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.     

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.     

High rate of transplacental infection and transmission of Neospora caninum following experimental challenge of cattle at day 210 of gestation

In order to investigate the pathogenesis of neosporosis following a primary infection in late pregnancy, cattle were subcutaneously challenged with 5 × 10⁸Neospora caninum (NC1 isolate) tachyzoites at day 210 of gestation and serial necropsies were then carried out at 14, 28, 42 and 56 days post-infection (dpi). No abortions occurred and all the foetuses were viable at the time of euthanasia. There was a high rate of vertical transmission, as parasites were detected by immunohistochemical labelling and PCR in all the foetuses from 28 dpi. Focal necrotic lesions were observed in the placentomes of the placenta from 28 dpi and showed resolution during later time points, denoted by infiltration of inflammatory cells at 42 dpi and fibrosis at 56 dpi. Foetuses at 28 and 42 dpi showed scarce and isolated lesions which are unlikely to represent a threat to foetal viability. No lesions were observed in the foetuses at 14 or 56 dpi suggesting control of the infection and resolution of the lesions by maternal and foetal immune responses. Once infection was established, it could not be cleared from the host and vertical transmission of the parasite occurred in all infected hosts. Parasite was detected in the placenta at 28 dpi, while in previous experimental infections of cattle at day 70 and 140 of gestation using the same challenge model, it was already present at day 14 post infection. This suggests that a change in the maternal immune response plays a crucial role in limiting the initial infection during the last term of pregnancy.     

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.     

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.     

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.     

奶牛新孢子虫病血清流行病学调查

本研究用酶联免疫吸附试验（ELISA）对采集自北京地区的94份奶牛血清（随机采集）和河北地区的55份奶牛血清（有流产史奶牛），进行Neospora caninum血清抗体检测。结果发现，北京地区随机采集的奶牛血清N．caninum抗体阳性率为18．1％（17／94），河北地区有流产史的奶牛N．caninum血清抗体阳性率为23．6％（13／55）。采用牛奶记录体系（DHI）对北京地区17头N．caninum血清抗体阳性牛进行了日产奶量、乳中蛋白率和乳脂率的测定，并与同群牛中134头阴性牛比较。结果表明，N．caninum血清抗体阳性牛日产奶量比阴性牛降低9．7％，乳中蛋白率和乳脂率分别降低20％和15．4％。初步证明N．caninum血清抗体阳性奶牛产奶量降低及奶品质的下降。对不同N．caninum抗体滴度阳性牛的泌乳期主要生产性能比较发现，其生产性能的变化与抗体滴度无明显相关性。     

Seroprevalence of Neospora caninum in dairy cattle in Bahia, Brazil.

Sera collected from 447 dairy cattle on 14 dairy farms were tested for Neospora caninum antibodies by use of an immunofluorescent antibody technique. Positive reactions with titres > or =1:200 were found in 63 (14.09%) of animals. Neospora positive sera were also tested for Toxoplasma gondii antibodies by using a commercial latex agglutination test. Antibodies to T. gondii were detected in 3 (4.76%) of 63 N. caninum positive sera. These results indicate that N. caninum infection is widespread among dairy cattle in Bahia state.     

Neospora caninum seroprevalence in dairy cattle in central Thailand

The seroprevalence, in dairy cattle, of antibodies to Neospora caninum, the relationship between seropositivity and age (heifer versus cow), the relationship of herd infection with herd size and the relationship of herd infection with the presence of dogs on the farm were studied. The study involved 549 cows and 82 dogs in 59 dairy herds in Nakhon Pathom, Thailand. A competitive enzyme-linked immunosorbent assay (cELISA) with NC-specific monoclonal antibody was used to detect the NC antibodies in the sera. Individual and herd seroprevalence of NC were 5.5% (30/549) and 34% (20/59), respectively. No significant relationships between NC seropositivity with the age of the cows (heifer versus cow; P > 0.05) and between herd infection and the presence of dogs on the farm (P > 0.05) were found. Herd size significantly affected herd infection (P < 0.05) with higher infection in large than small herds (> or = 21 versus < or = 20 cows). Of 12 cows with a history of abortion, one was seropositive to NC. The seroprevalence of NC antibodies in dogs was 1.2% (1/82). This is the first NC seroprevalence study in dogs in Thailand. It was concluded that Neospora infection was more common at the herd level rather than the individual level in Thailand and the presence of dogs on the farm was not related to the level of herd infection. Caution should be taken in the interpretation of serological tests from the farm dogs.     

Udder health in dairy cattle infected with Neospora caninum

Blood samples were collected from 3449 cows on 57 representative Ontario dairy herds during the summer of 1998 and analysed for antibody to Neospora caninum using an ELISA. Forty-eight herds (2742 cattle) contained at least one N. caninum-seropositive animal. Two composite milk samples were collected from all cattle: the first on the day of blood collection and the second 68 to 365 days later. All milk samples were submitted for bacteriological culture. Ontario Dairy Herd Improvement Corporation (DHI) data were available for 3162 cattle in the 57 herds at the time of bleeding. Furthermore, complete DHI data were available for 1658 cattle that were culled between 12 and 24 months following blood collection. Using a standardised ELISA sample-to-positive (S/P) cut-off of ≥0.45, the corrected seroprevalence was 8.2% overall and 10.1% within seropositive herds. At blood collection the odds of N. caninum-seropositive cows having a high linear score (≥4.0; equivalent to a somatic cell count ≥200,000 cells/ml) was 27% less than for seronegative animals. Similarly, at the time of culling, the odds of having a high linear score was 22% less in N. caninum-seropositive cattle. Overall, linear score was lower in N. caninum-seropositive cattle at culling. After controlling for herd, parity, days in milk, and the interval between collection of milk samples, the odds of N. caninum-seropositive cattle testing positive for an environmental pathogen (i.e. environmental Streptococcus species and coliforms) on the second milk sample was 56% less than for seronegative animals. The odds were 83% less at a higher ELISA S/P cut-off of ≥0.70. Finally, the odds of N. caninum-seropositive cattle developing a new infection with a major pathogen (environmental or contagious) were 60% less than seronegative cows using the higher ELISA S/P cut-off.     

Monensin use against Neospora caninum challenge in dairy cattle

Using a randomized controlled trial design, a randomly allocated intervention group of 15 cows received a slow-release bolus that delivered 100 days of monensin. The negative control group of 15 cows received a placebo bolus that was identical to the monensin bolus, except without the monensin. Two weeks after bolus administration, all cows were challenged with a 2 ml subcutaneous injection of a live tachyzoite suspension. Whole blood and serum samples were collected from each cow every week for the first month post-challenge, and then every 2 weeks for the next 2 months. The extracted DNA from whole blood was tested for the Nc-5 gene fragment of Neospora caninum using a quantitative real time polymerase chain reaction. Serum was tested for antibodies to N. caninum using the IDEXX ELISA. Cows treated with monensin boluses had a significantly lower humoral immune response than cows treated with placebo boluses at one time point post-challenge (week 4 post-challenge). However, when adjusting for repeated measures within cows, the P value for this humoral difference was 0.098. No DNA for N. caninum was detected in either group, likely due to study design features.     

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.     

Agreement between three serological tests for Neospora caninum in beef cattle.

During 1999, serum samples were collected from beef cows on pastures in western Canada. Some of the herds had a history of confirmed abortions associated with Neospora caninum infection. All these samples were initially analyzed using a single application of 1 common commercial enzyme-linked immunosorbent assay (ELISA) for antibodies to N. caninum. From these initial results, 239 positive and 250 negative samples were randomly selected for further testing. This group of samples was retested using the 3 commercially available ELISA tests for N. caninum as per the manufacturer's recommendations. The agreement between 2 of the ELISAs was good (K = 0.76); agreement of these 2 tests with the third test was much lower (K = 0.46 and 0.60). Quantitative agreement between tests measured by intraclass correlation coefficients was also acceptable between the first 2 tests but was almost zero when the first 2 tests were compared with the third. This information is necessary to understand the differences in seroprevalence reported in different regions from laboratories using different methods.     

Seroprevalence of Neospora caninum in beef cattle in northern Alberta

Blood samples were collected from 1806 pregnancy-tested cows from 174 herds at a northern Alberta auction mart in the fall of 1998. One hundred sixty-two (9.0%) of these samples were positive for antibodies to N. caninum. Thirty-five of 260 samples (13.5%) collected from the same region in the 1980s were also serologically positive for N. caninum.     

Molecular assessment of the transplacental transmission of Toxoplasma gondii,Neospora caninum,Brucella canis and Ehrlichia canis in dogs

Given the fact that numerous microbial species can be detected in pregnant female dogs, the objective of this study was to assess the transplacental transmission of Brucella canis, Ehrlichia canis, Neospora caninum and Toxoplasma gondii in stillborn puppies. This study involved 41 stillborn puppies, 78.6% of which were positive for T. gondii, 52.4% for N. caninum and 59.5% for B. canis. E. canis was not detected in any of the analyzed puppies. Pregnancy is an important physiological condition for the transmission of infectious agents to puppies and transplacental transmission may be epidemiologically relevant in the spread of these opportunistic agents.     

Nationwide seroprevalence of Neospora caninum among dairy cattle in Japan

Serum samples from 2420 clinically healthy dairy cattle, randomly selected from stored sera in 18 districts of Japan, were tested for the presence of Neospora caninum antibodies using an indirect fluorescent antibody test (titer > or =1:200). Nationwide seroprevalence is estimated at 5.7% (139/2420). Seropositive cattle were detected in all surveyed districts despite the evidence of confirmed case reports of bovine neosporosis, showing that N. caninum is widely distributed throughout Japan. Age-specific seroprevalence did not increase with cattle age, suggesting that Neospora infection is likely to be transmitted vertically rather than horizontally in Japan. Considering that N. caninum seropositive cows are thought to be more likely to abort, substantial fetal losses may be induced by N. caninum infection in Japan. Devising strategies are needed to reduce the economic impact on the Japanese dairy industry. This is the first study to investigate the nationwide seroprevalence of N. caninum in cattle in Asia.     

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.     

Efficacy of a Neospora caninum killed tachyzoite vaccine in preventing abortion and vertical transmission in dairy cattle

A clinical trial was undertaken to assess the efficacy of Bovilis(?) Neoguard, a killed Neospora caninum tachyzoite vaccine on 5 commercial dairy farms in New Zealand with a history of Neospora-associated abortion. Cattle were enrolled in the trial at 30-60 days of gestation and randomly allocated to treatment or control groups. Treatment consisted of 5 mL doses of Bovilis Neoguard administered subcutaneously at enrolment then 4 weeks later. Isotonic saline was administered to the control group. Of 2246 cattle enrolled in the trial, 10.7% of cows and 12.6% of heifers were seropositive to N. caninum. Sampling of a randomly selected proportion of enrolled animals 6 weeks after the second treatment showed that 188/232 (81.0%) vaccinated with Bovilis(?) Neoguard had seroconverted, while 11/130 (8.5%) cows and 10/36 (27.8%) heifers in the control group had seroconverted. Forty-eight vaccinated and 63 control animals aborted. On one farm 12.5% of control animals and 6.1% of vaccinated animals aborted (vaccine efficacy 0.61; p=0.03). On another farm with a high level of abortion 8.4% of control animals and 8.7% of vaccinates aborted. On the remaining 3 farms fewer abortions occurred than expected. A modified Poisson regression approach was used to calculate relative risks for abortion and vertical transmission. Overall vaccine efficacy was 0.25 (p=0.12). Heifer replacement calves from the animals enrolled in the trial were sampled for antibodies to N. caninum at 6-9 months of age. Fourteen of 17 calves from vaccinated, seropositive cows were seropositive as were 13/23 calves from seropositive cows in the control group. The interaction between dam serostatus and treatment group was significant (p=0.05) with vaccination increasing the risk of vertical transmission. It was concluded that vaccination after conception prevented 61% abortions in one of five herds and that vaccination may have increased the risk of early embryonic death.     

Estimation of Neospora caninum seroprevalence in dairy cattle from Normandy, France.

An epidemiological study was conducted in Orne (France) on randomly selected dairy herds (42 herds including 1,924 cows and heifers, which were at least 15 months old). The aim was primarily to estimate the seroprevalence of Neospora caninum infection from two blood samples per cow, using an enzyme-linked immunosorbent assay (ELISA) for N. caninum (one positive result indicating infection). The second aim was to test the association between some individual and herd factors and N. caninum seropositivity with a logistic model including a random term effect. The prevalence was estimated at 5.6% (107 seropositive animals). At least 27 of the 42 herds had one seropositive cow or heifer. The intra-herd seroprevalence varied from 1.1 to 8% for 18 positive herds (66.7%). Dogs were present in 36 farms and 104 of the 107 seropositive animals were exposed to them. The factors associated with individual seropositivity were the presence of cats (OR = 0.17; P < 0.001), dogs (OR = 4.35; P = 0.02), rabbits and/or ducks (OR = 2.10; P = 0.04), long calving periods (12 months) (OR = 0.44; P = 0.007), tethered housing (OR = 2.50; P = 0.01), somatic cell counts (200-400 x 10(3) cells/mL) (OR = 0.24; P < 0.001) and pond water supply (OR = 2.43; P = 0.04). In conclusion, the animal and intra-herd seroprevalences were low in dairy cows from Normandy, France.     

Recent advances in understanding the epidemiology of Neospora caninum in cattle

INTRODUCTION: Neospora caninum is the most frequently diagnosed cause of cattle abortion in New Zealand and is an important pathogen worldwide. The parasite has been found in aborted bovine foetuses and in puppies with neurological disease. Recent developments have provided new insights into the epidemiology of the disease that are reviewed in this article. TRANSMISSION: Transplacental infection is of major importance in the spread of N. caninum overseas. Most congenital infections result in the birth of apparently healthy calves. Seroepidemiological studies indicate introduced point-source infections are also likely, as do investigations of abortion outbreaks in cattle herds attributed to N. caninum. Horizontal transmission is an important determinant of the stability of infection prevalence in a herd. Two potential sources of horizontal transmission that have recently been reported are: via a canine definitive host and; cow-to-calf transmission through pooled colostrum or milk. Transmission parameters for the organism have been estimated using mathematical modeling, to explore the epidemiology and options for control of N. caninum infection in dairy cattle in the absence of precise epidemiological data. LIFE-CYCLE: Seroepidemiologic studies, combined with the knowledge that dogs can be a definitive host, provide evidence supporting a dog-cattle life-cycle. The importance of dogs in the epidemiology of bovine neosporosis is not yet clear. It is likely that N. caninum oocysts in dog faeces could serve as a source of infection for cattle and recent studies have shown that the canine and bovine isolatesflare the same organism. Infection of cattle orally exposed to oocysts has been demonstrated experimentally but not in nature. It is not known if other wild carnivores may serve as definitive hosts of N. caninum. CONCLUSION: Despite active research for over a decade, very little is known about the mechanisms of transmission of N. caninum. Vertical transmission is well documented but horizontal spread and the pathogenesis of abortion from this disease need further investigation. It is evident that, even with a very high probability of vertical transmission, some form of horizontal transmission is required for the disease to persist in a herd.