Shi-Bo Huang

Toxoplasmosis, caused by Toxoplasma gondii, remains an important zoonotic disease affecting both human and animal health, particularly in immunocompromised individuals and during pregnancy. Current treatments are unable to eliminate latent tissue cysts and are further limited by adverse effects and prolonged treatment regimens, highlighting the need for effective vaccines. In this study, we evaluated the virulence attenuation and protective efficacy of a CRISPR–Cas9-generated rhoptry protein 64 knockout strain (RHΔrop64) as a live attenuated vaccine candidate in Kunming mice. RHΔrop64 exhibited markedly reduced virulence, and mice tolerated doses of up to 1 × 10² tachyzoites without obvious clinical signs. Immunization provided substantial protection against acute challenge with the parental RHΔku80 and ToxoDB#9 (PYS) strains at 30 days post-vaccination and partial protection against type II (Pru) tachyzoites. In addition, all immunized mice survived following oral challenge with 10 or 40 Pru cysts and exhibited reduced brain cyst burdens among surviving mice. Vaccination also induced measurable humoral and cytokine responses, characterized by increased levels of T. gondii-specific IgG and cytokines that were maintained for at least 75 days post-vaccination. These results suggest that RHΔrop64 has the potential to serve as a live attenuated vaccine candidate, providing substantial, although incomplete, protection against acute challenge with the three T. gondii strains tested and being associated with reduced brain cyst burdens among surviving mice following chronic challenge. Further evaluation is required to determine its safety and applicability as a vaccine candidate for food-producing animals and cats.

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

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.

To date, more than 20 species in the genus Cyclospora have been reported. Among them, Cyclospora cayetanensis has been recognized as the causative agent of human cyclosporiasis, which is characterized by severe intestinal injury and prolonged diarrhea in patients with immune dysfunction. The presence of C. cayetanensis in cattle has been confirmed. To date, however, no surveillance data are available on the occurrence and prevalence of Cyclospora spp. in cattle in Shanxi Province, North China. In the present study, a total of 761 fecal samples collected from cattle in three representative counties (Qi, Jishan, and Shanyin) in this Province were examined for Cyclospora spp. by using a polymerase-chain-reaction–restriction-fragment-length polymorphism (PCR–RFLP) test based on the nuclear small subunit ribosomal RNA (SSU rRNA) gene. The prevalence of Cyclospora spp. in cattle was 2.1%, and region, age, sex, and breed were not identified to be risk factors. Molecular evolutionary analysis based on the SSU rRNA sequences revealed that all 12 of the isolates were relatively distant from the human pathogen C. cayetanensis; seven isolates were grouped with Cyclospora colobi, whereas the others were grouped with cattle Cyclospora spp. reported previously. Though C. cayetanensis was not detected in cattle in the present study, more investigations should be performed in human populations, other animal species, or cattle from other regions of Shanxi Province and other environmental sources from the One Health perspective.