Entamoeba spp. are common zoonotic intestinal protozoa, which can lead to serious intestinal diseases in both humans and animals through fecal–oral transmission, leading to significant economic losses and public health challenges. To reveal the prevalence of Entamoeba in sheep and cattle in Shanxi Province, North China, fecal samples were collected from 311 sheep, 392 dairy cattle, and 393 beef cattle from three representative counties in the northern, central, and southern regions of Shanxi Province. DNA was extracted from the fecal samples and amplified by PCR with primers targeting the nuclear small subunit ribosomal RNA (SSU rRNA) gene of Entamoeba spp., followed by the sequencing of the positive products. The overall infection rates of Entamoeba were 51.5% (160/311), 82.9% (325/392), and 79.1% (311/393) in sheep, dairy cattle, and beef cattle, respectively. Statistical analysis showed a significant correlation between the infection rate of Entamoeba and the location factor in sheep, dairy cattle, and beef cattle (p < 0.001). According to the obtained SSU rRNA sequences, several Entamoeba species, namely Entamoeba bovis, Entamoeba Ribosomal Lineage (RL) 2, Entamoeba RL4, and Entamoeba RL8, were identified. This study represents the first molecular survey of Entamoeba prevalence in sheep, beef cattle, and dairy cattle in Shanxi Province. The findings extend the geographical distribution of Entamoeba spp. and provide valuable scientific data for the prevention and control of amoebiasis in Shanxi Province.

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.

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.