A rhoptry protein, localizing in the bulb region of rhoptries, could induce protective immunity against Eimeria tenella infection.

Background: Rhoptry organelle proteins (ROPs) secreted by apicomplexan parasites play important roles during parasites invasion and survival in host cells, and are potential vaccine candidates against apicomplexan diseases. Eimeria tenella (E. tenella) is one of the most noteworthy apicomplexan species, which causes hemorrhagic pathologies. Although dozens of putative E. tenella ROP sequences are annotated, most ROP proteins are not well studied. Methods: In this study, an E. tenella ROP21 gene was identified and the recombinant EtROP21 protein (rEtROP21) was expressed in Escherichia coli. The developmental expression levels, localization, and protective efficacy against E. tenella infection in chickens were studied. Results: An EtROP21 gene fragment with an open reading frame (ORF) of 981 bp was obtained from the Beijing strain of E. tenella. The rEtROP21 has a molecular weight of approximately 50 kDa and was recognized by rEtROP21-immunized mouse serum. Two specific protein bands, about 43 KDa and 95 KDa in size, were detected in the whole sporozoite proteins using the rEtROP21-immunized chicken serum. RT-qPCR analysis of the E. tenella ROP21 gene (EtROP21) revealed that its mRNA levels were higher in merozoites and sporozoites than in sporulated and unsporulated oocysts. Immunofluorescence and immunoelectron analyses showed that the EtROP21 protein predominantly localizes in the bulb region of rhoptries distributed at anterior, posterior, and perinuclear regions of E. tenella sporozoites. Immunization and challenge experiments revealed that immunizing chickens with rEtROP21 significantly increased their average body weight gain while decreasing mean lesion score and oocyst output (P <0.05). When compared with the challenged control group, the rEtROP21-immunized group was associated with a significantly higher relative weight gain (90.2%) and a greater reduction in oocyst output (67%) (P <0.05). The anticoccidial index of the rEtROP21-immunized group was 163.2. Chicken serum ELISA revealed that the levels of the specific anti- rEtROP21 antibody, IFN-γ, and IL-4 were significantly higher in the rEtROP21-immunized group than in the challenged control group (P <0.05). Conclusion: These results indicate that rEtROP21 can induce a high level of specific immune response and it is a potential candidate for the development of vaccines against E. tenella infection in chickens.

Protective efficacy of Toxoplasma gondii GRA12 or GRA7 recombinant proteins encapsulated in PLGA nanoparticles against acute Toxoplasma gondii infection in mice.

Background: Toxoplasma gondii is an apicomplexan parasite that affects the health of humans and livestock, and an effective vaccine is urgently required. Nanoparticles can modulate and improve cellular and humoral immune responses. Methods: In the current study, poly (D, L-lactic-co-glycolic acid) (PLGA) nanoparticles were used as a delivery system for the T. gondii dense granule antigens GRA12 and GRA7. BALB/c mice were injected with the vaccines and protective efficacy was evaluated. Results: Mice immunized with PLGA+GRA12 exhibited significantly higher IgG, and a noticeable predominance of IgG2a over IgG1 was also observed. There was a 1.5-fold higher level of lymphocyte proliferation in PLGA+GRA12-injected mice compared to Alum+GRA12-immunized mice. Higher levels of IFN-g and IL-10 and a lower level of IL-4 were detected, indicating that Th1 and Th2 immune responses were induced but the predominant response was Th1. There were no significant differences between Alum+GRA7-immunized and PLGA+GRA7-immunized groups. Immunization with these four vaccines resulted in significantly reduced parasite loads, but they were lowest in PLGA+GRA12-immunized mice. The survival times of mice immunized with PLGA+GRA12 were also significantly longer than those of mice in the other vaccinated groups. Conclusion: The current study indicated that T. gondii GRA12 recombinant protein encapsulated in PLGA nanoparticles is a promising vaccine against acute toxoplasmosis, but PLGA is almost useless for enhancing the immune response induced by T. gondii GRA7 recombinant protein.

Molecular characterization and protective efficacy of vacuolar protein sorting 29 from Eimeria tenella.

Introduction: Vacuolar protein sorting 29 (VPS29) is a core component of the retromer-retriever complex and is essential for recycling numerous cell-surface cargoes from endosomes. However, there are no reports yet on VPS29 of Eimeria spp. Methods: Here, we cloned and prokaryotically expressed a partial sequence of Eimeria tenella VPS29 (EtVPS29) with RT-PCR and engineered strain of Escherichia coli respectively. The localization of the VPS29 protein in E. tenella sporozoites was investigated with immunofluorescence (IFA) and overexpression assays. And its protective efficacy against E. tenella infection was investigated in chickens with the animal protection test. Results: An EtVPS29 gene fragment with an ORF reading frame of 549 bp was cloned. The band size of the expressed recombinant protein, rEtVPS29, was approximately 39 kDa and was recognized by the chicken anti-E. tenella positive serum. EtVPS29 protein was observed widely distributing in the cytoplasm of E. tenella sporozoites in the IFA and overexpression assays. rEtVPS29 significantly increased average body weight gain and decreased mean lesion score and oocyst output in chickens. The relative weight gain rate in the rEtVPS29-immunized group was 62.9%, which was significantly higher than that in the unimmunized and challenged group (P < 0.05). The percentage of reduced oocyst output in the rEtVPS29 immunized group was 32.2%. The anticoccidial index of the rEtVPS29-immunized group was 144.2. Serum ELISA also showed that rEtVPS29 immunization induced high levels of specific antibodies in chickens. Discussion: These results suggest that rEtVPS29 can induce a specific immune response and is a potential candidate for the development of novel vaccines against E. tenella infections in chickens.

Molecular Detection and Genotyping of Toxoplasma gondii from Pigs for Human Consumption in Zhejiang and Jiangsu Provinces, Eastern China.

Toxoplasma gondii infections are common in humans and animals worldwide. Ingestion of raw or undercooked meat containing tissue cysts of T. gondii is one major source of transmission of this parasite. It is important to guarantee the meat quality of China since our pork industry produces about half of the world's pork. In this study, a total of 746 pig samples were collected from Zhejiang and Jiangsu provinces in eastern China, and examined for T. gondii infection by PCR amplification targeting B1 gene. In this study, we found that 57 of 746 (7.6%) pigs were positive for B1 gene, with 8.5% (48/562) in Zhejiang province and 4.9% (9/184) in Jiangsu province, respectively. The positive DNA samples were further genotyped at 11 genetic markers, including SAG1, 5'-and 3'-SAG2, alternative SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico through PCR-restriction fragment length polymorphism (PCR-RFLP) technology. Two genotypes (ToxoDB 9 and ToxoDB 10) of T. gondii were identified by PCR-RFLP in Zhejiang province. However, both genotypes were not determined from Jiangsu province, which is speculated on the low DNA concentration and the small number of samples. These results indicate that T. gondii infection is endemic in pigs in eastern China and may raise public food safety concerns, suggesting more interventions for T. gondii-related risks are needed in the future.

Enhancing Immune Responses to a DNA Vaccine Encoding Toxoplasma gondii GRA7 Using Calcium Phosphate Nanoparticles as an Adjuvant.

Toxoplasma gondii infects almost all warm-blooded animals, including humans. DNA vaccines are an effective strategy against T. gondii infection, but these vaccines have often been poorly immunogenic due to the poor distribution of plasmids or degradation by lysosomes. It is necessary to evaluate the antigen delivery system for optimal vaccination strategy. Nanoparticles (NPs) have been shown to modulate and enhance the cellular humoral immune response. Here, we studied the immunological properties of calcium phosphate nanoparticles (CaPNs) as nanoadjuvants to enhance the protective effect of T. gondii dense granule protein (GRA7). BALB/c mice were injected three times and then challenged with T. gondii RH strain tachyzoites. Mice vaccinated with GRA7-pEGFP-C2+nano-adjuvant (CaPNs) showed a strong cellular immune response, as monitored by elevated levels of anti-T. gondii-specific immunoglobulin G (IgG), a higher IgG2a-to-IgG1 ratio, elevated interleukin (IL)-12 and interferon (IFN)-γ production, and low IL-4 levels. We found that a significantly higher level of splenocyte proliferation was induced by GRA7-pEGFP-C2+nano-adjuvant (CaPNs) immunization, and a significantly prolonged survival time and decreased parasite burden were observed in vaccine-immunized mice. These data indicated that CaPN-based immunization with T. gondii GRA7 is a promising approach to improve vaccination.

Construction of a lentiviral T/A vector for direct analysis of PCR-amplified promoters.

The promoter plays an important role in the regulation of gene expression. To analyze a promoter's activity, we developed a novel lentiviral T/A vector that contains two reporter genes, a luciferase (Luc2) gene and a green fluorescent protein (Venus) gene, that are linked via an internal ribosome entry site (IRES2). To test the performance of this vector, phosphoglycerate kinase-1 (PGK) and elongation factor-1α (EF1α) promoters were amplified by PCR and inserted into this lentiviral T/A vector using T4 DNA ligase, yielding two promoter-reporter vectors: pLent-T-PGK and pLent-T-EF1α. When these vectors were transfected into 293T cells, we observed a higher level of Venus expression under a fluorescence microscopy in the case of pLent-T-EF1α as compared to pLent-T-PGK. The results of the luciferase reporter assay showed that the ratio of the promoter activities of EF1α and PGK was approximately 9:1. The two promoter-reporter vectors were also packaged as lentiviral particles to conduct promoter activity assay in cultured cells. The ratio of the promoter activities of EF1α and PGK was 4.23:1 when they were infected into 293T cells at a multiplicity of infection of 1. This value is comparable to that of a parallel experiment using the commercial luciferase reporter vector pGL4.10 with an activity ratio of 5.99:1 for EF1α and PGK. These results indicate that lentiviral T/A vector will be a useful tool for analysis of promoter activity and specificity.

[The advancement on mitochondrion of cryptosporidium].

Cryptosporidiosis is an important apicomplexan disease with medical and veterinary significance. There is still no effective drug for its control. Mitochondrion is an organelle which contains most protein and enzyme in eukaryotes, so the mitochondrion of Cryptosporidium may be a potential target of drugs. Recent studies provided evidence for a mitochondrial derived compartment in this parasite. But the organelle has some difference to that of other apicomplexan parasites. This organelle appears to lack its genome, and thus must be entirely dependent on nuclear-encoded proteins. This article reviews the evidence for the organelle in Cryptosporidium and its probable function.