Mining host candidate regulators of schistosomiasis-induced liver fibrosis in response to artesunate therapy through transcriptomics approach.

BACKGROUND: Artesunate (ART) has been reported to have an antifibrotic effect in various organs. The underlying mechanism has not been systematically elucidated. We aimed to clarify the effect of ART on liver fibrosis induced by Schistosoma japonicum (S. japonicum) in an experimentally infected rodent model and the potential underlying mechanisms. METHODS: The effect of ART on hepatic stellate cells (HSCs) was assessed using CCK-8 and Annexin V-FITC/PI staining assays. The experimental model of liver fibrosis was established in the Mongolian gerbil model infected with S. japonicum cercariae and then treated with 20 mg/kg or 40 mg/kg ART. The hydroxyproline (Hyp) content, malondialdehyde (MDA) content, superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities in liver tissue were measured and histopathological changes of liver tissues were observed. Whole-transcriptome RNA sequencing (RNA-seq) of the liver tissues was performed. Differentially expressed genes (DEGs) were identified using bioinformatic analysis and verified by quantitative PCR (qPCR) and western blot assay. RESULTS: ART significantly inhibited the proliferation and induce the apoptosis of HSCs in a dose-dependent manner. In vivo, Hyp content decreased significantly in the ART-H group compared to the model (MOD) group and GPX activity was significantly higher in the ART-H group than in the MOD group. Besides, ART treatment significantly reduced collagen production (p <0.05). A total of 158 DEGs and 44 differentially expressed miRNAs related to ART-induced anti-schistosomiasis liver fibrosis were identified. The qPCR and western blot results of selected DEGs were consistent with the sequencing results. These DEGs were implicated in key pathways such as immune and inflammatory response, integrin-mediated signaling and toll-like receptor signaling pathways. CONCLUSION: ART is effective against liver fibrosis using Mongolian gerbil model induced by S. japonicum infection. We identified host candidate regulators of schistosomiasis-induced liver fibrosis in response to ART through transcriptomics approach.

A novel loop-mediated isothermal amplification-lateral-flow-dipstick (LAMP-LFD) device for rapid detection of Toxoplasma gondii in the blood of stray cats and dogs.

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis and threatens warm-blooded animal and human health worldwide. Simple and applicable diagnostic methods are urgently needed to guide development of effective approaches for prevention of toxoplasmosis. Most molecular diagnostic tools for T. gondii infection require high technical skills, sophisticated equipment, and a controlled lab environment. In this study, we developed a loop-mediated isothermal amplification-lateral-flow-dipstick (LAMP-LFD) assay that specifically targets the 529 bp for detecting T. gondii infection. This novel portable device is universal, fast, user-friendly, and guarantees experimental sensitivity as well as low risk of aerosol contamination. Our LAMP-LFD assay has a detection limit of 1 fg of T. gondii DNA, and shows no cross-reaction with other parasitic pathogens, including Cryptosporidium parvum, Leishmania donovani, and Plasmodium vivax. We validated the developed assay by detecting T. gondii in DNA extracted from blood samples collected from 318 stray cats and dogs sampled from Deqing, Wenzhou, Yiwu, Lishui and Zhoushan cities across Zhejiang province, Eastern China. The LAMP-LFD device detected T. gondii DNA in 4.76 and 4.69% of stray cats and dogs, respectively. In conclusion, the developed LAMP-LFD assay is efficient, minimizes aerosol contamination, and is therefore suitable for detecting T. gondii across basic medical institutions and field settings.

TITLE: Un nouveau dispositif de bandelette à flux latéral d'amplification isotherme médiée par les boucles (LAMP-LFD) pour la détection rapide de Toxoplasma gondii dans le sang des chats et chiens errants. ABSTRACT: Toxoplasma gondii est un parasite protozoaire intracellulaire obligatoire qui provoque la toxoplasmose et menace la santé humaine et les animaux à sang chaud dans le monde entier. Des méthodes de diagnostic simples et applicables sont nécessaires de toute urgence pour guider le développement d'approches efficaces pour la prévention de la toxoplasmose. La plupart des outils de diagnostic moléculaire pour l'infection par T. gondii nécessitent des compétences techniques élevées, un équipement sophistiqué et un environnement de laboratoire contrôlé. Dans cette étude, nous avons développé un test par bandelettes à flux latéral d'amplification isotherme médiée par les boucles (LAMP-LFD) qui cible spécifiquement les 529 pb qui détectent une infection par T. gondii. Ce nouvel appareil portable est universel, rapide, convivial et garantit une sensibilité expérimentale ainsi qu'un faible risque de contamination par aérosol. Notre test LAMP-LFD a une limite de détection de 1 fg d'ADN de T. gondii et ne montre aucune réaction croisée avec d'autres pathogènes parasites, y compris Cryptosporidium parvum, Leishmania donovani et Plasmodium vivax. Nous avons validé le test en détectant T. gondii dans l'ADN extrait d'échantillons de sang prélevés sur 318 chats et chiens errants prélevés dans les villes de Deqing, Wenzhou, Yiwu, Lishui et Zhoushan dans la province du Zhejiang, dans l'est de la Chine. Le dispositif LAMP-LFD a détecté la prévalence de l'ADN de T. gondii chez respectivement 4,76 et 4,69% des chats et chiens errants. En conclusion, le test LAMP-LFD développé est efficace, minimise la contamination par les aérosols et convient donc à la détection de T. gondii dans les établissements médicaux simples et sur le terrain.

Early Detection of Trichinella spiralis DNA in Rat Feces Based on Tracing Phosphate Ions Generated During Loop-Mediated Isothermal Amplification.

Early diagnosis of trichinellosis is still difficult because of the lack of specific symptoms and limited window for serological detection. Here we established an assay based on tracing phosphate ions generated during loop-mediated isothermal amplification (LAMP) to detect Trichinella spiralis DNA in rat feces during its early stage of infection. By targeting a 1.6-kb repetitive element of Tri. spiralis, the assay was able to detect Tri. spiralis DNA in the feces of all infected rats as early as 1 day postinfection (dpi). The positive detection lasted to 7 dpi in the rats infected with 250 muscle larvae, and 21 dpi in the rats infected with 5,000 larvae. The assay was highly sensitive, and could detect 1.7 femtograms (fg) of Tri. spiralis DNA with high specificity, and with no cross reactivity with the DNA from Anisakis pegreffii, Gnathostoma spinigerum, Angiostrongylus cantonensis, Enterobius vermicularis, Schistosoma japonicum, and Trypanosoma evansi. Our present study provided a reliable technique for the early diagnosis of trichinellosis with the advantages of simplicity and speed, as well as high sensitivity and specificity.

The Virulence-Related MYR1 Protein of Toxoplasma gondii as a Novel DNA Vaccine Against Toxoplasmosis in Mice.

Toxoplasma gondii causes serious public health problems, but there is no effective treatment strategy against it currently. DNA vaccines have shown promising findings in this regard. MYR1 is a new virulence factor identified in T. gondii that may have potential as a DNA vaccine candidate. We constructed a recombinant eukaryotic plasmid, pVAX1-MYR1, as a DNA vaccine, injected it intramuscularly into BALB/c mice, and evaluated its immunoprotective effects. pVAX1-MYR1 immunization induced a sequential Th1 and Th2 T-cell response, as indicated by high levels of Th1 and mixed Th1/Th2 cytokines at 2 and 6 weeks after immunization, respectively. These findings were corroborated by the antibody assays too. In addition, increased levels of antigen-specific lymphocyte proliferation, CD4+ and CD8+ T lymphocytes, cytotoxic T lymphocyte activity and cytokine (IFN-γ, IL-12, and IL-10) production were also observed in the immunized mice. These findings showed that pVAX1-MYR1 stimulated humoral and cellular immune responses in the immunized mice. The increased production of IFN-γ and IL-12 was correlated with increased expression of the T-bet and p65 genes of the NF-κB pathway. However, no significant increase was observed in the level of IL-4. The survival of mice immunized with pVAX1-MYR1 was also significantly prolonged compared with the control group mice. Based on all the above findings, the current study proposes that pVAX1-MYR1 can induce a T. gondii-specific immune response and should therefore be considered as a promising vaccine candidate against toxoplasmosis. To the best of our knowledge, this is the first report to evaluate the immunoprotective value of an MYR1-based DNA vaccine against T. gondii.

Early Detection of Toxoplasma gondii Infection In Mongolian Gerbil By Quantitative Real-Time PCR.

Toxoplasmosis, caused by Toxoplasma gondii, is associated with several clinical syndromes, including encephalitis, chorioretinitis, and congenital infection. Toxoplasma gondii is a ubiquitous apicomplexan parasite found in both humans and animals. Mongolian gerbils, which are more susceptible to both high- and low-virulence Toxoplasma strains compared with mice, are considered useful models for assessing diagnosis and treatment methods for toxoplasmosis, as well as infection by and host defense to this organism. Here we established a quantitative real-time polymerase chain reaction (qPCR) method targeting the B1 gene for early and specific detection of T. gondii infection in Mongolian gerbil. The detection limit of the developed qPCR was approximately 1 T. gondii tachyzoite. This method was also applied to detect T. gondii genomic DNA in experimentally infected Mongolian gerbils, with positive results in blood (66.7%), liver (73.3%), lung (80.0%), spleen (80.0%), and peritoneal fluid (66.7%) samples as early as 1 day postinfection. Specificity tests confirmed no cross-reactivity with DNA templates of Neospora caninum, Cryptosporidium parvum, Eimeria tenella, Trypanosoma evansi, Schistosoma japonicum, Angiostrongylus cantonensis, and Strongyloides stercoralis. This study first reports the use of Mongolian gerbils as an animal model for early diagnosis of toxoplasmosis by qPCR.

DNA detection of Trypanosoma evansi: Diagnostic validity of a new assay based on loop-mediated isothermal amplification (LAMP).

Trypanosoma evansi (T. evansi) is the most widely spread pathogenic trypanosome in the world. The control of trypanosomiasis depends on accurate diagnosis and effective treatment. Focusing on the presence of T. evansi in Asia, we developed a detection assay based on tracing phosphate ions (Pi) generated during LAMP targeting the variant surface glycoprotein (VSG) gene of Rode Trypanozoon antigenic type 1.2 (RoTat 1.2 VSG). The diagnostic potential as well as the use of the assay as a test-of-cure method after berenil treatment, was assessed in mice at different time points of infection. In addition, 67 buffalo blood collected from Tongling county, Anhui province, as well as 42 cattle sera from the Shanghai area, were used to evaluate the diagnostic validity of the test. The detection limit of the novel LAMP assay was determined to be as low as 1 fg of T. evansi DNA, while the reaction time for the test was only 30min. Hence it outperforms both microscopy and PCR. In the test-of-cure assessment, successful berenil mediated cure could be confirmed within 48h after treatment. This offers a tremendous advantage over conventional antibody-based diagnostic tools in which successful cure only can be confirmed after months. In the cattle and buffalo screening, the LAMP was able to detect a false-negative determined sample, wrongly classified in a conventional microscopy and PCR screening. Finally, no cross-reactivity was observed with other zoonotic parasites, such as T. evansi type B, T. congolense, T. brucei, Schistosoma japonicum, Plasmodium falciparum, Leishmania donovani, Toxoplasma gondii and Angiostrongylus cantonensis. We conclude that the novel LAMP assay is sensitive, specific and convenient for field use, particularly in areas where infection incidence has become extremely low. The LAMP assay could be used as a tool for trypanosomiasis control and elimination strategies in areas where T. evansi Type A infections are causing a threat to livestock farming.

Immuno-Efficacy of a T. gondii Secreted Protein with an Altered Thrombospondin Repeat (TgSPATR) As a Novel DNA Vaccine Candidate against Acute Toxoplasmosis in BALB/c Mice.

Toxoplasma gondii (T.gondii) is distributed worldwide and infects most species of warm-blooded animals, including humans. Toxoplasmosis has serious consequences, especially in people with an impaired or immature immune system. Thus, an effective vaccine is urgently required. Secretory microneme proteins are essential for the adhesion and invasion of T. gondii. The gene encoding the microneme protein, T. gondii secreted protein with an altered thrombospondin repeat (TgSPATR), we constructed a recombinant eukaryotic plasmid, pVAX1-TgSPATR, as a DNA vaccine, injected it intramuscularly into BALB/c mice and evaluated the induced immune response. Lymphocyte proliferation assays, cytokine (IFN-γ, IL-2, IL-4, IL-10), and antibody determinations showed that mice immunized with pVAX1-TgSPATR produced humoral and mixed Th1/Th2 type cellular immune responses. The survival times of mice immunized with pVAX1-TgSPATR were also significantly prolonged (15.7 ± 1.42 days) compared with control groups, which died within 7 days of challenge (p < 0.05). The current study indicated that pVAX1-TgSPATR induce a T. gondii specific immune response and might be a promising vaccine candidate against toxoplasmosis. To the best of our knowledge, this is the first report to evaluate the immunoprotective value of TgSPATR against T. gondii.

Genome-wide miRNAs expression profiles of Schistosoma japonicum schistosomula in response to artesunate.

AIM: miRNAs play a significant role in pharmacogenomics and are likely to be important in the molecular mechanism of atesunate (ART) effects on Schistosoma japonicum. METHODS: We sequenced the RNAs using an Illumina (Solexa) DNA sequencer and compared the relative expression levels of the miRNAs in 10-day-old schistosomula from ART and the parallel control group. RESULTS: We characterized 95 known miRNAs from S. japonicum schistosomula individuals, including 38 novel miRNA families. Among the detectable 134 miRNAs differentially expressed (>2.0-fold change, p < 0.01) after ART treatment in schistosomula, a total of seven known or novel 3p- or 5p- derived S. japonicum miRNAs were characterized. We propose that sja-miR-125b may regulate the expression of ART metabolizing enzymes, glutathione synthetase or heme-binding protein 2 to help S. japonicum resists or adapts to drug stress and also ART may significantly inhibit sexual maturation of female worms mediated by mir-71b/2 miRNA cluster. CONCLUSION: This was the first comprehensive miRNAs expression profile analysis of S. japonicum in response to ART, and provides an overview of the complex network of the mechanism of action of ART on S. japonicum.

Quantitative proteomic analyses of Schistosoma japonicum in response to artesunate.

Artesunate (ART) has high prophylactic efficacy against Schistosoma japonicum infections and has been used to treat and prevent schistosomiasis in China since 1995. However, the molecular mechanism of ART's effects on S. japonicum remains unclear. Herein, we applied isobaric tagging reagents for relative and absolute quantification analyses coupled with two-dimensional liquid chromatography and tandem mass spectrometry to investigate the effect of ART on the proteome of S. japonicum in susceptible mice. 4529 proteins were quantified on the basis of 21,825 unique peptides. Comparative proteomic analyses revealed that 145, 228 and 185 proteins were significantly differentially expressed after ART treatment in schistosomula, juvenile and adult worms, respectively. Ninety proteins were differentially expressed between each two treatment groups in response to ART treatment: 67 proteins were associated with S. japonicum development/aging and 23 were specifically associated with ART treatment. Quantitative real-time PCR of selected genes verified the proteomic data. Gene ontology annotation and Kyoto encyclopedia of genes and genomes pathway mapping analysis showed that the majority of differentially expressed proteins were involved in stress/defense/detoxification, signal transduction, carbohydrate metabolism, amino acid metabolism, transcription/translation, and protein synthesis/assembly/degradation. Thirty-four of the proteins differentially expressed under ART treatment encoded hypothetical, uncharacterized proteins with unknown functions. This study obtained the first comprehensive protein expression profile of S. japonicum in response to ART, and provides a basis for a better understanding of the molecular mechanisms of ART effects on S. japonicum.

Molecular identification of Anisakis and Hysterothylacium larvae in marine fishes from the East China Sea and the Pacific coast of central Japan.

Anisakiasis is a human disease caused by the accidental ingestion of larvae belonging to the family Anisakidae. Three fish species, the small yellow croaker Pseudosciaena polyactis, the mackerel Pneumatophorus japonicus and the hairtail Trichiurus haumela are important source for food products in the East China Sea. The prevalence and the identification of Anisakidae larvae in these fishes will benefit the prevention and control of anisakiasis. In this study, fish samples were obtained from fish markers in the East China Sea and the Pacific coast of central Japan during April 2011 and July 2013. For species identification, the PCR-based restriction fragment length polymorphism (PCR-RFLP) analysis of the entire ITS region (ITS1, 5.8 S and ITS2) of nuclear ribosomal DNA (rDNA) was performed. In total, 2004 larvae were collected from 80 hairtail fish, 20 small yellow croaker, and 27 mackerel from the East China Sea and the Pacific coast of central Japan. High prevalence of Anisakidae larvae infection (116/122, 95.1%) was detected in the East China Sea. Seven species were identified belonging to the genera Anisakis (Nematoda: Anisakidae) and Hysterothylacium (Nematoda: Anisakidae). Anisakis pegreffii was the predominant species accounting for 84.8% of all larvae examined in East China Sea, while all Anisakidae larvae isolated from Japan were identified as Anisakis simplex sensu stricto (s.s.). In the East China Sea, A. simplex s.s. and Anisakis typica were 0.6% (4/619) and 1.5% (9/619) of the identified nematodes, respectively. Interestingly, one larva was identified as a recombinant genotype of A. simplex s.s. and A. pegreffii. In addition, four species of the genus Hysterothylacium, namely, Hysterothylacium amoyense (31/619, 5.0%), Hysterothylacium aduncum (10/619, 1.6%), Hysterothylacium fabri (21/619, 3.4%) and Hysterothylacium spp. (18/619, 2.9%) were also identified in the present study. This is a comprehensive epidemiological dataset for the family Anisakidae in the East China Sea. The identification of A. typica, recombinant genotype of A. simplex s.s. and A. pegreffii, H. amoyense and H. fabri is first reported in this area. The wide diversity and substantial geographical distributions of these nematodes will provide a foundation for future studies of Anisakidae family. The high prevalence of these nematodes in marine fishes off the East China Sea may pose considerable food safety problems, which is a potential cause of human anisakiasis.

A new surveillance and response tool: risk map of infected Oncomelania hupensis detected by Loop-mediated isothermal amplification (LAMP) from pooled samples.

Although schistosomiasis remains a serious health problem worldwide, significant achievements in schistosomiasis control has been made in the People's Republic of China. The disease has been eliminated in five out of 12 endemic provinces, and the prevalence in remaining endemic areas is very low and is heading toward elimination. A rapid and sensitive method for monitoring the distribution of infected Oncomelania hupensis is urgently required. We applied a loop-mediated isothermal amplification (LAMP) assay targeting 28S rDNA for the rapid and effective detection of Schistosoma japonicum DNA in infected and prepatent infected O. hupensis snails. The detection limit of the LAMP method was 100 fg of S. japonicum genomic DNA. To promote the application of the approach in the field, the LAMP assay was used to detect infection in pooled samples of field-collected snails. In the pooled sample detection, snails were collected from 28 endemic areas, and 50 snails from each area were pooled based on the maximum pool size estimation, crushed together and DNA was extracted from each pooled sample as template for the LAMP assay. Based on the formula for detection from pooled samples, the proportion of positive pooled samples and the positive proportion of O. hupensis detected by LAMP of Xima village reached 66.67% and 1.33%, while those of Heini, Hongjia, Yangjiang and Huangshan villages were 33.33% and 0.67%, and those of Tuanzhou and Suliao villages were 16.67% and 0.33%, respectively. The remaining 21 monitoring field sites gave negative results. A risk map for the transmission of schistosomiasis was constructed using ArcMap, based on the positive proportion of O. hupensis infected with S. japonicum, as detected by the LAMP assay, which will form a guide for surveillance and response strategies in high risk areas.

The virulence-related rhoptry protein 5 (ROP5) of Toxoplasma Gondii is a novel vaccine candidate against toxoplasmosis in mice.

Infections with the intracellular protozoan parasite Toxoplasma gondii pose a serious public health problem and are of great economic importance worldwide. The parasite rhoptry protein 5 (ROP5) has been implicated as a major virulence factor that reduces the accumulation of immunity-related GTPases (IRG) in parasitophorous vacuole membrane (PVM), which maintains PVM integrity and evades IFNγ-mediated killing by intracellular parasites. To study the immunoprotective value of ROP5, BALB/c mice were immunized with a recombinant form of the protein administered alone or in combination with another promising vaccine antigen, rSAG1. All mice vaccinated with the recombinant antigens developed a high level of specific antibody responses against soluble tachyzoite antigens (STAg), a statistically significant increase of the splenocyte proliferation response, and significant levels of IFN-γ and IL-2 production. In contrast to rSAG1, which only stimulated the release of IFN-γ and IL-2, rROP5 induced the specific production of IL-10, the Th2-type cytokine, in addition to IFN-γ and IL-2. These results demonstrated that rROP5 could induce significant cellular and humoral (Th1/Th2) immune responses. Moreover, mice immunized with rROP5 displayed a prolonged survival time against a lethal challenge with the T. gondii RH strain. Additionally, vaccination with the mixture of rROP5+rSAG1 resulted in higher levels of T. gondii-specific IgG antibodies and lymphocyte proliferative responses and conferred more efficient protection against T. gondii challenge compared to immunization with rROP5 or rSAG1 alone. Our studies show that recombinant ROP5 antigen may be a promising vaccine candidate against toxoplasmosis. To our knowledge, this is the first report to evaluate the immunoprotective value of ROP5.

Loop-mediated isothermal amplification (LAMP): early detection of Toxoplasma gondii infection in mice.

BACKGROUND: Toxoplasmosis is a widespread zoonotic parasitic disease that occurs in both animals and humans. Traditional molecular assays are often difficult to perform, especially for the early diagnosis of Toxoplasma gondii infections. Here, we established a novel loop-mediated isothermal amplification targeting the 529 bp repeat element (529 bp-LAMP) to detect T. gondii DNA in blood samples of experimental mice infected with tachyzoites of the RH strain. FINDINGS: The assay was performed with Bst DNA polymerase at 65°C for 1 h. The detection limit of the 529 bp-LAMP assay was as low as 0.6 fg of T. gondii DNA. The sensitivity of this assay was 100 and 1000 fold higher than that of the LAMP targeting B1 gene (B1-LAMP) and nested PCR targeting 529 bp repeat element (529 bp-nested PCR), respectively. The specificity of the 529 bp-LAMP assay was determined using the DNA samples of Trypanosoma evansi, Plasmodium falciparum, Paragonimus westermani, Schistosoma japonicum, Fasciola hepatica and Angiostrongylus cantonensis. No cross-reactivity with the DNA of any parasites was found. The assay was able to detect T. gondii DNA in all mouse blood samples at one day post infection (dpi). CONCLUSIONS: We report the following findings: (i) The detection limit of the 529 bp-LAMP assay is 0.6 fg of T. gondii DNA; (ii) The assay does not involve any cross-reactivity with the DNA of other parasites; (iii) This is the first report on the application of the LAMP assay for early diagnosis of toxoplasmosis in blood samples from experimentally infected mice. Due to its simplicity, sensitivity and cost-effectiveness for common use, we suggest that this assay should be used as an early diagnostic tool for health control of toxoplasmosis.

Loop-mediated isothermal amplification: rapid detection of Angiostrongylus cantonensis infection in Pomacea canaliculata.

BACKGROUND: Angiostrongylus cantonensis is a zoonotic parasite that causes eosinophilic meningitis in humans. The most common source of infection with A. cantonensis is the consumption of raw or undercooked mollusks (e.g., snails and slugs) harbouring infectious third-stage larvae (L3). However, the parasite is difficult to identify in snails. The purpose of this study was to develop a quick, simple molecular method to survey for A. cantonensis in intermediate host snails. FINDINGS: We used a loop-mediated isothermal amplification (LAMP) assay, which was performed using Bst DNA polymerase. Reactions amplified the A. cantonensis 18S rRNA gene and demonstrated high sensitivity; as little as 1 fg of DNA was detected in the samples. Furthermore, no cross-reactivity was found with other parasites such as Toxoplasma gondii, Plasmodium falciparum, Schistosoma japonicum, Clonorchis sinensis, Paragonimus westermani and Anisakis. Pomacea canaliculata snails were exposed to A. cantonensis first-stage larvae (L1) in the laboratory, and L3 were observed in the snails thirty-five days after infection. All nine samples were positive as determined by the LAMP assay for A. cantonensis, which was identified as positive by using PCR and microscopy, this demonstrates that LAMP is sensitive and effective for diagnosis. CONCLUSIONS: LAMP is an appropriate diagnostic method for the routine identification of A. cantonensis within its intermediate host snail P. canaliculata because of its simplicity, sensitivity, and specificity. It holds great promise as a useful monitoring tool for A. cantonensis in endemic regions.

Protective effect of DNA-mediated immunization with liposome-encapsulated GRA4 against infection of Toxoplasma gondii.

The dense granule protein 4 (GRA4) is a granular protein from Toxoplasma gondii, and is a candidate for vaccination against this parasite. In this study, the plasmid pcDNA3.1-GRA4 (pGRA4), encoding for the GRA4 antigen, was incorporated by the dehydration-rehydration method into liposomes composed of 16 mmol/L egg phosphatidylcholine (PC), 8 mmol/L dioleoyl phosphatidylethanolamine (DOPE), and 4 mmol/L 1,2-diodeoyl-3-(trimethylammonium) propane (DOTAP). C57BL/6 mice and BALB/c mice were immunized intramuscularly three times with liposome-encapsulated pGRA4 to determine whether DNA immunization could elicit a protective immune response to T. gondii. Enzyme-linked immunosorbent assay (ELISA) of sera from immunized mice showed that liposome-encapsulated pGRA4 generated high levels of IgG antibodies to GRA4. Production of primary interferon (IFN)-gamma and interleukin (IL)-2 in GRA4-stimulated splenocytes from vaccinated mice suggested a modulated Th1-type response. 72.7% of C57BL/6 mice immunized with liposome-encapsulated pGRA4 survived the challenge with 80 tissue cysts of ME49 strain, whereas C57BL/6 mice immunized with pGRA4 had only a survival rate of 54.5%. When immunized BALB/c mice were intraperitoneally challenged with 10(3) tachyzoites of the highly virulent RH strain, the survival time of mice immunized with liposome-encapsulated pGRA4 was markedly longer than that of other groups. Our observations show that liposome-encapsulated pGRA4 enhanced the protective effect against infection of T. gondii.