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PubMed1998

The major 36 kDa Neospora caninum tachyzoite surface protein is closely related to the major Toxoplasma gondii surface antigen.

The tachyzoites and the tissue cysts containing bradyzoites of Neospora caninum and Toxoplasma gondii, respectively, are difficult to distinguish morphologically. Specific antigens have been identified in T. gondii tachyzoites and bradyzoites, some of which are stage-specifically expressed, and different functions have been attributed to some of them. A tachyzoite stage-specifically expressed surface protein is the major surface antigen 1 (SAG1) which has been shown to be involved in host cell attachment and invasion. Previously we have identified a cell surface-associated glycoprotein (p36) in N. caninum tachyzoites. The full length coding sequence of the cDNA coding for p36 was determined, and analysis of the deduced amino acid sequence demonstrated that p36 is closely related to SAG1. p36 is encoded by a single copy gene which produces a transcript of 1.4 kb. Immunogold labeling of resin-embedded parasites using polyclonal antibodies affinity-purified on a recombinant p36 fusion protein expressed in Escherichia coli showed that this protein is located exclusively on the tachyzoite cell surface. As SAG1 in T. gondii, p36 is expressed in the tachyzoite stage, but is absent from bradyzoites. p36 is recognized by antibodies present in sera of cows experimentally infected with N. caninum tachyzoites.

Amino Acid Sequence↗
PubMed2000

Molecular characterization of a thrombospondin-related anonymous protein homologue in Neospora caninum.

Thrombospondin-related anonymous protein (TRAP) family members participate in attachment and invasion of host cells by apicomplexan parasites. A TRAP homologue in Neospora caninum strain Nc-1 (NcMIC2) was cloned, sequenced and found to be 61% identical (75% similar) at the amino acid level to Toxoplasma gondii MIC2 (TgMIC2). Similar to TgMIC2, the predicted amino acid sequence of NcMIC2 contains one integrin-like domain (I or A domain), five thrombospondin (TSP) repeats, a putative transmembrane spanning region and intracellular C-terminus, and was localized to micronemes by cryo-immunoelectron microscopy. The secretion of NcMIC2 was temperature dependent and was induced at or above 25 degrees C. The secreted form of NcMIC2 released into the medium was found to be proteolytically processed such that it lacked the C-terminal domain. Secretion of NcMIC2 was regulated by calcium, since several agents which raise intracellular calcium levels were shown to promote NcMIC2 secretion and chelation of [Ca(2+)](i) abrogated release. As a member of the growing family of apicomplexan TRAP proteins, NcMIC2 may play an important role in attachment and invasion by N. caninum into host cells.

Amino Acid Sequence↗
PubMed2001

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.

Animal Husbandry↗
PubMed2002

Factors associated with Neospora caninum serostatus in cattle of 20 specialised Costa Rican dairy herds.

Twenty-five specialised Costa Rican dairy farms (located in the Poás area) were used to determine neosporosis seroprevalence and the association of seropositivity with environmental and management factors. The farms involved were selected intentionally and all of them use VAMPP 5.1 (Veterinary Automated Management and Production Control Programme) as management-information system. Holstein-Friesian, Jersey and crosses between them were the most-frequent breeds in these herds. The number of females per farm varied from 41 to 296. Our cross-sectional study had two phases. In the first phase, we determined the presence or absence of seropositivity at herd level. For the second phase, all females in 20 seropositive farms were bled. Serum samples were tested for antibodies to Neospora caninum using an indirect enzyme-linked immunosorbent assay (ELISA). A questionnaire with factors mentioned in the literature was administered to the farmers. Logistic regression (LR with herd as random effect) was used to assess the relationships of the serostatus at the individual level with characteristics of the cows and environmental factors. In the first phase all herds had >20% seropositive females; therefore, all herds were eligible for the second phase. In the second phase, the overall prevalence was 39.7% (1191/3002), and within-herd prevalences were between 25.0 and 70.5%. Age 3-6 years, parity < or =2 of the dam of the cow, Jersey breed and lack of purposive sampling to diagnose abortive infectious disease were associated with positive serostatus; other management and environmental factors did not show significant associations. The lack of association between management and environmental factors with serostatus might be because all farms were exposed to a considerable number of potential factors. That all herds of this study were seropositive for neosporosis and the within-herd prevalence was considerable raises questions about how far the infection is spread in other dairy areas of Costa Rica.

Abortion, Veterinary↗
PubMed2002

Management factors related to seroprevalences to bovine viral-diarrhoea virus, bovine-leukosis virus, Mycobacterium avium subspecies paratuberculosis, and Neospora caninum in dairy herds in the Canadian Maritimes.

Bovine viral-diarrhoea (BVD), enzootic bovine leukosis (EBL), Johne's disease (JD), and neosporosis lower on-farm productivity, reduce export competitiveness, and increase consumer concerns regarding safety. Our purpose was to examine the relationship between 27 control practices and the estimated true seroprevalences for these four diseases for 2604 cattle in 90 dairy herds in the Maritimes provinces of Canada. Overall, 37.8, 20.4, 3.4, and 19.2% of all sampled cattle were truly exposed to the agents of BVD, EBL, JD, and neosporosis, respectively. The median within-herd true prevalences were 0, 9.3, 0, and 12.3%, respectively. Factor analysis reduced the 27 control practices to two highly correlated factors. Tobit-regression analyses determined that vaccination practices were associated with reduced prevalence of exposure for Bovine viral-diarrhoea and EBL. Also, farms that tended to purchase their dairy animals were associated with higher seroprevalence for Johnes' disease. Neither of these two factors was associated with the seroprevalence of Neospora caninum infection. The few routine biosecurity measures that were investigated in this study were generally not related to the seroprevalences of these farms.

Animal Husbandry↗
PubMed2003

Validation of a Neospora caninum iscom ELISA without a gold standard.

Neospora caninum is an intracellular parasite which causes abortion in cattle worldwide. One problem in the validation of the different methods for demonstration of this parasite is the lack of an appropriate gold standard. To validate an immunostimulating complex (iscom) enzyme-linked immunoassay (ELISA) used to detect antibodies to N. caninum, sera from 244 cattle in five Swedish dairy herds infected with N. caninum were analysed. The sera also were analysed by a standard indirect-fluorescent antibody test (IFAT). The results obtained by the two tests were compared using the Gibbs sampler. Gibbs sampling is a latent-class approach based on Bayesian statistics; neither test is assumed to be more correct in stating the true status of infection. The Gibbs sampler was run using both informative and non-informative prior probabilities. We also simulated different cut-offs in the iscom ELISA (providing data to inform selection of optimal cut-off values for different applications). The ELISA produced fewest incorrect test results over all at a cut-off value of 0.200. The sensitivity and specificity at this cut-off were 99 and 96%, respectively. The IFAT had a high specificity (99%) but a lower sensitivity (78%) than expected-confirming that the IFAT cannot be treated as a true gold standard. Sensitivity and specificity of the ELISA were presented in a two-graph receiver operating characteristic (TG-ROC) plot. Any cut-off between 0.150 and 0.300 will have both sensitivity and specificity > or =95%. Optical densities of < or =0.150 and > or =0.550 (or > or =0.350) were suggested as limits to rule out and rule in infection, respectively.

Animals↗
PubMed1999

Associations of Neospora caninum seropositivity with gestation number and pregnancy outcome in Danish dairy herds.

The prevalence and distribution of seropositivity towards the protozoan parasite Neospora caninum were studied in single blood samples from 1561 cows from 31 Danish dairy herds. Blood samples were analysed by an indirect enzyme-linked immunoassay and an indirect fluorescent-antibody test. Seroprevalence in 15 herds with previous abortions assigned to neosporosis ranged from 1% to 58%, with a mean frequency of 22%. In eight out of 16 herds without a history of N.caninum related abortions, no seroreactors were found. In the remaining eight herds, the seroprevalence ranged from 6% to 59%. The prevalence and distribution of seropositivity, gestation number prior to sampling, and breed were related to abortions and perinatal deaths using a random-effects logistic-regression model. Abortion risk was significantly increased in seropositive animals (OR = 3) and in > or = 2nd-gestation cows (OR = 3). Perinatal death was significantly influenced by gestation number and breed, but not by serostatus. Reproductive performance and culling risk of cows were not affected by serostatus. Seropositivity increased with "age" (i.e. gestation number) (P = 0.02). In open cows, seropositivity tended to decrease with distance from calving (P = 0.05). The proportion of seropositive pregnant cows increased with trimester (P = 0.02).

Abortion, Veterinary↗
PubMed2000

Progress in the serodiagnosis of Neospora caninum infections of cattle.

Neospora caninum is an apicomplexan protozoan that has become the focus of significant research attention worldwide. This organism infects a range of host species, including dogs, from which it was originally reported in 1984, but it is most important as a major cause of bovine abortion. As a result of the global importance of N. caninum, researchers have developed a number of serological tests to investigate the epidemiology of infection and disease. In this article, Robert Atkinson, Peter Harper, Michael Reichel and John Ellis consider progress made in the serodiagnosis of N. caninum.

Animals↗
PubMed2001

Protective efficacy of vaccination by recombinant vaccinia virus against Neospora caninum infection.

The recombinant vaccinia viruses expressing the surface protein of Neospora caninum tachyzoite, NcSAG1 or NcSRS2, were constructed. The vaccination with these recombinant viruses could protect effectively the parasite invasion in a mouse model system. The vaccine efficacy of NcSRS2 was higher than that of NcSAG1. The present study indicated that a high level of IgG1 Ab production to parasite is important for clearance of parasite at the early stage of infection and that T cell response has a crucial role for protection against the intracellular infection at the late stage of infection. The recombinant vaccinia viruses might be applicable as vaccine against N. caninum infection in a natural host.

Animals↗
PubMed2001

Prevention of vertical transmission of Neospora caninum in BALB/c mice by recombinant vaccinia virus carrying NcSRS2 gene.

Neospora caninum infection is the major cause of bovine abortion. To develop a vaccine against N. caninum infection, recombinant vaccinia viruses carrying NcSRS2 and NcSAG1 genes (vv/Nc-p43 and vv/Nc-p36, respectively) were constructed and were tested in a mouse model. Vaccination of dams with vv/Nc-p43 appeared to confer effective protection against vertical transmission to offspring, though that with vv/Nc-p36 only provided partial protection. Moreover, the vv/Nc-p43 vaccination provoked cellular immune responses and antibody production against N. caninum. In conclusion, it is expected that vv/Nc-p43 can be used as an effective live vaccine to prevent vertical transmission of N. caninum in natural hosts.

Animals↗
PubMed2000

Serological survey and first finding of Neospora caninum in Taiwan, and the detection of its antibodies in various body fluids of cattle.

A serological survey for antibodies against Neospora caninum in cattle, goats and farm dogs in Taiwan was carried out. Sera of 613 cattle from 25 dairy farms, 24 goats from six goat farms and 13 dogs from six dairy cattle farms were tested for antibodies against N. caninum using indirect fluorescence antibody test (IFAT). The same sera were also tested for antibodies against Toxoplasma gondii using latex agglutination test. Of the 613 cattle sera, 44.9% (275/613) were found to have antibodies against N. caninum. Among these 275 positive cattle, 77 also possessed antibodies against T. gondii. Nevertheless, 92 cattle which were negative for N. caninum showed antibodies against T. gondii. Of the 24 goat sera tested, none was found to be positive for N. caninum but 50% (12/24) were positive for T. gondii. Of the 13 farm dogs tested, three were found to possess antibodies against N. caninum, two of which tested negative for T. gondii antibodies. Besides sera, antibodies to N. caninum in cattle could be observed in the milk, vaginal secretion and saliva. However, the order of higher frequency of antibodies detection is in sera, milk, vaginal secretion and saliva. This is the first demonstration of the presence of antibodies to N. caninum in vaginal secretion and saliva of cattle. A 50microm cyst was observed in the brain of one of the 13 prednisolone-treated SPF ICR mice which had been peritoneally inoculated 4 months earlier with the brain homogenate of a serologically N. caninum positive but T. gondii negative cattle. Thus, we have confirmed for the first time the presence of N. caninum in Taiwan and also observed that it is widespread among dairy cattle and farm dogs.

Animals↗
PubMed2000

Neospora caninum seroprevalence and associated risk factors in beef cattle in the northwestern United States.

A Neospora caninum seroprevalence and risk factor survey of 2585 cows was conducted in 55 beef cow-calf herds located in five northwestern states of the USA. Blood samples were collected by private veterinary practitioners and management practices were surveyed using a mail questionnaire. Producers were randomly selected from those that employed these veterinarians to perform annual herd pregnancy examinations. Questions were asked about animal management, grazing and feeding, immunization and record keeping practices. Blood was collected from a systematically selected sample of cows in each herd, and age, origin, and pregnancy status were recorded. Blood samples were analyzed for antibodies against N. caninum antigen using a monoclonal antibody-based competitive inhibition (CI) ELISA. Overall seroprevalence was 24% and within herd seroprevalence ranged from 3 to 67% with a median of 19%. Within herd seroprevalence and mean inhibition percentage were different between the five states. Herds that managed their cows on range for summer grazing had lower seroprevalence than those that did not, while increased seroprevalence was associated with higher winter stocking density. Cows less than 3 years of age had higher CI ELISA inhibition percent values than cows greater than 6 years of age. No relationship was noted between serologic status and individual cow origin (purchased or raised), or pregnancy status at the time of sampling.

Animals↗
PubMed2000

The role of CD4(+) or CD8(+) T cells in the protective immune response of BALB/c mice to Neospora caninum infection.

The role of CD4(+) or CD8(+) T cells in the immune response of BALB/c mice against Neospora caninum infection was examined by using anti-CD4 and/or anti-CD8 monoclonal antibodies (mAbs). Mice were intraperitoneally inoculated with anti-CD4 and/or anti-CD8 mAbs before and after infection with N. caninum and observed for 30 days after infection. Most of the anti-CD4 mAb-treated mice and all of the anti-CD4 and anti-CD8 mAbs-treated mice died within 30 days post-infection (p.i.). In contrast, 100% of PBS-treated mice and 70% of anti-CD8 mAb-treated mice survived more than 30 days. When compared with phosphate-buffered saline (PBS)-treated mice, the weight of mice treated with mAbs tended to decrease. From these results CD4(+) T cells, but not CD8(+) T cells, have an important role for protection of mice against N. caninum infection. Serum antibody levels to N. caninum in infected-mice treated with anti-CD4 mAb or a mixture of anti-CD4 and anti-CD8 mAbs were lower than those in the infected mice treated with anti-CD8 mAb or PBS. The mice treated with anti-interferon-gamma (IFN-gamma) mAb produced high antibody levels to N. caninum, but all mice died within 18 days p.i. These results indicated that IFN-gamma is an important cytokine for protection against N. caninum infection at the early stage of infection. However, since CD4(+) T cells against N. caninum were essential to the production of specific antibody, these antibodies might have important roles in host protection at the later stage of infection.

Animals↗
PubMed2000

Isolation of Neospora caninum from the brain of a naturally infected adult dairy cow.

Neospora caninum was isolated from the brain of a 2-year-old dairy cow that had aborted confirmed N. caninum-infected fetuses on two occasions. The cow had an indirect fluorescent antibody titer of 1:1600 to N. caninum. The cow was killed 24 days after its second abortion and the brain was bioassayed for N. caninum in nude mice. Multifocal areas of perivascular cuffing and glial nodules were observed in the cerebrum and mesencephalon of the cow, but N. caninum was not identified in histological sections of the brain. All three nude mice inoculated with brain homogenate of the cow, developed emaciation and paralysis. Microscopical examination of the nude mice revealed systemic N. caninum infection with demonstrable tachyzoites in various organs. The parasites isolated from fresh mouse brain were transferred successfully into Vero cell cultures. PCR procedure on the purified tachyzoites obtained from the Vero cell cultures amplified the specific DNA sequence for N. caninum.

Animals↗
PubMed2001

Prevalence of antibodies to Neospora caninum and Toxoplasma gondii in gray foxes (Urocyon cinereoargenteus) from South Carolina.

Little is known about the epidemiology of Neospora caninum in wild mammal populations. It has been suggested that a sylvatic cycle exists for N. caninum. Dogs and potentially other canids are a definitive host for N. caninum. The present study was done to determine the prevalence of antibodies to N. caninum in a population of gray foxes (Urocyon cinereoargenteus) from a nonagricultural setting in South Carolina. We also determined the prevalence of antibodies to Toxoplasma gondii in these animals. Antibody levels were measured in direct agglutination tests using either N. caninum or T. gondii formalin-fixed tachyzoites as antigen. Four (15.4%) of the 26 gray foxes had titers to N. caninum. Titers to N. caninum were low being 1:25 in three gray foxes and 1:50 in the fourth gray fox. Antibodies to T. gondii were observed in 16 (61.5%) gray foxes. Titers to T. gondii were usually >1:50 and two gray foxes had titers of 1:1600. Results of this study indicate that gray foxes have more exposure to T. gondii than to N. caninum in this environment.

Age Factors↗
PubMed2001

Failure of dogs to shed oocysts after being fed bovine fetuses naturally infected by Neospora caninum.

Neospora caninum is a protozoan that causes abortion in cattle. The dog has recently been identified as a definitive host for N. caninum. To verify if bovine fetuses can infect dogs, nine 2-4-month-old dogs were fed bovine fetuses naturally infected by N. caninum. None of the dogs excreted oocysts, seroconverted, had clinical signs or lesions compatible with N. caninum infection. Additional studies will be necessary to determine the natural mode of infection of dogs by N. caninum.

Animals↗
PubMed2001

Neospora caninum infection associated with stillbirths in captive antelopes (Tragelaphus imberbis).

Neosporosis was diagnosed in full-term stillborn twin calves of captive antelopes (Tragelaphus imberbis) in a German zoo. In both calves a multifocal non-suppurative encephalitis was present and infection with Neospora (N.) caninum was confirmed by foetal serology and PCR. N. caninum-DNA was detected in brain, heart, lung, liver and spleen of a third full-term stillborn calf. Histopathological lesions were absent in this calf and antibodies against N. caninum were not detected in pleural fluid. Immunohistochemistry failed to demonstrate N. caninum antigen in all three calves.

Animals↗
PubMed2001

Isolation of Neospora caninum from the brain of a naturally infected dog, and production of encysted bradyzoites in gerbils.

Neospora caninum was isolated from the brain of an adult dog in Brazil. Cerebral tissue from the dog was inoculated into Mongolian gerbils. Gerbils were euthanized 3-4 months later and bradyzoite-containing tissue cysts were observed in their brains. N. caninum (designated NC-Bahia) was isolated in cell culture after inoculation with tissue cysts from the gerbils. The identity of the parasite was confirmed by immunohistochemical examination and polymerase chain reaction (PCR). Gerbils may be a useful alternative to immunosuppressed mice for isolation of N. caninum and for production of encysted bradyzoites.

Animals↗