Biochemical and molecular characterization of Campylobacter fetus isolates from bulls subjected to bovine genital campylobacteriosis diagnosis in Spain.

BACKGROUND: Bovine genital campylobacteriosis (BGC) is caused by Campylobacter fetus subsp. venerealis (Cfv) including its biovar intermedius (Cfvi). This sexually transmitted disease induces early reproductive failure causing considerable economic losses in the cattle industry. Using a collection of well-characterized isolates (n = 13), C. fetus field isolates (n = 64) and saprophytic isolates resembling Campylobacter (n = 75) obtained from smegma samples of breeding bulls, this study evaluated the concordance of the most used phenotypic (H2S production in cysteine medium and 1% glycine tolerance) and molecular (PCR) methods for the diagnosis of BGC and assessed possible cross-reactions in the molecular diagnostic methods. RESULTS: Characterization at the subspecies level (fetus vs. venerealis) of C. fetus isolated from bull preputial samples using phenotypic and molecular (PCR targeting nahE and ISCfe1) methods showed moderate concordance (κ = 0.462; CI: 0.256-0.669). No cross-reactions were observed with other saprophytic microaerophilic species or with other Campylobacter species that can be present in preputial samples. Whole genome sequencing (WGS) of discrepant isolates showed 100% agreement with PCR identification. For the differentiation of Cfv biovars, comparison of the H2S test (at 72 h and 5 days of incubation) and a PCR targeting the L-cysteine transporter genes showed higher concordance when H2S production was assessed after 5 days (72 h; κ = 0.553, 0.329-0.778 CI vs. 5 days; κ = 0.881, 0.631-1 CI), evidencing the efficacy of a longer incubation time. CONCLUSIONS: This study confirmed the limitations of biochemical tests to correctly identify C. fetus subspecies and biovars. However, in the case of biovars, when extended incubation times for the H2S test (5 days) were used, phenotypic identification results were significantly improved, although PCR-based methods produced more accurate results. Perfect agreement of WGS with the PCR results and absence of cross-reactions with non-C. fetus saprophytic bacteria from the smegma demonstrated the usefulness of these methods. Nevertheless, the identification of new C. fetus subspecies-specific genes would help to improve BGC diagnosis.

Comparative pangenomic analysis of Campylobacter fetus isolated from Spanish bulls and other mammalian species.

Campylobacter fetus comprises two closely related mammal-associated subspecies: Campylobacter fetus subsp. fetus (Cff) and Campylobacter fetus subsp. venerealis (Cfv). The latter causes bovine genital campylobacteriosis, a sexually-transmitted disease endemic in Spain that results in significant economic losses in the cattle industry. Here, 33 C. fetus Spanish isolates were whole-genome sequenced and compared with 62 publicly available C. fetus genomes from other countries. Genome-based taxonomic identification revealed high concordance with in silico PCR, confirming Spanish isolates as Cff (n = 4), Cfv (n = 9) and Cfv biovar intermedius (Cfvi, n = 20). MLST analysis assigned the Spanish isolates to 6 STs, including three novel: ST-76 and ST-77 for Cfv and ST-78 for Cff. Core genome SNP phylogenetic analysis of the 95 genomes identified multiple clusters, revealing associations at subspecies and biovar level between genomes with the same ST and separating the Cfvi genomes from Spain and other countries. A genome-wide association study identified pqqL as a Cfv-specific gene and a potential candidate for more accurate identification methods. Functionality analysis revealed variations in the accessory genome of C. fetus subspecies and biovars that deserve further studies. These results provide valuable information about the regional variants of C. fetus present in Spain and the genetic diversity and predicted functionality of the different subspecies.

Ovine placental explants: A new ex vivo model to study host‒pathogen interactions in reproductive pathogens.

Reproductive failure is one of the main performance constraints in ruminant livestock. Transmissible agents such as Toxoplasma gondii and Neospora caninum are commonly involved in the occurrence of abortion in ruminants, but little is known about the mechanisms involved. While in vivo models are optimal for the study of abortion pathogenesis, they have a high economic cost and come with ethical concerns. Unfortunately, alternative in vitro models fail to replicate the complex in vivo placental structure. To overcome the limitations of currently available models, we developed an ex vivo model based on the cultivation of fresh and cryopreserved sheep placental explants, enabling the biobanking of tissues. Reproducible and simple markers of tissue integrity (histology, RNA concentrations), viability (resazurin reduction), and functionality (synthesis of steroid hormones) were also investigated, allowing a clear quality assessment of the model. This work shows that, similar to fresh explants, tissues cryopreserved in ethylene glycol using slow freezing rates maintain not only their structure and function but also their receptivity to T. gondii and N. caninum infection. In addition, the findings demonstrate that explant lifespan is mainly limited by the culture method, with protocols requiring improvements to extend it beyond 2 days. These findings suggest that cryopreserved tissues can be exploited to study the initial host‒pathogen interactions taking place in the placenta, thus deepening the knowledge of the specific mechanisms that trigger reproductive failure in sheep. Importantly, this work paves the way for the development of similar models in related species and contributes to the reduction of experimental animal use in the future.

Whole-transcriptome analysis reveals virulence-specific pathogen-host interactions at the placenta in bovine neosporosis.

Research on bovine neosporosis has achieved relevant milestones, but the mechanisms underlying the occurrence of foetal death or protection against foetal death remain unclear. In a recent study, placentas from heifers challenged with the high-virulence isolate Nc-Spain7 exhibited focal necrosis and inflammatory infiltrates as soon as 10 days post-infection (dpi), although parasite detection was minimal. These lesions were more frequent at 20 dpi, coinciding with higher rates of parasite detection and the occurrence of foetal death in some animals. In contrast, such lesions were not observed in placentas from animals infected with the low-virulence isolate Nc-Spain1H, where the parasite was detected only in placenta from one animal at 20 dpi. This work aimed to study which mechanisms are triggered in the placentas (caruncles and cotyledons) of these pregnant heifers at early stages of infection (10 and 20 dpi) through whole-transcriptome analysis. In caruncles, infection with the high-virulence isolate provoked a strong proinflammatory response at 10 dpi. This effect was not observed in heifers infected with the low-virulence isolate, where IL-6/JAK/STAT3 signalling and TNF-alpha signalling via NF-κB pathways were down-regulated. Interestingly, the expression of E2F target genes, related to restraining the inflammatory response, was higher in these animals. At 20 dpi, more pronounced proinflammatory gene signatures were detectable in heifers infected with the high-virulence isolate, being more intense in heifers carrying dead fetuses. However, the low-virulence isolate continued without activating the proinflammatory response. In cotyledons, the response to infection with the high-virulence isolate was similar to that observed in caruncles; however, the low-virulence isolate induced mild proinflammatory signals at 20 dpi. Finally, a deconvolutional analysis of gene signatures from both placentome tissues revealed a markedly higher fraction of activated natural killers, M1 macrophages and CD8+ T cells for the high-virulence isolate. Therefore, our transcriptomic analysis supports the hypothesis that an intense immune response probably triggered by parasite multiplication could be a key contributor to abortion. Further studies are required to determine the parasite effectors that govern the distinct interactions of high- and low-virulence isolates with the host, which could help elucidate the molecular processes underlying the pathogenesis of neosporosis in cattle.

Characterization of Neospora caninum virulence factors NcGRA7 and NcROP40 in bovine target cells.

Bovine neosporosis is one of the major causes of reproductive failure in cattle worldwide, and differences in virulence between isolates have been widely shown. However, the molecular basis and mechanisms underlying virulence in Neospora caninum are mostly unknown. Recently, we demonstrated the involvement of NcGRA7 and NcROP40 in the virulence of N. caninum in a pregnant murine model using single knockout mutants in these genes generated by CRISR/Cas9 technology. In this study, the role of these proteins was investigated in two in vitro models using bovine target cells: trophoblast (F3 cell line) and monocyte-derived macrophages (BoMØ). The proliferation capacity of the single knockout mutant parasites was compared to the wild-type strain, the Nc-Spain7 isolate, using both cell populations. For the bovine trophoblast, no differences were observed in the growth of the defective parasites compared to the wild-type strain, neither in the proliferation kinetics nor in the competition assay. However, in naïve BoMØ, a significant decrease in the proliferation capacity of the mutant parasites was observed from 48 h pi onwards. Stimulation of BoMØ with IFN-γ showed a similar inhibition of tachyzoite growth in defective and wild-type strains in a dose-dependent manner. Finally, BoMØ infected with knockout parasites showed higher expression levels of TLR3, which is involved in pathogen recognition. These results suggest that NcGRA7 and NcROP40 may be involved in the manipulation of innate immune defense mechanisms against neosporosis and confirm the usefulness of the BoMØ model for the evaluation of N. caninum virulence mechanisms. However, the specific functions of these proteins remain unknown, opening the way for future research.

A new inactivated Tritrichomonas foetus vaccine that improves genital clearance of the infection and calving intervals in cattle.

Bovine trichomonosis is a sexually transmitted disease that is a primary cause of early reproductive failure in cattle. The aim of the present study was to develop a vaccine formulation based on Tritrichomonas foetus trophozoites inactivated by lyophilization and Quil-A-adjuvanted. The safety, immunogenicity and efficacy of this new vaccine formulation (Trichobovis®) administered by two routes (subcutaneous: SC, and intravulvar: IVU) were compared with a commercial vaccine (TrichGuard®) in a well-established experimental bovine model of genital T. foetus infection. The new vaccine was considered safe in cattle because only mild local reactions were found in the vaccination area, which disappeared 3 weeks after administration. Cows immunized with Trichobovis cleared the infection faster than the non-immunized/challenged group (27-28 vs. 60 days; P < 0.05). Not significant differences were observed with the commercial vaccine respect to the positive control group, or between SC and IVU routes. The new vaccine stimulated high serum anti-T. foetus IgG and genital IgA levels and generated an IgG booster effect similar to TrichGuard. IgA levels were associated with significantly earlier genital clearance of T. foetus in cows immunized with Trichobovis by SC route (G1A) or TrichGuard (G2). The strongest association was found in the group G1A on day 14 post-infection (p.i.) (r = -0.74) and in G2 on day 35 p.i. (r = -0.71). The efficacy of vaccination using Trichobovis on the reproductive performance was also investigated under field conditions in a herd where T. foetus was present. The calving intervals were significantly reduced by 45.2 days (P < 0.05), calves were born 28 days earlier (P < 0.05) and an increase of 8.7% in the calving rate (P > 0.05) was observed in the vaccinated group. These results demonstrate that Trichobovis improved the reproductive performance under field conditions in herds where T. foetus infection is present.

NcGRA7 and NcROP40 Play a Role in the Virulence of Neospora caninum in a Pregnant 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.

Prevalence of Bovine Genital Campylobacteriosis, Associated Risk Factors and Spatial Distribution in Spanish Beef Cattle Based on Veterinary Laboratory Database Records.

Bovine genital campylobacteriosis (BGC) is a sexually transmitted disease that causes early reproductive failure in natural breeding cattle that are managed extensively. The aim of this study was to assess the BGC prevalence in Spain from 2011 to 2019 using data collected cross-sectionally from the diagnostic reports issued by the SALUVET veterinary diagnostic laboratory from a total of 5,182 breeding bulls from 1,950 herds managed under "dehesa" systems (large herds within fenced pastures and all-year breeding season) or mountain systems (smaller herds with seasonal breeding management and grazing in communal mountain pastures). Infection was detected by PCR in 7.7 and 12.2% of the bulls and herds tested, respectively. The "dehesa" herd management system (OR = 2.078, P = < 0.001, 95% CI = 1.55-1.77), bovine trichomonosis status of the herd (OR = 1.606, P = 0.004, 95% CI = 1.15-2.22), and bulls ≥3 years old (OR = 1.392, P = 0.04, 95% CI = 1.01-1.92) were identified as risk factors associated with Campylobacter fetus venerealis infection. We also studied the high-risk areas for circulation of the infection in extensive beef cattle herds in Spain, showing four significant clusters in "dehesa" areas in the south-western provinces of the country and a fifth cluster located in a mountain area in northern Spain. The results obtained in the present study indicate that BGC is endemic and widely distributed in Spanish beef herds. Specifically, "dehesa" herds are at greater risk for introduction of Cfv based on relatively high local prevalence of the infection and the use of specific management practices.

Maternal and Foetal Cellular Immune Responses in Dams Infected With High- and Low- Virulence Isolates of Neospora caninum at Mid-Gestation.

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 by Neospora caninum isolate 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 of N. 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 to N. caninum infection at mid gestation. This work provides insights into how isolate virulence affects the maternal and foetal immune responses generated against N. caninum, which may influence the course of infection.

In vivo and in vitro models show unexpected degrees of virulence among Toxoplasma gondii type II and III isolates from sheep.

Toxoplasma gondii is an important zoonotic agent with high genetic diversity, complex epidemiology, and variable clinical outcomes in animals and humans. In veterinary medicine, this apicomplexan parasite is considered one of the main infectious agents responsible for reproductive failure in small ruminants worldwide. The aim of this study was to phenotypically characterize 10 Spanish T. gondii isolates recently obtained from sheep in a normalized mouse model and in an ovine trophoblast cell line (AH-1) as infection target cells. The panel of isolates met selection criteria regarding such parameters as genetic diversity [types II (ToxoDB #1 and #3) and III (#2)], geographical location, and sample of origin (aborted foetal brain tissues or adult sheep myocardium). Evaluations of in vivo mortality, morbidity, parasite burden and histopathology were performed. Important variations between isolates were observed, although all isolates were classified as "nonvirulent" (< 30% cumulative mortality). The isolates TgShSp16 (#3) and TgShSp24 (#2) presented higher degrees of virulence. Significant differences were found in terms of in vitro invasion rates and tachyzoite yield at 72 h post-inoculation (hpi) between TgShSp1 and TgShSp24 isolates, which exhibited the lowest and highest rates, respectively. The study of the CS3, ROP18 and ROP5 loci allelic profiles revealed only type III alleles in ToxoDB #2 isolates and type II alleles in the #1 and #3 isolates included. We concluded that there are relevant intra- and inter-genotype virulence differences in Spanish T. gondii isolates, which could not be inferred by genetic characterization using currently described molecular markers.

Isolation, Genotyping, and Mouse Virulence Characterization of Toxoplasma gondii From Free Ranging Iberian Pigs.

The present study aimed to isolate and perform molecular and phenotypic characterization of Toxoplasma gondii strains infecting Iberian pigs bred under semi-free conditions and destined for human consumption. Blood and heart tissue samples from 361 fattening pigs from 10 various herds selected in the main areas of Iberian pig production were collected at a slaughterhouse; the sera were tested for anti-T. gondii antibodies using a commercial indirect ELISA kit, and a mouse bioassay was carried out using heart muscle of seropositive individual representatives from each geographical location. Seventy-nine (21.9%) of the 361 animals tested positive for anti-T. gondii antibodies according to the serology test. Fifteen samples of myocardial tissue were subjected to bioassay and 5 isolates (TgPigSp1 to TgPigSp5) were obtained. The isolates were characterized by using 11 PCR-RFLP genetic markers; three isolates had a ToxoDB #3 genotype (3/5) and two isolates had a ToxoDB #2 genotype (2/5). The TgPigSp1 and TgPigSp4 isolates were selected for virulence in mice characterization as instances of each different RFLP-genotype found. The TgPigSp1 isolate (#2 genotype) was virulent in mice with notable cumulative mortality (87.5%) and morbidity rates (100%); the TgPigSp4 (#3) was nonvirulent and triggered mild clinical signs in 42.1% of seropositive mice. Infection dynamics and organ distribution of both isolates were analyzed; the data revealed significant differences, including substantially higher parasite load in the lung during the acute phase of infection, in mice infected with TgPigSp1 than in the case of TgPigSp4 (median parasite load 7.6 vs. 0 zoites/mg, respectively; p < 0.05). Furthermore, degrees of severity of detected histopathological lesions appeared to be related to higher parasite burdens. Taking into account the unexpectedly high mortality rate and parasite load associated with the clonal genotype III, which is traditionally considered nonvirulent in mice, the need for further investigation and characterization of the T. gondii strains circulating in any host in Europe is emphasized.

Correction to: Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks.

An amendment to this paper has been published and can be accessed via the original article.

Prevalence of intestinal parasite infections in stray and farm dogs from Spain.

Dogs play a potential role as reservoirs for zoonotic parasites, being especially problematic uncontrolled dog populations such as stray and farm dogs with access to populated areas. In order to investigate the prevalence of canine intestinal parasites in at-risk dog populations, we tested a total of 233 faecal samples shed by stray and dairy farm dogs from northern Spain. Telemann method was used to detect the presence of eggs and (oo)cysts of common dog intestinal parasites and Cryptosporidium was detected by PCR. One hundred and forty eight out of 233 samples (63.5%) were positive for at least one intestinal parasite, being Ancylostomidae (35.6%; 83/233) and Trichuris (35.2%; 82/233) the parasites most frequently identified. Cryptosporidium DNA was not detected in any of the faecal samples analysed. The overall prevalence was significantly higher in stray dogs than in farm dogs (72.5% vs 58.8%). Specifically, stray dogs had a significantly higher prevalence of Ancylostomatidae, Toxocara, Toxascaris and Taenidae. These dog populations are an important source of environmental contamination with intestinal parasite forms, which could be of significance to animal and human health.

Proteomic Characterization of Host-Pathogen Interactions during Bovine Trophoblast Cell Line Infection by Neospora caninum.

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.

Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks.

BACKGROUND: Toxoplasma gondii is a major cause of abortion in small ruminants and presents a zoonotic risk when undercooked meat containing cysts is consumed. The aim of the present study was to investigate the genetic diversity among the T. gondii strains circulating in ovine livestock in Spain. METHODS: Selected samples collected from abortion outbreaks due to toxoplasmosis (n = 31) and from chronically infected adult sheep at slaughterhouses (n = 50) in different Spanish regions were bioassayed in mice, aiming at parasite isolation. In addition, all original clinical samples and the resulting isolates were genotyped by multi-nested PCR-RFLP analysis of 11 molecular markers and by PCR-DNA sequencing of portions of the SAG3, GRA6 and GRA7 genes. RESULTS: As a result, 30 isolates were obtained from 9 Spanish regions: 10 isolates from abortion-derived samples and 20 isolates from adult myocardial tissues. Overall, 3 genotypes were found: ToxoDB#3 (type II PRU variant) in 90% (27/30) of isolates, ToxoDB#2 (clonal type III) in 6.7% (2/30), and ToxoDB#1 (clonal type II) in 3.3% (1/30). When T. gondii-positive tissue samples (n = 151) were directly subjected to RFLP genotyping, complete restriction profiles were obtained for 33% of samples, and up to 98% of the specimens belonged to the type II PRU variant. A foetal brain showed a clonal type II pattern, and four specimens showed unexpected type I alleles at the SAG3 marker, including two foetal brains that showed I + II alleles as co-infection events. Amplicons of SAG3, GRA6 and GRA7 obtained from isolates and clinical samples were subjected to sequencing, allowing us to confirm RFLP results and to detect different single-nucleotide polymorphisms. CONCLUSIONS: The present study informed the existence of a predominant type II PRU variant genotype (ToxoDB#3) infecting domestic sheep in Spain, in both abortion cases and chronic infections in adults, coexisting with other clonal (ToxoDB#1 and ToxoDB#2), much less frequent genotypes, as well as polymorphic strains as revealed by clinical sample genotyping. The use of multilocus sequence typing aided in accurately estimating T. gondii intragenotype diversity.

Neospora caninum infection induces an isolate virulence-dependent pro-inflammatory gene expression profile in bovine monocyte-derived macrophages.

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.

Crosstalk between Neospora caninum and the bovine host at the maternal-foetal interface determines the outcome of infection.

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.

Comparative tachyzoite proteome analyses among six Neospora caninum isolates with different virulence.

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.

Modeling the Ruminant Placenta-Pathogen Interactions in Apicomplexan Parasites: Current and Future Perspectives.

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.

Prevalence of intestinal parasite infections in stray and farm dogs from Spain / Prevalência de infecções por parasitos intestinais em cães errantes e de fazenda na Espanha

Abstract Dogs play a potential role as reservoirs for zoonotic parasites, being especially problematic uncontrolled dog populations such as stray and farm dogs with access to populated areas. In order to investigate the prevalence of canine intestinal parasites in at-risk dog populations, we tested a total of 233 faecal samples shed by stray and dairy farm dogs from northern Spain. Telemann method was used to detect the presence of eggs and (oo)cysts of common dog intestinal parasites and Cryptosporidium was detected by PCR. One hundred and forty eight out of 233 samples (63.5%) were positive for at least one intestinal parasite, being Ancylostomidae (35.6%; 83/233) and Trichuris (35.2%; 82/233) the parasites most frequently identified. Cryptosporidium DNA was not detected in any of the faecal samples analysed. The overall prevalence was significantly higher in stray dogs than in farm dogs (72.5% vs 58.8%). Specifically, stray dogs had a significantly higher prevalence of Ancylostomatidae, Toxocara, Toxascaris and Taenidae. These dog populations are an important source of environmental contamination with intestinal parasite forms, which could be of significance to animal and human health.

Resumo Os cães desempenham um importante papel como reservatório de parasitos zoonóticos, sendo especialmente problemáticas as populações descontroladas, como a de cães errantes e de fazenda, com acesso às áreas povoadas. Para investigar a prevalência de parasitos intestinais em populações caninas de risco, foram analisadas 233 amostras fecais provenientes de cães de fazendas leiteiras e errantes do norte da Espanha. O método Telemann foi utilizado para detectar ovos, cistos e oocistos dos parasitos caninos mais comuns e para a detecção de Cryptosporidium foi utilizada a técnica da PCR. Cento e quarenta e oito de 233 amostras analisadas (63,5%) foram positivas para pelo menos um parasito intestinal, sendo Ancyostomatidae (35,6%; 83/233) e Trichuris sp. (35,2%; 82/233) os parasitos identificados com maior frequência. O DNA de Cryptosporidium sp. não foi detectado em nenhuma das amostras fecais analisadas. A prevalência geral foi significativamente maior em cães errantes do que em cães de fazenda (72,5% vs 58,8%). Especificamente, os cães errantes tiveram prevalência maior para Ancylostomatidae, Toxocara, Toxascaris e Taenidae. Essas populações de cães são importantes fontes de contaminação ambiental, pois eliminam formas de vida desses parasitos, que podem ter impacto na saúde animal e humana.