The leishmaniases are multifactorial zoonotic diseases requiring a multidisciplinary One Health approach for diagnosis and control. For leishmaniasis diagnosis, here we describe production of a new recombinant protein based on a kinesin-related gene of Leishmania braziliensis (Lbk39), which shows 59% amino acid identity to the L. infantum homologue. The Lbk39 gene was synthesized, inserted into the pLEXSY-sat2 vector and transfected into L. tarentolae cells by electroporation. Culturing was carried out, and the secreted recombinant protein with a C-terminal histidine tag purified using nickel affinity chromatography on the culture supernatant, yielding a final product at 0.4 mg/mL. An indirect enzyme linked immunosorbent assay (ELISA) was standardised using sera from 74 Brazilian patients with cutaneous leishmaniasis and 11 with visceral leishmaniasis. Optimal ELISA conditions were established for the Lbk39 antigen in comparison with a crude extract from L. braziliensis. The sensitivity, specificity analysis and receiver operating characteristic (ROC) curve were determined with a significance level of 5%. The ROC curve showed a good accuracy with an area under curve (AUC) = 0.967, p < 0.001 (0.941–0.993) for CL patients and an AUC = 100 (100-100) for VL patients. The values of sensitivity and specificity were 88 and 98% for CL and 100 and 100% for VL, respectively. The study showed good production and expression of the target protein and has generated a potential new antigen for the diagnosis of leishmaniasis.

ResumoEste trabalho teve como objetivo caracterizar a composição e estrutura florística de um capão de floresta no município de Quatro Barras, inserido em Estepe do Primeiro Planalto Paranaense, relacionando relevo e solos. Com base na definição de compartimentos no ambiente, que considerou as formas e declividades das rampas e as características dos solos, foram instaladas parcelas para amostragem das árvores. As espécies responderam em diversidade e densidade às diferentes condições de drenagem dos Cambissolos e Gleissolos do capão. Podocarpus lambertii demonstrou elevada adaptabilidade a solos com diferentes regimes de saturação hídrica, enquanto Myrcia laruotteana apresentou relação estreita com solos saturados hidricamente. A compartimentação ambiental com base em geomorfologia e solos mostrou ser um quesito fundamental para a melhor compreensão da distribuição das espécies vegetais na paisagem. Palavras-chave: Composição e estrutura florística; solos e relevo; Floresta Ombrófila Mista. Abstract Toposequence segmentation and phytosocial characterization of a copse from a Mixed OmbrophilousForest. This work aim to characterize flora composition and structure of a copse, in Quatro Barras County, located in steppe area in first highland of Paraná, considering soil and topographic relief conditions. Based on the definition of compartments in the environment, that considered the forms and declivities of the slopes and the characteristics of soil, parcels for trees sampling had been installed. The species had answered in diversity and density to the different conditions of drainage of Cambissolos and Gleissolos of the copse. Podocarpus lambertii demonstrated high adaptability to soil with different moistures regimes, while Myrcia laruotteana presented narrow relation with water saturated soil. The environmental segmentation based on geomorphology and soil types showed to be fundamental aspect to better understand plants species distribution on the landscape.

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Oligosaccharides are broadly present on Leishmania cell surfaces. They can be useful for the leishmaniases diagnosis and also helpful in identifying new cell markers for the disease. The disaccharide Galα1-3Galβ is the immunodominant saccharide in Leishmania cell surface and is the unique non-reducing terminal glycosphingolipids structure recognized by anti-α-Gal. This study describes an enzyme-linked immunosorbent assay (ELISA) used to measure serum levels of anti-α-galactosyl (α-Gal) antibodies in patients with cutaneous leishmaniasis (CL). Optimal ELISA conditions were established and two neoglycoproteins (NGP) containing the Galα1-3Gal terminal fraction (Galα1-3Galβ1-4GlcNAc-HAS and Galα1-3Gal-HAS) and one Galα1-3Gal NGP analogue (Galα1-3Galβ1-3GlcNAc-HAS) were used as antigens. Means of anti-α-Gal antibody titres of CL patients were significantly higher (P < 0.05) than the healthy individuals for all NGPs tested. Sensitivity and specificity of all NGPs ranged from 62.2 to 78.4% and 58.3 to 96.7%, respectively. In conclusion, the NGPs can be used for CL diagnosis.

Visceral leishmaniosis is one of the most important zoonotic diseases on the planet and dogs are the main reservoir of canine visceral leishmaniosis (CVL) in endemic areas. They play an important role in human infection because in dogs the disease appears long time after infection, and they can move uncontrollably, contributing to disperse the parasite. To take the decision to treat the animals or for euthanasia, in an elimination programme, in order to reduce the parasitic load, it is necessary to diagnose correctly, having more effective tools. Our group has developed a new recombinant antigen-based kinesin-related gene of Leishmania braziliensis (Lbk39), which shows 59% amino acid identity to the L. infantum homologue. The Lbk39 gene was synthesized, inserted into the pLEXSY-sat2 vector and transfected into L. tarentolae cells by electroporation. The recombinant protein was secreted in the culture with a C-terminal histidine marker, purified, generating a product at 337.68 μg mL−1. A total of 152 sera from dog's endemic and non-endemic areas were used, being 78 positives and 75 negatives. The antigen Lbk39 showed 100% sensitivity and 96.1% specificity. We compared this antigen with other antigens such as total extract of the parasite, TRDPP, and our data indicate that Lbk39 has potential application in the diagnosis of CVL through antibody detection.

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