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Neospora caninum causes neosporosis, a leading cause of bovine abortion worldwide. Uruguay is a developing economy in South America that produces milk to feed seven times its population annually. Naturally, dairy production is paramount to the country’s economy, and bovine reproductive failure impacts it profoundly. Recent studies demonstrated that the vast majority of infectious abortions in dairy cows are caused by N. caninum. To delve into the local situation and contextualize it within the international standing, we set out to characterize the Uruguayan N. caninum strains. For this, we isolated four distinct strains and determined by microsatellite typing that these represent three unique genetic lineages, distinct from those reported previously in the region or elsewhere. An unbiased analysis of the current worldwide genetic diversity of N. caninum strains known, whereby six typing clusters can be resolved, revealed that three of the four Uruguayan strains group closely with regional strains from Argentina and Brazil. The remaining strain groups in an unrelated genetic cluster, suggesting multiple origins of the local strains. Microsatellite typing of N. caninum DNA from fetuses opportunistically collected from local dairy farms correlated more often with one of the isolates. Overall, our results contribute to further understanding of genetic diversity among strains of N. caninum both regionally and worldwide.

is a major apicomplexan pathogen responsible for significant reproductive losses in livestock, yet lacks effective therapeutics. Here, we identify and functionally characterize a previously unstudied OTU-like deubiquitinase (ncOTU; XP_003886403) as a key modulator of host-pathogen interactions. Sequence and structural analyses revealed conservation of the catalytic triad (D257, C260, H362) and a Y305-W315-G316 inhibition pocket analogous to viral OTU proteases. Recombinant ncOTU exhibited robust deubiquitinase activity and significantly reduced global ubiquitination levels in mammalian cells, preferentially targeting mono-ubiquitinated and low-molecular-weight substrates. Transcriptomic analysis demonstrated that ncOTU expression correlates with suppressed NF-κB signaling, type I interferon responses, and downstream antiviral effectors, while partially uncoupling upstream nucleic acid sensing pathways. Structure-based virtual screening and biochemical validation identified multiple small-molecule inhibitors targeting the conserved inhibition pocket. Dose-response analysis revealed submicromolar potency for ncOTUi-9 (IC = 0.1 μM), ncOTUi-8 (0.2 μM), and ncOTUi-19 (0.3 μM), whereas ncOTUi-3 showed lower activity (7.1 μM). Interaction analyses confirmed stable binding within the inhibition pocket, with more extensive contact networks correlating with increased potency. Collectively, these findings establish ncOTU as a functional deubiquitinase that contributes to evasion and highlight it as a promising therapeutic target for neosporosis.

is an apicomplexan parasite responsible for bovine neosporosis, a major cause of abortion in cattle worldwide. rhoptry protein 2 (NcROP2) has been identified as an essential factor in host cell invasion and parasitophorous vacuole formation, making it a potential target for disease control strategies. In this study, we generated knockout ( ) mutants using CRISPR/Cas9 technology to assess their role in parasite virulence. In a pregnant mouse model, parasites exhibited reduced virulence, as indicated by increased neonatal survival rates and lower parasite burden in the brain and attenuated clinical signs in the dams compared to the wild-type (Nc-Spain7) parental strain. Additionally, the mutants exhibited impaired proliferation and significantly induced the expression of interferon-stimulated genes in bovine monocyte-derived macrophages infected for 60 hours. Transcriptomic analysis further revealed a shift in parasite gene expression, with an upregulation of stress-related and bradyzoite markers. Functional assays confirmed that Δ parasites were less susceptible to IFN-γ-mediated inhibition and displayed an enhanced ability to convert to the semi-dormant bradyzoite stage. These findings highlight NcROP2 as a key virulence factor involved in immune evasion and parasite proliferation, providing new insights into infection pathogenesis and potential avenues for vaccine development.

Glutaredoxins (GRXs), important components of the intracellular thiol redox system, are involved in multiple cellular processes. In a previous study, we identified five GRXs in the apicomplexan parasite, Neospora caninum. In the present study, we confirmed that the GRXs S14 and C5 are located in the apicoplast, which suggests unique functions for these proteins. Although single-gene deficiency did not affect the growth of parasites, a double knockout (Δgrx S14Δgrx C5) significantly reduced their reproductive capacity. However, there were no significant changes in redox indices (GSH/GSSG ratio, reactive oxygen species and hydroxyl radical levels) in double-knockout parasites, indicating that grx S14 and grx C5 are not essential for maintaining the redox balance in parasite cells. Key amino acid mutations confirmed that the Cys203 of grx S14 and Cys253/256 of grx C5 are important for parasite growth. Based on comparative proteomics, 79 proteins were significantly downregulated in double-knockout parasites, including proteins mainly involved in the electron transport chain, the tricarboxylic acid cycle and protein translation. Collectively, GRX S14 and GRX C5 coordinate the growth of parasites. However, considering their special localization, the unique functions of GRX S14 and GRX C5 need to be further studied.

Neosporosis is a proven disease of farm animals and dogs caused by Neospora caninum. This cross-sectional study investigates N. caninum prevalence and seroprevalence among 268 dogs. Nc5 gene PCR was carried out on dog faeces and confirmed by sequencing. Seroprevalence was detected using an indirect fluorescent antibody test (IFAT). Three age groups, gender, locality (Amman, Irbid, and Zarqa Governorates), dog type (stray, pet, and breeding), place of living (indoor/outdoor), food type (raw/cooked), having diarrhoea, having abortion in the area, and having animals nearby were tested as independent variables for associations with positivity to N. caninum using univariate and multivariable logistic regression analyses. The true prevalence of N. caninum was 34.3% (95% CI 28.4, 40.5) using the Nc5 -PCR test. The true seroprevalence rate of N. caninum among dogs in Jordan was 47.9% (95% CI 41.4, 54.5) using IFAT. The sequenced isolates of Nc5 -PCR products ( n  = 85) matched three N. caninum strains, namely, NcHareGre ( n  = 70, 82.4%, 95% CI 72.6–89), NC MS2 ( n  = 14, 16.5%, 95% CI 9.3–26.1), and L218 ( n  = 1, 1.2%, 95% CI 0.03–6.4). The three strains were isolated previously from three different countries and continents. N. caninum shedding is associated with abortion among dogs and animals in the area (odds ratio = 3.6). In Amman and Zarqa, living indoors reduced seroprevalence at 0.45, 0.24, and 0.02 odds ratios, respectively. Jordan shares three molecular N. caninum strains with three different countries and continents.

We used immunohistochemistry and PCR to identify Neospora caninum in 6 infected marine mammal species, including 2 pups, that stranded in the northeastern Pacific Ocean. Our findings suggest the expansion of this parasite's host range to marine mammals, underscoring the effect of terrestrial pathogens that flow from land to sea.

Background Toxoplasma gondii and Neospora caninum are closely related protozoan parasites that are considered important causes of abortion in livestock, causing huge economic losses. Hunan Province ranks 12th in the production of beef and mutton in China. However, limited data are available on the seroprevalence, risk factors and molecular characterization of T. gondii and N. caninum in beef cattle and goats in Hunan province, China. Methods Sera of 985 beef cattle and 1147 goats were examined for the presence of specific antibodies against T. gondii using indirect hemagglutination test (IHAT) and anti- N. caninum IgG using competitive-inhibition enzyme-linked immunoassay assay (cELISA). Statistical analysis of possible risk factors was performed using PASW Statistics. Muscle samples of 160 beef cattle and 160 goats were examined for the presence of T. gondii DNA (B1 gene) and N. caninum DNA (Nc-5 gene) by nested PCR. The B1 gene-positive samples were genotyped at 10 genetic markers using the multilocus nested PCR-RFLP (Mn-PCR-RFLP). Results Specific IgG against T. gondii were detected in 8.3% (82/985) and 13.3% (153/1147) and against N. caninum in 2.1% (21/985) and 2.0% (23/1147) of the beef cattle and goats, respectively. Based on statistical analysis, the presence of cats, semi-intensive management mode and gender were identified as significant risk factors for T. gondii infection in beef cattle. Age was a significant risk factor for T. gondii infection in goats ( P  < 0.05), and age > 3 years was a significant risk factor for N. caninum infection in beef cattle ( P  < 0.05). PCR positivity for T. gondii was observed in three beef samples (1.9%; 3/160) and seven chevon samples (4.4%; 7/160). Genotyping of PCR positive samples identified one to be ToxoDB#10. The N. caninum DNA was observed in one beef sample (0.6%; 1/160) but was negative in all chevon samples. Conclusions To our knowledge, this is the first large-scale serological and molecular investigation of T. gondii and N. caninum and assessment of related risk factors in beef cattle and goats in Hunan Province, China. The findings provide baseline data for executing prevention and control of these two important parasites in beef cattle and goats in China. Graphical Abstract

Sarcocystis neurona, Neospora spp. and Toxoplasma gondii are related protozoans; they were considered the same parasite until 1970s. Two of these parasites, S. neurona and Neospora spp. are associated with a neurological syndrome in horses, called equine protozoal myeloencephalitis (EPM). The diagnosis and treatment of EPM are difficult. Most cases of EPM are related to S. neurona while only a few are due to Neospora spp. infections. There are two species of Neospora, Neospora caninum that has a wide host range and Neospora hughesi that has been found only in horses. Currently, T. gondii is not considered as a cause of EPM in horses, although it causes neurological illness in many other hosts, including humans. The present review provides an update on history, life cycle, diagnosis and treatment of these three infections in horses.

Neosporosis primarily affects cattle and dogs and is not currently considered a zoonotic disease. Toxoplasmosis is a zoonosis with a worldwide distribution that is asymptomatic in most cases, but when acquired during pregnancy, it can have serious consequences. The seropositivity rates determined by the indirect fluorescent antibody test for Neospora caninum ( N. caninum ) and Toxoplasma gondii ( T. gondii ) were 24.3% (49 samples) and 26.8% (54 samples), respectively. PCR positivity for N. caninum was observed in two samples of cord blood (1%) using the Nc5 and ITS1 gene, positivity for T. gondii was observed in 16 samples using the primer for the B1 gene (5.5% positivity in cord blood and 2.5% positivity in placental tissue). None of the samples showed structures characteristic of tissue cysts or inflammatory infiltrate on histopathology. Significant associations were observed only between N. caninum seropositivity and the presence of domestic animals (p = 0.039) and presence of dogs (p = 0.038) and between T. gondii seropositivity and basic sanitation (p = 0.04). This study obtained important findings regarding the seroprevalence and molecular detection of N. caninum and T. gondii in pregnant women; however, more studies are necessary to establish a correlation between risk factors and infection.

Toxoplasma gondii, Neospora spp., Sarcocystis spp., Hammondia spp. and Besnoitia besnoiti are genetically related cyst-forming coccidia. Serology is frequently used for the identification of T. gondii, Neospora spp. and B. besnoiti-exposed individuals. Serologic cross-reactions occur in different tests among animals infected with T. gondii and H. hammondi, as well as among animals infected by T. gondii and N. caninum. Infections caused by N. caninum and N. hughesi are almost indistinguishable by serology. Neospora caninum, B. besnoiti and Sarcocystis spp. infections in cattle show some degree of serologic cross-reactivity. Antibody cross-reactivity between Neospora spp. and H. heydorni-infected animals is suspected, but not proven to occur. We review serologic cross-reactivity among animals and/or humans infected with T. gondii, Neospora spp., Sarcocystis spp., Hammondia spp. and B. besnoiti. Emphasis is laid upon antigens and serological methods for N. caninum diagnosis which were tested for cross-reactivity with related protozoa. Species-specific antigens, as well as stage-specific proteins have been identified in some of these parasites and have promising use for diagnosis and epidemiological surveys.