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PubMed1999

Effect of polyclonal antisera developed against dense granule-associated Neospora caninum proteins on cell invasion and development in vitro by N. caninum tachyzoites.

The effects of polyclonal antisera to 2 recombinant dense granule (DG) antigens of Neospora caninum on invasion and development by N. caninum tachyzoites in baby hamster kidney cell cultures were examined. In immunofluorescent antibody tests, the anti-DG sera, at dilutions of 1:100 to 1:500, reacted intensely with individual intracellular tachyzoites and groups of tachyzoites enclosed in parasitophorous vacuoles at 2 and 52 h p.i., respectively. Tachyzoites suspended in diluted anti-DG sera and inoculated immediately into the cell cultures invaded cells in significantly fewer numbers (53-68% fewer by 2 h p.i.) than tachyzoites suspended in similarly diluted normal rabbit serum. In contrast, tachyzoites suspended in anti-Eimeria tenella sporozoite serum invaded cells as efficiently as those suspended in normal rabbit serum. Addition of anti-DG sera at the time of inoculation of tachyzoites, or to the cell cultures after the parasites had entered cells, had little effect on either the percentage or rate of their subsequent development.

Animals↗
PubMed2000

Isolation, characterization and expression of a GRA2 homologue from Neospora caninum.

A cDNA library derived from mRNA of tachyzoites of Neospora caninum (NC-Liverpool strain) was screened with antisera from a cow naturally infected with N. caninum. The DNA sequence of 1 recombinant isolated predicted a significant protein sequence homology of the gene product to the 28 kDa (GRA2) antigen of Toxoplasma gondii. Studies on the N. caninum gene coding for this antigen demonstrated the presence of a single intron flanked by 2 exons; the gene was also highly expressed in culture-derived tachyzoites. The antigen was expressed in Escherichia coli; when injected into mice it stimulated the production of antibodies which detected a 29 kDa antigen of N. caninum. Secondary structure predictions made for the N. caninum protein showed support for several amphipathic helices separated by loops and turns. The available evidence indicates maintenance of protein secondary structure, and not DNA or amino acid sequence, has occurred during the evolution of GRA2 proteins in N. caninum and T. gondii.

Amino Acid Sequence↗
PubMed2000

Neospora caninum-associated abortion in cattle: the time of experimentally-induced parasitaemia during gestation determines foetal survival.

The parasite, Neospora caninum is an important cause of abortion in cattle. It is transmitted vertically or horizontally and infection may result in abortion or the birth of a live, healthy but infected calf at full-term. Only a proportion of infected cattle abort and the pathogenesis of abortion is not understood. Groups of cattle were infected with 10(7) N. caninum tachyzoites intravenously at different times relative to gestation. Intravenous inoculation was chosen to reproduce the putative haematogenous spread of N. caninum following either recrudescence of endogenous infection or de novo infection. In all cattle, infection was accompanied by high gamma-interferon and lymphoproliferative responses, and a biased IgG2 response indicating that N. caninum infection is accompanied by a profound Th1 helper T cell-like response. Infection at 10 weeks gestation resulted in foetopathy and resorption of foetal tissues 3 weeks after infection in 5 out of 6 cows. Infection at 30 weeks gestation resulted in the birth of asymptomatic, congenitally-infected calves at full term in all 6 cows, whereas the 6 cows infected before artificial insemination gave birth to live, uninfected calves. These results suggest that the reason some cows abort is related to the time during gestation when they become infected or an existing infection recrudesces.

Abortion, Veterinary↗
PubMed1998

Characterization of Neospora caninum iscom antigens using monoclonal antibodies.

Neospora caninum is a cyst-forming coccidian parasite which in cattle can cause abortion and birth of feeble calves. For the serological diagnosis of neosporosis, antibodies directed to the parasite can be demonstrated by ELISA, utilizing N. caninum proteins incorporated into iscoms (immunostimulating complexes) as antigen. Electrophoretic and immunoblotting studies had earlier revealed that N. caninum iscoms contained a restricted number of proteins compared with soluble parasite extracts, but the cellular origin of the incorporated proteins has not yet been determined. In the present study, monoclonal antibodies were raised against N. caninum iscoms. Six of these, named Ncmab-4, 7, 10, 13, 17 and 24, were used to characterize the N. caninum iscom antigen. The nature of the reactive epitopes and their localization within N. caninum tachyzoites were determined by means of immunological methods, including immunoblot, IFAT and immunogold electron microscopy. The apparent molecular weights of the dominant iscom antigens were found to be 18, 30, 32, 41 and 61 kDa. While the 61 kDa antigen was located intracellularly, the others were found on the parasite surface as well as within distinct intracellular compartments.

Animals↗
PubMed1998

Cellular immune responses in cattle experimentally infected with Neospora caninum.

Neospora caninum has recently been identified as an important cause of infectious abortion in cattle. The parasite is closely related to Toxoplasma gondii, but the two species are antigenically distinct. To examine cell proliferative responses and the induction of IFN-gamma in experimentally infected cattle, four 2-4 months old calves were subcutaneously inoculated with N. caninum tachyzoites. Peripheral blood mononuclear cells were collected regularly and stimulated in vitro with a crude lysate of N. caninum or T. gondii tachyzoites. Significant proliferative responses to N. caninum antigen were recorded in all calves from days 4-6 postinoculation. This response was accompanied by production of high levels of IFN-gamma. Although the calves remained seronegative to T. gondii, while seroconverting to N. caninum, stimulation with T. gondii lysate resulted in cell proliferation of a similar magnitude as that obtained using the N. caninum lysate. However, the T. gondii lysate appeared less effective than the N. caninum lysate to stimulate IFN-gamma production. Cells taken from uninfected control animals did not show any significant proliferation to either N. caninum or T. gondii antigen and no IFN-gamma was produced. These results suggest that the two parasites may possess cross-reacting T-cell epitopes, but that the T cells specific for N. caninum may have a different functional capacity. This highlights the need to investigate the antigen specificity and cytokine profile of T cells from infected animals to help understand their role in immunity to N. caninum.

Animals↗
PubMed1999

Susceptibility of B-cell deficient C57BL/6 (microMT) mice to Neospora caninum infection.

Neospora caninum is a coccidian parasite of veterinary importance by causing abortion or stillbirth in cattle among other problems in diverse animal species. We assessed an experimental murine model for its suitability to study the immune response to N. caninum infection. Thus, wild-type (wt) C57BL/6 mice and B-cell (and consequently antibody)-deficient microMT mice were infected with N. caninum tachyzoites and sacrificed at days 10, 24 and 29-44 post infection (dpi). Various organs were collected for parasitological and pathological analysis, spleen and serum for immunological investigations. Splenocytes were in vitro-stimulated with N. caninum (NC)- and T. gondii-antigens for assessing T cell proliferation and cytokine production. While wt mice were resistant to disease, microMT mice died starting from 29 dpi onwards. Histological examination of brain tissue from microMT mice exhibited a high infection intensity with multifocal necrotic cerebral lesions, which were absent in the brains of wt mice. NC antigen-stimulated spleen cells of both wt and microMT mice infected with N. caninum showed a marked proliferative depression at 10 dpi. At 24 dpi, this immunosuppression was still maintained in microMT mice whereas it was restored in wt mice. Stimulated splenocytes of infected microMT mice secreted significantly less IFN-gamma and less IL-10 than corresponding wt splenocytes. For IL-10, this difference increased with time. The susceptibility of microMT mice appeared associated to B-cell deficiency, allowing the persistent spread of the parasite causing immunosuppression and finally resulting in a lethal outcome of infection.

Animals↗
PubMed2001

A previous infection with Toxoplasma gondii does not protect against a challenge with Neospora caninum in pregnant sheep.

Sheep immunized with Toxoplasma gondii (Toxovax) prior to pregnancy were tested for their ability to withstand a challenge at 90 days gestation with 107 Neospora caninum (NC1) tachyzoites. The antibody responses in sheep following immunization with T. gondii were specific for T. gondii whereas peripheral blood mononuclear cells responded to both T. gondii and N. caninum antigen in vitro. This suggested that there was induction of crossreactive immune recognition in the sheep, at least at the cellular level. Following challenge of sheep at mid-gestation with N. caninum, no febrile responses were recorded in the group of sheep which had previously received Toxovax while significant febrile responses were recorded in the group of sheep which received N. caninum challenge alone. Antibody responses to N. caninum developed in all sheep following N. caninum challenge and antibody responses to T. gondii were boosted in the group of sheep which had previously been immunized with Toxovax. No antibodies to T. gondii were observed in the sheep which received the N. caninum challenge alone. Peripheral blood mononuclear cells from both groups of sheep responded to T. gondii and N. caninum antigen in vitro and interferon gamma was present in the cell-free supernatant from activated cells. However despite evidence of the induction of crossreactive immunity between T. gondii and N. caninum, this was not sufficient to prevent foetal death. The group of sheep which had received Toxovax prior to pregnancy and the group of sheep which only received the N. caninum challenge experienced 100% foetal death compared with 0% in the unchallenged control group. Vaccination prior to pregnancy with Toxovax did protect against foetal death following oral challenge at 90 days with 2000 T. gondii oocysts which caused 100% foetal death in a control challenge group.

Animals↗
PubMed2002

Mechanisms of apoptosis in murine fibroblasts by two intracellular protozoan parasites, Toxoplasma gondii and Neospora caninum.

Studies to clarify the mechanisms of apoptosis in host cells, A31 (BALB/3T3 clone A31 fibroblasts), caused by two intracellular protozoan parasites, Toxoplasma gondii and Neospora caninum, were carried out in an in vitro system. The viability of N. caninum-infected cells was significantly reduced following treatment with mouse interferon (IFN)-gamma. By contrast, mouse IFN-gamma treatment had no significant effect on the induction of apoptosis in T. gondii-infected cells. Apoptosis of N. caninum-infected and mouse IFN-gamma-treated cells was shown to be associated with increased DNA fragmentation, and increased caspase-3 and -8 activity, and the administration of caspase-3 and -8 inhibitors inhibited cell death. FasL expression was clearly induced by N. caninum-infection and IFN-gamma treatment compared to the T. gondii-infected cells and the uninfected control with or without IFN-gamma treatment. The reduction in host-cell viability was prevented with the addition of antimouse FasL monoclonal antibody (mAb). Moreover, TUNEL analyses indicated that apoptosis was induced by the treatment with Fas mAb in both T. gondii and N. caninum-infected cells. These results suggest that the Fas/FasL pathway may play a crucial role in the induction of apoptosis in N. caninum- and T. gondii-infected cells mediated by IFN-gamma.

Animals↗
PubMed2002

Necrotizing cerebellitis due to Neospora caninum infection in an old dog.

A severe, necrotizing, non-suppurative inflammation of the cerebellum associated with Neospora caninum infection was identified in a 14-year-old male Labrador Retriever. On presentation, clinical signs included mild depression and head tremor, marked ataxia of both thoracic and pelvic limbs, and abnormal postural reactions. In the central nervous system, inflammatory lesions were mainly restricted to the cerebellar leptomeninges and cerebellar cortex, which appeared necrotic and atrophic. Protozoal organisms were positively stained with an anti-N. caninum antibody in an immunohistochemical procedure.

Animals↗
PubMed2003

Reproductive losses due to Neospora caninum in a beef herd in Argentina.

Reproductive losses in a beef herd of 857 heifers with a pregnancy rate of 86.3% are described. After pregnancy testing, 69 abortions were seen during a 3 month period. Before calving season, three heifers had delivered pre-mature non-viable calves. Serum samples from 58 of 69 aborted heifers were available for serological tests. In order to compare the seroprevalence in non-aborted vs. aborted heifers, 214 pregnant animals were bleed during the abortion storm. In addition, blood samples were collected from two heifers with pre-mature calves and from 16 heifers with their calves prior to colostrum intake. All available serum samples were tested for Neospora caninum antibodies using an indirect fluorescence antibody test (IFAT). Fifty-nine of 290 (20.3%) evaluated heifers were seropositive. Heifers that aborted and heifers with pre-mature calves were more likely to be seropositive than pregnant heifers and heifers with normal calves [odds ratio (OR), 12.01; 95% CI, 6.18-23.30]. Vaginal mucus from four aborted heifers, and samples from two aborted foetuses and three pre-mature calves were available. Laboratory tests for Tritrichomonas foetus, bacterial and viral isolation, and histological examination were performed. Culture from vaginal mucus and foetal samples were negative. Histological lesions consistent with neosporosis and positive immunohistochemistry (IHC) to N. caninum were found in one aborted foetus and in one pre-mature calf. It is the first description of reproductive losses because of N. caninum in beef herds in Argentina.

Abortion, Veterinary↗
PubMed2003

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.

Animals↗
PubMedDate not supplied

Use of a serum-free medium to produce in vitro Neospora caninum and Toxoplasma gondii tachyzoites on Vero cells.

Neospora caninum and Toxoplasma gondii are cyst-forming coccidian parasites of human and veterinary clinical relevance. In vitro cultivation of the protozoans using Vero cells is usually performed in order to produce antigenic materials. Quantitative and qualitative comparisons of Vero cells grown in RPMI medium supplemented either with foetal calf serum (FCS), horse serum (HS) or a specific serum-free additive (DefCell) were performed. A serum-free cell culture system used to propagate N. caninum (NC-1 isolate) and T. gondii tachyzoites (Rh stain) were compared with the other two cell culture systems. FCS supplemented media was found to be more effective than the others in promoting Vero cells and N. caninum tachyzoites. However, it was found unable to support adequate T. gondii tachyzoite proliferation. Vero cells, T. gondii and N. caninum tachyzoite production gave similar growth patterns with either HS or DefCell supplemented media. Defcell was considered as a good alternative to supplement culture medium.

Animals↗
PubMedDate not supplied

Survival, immune responses and tissue cyst production in outbred (Swiss white) and inbred (CBA/Ca) strains of mice experimentally infected with Neospora caninum tachyzoites.

The present work compared inbred (CBA/Ca) and outbred (Swiss white) strains of mice for their capacity to cope with a Neospora caninum infection and to consistently produce tissue cysts. In each experiment Swiss white and CBA/Ca mice were given three different doses of NC-1 tachyzoites. Lymphoproliferative and humoral responses as well as cytokine production were evaluated eight weeks after infection (PI) whereas tissue cyst production and histopathology were assessed 4, 6 and 10 weeks PI in immunosuppressed mice. Tissue cysts were observed 10 weeks after infection only in CBA/Ca mice receiving the two highest inoculum doses. Furthermore this strain showed the highest specific lymphoproliferative response. A mixed cytokine response with elevated IFN-gamma and fairly low IL-4 and IL-10 secretion was recorded. In both strains, no lesions were observed in the tissues of infected mice. This study indicates that CBA/Ca female mice infected with 5 x 10(6) NC-1 tachyzoites represent a useful model for the study of specific maternal immune responses in pregnant animals.

Animals↗
PubMedDate not supplied

Finding of Neospora caninum in the wild brown rat (Rattus norvegicus).

Nine rats (16.4%) out of 55 (Rattus norvegicus) from cattle farms were seropositive to Neospora caninum. Two of the seropositive rats were also PCR positive but all were negative by immunohistochemistry and PAS staining. The brains of all the captured rats were homogenized and initially inoculated intraperitoneally into nude mice or into SPF ICR mice, which had been immunosuppressed with prednisolone. One mouse that was inoculated with brain material from a seropositive rat became infected with N. caninum, as demonstrated by the presence of a tissue cyst in the brain and confirmed by immunohistochemistry and PCR. This is the first finding of N. caninum in naturally infected farm rats. The findings show that natural N. caninum infection occurs in wild brown rats and thus rats may serve as a reservoir for the protozoan on the cattle farm.

Animals↗
PubMed2001

Neosporosis in Mexican dairy herds: lesions and immunohistochemical detection of Neospora caninum in fetuses.

Of 211 aborted bovine fetuses collected from Mexican dairy herds between January 1996 and March 1999, 73 showed microscopical lesions consistent with neosporosis. Of these 73 fetuses, 58 (79%) showed lymphocytic myocarditis, 39 (53%) showed microgliosis and multifocal necrosis in the brain, 39 (53%) showed lymphocytic hepatitis, and 19 (26%) showed lymphocytic myositis. Immunohistochemical examination of brain, myocardium and liver from 53 of the same 73 fetuses demonstrated Neospora caninum antigens in 41 (77%), of which 19 (46%) gave positive results in one of the three sites, 15 (37%) in two, and seven (17%) in three. The results indicated the presence of neosporosis in a number of the main dairy farming regions of Mexico.

Abortion, Veterinary↗
PubMed1999

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

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

Animals↗
PubMed2001

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.

Animals↗
PubMed2000

Neospora caninum and bovine virus diarrhoea virus infections in Swedish dairy cows in relation to abortion.

The protozoan parasite Neospora caninum and bovine virus diarrhoea virus (BVDV) are recognized as important causes of bovine abortion and congenital disease worldwide. In this study, serological investigations were performed to estimate the prevalence of N. caninum infection in Swedish dairy cattle, to assess to what extent it may affect abortion rates, and to determine possible effects of coinfection with BVDV. The overall N. caninum seroprevalence in Swedish dairy cows was estimated at 2% (16/780), and the BVDV seroprevalence was 32% (249/780). Among aborting cows from herds with abortion problems, 7% (26/378) had antibodies to N. caninum and 42% (153/378) to BVDV. Seventeen of the N. caninum positive animals also had antibodies to BVDV. There was a statistically significant (P = 0.013) association between presence of antibodies to N. caninum and BVDV. In a case-control study comprising sera from cows in herds without recognized abortion problems, 6% (5/89) and 1% (1/89) of sera from aborting and non-aborting cows, respectively, had antibodies to N. caninum. Two of the N. caninum seropositive aborting cows also had antibodies to BVDV. These results confirm that N. caninum infection is associated with bovine abortion in Sweden and also indicate that there might be concurrent effects of N. caninum and BVDV. It is concluded that Swedish dairy cows have a low prevalence of N. caninum infection which is favourable in relation to possible future control programmes.

Abortion, Veterinary↗