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Background: Molecular genetic markers are one of the most informative and widely used genome features in clinical and environmental diagnostic studies. A polymerase chain reaction (PCR)-based molecular marker is very attractive because it is suitable to high throughput automation and confers high specificity. However, the design of taxon-specific primers may be difficult and time consuming due to the need to identify appropriate genomic regions for annealing primers and to evaluate primer specificity. Results: Here, we report the development of a Tool for Identification of Primers for Multiple Taxa (TipMT), which is a web application to search and design primers for genotyping based on genomic data. The tool identifies and targets single sequence repeats (SSR) or orthologous/taxa-specific genes for genotyping using Multiplex PCR. This pipeline was applied to the genomes of four species of Leishmania (L. amazonensis, L. braziliensis, L. infantum and L. major) and validated by PCR using artificial genomic DNA mixtures of the Leishmania species as templates. This experimental validation demonstrates the reliability of TipMT because amplification profiles showed discrimination of genomic DNA samples from Leishmania species. Conclusions: The TipMT web tool allows for large-scale identification and design of taxon-specific primers and is freely available to the scientific community at http://200.131.37.155/tipMT/. [ABSTRACT FROM AUTHOR]

Background: Trypanosoma cruzi is a protist parasite that causes Chagas disease. Several proteins that are essential for parasite virulence and involved in host immune responses are anchored to the membrane through glycosylphosphatidylinositol (GPI) molecules. In addition, T. cruzi GPI anchors have immunostimulatory activities, including the ability to stimulate the synthesis of cytokines by innate immune cells. Therefore, T. cruzi genes related to GPI anchor biosynthesis constitute potential new targets for the development of better therapies against Chagas disease. Methodology/Principal Findings: In silico analysis of the T. cruzi genome resulted in the identification of 18 genes encoding proteins of the GPI biosynthetic pathway as well as the inositolphosphorylceramide (IPC) synthase gene. Expression of GFP fusions of some of these proteins in T. cruzi epimastigotes showed that they localize in the endoplasmic reticulum (ER). Expression analyses of two genes indicated that they are constitutively expressed in all stages of the parasite life cycle. T. cruzi genes TcDPM1, TcGPI10 and TcGPI12 complement conditional yeast mutants in GPI biosynthesis. Attempts to generate T. cruzi knockouts for three genes were unsuccessful, suggesting that GPI may be an essential component of the parasite. Regarding TcGPI8, which encodes the catalytic subunit of the transamidase complex, although we were able to generate single allele knockout mutants, attempts to disrupt both alleles failed, resulting instead in parasites that have undergone genomic recombination and maintained at least one active copy of the gene. Conclusions/Significance: Analyses of T. cruzi sequences encoding components of the GPI biosynthetic pathway indicated that they are essential genes involved in key aspects of host-parasite interactions. Complementation assays of yeast mutants with these T. cruzi genes resulted in yeast cell lines that can now be employed in high throughput screenings of drugs against this parasite. Author Summary: Chagas disease, considered one of the most neglected tropical diseases, is caused by the blood-borne parasite Trypanosoma cruzi and currently affects about 8 million people in Latin America. T. cruzi can be transmitted by insect vectors, blood transfusion, organ transplantation and mother-to-baby as well as through ingestion of contaminated food. Although T. cruzi causes life-long infections that can result in serious damage to the heart, the two drugs currently available to treat Chagas disease, benznidazole and nifurtimox, which have been used for more than 40 years, have proven efficacy only during the acute phase of the disease. Thus, there is an urgent need to develop new drugs that are more targeted, less toxic, and more effective against this parasite. Here we described the characterization of T. cruzi genes involved in the biosynthesis of GPI anchors, a molecule responsible for holding different types of glycoproteins on the parasite membrane. Since GPI anchored proteins are essential molecules T. cruzi uses during infection, besides helping understand how this parasite interacts with its host, this work may contribute to the development of better therapies against Chagas disease. [ABSTRACT FROM AUTHOR]

Background: Trypanosoma cruzi is a protist parasite that causes Chagas disease. Several proteins that are essential for parasite virulence and involved in host immune responses are anchored to the membrane through glycosylphosphatidylinositol (GPI) molecules. In addition, T. cruzi GPI anchors have immunostimulatory activities, including the ability to stimulate the synthesis of cytokines by innate immune cells. Therefore, T. cruzi genes related to GPI anchor biosynthesis constitute potential new targets for the development of better therapies against Chagas disease. Methodology/Principal Findings: In silico analysis of the T. cruzi genome resulted in the identification of 18 genes encoding proteins of the GPI biosynthetic pathway as well as the inositolphosphorylceramide (IPC) synthase gene. Expression of GFP fusions of some of these proteins in T. cruzi epimastigotes showed that they localize in the endoplasmic reticulum (ER). Expression analyses of two genes indicated that they are constitutively expressed in all stages of the parasite life cycle. T. cruzi genes TcDPM1, TcGPI10 and TcGPI12 complement conditional yeast mutants in GPI biosynthesis. Attempts to generate T. cruzi knockouts for three genes were unsuccessful, suggesting that GPI may be an essential component of the parasite. Regarding TcGPI8, which encodes the catalytic subunit of the transamidase complex, although we were able to generate single allele knockout mutants, attempts to disrupt both alleles failed, resulting instead in parasites that have undergone genomic recombination and maintained at least one active copy of the gene. Conclusions/Significance: Analyses of T. cruzi sequences encoding components of the GPI biosynthetic pathway indicated that they are essential genes involved in key aspects of host-parasite interactions. Complementation assays of yeast mutants with these T. cruzi genes resulted in yeast cell lines that can now be employed in high throughput screenings of drugs against this parasite. [ABSTRACT FROM AUTHOR]

The laboratorial diagnosis of leishmaniasis is based on parasitological methods, which are invasive, present high cost, require laboratorial infrastructure and/or trained professionals; as well as by immunological methods, which usually present variable sensitivity and/or specificity, such as when they are applied to identify asymptomatic cases and/or mammalian hosts presenting low levels of antileishmanial antibodies. As consequence, new studies aiming to identify more refined antigens to diagnose visceral (VL) and tegumentary (TL) leishmaniasis are urgently necessary. In the present work, the Leishmania eukaryotic elongation factor-1 beta (EF1b) protein, which was identified in L. infantum protein extracts by antibodies in VL patients' sera, was cloned and its recombinant version (rEF1b) was expressed, purified and tested as a diagnostic marker for VL and TL. The post-therapeutic serological follow-up was also evaluated in treated and untreated VL and TL patients, when anti-rEF1b antibody levels were measured before and after treatment. Results showed that rEF1b was highly sensitive and specific to diagnose symptomatic and asymptomatic canine VL, as well as human TL and VL. In addition, low cross-reactivity was observed when sera from healthy subjects or leishmaniasis-related diseases patients were tested. The serological follow-up showed also that rEF1b-specific antibodies declined significantly after treatment, suggesting that this protein could be also evaluated as a prognostic marker for human leishmaniasis. Image 1 • The eukaryotic elongation factor-1 was tested to diagnose leishmaniasis. • Canine and human serological panels were used in the ELISA experiments. • Sensitivity and specificity values of 100% were found in both sera classes. • The post-treatment serological follow-up specific to protein was evaluated. • Results showed that specific antibodies declined significantly after the end of treatment. [ABSTRACT FROM AUTHOR]

Background and purpose: Lead (Pb) is used as a flux in the glazing process of pottery utensils in several regions of the world. It can affect the nervous and hematopoietic systems; in addition, it is classified as a probable human carcinogen. This work aims to evaluate Pb exposure of potters and describe the main determinants of elevated blood Pb (PbB) levels in this group of workers. Methods: This is a cross-sectional study with potters of Maragogipinho Village, Bahia, Brazil, of both sexes, aged 16–72 years (n = 85). Non-exposed workers of the same age range residing in the urban area of Aratuípe town were also recruited (n = 50). We evaluated Pb dust deposition rates (PbDrt) in pottery workshops and PbB levels. All Pb measurements were performed by Graphite Furnace Atomic Absorption Spectrometry. Results: The median of PbB (min–max) and geometric mean (SD) PbDrt for the exposed group were 7.9 (0.9–49.8) µg/dL and 1463 (± 290,000) μg/m2/30 days, respectively. For the control group, levels were 1.5 (0.1–19.8) μg/dL and 82 (46) μg/m2 30 days, respectively. Conclusion: The data found showed an excessive exposure among artisans, exceeding occupationally safe levels and those reported in the literature. It is important to implement occupational hygiene measures and improvements of the working conditions of these labors, especially the replacement of lead oxide in the pottery-glazing process. [ABSTRACT FROM AUTHOR]

Treatment for visceral leishmaniasis (VL) is hampered mainly by the toxicity and/or high cost of antileishmanial drugs. What is more, variability on sensitivity and/or specificity of diagnostic tests hinders effective disease management. In this context, prophylactic vaccination should be considered as a strategy to prevent disease. In the present study, immunogenicity of the Leishmania eukaryotic Elongation Factor-1 beta (EF1b) protein, classified as a Leishmania virulence factor, was evaluated in vitro and in vivo and tested, for the first time, as a vaccine candidate against Leishmania infantum infection. The antigen was administered as DNA vaccine or as recombinant protein (rEF1b) delivered in saponin. BALB/c mice immunization with a DNA plasmid and recombinant protein plus saponin induced development of specific Th1-type immunity, characterized by high levels of IFN-γ, IL-12, GM-CSF, both T cell subtypes and antileishmanial IgG2a isotype antibodies, before and after infection. This immunological response to the vaccines was corroborated further by parasitological analysis of the vaccinated and then challenged mice, which showed significant reductions in the parasite load in their liver, spleen, bone marrow and draining lymph nodes, when compared to the controls. Vaccination using rEF1b/saponin induced a more robust Th1 response and parasitological protection when compared to the DNA vaccine. Furthermore, in vitro analysis of lymphoproliferation, IFN-γ and IL-10 levels in human PBMC cultures showed as well development of a specific Th1-type response. In conclusion, data suggest that EF1b could be a promising vaccine candidate to protect against L. infantum infection. Image 1 • The eukaryotic elongation factor-1 beta protein was tested against L. infantum. • The antigen was administered as DNA vaccine or recombinant protein plus adjuvant. • Vaccinated mice mounted a Th1-type immune response before and after infection. • Lower parasite burden was found in distinct organs using both vaccination schedules. • The recombinant protein plus adjuvant induced better response than DNA vaccine. [ABSTRACT FROM AUTHOR]