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The apicomplexan parasite Neospora caninum is an important causative agent of congenital neosporosis, resulting in abortion, birth of weak offspring and neuromuscular disorders in cattle, sheep, and many other species. Among several compound classes that are currently being developed, two have been reported to limit the effects of congenital neosporosis: (i) bumped kinase inhibitors (BKIs) target calcium dependent protein kinase 1 (CDPK1), an enzyme that is encoded by an apicoplast-derived gene and found only in apicomplexans and plants. CDPK1 is essential for host cell invasion and egress; (ii) endochin-like quinolones (ELQs) are inhibitors of the cytochrome bc1 complex of the mitochondrial electron transport chain and thus inhibit oxidative phosphorylation. We here report on the in vitro and in vivo activities of BKI-1748, and of ELQ-316 and its respective prodrugs ELQ-334 and ELQ-422, applied either as single-compounds or ELQ-BKI-combinations. In vitro, BKI-1748 and ELQ-316, as well as BKI-1748 and ELQ-334, acted synergistically, while this was not observed for the BKI-1748/ELQ-422 combination treatment. In a N. caninum-infected pregnant BALB/c mouse model, the synergistic effects observed in vitro were not entirely reproduced, but 100% postnatal survival and 100% inhibition of vertical transmission was noted in the group treated with the BKI-1748/ELQ-334 combination. In addition, the combined drug applications resulted in lower neonatal mortality compared to treatments with single drugs., Graphical abstract Image 1, Highlights • BKI-1748 is a bumped kinase inhibitor with anti-Neospora activity in vitro and in vivo • Endochin-like quinolones target the cytochrome bc1 complex and exhibit anti-Neospora properties. • Single and combined applications of these two compound classes do not interfere in pregnancy outcome in mice • BKI-1748/ELQ-334 combination treatment results in 100% postnatal survival and blocks vertical transmission in the neosporosis mouse model

The intraspecific variability among Neospora caninum isolates in their in vitro behaviour and in vivo virulence has been widely studied. In particular, transcriptomic and proteomic analyses have shown a higher expression/abundance of specific genes/proteins in high-virulence isolates. Consequently, the dense granule protein NcGRA7 and the rhoptry protein NcROP40 were proposed as potential virulence factors. The objective of this study was to characterize the role of these proteins using CRISPR/Cas9 knockout (KO) parasites in a well-established pregnant BALB/c mouse model of N. caninum infection at midgestation. The deletion of NcGRA7 and NcROP40 was associated with a reduction of virulence, as infected dams displayed milder clinical signs, lower parasite burdens in the brain, and reduced mortality rates compared to those infected with the wild-type parasite (Nc-Spain7). Specifically, those infected with the NcGRA7 KO parasites displayed significantly milder clinical signs and a lower brain parasite burden. The median survival time of the pups from dams infected with the two KO parasites was significantly increased, but differences in neonatal mortality rates were not detected. Overall, the present study indicates that the disruption of NcGRA7 considerably impairs virulence in mice, while the impact of NcROP40 deletion was more modest. Further research is needed to understand the role of these virulence factors during N. caninum infection.

Neospora caninum is an apicomplexan protozoan and a major cause of abortion in cattle worldwide. In the Argentinian Humid Pampa, bovine neosporosis causes severe economic losses. Despite this, information on the genetic structure of N. caninum in this region is limited. Therefore, this study aimed to genetically characterize N. caninum isolates associated with bovine abortion in the Humid Pampa region. For this purpose, spontaneous bovine fetal tissues submitted for diagnosis to the Veterinary Diagnostic Service at INTA Balcarce during 2008-2019 were assessed by PCR, indirect fluorescent antibody test (IFAT), and histologic analysis. PCR-positive samples were tested by multilocus microsatellite genotyping (MLGs) using 9 microsatellite markers. Thirty-one different genotypes were identified from 32 samples with at least seven markers. Argentinian MLGs were grouped into two clonal clusters when analyzed using eBURST network and principal coordinate analysis. No segregation based on the year of collection, animal biotype, or geographic origin was observed. In addition, the presence of linkage disequilibrium supported the clonal propagation of Argentinian MLGs. One Argentinian subpopulation was associated with isolates from Spain, Uruguay, Brazil, and Mexico, and the other one was linked to isolates from Scotland, Spain, and Germany. These findings reveal the presence of two clonal subpopulations of N. caninum in the Humid Pampa.

Rodents and other micromammals constitute important reservoirs of infectious diseases; their role in the life cycle of apicomplexan parasites such as Toxoplasma gondii, Neospora caninum, and Sarcocystis spp. still needs clarification. In the present study, we analyzed by PCR and Sanger sequencing methods the presence of specific parasite DNA within brain and heart tissues of 313 individuals of five synanthropic small mammal species (Apodemus sylvaticus, Mus spretus, M. musculus, Rattus rattus, and Crocidura russula) collected in Barcelona metropolitan area (NE Spain). In addition, PCR-RFLP and microsatellites were also used as tools for genotypic characterization of T. gondii and N. caninum, respectively. Specific DNA of T. gondii, N. caninum, and Sarcocystis spp. was detected in 0.3% (n = 1), 1.3% (n = 4), and 3.8% (n = 12) of the animals, respectively. No mixed infections were observed. Crocidura russula stood out as the main host for Sarcocystis spp. Toxoplasma gondii-specific DNA detected in a house rat was genetically characterized by PCR-RFLP, presenting type II and III alleles (SAG1 [II], SAG3 [II], GRA6 [II], c22-8 [III], Apico [III]). Also, unsuccessful DNA sequencing and microsatellite typing were attempted in N. caninum-positive samples, which suggested a lack of PCR specificity and open avenues to speculate the host competence of rodents for N. caninum. Likewise, Sarcocystis spp. identity was studied by alignment and phylogenetic analyses of cox1 and 28S rRNA sequences from the 14 positive samples. It resulted in at least three unknown organisms closely similar (95.7-100% cox1-sequence homology) to Sarcocystis pantherophisi from the Eastern rat snake (Pantherophis alleghaniensis) (KU891603), suggesting together with 28S rRNA sequences analyses, three Sarcocystis sp. with a life cycle conformed by rodents as intermediate host (IH) and snakes as definitive hosts (DH) infecting the periurban micromammals surveyed. Prevalence figures found in this first survey carried out in Spain agree with other international studies focused on periurban areas. Further surveys should be conducted in farms and their surroundings in order to unravel the role of wild micromammals in the epidemiology of such protozoan parasites affecting our livestock, and therefore human population.

The biological variability among Neospora caninum isolates has been widely shown, however, the molecular basis that determines this diversity has not been thoroughly elucidated to date. The latest studies have focused on a limited number of isolates. Therefore, the goal of the present study was to compare the proteome of a larger number of N. caninum isolates with different origins and virulence. Label-free LC-MS/MS was used to investigate the tachyzoite proteomic differences among Nc-Bahia, Nc-Spain4H and Nc-Spain7, representing high virulence isolates and Nc-Ger6, Nc-Spain2H and Nc-Spain1H, representing low virulence isolates. Pairwise comparisons between all isolates and between high virulence and low virulence groups identified a subset of proteins with higher abundance in high virulence isolates. These proteins were involved in energy and redox metabolism, and DNA/RNA processing, which might determine the faster growth rates and parasite survival of the high virulence isolates. Highlighted proteins included a predicted member of the rhoptry kinase family ROP20 specific for N. caninum, Bradyzoite pseudokinase 1 and several dense granule proteins. DNA polymerase, which was more abundant in all high virulence isolates in all comparisons, might also be implicated in virulence. These results reveal insights into possible mechanisms involved in specific phenotypic traits and virulence in N. caninum, and the relevance of these candidate proteins for N. caninum virulence deserves further investigation.

The Neospora caninum Calcium-dependent protein kinase 1 (NcCDPK1) inhibitor BKI-1294 had demonstrated excellent efficacy in a pregnant mouse model of neosporosis, and was also highly efficacious in a pregnant sheep model of toxoplasmosis. In this work, we present the efficacy of BKI-1294 treatment (dosed 5 times orally every 48 h) starting 48 h after intravenous infection of sheep with 105 Nc-Spain7 tachyzoites at mid-pregnancy. In the dams, BKI-1294 plasma concentrations were above the IC50 for N. caninum for 12–15 days. In treated sheep, when they were compared to untreated ones, we observed a minor increase in rectal temperature, higher IFNγ levels after blood stimulation in vitro, and a minor increase of IgG levels against N. caninum soluble antigens through day 28 post-infection. Additionally, the anti-NcSAG1 and anti-NcSAG4 IgGs were lower in treated dams on days 21 and 42 post-infection. However, BKI-1294 did not protect against abortion (87% foetal mortality in both infected groups, treated and untreated) and did not reduce transplacental transmission, parasite load or lesions in placentomes and foetal brain. The lack of foetal protection was likely caused by short systemic exposure in the dams and suboptimal foetal exposure to this parasitostatic drug, which was unable to reduce replication of the likely established N. caninum tachyzoites in the foetus at the moment of treatment. New BKIs with a very low plasma clearance and good ability to cross the blood-brain and placental barriers need to be developed., Graphical abstract Image 1, Highlights • BKI-1294 triggers a transiently decrease in anti-N. caninum IgG levels in sheep. • BKI-1294 does not protect against abortion and vertical transmission of N. caninum. • The efficacy of BKI-1294 is different in pregnant mice and sheep models of neosporosis.

Bovine neosporosis is currently considered one of the main causes of abortion in cattle worldwide and the outcome of the infection is, in part, determined byNeospora caninumisolate virulence. However, the dam and foetal immune responses associated with this factor are largely unknown. We used a model of bovine infection at day 110 of gestation to study the early infection dynamics (10- and 20-days post-infection, dpi) after experimental challenge with high- and low-virulence isolates ofN. caninum(Nc-Spain7 and Nc-Spain1H, respectively). In the present work, dam peripheral cellular immune responses were monitored twice a week from -1 to 20 dpi. At different time points, IFN-γ and IL-4 production was investigated in stimulated dam blood and the percentage of monocytes, NK cells, B cells and T cells (CD4+, CD8+ and γδ) in peripheral blood mononuclear cells (PBMC) were determined by flow cytometry. In addition, maternal iliofemoral lymph nodes and foetal spleen and thymus were collected at 10 and 20 dpi for the study of the same cell subpopulations. Peripheral immune response dynamics were similar after the infection with both isolates, with a significant increase in the percentage of CD4+ T cells at 6 and 9 dpi in PBMC, coincident with the higher levels of IFN-γ and IL-4 release. However, the levels of IFN-γ were significantly higher and an increase in CD8+ T cells at 9, 13 and 20 dpi was observed in the dams infected with Nc-Spain7. Nc-Spain1H infection induced higher IL4 levels in stimulated blood and a higher CD4+/CD8+ ratio in PBMC. The analysis of the maternal iliofemoral lymph node showed a significant enhancement in the percentage of NK, CD4+ and CD8+ T cells for the animals infected with the highly virulent isolate and euthanized at 20 dpi. Regarding the foetal responses, the most remarkable result was an increase in the percentage of monocytes at 20 dpi in the spleen of foetuses from both infected groups, which suggests that foetuses were able to respond toN. caninuminfection at mid gestation. This work provides insights into how isolate virulence affects the maternal and foetal immune responses generated againstN. caninum, which may influence the course of infection.

Neospora caninum and Toxoplasma gondii are one of the main concerns of the livestock sector as they cause important economic losses in ruminants due to the reproductive failure. It is well-known that the interaction of these parasites with the placenta determines the course of infection, leading to fetal death or parasite transmission to the offspring. However, to advance the development of effective vaccines and treatments, there are still important gaps on knowledge on the placental host-parasite interactions that need to be addressed. Ruminant animal models are still an indispensable tool for providing a global view of the pathogenesis, lesions, and immune responses, but their utilization embraces important economic and ethics restrictions. Alternative in vitro systems based on caruncular and trophoblast cells, the key cellular components of placentomes, have emerged in the last years, but their use can only offer a partial view of the processes triggered after infection as they cannot mimic the complex placental architecture and neglect the activity of resident immune cells. These drawbacks could be solved using placental explants, broadly employed in human medicine, and able to preserve its cellular architecture and function. Despite the availability of such materials is constrained by their short shelf-life, the development of adequate cryopreservation protocols could expand their use for research purposes. Herein, we review and discuss existing (and potential) in vivo, in vitro, and ex vivo ruminant placental models that have proven useful to unravel the pathogenic mechanisms and the host immune responses responsible for fetal death (or protection) caused by neosporosis and toxoplasmosis.

This is a review of the development of bumped-kinase inhibitors (BKIs) for the therapy of One Health parasitic apicomplexan diseases. Many apicomplexan infections are shared between humans and livestock, such as cryptosporidiosis and toxoplasmosis, as well as livestock only diseases such as neosporosis. We have demonstrated proof-of-concept for BKI therapy in livestock models of cryptosporidiosis (newborn calves infected with Cryptosporidium parvum), toxoplasmosis (pregnant sheep infected with Toxoplasma gondii), and neosporosis (pregnant sheep infected with Neospora caninum). We discuss the potential uses of BKIs for the treatment of diseases caused by apicomplexan parasites in animals and humans, and the improvements that need to be made to further develop BKIs.

Despite the importance of bovine neosporosis, relevant knowledge gaps remain concerning the pathogenic mechanisms of Neospora caninum. Infection of the placenta is a crucial event in the pathogenesis of the disease, however, very little is known about the relation of the parasite with this target organ. Recent studies have shown that isolates with important variations in virulence also show different interactions with the bovine trophoblast cell line F3 in terms of proliferative capacity and transcriptome host cell modulation. Herein, we used the same model of infection to study the interaction of Neospora with these target cells at the proteomic level using LC-MS/MS over the course of the parasite lytic cycle. We also analysed the proteome differences between high- (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates. The results showed that mitochondrial processes and metabolism were the main points of Neospora-host interactions. Interestingly, Nc-Spain1H infection showed a higher level of influence on the host cell proteome than Nc-Spain7 infection.

Background Neospora caninum is an obligate intracellular parasite, and its ability to survive inside host immune cells may be a key mechanism for the establishment of infection in cattle. In vitro studies carried out by our group have shown that N. caninum is able to replicate in bovine macrophages (MØs), alter their microbicidal mechanisms and exploit their motility. Furthermore, host-cell control seems to be isolate virulence-dependent. Methods To investigate the molecular basis underlying the innate responses in MØs against N. caninum and the mechanisms of parasite manipulation of the host cell environment, the transcriptome profile of bovine monocyte-derived MØs infected with high-virulence (Nc-Spain7) or low-virulence (Nc-Spain1H) N. caninum isolates was studied. Results Functional enrichment revealed upregulation of genes involved in chemokine signalling, inflammation, cell survival, and inhibition of genes related with metabolism and phagolysosome formation. MØs activation was characterized by the induction of a predominantly M1 phenotype with expression of TLR2, TLR3 and TLR9 and activation of the NF-ƙB signalling pathway. Heat-killed N. caninum tachyzoites failed to activate NF-ƙB, and to inhibit lysosomal activity and apoptosis, which indicates active modulation by the parasite. The FoxO signalling pathway, Th1-Th2 differentiation, glycosaminoglycan degradation and apoptosis were pathways enriched only for low virulent Nc-Spain1H infection. In addition, Nc-Spain1H infection upregulated the IL12A and IL8 pro-inflammatory cytokines, whereas IL23 was downregulated by high virulent Nc-Spain7. Conclusions This study revealed mechanisms implicated in the recognition of N. caninum by bovine MØs and in the development of the subsequent immune response. NF-ƙB seems to be the main signalling pathway implicated in the pro-inflammatory bovine MØs response against this pathogen. Apoptosis and phagolysosome maturation are processes repressed by N. caninum infection, which may guarantee its intracellular survival. The results also indicate that Nc-Spain7 may be able to partially circumvent the pro-inflammatory response whereas Nc-Spain1H induces a protective response to infection, which may explain the more efficient transmission of the high-virulence Nc-Spain7 isolate observed in vivo.

19 páginas, 9 figuras, 1 tabla., Neospora caninum is an apicomplexan cyst-forming parasite that is considered one of the main causes of abortion. The pathogenic mechanisms associated with parasite virulence at the maternal-foetal interface that are responsible for the outcome of infection are largely unknown. Here, utilizing placentomes from cattle experimentally infected with high-virulence (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates, we studied key elements of the innate and adaptive immune responses, as well as components of the extracellular matrix (ECM), at 10 and 20 days post-infection (dpi). The low-virulence isolate elicited a robust immune response characterized by upregulation of genes involved in pathogen recognition, chemokines and pro-inflammatory cytokines, crucial for its adequate control. In addition, Nc-Spain1H triggered the expression of anti-inflammatory cytokines and other mechanisms implicated in the maintenance of ECM integrity to ensure foetal survival. In contrast, local immune responses were initially (10 dpi) impaired by Nc-Spain7, allowing parasite multiplication. Subsequently (20 dpi), a predominantly pro-inflammatory Th1-based response and an increase in leucocyte infiltration were observed. Moreover, Nc-Spain7-infected placentomes from animals carrying non-viable foetuses exhibited higher expression of the IL-8, TNF-α, iNOS and SERP-1 genes and lower expression of the metalloproteases and their inhibitors than Nc-Spain7-infected placentomes from animals carrying viable foetuses. In addition, profound placental damage characterized by an alteration in the ECM organization in necrotic foci, which could contribute to foetal death, was found. Two different host-parasite interaction patterns were observed at the bovine placenta as representative examples of different evolutionary strategies used by this parasite for transmission to offspring., This work was supported by the Spanish Ministry of Economy and Competi‑ tiveness (AGL2013-44694-R and AGL2016-75935-C2-1-R) and the Community of Madrid (PLATESA2-CM P2018/BAA-4370). Laura Jiménez-Pelayo was fnan‑ cially supported by a fellowship from the University Complutense of Madrid (including two research stays in 2017 and 2018) and Marta García-Sánchez was fnancially supported through a grant from the Spanish Ministry of Economy and Competitiveness (BES-2014-070723). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

5 páginas, 2 tablas, 2 figuras., Neosporosis and besnoitiosis, caused by cyst-forming protozoa Neospora caninum and Besnoitia besnoiti, respectively, are parasitic infestations of livestock in Israel. These parasites cause significant economic losses in cattle due to reproductive and productive disorders. Both parasites have been detected in several wild ruminant species throughout other regions of the world, while the existence of a sylvatic life cycle in Israel remains uncertain. Thus, a wide panel of 871 sera from two wild carnivores and nine wild ruminant species were tested. All sera were first analysed by MAT for an initial screening and positive samples were confirmed a posteriori by Western blot. Additionally, a complementary IFAT was used for the detection of antibodies against N. caninum. Neospora antibodies were present in six out of the 11 species investigated, whereas Besnoitia antibodies were undetected. Golden jackal, red fox, addax, Arabian oryx, Persian fallow deer, mouflon, mountain gazelle, Nubian ibex, scimitar horned oryx and water buffalo were seropositive against N. caninum infection by IFAT and/or MAT. Moreover, the presence of Neospora spp.-specific antibodies was confirmed by Western blot in golden jackal (6/189; 3.2%), red fox (1/75; 1.3%), Persian fallow deer (13/232; 5.6%), mouflon (1/15; 16.7%), Nubian ibex (22/ 55; 40%) and water buffalo (12/18; 66.7%). Addax (1/49) and water buffalo (1/18) were MAT-seropositive against B. besnoiti but were seronegative by Western blot. Hence, Neospora sylvatic cycle is present in Israel and may cross over to a domestic life cycle. In contrast, wildlife species investigated are unlikely to present a risk of transmitting Besnoitia to livestock in Israel., Part of this work was supported by a research project from the Spanish Ministry of Science and Innovation (AGL 2010-20561) and by The Israeli Services and Animal Health (Ministry of Agriculture and Rural Development). Daniel Gutierrez Exposito was financially supported by the Ministry of Science and Innovation (grant no. BES-2011-043753). The authors would like to thank the rangers of the Israel Nature & Parks Authority for their cooperation. Finally, we also acknowledge Elena Blinder, Ricardo Wolkomirsky and Noam Sugar for their excellent technical assistance.

Herein we describe, to our knowledge for the first time the use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9 (CRISPR/Cas9) system for genome editing of Neospora caninum, an apicomplexan parasite considered one of the main causes of abortion in cattle worldwide. By using plasmids containing the CRISPR/Cas9 components adapted to the closely related parasite Toxoplasma gondii, we successfully knocked out a green fluorescent protein (GFP) in an Nc-1 GFP-expressing strain, and efficiently disrupted the NcGRA7 gene in the Nc-Spain7 isolate by insertion of a pyrimethamine resistance cassette. The successful use of this technology in N. caninum lays the foundation for an efficient, targeted gene modification tool in this parasite.

15 páginas, 2 tablas, 7 figuras., Experimental infections in pregnant sheep have been focused on studying the effect of the time of challenge on the outcome of N. caninum infection, whereas the impact of the dose and route of challenge has not been studied in depth. Therefore, clinical outcome, immune responses, parasite detection and burden, and lesion severity in placental tissues and foetal brains were investigated in 90-day-pregnant sheep inoculated intravenously with 10(5) (G1), 10(4) (G2), 10(3) (G3), or 10(2) (G4) tachyzoites or subcutaneously with 10(4) (G5) tachyzoites of the virulent Nc-Spain7 isolate and an uninfected group (G6). Comparing challenge doses, G1 was the only group that had 100% abortion. Likewise, IFN gamma levels in G1 increased earlier than those in other intravenously infected groups, and IgG levels on day 21 post-infection (pi) were higher in G1 than those in other intravenously infected groups. Concerning vertical transmission, G1 shows a higher parasite burden in the foetal brain than did G2 and G3. Comparing routes of administration, no differences in foetal survival rate or parasite load in the foetal brain were found. Although G2 had higher IFN gamma levels than G5 on day 10 pi, no differences were found in humoral immune responses. Because the outcome after intravenous infection with 10(5) tachyzoites was similar to that observed after intravenous infection with 10(6) tachyzoites used in a previous work (100% abortion and vertical transmission), we conclude that it may be reasonable to use 10(5) tachyzoites administered by the intravenous route in further experiments when assessing drugs or vaccine candidates., We gratefully acknowledge Neus Callizo Esteve from Clinical Veterinary Hospital (Complutense University of Madrid, Spain) and Jose Maria Gonzalez (University of Zaragoza, Spain) for their excellent technical assistance. The Animal Experimentation Service (SEA) at the University of Zaragoza is acknowledged for using their facilities to carry out the reproduction programme. Roberto Sanchez Sanchez is supported by a fellowship from the Spanish Ministry of Education, Culture and Sports (MECD) as a part of the Programme of Training of University Teaching Staff (FPU, Grant Number FPU13/03438). This work was supported by the Ministry of Economy and Competitiveness (AGL2016-75935-C2) and Community of Madrid, Spain (PLATESA, S2013/ABI2906).

13 páginas, 2 tablas, 7 figuras., Neospora caninum is one of the main causes of abortion in cattle, and recent studies have highlighted its relevance as an abortifacient in small ruminants. Vaccines or drugs for the control of neosporosis are lacking. Bumped kinase inhibitors (BKIs), which are ATP-competitive inhibitors of calcium dependent protein kinase 1 (CDPK1), were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. We here present the pharmacokinetics, safety and efficacy of BKI-1553 in pregnant ewes and foetuses using a pregnant sheep model of N. caninum infection. BKI-1553 showed exposure in pregnant ewes with trough concentrations of approximately 4 mu M, and of 1 mu M in foetuses. Subcutaneous BKI-1553 administration increased rectal temperatures shortly after treatment, and resulted in dermal nodules triggering a slight monocytosis after repeated doses at short intervals. BKI-1553 treatment decreased fever in infected pregnant ewes already after two applications, resulted in a 37-50% reduction in foetal mortality, and modulated immune responses; IFN. levels were increased early after infection and IgG levels were reduced subsequently. N. caninum was abundantly found in placental tissues; however, parasite detection in foetal brain tissue decreased from 94% in the infected/untreated group to 69-71% in the treated groups. In summary, BKI-1553 confers partial protection against abortion in a ruminant experimental model of N. caninum infection during pregnancy. In addition, reduced parasite detection, parasite load and lesions in foetal brains were observed., We gratefully acknowledge Jacobo Sampere from the SALUVETgroup (Complutense University of Madrid, Spain), Teresa Navarro andJosé María González (University of Zaragoza, Spain), Isabel Ayala,Lucas Troya, Clara Colmenero, Dolores González, Natalia Peral,Verónica Pérez, Ignacio Gómez and Isabel Santiago from the Clinical Veterinary Hospital (Complutense University of Madrid, Spain) for their excellent technical assistance, and also Rama Subba Rao Vidadala and Dustin Maly (Department of Chemistry, University of Washington) fortheir assistance in further purification of the study drug. The AnimalExperimentation Service (SEA) at the University of Zaragoza is ac-knowledged for providing their facilities to carry out the reproduction programme. Roberto Sánchez Sánchez is supported by a fellowship from the Spanish Ministry of Education, Culture and Sports (MECD), asa part of the Program of Training of University Teaching Staff (FPU,grant number FPU13/03438). Patricia Vázquez has a Juan de la Cierva-Formación post-doctoral contract (FJCI-2014-20982) from the Spanish Ministry of Economy and Competitiveness (MINECO). Andrew Hemphill is supported by the Swiss National Science Foundation grant(No. 310030 165782). This work was supported by the Public Health Service, National Institutes of Health, Bethesda, MD (grants R01 AI111341 and R01 HD 080670), the U.S. Department of Agriculture(grant 2014-67015-22106) and the Community of Madrid, Spain(PLATESA, S2013/ABI2906).

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.

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a chronic and debilitating disease that causes systemic and skin manifestations and sterility in bulls. Neither treatments nor vaccines are currently available. In the search for therapeutic candidates, calcium-dependent protein kinases have arisen as promising drug targets in other apicomplexans (e.g. Neospora caninum, Toxoplasma gondii, Plasmodium spp. and Eimeria spp.) and are effectively targeted by bumped kinase inhibitors. In this study, we identified and cloned the gene coding for BbCDPK1. The impact of a library of nine bumped kinase inhibitor analogues on the activity of recombinant BbCDPK1 was assessed by luciferase assay. Afterwards, those were further screened for efficacy against Besnoitiabesnoiti tachyzoites grown in Marc-145 cells. Primary tests at 5µM revealed that eight compounds exhibited more than 90% inhibition of invasion and proliferation. The compounds BKI 1294, 1517, 1553 and 1571 were further characterised, and EC99 (1294: 2.38µM; 1517: 2.20µM; 1553: 3.34µM; 1571: 2.78µM) were determined by quantitative real-time polymerase chain reaction in 3-day proliferation assays. Exposure of infected cultures with EC99 concentrations of these drugs for up to 48h was not parasiticidal. The lack of parasiticidal action was confirmed by transmission electron microscopy, which showed that bumped kinase inhibitor treatment interfered with cell cycle regulation and non-disjunction of tachyzoites, resulting in the formation of large multi-nucleated complexes which co-existed with viable parasites within the parasitophorous vacuole. However, it is possible that, in the face of an active immune response, parasite clearance may occur. In summary, bumped kinase inhibitors may be effective drug candidates to control Besnoitiabesnoiti infection. Further in vivo experiments should be planned, as attainment and maintenance of therapeutic blood plasma levels in calves, without toxicity, has been demonstrated for BKIs 1294, 1517 and 1553.

Neospora caninum is one of the most efficient transplacentally transmitted pathogens in cattle and is a cause of abortion in this domestic species. The invasion and proliferation of Neospora caninum in the placenta and its dissemination to the foetus are crucial events in the outcome of an infection. In the bovine placenta, the placentomes are formed by maternal caruncles, which are delimited by a maternal epithelium and foetal cotyledons, which are delimited by an epithelial layer named the trophoblast. These epithelia form a physical barrier against foetal infection. Furthermore, trophoblast cells act as an innate immune defence at the foetal-maternal interface. Neospora caninum invades and proliferates in trophoblast cells in vitro, but it is unknown whether host cell modulation events, which affect the immune response and other processes in the trophoblast, occur. In this work, we investigated the transcriptomic modulation by Neospora caninum infection in the bovine trophoblast cell line F3. In addition, two Neospora caninum isolates with marked differences in virulence, Nc-Spain1H and the Nc-Spain7, were used in this study to investigate the influence of these isolates in F3 modulation. The results showed a clear influence on extracellular matrix reorganisation, cholesterol biosynthesis and the transcription factor AP-1 network. Interestingly, although differences in the transcriptome profiles induced by each isolate were observed, specific isolate-modulated processes were not identified, suggesting very similar regulation in both isolates. Differential expression of the N. caninum genes between both isolates was also investigated. Genes involved in host cell attachment and invasion (SAG-related and microneme proteins), glideosome, rhoptries, metabolic processes, cell cycle and stress response were differentially expressed between the isolates, which could explain their variability. This study provides a global view of Neospora caninum interactions with bovine trophoblast cells and of the intra-specific differences between two Neospora caninum isolates with biological differences.

Exposure of Neospora caninum tachyzoites to BKI-1294 in vitro results in the formation of long-lived multinucleated complexes (MNCs). However, in vivo treatment of BALB/c mice with BKI-1294 shortly after N. caninum infection during pregnancy was safe and profoundly reduced pup mortality and vertical transmission. We hypothesized that the formation of MNCs could trigger immune responses that contribute to BKI efficacy in vivo. In this study, mice were first vaccinated with a sublethal dose of N. caninum tachyzoites and were treated with BKI-1294. We then investigated the effects of these treatments after mating and re-infection during pregnancy. Effects on fertility, pup survival, vertical transmission, and parasite load in dams were evaluated. Cytokines in sera or splenocyte culture supernatants were assessed by either ELISA or the Luminex™ 200 system, and humoral immune responses against tachyzoite and MNC antigens were compared by ELISA, Western blotting and immunoproteomics. Our results showed that BKI-1294 treatment of live-vaccinated mice reduced the cerebral parasite load in the dams, but resulted in higher neonatal pup mortality and vertical transmission. In live-vaccinated mice, cytokine levels, most notably IFN-y, IL-10, and IL-12, were consistently lower in BKI-1294 treated animals compared to non-treated mice. In addition, comparative Western blotting identified two protein bands in MNC extracts that were only recognized by sera of live-vaccinated mice treated with BKI-1294, and were not found in tachyzoite extracts. We conclude that treatment of live-vaccinated mice with BKI-1294 influenced the cellular and humoral immune responses against infection, affected the safety of the live-vaccine, and decreased protection against re-infection and vertical transmission during pregnancy.

Neospora caninum is an apicomplexan protozoan responsible for abortion in ruminants. The present study aimed to diagnose an abortion from an Anglo Nubian goat from a dairy herd located in Buenos Aires province, Argentina. The goat delivered a fetus of approximately 3 months gestation that was studied by indirect fluorescent antibody test (IFAT), Immunoblot (IB), histopathology (HP), immunohistochemistry (IHC), and molecular assays (PCR, sequencing and microsatellite genotyping). Interferon gamma knock-out mice were inoculated with a pool of tissues for isolation attempts. The mother had IFAT titers of 1:3200 and 1:400 for N. caninum and Toxoplasma gondii, respectively, as well as positive IB reactions, whereas the fetus was seronegative to both parasites by IFAT and IB. The fetus had severe multifocal necrotizing myocarditis and hepatitis, moderate interstitial pneumonia, and nephritis. Myocardium sample resulted positive by IHC, evidencing clusters of N. caninum tachyzoites within myocardiocytes associated with histopathological lesions. Neospora caninum-DNA was detected by PCR in heart, liver, lungs, kidney, and muscle from the fetus, and was negative for T. gondii by PCR. NC-5 and 18 S rRNA gene fragment sequences showed 100% identity with N. caninum. Inoculated mice bled 30 days post-inoculation resulted seronegative to N. caninum and T. gondii by IFAT, and showed no clinical signs. Multilocus-microsatellite genotyping revealed a genetic profile that differed from previously reported N. caninum genotypes, with unique MS21 and MS10 alleles. These findings indicate that N. caninum was efficiently transmitted from the mother to the fetus. We report the first case of direct detection of N. caninum in a goat fetus in Argentina and N. caninum microsatellite genotyping in naturally infected goat. Fil: Campero, Lucía María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina Fil: Gos, Maria Laura. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina Fil: Moore, Dadin Prando. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Regidor Cerrillo, Javier. Universidad Complutense de Madrid; España Fil: Unzaga, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina Fil: Moré, Gastón Andrés. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina Fil: Ortega Mora, Luis Miguel. Universidad Complutense de Madrid; España Fil: Venturini, María Cecilia. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina

Neospora caninum is considered one of the main causes of abortion in cattle but can also cause abortion in sheep. There is limited knowledge of the N. caninum population infecting sheep, and only one N. caninum isolate from a pregnant sheep from Japan has been reported. This study describes the in vitro isolation and genetic characterization of two new sheep isolates of N. caninum implicated in ovine reproductive failure. We used IFN-γ-knockout mice inoculated with PCR-positive brain homogenates from two clinically healthy but congenitally infected lambs at 4.5 months of age for parasite isolation. The lambs were born to dams from a sheep farm that had experienced pregnancy failure caused by N. caninum in successive generations. Tachyzoites were microscopically visualized in peritoneal flushes from all inoculated mice and were also observed in MARC-145 cell cultures within one week after inoculation with peritoneal flushes. Two N. caninum isolates, Nc-Spain11 and Nc-Spain12, were obtained from each lamb. The genotyping of the Nc-Spain11 and Nc-Spain12 isolates based on 9 microsatellite markers showed identical multilocus genotype (MLG). Comparison between a previous N. caninum genotype dataset including 80 MLGs from Argentinean, Spanish, Mexican, German and Scottish bovine isolates and the Japanese sheep isolate showed that the Nc-Spain11 and Nc-Spain12 MLG was unique and differed from the other MLGs. eBURST analyses showed that the Nc-Spain11 and Nc-Spain12 MLG was genetically clustered with other bovine MLGs and one ovine MLG, and the nearest genetic relationship was with an MLG from a bovine abortion collected in the same geographical area of Galicia.

Neospora caninum , a cyst-forming apicomplexan parasite, is a leading cause of neuromuscular diseases in dogs as well as fetal abortion in cattle worldwide. The importance of the domestic and sylvatic life cycles of Neospora , and the role of vertical transmission in the expansion and transmission of infection in cattle, is not sufficiently understood. To elucidate the population genomics of Neospora , we genotyped 50 isolates collected worldwide from a wide range of hosts using 19 linked and unlinked genetic markers. Phylogenetic analysis and genetic distance indices resolved a single genotype of N. caninum . Whole-genome sequencing of 7 isolates from 2 different continents identified high linkage disequilibrium, significant structural variation, but only limited polymorphism genome-wide, with only 5,766 biallelic single nucleotide polymorphisms (SNPs) total. Greater than half of these SNPs (∼3,000) clustered into 6 distinct haploblocks and each block possessed limited allelic diversity (with only 4 to 6 haplotypes resolved at each cluster). Importantly, the alleles at each haploblock had independently segregated across the strains sequenced, supporting a unisexual expansion model that is mosaic at 6 genomic blocks. Integrating seroprevalence data from African cattle, our data support a global selective sweep of a highly inbred livestock pathogen that originated within European dairy stock and expanded transcontinentally via unisexual mating and vertical transmission very recently, likely the result of human activities, including recurrent migration, domestication, and breed development of bovid and canid hosts within similar proximities.

Intraspecific differences in biological traits between Neospora caninum isolates have been widely described and associated with variations in virulence. However, the molecular basis underlying these differences has been poorly studied. We demonstrated previously that Nc-Spain7 and Nc-Spain1H, high- and low-virulence isolates, respectively, show different invasion, proliferation and survival capabilities in bovine macrophages (boMØs), a key cell in the immune response against Neospora, and modulate the cell immune response in different ways. Here, we demonstrate that these differences are related to specific tachyzoite gene expression profiles. Specifically, the low-virulence Nc-Spain1H isolate showed enhanced expression of genes encoding for surface antigens and genes related to the bradyzoite stage. Among the primary up-regulated genes in Nc-Spain7, genes involved in parasite growth and redox homeostasis are particularly noteworthy because of their correlation with the enhanced proliferation and survival rates of Nc-Spain7 in boMØs relative to Nc-Spain1H. Genes potentially implicated in induction of proinflammatory immune responses were found to be up-regulated in the low-virulence isolate, whereas the high-virulence isolate showed enhanced expression of genes that may be involved in immune evasion. These results represent a further step in understanding the parasite effector molecules that may be associated to virulence and thus to disease traits as abortion and transmission.

Recent studies have revealed extensive genetic variations among Neospora caninum, a cyst-forming protozoan parasite that is one of the main causes of bovine abortion in the cattle industry worldwide. Previous genetic studies based on multilocus microsatellite genotyping (MLGs) of different Ibero-American populations showed a high genetic diversity. These studies provided clear clues of a predominant clonal propagation in cattle and population sub-structuring partially associated with geographical origin. Although, these reports were limited to a reduced number of countries. In this study, the N. caninum isolates from aborted bovine fetuses and stillbirths and a goat abortion from Northern Italy were investigated genetically using 9 microsatellite markers. Complete or nearly complete isolate profiles were obtained from 30 fetuses and stillbirths. An extensive genetic diversity was also found in this Italian N. caninum population. The study of genetic relationships among Italian MLGs using network (eBURST) and principal component analyses based on the allele-sharing coefficient (PCoA) showed different clonal subpopulations disseminated throughout Northern Italy without apparent segregation depending on the geographic origin, cattle breed, or time of collection. The presence of linkage disequilibrium supports a predominant clonal propagation of Italian N. caninum. In addition, most of Italian MLGs segregated from other global populations including Spain, Argentina, Mexico, Brazil, Germany, and Scotland, suggesting the existence of specific N. caninum subpopulations in the Northern Italy and different subpopulations of N. caninum circulating in Europe.

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., Facultad de Ciencias Veterinarias

Background Bovine neosporosis, one of the main causes of reproductive failure in cattle worldwide, poses a challenge for the immune system of pregnant cows. Changes in the Th-1/Th-2 balance in the placenta during gestation have been associated with abortion. Cotyledon and caruncle cell layers form the maternal-foetal interface in the placenta and are able to recognize and induce immune responses against Neospora caninum among other pathogens. The objective of the present work was to elucidate the immunomodulation produced by high- (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates of N. caninum in bovine trophoblast (F3) and caruncular cells (BCEC-1) at early and late points after infection. Variations in the mRNA expression levels of toll-like receptor-2 (TLR-2), Th1 and Th2 cytokines (IL-4, IL-10, IL-8, IL-6, IL-12p40, IL-17, IFN-γ, TGF-β1, TNF-α), and endothelial adhesion molecules (ICAM-1 and VCAM-1) were investigated by RT-qPCR, and protein variations in culture supernatants were investigated by ELISA. Results A similar pattern of modulation was found in both cell lines. The most upregulated cytokines in infected cells were pro-inflammatory TNF-α (P

Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, represents one of the main causes of abortion in cattle. Macrophages (MØs) are mediators of the innate immune response against infection and likely one of the first cells encountered by the parasite during the host infection process. In this study, we investigated in vitro how high or low virulent isolates of N. caninum (Nc-Spain7 and Nc-Spain1H, respectively) interact with bovine monocyte-derived MØs and the influence of the isolate virulence on the subsequent cellular response. Both isolates actively invaded, survived and replicated in the MØs. However, Nc-Spain7 showed a higher invasion rate and a replication significantly faster, following an exponential growth model, whereas Nc-Spain1H presented a delayed replication and a lower growth rate without an exponential pattern. N. caninum infection induced a hypermigratory phenotype in bovine MØs that was characterized by enhanced motility and transmigration in vitro and was accompanied by morphological changes and abrogated extracellular matrix degradation. A significantly higher hypermotility was observed with the highly virulent isolate Nc-Spain7. Nc-Spain1H-infected MØs showed elevated reactive oxygen species (ROS) production and IL12p40 expression, which also resulted in increased IFN-γ release by lymphocytes, compared to cells infected with Nc-Spain7. Furthermore, IL-10 was upregulated in MØs infected with both isolates. Infected MØs exhibited lower expression of MHC Class II, CD86, and CD1b molecules than uninfected MØs, with non-significant differences between isolates. This work characterizes for the first time N. caninum replication in bovine monocyte-derived MØs and details isolate-dependent differences in host cell responses to the parasite.

5 páginas, 1 tabla, 1 figura., The cyst-forming protozoan parasite Neospora caninum is one of the main causes of bovine abortion worldwide and is of great economic importance in the cattle industry. Recent studies have revealed extensive genetic variation among N. caninum isolates based on multilocus microsatellite genotyping. Currently, the most extensive study reported is based on the N. caninum genotyping of 96 samples from four countries on two continents (Spain, Argentina, Germany and Scotland) that demonstrate different clusters of multilocus genotypes (MLGs) implicated in cattle abortions as well as the population sub structuring of N. caninum, which is partially associated with the geographical origin. The aim of this study was to genotype N. caninum from aborted bovine foetuses that originated from Mexico within the region of Aguascalientes and to investigate their genetic diversity. Parasite DNA was detected in 27 out of the 63 analysed foetuses recovered from 10 different herds. Complete or nearly complete profiles based on 9 microsatellite markers were obtained from 11 samples. Diverse N. caninum MLGs were implicated in the occurrence of abortion in each herd. All of the Mexican MLGs differed from the MLGs previously determined for the Argentinean, Spanish, German and Scottish N. caninum populations. The Mexican MLGs failed to cluster by eBURST analyses. The MLG relationships using PCoA showed a close genetic relationship between the Spanish population and a portion of the Mexican population, but a more distant genetic relationship with the Argentinean genotypes. These results demonstrate the genetic diversity of N. caninum in the studied areas that differed from other populations of N. caninum around the world. (C) 2016 Elsevier B.V. All rights reserved., This project was financed by DGEST-SEP 3503.10-P and CYTED (Iberoamerican Science and Technology Programme for Development) (113RT0469).

Neospora caninum is worldwide recognized as one of the major abortive pathogens in cattle. Although cases of abortion are also registered in sheep and goats, information and epidemiological data on neosporosis in small ruminants are usually scant. In Italy, but few exceptions, data are limited to spare reports of abortions. This survey was aimed to i) update information on N. caninum infection in sheep and goats from north-eastern Italy; ii) analyze associated individual and flock risk factors and iii) compare N. caninum positivity with previously obtained data on Toxoplasma gondii. Four hundred and fourteen goats and 428 sheep from 39 flocks in Lombardy region were enrolled. Blood samples were collected and analyzed by an in-house ELISA followed by a confirmatory Western Blot. The test resulted positive for 5.7% of goats and 19.3% of sheep. As to farms, 32.1% and 89.4% of caprine and ovine flocks showed positive, respectively. Managerial variables, including rearing system and farm size, affected N. caninum infection. Small, family-run farms were at higher risk rather than intensive caprine farms or transhumant sheep flocks. Outcomes from the survey mainly indicate that these farms should implement their care about proper sanitary measures and monitor the spread of the infection among small ruminants.

Toxoplasma gondii and Neospora caninum are two major abortifacient protozoans in domestic small ruminants and cattle, respectively, and they also parasitize a wide range of wildlife. Numerous serosurveys have been conducted in wild ruminants worldwide. However, the potential effect of different ecosystems and management practices on these infections has not been investigated. We studied the prevalence of antibodies to T. gondii and N. caninum in wild ruminants between 2007 and 2012 from four national wildlife reserves: three open space reserves in northwest Spain (Ancares, Mampodre, and Riaño) and a fenced reserve in central Spain (Quintos de Mora). Sera from roe deer ( Capreolus capreolus ) and chamois ( Rupicapra rupicapra ) were collected in Ancares (roe deer), Mampodre (both species), and Riaño (both species), whereas red deer ( Cervus elaphus ) sera were collected only in Quintos de Mora. The results of immunofluorescence antibody tests showed a T. gondii antibody prevalence significantly higher in red deer (13%; 17/131) than in roe deer (2%; 5/228) and chamois (4%; 6/149) (P0.05, Fisher's exact test). Moreover, N. caninum -specific antibodies were only detected in 1% of animals (2/131 red deer, 2/228 roe deer, and 2/149 chamois). Management measures were implemented in the Quintos de Mora reserve and T. gondii antibody prevalence in red deer decreased from 13% to 2% after 5 yr. In contrast, N. caninum antibody prevalences were very low (2%) over the years. The results suggest a low frequency of sylvatic life cycles in the hunting reservations studied, so interconnection between sylvatic and domestic life cycles is unlikely. Regardless, a sustainable exploitation of natural resources in wildlife reserves may help to reduce the prevalence of T. gondii infection.

Parameters such as pathogen dose and inoculation route are paramount in animal models when studying disease pathogenesis. Here, clinical findings, including foetal mortality, parasite transmission rates and lesion severity, and immune responses were evaluated in Asturiana pregnant heifers at day 110 of gestation challenged with a virulent (Nc-Spain7) Neospora caninum isolate. Four different doses of parasite tachyzoites were inoculated intravenously (IV1, 107 parasites, n = 6; IV2, 105, n = 6; IV3, 103, n = 6; and IV4, 102, n = 5), and the subcutaneous (SC) inoculation route was also assessed for the dose of 105 tachyzoites (SC, n = 6). In addition, a control group (n = 4 pregnant heifers) was evaluated. Foetal death was observed in all infected groups from 25 to 62 days post-infection, varying with the dose (IV1:4/6, IV2:3/6; IV4:2/5, IV3:1/6), and was three times less frequently associated with the SC route than IV inoculation (1/6 vs. 3/6). A dose-dependent effect for parasite loads in placental and foetal brain tissues was also detected. After SC challenge, a reduced number of tachyzoites were able to reach foetal brain tissues, and no lesions were observed. In calves, specific IgG responses in precolostral sera were mainly associated with high-dose groups (IV1 [100.0%] and IV2 [66.7%]), and cerebral parasite DNA detection was scarce (3/18). In dams, IFN-γ production and the dynamics of anti-N. caninum IgG antibodies varied with the dose, and the cell-mediated immune response was also found to be route-dependent. Our results confirm the influence of parasite dose and inoculation route on the outcome and dynamics of bovine neosporosis at mid-gestation., Patricia Vázquez had a Juan de la Cierva-Formación post-doctoral contract (FJCI-2014-20982) from the Spanish Ministry of Economy and Competitiveness (MINECO). Laura Jiménez-Pelayo was founded by UCM-Santander and Marta García-Sánchez was founded by MINECO (BES-2014-070723). Alicia Román-Trufero was supported by a 4-year FPI-INIA fellowship from the Spanish National Institute for Agriculture and Food Research and Technology (INIA).

OprI is an outer membrane lipoprotein from Pseudomonas aeruginosa, and when fused to a recombinant antigen, will exert adjuvant properties by engaging Toll-like receptor 2, leading to dendritic cell activation. Previous studies have shown that the Neospora caninum (Nc) antigens NcPDI, NcROP2 and NcROP40 are implicated in host cell interactions and are promising vaccine candidates. In two independent experiments, the efficacy of a polyvalent vaccine formulation composed of OprI-NcPDI, OprI-NcROP2 and OprI-NcROP40 (collectively named O-Ags) was assessed in non-pregnant and pregnant Balb/c mouse models challenged with tachyzoites of the high-virulence isolate Nc-Spain7. Parameters that were investigated were clinical signs, fertility, parasite burden in adult mice, humoral and cellular immune responses at different time-points prior to and after challenge infection, vertical transmission and post-natal survival of offspring mice, all to explore potential correlations with efficacy. Vaccination of mice with O-Ags induced a mixed Th1/Th2 immune response in adult mice and led to significantly increased protection against cerebral infection. Vaccination with O-Ags also resulted in reduced vertical transmission, and postnatal disease in offspring was significantly inhibited at a rate not observed in mice infected with a high-virulence isolate to date. However, O-Ags mixed with TLR ligands targeting TLR3 and TLR7, which are known to induce clear Th1-biased responses, or vaccination with OprI fused to the non-N. caninum antigen ovalbumin (OprI-OVA) did not confer protection.

Low antibody titers to Neospora caninum have been reported in humans, but infection has not been confirmed. We used N. caninum-specific PCR to test 600 clinical samples from patients with toxoplasmosis signs but Toxoplasma gondii-negative PCR results. We did not detect N. caninum DNA, demonstrating it is an unlikely opportunistic zoonotic agent.

Bovine besnoitiosis control remains a challenge because the disease continues to spread and control relies solely on accurate diagnosis coupled to management measures. However, recent studies have reported that routinely used ELISAs may raise a high number of false-positive results. Herein, cross-reactions between Besnoitia besnoiti antigens and anti-Neospora caninum and/or anti-Sarcocystis spp.-specific antibodies were studied in an in house ELISA since N. caninum and Sarcocystis spp. are closely related parasites, and both infections are highly prevalent in cattle worldwide. The serum panel was composed of the following categories: sera from B. besnoiti-seronegative (n=75) and -seropositive cattle (n=66), B. besnoiti-based-ELISA false-positive reactors (n=96) together with N. caninum (n=36) and Sarcocystis spp. (n=42) -seropositive reference cattle sera. B. besnoiti tachyzoite based western blot (WB) results classified animals as seropositive or seronegative. Sera were analyzed for the detection of anti-N. caninum by WB and ELISA and anti-Sarcocystis spp.-specific antibodies by WB and IFAT. Those samples recognizing a Sarcocystis spp. 18-20kDa antigenic region and N. caninum 17-18kDa immunodominant antigen were considered to be Sarcocystis spp. and N. caninum seropositive, respectively. The category of B. besnoiti based-ELISA false-positive reactors showed the highest number of sera with specific anti-Sarcocystis spp. and anti-N. caninum antibodies (74%; 71/96), followed by the N. caninum-seropositive cattle category (52.8%; 19/36). In contrast, few B. besnoiti-seronegative and -seropositive cattle showed antibodies against Sarcocystis spp. and N. caninum (10.7%; 8/75 and 1.5%; 1/66), respectively). This study revealed that B. besnoiti false-positive ELISA results were associated not only with the presence of anti-N. caninum and anti-Sarcocystis spp. antibodies (χ2: 78.36; p

Pathogenesis of bovine neosporosis is determined by different host- and parasite-dependent factors, including isolate virulence. A previous study identified that several Neospora caninum tachyzoite proteins were more abundant in virulent isolates, Nc-Liv and Nc-Spain7, compared with the low-virulent isolate Nc-Spain1H. Herein, we explored differences in the immunomes of these three isolates. Protein extracts from the Nc-Liv, Nc-Spain1H and Nc-Spain7 isolates were analysed in a 3×3 design by 2-DE immunoblot using sera from experimentally infected mice with these same three isolates. All isolates displayed a highly similar antigenic pattern when they were assessed using the same serum. Most of the reactive spots were located in the acidic region (pH 3-7) and grouped in 3 antigenic areas (250-70, 45-37 and 35-15 KDa). Differences found in the immunome depended on the sera used, regardless of the extract employed. In this sense, sera from Nc-Liv and Nc-Spain7 infected mice recognized the highest number of antigens, followed by Nc-Spain1H infected mice sera. In fact, 4 proteins identified by MS were not consistently detected in each isolate extract by sera from low-virulent Nc-Spain1H-infected mice: serine-threonine phosphatase 2C and superoxide dismutase (related to metabolism), gliding associated protein GAP45 (related to tachyzoites invasion), and NcGRA1 (located on dense granules). Similarly, 4 non-identified spots and another 2 spots chains located in 45-37 kDa area were not detected by this pooled sera. Variations between virulent Nc-Spain7 and Nc-Liv were limited to the absence of recognition by sera from Nc-Spain7-infected mice of GAP45 and the spot chains located in the 45-37 kDa area. These results suggest that variations in the immunome profiles rely on the immune response induced by each isolate and that these differentially recognized antigens could be investigated as putative virulence markers of neosporosis.

Highlights • Buparvaquone inhibits proliferation of Neospora caninum at nanomolar concentrations. • In vitro, the drug acts mainly parasitostatic. • Parasiticidal effects occur at µmolar concentrations after extended periods of time. • Buparvaquone acts slowly as evidenced by transmission electron microscopy. • Buparvaquone prevents clinical signs of acute neosporosis in mice., Graphical Abstract, The naphthoquinone buparvaquone is currently the only drug used against theileriosis. Here, the effects of buparvaquone were investigated in vitro and in an experimental mouse model for Neospora caninum infection. In 4-day proliferation assays, buparvaquone efficiently inhibited N. caninum tachyzoite replication (IC50 = 4.9 nM; IC100 = 100 nM). However, in the long term tachyzoites adapted and resumed proliferation in the presence of 100 nM buparvaquone after 20 days of cultivation. Parasiticidal activity was noted after 9 days of culture in 0.5 µM or 6 days in 1 µM buparvaquone. TEM of N. caninum infected fibroblasts treated with 1 µM buparvaquone showed that the drug acted rather slowly, and ultrastructural changes were evident only after 3–5 days of treatment, including severe alterations in the parasite cytoplasm, changes in the composition of the parasitophorous vacuole matrix and a diminished integrity of the vacuole membrane. Treatment of N. caninum infected mice with buparvaquone (100 mg/kg) either by intraperitoneal injection or gavage prevented neosporosis symptoms in 4 out of 6 mice in the intraperitoneally treated group, and in 6 out of 7 mice in the group receiving oral treatment. In the corresponding controls, all 6 mice injected intraperitoneally with corn oil alone died of acute neosporosis, and 4 out of 6 mice died in the orally treated control group. Assessment of infection intensities in the treatment groups showed that, compared to the drug treated groups, the controls showed a significantly higher parasite load in the lungs while cerebral parasite load was higher in the buparvaquone-treated groups. Thus, although buparvaquone did not eliminate the parasites infecting the CNS, the drug represents an interesting lead with the potential to eliminate, or at least diminish, fetal infection during pregnancy.

We carried out an inter-laboratory trial to compare the serological tests commonly used for the detection of specific Neospora caninum antibodies in cattle in Ibero- American countries. A total of eight laboratories participated from the following countries: Argentina (n = 4), Brazil (n = 1), Peru (n = 1), Mexico (n = 1), and Spain (n = 1). A blind panel of well-characterized cattle sera (n = 143) and sera representative of the target population (n = 351) was tested by seven in-house indirect fluorescent antibody tests (IFATs 1–7) and three enzyme-linked immunosorbent assays (ELISAs 1–3; two in-house and one commercial). Diagnostic performance of the serological tests was calculated and compared according to the following criteria: (1) the BPre-test information,^ which uses previous epidemiological and serological data; (2) the BMajority of tests,^ which classifies a serumas positive or negative according to the results obtained by most tests evaluated. Unexpectedly, six tests showed either sensitivity (Se) or specificity (Sp) values lower than 90%. In contrast, the best tests in terms of Se, Sp, and area under the ROC curve (AUC) values were IFAT 1 and optimized ELISA 1 and ELISA 2. We evaluated a high number of IFATs, which are the most widely used tests in Ibero-America. The significant discordances observed among the tests regardless of the criteria employed hinder control programs and urge the use of a common test or with similar performances to either the optimized IFAT 1 and ELISAs 1 and 2., Facultad de Ciencias Veterinarias

Background: This work studied the natural infection of Neospora caninum during the first gestation of heifers in a dairy farm in animals consuming a ration contaminated naturally with Zearalenone (ZEA), and to find out effect of mycotoxin in the levels of estrogen (E) and progesterone (P4) and that`s relation to the infection to N. caninum and in the abortions. Methods: The study was conducted in a dairy farm located in El Llano municipality, in Aguascalientes, Mexico, in 2013. Two groups were formed, the group “A” with 20 seronegative animals to N. caninum, and group “B” with 20 seropositive. Once a month was determined the levels of total IgG to N. caninum, the serum concentration of E and P4, and the level of ZEA in the ration; in cases of abortion, fetal brain samples were taken to identify the presence of N. caninum DNA. Results: In group “A”, was observed two subgroups: seronegative (60%) and seroconverted (40%), and three abortions. In group “B”, all animals maintain their serostatus, and three animals aborted. All abortions were positive for N. caninum DNA. The level of ZEA in the ration has an average of 426 µg/kg; during the gestation did not identify that animals suffer any alteration in the levels of E or P4. No statistical differences among the studied variables (levels of E and P) in time (nine months of gestation) were detected. It was not identified any interaction with the natural exposure to ZEA intake in any of the groups under study. Conclusion: The chronic ingestion of ZEA does not affect serum concentrations of E and P4 during gestation of heifers under study and cannot be related to the infection for N. caninum and the abortion.

Neospora caninum is one of the main causes of transmissible abortion in cattle. Intraspecific variations in virulence have been widely shown among N. caninum isolates. However, the molecular basis governing such variability have not been elucidated to date. In this study label free LC-MS/MS was used to investigate proteome differences between the high virulence isolate Nc-Spain7 and the low virulence isolate Nc-Spain1H throughout the tachyzoite lytic cycle. The results showed greater differences in the abundance of proteins at invasion and egress with 77 and 62 proteins, respectively. During parasite replication, only 19 proteins were differentially abundant between isolates. The microneme protein repertoire involved in parasite invasion and egress was more abundant in the Nc-Spain1H isolate, which displays a lower invasion rate. Rhoptry and dense granule proteins, proteins related to metabolism and stress responses also showed differential abundances between isolates. Comparative RNA-Seq analyses during tachyzoite egress were also performed, revealing an expression profile of genes associated with the bradyzoite stage in the low virulence Nc-Spain1H isolate. The differences in proteome and RNA expression profiles between these two isolates reveal interesting insights into likely mechanisms involved in specific phenotypic traits and virulence in N. caninum. Significance The molecular basis that governs biological variability in N. caninum and the pathogenesis of neosporosis has not been well-established yet. This is the first study in which high throughput technology of LC-MS/MS and RNA-Seq is used to investigate differences in the proteome and transcriptome between two well-characterized isolates. Both isolates displayed different proteomes throughout the lytic cycle and the transcriptomes also showed marked variations but were inconsistent with the proteome results. However, both datasets identified a pre-bradyzoite status of the low virulence isolate Nc-Spain1H. This study reveals interesting insights into likely mechanisms involved in virulence in N. caninum and shed light on a subset of proteins that are potentially involved in the pathogenesis of this parasite.

Background Neospora caninum, one of the main causes of abortion in cattle, is very effective at crossing the placental barrier and placental damage is crucial in the pathogenesis of abortion. Bovine trophoblast and caruncular cell layers are key cellular components in the maternal-foetal interface in placentomes, playing a fundamental role in placental functionality. Methods We studied tachyzoite adhesion, invasion, proliferation and egress of high- (Nc-Spain7) and low- (Nc-Spain1H) virulence N. caninum isolates in established cultures of bovine caruncular epithelial (BCEC-1) and trophoblast (F3) cells. The parasite invasion rate (pInvR) and the cell infection rate (cInfR) were determined by immunostaining plaque assay at different time points and multiplicities of infection (MOIs), respectively. In addition, tachyzoite growth kinetics were investigated using real-time PCR (qPCR) analysis and immunostaining plaque assay at different times. Results Neospora caninum invaded and proliferated in both cell lines. The pInvR was higher in F3 compared to BCEC-1 cells for the Nc-Spain7 isolate (P

SUMMARYRecent work has highlighted and enumerated the economic annual losses due to Neospora caninum abortions worldwide, which should provide strong motivation for the control of bovine neosporosis. However, with the recent withdrawal from sale of the only commercially available vaccine, control options for N. caninum have become more restricted. While researchers continue to work on developing alternative efficacious vaccines, what are the control options presently available for the cattle industries? At the practical level, recommendations for ‘Test-and-cull’, or ‘not breeding from seropositive dams’ stand diametrically opposed to alternative options put forward that suggest a primary producer is better advised to keep those cows in the herd that are already seropositive, i.e. assumed to be chronically infected, and indeed those that have already aborted once. Treatment with a coccidiostat has been recommended as the only economically viable option, yet no such treatment has gained official, regulatory approval. Dogs are central to the life cycle of N. caninum and have repeatedly been associated with infection and abortions in cattle by epidemiological studies. Knowledge and understanding of that pivotal role should be able to be put to use in control programmes. The present review canvasses the relevant literature for evidence for control options for N. caninum (some of them proven, many not) and assesses them in the light of the authors’ knowledge and experience with control of N. caninum.

Recent publications have calculated the considerable losses inflicted by N. caninum oncattle industries world-wide. Vaccination, at least at this stage, appears to be the only viable intervention strategy that appears feasible. The development of a live vaccine against neosporosis should be pursued, in order that its benefits can be transferred as soon as possible to veterinarians and farmers in order to prevent the reproductive, productive and economic losses caused by N. caninum. Fil: Reichel, Michael P.. University Of Adelaide; Australia Fil: Moore, Dadin Prando. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Hemphill, Andrew. University Of Berne. Faculties Of Veterinary And Medicine. Institute Of Parasitology; Suiza Fil: Ortega Mora, Luís Miguel. Universidad Complutense de Madrid. Facultad de Veterinaria; España Fil: Dubey, J. P.. United States Department of Agriculture; Argentina Fil: Ellis, John. University of Technology; Australia

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.

SUMMARYThe biological and genetic diversity ofNeospora caninumis very limited because of availability of only a few viable isolates worldwide. This study describes the isolation and biological and molecular characterization of a new viable isolate ofN. caninum(NC-SP1), from a cattle in Brazil. Approximately 400 g of brain from a naturally infected adult male cattle from an abattoir was fed to a 2-month-old dog.Neospora-like oocysts were observed on day 7 post-inoculation (PI) and the duration of oocyst shedding was 14 days. The DNA obtained from oocysts was characterized molecularly and the final sequence was 99% identical to homologous sequences ofN. caninumavailable in GenBank®. For bioassay, gerbils (Meriones unguiculatus) were orally inoculated with 10 100 and 1000 oocysts; all gerbils remained clinically normal but developedN. caninumantibodies 14 days PI. Cell culture isolation was successful using the brain homogenate from one of the gerbils and tachyzoites were observed 24 days PI. Microsatellite genotyping revealed a unique genetic profile for this new reference isolate.

SUMMARYThe 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.

10 páginas, 2 tablas, 3 figuras.--This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated., Here, we assessed outcome of experimental infection by Neospora caninum in goats intravenously inoculated with 10 tachyzoites of the Nc-Spain7 isolate at 40 (G1), 90 (G2) and 120 (G3) days of gestation. Infected goats had fever between 5 and 9 days post inoculation (dpi); all were seropositive at the time of abortion/birth. Foetal death occurred in G1 from 10 to 21 dpi (n = 7) and in G2 from 27 to 35 dpi (n = 4). Goats in G2 also had seropositive stillbirth (n = 1) and healthy kids (n = 2). G3 goats (n = 7) had 3 seropositive and 3 seronegative weak kids, and 2 seronegative healthy kids. Parasite DNA detection in placentomes was 100% in G2, 85.7% in G3 and in G1 was detected only in placentomes from the goats with foetal losses from 17 dpi (100%). Parasites were detected in foetal/kid brain (>85.7%) and liver (≥50%) of G2 and G3, and in G1 after 17 dpi (100%). The highest parasite loads were detected in the placentomes of G1 from 17 dpi and G2, and in foetal tissues of G1 from 17 dpi and G3. Multifocal necrotic lesions were observed in the placentas of the three groups, but they were larger and more frequent in G1 and G2. Similar lesions were observed in foetal tissues, but they were more frequent in G3. These findings suggest that, as observed in cattle and sheep, the clinical consequences of N. caninum in pregnant goats are dependent in part on the time of gestation when animals were infected., The authors acknowledge CNPq (National Council for Scientific and Technological Development) for financial support (Process 400405/2012-1). Support from CYTED (Iberoamerican science and technology programme for development) (113RT0469) and CAPES (Brazilian Federal Agency for Support and Evaluation of Graduate Education) (PHBP14/00081) is also gratefully acknowledged.

13 páginas, 4 Figuras.--Creative Commons Attribution License, Besides its importance in cattle, Neospora caninum may also pose a high risk as abortifacient for small ruminants. We have recently demonstrated that the outcome of experimental infection of pregnant sheep with 106 Nc-Spain7 tachy‑ zoites is strongly dependent on the time of gestation. In the current study, we assessed peripheral and local immune response in those animals. Serological analysis revealed earlier and higher IFN-γ and IgG responses in ewes infected at early (G1) and mid (G2) gestation, when abortion occurred. IL-4 was not detected in sera from any sheep. Inflam‑ matory infiltrates in the placenta mainly consisted of CD8+ and, to a lesser extent, CD4+ T cells and macrophages (CD163+). The infiltrate was more intense in sheep infected at mid-gestation. In the foetal mesenchyme, mostly free tachyzoites were found in animals infected at G1, while those infected in G2 displayed predominantly particulate antigen, and parasitophorous vacuoles were detected in sheep infected at G3. A similar pattern of placental cytokine mRNA expression was found in all groups, displaying a strengthened upregulation of IFN-γ and IL-4 and milder increases of TNF-α and IL-10, reminiscent of a mixed Th1 and Th2 response. IL-12 and IL-6 were only slightly upregu‑ lated in G2, and TGF-β was downregulated in G1 and G2, suggestive of limited T regulatory (Treg) cell activity. No significant expression of TLR2 or TLR4 could be detected. In summary, this study confirms the pivotal role of systemic and local immune responses at different times of gestation during N. caninum infection in sheep, We gratefully acknowledge Esperanza Selva from the SALUVET group (Complutense University of Madrid, Spain), Mario Ferrero, Melchor Molero, Iván Panero, Miguel Fernández and Carmen Espiniella from IGM (CSIC-ULE) and Idoia Badiola, from Agropal S. C. for their excellent technical assistance. D. Arranz is supported by a fellowship from the Spanish Ministry of Economy and Competitiveness (MINECO), as part of the Program of Training of Researcher Staff (FPI, grant number BES-2011-043433). This work was supported by the Swiss National Foundation (SNF grant No. 310030_146162), research grant LE253U13 from Junta de Castilla y León (Spain) and the Iberoamerican Science and Technology Programme for Development (CYTED, 113rt0469).

Neospora caninum can cause reproductive failure in goats. However, the pathogenesis of neosporosis in this domestic species remains largely unknown. We recently demonstrated that the outcome of experimental infection by N. caninum in pregnant goats is highly dependent on the time of gestation, during which infection occurs. In the present study, we examined the peripheral and placental immune responses in these groups of goats infected with 106 tachyzoites of the Nc-Spain7 isolate at early (G1, at day 40 of gestation, dg), mid (G2, 90 dg) and late (G3, 120 dg) gestation, together with a group of non-infected goats as a control group (G4). Seroconversion was observed as early as day 10 post-infection (pi) in all goats from G1 that aborted earlier (10-11 pi). The remaining infected goats had seroconverted by day 14 pi. Similar IFN-γ kinetic profiles were found in sera from goats in G1 and G2 with a significant increase in the IFN-γ levels on days 7 and 10 pi. This increase was not observed in G3. A similar pattern of placental cytokine expression was found in all infected groups. IFN-γ and IL-4 showed the highest increase, followed by a weaker up-regulation in TNF-α and IL-10. The lowest up-regulation was observed for IL-12 expression. In summary, this study provides information regarding the dynamics of immune responses and their relationship with the outcome of N. caninum infection in goats during gestation.