Predictive factors for Leishmania infantum infection in dogs examined at a veterinary teaching hospital in Teresina, State of Piau��, Brazil

Abstract: INTRODUCTION: In Brazil, culling of seropositive dogs is one of the recommended strategies to control visceral leishmaniasis. Since infectiousness is correlated with clinical signs, control measures targeting symptomatic dogs could be more effective. METHODS: A cross-sectional study was carried out among 1,410 dogs, predictive models were developed based on clinical signs and an indirect immunofluorescence antibody test. RESULTS: The validated predictive model showed sensitivity and specificity of 86.5% and 70.0%, respectively. CONCLUSIONS: Predictive models could be used as tools to aid control programs in focusing on a smaller fraction of dogs contributing more to infection dissemination.

Comportamento biol��gico "in vitro" de "novas esp��cies" de Leishmania no Velho Mundo

As leishmanioses s��o um grupo de doen��as causadas pelo parasita protozo��rio Leishmania sp. Na Bacia mediterr��nica, Leishmania infantum, �� a principal esp��cie causadora de leishmaniose visceral, a forma mais severa da doen��a, sendo L. major um dos agentes etiol��gicos da leishmaniose cut��nea. Apesar de se considerar que estes parasitas t��m uma reprodu����o essencialmente clonal, nos ��ltimos 20 anos tem vindo a ser descrita a recombina����o gen��tica entre diferentes estirpes e esp��cies, com ocorr��ncia de h��bridos naturais, quer no Velho quer no Novo Mundo. Recentemente, em Portugal, foram isoladas e identificadas pela primeira vez, estirpes h��bridas de L. infantum/L. major. O presente estudo teve como principais objetivos, a pesquisa de ���novas esp��cies��� de Leishmania e a an��lise do comportamento ���in vitro��� de estirpes parentais e h��bridas de L. infantum e L. major. Numa primeira parte do trabalho efetuou-se a cultura e pesquisa de DNA de Leishmania sp., em amostras de sangue medular de 229 c��es provenientes de uma regi��o end��mica de Portugal, utilizando diferentes marcadores moleculares (kDNA, ITS1 e SSU rRNA) e protocolos de PCR. N��o foi encontrado DNA de esp��cies h��bridas, tendo-se no entanto, identificado DNA de Leishmania sp. em 45,85% (105/229) das amostras, incluindo c��es sem sinais cl��nicos. Na segunda parte do trabalho, realizaram-se diversos ensaios ���in vitro��� com estirpes h��bridas naturais L. infantum/L. major e parentais L. infantum e L. major. Em condi����es normais de crescimento, observou-se um padr��o de crescimento distinto para cada estirpe estudada. Em condi����es de ���stress��� oxidativo, destacou-se uma diferen��a significativa entre as duas estirpes h��bridas estudadas. Em condi����es de ���stress��� nutricional, as estirpes n��o apresentaram diferen��as entre si. Ap��s avalia����o da suscetibilidade das estirpes na presen��a de Anfotericina B, todas se mostraram suscet��veis, com concentra����es inibit��rias (CI50) entre 0.21 e 1.15 ��g/mL. Ap��s infe����o em linhas celulares monoc��ticas, n��o se verificaram diferen��as estatisticamente significativas na taxa e intensidade de infe����o das estirpes h��bridas em compara����o ��s putativas parentais. Os resultados obtidos, contribu��ram para um melhor conhecimento sobre o comportamento biol��gico destas estirpes h��bridas naturais L. infantum/L. major. Estas demonstraram um comportamento ���in vitro��� interm��dio, relativamente ��s estirpes parentais. Estes resultados poder��o servir de base para o desenvolvimento de outros estudos com estas ���novas esp��cies���, nomeadamente estudos de patogenicidade ���in vivo��� e o papel de biomarcadores de virul��ncia, que permitam um potencial progn��stico da infe����o e avalia����o do seu risco epidemiol��gico.

Estudo dos fleb��tomos (Diptera, Phlebotominae), vectores de Leishmania sp. No Concelho de Torres Novas, Portugal

Os fleb��tomos s��o insectos vectores de v��rios agentes patog��nicos, dos quais se destacam os protozo��rios do G��nero Leishmania. Em Portugal, as leishmanioses, canina e humana, s��o causadas por L. infantum, sendo o c��o o principal reservat��rio e Phlebotomus perniciosus e P. ariasi os vectores comprovados do parasita. S��o conhecidos tr��s focos de doen��a, mas casos de leishmaniose canina t��m sido reportados em outras regi��es nas quais se desconhecem as esp��cies flebotom��nicas presentes e respectivas taxas de infec����o. Neste trabalho, efectuou-se a primeira prospec����o flebotom��nica no Concelho de Torres Novas, Distrito de Santar��m, localizado na regi��o Centro de Portugal. Os principais objectivos foram determinar a fauna flebotom��nica do Concelho, os aspectos bioecol��gicos, as taxas de infec����o por Leishmania e os factores de risco para a transmiss��o vectorial. De Junho a Novembro de 2010, 275 bi��topos foram prospectados com armadilhas CDC. As capturas foram realizadas em 91 localidades, nas 17 freguesias do Concelho, e inclu��ram habitats dom��sticos, peridom��sticos e silv��ticos. Os exemplares capturados foram identificados morfologicamente, as f��meas utilizadas para detec����o molecular de DNA de Leishmania e identifica����o das refei����es sangu��neas. An��lises de regress��o simples e m��ltipla foram utilizadas para avalia����o dos factores de risco para a presen��a das v��rias esp��cies flebotom��nicas. Testes n��o param��tricos foram usados para comparar densidades. Dos 1262 fleb��tomos capturados, quatro esp��cies foram assinaladas com as seguintes abund��ncias relativas: P. perniciosus 73,69%, P. ariasi 8,16%, P. sergenti 6,58% e Sergentomyia minuta 11,57%. Em 82% das localidades prospectadas foi detectada pelo menos uma esp��cie flebotom��nica e em 71,4% destas foi capturada pelo menos uma das esp��cies comprovadamente vectoras de L. infantum. P. perniciosus foi assinalado em todas as 17 freguesias do Concelho. Os factores de risco identificados foram: temperaturas elevadas e humidades relativas baixas, locais abrigados e aus��ncia de vento forte, presen��a de pinheiros como vegeta����o dominante, bi��topos peridom��sticos, particularmente currais de ovelhas e coelheiras, ou na proximidade de ovelhas, aves de capoeira e ninhos com andorinhas. A taxa de infec����o flebotom��nica por L. infantum foi de 4% para P. ariasi e de 0,32% para o total de f��meas capturadas. A maioria das f��meas para as quais se identificou a origem da refei����o sangu��nea pertencia a P. perniciosus. Esta esp��cie apresentou um comportamento oportunista, alimentando-se numa grande variedade de hospedeiros vertebrados. A elevada abund��ncia e distribui����o das esp��cies vectoras, juntamente com a seropreval��ncia de Leishmania nos c��es do Distrito (5-10%), e a captura de uma f��mea gr��vida de P. ariasi (infectante), sugerem que o Concelho de Torres Novas �� um foco de leishmaniose no pa��s. A maior abund��ncia relativa de P. sergenti, comparando com prospec����es realizadas noutras ��reas da regi��o Centro de Portugal, sugere que este potencial vector esteja a expandir-se para latitudes mais elevadas, aumentando o risco de introdu����o de L. tropica no territ��rio, por contacto com imigrantes ou viajantes infectados de ��reas end��micas. A monitoriza����o flebotom��nica, e dos hospedeiros vertebrados, dever�� ser continuada no Concelho para que medidas eficazes de controlo possam ser definidas e implementadas.

Isolation of an enriched plasma membrame subpellicular microtubule fraction of Leishmania mexicana amazonensis

A cell fractionation procedure previously developed for Trypanosoma cruzi was applied to isolated the plasma membrane of promastigotes of Leishania mexicana amazonensis. The cell, swollen in an hypotonic mediun, were disrupted in the presence of a nonionic detergent and the membrane fraction isolated by differencial centrifugation. Electron microscopy showed that the fraction consisted of pieces of the plasma membrane associated with subpellicular microtubules. It was also shown that this fraction is able to induce cell-mediated immune response in mice.

Factores dermicos que condicionan la infeccion de Lutzomyia townsendi (Ortiz, 1959) por Leishmania spp. de Venezuela

Se estudia la susceptibilidad de Lutzomyia townsendi a la infecci��n con Leishmania spp. sobre lesiones experimentales de hamsteres. Se estudia la frecuencia y distribuci��n de los amastigotos en la dermis, relacion��ndola con la profundidad que alcanza el estilete bucal del insecto. Una correlaci��n positiva, con significante coeficiente de correlaci��n, se establece entre la frecuencia de los par��sitos a una profundidad de 100-150 nm en la dermis y el ��xito de la infecci��n de los fleb��tomos.

Atypical epidermal alterations in chronic Leishmania mexicana mexicana lesions fo C3H mice

C3H mice chronically infected with Leishmania m. mexicana, and in some groups treated with BCG or levamisole, presented atypical epidermal alterations, including pseudoepitheliomatous hyperplasia, hyperkeratosis and dysplasia. These alterations increased in frequency and intensity during the course of infection, but were not related to lesion size or tissue parasite load. Age matched normal, BCG and levamisole treated control mice, examined simultaneously, did not show epidermal modifications. In infected mice the dermis and hypodermis presented an inflammatory infiltrate of histiocytes, lymphocytes and plasma cells, accompanied at times by neutrophils and eosinophils, which did not vary with duration of infection.

Interaction of avirulent Leishmania species with rat peritoneal macrophages

An "in vitro" system has been developed for study of host cell-parasite interaction in visceral and cutaneous leishmaniasis. Avirulent promastigotes of L. brasiliensis and L. donovani, from strains originally isolated from human cases and mantained by serial culture in Davis' Medium were allowed to infect cultured macrophages from rat peritoneal exudate. Challenge of the macrophages by parasites took place in 199 medium, at 33��C for L. brasiliensis and at 37��C for L. donovani. Although the rat is resistant to infections by Leishmania spp., the promastigotes not only invaded the host cells, but transformed into amastigotes and later mutiplied, from 10 min after challenge to 24 hours later.

Host factors influencing outcome of Leishmania mexicana infection in mice

Studies were undertaken to determine the influence of several host-related parameters on the course of Leishmania mexicana mexicana infection in inbred C57B1/10 (C57) and outbred albino (OA) mice. An important influence of the following variables was demonstrated: Host strain: lesions in C57s were significantly less variable in size and outcome than those of OAs under the conditions studied and even when persistent developed at a slower rate. Host age: Subcutanous injection of 2 x 10 [raised to the power of 4] to 2 x 10 [raised to the power of 6] amastigotes into the dorsum of the rear paw produced significantly larger lesions which healed more slowly in 2 mo. old C57s than in 4 mo. old mice. Reduced healing ability was observed in older (8 mo. old) female C57s, and low mortality occurred after 15 months of age in infected mice of both sexes. Lesion site: Following amastigote infection, lesions in paws of most C57s regress within 15 - 25 wks. In contrast, perinasal legions produced with the same number of parasites tend to persist for the life of the animal as slowly spreading irregular nodules. In animals infected in both locations, each lesion site behaves similarly to that in singly infected animals of the same age, i.e. regression in the two sites is independent. Our results indicate that while host strain may strongly influence infection outcoem, such variables as lesion site and host age play important roles and may explain, in part, reported inter- and intraexperimental variability in responses of murine hosts to a given leishmanial parasite.

Functional analysis of the murine T lymphocyte immune response to a protozoan parasite, Leishmania tropica

The results presented in this review summarize a seirs of experiments designed to characterize the murine T cell imune response to the protozoan parasite Leishmania tropica. Enriched T cell populations and T cell clones specific for L. tropica antigens were derived from lymph nodes of primed mice and maintained in continous culture in vitro. These T lymphocytes were shown (A) to express the Lyt 1+ 3- cell surface phenotype, (B) to proliferate specifically in vitro in response to parasite antigens, together with a source of irradiated syngeneic macrophages, (C) to transfer antigen-specific delayed-type hypersensitivity (DTH) responses to normal syngeneic mice, (D) to induce specific activation of parasitized macrophages in vitro resulting in the destruction of intracellular parasites, (E) to provide specific helper activity for antibody responses in vitro in a hapten-carrier system. Protection studies using these defiened T cell populations should allow the characterization of parasite antigen(s) implicated in the induction of cellular immune responses beneficial for the host.

Interactions between Leishmania mexicana mexicana promastigotes and amastigotes and murine peritoneal macrophages in vitro

Unstimulated adherent mouse peritoneal cells were cultured in vitro and infected with equal numbers of a single strain of Leishmania m. mexicana amastigotes (AM), virulent promastigotes (VP), avirulent promastigotes (AVP) and fixed promastigotes (FP). Duplicate May-Gr��nwald-Giemsa stained coverslips were examined at time intervals up to 13 days. By 3 hr post infection, the number of macrophages containing parasites varied between 60.5% (VP) and 84% (AM) for macrophages exposed to living parasites, compared to 6.5% for macrophages exposed for FP. However, variable numbers of parasites showed degenerative changes by 3 hr, and the number of macrophages containing morphologically intact parasites varied significantly between cells infected with AM (84%) and those infected with VP (42%) or AVP(40%). The mean number on intacte parasites/macrophage also differed significantly between AM-infected cells and living or fixed promastigotes-infected cells. Quantitation of intact and degenerated parasites indicated parasite multiplication, as well as destruction, in VP-infected cells and parasite survival and multiplication in AM-infecte monolayers; in contrast no evidence of parasite multiplication was seen in AVP-infected cells. Changes in the mono layer itself (cell loss and macrophage vacuolization) were also evaluated. These results suggest that crucial events determining the outcome of infection occur in the host-parasite relationship during the fist 24 hours of infection. These events are apparently influenced not only by parasite or host strain but by environmentally induced variation within a given strain.

Reproductive isolation between different forms of Lutzomyia longipalpis (Lutz & Neiva), (Diptera: Psychodidae), the vector of Leishmania donovani chagasi Cunha & Chagas and its significance to kala-azar distribution in South America

The males of the sandfly Lutzomyia longipalpis occur in two forms, one which bears a single pair of pale spots on tergite 4 and another in which an additional pair of spots characterizes tergite 3. In crosses between laboratory reared stocks of the two forms originating from allopatric and sympatric sites in Brazil nearly all males of one form fail to inseminate females of the other. In addition, insemination failure between some allopatric populaytions of Lu. longipalpis with similar tergal spot patterns is recorded, indicating the existence of additional forms in an apparent species complex. The possibility that Lu. longipalpis sensu latu represents more than a single taxon is discussed and the relevance of these findings to future epidemiological studies on kala-azar is considered.

Leishmania infections in Lutzomyia longipalpis (Diptera: Psychodidae) on the Island of Sao Luis, Maranhao State, Brazil

Dissection of Lutzomyia longipalpis, captured in the S��o Luis focus of visceral Leishmaniasis revealed a 1.8% promastigote infection rate.

Natural infection of the opossum Didelphis albiventris (Marsupialia, Didelphidae) with Leishmania donovani, in Brazil

An opossum, Didelphis albiventris, from Jacobina, bahia State, was found naturally infected with Leishmania donovani, being the first non-canid wild mammal to be detected with agent of kala-azar in the New World.

Lymph node cell responsiveness in BALB/C mice infected with Leishmania mexicana

In the present study we measured the blastogenic response of lymph node cells from BALB/c mice infected with Leishmania mexicana throughout the course of infection. Results showed that infected mice displayed normal blastogenic responses in the lymph nodes until twenty weeks of infection. Thereafter, there was a gradual suppression. Comparison of the immunoresponsiveness in the spleen and lymph nodes, revealed normal responses in the lymph nodes several weeks after suppression in the spleen had occurred. Suppression of blastogenic responses in the lymph nodes was related to an adherent macrophage-like cell which actively suppressed normal proliferative responses to mitogens.