Coinfection of Leishmania chagasi with Toxoplasma gondii, Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) in cats from an endemic area of zoonotic visceral leishmaniasis

The aim of the present study was to determine the coinfection of Leishmania sp. with Toxoplasma gondii, Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) in a population of cats from an endemic area for zoonotic visceral leishmaniasis. An overall 66/302 (21.85%) cats were found positive for Leishmania sp., with infection determined by direct parasitological examination in 30/302(9.93%), by serology in 46/302(15.23%) and by both in 10/302 (3.31%) cats. Real time PCR followed by amplicon sequencing successfully confirmed Leishmania infantum (syn Leishmania chagasi) infection. Out of the Leishmania infected cats, coinfection with FIV was observed in 12/66(18.18%), with T. gondii in 17/66 (25.75%) and with both agents in 5/66(7.58%) cats. FeLV was found only in a single adult cat with no Leishmania infection. A positive association was observed in coinfection of Leishmania and FIV (p < 0.0001), but not with T. gondii (p > 0.05). In conclusion, cats living in endemic areas of visceral leishmaniasis are significantly more likely to be coinfected with Fly, which may present confounding clinical signs and therefore cats in such areas should be always carefully screened for coinfections. (C) 2012 Elsevier B.V. All rights reserved.

Molecular detection of Leishmania sp. in cats (Felis catus) from Andradina Municipality, São Paulo State, Brazil

The aim of this work was to molecularly detect Leishmania species in 52 cats from Andradina Municipality, São Paulo State, Brazil. The direct parasitological test was performed by using imprints of poplited lymph node, bone marrow and spleen to verify amastigote forms of Leishmania spp. The samples that were positive parasitological tests were subjected to molecular analysis (PCR) and sequencing. Infection was detected for 5.76% (3/52) of the examined cats and two had presence of amastigote forms of Leishmania spp. in lymph nodes. Polymerase chain reaction (PCR) of kinetoplast minicircle DNA, indicated positive amplification for samples of spleen and lymph nodes and the sequencing resulted in 97% similarity with Leishmania (L) chagasi. This study proved the occurrence of infection with Leishmania (L) chagasi in felines from Andradina municipality, São Paulo State. (C) 2010 Elsevier B.V. All rights reserved.

Toxoplasma gondii and Leishmania spp. infection in captive crab-eating foxes, Cerdocyon thous (Carnivora, Canidae) from Brazil

The zoonoses toxoplasmosis and leishmaniasis are important worldwide and also affect wild animals. Thus, the present study aimed to assess the prevalence of Leishmania spp. and Toxoplasma gondii antibodies in 52 serum samples from captive crab-eating foxes (Cerdocyon thous) kept in 17 zoos in São Paulo State, Brazil. Modified agglutination test (MAT, for toxoplasmosis) and indirect fluorescent antibody test (IFAT, for toxoplasmosis and leishmaniasis) were employed with heterologous anti-dog immunoglobulin. Antibodies to T. gondii were found in 19.2% animals, with an almost perfect concordance (kappa = 0.86; standard error = 9.31%; CI95% = 68.25-104.76%; P < 0.0001) and a strong correlation coefficient (rs = 0.87; P < 0.0001), which allows the use of heterologous anti-dog immunoglobulin to perform IFAT for toxoplasmosis in crab-eating foxes. No sample was positive for Leishmania spp. Toxoplasmosis infection occurs in wild animals from the studied Brazilian zoos, which indicates a probable environmental contamination, highlighting the importance of appropriate zoo management and the action of the parasite as a sentinel to human infection. (C) 2009 Elsevier B.V. All rights reserved.

Leishmania spp. and/or Trypanosoma cruzi diagnosis in dogs from endemic and nonendemic areas for canine visceral leishmaniasis

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

Clinical and parasitological evaluation of dogs naturally infected by Leishmania (Leishmania) chagasi submitted to treatment with meglumine antimoniate

Aiming to evaluate the efficacy of the treatment of canine visceral leishmaniasis, to verify the occurrence of a possible disease relapse, and to search for the presence of the parasites after the end of the treatment, seven dogs naturally infected by Leishmania (Leishmania) chagasi were used. The dogs were subjected to a treatment with 75 mg/kg meglumine antimoniate subcutaneously every 12 h for 21 days, and followed-up for a period of 6 months. During the whole experimental period the animals wore deltamethrin collars and were kept in a screened kennel to avoid reinfection. Lymph node and bone marrow aspiration biopsy was carried out to search for the parasite at seven moments: before the treatment, 30, 60, 90, 120 150 and 180 days after the start of the treatment. After the end of the experiment all dogs were humanely euthanized. Then, spleen and liver imprints and ill vitro cultures were carried out to search for amastigote forms of the parasite. During the treatment all animals presented remission of symptoms. However, two dogs were observed to present new symptoms in the course of the experiment. At the end of the experiment, the presence of amastigote forms of the parasite was evidenced in five of the seven dogs. This enabled us to conclude that the treatment promoted clinical cure but did not eliminate the parasites completely. (c) 2006 Elsevier B.V. All rights reserved.

Ocorrência de Leishmania spp. em felinos do município de Araçatuba, SP

Este trabalho teve como objetivo comparar a ocorrência de Leishmania spp. em gatos por dois métodos (citológico e sorológico), bem como associar a ocorrência deste protozoário com as variáveis sexo, idade e raça. Amostras séricas de 283 felinos domésticos foram testadas pela Reação de Imunofluorescência Indireta (RIFI), e o exame parasitológico direto de linfonodos também foi realizado para a verificação da positividade para Leishmania spp. Ocorrência de 0,7% (2/283) foi observada nos felinos examinados, por meio de imprint de linfonodos e nenhum animal apresentou títulos de anticorpos para Leishmania spp. As duas fêmeas positivas eram sem raça definida, sendo uma jovem e outra adulta. Por meio dos resultados obtidos, não foi constatada diferença estatisticamente significante em relação às variáveis sexo, raça e idade nos gatos desta pesquisa (p > 0,05). Ocorrência de Leishmania spp. nos gatos deste estudo foi baixa. Devido a esta baixa incidência sugere-se que estes não assumem importância epidemiológica na área do estudo.

Anti-leishmania antibodies in cerebrospinal fluid from dogs with visceral leishmaniasis

Visceral leishmaniasis in Brazil is caused by Leishmania (Leishmania) chagasi and the dog is its most important reservoir. The clinical features in dogs include loss of weight, lymphadenopathy, renal failure, skin lesions, fever, hypergammaglobulinemia, hepatosplenomegaly, anemia, and, rarely, neurological symptoms. Most infected animals develop active disease, characterized by high anti-leishmania antibody titers and depressed lymphoproliferative ability. Antibody production is not primarily important for protection but might be involved in the pathogenesis of tissue lesions. An ELISA test was used to determine if there is an association between neurological symptoms and the presence of anti-L. chagasi antibodies in cerebrospinal fluid (CSF). Thirty serum and CSF samples from symptomatic mixed breed dogs (three with neurological symptoms) from a region of high incidence of visceral leishmaniasis in Brazil were examined for antibody using total parasite antigen and anti-dog IgG peroxidase conjugate. A high level of L. chagasi antibodies was observed in sera (mean absorbance ± SD, 1.939 ± 0.405; negative control, N = 20, 0.154 ± 0.074) and CSF (1.571 ± 0.532; negative control, N = 10, 0.0195 ± 0.040) from all animals studied. This observation suggests that L. chagasi can cause breakdown of filtration barriers and the transfer of antibodies and antigens from the blood to the CSF compartment. No correlation was observed between antibody titer in CSF and neurological symptoms.

Qualidade espermática de sêmen de cães naturalmente infectados por Leishmania sp:

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

Use of crude, FML and rK39 antigens in ELISA to detect anti-Leishmania spp. antibodies in Felis catus

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

Glial reactivity in dogs with visceral leishmaniasis: correlation with T lymphocyte infiltration and with cerebrospinal fluid anti-Leishmania antibody titres

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

Leishmune (R) vaccine blocks the transmission of canine visceral leishmaniasis - Absence of Leishmania parasites in blood, skin and lymph nodes of vaccinated exposed dogs

Leishmune (R) vaccine is the first licensed vaccine against canine visceral leishmaniasis. It contains the Fucose-Mannose-ligand (FML) antigen of Leishmania donovani. The potential Leishmune (R) vaccine effect on the interruption of the transmission of the disease, was assayed by monitoring, in untreated (n = 40) and vaccinated dogs (n = 32) of a Brazilian epidemic area: the kala-azar clinical signs, the FML-seropositivity and the Leishmania parasite evidence by immunohistochemistry of skin and PCR for Leishmanial DNA of lymph node and blood samples. on month I I after vaccination, untreated controls showed: 25% of symptomatic cases, 50% of FML-seropositivity, 56.7% of lymph node PCR, 15.7% of blood PCR and 25% of immunohistochemical positive reactions. The Leishmune (R)-vaccinated dogs showed 100% of seropositivity to FML and a complete absence of clinical signs and of parasites (0%) in skin, lymph node and blood PCR samples (P < 0.01). The positivity in FML-ELISA in untreated dogs significantly correlates with the PCR in lymph node samples (p < 0.001) and with the increase in number of symptoms (p = 0.006) being strong markers of infectiousness. The absence of symptoms and of evidence of Leishmania DNA and parasites in Leishmune (R)-vaccinated animals indicates the non-infectious condition of the Leishmune (R)-vaccinated dogs. (c) 2005 Elsevier Ltd. All rights reserved.

Distinct mitochondrial HSP70 homologues conserved in various Leishmania species suggest novel biological functions

We report the identification of two distinct homologues of the 70-kDa mitochondrial heat shock protein (mtHSP70) from Leishmania chagasi/Leishmania infantum (Lc2.1 and Lc2.2). in Leishmania species, multiple genes encoding Lc2.2 are present whilst single genes encode Lc2.1. Strikingly, genes encoding Lc2.1-like proteins are absent from Trypanosoma species. Lc2.2 is characterized by a poly-glutamine rich C-terminus, absent from Lc2.1 or mtHSP70 homologues outside the trypanosomatids. Lc2.1 displays unique substitutions within its peptide-binding domain which modify amino acids strictly conserved in cytoplasmic and mitochondrial HSP70 proteins alike. Affinity purified antibodies recognize mainly a single protein in extracts from promastigotes/epimastigotes of various Leishmania/Trypanosoma species. Upon differentiation of Leishmania amazonensis into amastigotes a second protein (presumably Lc2.1) is induced and becomes the predominant mtHSP70 homologue expressed. Subcellular localization of these proteins was investigated and ratified a distribution throughout the mitochondrial matrix. Our results imply novel mtHSP70 functions which evolved within the genus Leishmania. (C) 2008 Elsevier B.V. All rights reserved.

LaTBP1: A Leishmania amazonensis DNA-binding protein that associates in vivo with telomeres and GT-rich DNA using a Myb-like domain

Different species of Leishmania can cause a variety of medically important diseases, whose control and treatment are still health problems. Telomere binding proteins (TBPs) have potential as targets for anti-parasitic chemotherapy because of their importance for genome stability and cell viability. Here, we describe LaTBP1 a protein that has a Myb-like DNA-binding domain, a feature shared by most double-stranded telomeric proteins. Binding assays using full-length and truncated LaTBP1 combined with spectroscopy analysis were used to map the boundaries of the Myb-like domain near to the protein only tryptophan residue. The Myb-like domain of LaTBP1 contains a conserved hydrophobic cavity implicated in DNA-binding activity. A hypothetical model helped to visualize that it shares structural homology with domains of other Myb-containing proteins. Competition assays and chromatin immunoprecipitation confirmed the specificity of LaTBP1 for telomeric and GT-rich DNAs, suggesting that LaTBP1 is a new TBP. (C) 2007 Elsevier B.V. All rights reserved.

LaRbp38: A Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs

Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38 kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA and to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function. (C) 2007 Elsevier B.V. All rights reserved.

Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres. (C) 2007 Elsevier B.V. All rights reserved.