Xiao Zhang

Coccidiosis, caused by Eimeria protozoa remains a major economic burden for the global poultry industry. Live-vector vaccines represent a promising alternative strategy for coccidiosis prevention; effective live-vector anticoccidial vaccines rely on two key prerequisites: a safe and efficient delivery vector and well-defined protective parasite antigens. Attenuated Salmonella typhimurium is an appealing oral live-delivery platform, capable of delivering heterologous antigens while triggering a robust intestinal mucosal immune response. Apical membrane antigen 1 (AMA1) participates in the invasion process of Eimeria tenella and serves as a promising protective vaccine candidate. In this study, bioinformatic tools were applied to identify the extracellular domain of EtAMA1, and a recombinant attenuated S. typhimurium X4550 (EtAMA1) vaccine strain was constructed. We first assessed the biosafety of this engineered strain. Subsequently, oral immunization and E. tenella challenge trials in chickens were performed. Protective parameters including body weight gains, oocyst excretion, cecal lesion scores, and the anticoccidial index (ACI) were evaluated. Serum IgG and secretory immunoglobulin A (sIgA), as well as peripheral blood lymphocyte proportions and transformation rates, were further detected to characterize humoral, mucosal, and cellular immune responses induced by vaccination. The results showed that the recombinant X4550 (EtAMA1) strain exhibited favorable safety performance. Vaccinated chickens displayed significantly mitigated body weight loss (P<0.001), alleviated cecal pathological lesions, and markedly reduced oocyst excretion (P<0.001), yielding an ACI value of 171.14. Moreover, significantly elevated levels of serum IgG and intestinal sIgA (P<0.001), accompanied by increased peripheral blood lymphocyte proportion and transformation rate (P<0.001), were observed in immunized animals. Overall, the recombinant Salmonella X4550(EtAMA1) strain exhibits good immunogenicity and safety. It elicits integrated humoral, mucosal, and cellular immune responses and provides an effective candidate strategy for the prevention and control of coccidiosis in chickens.

Serpin protease inhibitors (SERPIN) in protozoa play crucial roles in various biological processes, including the invasion of host cells. However, the precise roles and molecular mechanisms underlying SERPIN-mediated invasion of parasite remain poorly understand. In this study, we provide evidence that surface-expressed Eimeria tenella SERPIN1 (EtSERPIN1) on sporozoites is involved in adhesion and invasion processes. To elucidate the molecular target responsible for mediating EtSERPIN1-induced invasion, we utilized GST pull-down and yeast two-hybrid assays to identify host cell membrane proteins interacting with EtSERPIN1. Our findings revealed an interaction between EtSERPIN1 and a membrane protein called annexin A2 (ANXA2). Furthermore, recombinant GgANXA2 was shown to bind to the sporozoite surface. Pre-treatment with anti-GgANXA2-specific antibody or recombinant GgANXA2 protein significantly inhibited EtSERPIN1 binding to host cells. Pre-incubation with recombinant GgANXA2 also reduced sporozoite infection in both DF-1 cells and chickens. These results suggest that the interaction between EtSERPIN1 and GgANXA2 plays a critical role in both adhesion and invasion processes of E. tenella sporozoites. Finally, we investigated the impact of recombinant GgANXA2 and EtSERPIN1 proteins on E. tenella infection. Our results demonstrate that incubation with GgANXA2 protein significantly attenuated sporozoite infectivity, as evidenced by a significant reduction in parasite burden within the chicken cecum. Immunization with recombinant EtSERPIN1 exhibited potent anti-E. tenella activity, with higher body weight gains, lower cecal lesions and oocyst output, as well as elevated levels of cecal mucosa antibodies. These findings suggest that targeting GgANXA2 through EtSERPIN1 mediates adhesion and invasion processes of E. tenella, highlighting its potential as a novel therapeutic target.

Purpose Recent years have seen increased focused on developing new anti-parasitic drugs that are both highly effective and low in toxicity, as the widespread use of existing anti-parasitic drugs has raised growing concerns. Natural products have gained significant interest due to their diverse biological activities with minimal toxic side effects. Our previous studies have already demonstrated the good anti-E. tenella effect of Perillyl Alcohol. To further investigate its efficacy against other protozoa, we selected Toxoplasma gondii as the researchsubject. Methods In this study, we utilized the CCK-8 assay in vitro to assess the cytotoxicity of Perillyl Alcohol on DF-1 cells. The impact of Perillyl Alcohol of T. gondii tachyzoite invasion and intracellular proliferation were investigated in vitro. In vivo, we evaluated the effect of Perillyl Alcohol on the pathogenicity of T. gondii, including host survival time, liver and spleen tissue damage, cysts formed in the brain. Furthermore, we analyzed the expression levels of isoprenylation-related genes using quantitative PCR (qPCR). Results The half maximal inhibitory concentration (CC50) of Perillyl Alcohol against DF-1 cells was determined to be 525.0. In vitro studies showed that treatment with Perillyl Alcohol effectively inhibited the invasion rate and intracellular proliferation of T. gondii tachyzoite. The half maximal inhibitory concentration (IC50) was calculated to be 314.3, and further analysis yielded a selectivity index (SI) of 1.67. In vivo, Perillyl Alcohol treatment prolonged the survival time and increased the survival rate of T. gondii-infected mice, while reducing the parasite burden in liver and spleen tissues. It also demonstrated a certain protective effect against T. gondii-induced tissue damage, including effectively alleviating hepatosplenomegaly and mitigating the elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels induced by hepatic injury. Building on this foundation, we further explored the impact of Perillyl Alcohol on the formation of brain cysts and found that it could significantly reduce the number of brain cysts induced by the Pru strain of T. gondii infection. After treatment with Perillyl Alcohol, the expression levels of isoprenylation-related enzymes Tgmecs, Tgdxr, and Tghdr were significantly reduced. Conclusions These results demonstrate that Perillyl Alcohol may suppress the growth of T. gondii by significantly inhibiting the expression of enzymes involved in isoprenoid biosynthesis.

Protozoan parasites such as Eimeria tenella and Toxoplasma gondii pose significant health challenges in livestock and humans. The limited treatment options and rising drug resistance underscore the urgent need for new therapies. This study investigates myrcene, a monoterpene hydrocarbon classified for its antiprotozoal potential against E. tenella and T. gondii infections. Initially, we examined its effect on the sporulation process of E. tenella oocysts in vitro and its anti-E. tenella activity in vivo. Myrcene significantly reduced the sporulation rate of E. tenella oocysts at 3 and 4 mg/kg. In vivo experiments demonstrated that treatment with 4 mg/kg myrcene significantly reduced E. tenella load and oocyst output, as well as cecal lesion and weight loss caused by E. tenella infection, showing moderate anti-E. tenella activity, with an Anticoccidial Index (ACI) of 161.4. Furthermore, we investigated the anti-T. gondii activity of myrcene both in vitro and in vivo. In vitro studies showed that treatment with myrcene effectively inhibited the invasion rate and intracellular proliferation ability of T. gondii tachyzoite in DF-1 cells in a dose-dependent manner. In vivo administration prolonged the survival time in T. gondii-infected mice, suggesting notable protective effects. Additionally, it mitigated T. gondii-induced hepatosplenic toxicity by reducing parasite load in the liver and spleen, and ameliorating liver function as evidenced by decreased serum transaminase levels. In conclusion, the findings demonstrate promising anti-E. tenella and anti-T. gondii activity exhibited by myrcene warranting further exploration into its mechanisms and potential therapeutic applications.