BackgroundToxoplasmosis 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.FindingsThe 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).ConclusionsWe 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.

No abstract

No abstract

BackgroundAngiostrongylus 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.FindingsWe 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.ConclusionsLAMP 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.

No abstract

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.

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 .

Abstract: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)-γ 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.