RPA-1 from Leishmania amazonensis (LaRPA-1) structurally differs from other eukaryote RPA-1 and interacts with telomeric DNA via its N-terminal OB-fold domain

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Canine osteomyelitis caused by Leishmania sp

Leishmaniasis is a disease emerging or re-emerging worldwide distribution (Sousa, 2008), a major impact on public health. The canine visceral leishmaniasis is an infectious parasitic zoonosis of worldwide distribution (Troncarelli, 2009), caused by a flagellate protozoan called Leishmania chagasi (Costard, 2009). Osteomyelitis can develop when the parasite reaches the bone tissue of the host via blood or continuity of adjacent soft tissue infection (Baltenperger, 2004). The histopathology of the lesions has 100% specificity when viewing the amastigote form of the parasite (Feitosa et al., 2000). A dog breed poodle, female, five years, with chronic lameness four months ago was attended by a veterinary, proceeded to the general clinical examination, radiographic evaluation of the hindquarters and the laboratory tests of enzyme immunoassay (ELISA) and indirect immunofluorescence (RIFI) for Leishmania sp. On examination, besides the enlargement of lymph nodes, the animal showed pain on flexion and extension of hind limbs. In radiographic lytic bone lesions were observed in bilateral ischial board and greater trochanter of the femur, suggestive of osteomyelitis. In specific laboratory tests for diagnosis of leishmaniasis ELISA reagent and RIFI reagent 1:40. As recommended by the Ministry of Health, the animal was euthanized. No macroscopic findings were reported during the necropsy, usually associated with leishmaniasis. The ischium bone fragments were sent for histopathological examination. There was intense proliferation of mononuclear inflammatory cells, mainly macrophages and lymphocytes. Amastigotes of Leishmania sp, were identified in the cytoplasm of some macrophages and bone tissue. In endemic areas for canine leishmaniasis, dogs with a history of intermittent lameness, and radiographic lytic bone lesions suggestive of osteomyelitis should be directed to realization of the histopathology and serologic tests for the differential diagnosis of Leishmania sp. Even without evidence of cutaneous or visceral lesions, usually associated with this disease.

Obtenção de uma nova proteína recombinante componente do complexo telemérico LAGT1 de Leishmania Amazonensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Síntese e avaliação biológica de bioisósteros de nitrofural ativos contra Leishmania amazonensis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo das células mononucleares e polimorfonucleares no intestino de cães naturalmente infectados por leishmania infantum

Pós-graduação em Ciência e Tecnologia Animal - FEIS

Caracterização das espécies de leishmania em sangue periférico de cães por PCR-RFLP NA área endêmica de Bauru/SP

Pós-graduação em Ciência Animal - FMVA

Study on coinfecting vector-borne pathogens in dogs and ticks in Rio Grande do Norte, Brazil

Since dogs presenting several vector borne diseases can show none or nonspecific clinical signs depending on the phase of infection, the assessment of the particular agents involved is mandatory. The present study aimed to investigate the presence of Babesia spp., Ehrlichia spp., Anaplasma spp., Hepatozoon spp. and Leishmania spp. in blood samples and ticks, collected from two dogs from Rio Grande do Norte showing suggestive tick-borne disease by using molecular techniques. DNA of E. canis, H. canis and L. infantum were detected in blood samples and R. sanguineus ticks collected from dogs. Among all samples analyzed, two showed the presence of multiple infections with E. canis, H. canis and L. infantum chagasi. Here we highlighted the need for molecular differential diagnosis in dogs showing nonspecific clinical signs.

Serological detection of Toxoplasma gondii, Leishmania infantum and Neospora caninum in cats from an area endemic for leishmaniasis in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pathology of dogs in Campo Grande, MS, Brazil naturally co-infected with Leishmania infantum and Ehrlichia canis

Different parasites that commonly occur concomitantly can influence one another, sometimes with unpredictable effects. We evaluated pathological aspects of dogs naturally co-infected with Leishmania infantum and Ehrlichia canis. The health status of the dogs was investigated based on histopathological, hematological and biochemical analyses of 21 animals infected solely with L. infantum and 22 dogs co-infected with L. infantum and E. canis. The skin of both groups showed chronic, predominantly lymphohistioplasmacytic inflammatory reaction. The plasmacytosis in the lymphoid tissues was likely related with the hypergammaglobulinemia detected in all the dogs. The disorganization of extracellular matrix found in the reticular dermis of the inguinal region and ear, characterized by the substitution of thick collagen fibers for thin fibers, was attributed to the degree of inflammatory reaction, irrespective of the presence of parasites. In addition, the histopathological analysis revealed that twice as many dogs in the co-infected group presented Leishmania amastigotes in the ear skin than those infected solely with Leishmania, increasing the possibility of becoming infected through sand fly vectors. Our findings highlight the fact that the health of dogs infected concomitantly with L. infantum and E. canis is severely compromised due to their high levels of total plasma protein, globulins, alkaline phosphatase and creatine kinase, and severe anemia.

First detection of Leishmania spp. DNA in Brazilian bats captured strictly in urban areas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The 2',4'-dihydroxychalcone could be explored to develop new inhibitors against the glycerol-3-phosphate dehydrogenase from Leishmania species

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Leishmania sp. amastigotes identification in canine transmissible venereal tumor

Leishmaniasis is a vector-borne disease with Leishmania chagasi being the etiological agent of canine visceral leishmaniasis in South America. Canine venereal tumor is a transplantable round cell tumor of histiocytic origin which is mostly observed in sexually active male and female intact dogs. It has been shown that Leishmania amastigotes have higher tropism for the canine male genital tract tissues and venereal leishmaniasis transmission has been documented in dogs but, to date, a canine venereal tumor-dependent transmission route has not been fully demonstrated. In this report, a 10-year-old, mixed breed, intact female dog presented a vaginal venereal transmissible tumor but no other clinical abnormalities otherwise. Unexpectedly, tumor tissue imprint smears examination revealed Leishmania sp. amastigotes within infiltrating macrophages. In addition to the cytological direct identification, the protozoan was confirmed within the neoplastic tissue by means of immunohistochemistry and polymerase chain reaction. This report illustrates an asymptomatic Leishmania sp. infection that may have started on or from the canine venereal tumor tissue, the latter option further supporting previous evidence of such an alternative vector-independent route of transmission for canine visceral leishmaniasis in areas where these diseases coexist.

Analysis of the protective potential of antigens released by Leishmania (Viannia) shawi promastigotes

Leishmania (Viannia) shawi causes cutaneous lesions in humans. Parasite antigens conferring significant protection against American tegumentar leishmaniosis (ATL) might be important for the development of effective vaccine. Therefore, this work evaluates the protective effect of antigenic fractions released by L. shawi. Antigens released by promastigotes to culture medium were concentrated and isolated by SDS-PAGE. The three main fractions LsPass1 (>75 kDa), LsPass2 (75-50 kDa) and LsPass3 (<50 kDa) were electro-eluted according with their molecular mass. Immunized BALB/c mice were challenged with L. shawi promastigotes and the course of infection monitored during 5 weeks. LsPass1-challenged mice showed no protection, however, a strong degree of protection associated to smaller lesions and high expression of IFN-gamma and TNF-alpha by CD4(+) T, CD8(+) T and double negative CD4CD8 cells was achieved in LsPass3-challenged mice. Furthermore, LsPass2-challenged mice showed an intermediated degree of protection associated to high levels of IFN-gamma, IL-4 and IL-10 mRNA. In spite of increased expression of IFN-gamma and TNF-alpha, high amounts of IL-4 and IL-10 mRNA were also detected in LsPass3-challenged mice indicating a possible contribution of these cytokines for the persistence of a residual number of parasites that may be important in inducing long-lasting immunity. Therefore, LsPass3 seems to be an interesting alternative that should be considered in the development of an effective vaccine against ATL.

In silico identification of conserved intercoding sequences in Leishmania genomes: Unraveling putative cis-regulatory elements

In silico analyses of Leishmania spp. genome data are a powerful resource to improve the understanding of these pathogens' biology. Trypanosomatids such as Leishmania spp. have their protein-coding genes grouped in long polycistronic units of functionally unrelated genes. The control of gene expression happens by a variety of posttranscriptional mechanisms. The high degree of synteny among Leishmania species is accompanied by highly conserved coding sequences (CDS) and poorly conserved intercoding untranslated sequences. To identify the elements involved in the control of gene expression, we conducted an in silico investigation to find conserved intercoding sequences (CICS) in the genomes of L major, L infantum, and L braziliensis. We used a combination of computational tools, such as Linux-Shell, PERL and R languages, BLAST, MSPcrunch, SSAKE, and Pred-A-Term algorithms to construct a pipeline which was able to: (i) search for conservation in target-regions, (ii) eliminate CICS redundancy and mask repeat elements, (iii) predict the mRNA's extremities, (iv) analyze the distribution of orthologous genes within the generated LeishCICS-clusters, (v) assign GO terms to the LeishCICS-clusters. and (vi) provide statistical support for the gene-enrichment annotation. We associated the LeishCICS-cluster data, generated at the end of the pipeline, with the expression profile oft. donovani genes during promastigote-amastigote differentiation, as previously evaluated by others (GEO accession: GSE21936). A Pearson's correlation coefficient greater than 0.5 was observed for 730 LeishCICS-clusters containing from 2 to 17 genes. The designed computational pipeline is a useful tool and its application identified potential regulatory cis elements and putative regulons in Leishmania. (C) 2012 Elsevier B.V. All rights reserved.

Isolation and biochemical, functional and structural characterization of a novel L-amino acid oxidase from Lachesis muta snake venom

The aim of this study was the isolation of the LAAO from Lachesis muta venom (LmLAAO) and its biochemical, functional and structural characterization. Two different purification protocols were developed and both provided highly homogeneous and active LmLAAO. It is a homodimeric enzyme with molar mass around 120 kDa under non-reducing conditions, 60 kDa under reducing conditions in SDS-PAGE and 60852 Da by mass spectrometry. Forty amino acid residues were directly sequenced from LmLAAO and its complete cDNA was identified and characterized from an Expressed Sequence Tags data bank obtained from a venom gland. A model based on sequence homology was manually built in order to predict its three-dimensional structure. LmLAAO showed a catalytic preference for hydrophobic amino acids (K-m of 0.97 mmol/L with Leu). A mild myonecrosis was observed histologically in mice after injection of 100 mu g of LmLAAO and confirmed by a 15-fold increase in CK activity. LmLAAO induced cytotoxicity on AGS cell line (gastric adenocarcinoma, IC50: 22.7 mu g/mL) and on MCF-7 cell line (breast adenocarcinoma, IC50:1.41 mu g/mL). It presents antiparasitic activity on Leishmania brasiliensis (IC50: 2.22 mu g/nnL), but Trypanosoma cruzi was resistant to LmLAAO. In conclusion, LmLAAO showed low systemic toxicity but important in vitro pharmacological actions. (C) 2012 Elsevier Ltd. All rights reserved.