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PubMed2006

Plasma pregnancy-associated glycoprotein-1 (PAG-1) concentrations during gestation in Neospora-infected dairy cows.

The aim of this study was to determine whether plasma pregnancy-associated glycoprotein-1 (PAG-1) concentrations in pregnancy are affected by persistent Neospora caninum infection in dairy cows. The data analyzed were derived from 22 multiparous cows: 16 N. caninum-seropositive and 6 N. caninum-seronegative animals (used as controls). Three of the 16 seropositive cows aborted during the study period and the corresponding data were analyzed separately. Pregnancy diagnoses were performed on day 40 post-insemination by transrectal ultrasound, and by palpation per rectum on days 90, 120, 150, 180 and 210. Blood samples were collected from each animal immediately before each pregnancy diagnosis, and then at parturition or at the time of abortion detection. Plasma was tested for antibodies against N. caninum and PAG-1 concentrations were determined by radioimmunoassay. In non-aborting animals, the effects of neosporosis (seropositive versus seronegative), N. caninum antibody levels, semen providing bull, sex of the newborn, and day of gestation on PAG-1 concentrations were evaluated by GLM repeated measures analysis of variance. The effect of the gestation period (first half versus second half) on the N. caninum antibody titer was established by the Student's t-test in seropositive cows. A significant positive effect of gestation day on PAG-1 concentrations was observed (d.f.=6; F=12.6; P<0.0001). For all cows, PAG-1 concentrations increased steadily during the course of gestation, with peak concentrations recorded at parturition. Neosporosis (P=0.493), N. caninum antibody levels (P=0.921), sex of the newborn (P=0.856) and semen providing bull (P=0.087) had no effect on plasma PAG-1 concentration. There was a significant 52% increase (P<0.0001) in N. caninum antibody titers during the second half of gestation compared to the first half. The fates of the three aborting cows were abortion on gestation day 215 in one, and fetus mummification diagnosed on gestation days 180 and 210, respectively, in the remaining two cows. A luteolytic dose of prostaglandin was applied 30 days after mummification diagnosis in these last two cows, and fetus expulsion was detected on days 215 and 250, respectively. Two of the aborted fetuses were submitted to laboratory analysis and the presence of N. caninum was confirmed by specific PCR. In the cows with a mummified fetus, PAG-1 concentrations were low or undetectable when the diagnosis was made. These findings suggest that N. caninum infection has no effect on placental function in chronically infected, cows not suffering abortion, while PAG-1 measurements in aborting animals provide a useful indication of feto-placental status.

Animals↗
PubMed2006

Diagnostic survey of bovine abortion with special reference to Neospora caninum infection: importance, repeated abortion and concurrent infection in aborted fetuses in Southern Brazil.

The protozoa Neospora caninum is an important cause of bovine abortion world-wide. The objective of this survey was to determine the distribution pattern of infectious abortion in Southern Brazil with special reference to N. caninum infection. A total of 161 bovine aborted fetuses from 149 farms were analysed during a 1.5 year period. The cause of abortion was identified in 51.5% of cases. Overall, 23% (37/161) of the fetuses were considered to be infected with N. caninum. Bacterial infection accounted for 17.4% (28/161) of cases, fungal infection for 3.1% (5/161) of cases and viral aetiology for 1.8% (3/161). Six fetuses had concurrent infection with N. caninum and Leptospira spp. Data from 111 fetuses and the respective aborted cows were analysed to investigate the association between previous abortion and current N. caninum infection. The prevalence of N. caninum-infected fetuses from cows with and without a history of previous abortion was 44% (11/25) and 24.4% (21/86), respectively. Cows aborting a N. caninum-infected fetuses were 2.4 times more likely to have aborted previously than cows aborting for other reasons (95% CI of odds ratio=0.9-6.8, P=0.06).

Abortion, Veterinary↗
PubMed2006

Toxoplasma gondii and Neospora caninum infections of bovine endothelial cells induce endothelial adhesion molecule gene transcription and subsequent PMN adhesion.

Toxoplasma gondii and Neospora caninum are important, closely related coccidian parasites infecting a broad spectrum of hosts and host cells. Infections underly a complex immunological regulation; however, little is known on innate immune reactions to these parasites. To investigate interactions between infected cells and polymorphonuclear neutrophil cells (PMN), PMN adhesion to tachyzoite-infected bovine umbilical vein endothelial cells (BUVECs) under physiological flow conditions and adhesion molecule (E-selectin, P-selectin, VCAM-1, ICAM-1) gene transcription in infected BUVECs were examined in vitro for 72h post-infection (p.i.). BUVECs were rapidly invaded by T. gondii and N. caninum; in general 10-15% of the cells became infected. Tachyzoites were released from 24 and 48h p.i. onwards, for T. gondii and N. caninum, respectively. PMN adhesion to infected cell layers increased early (4h) after infection with both parasites, reached maximum levels 16-24h p.i., but remained enhanced throughout the observation period. PMN adhered to both, infected and non-infected cells within one cell layer, suggesting parasites induced paracrine activation of the BUVECs. Semiquantitative Realtime RT-PCR showed upregulated transcription of the E- and P-selectin genes in BUVECs within 1h p.i. and of ICAM-1 and VCAM-1 genes within 2h p.i. Maximum transcript levels were observed at 4-6h p.i.; the 24h p.i. gene transcription had declined to control levels. In general, T. gondii more strongly induced PMN adhesion and adhesion molecule gene transcription than N. caninum. The data suggest an effective role of PMN in innate immune reactions to these parasites.

Animals↗
PubMed2006

Astroglial cells in primary culture: a valid model to study Neospora caninum infection in the CNS.

The protozoan Neospora caninum has a veterinary importance because it causes abortion in cattle and neuromuscular alterations in dogs. We infected rat astrocytes, in vitro, with different concentrations of N. caninum. Astrocytes responded to infection by producing the pro-inflammatory cytokine TNF-alpha and the neurotoxic-free radical NO, 24 and 72 h post-infection. These data suggest that astrocytes, which are essential for brain function, are targets for the parasite and this represents a practical and valid model to study the effects of N. caninum on the CNS.

Animals↗
PubMed2005

Effects of seropositivity for bovine leukemia virus, bovine viral diarrhoea virus, Mycobacterium avium subspecies paratuberculosis, and Neospora caninum on culling in dairy cattle in four Canadian provinces.

The purpose of this research was to determine the effects of seropositivity for exposure to bovine leukemia virus (BLV), bovine viral diarrhoea virus (BVDV), Mycobacterium avium subspecies paratuberculosis (MAP) and Neospora caninum (NC) on overall and reason-specific culling in Canadian dairy cattle. Serum samples from, approximately, 30 randomly selected cows from 134 herds were tested for antibodies against BLV, MAP and NC using commercially available ELISA test kits, while 5 unvaccinated cattle over 6 months of age were tested for antibodies to bovine viral diarrhoea virus (BVDV). For analyzing the time (in days) to culling of cows after the blood testing, a two-step approach was utilized, non-parametric (Kaplan-Meier survival graphs) visualization and then semi-parametric survival modelling (Cox proportional hazards model), while controlling for confounding variables and adjusting for within herd clustering. For all reasons of culling, MAP-seropositive cows had a 1.38 (1.05-1.81, 95% C.I.) times increased hazard of culling compared to MAP-seronegative cows. Seropositivity for the other pathogens was not associated with an increased risk of overall culling. Among cows that were culled because of either decreased reproductive efficiency or decreased milk production or mastitis, MAP-seropositive cows were associated with 1.55 (1.12-2.15, 95% C.I.) times increased hazard compared to MAP-seronegative cows. Among cows that were culled because of reproductive inefficiency, NC-seropositive cows had a 1.43 (1.15-1.79, 95% C.I.) times greater hazard than NC-seronegative cows. Among cows that were culled because of decreased milk production, cows in BVDV-seropositive herds had a 1.86 (1.28-2.70, 95% C.I.) times increased hazard compared to cows in BVDV-seronegative herds. BLV-seropositive cows did not have an increased risk of reason-specific culling as compared to BLV-seronegative cows. No significant interaction on culling among seropositivity for the pathogens was detected, but only a limited number of cows tested seropositive for multiple pathogens. Results from our research will help in better understanding the economic impacts of these pathogens and justification for their control.

Animals↗
PubMed2003

A role for balance of interferon-gamma and interleukin-4 production in protective immunity against Neospora caninum infection.

A suitable balance in the production of Th1/Th2-type cytokines has a crucial role in the control of microbial infections. We investigated cytokine production patterns and effects during Neospora caninum infection, based on two mouse models and an in vitro system. In the acute infection of N. caninum, BALB/c-background IFN-gamma-deficient mice that were sensitive to the N. caninum infection showed high levels of IL-10 production, whereas significant levels of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production were observed in resistant wild type mice. BALB/c mice vaccinated with recombinant vaccinia virus expressing N. caninum surface protein NcSRS2 resisted parasite spread throughout the body, low levels of IFN-gamma production and high levels of IL-4 production were observed compared to unvaccinated animals. The treatment of N. caninum-infected cells with IFN-gamma or IL-10 decreased the host-cell viability in an in vitro system using mouse macrophage J774A.1 cells. On the other hand, IL-4, but not IL-10 administration, increased the viability of N. caninum-infected and IFN-gamma-treated cells. In the light of the balance of Th1/Th2-type cytokine production, an IFN-gamma/IL-4 balance may have a crucial role for the control of cellular responses against the parasite invasion.

Animals↗
PubMed2003

Neospora caninum in sheep: a herd case report.

Neospora caninum was detected by means of PCR in the brain of 4 out of 20 aborted fetuses in a flock of 117 sheep exhibiting a persistent abortion problem, and N. caninum tissue cysts were furthermore found in encephalitic lesions in one of the PCR-positive fetuses. Toxoplasma gondii was detected as aborting agent in another 3 out of 20 fetuses. Antibodies to N. caninum (by indirect fluorescence antibody test (IFAT)) were found in 10.3% of 117 ewes and antibodies for T. gondii were found in 97.4% of 117 ewes. Other organisms associated with abortion were Chlamydia psittaci in three fetuses and Pasteurella multocida in one fetus. This is the first report of N. caninum associated abortion in naturally infected sheep.

Aborted Fetus↗
PubMed2003

Detection of Neospora caninum in semen of bulls.

In cattle, transplacental infection is the main route of Neospora caninum transmission, but postnatal transmission by the oral uptake of sporozoite-containing oocysts shed by dogs may also be possible. Other routes of horizontal transmission, such as the venereal route, have not been investigated. In this study, we evaluated the presence of N. caninum DNA by a nested-PCR in fresh non-extended semen and frozen extended semen straws of five Holstein-Friesian bulls with naturally-acquired neosporosis. The infection status was assessed by an immunofluorescent antibody test (IFAT) and confirmed by immunoblotting (IB). Because of inhibitory components of semen, a protocol was developed to purify N. caninum DNA from bovine semen. Sporadically, N. caninum DNA was detected in non-extended fresh semen samples and frozen extended semen straws of the five seropositive bulls. In all positive samples, specific DNA was consistently found in the cell fraction of semen and not in seminal plasma. The parasite mean load in positive fresh semen samples determined by a real-time PCR was low oscillating between 1 and 2.8 parasites/ml of semen (maximum parasite load detected in one sample was 7.5 parasites/ml of semen). In parallel, another three similar but uninfected bulls acted as controls and no N. caninum DNA was amplified in any of their fresh and straw semen samples assayed. Whether venereal transmission plays a role in the spread of bovine neosporosis needs to be determined.

Animals↗
PubMed2003

Serodiagnosis of Neospora caninum infection in cattle by enzyme-linked immunosorbent assay with recombinant truncated NcSAG1.

Neospora caninum is a veterinary medically important pathogen capable of causing abortion in cattle and neuromuscular paralysis in dogs. The surface antigen 1 of N. caninum (NcSAG1) is an important candidate for the development of a diagnostic reagent for neosporosis. In order to establish an effective diagnostic method, the gene encoding truncated NcSAG1 (NcSAG1t) lacking a signal peptide and C-terminal hydrophobic regions was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The purified GST-NcSAG1t was tested in an enzyme-linked immunosorbent assay (ELISA) for the detection of N. caninum antibodies in cattle. The ELISA with GST-NcSAG1t clearly differentiated between immunofluorescent antibody test (IFAT)-positive and -negative sera from cattle. In addition, the ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Toxoplasma gondii. Field serum samples collected from cattle in Brazil were examined for the diagnosis of neosporosis by using the ELISA. Of the 197 samples analyzed, 66 (33.5%) samples were positive for antibodies to N. caninum. Of the 66 ELISA-positive samples, 60 (90%) samples were confirmed as positive by Western blot analysis with whole parasite antigens. These results suggest that the recombinant NcSAG1t could be a reliable reagent for use as an antigen in ELISA for the serodiagnosis of N. caninum infection in cattle.

Animals↗
PubMed2004

Adaptation of a commercial ELISA for the detection of antibodies against Neospora caninum in bovine milk.

The aim of the present study was to determine whether a commercially available ELISA could be used to examine bovine milk for antibodies against Neospora caninum. In an initial titration experiment, a milk dilution of 1:2 was found optimal to obtain milk results that were linearly correlated to those obtained with corresponding sera. This dilution was then used to examine 791 milk samples from N. caninum infected herds in the commercial ELISA. Milk results of individual animals were compared with those obtained by the same ELISA for the corresponding serum samples. The linear correlation between milk and serum antibody results of individual animals was characterized by R2 = 0.702. Multiple linear regression indicated that the later the stage of lactation at which an animal was sampled, the higher the milk ELISA result was as compared to the serum ELISA result. The examination of the two-graph receiver operating characteristics revealed an optimal cut-off of 0.261 to obtain similar results in the examination of milk and serum. With this cut-off, the test had a sensitivity and specificity relative to the serum results of 90%. The milk-based commercial ELISA classified more aborting dams as positive than the serum-based ELISA with this cut-off. The milk ELISA may be a valuable tool to assess the herd status with regard to abortion caused by N. caninum.

Abortion, Veterinary↗
PubMed2004

Infection by Neospora caninum associated with bovine herpesvirus 1 and bovine viral diarrhea virus in cattle from Minas Gerais State, Brazil.

Neospora caninum is a canine parasite which is considered a significant cause of bovine abortion. Two cattle herd groups were serologically studied with the objective of studying the prevalence of infection by N. caninum associated with BHV1 and BVDV infections. In group I, 15 dairy herds (476 samples) naturally infected by the three infectious agents were analyzed,. In group II, three dairy herds (100 samples) of cows vaccinated for two viruses were analyzed, in order to determine the infection prevalence by N. caninum. In the first group, an infection prevalence of 12.61, 34 and 28.3% was determined for N. caninum BHV1 and BVDV, respectively. In the second group, a seropositive prevalence of 46, 85 and 76%, respectively, was determined for N. caninum, BVH1 and BVDV. In the first group, the virus and N. caninum had shown in the first group 4.41% positive samples in association with BVH1, 3.15% with BVDV, and 8.41% with BVH1 and BVDV.

Animals↗
PubMed2004

Immune response and antigen recognition in non-pregnant ewes experimentally infected with Neospora caninum tachyzoites.

The cellular and humoral responses as well as the antigen recognition during the acute stage of a Neospora caninum (NC) infection were investigated in non-pregnant ewes. The experimentally infected ewes developed specific lymphoproliferative and humoral responses within 2 weeks post-infection (PI). The magnitude of the cellular response showed large variations between animals. A significant decrease in the proliferative response to Con A mitogen and N. caninum, Toxoplasma gondii (TG) antigens was recorded on day 21 post-infection (PI). The humoral response and the pattern of antigen recognition were similar among infected ewes. Proteins of 44, 42, 40, 39 and 28 kDa were intensively recognized by the infected animals during the experiment. The 42 and 28 kDa antigens should be considered as useful for the diagnostic of N. caninum infection, as the intensity of recognition infection of the other antigens had decreased markedly 8 weeks post-infection. For some antigens a sequential recognition was recorded. The 59, 54 and 38-37 kDa proteins were frequently recognized by infected sera during the first weeks of the infection, but recognition of these antigens was absent or rare at the end of the experiment. These antigens could be related to the acute stage of the infection.

Acute Disease↗
PubMed2005

Isolation of Neospora caninum from naturally infected white-tailed deer (Odocoileus virginianus).

Attempts were made to isolate Neospora caninum from naturally infected white-tailed deer (Odocoileus virginianus). A total of 110 deer killed during the 2003 hunting season in Virginia region were used for the isolation of N. caninum. Of these, brains from 28 deer that had NAT titer of 1:200 were inoculated into interferon-gamma gene knock out (KO) mice. N. caninum was isolated from the tissues of three deer and all three isolates were mildly virulent to KO mice. Only one of the isolates could be adapted to in vitro growth. Protozoa in the tissues of KO mice reacted with N. caninum-specific polyclonal antibodies and N. caninum DNA was demonstrated in infected tissues by PCR assays; sequences of portions of the ITS-1 and gene 5 loci were identical to those in the public database. This is the first record of in vitro isolation of N. caninum from white-tailed deer and lends credence to the white-tailed deer as an intermediate host for this parasite.

Agglutination Tests↗
PubMed2004

Vertical transmission of Neospora caninum in BALB/c mice in both acute and chronic infection.

To examine the frequency of congenital infection by Neospora caninum, BALB/c mice were inoculated intraperitoneally with tachyzoites of N. caninum either during pregnancy (Group 1) or 4 weeks or more before pregnancy (Group 2). Further, the mice inoculated during pregnancy were bred at 4 weeks or more after delivery to form Group 3. Congenital transmission was observed in 76% of the neonates of the mice in Group 1 and in 50% of the neonates of the mice in Group 2. Interestingly, congenital transmission was observed in 86% of the neonates from Group 3. These results suggest that chronically-infected BALB/c mice efficiently transmit N. caninum infection to their offspring.

Animals↗
PubMed2004

Prevalence of anti-Neospora caninum antibodies in dogs from urban, periurban and rural areas of the city of Uberlândia, Minas Gerais--Brazil.

In Brazil there are few reports on the prevalence of anti-Neospora caninum antibodies in dogs from urban, periurban and rural areas. Serum samples from 450 dogs, 300 from urban, 58 from periurban and 92 from rural areas, were tested for the detection of anti-N. caninum IgG antibodies using IFAT: indirect fluorescent antibody test (IFAT, > or =50). Antibodies were observed in 63 (14%) of the 450 serum samples analyzed, with 32 (10.7%) in dogs coming from the urban area, 11 (18.9%) from the periurban area and 20 (21.7%) from the rural area. Statistical differences were seen between the occurrences in animals from the urban area and those of the rural area (P = 0.01). The antibody titers found were: 1:50 in 20 dogs, 1:100-1:800 in 41 dogs, and 1:3200 in two dogs. In the female dogs, a smaller prevalence of dogs with antibodies was observed in those from the urban area (7.5%) in comparison with those of the rural (21.0%) (P = 0.05) and periurban (23.3%) (P = 0.01) areas. There were growing levels of antibody prevalence with the increase in age of the dogs in all three areas studied. Although this increase was not significant, it indicates a tendency towards more infections with age, suggesting post-natal exposure to N. caninum. However, a significant difference (P = 0.05) was observed in the occurrence of anti-N. caninum antibodies in dogs with ages = 2 years in urban (13.1% urban) versus rural environments (27.1% rural). Among the other age brackets studied the difference was not significant. The results confirm the presence of N. caninum in the region and reveal the important role of dogs in the parasite's epidemiology.

Age Factors↗
PubMed2004

The use of PCR to detect Neospora caninum DNA in the blood of naturally infected cows.

Twelve 2-year old heifers in their fifth month of gestation when pregnancy tested were used in this study. Six heifers aborted at approximately 4 months of gestation and had blood samples drawn less than 6 weeks after the abortions were identified. Blood samples were also drawn from three sero-positive pregnant and three sero-negative pregnant heifers. DNA was isolated from the samples and a 350 bp fragment of the Nc-5 gene was PCR amplified using primer pair Np21+ and Np6+. Also, the Internal Transcribed Spacer 1 (ITS1) was PCR amplified using Tim 3 and Tim 11 primer pair. The Nc-5 gene fragment was cloned, sequenced and the sequence BLAST-tested. Similarly, the ITS1 product was sequenced and BLAST-tested. The BLAST test results revealed that Neospora caninum DNA was present in these blood samples indicating that polymerase chain reaction can be used in the detection of N. caninum DNA in the blood of sero-positive cows.

Abortion, Veterinary↗
PubMed2004

Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in ovine from São Paulo State, Brazil.

Serum samples from 597 sheep from São Paulo State, in the southeastern region of Brazil, were tested to determine the prevalence of antibodies directed against Toxoplasma gondii (> or = 1:64) and Neospora caninum (> or = 1:50) using the indirect fluorescent antibody test (IFAT). The animals were divided into three groups based on their age: < or = 1 year, 1-4 years, and > or = 4 years. Antibodies to T. gondii were observed in 34.7% of the samples with titers ranging from 64 to 16,384 and IgG antibodies directed against N. caninum were observed in 9.2%, with titers ranging from 50 to 3200. Only 3.5% of the sheep were positive for both agents. All farms had at least one positive animal for T. gondii, and 26 of the 30 farms had at least one positive animal for N. caninum. An association between seroprevalence and age was observed for T. gondii (P = 0.001), but not to N. caninum (P = 0.343). It was not possible to associate seroprevalence to T. gondii and the presence of domestic or feral cats, since in all farms there was at least one positive sheep. There was no association between seropositivity to N. caninum and the presence of domestic (P = 1.000) and feral dogs (P = 0.550).

Age Factors↗
PubMed2004

Sero-epidemiological survey of Neospora caninum infection in dogs in north-eastern Italy.

Risk factors associated with Neospora caninum seroprevalence in north-eastern Italy in healthy dogs were assessed. Antibodies to N. caninum were found in 10.9% of 707 kennel and owned dogs by a commercial competitive ELISA (VMRD Inc.). All dogs were negative for Leishmania infantum by indirect fluorescent antibody test indicating no cross reactivity or association between the two protozoa in this area. Seroprevalence association with breed and age of dogs and other factors are discussed.

Animals↗