Evidence of Leishmania infantum infection in rabbits (Oryctolagus cuniculus) in a natural area in Madrid, Spain

Leishmaniasis is one of most important neglected zoonosis and remains endemic in at least 88 developing countries in the world. In addition, anthropogenic environmental changes in urban areas are leading to its emergency world-wide. Zoonotic leishmaniasis control might only be achieved by an integrated approach targeting both the human host and the animal reservoirs, which in certain sylvatic cycles are yet to be identified. Recently, hares have been pointed out as competent reservoirs of Leishmania infantum in Spain, but the role of other lagomorphs has not been clarified. Here, 69 rabbits (Oryctolagus cuniculus) from a natural area in Madrid in which a high density was present were analyzed using indirect (immunofluorescence antibody test, IFAT) and direct (PCR, culture) techniques. Fifty-seven (82.6%) of the animals were positive to at least one technique, with IFAT yielding the highest proportion of positive samples. L. infantum was isolated in 13% animals demonstrating the occurrence of infection in this setting. Our results suggest that rabbits could play a role of competent reservoir of L. infantum and demonstrate that the prevalence of infection is high in the analyzed area.

A prospective study on the dynamics of the clinical and immunological evolution of human Leishmania (L.) infantum chagasi infection in the Brazilian Amazon region

This prospective study was carried out from October 2003 to December 2005 and involved a cohort of 946 individuals of both genders, aged 1-89 years, from an endemic area for American visceral leishmaniasis (AVL), in Para State, Brazil. The aim of the study was to analyze the dynamics of the clinical and immunological evolution of human Leishmania ( L.) infantum chagasi infection represented by the following clinical-immunological profiles: asymptomatic infection (AI); symptomatic infection (SI = AVL); subclinical oligosymptomatic infection (SOI); subclinical resistant infection (SRI); and indeterminate initial infection (III). Infection diagnosis was determined by the indirect fluorescent antibody test and leishmanin skin test. In total, 231 cases of infection were diagnosed: the AI profile was the most frequent (73.2%), followed by SRI (12.1%), III (9.9%), SI (2.6%) and SOI (2.2%). The major conclusion regarding evolution dynamics was that the III profile plays a pivotal role from which the cases evolve to either the resistant, SRI and AI, or susceptible, SOI and SI, profiles; only one of the 23 III cases evolved to SI, while most evolved to either SRI (nine cases) or SOI (five cases) and eight cases remained as III. (C) 2010 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

A cross-sectional study on the clinical and immunological spectrum of human Leishmania (L.) infantum chagasi infection in the Brazilian Amazon region

The objectives of this study were to identify individuals with symptomatic and/or asymptomatic infection due to Leishmania (L.) infantum chagasi; to study the two types of infection, both clinically and immunologically, and to determine the prevalence rate of infection at the beginning of the study. This was a cross-sectional study with a cohort of 946 individuals, of both genders, from the age of 1 year, living in the municipality of Barcarena, PA, Brazil, an area endemic for American visceral leishmaniasis (AVL). The leishmanin skin test (LST) and the indirect fluorescent test (IFAT), were used for the diagnosis of infection. One hundred and twenty cases of infection were diagnosed, with a prevalence rate of 12.6%; eight cases showed high seroreactivity (1280-10240, IgG) in IFAT and no LST reaction; four of these cases were typical AVL and four had subclinical oligosymptomatic infection. Using two immunological methods with a clinical examination of the infected individuals enabled the identification of five clinical-immunological profiles which may promote a better understanding of the interaction between L. (L.) i. chagasi and the human immune response: asymptomatic infection (AI) 73.4%; subclinical resistant infection (SRI) 15%; subclinicat oligosymptomatic infection (SOI) 3%; symptomatic infection (AVL) 3% and indeterminate initial infection (III) 5%. (C) 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Susceptibility of peritoneal macrophage from different species of neotropical primates to Ex vivo Leishmania (L.) infantum chagasi-infection

Este estudo examinou a susceptibilidade do macrófago peritoneal (PM) dos primatas neotropicais: Callithrix jacchus, Callithrix penicillata, Saimiri sciureus, Aotus azarae infulatus e Callimico goeldii para a infecção ex vivo por Leishmania (L.) infantum chagasi, o agente etiológico da leishmaniose visceral americana (LVA), como método de triagem para avaliar o potencial desses primatas como modelo de estudo da LVA. A susceptibilidade do PM para a infecção foi investigada através do índice de infecção do PM (PMI) a intervalos de 24, 72 horas e, ainda, pela média dessas taxas (FPMI), assim como, pelas respostas do TNF-α, IL-2 (ELISA de captura) e óxido nítrico (NO) (método de Griess). Às 24hs da infecção experimental, o PMI do primata A. azarae infulatus (128) foi maior que aqueles de C. penicillata (83), C. goeldii (78), S. sciureus (77) e C. jacchus (55). Às 72hs, houve uma redução significativa do PMI de quatro primatas: A. azarae infulatus (128/37), C. penicillata (83/38), S. sciureus (77/38) e C. jacchus (55/12), com exceção de C. goeldii (78/54). O FPMI dos primatas A. azarae infulatus (82.5) e C. goeldii (66) foi maior que do primata C. jacchus (33.5), porém, não foi maior que dos primatas C. penicillata (60.5) e S. sciureus (57.5). A resposta do TNF-α foi mais regular nos quatro primatas que reduziram o PMI no intervalo de 24-72hs: C. jacchus (145/122 pg/µL), C. penicillata (154/130 pg/µL), S. sciureus (164/104 pg/µL) e A. azarae infulatus (154/104 pg/µL), com exceção de C. goeldii (38/83 pg/µL). A resposta de IL-12 foi, principalmente, marcante nos primatas A. azarae infulatus e C. goeldii, os quais apresentaram as maiores taxas do FPMI, e a resposta do NO foi maior no primata C. goeldii, em especial no intervalo de 72hs. Estes achados sugerem, fortemente, que estes primatas neotropicais parecem ter desenvolvido mecanismos resistentes de resposta imune inata capaz de controlar o crescimento intracelular da infecção por L. (L.) i. chagasi no macrófago, o que não encoraja o uso destes primatas como modelo de estudo da LVA.

Susceptibility of Cebus apella monkey (Primates: Cebidae) to experimental Leishmania (L.) infantum chagasi-infection

Na Amazônia Brasileira, o macaco Cebus apella (Primata: Cebidae) tem sido associado com o ciclo enzoótico da Leishmania (V.) shawi, um parasito dermotrópico causador da Leishmaniose Tegumentar Americana (LTA). Ele tem sido também empregado com sucesso como modelo experimental para estudo da leishmaniose tegumentar. Neste trabalho, foi investigada sua susceptibilidade à infecção experimental por Leishmania (L.) infantum chagasi, o agente etiológico da Leishmaniose Visceral Americana (LVA). Foram usados dez espécimes de C. apella oito adultos e dois jovens, quatro machos e seis fêmeas, todos nascidos e criados em cativeiro. Dois protocolos de infecção experimental foram feitos: i) seis macacos foram inoculados por via intradérmica (ID), na base da cauda com 2x106 formas promastigotas em fase estacionária de crescimento; ii) outros quatro macacos foram inoculados com 3x107 formas amastigotas de infecção visceral de hamsteres por duas vias diferentes: a) dois por via intravenosa (IV) e, b) outros dois pela via intraperitoneal (IP). A avaliação da infecção incluiu parâmetros: clínico: exame físico do abdômen, peso e temperatura corporal; b) parasitológico: aspirado de medula óssea por agulha para procura de amastigotas (esfregaço corado por Giemsa) e formas promastigotas (meio de cultura); c) imunológico: Reação de Imunofluorescência Indireta (RIFI) e, resposta de hipersensibilidade tardia (DTH). Nos seis macacos inoculados ID (formas promastigotas) todos os parâmetros de avaliação da infecção foram negativos durante o período de 12 meses. Entre os quatro macacos inoculados com formas amastigotas, dois inoculados IV mostraram parasitos na medula óssea do primeiro ao sexto mês p.i. e em seguida houve a resolução da infecção, no entanto os outros dois inoculados IP foram totalmente negativos. Esses quatro macacos apresentaram resposta específica de anticorpo IgG desde o terceiro mês p.i. (IP: 1/80 e IV: 1/320) até o décimo segundo mês (IP: 1/160 e IV: 1/5120). A conversão DTH ocorreu em apenas um macaco inoculado IV com uma forte reação na pele (30 mm). Considerando esses resultados, nós não recomendamos o uso do macaco C. apella como modelo animal para estudo da LVA.

Canine visceral leishmaniasis due to Leishmania (L.) infantum chagasi in Amazonian Brazil: comparison of the parasite density from the skin, lymph node and visceral tissues between symptomatic and asymptomatic, seropositive dogs

A leishmaniose visceral canina (LVC) é reconhecida pelas características clínicas da doença e é altamente letal. A infecção, entretanto, pode ser totalmente assintomática em alguns cães soropositivos, o que tem levantado questão polêmica sobre a possibilidade desses animais, serem ou não uma fonte importante da infecção para o flebotomíneo, Lutzomyia longipalpis, o principal vetor da leishmaniose visceral americana (LVA). Neste estudo foram examinados 51 cães com LVC aguda, provenientes de área endêmica de LVA no Estado do Pará, Brasil, e a carga parasitária, formas amastigotas de, na pele, linfonodo poplíteo e vísceras (fígado e baço) foi comparada com a de nove cães assintomáticos soropositivos (IFAT-IgG). Fragmentos de biópsia desses tecidos obtidos post-mortem foram processados para análise através de imunohistoquímica, usando um anticorpo policlonal contra Leishmania sp. Os testes do Qui-quadrado (X²) e Mann Whitney foram usados para avaliar as médias da densidade de macrófagos infectados (p < 0,05). Os resultados mostraram que não houve diferença (p > 0,05) na densidade de macrófagos infectados da pele (10,7/mm² x 15,5/mm²) e do linfonodo (6,3/mm² x 8,3/mm²) entre cães assintomáticos e sintomáticos. Entretanto, a densidade de macrófagos infectados da víscera de cães sintomáticos (5,3/mm²) foi maior (p < 0,05) que a de cães assintomáticos (1,4/mm²). Estes resultados sugerem, fortemente, que cães naturalmente infectados por L. (L.) i. chagasi, assintomáticos ou sintomáticos, podem servir como fonte de infecção, principalmente, considerando-se que a densidade de macrófagos infectados da pele (10,7/mm² x 15,5/mm²), local onde o flebotomíneo vetor Lu. longipalpis realiza a hematofagia, foi maior (p < 0,05) que as do linfonodo (6,3/mm² x 8.3/mm²) e vísceras (1,4/mm2x 5,3/mm2).

MOLECULAR DETECTION OF Leishmania IN PHLEBOTOMINE SAND FLIES IN A CUTANEOUS AND VISCERAL LEISHMANIASIS ENDEMIC AREA IN NORTHEASTERN BRAZIL

Several phlebotomine sand fly species have been regarded as putative or proven vectors of parasites of the genus Leishmania in Brazil, but data for the northeastern region remains incipient. In this study, a total of 600 phlebotomine sand flies were grouped in pools of 10 specimens each and tested by a Leishmania genus-specific PCR and by a PCR targeting Leishmania (Leishmania) infantum. Fourteen out of 60 pools were positive by the genus-specific PCR, being five pools of L. migonei, seven of L. complexa, one of L. sordellii and one of L. naftalekatzi, which correspond to a minimal infection rate of 2.3% (14/600). Our results, associated with their known anthropophily and their abundance, suggest the participation of L. migonei and L. complexa as vectors of Leishmania in northeastern Brazil. Remarkably, this is the first time in this country that the detection of Leishmania DNA in L. sordellii and L. naftalekatzi has been reported, but future studies are necessary to better understand the significance of these findings.

Epidemiovigilância das infeções por Leishmania spp. no efetivo cinotécnico da Guarda Nacional Republicana e em mesocarnívoros silvestres

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Comportamento biológico "in vitro" de "novas espécies" de Leishmania no Velho Mundo

As leishmanioses são um grupo de doenças causadas pelo parasita protozoário Leishmania sp. Na Bacia mediterrânica, Leishmania infantum, é a principal espécie causadora de leishmaniose visceral, a forma mais severa da doença, sendo L. major um dos agentes etiológicos da leishmaniose cutânea. Apesar de se considerar que estes parasitas têm uma reprodução essencialmente clonal, nos últimos 20 anos tem vindo a ser descrita a recombinação genética entre diferentes estirpes e espécies, com ocorrência de híbridos naturais, quer no Velho quer no Novo Mundo. Recentemente, em Portugal, foram isoladas e identificadas pela primeira vez, estirpes híbridas de L. infantum/L. major. O presente estudo teve como principais objetivos, a pesquisa de “novas espécies” de Leishmania e a análise do comportamento “in vitro” de estirpes parentais e híbridas 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 cães provenientes de uma região endémica de Portugal, utilizando diferentes marcadores moleculares (kDNA, ITS1 e SSU rRNA) e protocolos de PCR. Não foi encontrado DNA de espécies híbridas, tendo-se no entanto, identificado DNA de Leishmania sp. em 45,85% (105/229) das amostras, incluindo cães sem sinais clínicos. Na segunda parte do trabalho, realizaram-se diversos ensaios “in vitro” com estirpes híbridas naturais L. infantum/L. major e parentais L. infantum e L. major. Em condições normais de crescimento, observou-se um padrão de crescimento distinto para cada estirpe estudada. Em condições de “stress” oxidativo, destacou-se uma diferença significativa entre as duas estirpes híbridas estudadas. Em condições de “stress” nutricional, as estirpes não apresentaram diferenças entre si. Após avaliação da suscetibilidade das estirpes na presença de Anfotericina B, todas se mostraram suscetíveis, com concentrações inibitórias (CI50) entre 0.21 e 1.15 μg/mL. Após infeção em linhas celulares monocíticas, não se verificaram diferenças estatisticamente significativas na taxa e intensidade de infeção das estirpes híbridas em comparação às putativas parentais. Os resultados obtidos, contribuíram para um melhor conhecimento sobre o comportamento biológico destas estirpes híbridas naturais L. infantum/L. major. Estas demonstraram um comportamento “in vitro” intermédio, relativamente às estirpes parentais. Estes resultados poderão servir de base para o desenvolvimento de outros estudos com estas “novas espécies”, nomeadamente estudos de patogenicidade “in vivo” e o papel de biomarcadores de virulência, que permitam um potencial prognóstico da infeção e avaliação do seu risco epidemiológico.

The molecular detection of different Leishmania species within sand flies from a cutaneous and visceral leishmaniasis sympatric area in Southeastern Brazil

Over the last 20 years, there has been an increase in the number of leishmaniasis cases in Brazil. Belo Horizonte (BH) is one of the most highly populated Brazilian cities that is affected by visceral leishmaniasis (VL). The health services in BH are coordinated by a central nucleus that is subdivided into nine sanitary districts. Historically, the highest level of human VL cases was found in the northeast sanitary district (NSD). The objective of our study was to detect Leishmania infection in the phlebotomine sand flies collected in the NSD by dissection and molecular approaches. Following the occurrence of human VL cases in 2005, entomological captures were performed from July 2006-June 2007. Out of the 245 sand flies dissected, only three Lutzomyia longipalpis spp contained flagellates. The female sand flies were grouped into 120 pools according to date, collection site and species, with approximately 10 individual sand flies in each pool. Subsquently, the DNA was extracted and Leishmania spp and other parasites were detected and identified by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorfism. Leishmania infantum was present in at least 19% of the Lu. longipalpis collected, in 3.8% of the Nyssomiya whitmani collected, in 33.3% of the Evandromiya termitophila collected and in 14.3% of the Nyssomiya intermedia collected. When the females of the cortelezzii complex were compared with each other, 3.2% of the females were infected with Leishmania braziliensis, whereas 3.2% of the females were infected with trypanosomatids.

An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens

Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs) to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.

Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area, the Algarve Region in Portugal

The Algarve Region (AR) in southern Portugal, which is an international tourist destination, has been considered an endemic region of zoonotic leishmaniasis caused by Leishmania infantum since the 1980s. In the present study, phlebotomine and canine surveys were conducted to identify sandfly blood meal sources and to update the occurrence of Leishmania infection in vectors and dogs. Four sandfly species were captured: Phlebotomus perniciosus, Phlebotomus ariasi, Phlebotomus sergenti and Sergentomyia minuta. In one P. perniciosus female, L. infantum DNA was detected. Blood meal tests showed that this species had no host preferences and was an opportunistic feeder. An overall canine leishmaniasis (CanL) seroprevalence of 16.06% was found; the seroprevalence was 3.88% in dogs housed in kennels and 40.63% in dogs that attended veterinary clinics. The simultaneous occurrence of dogs and P. perniciosus infected with L. infantum in the AR indicates that the region continues to be an endemic area for CanL. Our results reinforce the need for the systematic spatial distribution of phlebotomine populations and their Leishmania infection rates and the need to simultaneously perform pathogen monitoring in both invertebrate and vertebrate hosts to investigate the transmission, distribution and spreading of Leishmania infection.

Sandflies (Diptera: Psychodidae) associated with opossum nests at urban sites in southeastern Brazil: a risk factor for urban and periurban zoonotic Leishmania transmission?

Sandflies associated with opossum nests are reported for the first time in the yards of residences located in the urban area of the municipality of Monte Mor, situated in the metropolitan region of Campinas, state of São Paulo, Brazil. Eleven specimens of Evandromyia cortelezzii and one of Evandromyia lenti were captured in two Didelphis albiventris nests. Ev. cortelezzii is considered a secondary vector species for the transmission of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum in the Neotropics. This association may contribute to the introduction, establishment and maintenance of urban and periurban zoonotic transmission outbreaks of Leishmania and should therefore be investigated further.

Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection

In Brazil, human and canine visceral leishmaniasis (CVL) caused byLeishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA), decreased sensitivity (83.3%) and increased specificity (92.5%) were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL.

Molecular and serological detection of Leishmania spp. in captive wild animals from Ilha Solteira, SP, Brazil

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