Molecular detection of coccidian Apicomplexa Parasites isolated from wild crab-eating and pampas foxes through novel TaqMan™ probes: a contribution to their molecular epidemiology.

Neospora caninum, Toxoplasma gondii and Hammondia spp. are coccidian parasites similar in morphology. Molecular techniques are necessary to detect parasite DNA isolated from stool samples in wild canids because they were reported as definitive hosts of N. caninum life cycle. The objective of this study was to develop a highly sensitive and accurate molecular method for the identification of coccidian Apicomplexa parasites in crab-eating fox (Cerdocyon thous) and pampas fox (Lycalopex gymnocercus). Tissue samples from road-killed animals (pampas fox = 46, crab-eating fox = 55) and feces (pampas fox = 84, crab-eating fox = 2) were collected, and species were diagnosed through molecular assay. PCR was used for the amplification of a fragment of the coccidian Apicomplexa nss-rRNA gene. Additionally, we developed a novel real-time PCR TaqMan™ probe approach to detect T. gondii- Hammondia spp. and N. caninum. This is the first report of N. caninum DNA in pampas fox feces (n = 1), thus it was also detected from pampas fox tissues (n = 1). Meanwhile, T. gondii was found in tissues of pampas (n = 1) and crab-eating (n = 1) foxes and H. triffittae in one crab-eating fox tissue. Despite the low percentage (2.5%) of positive samples, the molecular method developed in this study proved to be highly sensitive and accurate allowing to conduct an extensive monitoring analysis for these parasites in wildlife.

Function of Neospora caninum dense granule protein 7 in innate immunity in mice.

The intracellular parasite Neospora caninum can parasitize all nucleated cells of the host. Dense granule proteins (GRAs) secreted by dense granules are an important material involved in the formation of parasitophorous vacuoles (PVs), which facilitate parasite survival and replication in host cells. Due to the secretory and immune properties of NcGRA7, it is considered to be a promising serodiagnosis marker and an effective neosporosis vaccine candidate. However, the intracellular regulatory mechanisms involved in NcGRA7-induced host responses have rarely been examined. Here, we used the CRISPR/Cas9 genome editing system to obtain a NcGRA7 knockout strain (ΔNcGRA7) and a NcGRA7 complementary strain (iΔNcGRA7) to study their function. We found that ΔNcGRA7 exhibited slower growth in vitro and weakened virulence in mice compared with Nc1 and iΔNcGRA7. All parasite strains can stimulate host immune cells to produce IFN-γ, and the amount of IFN-γ production stimulated by Nc1 was significantly higher than that stimulated by ΔNcGRA7. The transcription levels of the cellular immune factors GBP1, GBP2, IRGa6, and IRGb6 were significantly higher after stimulation with ΔNcGRA7 parasites than after stimulation with Nc1. Furthermore, ΔNcGRA7 infection resulted in greater IRGa6 recruitment to the PVM than Nc1 infection. ΔNcGRA7 parasites were more easily cleared by macrophages than Nc1 parasites. Collectively, these results showed that NcGRA7 plays an important role in regulating the immune factors of mice and the aggregation of IRGa6 at the PVM, which affects the pathogenicity of N. caninum.

Experimental infection of tachyzoites of the NC1 strain of Neosporacaninum in female swine.

Neospora caninum is a protozoan that can cause reproductive problems in several animal species. Although N. caninum infection has been reported in swine, the pathogenesis and clinical signs are not fully known in this species. The objective of this work was to evaluate the effect of experimental infection with tachyzoites of the N. caninum strain Nc1 in swine matrices at different stages of gestation. For that purpose, 12 gilts, seronegative for N. caninum and T. gondii, were selected and allocated into four groups of three animals each. Animals in group A were not inoculated (control) and animals in groups B, C, and D were inoculated intravenously with of 2.9 × 107 tachyzoites, 30 days before conception, and at 45 and 90 days of gestation, respectively. Temperature, heart rate, blood, saliva, and vaginal mucus samples from the animals were collected periodically until the time of delivery for the investigation of IgG and IgM antibodies against N. caninum using IFAT and PCR to detect the parasite DNA. All gilts sero-converted from 5 and 7 DPI (days postinoculation) to IgM and IgG, respectively. Two gilts showed hypothermia on the 5th and 7th DPI, and five inoculated animals had leukocytosis on the 7th DPI. It was possible to detect DNA of N. caninum in samples of saliva (33/84), vaginal mucus (17/84), and blood (2/84). Based on serology (IgM) and PCR, three animals in group B showed evidence of reappearance of the infection during pregnancy. It is concluded that N. caninum can cause clinical signs in infected swine females, in addition to indicating saliva as a suitable diagnostic biological material for the detection of N. caninum DNA in this animal species.

Early Neospora caninum infection dynamics in cattle after inoculation at mid-gestation with high (Nc-Spain7)- or low (Nc-Spain1H)-virulence isolates.

Early Neospora caninum infection dynamics were investigated in pregnant heifers intravenously inoculated with PBS (G-Control) or 107 tachyzoites of high (G-NcSpain7)- or low (G-NcSpain1H)-virulence isolates at 110 days of gestation. Serial culling at 10 and 20 days post-infection (dpi) was performed. Fever was detected at 1 dpi in both infected groups (P < 0.0001), and a second peak was detected at 3 dpi only in G-NcSpain7 (P < 0.0001). At 10 dpi, Nc-Spain7 was detected in placental samples from one animal related to focal necrosis, and Nc-Spain7 transmission was observed, although no foetal lesions were associated with this finding. The presence of Nc-Spain1H in the placenta or foetuses, as well as lesions, were not detected at 10 dpi. At 20 dpi, G-NcSpain7 animals showed almost 100% positive placental tissues and severe focal necrosis as well as 100% transmission. Remarkably, foetal mortality was detected in two G-NcSpain7 heifers. Only one animal from G-NcSpain1H presented positive placental samples. No foetal mortality was detected, and lesions and parasite transmission to the foetus were not observed in this group. Finally, 100% of G-NcSpain7 heifers at 20 dpi presented specific antibodies, while only 60% of G-NcSpain1H animals presented specific antibodies at 20 dpi. In addition, earlier seroconversion in G-Nc-Spain7 was observed. In conclusion, tachyzoites from Nc-Spain7 reached the placenta earlier and multiplied, leading to lesion development, transmission to the foetus and foetal mortality, whereas Nc-Spain1H showed delayed infection of the placenta and no lesional development or transmission during early infection.

The pathogenicity of two Neospora caninum goat strains in a BALB/c mouse model.

Goats are frequently described as an intermediate host for the protozoan Neospora caninum, manifesting the disease mainly by recurrent abortions with placentitis and encephalitis in fetuses. Several reports of natural and experimental infections in cattle and mice show differences in the immune response, and the outcome of the infection can be variable depending on the species affected and by the behavior of the infective strain. This study describes for the first time two Neospora caninum strains isolated from naturally infected goats from the state of Minas Gerais, Brazil. One placenta and one brain from different goats were processed for a first bioassay in gerbils (Meriones unguiculatus). Subsequently, a second bioassay was performed by inoculating the processed brain samples from gerbils into Interferon gamma (IFN-γ) knockout mice (KO mice). Tachyzoites collected from the peritoneal fluid of the KO mice were inoculated into VERO cell monolayers, where they presented a very slow growth rate. The tachyzoites were also inoculated into BALB/c mice with a dose of 106 tachyzoites per animal. After a 5-week follow up, the animals infected with both of the strains developed a strong polarized Th1 response with increased serum and spleen gene expression levels of pro-inflammatory cytokines (mainly IFN-γ and TNF-α) in the first week. Tissue lesions were mild in the animals infected with both strains. Despite the strong immune response preventing an infection in the visceral organs, the parasite was able to reach the brain, causing progressive brain lesions from the second to fifth week post infection. The NC-goat1-infected mice presented with severe meningoencephalitis, but the NC-goat2-infected animals had considerable histological brain lesions only at week 5. Immunohistochemical analysis of the mouse brains revealed a different pattern of inflammatory cells compared to the naturally infected goats. A severe inflammatory infiltrate of CD3+ T lymphocytes was found in the NC-goat1-infected mice. A more discrete infiltrate of CD3+ T cells was found in the NC-goat2-infected animals. Additionally, IBA1 IHC revealed an intense microglial reaction and monocyte perivascular cuffs in the NC-goat1-infected animals and lower microglia/monocyte infiltrates in the NC-goat2-infected mice. This work contributes knowledge on the pathogenicity of new Neospora caninum strains in mice, comparable with other well-established mouse models of the disease, and demonstrates the importance of studying goats as an intermediate host of this parasite.

Immune response to Neospora caninum live tachyzoites in prepubertal female calves.

The aim of the present study was to characterize the specific immune response in prepubertal female calves inoculated with Neospora caninum. Forty-eight N. caninum-seronegative 6-month-old Angus female calves were randomly allocated into two groups: group A calves were inoculated subcutaneously (sc) with 1 × 106 tachyzoites of the low virulence NC-Argentina LP1 isolate in sterile phosphate-buffered saline (PBS); group B calves were mock inoculated sc with sterile PBS. Calves from group A developed a specific immune response characterized by the production of IgG antibodies and the expression of IFN-γ and TNF-α cytokines. Animals did not present any febrile reaction or reactions at the site of inoculation. Although chronic N. caninum infection was developed in 50% of calves of group A after inoculation, according to the presence of antibodies against rNc-SAG4, antigen characteristic of bradyzoites, N. caninum antibodies dropped below the cut-off of ELISA from day 210 post-inoculation onwards. Future trials using the same group of inoculated animals will allow the characterization of the evolution of the immune response during pregnancy and to determine whether the immunization with the local isolate is able to prevent congenital transmission and to protect against heterologous challenges.

Neospora caninum Dense Granule Protein 7 Regulates the Pathogenesis of Neosporosis by Modulating Host Immune Response.

Neospora caninum is a protozoan parasite closely related to Toxoplasma gondii Neosporosis caused by N. caninum is considered one of the main causes of abortion in cattle and nervous-system dysfunction in dogs, and identification of the virulence factors of this parasite is important for the development of control measures. Here, we used a luciferase reporter assay to screen the dense granule proteins genes of N. caninum, and we found that NcGRA6, NcGRA7, and NcGRA14 are involved in the activation of the NF-κB, calcium/calcineurin, and cAMP/PKA signals. To analyze the functions of these proteins and Neospora cyclophilin, we successfully knocked out their genes in the Nc1 strain using plasmids containing the CRISPR/Cas9 components. Among the deficient lines, the NcGRA7-deficient parasites showed reduced virulence in mice. An RNA sequencing analysis of infected macrophage cultures showed that NcGRA7 mainly regulates the host cytokine and chemokine production. The levels of gamma interferon in the ascites fluid, CXCL10 expression in the peritoneal cells, and CCL2 expression in the spleen were lower 5 days after infection with the NcGRA7-deficient parasite than after infection with the parental strain. The parasite burden and the degree of necrosis in the brains of mice infected with the NcGRA7-deficient parasite were also lower than in those of the parental strain. Collectively, our data suggest that both the NcGRA7-dependent activation of the inflammatory response and the parasite burden are important in Neospora virulence.IMPORTANCENeospora caninum invades and replicates in a broad range of host species and cells within those hosts. The effector proteins exported by Neospora induce its pathogenesis by modulating the host immunity. We show that most of the transcriptomic effects in N. caninum-infected cells depend upon the activity of NcGRA7. A deficiency in NcGRA7 reduced the virulence of the parasite in mice. This study demonstrates the importance of NcGRA7 in the pathogenesis of neosporosis.

Isolation of Neospora caninum from kidney and brain of a bovine foetus and molecular characterization in Brazil.

Bovine neosporosis has become a disease of international concern as it is among the main causes of abortion in cattle. Viable N. caninum has been isolated from brains of fetuses and neonatal calves, and there is no report of isolation of tachyzoites from kidney. Also, detailed information about the genetic diversity of N. caninum is scarce. N. caninum tachyzoites were isolated from the kidney and the brain of an aborted 4-month-old bovine foetus. The parasite was confirmed to be N. caninum by PCR. The tachyzoites of the new isolate, named BNC-PR4, were propagated in Vero cell cultures. Pathogenicity of the parasite was examined in BALB/c mice. Mice inoculated intraperitoneally with BNC-PR4 failed to yield clinical signs of disease and did not induce severe brain lesions, suggesting a bovine isolate with low virulence. The N. caninum-positive DNA sample was further analyzed by multilocus microsatellite (MS) genotyping for MS4, MS5, MS6A, MS6B, MS7, MS8, MS10, MS12, and MS21. Multilocus-microsatellite genotyping revealed a unique genetic profile that differed from previously reported isolates.

Immune response in bovine neosporosis: Protection or contribution to the pathogenesis of abortion.

Neospora caninum is a protozoan parasite with a preference for cattle and dogs as hosts. When N. caninum infection occurs in cattle it induces abortion, bovine neosporosis being a main cause of abortion worldwide. In dairy cattle, the economic burden of neosporosis-associated abortion is so great that it might results in closure of a farm. However, not all infected cows abort and it is not yet understood why this occurs. At present there is no effective treatment or vaccine. This review provides insights on how immune response against the parasite determines protection or contribution to abortion. Aspects on markers of risk of abortion are also discussed. Humoral immune responses are not protective against N. caninum but seropositivity and antibody level can be good markers for a diagnosis of bovine neosporosis and its associated abortion risk. In addition, humoral mechanisms against N. caninum infection and abortion differ in pure-breed and cross-breed pregnant dairy and beef cattle. Concentrations of Pregnancy Associated glycoprotein -2 (PAG-2) can also be used to predict abortion. A partially protective immune response encompasses increased IFN-γ expression, which has to be counterbalanced by other cytokines such as IL-12 and IL-10, especially towards the end of pregnancy. Although IFN-γ is required to limit parasite proliferation a critical threshold of the IFN-γ response is also required to limit adverse effects on pregnancy. In clinical terms, it may be stated that IFN-γ production and cross-breed pregnancy can protect Neospora-infected dairy cows against abortion.

Risk factors for Neospora caninum infection in dairy cattle and their possible cause-effect relation for disease.

Neospora caninum causes reproductive problems in cattle such as abortion, premature birth, retention of fetal membranes, and metritis. Therefore, this study aimed to verify possible risk factors for N. caninum infection in dairy cattle and their cause-effect relation to neosporosis. Serum samples of 1518 dairy cows from the West of Santa Catarina State, Southern Brazil were analyzed by indirect immunofluorescence assay (IFA) for N. caninum, where 466 were found to be positives (30.69%-CI95%; 28.3-33.0). In addition, an epidemiological survey was conducted in order to verify possible risk factors for neosporosis and their relation to the disease. The presence of dogs in the farm was strongly associated with IFA positive results for N. caninum, and lack of history for neosporosis in the farm increased the chances of positivity in 66%. It was found a significant cause-effect relation between the occurrence of reproductive problems and the presence of antibodies against N. caninum (p = 0.05). It is possible to conclude that N. caninum is widely distributed in dairy farms of the Western part of Santa Catarina state, Brazil, and that the occurrence of reproductive problems is directly related to the disease with the presence of dogs as a risk factor for N. caninum infection.

Foetal death in naive heifers inoculated with Neospora caninum isolate Nc-Spain7 at 110 days of pregnancy.

Neospora caninum infection is a leading cause of abortion in cattle worldwide. The pathogenesis of bovine neosporosis, particularly during the second term of gestation when most abortions occur in naturally infected dams, is poorly understood. In the present study foetal death was observed in 3 of 6 experimentally infected dams at 110 days of gestation after 6 weeks of experimental period. All experimental heifers were febrile between 3 and 5 days post infection (dpi). Inoculated dams seroconverted by 3-4 weeks post-infection with higher mean antibody titres in aborting dams compared to non-aborting heifers, although not significantly (p > 0.05). Neospora caninum DNA was detected in all infected foetuses and placentas, and three infected foetuses also had N. caninum antibodies. The parasite burden was higher in the brain of dead/aborted foetuses than in live foetuses. Interestingly, high IFN-γ production was detected in foetal fluids of a dead foetus found upon euthanasia of its dam, while no IFN-γ was observed in amniotic, allantoic and/or foetal fluids in the three infected foetuses that were alive upon maternal euthanasia. The present study confirms that the infection of dams on gestation day 110 with 10(7) tachyzoites of the Nc-Spain7 isolate causes abortion. The fact that some infected dams aborted and some did not is relevant to the understanding of N. caninum pathogenesis of abortion in naturally infected cows.

Negative Impact of Hypoxia on Tryptophan 2,3-Dioxygenase Function.

Tryptophan is an essential amino acid for hosts and pathogens. The liver enzyme tryptophan 2,3-dioxygenase (TDO) provokes, by its ability to degrade tryptophan to N-formylkynurenine, the precursor of the immune-relevant kynurenines, direct and indirect antimicrobial and immunoregulatory states. Up to now these TDO-mediated broad-spectrum effector functions have never been observed under hypoxia in vitro, although physiologic oxygen concentrations in liver tissue are low, especially in case of infection. Here we analysed recombinant expressed human TDO and ex vivo murine TDO functions under different oxygen conditions and show that TDO-induced restrictions of clinically relevant pathogens (bacteria, parasites) and of T cell proliferation are abrogated under hypoxic conditions. We pinpointed the loss of TDO efficiency to the reduction of TDO activity, since cell survival and TDO protein levels were unaffected. In conclusion, the potent antimicrobial as well as immunoregulatory effects of TDO were substantially impaired under hypoxic conditions that pathophysiologically occur in vivo. This might be detrimental for the appropriate host immune response towards relevant pathogens.

Comparative genomics of the apicomplexan parasites Toxoplasma gondii and Neospora caninum: Coccidia differing in host range and transmission strategy.

Toxoplasma gondii is a zoonotic protozoan parasite which infects nearly one third of the human population and is found in an extraordinary range of vertebrate hosts. Its epidemiology depends heavily on horizontal transmission, especially between rodents and its definitive host, the cat. Neospora caninum is a recently discovered close relative of Toxoplasma, whose definitive host is the dog. Both species are tissue-dwelling Coccidia and members of the phylum Apicomplexa; they share many common features, but Neospora neither infects humans nor shares the same wide host range as Toxoplasma, rather it shows a striking preference for highly efficient vertical transmission in cattle. These species therefore provide a remarkable opportunity to investigate mechanisms of host restriction, transmission strategies, virulence and zoonotic potential. We sequenced the genome of N. caninum and transcriptomes of the invasive stage of both species, undertaking an extensive comparative genomics and transcriptomics analysis. We estimate that these organisms diverged from their common ancestor around 28 million years ago and find that both genomes and gene expression are remarkably conserved. However, in N. caninum we identified an unexpected expansion of surface antigen gene families and the divergence of secreted virulence factors, including rhoptry kinases. Specifically we show that the rhoptry kinase ROP18 is pseudogenised in N. caninum and that, as a possible consequence, Neospora is unable to phosphorylate host immunity-related GTPases, as Toxoplasma does. This defense strategy is thought to be key to virulence in Toxoplasma. We conclude that the ecological niches occupied by these species are influenced by a relatively small number of gene products which operate at the host-parasite interface and that the dominance of vertical transmission in N. caninum may be associated with the evolution of reduced virulence in this species.

Neospora caninum infection during early pregnancy in cattle: how the isolate influences infection dynamics, clinical outcome and peripheral and local immune responses.

This work studies the influence of Neospora caninum intra-species diversity on abortion outcome, infection dynamics in terms of parasite dissemination and peripheral-local immune responses in pregnant cattle. Animals were intravenously inoculated at day 70 of pregnancy with 107 tachyzoites of two isolates showing marked differences in virulence in vitro and in pregnant mouse models: Nc-Spain7, a high virulence isolate, and Nc-Spain8, a low-to-moderate virulence isolate. After inoculation, pregnancy was monitored, and dams were culled when foetal death was detected. Foetal mortality occurred in all infected heifers between days 24 and 49 post-infection (pi), however, it was detected sooner in Nc-Spain7-infected animals (median day = 34) than those inoculated with Nc-Spain8 (median day = 41) with a trend towards significance (P < 0.11). Similar histological lesions were observed in placentomes and in most of the foetuses from the two infected groups. However, parasites were more frequently detected in the placenta and foetuses by PCR and in the foetal brain by immunohistochemistry in Nc-Spain7-infected animals. Specific antibodies were detected starting at day 13 post-infection in all infected cattle, with higher IgG levels in Nc-Spain7-infected group. IFN-γ and IL-4 profiles also varied between infected groups in PBMC stimulation assays. Infected animals showed significant increases in their cytokine mRNA levels (IFN-γ, IL-4, IL-10, IL-12p40 and TNF-α) in the caruncle at time of foetal death. Differences between the infected groups were also observed for cytokine profiles. These results demonstrate the influence of the N. caninum isolate on foetal death outcome, infection dynamics and immune responses in cattle.

Clinical outcome and vertical transmission variability among canine Neospora caninum isolates in a pregnant mouse model of infection.

We compared the clinical outcome and vertical transmission of six canine Neospora caninum isolates using a pregnant BALB/c model. Four of the isolates were obtained from oocysts of naturally infected dogs (Nc-Ger2, Nc-Ger3, Nc-Ger6 and Nc-6Arg) and two were from diseased dogs with neurological signs (Nc-Bahia and Nc-Liv). The dams were inoculated with 2×106 tachyzoites of each isolate at day 7 of pregnancy. Morbidity, mortality and the antibody responses were evaluated in both the dams and the offspring, as was parasite transmission to the progeny. The mortality rates varied from 100% in Nc-Bahia and Nc-Liv-infected pups to 19% or less for those infected with the isolates from oocysts. The vertical transmission rates varied from 9 to 53% for N. caninum from oocysts, compared with 100% for the Nc-Liv and Nc-Bahia isolates. All dams showed specific IgG responses against tachyzoite and rNc-GRA7 antigens, confirming Neospora infection. The highest IgG levels were detected in mice inoculated with the Nc-Liv and Nc-Bahia isolates. These results demonstrate marked differences in virulence between the N. caninum isolates obtained from oocysts and neurologically affected dogs. This variability could help us to explain the differences in the outcome of the infection in definitive and intermediate hosts.

Pathogenicity of Nc-Bahia and Nc-1 strains of Neospora caninum in experimentally infected cows and buffaloes in early pregnancy.

Neospora caninum is a protozoan parasite known as an important cause of bovine abortion worldwide. Little is currently known about how different strains of N. caninum vary in their pathogenicity. In this study, we compared a Brazilian strain, Nc-Bahia, with the first isolate of this coccidian, Nc-1. Eight cows and seven buffaloes were submitted to fixed-time artificial insemination protocols for a better control of pregnancy. Group 1 was inoculated with Nc-Bahia (n = 8; five cows and three buffaloes), and Group 2 was inoculated with Nc-1 (n = 5; two cows and three buffaloes). One nonpregnant female of each species was left uninfected as sentinel controls for potential environmental infection. All inoculated animals received 5 × 10(8) tachyzoites of N. caninum, by intravenous route, on the 70th day of gestation. Uninfected animals remained seronegative throughout the experiment, indicating no exogenous infection, whereas all inoculated animals became seropositive to N. caninum. In Group 1, abortion was found in only one cow on 42 days postinfection (dpi; frequency of abortion = 12.5%), whilst all animals from Group 2 aborted on 35 dpi (frequency of abortion = 100%). Parasite DNA was detected by seminested PCR in maternal, foetal and placental tissues, confirming vertical transmission in Groups 1 and 2, although histological lesions had different frequencies and degrees of severity between the groups. There was evidence of lower pathogenicity of Nc-Bahia compared to Nc-1 when used in experimental infection, as it caused fewer abortions, as well as less frequent and milder histological lesions. This was the first time Nc-Bahia has been used for experimental infection.

Effect of vaccination of cattle with the low virulence Nc-Spain 1H isolate of Neospora caninum against a heterologous challenge in early and mid-gestation.

Live vaccines have emerged as one of the most potentially cost-effective measures for the control of bovine neosporosis. Previous studies have shown that Nc-Spain 1H is a naturally attenuated isolate of Neospora caninum and that immunisation with live Nc-Spain 1H tachyzoites generated a protective immune response in mice. The aim of this study was to evaluate the safety and efficacy of immunisation in cattle. N. caninum-seronegative heifers were immunised subcutaneously twice with 107 live Nc-Spain 1H tachyzoites prior to artificial insemination. No adverse reactions or negative effects on reproductive parameters were recorded following immunisation. In immunised and non-challenged heifers, no foetal deaths were observed, and none of the calves was congenitally infected. The efficacy against N. caninum-associated foetal death and vertical transmission was determined after challenge with high doses of the Nc-1 isolate at 70 and 135 days of gestation, respectively. After the challenge in early gestation, the immunisation induced a protection of 50% against foetal death. In addition, the microsatellite analysis performed in PCR-positive tissue samples from foetuses that died after challenge infection showed that the profiles corresponded to the challenge isolate Nc-1. A degree of protection against vertical transmission was observed after challenge at mid-gestation; calves from immunised heifers showed significantly lower pre-colostral Neospora-specific antibody titres than calves from the non-immunised/challenge group (P < 0.05). Strong antibody and interferon gamma responses were induced in the immunised heifers. This study indicates that the immunisation before pregnancy with the Nc-Spain 1H vaccine isolate appeared to be safe and reduced the occurrence of N. caninum-associated abortion and vertical transmission in experimentally infected cattle. In light of these encouraging results, the next step for testing this live attenuated candidate should be the assessment of its efficacy and safety in naturally infected cattle.

Specific antibody responses against Neospora caninum recombinant rNcGRA7, rNcSAG4, rNcBSR4 and rNcSRS9 proteins are correlated with virulence in mice.

The intraspecific diversity of Neospora caninum is a determinant for in vivo parasite virulence and in vitro parasite behaviour. The relationship between isolate virulence and specific antibody responses against key parasite proteins has not been well characterized. The response kinetics and the differences in specific anti-rNcGRA7, -rNcSAG4, -rNcBSR4 and -rNcSRS9 antibody levels were analysed by recombinant protein-based ELISA in groups of mice inoculated with 10 different N. caninum isolates that differ in their virulence. The majority of the virulence parameters analysed correlated with the specific antibody levels against the 4 recombinant proteins. The antibodies developed against the highly immunogenic protein NcGRA7 were significantly higher in mice inoculated with high virulence isolates than in those inoculated with low-to-moderate virulence isolates in both non-pregnant and pregnant mouse models. Moreover, these levels were correlated with the anti-N. caninum IgG1 and IgG2a responses and the in vitro tachyzoite yield at 56 h. The antibodies directed against the bradyzoite-specific proteins were not detected in a non-pregnant mouse model. However, some seropositive mice were found in groups inoculated with high virulence isolates in a pregnant mouse model. NcGRA7 and NcSAG4 are proteins clearly correlated with virulence, and to a lesser extent NcBSR4 and NcSRS9 proteins. Moreover, antibodies to bradyzoite-specific proteins appear to also be related to virulence in mice. Further analyses should be performed in order to verify the usefulness of these proteins as predictive markers for virulence in an experimental bovine model of neosporosis.

Mice congenitally infected with low-to-moderate virulence Neospora caninum isolates exhibited clinical reactivation during the mating period without transmission to the next generation.

Endogenous transplacental transmission (EnTT) is the major transmission route of Neospora caninum in cattle. Thus, the development of a standardised experimental model of EnTT is needed for more appropriate testing of parasite biology and control strategies. A recent study reported up to 40-50% EnTT rates in chronically infected dams with either high or low-to-moderate virulence isolates, although low fertility rates were observed in dams inoculated with the high virulence isolate. Therefore, low-to-moderate virulence N. caninum isolates (Nc-Spain 3H; G1 and Nc-Spain 8; G2) that previously showed high TT rates versus low mortality and morbidity rates in a congenital mouse model were inoculated into BALB/c dams (first generation). The new approach followed in the present study aimed to start with a high number of congenitally infected mice (second generation), which allowed a more efficient EnTT from congenitally infected dams to their progeny (third generation). Interestingly, a reactivation of infection occurred in several congenitally infected non-pregnant females (second generation) from both infected groups. This fact was evidenced by neosporosis-associated clinical signs after mating accompanied by an increase of specific antibody levels (IgG1, IgG2a and specific antibodies against rNcGRA7) (P<0.0001; one-way ANOVA). Moreover, a higher number of PCR-positive mice compared to pregnant females were observed (P<0.05; Fisher's exact test). These results support the hypothesis that only mice without clinical signs and with a low parasite burden in the brain became pregnant, which may explain the posterior failure in inducing EnTT from the second to the third generation. These findings confirm that this mouse model is not a suitable experimental EnTT model for testing the efficacy of drugs and vaccine candidates against EnTT. The employment of other putative suitable species with a similar placenta structure, such as small ruminants, should be taken into consideration.

Neospora caninum NC-6 Argentina induces fetopathy in both serologically positive and negative experimentally inoculated pregnant dams.

Neospora caninum infection is a major cause of abortion in cattle. The objectives of this study were to genetically characterize the N. caninum NC-6 Argentina isolate using a multilocus microsatellite analysis approach and to study its biological behavior by experimental inoculations into seronegative and seropositive pregnant cattle, evaluating the humoral and cellular immune response elicited and the occurrence of transplacental transmission and fetopathy. Pregnant cows (65 days of gestation) seropositive and seronegative to N. caninum were intravenously inoculated with tachyzoites of the NC-6 Argentina N. caninum strain and slaughtered at 108 ± 2 days of gestation. Serum samples were analyzed for N. caninum antibodies by indirect fluorescent antibody test. The cellular immune response was analyzed by detection of gamma interferon (γIFN) production in blood cells. Tissue samples from dams, fetuses, and placental cotyledons were processed by histopathological and immunohistochemical techniques and examined for N. caninum DNA by PCR. Positive DNA samples were further analyzed by multilocus microsatellite typing for N. caninum. Inoculated animals had significantly higher N. caninum antibody titers and γIFN production than control animals. One seropositive inoculated cow aborted, one seronegative cow had a non-viable fetus, and the remaining fetuses from the experimentally inoculated dams had histopathologic lesions. The PCR was positive in 3/4 fetuses from seronegative inoculated cows and in 2/3 fetuses from seropositive inoculated cows. Multilocus microsatellite analysis revealed that the N. caninum DNA present in fetuses and placentas had an identical pattern to NC-6 Argentina strain. The NC-6 Argentina strain proved to be able to cross the placenta and to induce fetopathy in both the seropositive and seronegative dams.