The Hus1 homologue of Leishmania major encodes a nuclear protein that participates in DNA damage response

The protozoan parasite Leishmania presents a dynamic and plastic genome in which gene amplification and chromosome translocations are common phenomena. Such plasticity hints at the necessity of dependable genome maintenance pathways. Eukaryotic cells have evolved checkpoint control systems that recognize altered DNA structures and halt cell cycle progression allowing DNA repair to take place. In these cells, the PCNA-related heterotrimeric complex formed by the proteins Hus1, Rad9, and Rad1 is known to participate in the early steps of replicative stress sensing and signaling. Here we show that the Hus1 homolog of Leishmania major is a nuclear protein that improves the cell capability to cope with replicative stress. Overexpression of LmHus1 confers resistance to the genotoxic drugs hydroxyurea (HU) and methyl methanesulfonate (MMS) and resistance to HU correlates to reduced net DNA damage upon LmHus1 expression. (C) 2011 Elsevier B.V. All rights reserved.

The influence of glutathione modulators on the course of Leishmania major infection in susceptible and resistant mice

Glutathione (GSH) has an important dual role in parasite-host relationship in Leishmania major infection. Our previous studies showed that both antioxidant systems, glutathione and trypanothione/trypanothione reductase, participate in the protection of Leishmania against the toxic effect of nitrogen-derived reactive species. On the other hand, GSH also is very important to the modulation of the effective immune response, inducting NO production and leishmanicidal activity of macrophages. In the present study, we investigated the role of host GSH during the course of L. major infection, analysing the size of footpad lesions and parasite load from mice treated with two GSH modulators, N-acethyl-L-cysteine (NAC) and buthionine sulphoximine (BSO). Resistant mice treated with BSO, which depletes GSH develop exacerbated lesions, but only harbour higher parasite load in their lesions 2 weeks post-infection. Although the NAC treatment does not affect the footpad lesions development in susceptible BALB/c mice, it significantly reduced the tissue parasitism in the lesions throughout the course of infection. Interestingly, the treatment with BSO did not change the course of L. major infection on susceptible mice when compared with nontreated mice. These results suggest that GSH is an important antioxidant modulator during anti-Leishmania immune response in vivo.

Occurrence of antibodies anti-Neospora caninum, anti-Toxoplasma gondii, and anti-Leishmania chagasi in serum of dogs from Para State, Amazon, Brazil

A cross-sectional study was conducted to determine the occurrence of anti-Toxoplasma gondii, anti-Neospora caninum, and anti- Leishmania chagasi antibodies in dogs of the state of Para, Brazil. For this purpose, 129 blood samples were collected from dogs of different ages and gender. Samples of 72 dogs were collected from 39 rural properties from 19 municipalities, and 57 samples were from stray dogs, collected after captivity by the Center of Zoonosis Control from the municipality of Santar,m. The sera were analyzed for anti-T. gondii and anti-N. caninum antibodies by indirect fluorescent antibody tests with cutoff values of 1:16 and 1:50, respectively. For the presence of L. chagasi antibodies, enzyme-linked immunosorbent assay was used and positive results were confirmed by immunochromatographic method using the recombinant antigen K39. Of the total of 129 dogs, 90 (69.8%) were positive for T. gondii, 16 (12.4%) for N. caninum, and 30 (23.3%) for L. chagasi. Antibodies for all three parasites were found simultaneously in seven dogs (5.4%), mostly in urban dogs (six of seven). No association was observed related to gender and location (urban or rural) of dogs and occurrence of N. caninum and T. gondii antibodies although, regarding L. chagasi, higher prevalence was found in females (P < 0.02) and in dogs from urban location (P < 0.001). From the 39 farms, in 30 (76.9%) at least one dog was positive for T. gondii or N. caninum or both. Higher occurrence of Leishmania antibodies was observed in N. caninum-negative dogs (P < 0.05).

Transovarial passage of Leishmania infantum kDNA in artificially infected Rhipicephalus sanguineus

Phlebotomine sand flies are the only proven biological vectors of Leishmania parasites. However, Rhipicephalus sanguineus ticks have long been suspected to transmit Leishmania infantum in studies carried out in laboratory and natural conditions. In the present study, 5 mu l of L. infantum promastigotes (1 x 10(6) cells per ml) was injected into the hemocel through the coxa 1 of four engorged females (F1, F2, F3 and F4). Control ticks (F5 and F6) were injected with sterile phosphate-buffered saline (PBS) using the same procedure. Then, these females, their eggs, and the originated larvae were tested by real time polymerase chain reaction (real-time PCR) for the presence of L. infantum kinetoplast DNA (kDNA). Females and eggs were tested after the end of the oviposition period (about 5 weeks post-inoculation) whereas larvae were tested about 4 months after the inoculation of females. All artificially infected females were positive for L. infantum kDNA. In addition, two pools of eggs (one from F2 and other from F4) and four pools of larvae (one from each F1 and F4 and two from F2) were positive for L infantum kDNA. These results showed, for the first time, the transovarial passage of L. infantum kDNA in R. sanguineus ticks, thus suggesting that the transovarial transmission of L. infantum protozoa in ticks is worth to be investigated. (C) 2010 Elsevier Inc. All rights reserved.

Canine visceral leishmaniasis: Performance of a rapid diagnostic test (Kalazar Detect (TM)) in dogs with and without signs of the disease

Current visceral leishmaniasis (VL) control programs in Brazil include the infected dog elimination but, despite this strategy, the incidence of human VL is still increasing. One of the reasons is the long delay between sample collection, analysis, control implementation and the low sensitivity of diagnostic tests. Due to the high prevalence of asymptomatic dogs, the diagnosis of these animals is important considering their vector infection capacity. Hence, a rapid and accurate diagnosis of canine visceral leishmaniasis is essential for an efficient surveillance program. In this study we evaluated the performance of rK39 antigen in an immunochromatographic format to detect symptomatic and asymptomatic Leishmania chagasi infection in dogs and compared the results with those using a crude antigen ELISA. The sensitivity of rK39 dipstick and ELISA were 83% vs. 95%, respectively, while the specificity was both 100%. Our results also demonstrated that the dipstick test was able to detect infected dogs presenting different clinical forms. (C) 2008 Elsevier B.V. All rights reserved.

Stability of nest range, home range and movement of the northern bettong (Bettongia tropica) following moderate-intensity fire in a tropical woodland, north-eastern Queensland

Nest use, home-range characteristics and nightly movements by the northern bettong (Bettongia tropica) were examined before and after a low- to moderate-intensity fire in sclerophyll woodland in north-eastern Australia using radio-telemetry. In all, 23 animals were radio-tracked at three-month intervals between February 1995 and May 1996. During November 1995 a low- intensity experimental fire burned the entire home range of most animals. The northern bettong appeared fairly catholic in choice of nest site, with a variety of nest locations and nesting materials used. Prior to the fire, nests were generally located in areas of dense cover, such as the skirts of grass trees (46%) or grass close to a log (29%). After fire removed most ground cover in the nesting areas of most animals, bettongs used remaining shelter such as boulder piles (45%), recently fallen trees (8%) and patches of unburnt vegetation (21%). Nest areas (10.1 ha) of males were significantly larger than those of females (5.4 ha). Home ranges of both sexes were large (59 ha) and most ranges lacked distinct core areas, suggesting that bettongs used all parts of their home ranges equally. High mean rates of nightly movement by the northern bettong indicated that large distances were moved within home ranges during nightly foraging. No significant fire-related changes were detected in home-range size, home-range location, nest-area location or mean rates of nightly movement, suggesting that the northern bettong is well adapted to the low- and medium-intensity fires that characterise its habitat.

Infecção experimental de Calomys callosus (Rodentia-Cricetidae) com Leishmania donovani chagasi (Laison, 1982)

Foi descrita a infecção experimental em Calomys callosus com uma cepa de Leishmania donovani chagasi de caso humano. Um grupo de 22 roedores foi inoculado por via intraperitoneal com 0,1 ml de um macerado de baço em salina, rico em amastigotas. Esses animais foram sacrificados três meses após as inoculações, tendo sido realizado: cultura "in vitro" em meio acelular (LIT e NNN) e esfregaços, corados pelo Giemsa, de fígado, baço, medula óssea e sangue; cortes histológicos corados com hematoxilina-eosina de fígado e baço. Os resultados para fígado e baço foram: 67% de positividade nas culturas "in vitro"; esfregaços ricos em amastigotas intra e extra celular (inclui medula óssea); reações teciduais traduzidas por hepatomegalia com proliferação das células de Kupffer; reação granulomatosa das áreas portais, esplenomegalia com reações granulomatosas, abundância de formas amastigotas. Os resultados para o sangue foram negativos em todas as investigações.

Resposta imune na infecção por Leishmania infantum em modelo murino

As leishmanioses são doenças causadas por protozoários do género Leishmania que são parasitas intracelulares obrigatórios das células fagocíticas. O objectivo deste estudo foi caracterizar a infecção por Leishmania infantum em murganhos BALB/c inoculados por via intradérmica, analisando a evolução do parasitismo e as respostas imunitárias desenvolvidas. A carga parasitária foi determinada por PCR em tempo real. Foram detectados parasitas desde o 7º dia pós-infecção, verificando-se a disseminação visceral do parasita ao 56º dia pós-infecção. Os linfócitos dos animais do grupo infectado proliferaram em resposta à estimulação antigénica, enquanto que os macrófagos peritoneais produziram nitritos na presença do antigénio. Estes resultados demonstraram que os murganhos BALB/c inoculados por via intradérmica constituem um bom modelo experimental de leishmaniose visceral.

Leishmania braziliensis: aislamiento de lesiones por inoculación de hámsteres con o sin adición de lisado de glándulas salivares de Lutzomyia youngi

Homogeneizados de biopsias de lesiones cutáneas de 50 casos de leishmaniasis tegumentaria de Trujillo, Venezuela, han sido inoculados en hámsteres machos. Se ha comparado la infectvidad de Leishamania braziliensis, de homogeneizados simples, con la de los mezclados con lisado de glándula salival de Lutzomyia youngi, registrandose un 58,5% de infecciones para una media de 12 semanas de prepatencia con los homogeneizados simples, contra 92% de infecciones con una media de 3 semanas de prepatencia, cuando cada uno de los inóculos de homogeneizado se mezcló con lisado equivalente al de una glándula salival de flebótomo.

Excreción de promastigotos de Leishmania pifanoi por Lutzomyia youngi experimentalmente infectada

Se describe el desarrollo poblacional promastigótico de Leishmania pifanoi en Lutzomyia youngi experimentalmente infectada y mantenida con sacarosa al 50% bajo condiciones constantes de temperatura y humedad. Se reconocen dos etapas para la diferenciación y el crecimiento de los parásitos entre las dos y ciento veinte horas postprandiales. Hasta 48 horas tiene lugar la diferenciación pleomórfica de amastigotos en promastigotos cortos, que se multiplican por división binaria hasta las 60 horas, cuando ocurre la ruptura de la membrana peritrófica. La segunda etapa tiene lugar entre las 72 y 96 horas cuando algunos parásitos migran hacia la válvula esofágica y los demás parásitos libres son excretados en gotitas fecales como promastigotos grandes y activos. Las primeras gotitas excretadas dan reacción positiva a glucosa o contienen cristales de urato. El exceso de promastigotos de la segunda fase de desarrollo es eliminado en las últimas excretas que dan reacción positiva con las pruebas Hemoscreen y Biuret para proteínas totales y también para glucosa, y constituyen el 82% del total de gotas excretadas. La excreción de parásitos por Lu. youngi es fase normal del desarrollo de L. pifanoi en un vector.

Detecção de DNA de Leishmania braziliensis em pacientes de leishmaniose tegumentar americana

Foi realizado diagnóstico para leishmaniose tegumentar americana a partir de sangue de pacientes residentes em dois municípios endêmicos do estado de Pernambuco. O DNA de 119 amostras de sangue foi extraído e submetido a reação em cadeia da polimerase. Utilizaram-se primers do minicírculo do DNA do cinetoplasto (kDNA) de Leishmania braziliensis, circulante em Pernambuco, cuja seqüência-alvo gera um fragmento de 750 pares de bases. No total 58 (48,7%) indivíduos apresentaram amplificação positiva e 61 (51,3%) negativa. Das amostras positivas para a PCR, 37 (≅ 64%) pertenciam a indivíduos tratados e sem lesão. Conclui-se que a técnica de PCR é eficaz para identificar o DNA de leishmânia em material de biópsias e em sangue venoso.

Clonagem funcional como metodologia na identificação de marcadores de virulência de Leishmania (Viannia) braziliensis

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia.

Leishmaniose cutânea na Amazônia: registro do primeiro caso humano de infecção mista, determinado por duas espécies distintas de Leishmnias: Leishmania brasiliensis e Leishmania mexicana amazonensis

Fez-se o registro, na Amazônia, do primeiro caso humano de infecção cutânea mista determinada por duas espécies distintas de Leishmania: a Leishmania braziliensis braziliensis e a Leishmania mexicana amazonensis. As duas amostras, em questão, foram isoladas de lesões distintas de um mesmo paciente, e a caracterização das espécies foi feita com base em observações de infecção experimental em hamsters, comportamento em meios artificiais de cultura, desenvolvimento de infecção experimental em Lutzomyia longipalpis, e eletroforese de isoenzimas em gel de amido. Conclui-se ser de interesse o achado que, combinado com o fato já conhecido de ausência de imunidade cruzada entre a maioria das leishmânias, sugere a necessidade do emprego de uma vacina polivalente para a região.

Anti-leishmania igA immunoenzymatic assay in mucocutaneous leishmaniasis (Preliminary report)

The Authors describe an anti-Leishmania IgA-ELISA assay in mucocutaneous leishmaniasis. Increased titers were found in leishmaniasis patients, mainly in the first and second year of infection and in deep mycoses patients showing either mucosal involvement or widespread disease.