Short-term culture adaptation of Toxoplasma gondii archetypal II and III field isolates affects cystogenic capabilities and modifies virulence in mice.

Most Toxoplasma gondii research has been carried out using strains maintained in the laboratory for long periods of time. Long-term passage in mice or cell culture influences T. gondii phenotypic traits such as the capability to produce oocysts in cats and virulence in mice. In this work, we investigated the effect of cell culture adaptation in the short term for recently obtained type II (TgShSp1 (Genotype ToxoDB#3), TgShSp2 (#1), TgShSp3 (#3) and TgShSp16 (#3)) and type III (#2) isolates (TgShSp24 and TgPigSp1). With this purpose, spontaneous and alkaline stress-induced cyst formation in Vero cells during 40 passages, from passage 10 (p10) to 50 (p50), and isolate virulence at p10 versus p50 were studied using a harmonized bioassay method in Swiss/CD1 mice. T. gondii cell culture maintenance showed a drastic loss of spontaneous and induced production of mature cysts after ≈25-30 passages. The TgShSp1, TgShSp16 and TgShSp24 isolates failed to generate spontaneously formed mature cysts at p50. Limited cyst formation was associated with an increase in parasite growth and a shorter lytic cycle. In vitro maintenance also modified T. gondii virulence in mice at p50 with events of exacerbation, increasing cumulative morbidity for TgShSp2 and TgShSp3 isolates and mortality for TgShSp24 and TgPigSp1 isolates, or attenuation, with absence of mortality and severe clinical signs for TgShSp16, and better control of the infection with the lowest parasite and cyst burdens in lungs and brain for the TgShSp1 isolate. The present findings show deep changes in relevant phenotypic traits in laboratory-adapted T. gondii isolates and open new discussion about their use for inferring keys to parasite biology and virulence.

A comparative study of eight serological methods shows that spike protein-based ELISAs are the most accurate tests for serodiagnosing SARS-CoV-2 infections in cats and dogs.

Introduction: Coronavirus disease 2019 (COVID-19) is an infectious zoonotic disease caused by SARS-CoV-2. Monitoring the infection in pets is recommended for human disease surveillance, prevention, and control since the virus can spread from people to animals during close contact. Several diagnostic tests have been adapted from humans to animals, but limited data on the validation process are available. Methods: Herein, the first comparative study of six "in house" and two commercial serological tests developed to monitor SARS-CoV-2 infection in pets was performed with a well-coded panel of sera (61 cat sera and 74 dog sera) with a conservative criterion (viral seroneutralisation and/or RT-qPCR results) as a reference. Four "in house" tests based on either the RBD fragment of the spike protein (RBD-S) or the N-terminal fragment of the nucleoprotein (N) were developed for the first time. The analytical specificity (ASp) of those tests that showed the best diagnostic performance was assessed. The validation included the analysis of a panel of sera obtained pre-pandemic from cats and dogs infected with other coronaviruses to determine the analytical Sp (17 cat sera and 41 dog sera). Results and discussion: ELISAS based on the S protein are recommended in serosurveillance studies for cats (RBD-S SALUVET ELISA, ELISA COVID UNIZAR and INgezim® COVID 19 S VET) and dogs (INgezim® COVID 19 S VET and RBD-S SALUVET ELISA). These tests showed higher diagnostic sensitivity (Se) and DSp in cats (>90%) than in dogs. When sera obtained prior to the pandemic and from animals infected with other coronaviruses were analyzed by RBD-S and N SALUVET ELISAs and INgezim® COVID 19 S VET, a few cross reactors or no cross reactions were detected when dog and cat sera were analyzed by tests based on the S protein, respectively. In contrast, the number of cross reactions increased when the test was based on the N protein. Thus, the use of tests based on the N protein was discarded for serodiagnosis purposes. The results obtained revealed the most accurate serological tests for each species. Further studies should attempt to improve the diagnostic performance of serological tests developed for dogs.

Archetypal type II and III Toxoplasma gondii oocysts induce different immune responses and clinical outcomes in experimentally infected piglets.

Livestock animals, such as swine, are an important source of Toxoplasma gondii in the human population. Currently, there is limited knowledge regarding the potential influence that the T. gondii genotype might exert on establishing infection in swine. Herein, we investigated the role of 2 T. gondii isolates, type II and III, representative of the genotypes circulating in Europe, in the immune responses and infection dynamics in piglets. Recently obtained oocysts (103) from the T. gondii field isolates TgShSp1 (type II, ToxoDB genotype #3) and TgShSp24 (type III, #2) were used for oral infection. Thirteen 50-day-old female piglets of the Landrace-Large White crossbreed were randomly allocated into three different groups: Group 1 (G1, n=5), inoculated with TgShSp1; Group 2 (G2, n=5), inoculated with TgShSp24; and Group 3 (G3, n=3), a non-infected control group. Clinical signs were monitored daily until 42 days post-infection (dpi) when piglets were euthanized. Blood samples were collected weekly to test the cellular immune response in parasite-stimulated peripheral blood and specific IgG, IgG1 and IgG2, responses in sera. Parasite distribution and burden were evaluated in target tissues using a mouse bioassay and quantitative RT-PCR (qPCR). Apathy and a moderate decrease in feed consumption were observed in G1 and G2 piglets between 5 and 8 dpi, coinciding with fever (>40°C). G2 piglets had higher temperatures for a longer duration. Using mouse bioassay and qPCR, the detection frequency was higher in G2 vs. G1, and the highest parasite burdens in target tissues were also found in G2. Seroconversion was detected at 14 dpi in both infected groups, but higher antibody levels were observed in G2 piglets. Cytokine analyses revealed the production of IL-8, IL-1ß and IFN-ɤ from 7 dpi in both infected groups. Moreover, IL-12 was produced from 7 dpi in G1 and from 14 dpi in G2. Levels of IL-8 were higher in G2, but IL-1ß, IL-12 and IFN-ɤ were higher in G1 at 14 dpi. This cytokine profile reveals a predominant proinflammatory response that could be involved in limiting T. gondii infection in piglets, although it is more efficient against TgShSp1 type II-driven infection.

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.

Parasitemia and Associated Immune Response in Pregnant and Non-Pregnant Beef Cows Naturally Infected With Neospora caninum.

The aim of this longitudinal study was to characterize the parasitemia of Neospora caninum and the associated immunological parameters in naturally infected beef cows for 10 months. The following groups were established: Neospora caninum seropositive pregnant cows (+Preg, n = 7), seropositive non-pregnant cows (+Npreg, n = 7), seronegative pregnant cows (-Preg, n = 4), and seronegative non-pregnant cows (-Npreg, n = 4). Several samples were obtained for absolute and relative leukocyte counting, cytokines IL-10, IL-12, α-TNF, and γ-IFN quantification, specific IgG, IgG1, and IgG2 and avidity and N. caninum DNA molecular detection and quantification. The +Preg group had a higher frequency and concentration of N. caninum DNA in PBMC in the last third of pregnancy compared to +Npreg (p <0.05), with 22 and 8% of detection, respectively. Parasitemia correlated positively with IgG titers and negatively with IgG1/IgG2 ratio (p <0.05). On day 222 of the assay, the +Preg group had the lowest total leukocyte counting (p <0.05). The +Preg group had a higher concentration of IgG and higher avidity in the last third of gestation compared to +Npreg (p <0.05). Avidity correlated with total IgG and IgG2 (p <0.05). All +Preg cows gave birth to clinically healthy but seropositive calves before colostrum intake, therefore, the congenital transmission was 100% efficient. Only a complete N. caninum genotype from a placenta and a partial genotype from cow #3 of the group +Preg were achieved by multilocus microsatellite analysis. Overall, N. caninum parasitemia is frequent in seropositive beef cows during the last third of gestation. This correlates with higher antibody levels and a decrease in total leukocyte counting. The precise timing of the parasitemia may be used for diagnosis purposes and/or for design strategies to avoid vertical transmission. Further studies are needed to identify the immune molecular mechanisms that favor parasitemia during gestation in chronically infected cattle.

SARS-CoV-2 Infection in One Cat and Three Dogs Living in COVID-19-Positive Households in Madrid, Spain.

In this study, we describe SARS-CoV-2 infection dynamics in one cat and three dogs from households with confirmed human cases of COVID-19 living in the Madrid Community (Spain) at the time of expansion (December 2020 through June 2021) of the alpha variant (lineage B.1.1.7). A thorough physical exam and nasopharyngeal, oropharyngeal, and rectal swabs were collected for real-time reverse-transcription PCR (RT-qPCR) SARS-CoV-2 testing on day 0 and in successive samplings on days 7, 14, 21, and 47 during monitoring. Blood was also drawn to determine complete blood counts, biochemical profiles, and serology of the IgG response against SARS-CoV-2. On day 0, the cat case 1 presented with dyspnea and fever associated with a mild bronchoalveolar pattern. The dog cases 2, 3, and 4 were healthy, but case 2 presented with coughing, dyspnea, and weakness, and case 4 exhibited coughing and bilateral nasal discharge 3 and 6 days before the clinical exam. Case 3 (from the same household as case 2) remained asymptomatic. SARS-CoV-2 detection by RT-qPCR showed that the cat case 1 and the dog case 2 exhibited the lowest cycle threshold (Ct) (Ct < 30) when they presented clinical signs. Viral detection failed in successive samplings. Serological analyses revealed a positive IgG response in cat case 1 and dog cases 3 and 4 shortly after or simultaneously to virus shedding. Dog case 2 was seronegative, but seroconverted 21 days after SARS-CoV-2 detection. SARS-CoV-2 genome sequencing was attempted, and genomes were classified as belonging to the B.1.1.7 lineage.

Dynamics of Neospora caninum-Associated Abortions in a Dairy Sheep Flock and Results of a Test-and-Cull Control Programme.

Neospora caninum is an apicomplexan parasite that can cause abortions and perinatal mortality in sheep. Although ovine neosporosis has been described worldwide, there is a lack of information about the relationship between N. caninum serostatus and the reproductive performance. In this study, we described the infection dynamics in a dairy sheep flock with an abortion rate up to 25% and a N. caninum seroprevalence of 32%. Abortions were recorded in 36% and 9% of seropositive and seronegative sheep, respectively. Seropositive sheep were more likely to abort twice (OR = 4.44) or three or more times (OR = 10.13) than seronegative sheep. Endogenous transplacental transmission was the main route of transmission since 86% of seropositive sheep had seropositive offspring. Within dams that had any abortion, seropositive sheep were more likely than seronegative ones to have female descendants that aborted (OR = 8.12). The slight increase in seropositivity with the age, the low percentage of animals with postnatal seroconversion or with low avidity antibodies, and the seropositivity of one flock dog, indicated that horizontal transmission might have some relevance in this flock. A control programme based on selective culling of seropositive sheep and replacement with seronegative animals was effective in reducing the abortion rate to 7.2%.

Morphometric study of encephalic lesions in aborted bovine fetuses naturally infected by two subpopulations of Neospora caninum.

Neospora caninum is a major reproductive disease in cattle worldwide. In the Argentinian Humid Pampa, the seroprevalence, incidence of abortions, and economic losses due to neosporosis are considerably higher in dairy than in beef cattle. Despite this, we recently demonstrated that N. caninum subpopulations are indistinctly distributed in both dairy and beef production systems. The association between genotypic characteristics defined by microsatellite analysis and the virulence of the different strains-particularly with regard to the severity and extension of histological lesions-is largely unknown. Herein, we used a morphometric approach to analyze encephalic lesions in 62 bovine fetuses spontaneously infected by N. caninum. Morphometric parameters (average size of focal lesions, number of foci/cm2 and the percentage of the section affected by lesions) were compared according to the N. caninum subpopulations found in our previous microsatellite genotyping analysis, animal biotype (beef versus dairy), and fetal age (second stage of gestation versus third stage). The average size of the lesions differed significantly among fetuses with different gestational ages; however, no significant differences among animal biotypes or genotypic patterns were found. Further research into the genetic, molecular, and husbandry factors that could account for this greater impact in Argentinian dairy herds is needed.

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.

Microsatellite genotyping reveals extensive genetic diversity in bovine Neospora caninum from the humid Pampa region in Argentina.

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.

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.