Search Results (239)

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that poses serious risks to immunocompromised individuals and the global livestock industry. Our previous study showed that AP2X-1 regulates stage conversion and virulence of T. gondii; however, whether AP2X-1-deficient strains can serve as live-attenuated vaccine candidates is unknown. Here, we evaluated the in vivo virulence, protective efficacy, and induced immunity of the ap2X-1 knockout strain PruΔap2X-1. In Kunming mice, infection with even the highest tested dose (5 × 10⁶ tachyzoites) of PruΔap2X-1 resulted in 100% survival without detectable brain cysts. Vaccination with 10⁶ PruΔap2X-1 tachyzoites conferred complete protection (100% survival) against acute challenge with homologous type II Pru or heterologous ToxoDB#9 PYS strains, whereas control mice exhibited only 0% to 16.7% survival. Moreover, immunized mice orally challenged with Pru cysts showed 100% survival, and no brain cysts were detected under the conditions tested, compared with only 30% survival in control mice challenged with 10 cysts and no survival with 40 cysts. The vaccination induced a Th1-biased response as evidenced by significantly elevated T. gondii-specific total IgG, IgG2a, and the Th1 cytokines IFN-γ and IL-2, along with a significantly reduced IgG1/IgG2a ratio. Collectively, the attenuated PruΔap2X-1 strain generates strong protective immunity against acute and chronic infection with low-virulence strains (the type II Pru and ToxoDB#9 PYS), as well as partial protection against the hypervirulent type I RH strain, positioning it as a potential candidate for a live-attenuated vaccine, with a favorable safety profile observed under the experimental conditions. Full article

Cryptosporidium parvum is an important apicomplexan parasite that causes severe diarrheal disease in children and immunocompromised individuals. However, the structural basis for the limited immunogenicity of its T-cell epitopes remains poorly understood. This study integrates structural biology and immunological approaches to elucidate the molecular basis underlying the non-immunogenicity of KAV9, a Cp23-derived epitope with the sequence KAVKNPAPI. Biophysical analyses demonstrated that KAV9 forms a high-affinity complex with H-2D^b, with an IC₅₀ of 7.83 nM, and exhibits higher thermal stability (T_m = 59.16 °C) than the immunodominant LCMV gp33 epitope (T_m = 51.15 °C). Despite strong pMHC binding and high pMHC stability, in vivo peptide immunization failed to elicit a detectable KAV9-specific CD8⁺ T-cell response. Crystal structure analysis revealed that KAV9 is tightly accommodated within the H-2D^b binding groove through an extensive hydrogen-bond network. However, its distinct peptide conformation, particularly involving P4-Lys and the proline residues at P6 and P8, markedly reshapes the TCR-exposed surface compared to gp33. AlphaFold3 (AF3) modeling further suggested that these structural deviations disrupt critical hydrogen-bond interactions with the T-cell receptor (TCR) CDR3 loops, thereby eliminating contacts required for TCRβ engagement. Sequence analysis revealed that KAV9 is highly conserved across multiple Cryptosporidium species, suggesting a conserved structural feature associated with limited T-cell recognition. Together, these findings demonstrate that strong MHC binding and pMHC stability are insufficient to ensure CD8⁺ T-cell immunogenicity. Instead, the topology of the TCR-accessible peptide surface represents a critical determinant of epitope immunogenicity, with significant implications for epitope selection and vaccine design against cryptosporidiosis. Full article

Bumped kinase inhibitors are safe with promising efficacy against apicomplexan parasites. The 5-aminopyrazole-4-carboxamide BKI-1708 effectively inhibited vertical transmission of Toxoplasma gondii and significantly reduced the cerebral parasite loads in experimentally infected pregnant mice. In vitro experiments revealed that exposure of T. gondii tachyzoites to BKI-1708 induces the formation of intracellular multinucleated complexes called “baryzoites”, exhibiting increased expression of bradyzoite-stage proteins while still displaying classical tachyzoite markers. Differential affinity chromatography of T. gondii extracts identified numerous BKI-1708-binding proteins involved in invasion/egress, redox homeostasis, and RNA processing. To understand the transcriptional implications of BKI-1708 treatment on T. gondii tachyzoites and human foreskin fibroblast host cells, T. gondii-infected host cells, either treated with BKI-1708 or untreated, were subjected to dual RNA-seq analysis. BKI-1708 induced a significant transcriptional remodeling in the parasite, with an enrichment in pathways related to translation, RNA metabolism, and stress responses. In contrast, host–cell transcriptional changes were more limited, with transcripts related to metabolic and detoxification programs upregulated in uninfected fibroblasts, and increased transcription of immune, lysosomal, and glycan degradation pathways in infected fibroblasts. These findings suggest that BKI-1708 modulates the transcriptome in a predominantly parasite-specific manner, disrupting essential biological processes in T. gondii while largely preserving host cell function. Full article

Sarcocystis spp. are apicomplexan parasites with an obligatory two-host prey–predator life cycle. While the intermediate hosts (IHs) of many species are well known, the definitive hosts (DH) of numerous Sarcocystis spp., especially those involving wildlife predators, remain poorly understood. In the current study, Sarcocystis species richness was investigated in the intestines of 69 red foxes (Vulpes vulpes) from Lithuania. Intestinal samples were examined by light microscopy and nested PCR targeting the cytochrome c oxidase I gene (cox1) sequence of species associated with ungulate IH and the internal transcribed spacer I (ITS1) region of species using birds as IH, followed by Sanger sequencing and phylogenetic analyses. Red foxes harboured a broad spectrum of Sarcocystis species, with those utilising ungulate IHs being considerably more common than species associated with birds. Seven species, Sarcocystis alces, Sarcocystis bertrami, Sarcocystis capreolicanis, Sarcocystis hjorti, Sarcocystis iberica, Sarcocystis linearis, and Sarcocystis morae, were identified in red foxes for the first time. Nine previously reported species, Sarcocystis albifronsi, Sarcocystis arieticanis, Sarcocystis capracanis, Sarcocystis cruzi, Sarcocystis gracilis, Sarcocystis miescheriana, Sarcocystis pilosa, Sarcocystis rileyi, and Sarcocystis tenella, were also detected. In addition, two potentially novel Sarcocystis taxa were identified and warrant further investigation. These findings highlight the role of red foxes as an important DH and provide a valuable framework for comparative studies of Sarcocystis distribution in predatory mammals. Full article

Bumped kinase inhibitor 1708 (BKI-1708), previously demonstrated to target apicomplexan kinases and CDPK1 and MAPKL1, exhibits remarkable activity against Toxoplasma gondii infection both in vitro and in vivo. Notably, BKI-1708 does not affect the viability of mammalian cells. Upon exposure to BKI-1708, T. gondii tachyzoites form large multinucleated complexes named baryzoites and remain trapped within host cells. In this study, proteins binding to BKI-1708 were identified in soluble extracts of T. gondii ME49 tachyzoites and human foreskin fibroblasts (HFF) using differential affinity chromatography coupled to mass spectrometry (DAC-MS). Beyond kinases, secondary interactions in T. gondii involved the binding of proteins associated with cell division, cytoskeleton, vesicular trafficking, secretory organelles, and transcriptional and translational regulators. In non-infected HFFs, BKI-1708 interactors included cytoskeletal regulators along with multiple RNA/DNA-binding proteins. Upon infection, this profile shifted, with cytoskeletal components no longer detected, while nucleic acid-binding proteins remained present, consistent with infection-induced chromatin and transcriptional remodeling. These results suggest that multi-target interference could contribute to the impaired cytokinesis and the formation of multinucleated baryzoites, aligning with the concept that antiprotozoal drugs exert efficacy through coordinated perturbation of multiple cellular processes rather than a single dominant target. Full article

Sarcocystis spp. are apicomplexan parasites that form sarcocysts mainly in the muscles or central nervous system of intermediate hosts and sporocysts in the intestines of definitive hosts. Three species, Sarcocystis falcatula, Sarcocystis calchasi and Sarcocystis halieti, are potentially pathogenic to their intermediate hosts. Over the past two decades, we have examined 72 thrushes across four species (redwing (Turdus iliacus), common blackbird (Turdus merula), song thrush (Turdus philomelos), and fieldfare (Turdus pilaris)) for sarcocysts to better understand their role as intermediate hosts. Sarcocysts were detected in 28 individuals (38.9%). Most sarcocysts observed by light microscopy were of a single morphological type consistent with Sarcocystis turdusi. A molecular analysis of ITS1 sequences confirmed the presence of S. turdusi in the common blackbird, song thrush, and fieldfare, establishing the latter two bird species as new intermediate hosts. In contrast, cox1 was not a sufficiently variable locus for species differentiation. Additionally, a single sarcocyst with a smooth cyst wall, distinct from S. turdusi, was detected in a common blackbird and identified as S. halieti based on ITS1 sequence analysis. This atypical host record represents an isolated finding in a long-term dataset. Further sampling is required to confirm its epidemiological significance. Full article

Congenital toxoplasmosis, caused by the obligatory intracellular apicomplexan protozoan Toxoplasma gondii, can lead to severe complications during pregnancy, including fetal malformations and spontaneous abortion. In the present study, the anti-T. gondii effects of the natural flavonoid quercetin were evaluated using in vitro, ex vivo, and in silico models. Cell viability and intracellular proliferation of the parasite were determined via colorimetric assays. The lipid droplet assay was analyzed using Nile Red staining, the antioxidant and oxidative stress parameters were determined by biochemical assays, and the cytokine levels were quantified by immunoassays. Our results demonstrated that non-cytotoxic concentrations of quercetin (CC₅₀ > 100 μM) significantly inhibited parasite proliferation (IC₅₀ = 14.10 ± 2.83 μM; SI > 7.09) in an irreversible manner. Quercetin impairs parasite adhesion, invasion, and reinfection capacity. In parallel, quercetin reduced lipid droplet accumulation, restored antioxidant balance by modulating redox biomarkers, and regulated cytokine production, notably increasing IL-4, IL-6, and IL-8 levels. Corroborating the in vitro findings, quercetin significantly reduced T. gondii proliferation in human placental villous explants while preserving tissue architecture and viability. In silico analyses revealed that quercetin binds to the active site of T. gondii hypoxanthine-guanine phosphoribosyltransferase (TgHGPRT) and exhibits favorable pharmacokinetic and drug-likeness properties. Full article

Toxoplasma gondii is an apicomplexan parasite that causes toxoplasmosis, a widespread zoonotic disease leading to serious public health concerns and economic losses to animal husbandry. Currently, highly effective vaccines against toxoplasmosis remain unavailable. This study aimed to investigate the safety, immunogenicity, and protective efficacy of the gra47-deficient mutant strain PruΔgra47 as a live-attenuated vaccine candidate. We evaluated the virulence of PruΔgra47 in a mouse model, determined the optimal immunization dose, and measured serum antibody levels and cytokine profiles. Then, mice immunized with PruΔgra47 were challenged with different T. gondii strains to assess protection against acute and chronic infection. PruΔgra47 displayed significantly attenuated virulence and its ability to form cysts was weakened. Vaccination with 5 × 10⁶ tachyzoites elicited predominantly Th1-skewed immune responses. Immunization with PruΔgra47 provided complete protection against challenge infection with relatively low-virulent PYS strain and homologous Pru strain, prolonged survival against the highly virulent RH strain, and achieved a 90% survival rate with reduced brain cyst burden under chronic challenge. In conclusion, PruΔgra47 is relatively safe and immunogenic in the murine model, and is worth being evaluated in food-producing animals and cats. Full article

Neospora caninum 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 (IC₅₀ = 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. Full article

Colpodella spp. are phylogenetically related to apicomplexans such as Plasmodium spp., Babesia spp., and Cryptosporidium spp. Colpodella spp. are free-living protists that prey on bodonids, ciliates, and algae using myzocytosis. Colpodella spp. cause human and animal infections known as colpodellosis, with transmission via ticks across different geographic areas on different continents. Colpodella spp. DNA has been detected in ticks, the biting fly Stomoxys indicus and vertebrate samples using polymerase chain reaction (PCR). Ticks transmit zoonotic pathogens, and the identification of Colpodella spp. in animals poses a major public health risk due to human and animal encounters exposing humans to tick bites. However, it is unclear if ticks are confirmed vectors for Colpodella spp., since tick vector competence and capacity for Colpodella spp. transmission has not been experimentally demonstrated. Human cases of colpodellosis have involved three cases of blood infection, a fourth case of tickborne infection, and a fifth case of urinary tract infection. In this narrative review, the occurrence of Colpodella spp. in ticks that transmit zoonotic pathogens will be reviewed. Differences in the disease presentations and symptoms of colpodellosis in tickborne infections will be discussed. The pattern of Colpodella spp. coinfections with piroplasms and Cryptosporidium spp. will be evaluated. The pressing need for morphological identification of Colpodella spp. to assist proper characterization of the different species identified in arthropods and vertebrate hosts will be highlighted. Full article

Toxoplasma gondii and Neospora caninum are apicomplexan parasites infecting cattle, with implications for public health and livestock productivity, respectively. Since effective vaccines against these parasites are not currently available, identifying epidemiological factors associated with infection is important for improving control strategies. This study aimed to estimate the seroprevalence of both parasites and to identify factors associated with seropositivity in intensive dairy cattle in central Chile. A cross-sectional study was conducted using serum samples from 567 cattle, analyzed by ELISA. Epidemiological data were collected through semi-structured surveys, and associations with seropositivity were evaluated using multivariable logistic regression models, including mixed-effects models to account for farm-level clustering. Seroprevalence was 7.6% for T. gondii and 22.4% for N. caninum. For T. gondii, factors associated with seropositivity included older age categories (OR = 7.09; 11.25) and the presence of dogs in pens (OR = 6.07). For N. caninum, straw bedding use (OR = 5.13) and cat presence (OR = 6.32) were associated with higher odds of seropositivity. An additional association with lower N. caninum seropositivity was observed for BCG vaccination (OR = 0.24). These findings provide updated epidemiological data for dairy cattle in Chile. The association observed with BCG vaccination should be interpreted cautiously, as the study design does not permit causal inference. Full article

Neospora caninum is an apicomplexan parasite with a broad geographical distribution and a complex life cycle. It can cause infectious abortions in a variety of animals in the major livestock-producing nations, resulting in huge economic losses to the livestock industry. Shanxi Province in north China is one of China’s important livestock-producing provinces, but the data on the prevalence of N. caninum in sheep and goats in this province was not available prior to the present investigation. To fulfill this gap in our knowledge, serum samples were collected from 504 sheep and 300 goats across 11 cities representing three distinct geographical regions of Shanxi Province. A commercially available indirect enzyme-linked immunosorbent assay (iELISA) kit was used to determine the N. caninum prevalence by detecting N. caninum-specific IgG antibodies. Results showed that a total of 24 sheep (4.8%; 95% CI: 2.9–6.6) and 8 goat samples (2.7%; 95% CI: 0.8–4.5) tested positive for N. caninum antibodies. Geographical distribution was identified as the predominant risk factor influencing N. caninum infection in sheep and goats in Shanxi Province, with seroprevalence of N. caninum ranging from 0% to 16.7% across different sampling sites. This study reports the seroprevalence of N. caninum infection in sheep and goats in Shanxi Province for the first time, providing baseline data for the prevention and control of N. caninum infection in this northern province of China. Full article

A new intracellular parasite of the phylum Apicomplexa is described infecting the digestive gland of the gastropod Bulla striata from the south coast of Portugal. Only merogonial stages enclosed within parasitophorous vacuoles were observed by light and electron microscopy. The meront cytoplasm contained lipid droplets, rough endoplasmic reticulum cisternae, and several round or oval electron-dense microbodies closely associated with amylopectin granules, suggesting that these microbodies may represent glycosomes. Mitochondria or related organelles were not identified. A reticulum of branched tubules extending from the parasitophorous vacuole membrane was observed, likely increasing the surface available for metabolite exchange between parasite and host cell. Merozoites resulting from meront division were present within the parasitophorous vacuoles. They were elongated and slightly curved, measuring 7–8 µm in length and about 2 µm in width, and possessed an apical complex comprising numerous rod-shaped micronemes, rhoptries, and a conoid. Phylogenetic analyses based on a partial 18S rDNA sequence placed this parasite within the coccidian lineage, at the base of the ichthyocolid clade, a recently recognized group of apicomplexans previously known from fish blood cells. This finding expands the host range of ichthyocolids to gastropods and provides the first ultrastructural observations of this lineage, although only of merogonic stages. Full article

Toxoplasma gondii and Neospora caninum are closely related apicomplexan parasites of veterinary and public health importance. T. gondii is a zoonotic pathogen for which cats are the definitive host, whereas N. caninum is a major cause of reproductive losses in cattle, with dogs acting as the definitive host. Data on exposure in pet animals in Thailand remain limited. This study investigated seroprevalence and associated risk factors of T. gondii and N. caninum in pet cats and dogs in Pathum Thani Province, an urban area adjacent to Bangkok. Between June 2020 and July 2021, serum samples were collected from 169 owned animals, including 86 cats and 83 dogs, participating in a mobile sterilization program. Antibodies were detected using the indirect fluorescent antibody test (IFAT), and animal characteristics, behaviors, and environmental factors were obtained via owner questionnaires. Serological evidence of exposure to both parasites was detected. Antibodies against T. gondii were detected in 4.73% (8/169) of animals, including 4.65% (4/86) of cats and 4.82% (4/83) of dogs. For N. caninum, the overall seroprevalence was 10.06% (17/169), with a higher prevalence in dogs (15.66%, 13/83) than in cats (4.65%, 4/86). No significant risk factors were identified for T. gondii or N. caninum infection in either cats or dogs (p > 0.05). Full article

Bumped kinase inhibitors (BKIs) have demonstrated safety and promising efficacy against various apicomplexan pathogens both in vitro and in vivo, but do not act parasiticidal in vitro. In the closely related cyst-forming coccidians Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti, treatments with BKI-1708 induce the conversion of intracellular tachyzoites into atypical multinucleated complexes named “baryzoites”. In this study, we comparatively assessed tachyzoites and baryzoites of all three species with respect to ultrastructure, differential antigen expression by immunofluorescence, and overall differential protein expression by MS-proteomics. TEM demonstrated common, but also distinguishing, structural features in baryzoites of the three species. They contained newly formed zoites, unable to complete cytokinesis, and thus they were trapped intracellularly. An electron-dense cyst wall-like structure was found only in T. gondii baryzoites. Species-specific differences in antigen expression were observed by immunofluorescence. Comparative proteomic analysis of baryzoites versus tachyzoites revealed a downregulation of ribosomal proteins, proteins associated with secretory organelles, as well as of transcription and translation factors in baryzoites across all species. Bradyzoite-specific markers were upregulated only in T. gondii baryzoites. Two alveolin-domain filament proteins and a hypothetical protein (TGME49_236950, NCLIV_050850, BESB_060040) were detected at higher abundance in all three species. Thus, baryzoites exhibit distinct phenotypic and proteomic profiles, with ambiguous expression of tachyzoite and bradyzoite antigens, suggesting a reversible response to stress rather than progression into a fully differentiated form. Full article