Evaluation of recombinant antigens in combination and single formula for diagnosis of feline toxoplasmosis.

Cats are the only definitive hosts of Toxoplasma gondii and constitute an essential source of infection to all warm blooded animals and humans. Diagnosis of T. gondii infection in cats is fundamental for proper management and control of infection in humans and animals. In the current study, we have evaluated the diagnostic performance of tachyzoite lysate antigen (TLA) and different T. gondii recombinant antigens including surface antigen 2 (SAG2), dense granule proteins 2, 6, 7, 15 (GRA2, GRA6, GRA7, GRA15) and microneme 10 protein (MIC10) in immunoglobulin G enzyme linked-immunosorbent assay (IgG ELISA) using cat serum samples, with reference to latex agglutination test (LAT). Remarkably, TLA showed better performance than other recombinant antigens in IgG ELISAs as compared to LAT, with concordance and Kappa values of 94.27% and 0.93, respectively. Furthermore, to improve the reactivity of the recombinant antigens, we have developed IgG ELISAs using different combinations with these recombinant antigens. Strikingly, a combination of SAG2 and GRAs has relatively similar performance as TLA evidenced by concordance and Kappa values of 94.27% and 0.81, respectively. The developed ELISA with a combination of recombinant antigens can be used as a promising diagnostic tool for routine testing of T. gondii infection and mass screening in cats. The major advantages of this assay are the high sensitivity and specificity, lower cost, safer production and easiness of standardization in various laboratories worldwide.

New Molecules in Babesia gibsoni and Their Application for Diagnosis, Vaccine Development, and Drug Discovery

Babesia gibsoni is an intraerythrocytic apicomplexan parasite that causes piroplasmosis in dogs. B. gibsoni infection is characterized clinically by fever, regenerative anemia, splenomegaly, and sometimes death. Since no vaccine is available, rapid and accurate diagnosis and prompt treatment of infected animals are required to control this disease. Over the past decade, several candidate molecules have been identified using biomolecular techniques in the authors' laboratory for the development of a serodiagnostic method, vaccine, and drug for B. gibsoni. This review article describes newly identified candidate molecules and their applications for diagnosis, vaccine production, and drug development of B. gibsoni.

HlSRB, a Class B scavenger receptor, is key to the granulocyte-mediated microbial phagocytosis in ticks.

Ixodid ticks transmit various pathogens of deadly diseases to humans and animals. However, the specific molecule that functions in the recognition and control of pathogens inside ticks is not yet to be identified. Class B scavenger receptor CD36 (SRB) participates in internalization of apoptotic cells, certain bacterial and fungal pathogens, and modified low-density lipoproteins. Recently, we have reported on recombinant HlSRB, a 50-kDa protein with one hydrophobic SRB domain from the hard tick, Haemaphysalis longicornis. Here, we show that HlSRB plays vital roles in granulocyte-mediated phagocytosis to invading Escherichia coli and contributes to the first-line host defense against various pathogens. Data clearly revealed that granulocytes that up-regulated the expression of cell surface HlSRB are almost exclusively involved in hemocyte-mediated phagocytosis for E. coli in ticks, and post-transcriptional silencing of the HlSRB-specific gene ablated the granulocytes' ability to phagocytose E. coli and resulted in the mortality of ticks due to high bacteremia. This is the first report demonstrating that a scavenger receptor molecule contributes to hemocyte-mediated phagocytosis against exogenous pathogens, isolated and characterized from hematophagous arthropods.

Scavenger receptor mediates systemic RNA interference in ticks.

RNA interference is an efficient method to silence gene and protein expressions. Here, the class B scavenger receptor CD36 (SRB) mediated the uptake of exogenous dsRNAs in the induction of the RNAi responses in ticks. Unfed female Haemaphysalis longicornis ticks were injected with a single or a combination of H. longicornis SRB (HlSRB) dsRNA, vitellogenin-1 (HlVg-1) dsRNA, and vitellogenin receptor (HlVgR) dsRNA. We found that specific and systemic silencing of the HlSRB, HlVg-1, and HlVgR genes was achieved in ticks injected with a single dsRNA of HlSRB, HlVg-1, and HlVgR. In ticks injected first with HlVg-1 or HlVgR dsRNA followed 96 hours later with HlSRB dsRNA (HlVg-1/HlSRB or HlVgR/HlSRB), gene silencing of HlSRB was achieved in addition to first knockdown in HlVg-1 or HlVgR, and prominent phenotypic changes were observed in engorgement, mortality, and hatchability, indicating that a systemic and specific double knockdown of target genes had been simultaneously attained in these ticks. However, in ticks injected with HlSRB dsRNA followed 96 hours later with HlVg-1 or HlVgR dsRNAs, silencing of HlSRB was achieved, but no subsequent knockdown in HlVgR or HlVg-1 was observed. The Westernblot and immunohistochemical examinations revealed that the endogenous HlSRB protein was fully abolished in midguts of ticks injected with HlSRB/HlVg-1 dsRNAs but HlVg-1 was normally expressed in midguts, suggesting that HlVg-1 dsRNA-mediated RNAi was fully inhibited by the first knockdown of HlSRB. Similarly, the abolished localization of HlSRB protein was recognized in ovaries of ticks injected with HlSRB/HlVgR, while normal localization of HlVgR was observed in ovaries, suggesting that the failure to knock-down HlVgR could be attributed to the first knockdown of HlSRB. In summary, we demonstrated for the first time that SRB may not only mediate the effective knock-down of gene expression by RNAi but also play essential roles for systemic RNAi of ticks.

Toxoplasma gondii: DNA vaccination with genes encoding antigens MIC2, M2AP, AMA1 and BAG1 and evaluation of their immunogenic potential.

A combination of antigenic regions of microneme proteins have been previously reported as being protective against chronic toxoplasmosis. In this work, we evaluated immune responses induced by immunizing BALB/c and C57BL/6 mice intradermally with plasmid DNA encoding the protein sequences of Toxoplasma gondii AMA1, MIC2, M2AP and BAG1. Mice immunized with the AMA1 gene developed high levels of serum IgG2a and c antibodies as well as cellular immune responses associated with IFN-gamma synthesis suggesting a modulated Th1 type of response. Immunization with the AMA1 gene resulted in a partial but significant protection against the acute phase of toxoplasmosis compared to MIC2, M2AP and BAG1 genes. Therefore, the AMA1 gene appears to generate a strong specific immune response and also provides effective protection against toxoplasmosis more than the MIC2, M2AP and BAG1 genes.

Genetic characterization of GRA6 genes from Toxoplasma gondii from pigs in Okinawa, Japan.

Toxoplasma gondii from pigs in Okinawa Prefecture was characterized by nested PCR-restriction fragment length polymorphism (RFLP) and DNA sequence analysis of the dense granule antigen GRA6 gene. By nested PCR, parasite DNA was detected in 33 out of 91 lymph node samples with lesions similar to those found in toxoplasmosis samples that had been collected from pigs at an abattoir. RFLP analysis with MseI was successfully conducted in 29 of 33 PCR-positive samples to group the isolates into one of the three genotypes of T. gondii. Genotyping of the 29 studied samples rendered the following results: 13 of type I (44.8%), 14 of type II (48.3%), and 2 of type III (6.9%). The GRA6 genes of 12 Okinawa isolates were cloned and sequenced. Nine new nucleotide sequences were found, and nucleotide substitutions specific for the Okinawa isolates were found at 13 positions. Phylogenetic analysis indicated that all GRA6 sequences were divided into one of the 3 main groups, and Okinawa isolates of GRA6 genotypes II and III seemed to be closely related to the Beverley strain and the NED strain, respectively. The results from this study may provide basic and useful information for the analysis of the molecular epidemiology of T. gondii infection within Japan.

Characterisation of Toxoplasma gondii engineered to express mouse interferon-gamma.

Recent studies have shown the feasibility of using Toxoplasma gondii as an expression system for heterologous protein. For better understanding of the mechanism of interferon-gamma (IFN-gamma) dependent immunity to T. gondii, the parasites were stably transfected with IFN-gamma gene, under control of the GRA1 promoter. Immunofluorescence analyses showed that recombinant mouse IFN-gamma localised to discrete punctuate structures consistent with dense granules and secreted into the vacuolar space. The production of IFN-gamma was detectable in both extracellular parasites and the parasite-infected cells. Growth of the recombinant parasites was inhibited in the mouse macrophage cell line (J774A.1 cells), but not in monkey kidney adherent fibroblasts (Vero cells), demonstrating the species-specificity of IFN-gamma. Addition of anti-mouse IFN-gamma antibody resulted in growth recovery of the recombinant parasites, suggesting that IFN-gamma, secreted from the parasitised host cells across the parasitophorous vacuole membrane, acted in a paracrine manner. Reverse transcription (RT)-PCR analysis revealed significant expression of inducible nitric oxide synthase mRNA and high levels of nitric oxide production in recombinant parasite-infected J774A.1 cells. A competitive inhibitor of the L-arginine-dependent effector pathway, N(G)-monomethyl-L-arginine, inhibited the reduction of recombinant parasite growth in J774A.1 cells. Taken together, our data suggest that the T. gondii expression system may provide a new tool for cytokine gene expression and that parasites engineered to express a cytokine gene may be rationally designed for use in studies on immune responses to T. gondii.