Neospora caninum is an obligate intracellular parasitic protozoan that can cause abortions in cattle and pose considerable economic losses to the cattle industry. As a major livestock province, little is known of N. caninum infection in cattle in Shanxi Province, north China. In order to investigate the seroprevalence of N. caninum in cattle in Shanxi Province, 978 cattle serum samples were collected from 11 cities in three representative geographical locations in Shanxi Province, and the N. caninum-specific IgG antibodies were examined using an indirect enzyme linked immunosorbent assay (ELISA) kit commercially available. The results showed that 133 of the 978 examined cattle serum samples (13.60%, 95% CI = 11.45–15.75) were positive for N. caninum antibodies, and the seroprevalence in different cities ranged from 0 to 78.89%. The geographical location and management mode were the risk factors associated with N. caninum infection in cattle herds in Shanxi Province. Cattle in Northern and Central Shanxi Province as well as cattle whose management mode is that of large-scale cattle farming companies are more susceptible to N. caninum infection. This was the first large-scale survey of N. caninum seroprevalence and assessment of associated risk factors in cattle in Shanxi Province, which provided baseline information for the prevention and control of N. caninum infection in cattle in Shanxi Province, north China.

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

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.