 and 2 days. Three fetuses had tested positive for neosporosis using histopathology and immunohistochemistry. In the third model, three dogs were fed pieces of raw tissue from two neonatal calves that had not received colostrum, and which had tested positive for antibodies against N. caninum. The brains of these calves were positive for neosporosis by histopathology and immunohistochemistry. In all three models, none of the dogs excreted oocysts of N. caninum, developed intestinal parasites or seroconverted.     

Neospora caninum serostatus and milk production of Holstein cattle

OBJECTIVE: To determine whether Neospora caninum serostatus was associated with milk production among Holstein cattle in Ontario. DESIGN: Case-control study and cross-sectional observational study. ANIMALS: 3,702 Holstein cows in 83 herds (case-control study) and 3,162 Holstein cows in 57 herds. PROCEDURE: Herds in the case-control study were grouped on the basis of N. caninum abortion status. Herds in the observational study were considered representative of Ontario dairy herds. The N. caninum serostatus of individual cows was determined with a kinetic ELISA. Milk production was modeled to compare seropositive with seronegative animals while controlling for parity, days since parturition, and herd clustering. RESULTS: In the case-control study, 305-day milk production of seropositive cows was significantly less than milk production of seronegative cows in herds with abortions attributable to N. caninum infection and in herds with abortions attributable to pathogens other than N. caninum, but not in herds without abortion problems. In the observational study, 305-day milk production for seropositive cows was not significantly different from milk production of seronegative cows. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the association between N. caninum serostatus and milk production in Ontario Holstein dairy cattle may depend on abortion status of the herd. In herds with abortion problems, regardless of cause, N. caninum-seropositive cattle produced less milk, whereas in herds without abortion problems, N. caninum-seropositive cattle produced the same amount of milk as seronegative cattle.     

Quantification of vertical and horizontal transmission of Neospora caninum infection in Dutch dairy herds

Ninety-six of 108 randomly selected Dutch dairy herds had one or more cows with a positive serostatus for N. caninum. In these 96 herds, we have quantified the probabilities of vertical transmission (VT) and horizontal transmission (HT) of N. caninum infection by combining serostatus and pedigree data in 4091 dam-daughter pairs. The probability of animals infected by vertical transmission during pregnancy (Prob(VT)) was calculated as the proportion of seropositive daughters among daughters of seropositive dams. The probability of animals infected by horizontal transmission (Prob(HT)) was the proportion of seropositive daughters among daughters of seronegative dams. These probabilities were calculated after the frequencies of observed dam-daughter combinations were corrected for (1) imperfect test-characteristics, (2) underestimation of horizontal transmission in situations that seronegative dams were horizontally infected after the birth of their daughters and (3) overestimation of vertical transmission in situations that seronegative daughters born from seropositive dams were horizontally infected. The incidence rate for horizontal transmission was calculated based on Prob(HT) and the average age of the animals in these herds. Based on the analysis of dam-daughter serology, Prob(VT) was 61.8% (95% CI: 57.5-66.0%) and Prob(HT) was 3.3% (95% CI: 2.7-3.9%). After adjusting the observed frequencies for imperfect test-characteristics, underestimation of horizontal transmission and overestimation of vertical transmission, Prob(VT) decreased to 44.9% (95% CI: 40.0-49.9%) while Prob(HT) increased to 4.5% (95% CI: 3.9-5.2%). Prob(HT) corresponded with an incidence rate for horizontal transmission of 1.4 (95% CI: 1.2-1.7) infections per 100 cow-years at risk. When stratifying herds for the presence of farm dogs, Prob(HT) was higher (5.5% (95% CI: 4.6-6.4%)) in herds with farm dogs than in herds without farm dogs (2.3% (95% CI: 1.5-3.4%)). When stratifying for within-herd seroprevalence, Prob(HT) was higher (10.3% (95% CI: 8.6-12.2%)) in herds with high (> or =10%) within-herd seroprevalence compared with herds with low (<10%) within-herd seroprevalence (2.0% (95% CI: 1.5-2.6%)). Although there was this relation between Prob(HT) and within-herd seroprevalence (crude OR(PREV) = 5.7 (95% CI: 4.0-7.9)), in herds without farm dogs, this relationship was no longer statistical significant (OR(PREV|DOG-) = 1.9 (95% CI: 0.7-5.5)). It indicated that the association between seroprevalence and Prob(HT) depended largely on the presence of farm dogs. In addition, when looking for the presence of specific age-groups with significantly higher seroprevalence compared with the rest of the herd, there were 7 herds in which two or more horizontally-infected animals were present in specific age-groups. This was an indication of a recent point-source exposure to N. caninum. These results reiterate the current control strategies to apply strict dog-management measures as well as to minimize within-herd seroprevalence by monitoring serostatus of animals.