Sarcocystis species are intracellular coccidian protozoans that can infect a range of animals and humans and cause public health problems and economically significant losses. Sarcocystosis in sheep (Ovis aries) can cause abortion, neurological symptoms, and even death and results in significant economic losses to the livestock industry. To date, however, it is yet unknown whether sheep in Shanxi Province, north China, are infected with Sarcocystis spp. The purpose of this study was to investigate the prevalence of Sarcocystis spp. in sheep in Shanxi Province. Thus, 582 muscle samples of sheep were purchased from farmers’ markets from ten representative counties in Shanxi Province, north China, and examined for the presence and prevalence of Sarcocystis spp. by PCR amplification of the mitochondrial cytochrome c oxidase subunit I (cox1) gene. Of the examined 582 mutton samples, 197 samples (33.85%) were Sarcocystis-positive and were sequenced. Of the obtained 197 cox1 sequences, 196 sequences showed nucleotide similarity of 98.56–99.81% with those of S. tenella, and the remaining one cox1 sequence showed nucleotide similarity of 99.71% with that of S. arieticanis. Two representative cox1 sequences of S. tenella (accession nos. PQ189447 and PQ189448) have 99.52% and 99.61% identity with S. tenalla (KC209725) and S. tenalla (MK419984), respectively. The sequence of S. arieticanis (accession no. PQ165949) obtained in this study has 99.71% identity with S. arieticanis (MK419975). This present study documents the occurrence and prevalence of Sarcocystis spp. in sheep in Shanxi Province for the first time, which enriches the data on the distribution of Sarcocystis spp. in sheep in China and has implications for the control of sheep sarcocystosis.

Toxoplasma gondii is a globally prevalent parasite capable of establishing lifelong infections, which can have severe consequences in immunocompromised individuals and developing fetuses. GRAs are essential secretory effectors that facilitate nutrient acquisition, modulate host immune responses, and support intracellular survival. In this study, we characterized four putative GRAs (GRA85–88) that co-localize with GRA12 in both tachyzoite and bradyzoite stages. Using CRISPR-Cas9-mediated homologous recombination, we successfully generated knockout strains in both type I RH and type II Pru backgrounds. Phenotypic analysis revealed that GRA85, GRA87, and GRA88 were not individually required for parasite replication, invasion, or virulence. However, deletion of gra86 (PruΔgra86) resulted in a significant reduction in virulence and fewer brain cysts in chronically infected mice, although in vitro growth remained unaffected. Transcriptomic profiling of PruΔgra86 revealed downregulation of bradyzoite–related genes and upregulation of GRAs involved in host interaction. Additionally, in vitro differentiation assays showed impaired bradyzoite development in the absence of GRA86. These findings from murine models and in vitro phenotypic assays highlight GRA86 as a regulator of chronic infection and stage conversion, positioning it as an important player in T. gondii pathogenesis and a promising target for therapeutic intervention.

Toxoplasma gondii is one of the most widely distributed intracellular parasites worldwide, which can infect humans and a wide range of warm-blooded animals including sheep, with felines serving as its definitive host. T. gondii infection in sheep can lead to premature births, abortions and stillbirths, causing significant economic losses to the sheep industry. Sheep farming has become a key pillar of the agricultural economy in Shanxi Province, North China, but little is known about T. gondii infection in sheep in this province. In the present study, a total of 755 sheep tissue samples (682 muscle tissue samples and 73 lymphatic tissue samples) were collected from different markets in 10 different cities of Shanxi Province. The genomic DNA of all samples was extracted and the B1 gene of T. gondii was amplified by PCR. The B1 gene-positive samples were genotyped at 12 genetic markers employing the multilocus nested PCR-restriction fragment length polymorphism (Mn-PCR-RFLP). The molecular prevalence of T. gondii infection in sheep tissues in Shanxi Province was 20.5% (155/755). The T. gondii genotype ToxoDB#9 was identified in one positive T. gondii sample, with complete genotyping at all 12 genetic markers based on Mn-PCR-RFLP. This is the first report of molecular prevalence and genotype of T. gondii infection in sheep in Shanxi Province. These results reveal the widespread distribution of T. gondii in sheep in Shanxi, which is of significant public health importance.

Background: Rhoptry proteins (ROPs) are secreted effectors that play important roles in the virulence of Toxoplasma gondii by facilitating host cell invasion and immune modulation. Although many ROPs have been predicted, their specific functions remain largely unexplored. This study investigates the roles of 11 previously uncharacterized ROPs in T. gondii biology, with a focus on their contributions to virulence. Methods: Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9)–mediated genome editing was employed to generate epitope-tagged and knockout mutants for each candidate ROP in the T. gondii RHΔ ku80 strain. Subcellular localization was determined via immunofluorescence microscopy in both tachyzoite and bradyzoite stages. In vitro assays assessed parasite invasion, replication, egress, and plaque formation. In vivo virulence was evaluated in mouse infection models. To explore molecular mechanisms underlying virulence attenuation, we performed transcriptomic profiling of RHΔ rop64 and RHΔ rop65 knockout strains. Results: All 11 candidate ROPs exhibited rhoptry localization in both tachyzoite and bradyzoite stages. Despite no apparent in vitro growth defects, deletion of ROP64 and ROP65 led to significant attenuation of virulence in mice, with ROP64 showing the most pronounced effect. Transcriptome analysis revealed downregulation of key immune-modulatory genes, including ROP5 , ROP39 , TgIST , and PLP1 . In addition, RHΔ rop64 exhibited broader suppression of ROPs than RHΔ rop65 , suggesting it has a more pronounced role in immune modulation. Conclusions: ROP64 and ROP65 are critical to T. gondii virulence, likely through modulation of the parasite's immune-evasive machinery. Their regulatory influence on effector expression underscores their importance in host adaptation. Importantly, the RHΔ rop64 mutant displays characteristics of an attenuated strain with potential for vaccine development against toxoplasmosis. Graphical

Dense granule protein 47 (GRA47) of Toxoplasma gondii, which localizes to the parasitophorous vacuole membrane (PVM) and the parasitophorous vacuole (PV), is critical for PVM integrity, nutrient permeability, and parasite virulence. However, the global transcriptional response to GRA47 loss remains unexplored. High-throughput RNA sequencing was performed to compare transcriptomic profiles of a GRA47 knockout strain (RHΔgra47) and its parental wild-type RH strain. Differentially expressed genes (DEGs) were identified and analyzed by Gene Ontology (GO) and KEGG enrichment, with selected DEGs validated by reverse transcription quantitative real-time PCR (RT-qPCR). A total of 285 DEGs were identified, comprising 218 upregulated and 67 downregulated genes. Upregulated DEGs were enriched in autophagy, mitophagy, ribosome biogenesis, and GPI anchor biosynthesis pathways, along with nutrient starvation response terms, which may suggest potential roles for the activation of stress response and compensatory metabolic programs. Downregulated DEGs were enriched in host cell entry, rhoptry components, membrane-related terms, and the Hedgehog signaling pathway, a pattern that could suggest a possible association with resource allocation strategy. This first transcriptomic analysis of GRA47 loss in T. gondii identifies transcriptional changes that point to a possible resource allocation strategy prioritizing parasite survival over proliferation and offers a foundational resource for investigating compensatory mechanisms and developing novel intervention strategies. This study reports transcriptional changes only; further experiments are needed to confirm protein-level effects and biological function.

Toxoplasma gondii is a widespread intracellular parasite posing serious risks to immunocompromised individuals and pregnant women, and causing significant economic losses to the livestock industry. Current clinical drugs mainly target acute-stage tachyzoites but are ineffective against chronic cysts, necessitating new interventions. The parasite’s sexual stage in the feline intestine remains poorly understood but is critical for transmission. Here, based on our unpublished weighted gene co-expression network analysis of single-cell transcriptomic data of cat intestine after infection with T. gondii, we selected five previously uncharacterized genes, including three CCCH-type zinc finger proteins (TGME49_257130, TGME49_500318, TGME49_313740) and two subtilisin-like proteases (TGME49_248760/sub7, TGME49_235950/sub8). Using the CRISPR-Cas9 system, individual knockout strains were generated from the type II Pru strain. And, we performed phenotypic assessments at this stage as an essential validation to confirm neutrality before feline infection experiments. Phenotypic analyses revealed that none of the knockout strains showed significant differences in vitro growth, replication, egress, in vivo virulence, or cyst formation from the wild-type strain, corroborating the transcriptional prediction. Publicly available ToxoDB transcriptomic data confirmed that four of these genes were highly expressed at the oocyst or feline enteroepithelial stages, but barely expressed in tachyzoites and bradyzoites, consistent with our phenotypic observations. Collectively, these findings indicate that these genes are dispensable for asexual development, while their expression pattern suggests potential involvement in sexual reproduction, a hypothesis that warrants direct experimental validation. This study provides validated knockout strains and candidate genes, establishing an experimental foundation for future functional studies on Toxoplasma sexual development and transmission-blocking strategies.