Experimental infection of Phlebotomus perniciosus by bioluminescent Leishmania infantum using murine model and artificial feeder

Leishmaniasis is a vector-borne disease that is transmitted by sandflies and caused by obligate intracellular protozoa of the genus Leishmania. In the present study, we carried out a screening on the experimental infection of Phlebotomus pernioucus by bioluminescent Leishmania infantum using murine model and artificial feeder. We developed a real-time polymerase chain reaction (RT-PCR)-based method to determine individually the number of Leishmania promastigotes fed by infected flies. Among 1840 new emerged female sand flies, 428 were fed on the infected mice. After their death, they were analysed individually by RT-PCR. Our results demonstrated just a single Leishmania positive female at sixth day post meal. A total of 1070 female sand flies were exposed in contact with artificial feeder containing the human blood with two different quantities of Leishmania parasites: 2.106/mL and 1.107/mL. A blood meal including 1.107/mL LUC-promastigotes was proposed to 270 females and 75 (28%) flies were engorged. Among them, 44 (59%) were positive by RT-PCR analysis, with a relative average of 50551 Leishmania parasites. In case of blood feeding of females with 2.106/mL promastigotes, 57 out of 800 (7%) females succeed to feed from artificial feeder which 22 (39%) were positive with a relative average of 6487 parasites.

Avaliação da susceptibilidade de Lutzomyia migonei (Diptera: Psychodidae) ao desenvolvimento de Leishmania (Leishmania) infantum

Leishmania (Leishmania) infantum é o agente etiológico da leishmaniose visceral (LV) mais disseminado no mundo, com taxas de mortalidade significativas em casos humanos. Na América Latina, este parasito é transmitido principalmente por Lutzomyia longipalpis, entretanto, o papel de Lutzomyia migonei como um potencial vetor deste protozoário tem sido discutido. Investigações laboratoriais e de campo têm contribuído para esta hipótese, no entanto, a prova da competência vetorial de L. migonei ainda não foi fornecida. Neste estudo, foi avaliada pela primeira vez a susceptibilidade de L. migonei para duas cepas de L. (L.) infantum e realizada a comparação com o desenvolvimento em L. longipalpis. A colônia de L. migonei foi estabelecida na Faculdade de Ciências da Charles University em Praga, República Theca de espécimes capturados no município de Baturité, estado do Ceará...

Leishmania (Leishmania) infantum is the most widespread etiological agent of visceral leishmaniases (VL) in the world, with significant mortality rates in human cases. In Latin America, this parasite is primarily transmitted by Lutzomyia longipalpis, but the role of Lutzomyia migonei as a potential vector for this protozoan has been discussed. Laboratory and field investigations have contributed to this hypothesis. However, proof of the vector competence of L. migonei has not yet been provided. In this study, we evaluate for the first time the susceptibility of L. migonei to two L. (L.) infantum strains and compared with development of L. longipalpis. Colony of L. migonei was established at Faculty of Science, Charles University in Prague from specimens captured in Baturité municipality, Ceará state...

Estudo da coinfecção Leishmania infantum e Ehrlichia canis em cães numa área endêmica para leishmaniose visceral canina / Study co infection Leishmania infantum and Ehrlichia canis in dogs in an endemic area for canine visceral leishmaniasis

A Erliquiose Monocítica Canina (EMC) e a Leishmaniose Visceral Canina (LVC) são duas doenças, transmitidas por vetores, com ampla distribuição mundial. Os agentes causadores dessas doenças são Ehrlichia canis e Leishmania infantum, respectivamente. Enquanto a EMC é transmitida por carrapatos, principalmente Rhipicephalus sanguineus, a Leishmania é inoculada no hospedeiro através do inseto-vetor da subfamilia Phlebotominae. A maioria dos sinais físicos são comuns às duas enfermidades, o que dificulta o diagnóstico clínico e o tratamento, especialmente em áreas endêmicas. Poucos são os estudos relacionados à coinfecção da erliquiose e leishmaniose caninas no Brasil. No Piauí, particularmente em Teresina, ainda não se tem dados concretos sobre a prevalência dessas afecções.Os sinais físicos variam com a severidade da infecção, a resposta imune do hospedeiro e a presença de coinfecção. Assim, buscamos avaliar cães, independente dos sinais físicos, atendidos no hospital universitário e clínicas particulares da cidade de Teresina, Piauí quanto a frequência de erliquiose e leishmaniose caninas no período de março de 2012 a setembro de 2014...

The Canine Monocytic Ehrlichiosis (CME) and Canine Visceral Leishmaniasis (CVL) are the two vector-borne diseases with worldwide distribution. The causative agents of these diseases are Ehrlichia canis and Leishmania infantum, respectively. While CME is transmitted by ticks, especially Rhipicephalus sanguineus, Leishmania is inoculated into the host through the insect vector of the subfamily Phlebotominae. Most clinical signs are common to both conditions, which hinders the clinical diagnosis and treatment, especially in endemic areas. There are few studies related to the coinfection of canine ehrlichiosis and leishmaniasis in Brazil. In Piaui, particularly in Teresina, there is not yet concrete data on the prevalence of these diseases. The clinical signs vary according to the severity of the infection, to the host immune response and to the presence of coinfection. Thus, we sought to evaluate dogs, regardless of clinical signs, seen at university hospital and private clinics in the city of Teresina, Piauí, for the frequency of canine ehrlichiosis and leishmaniasis from March 2012 to September 2014...

Análisis cuantitativo del crecimiento y cambio morfométrico en poblaciones de Leishmania chagasi y Trypanosoma cruzi mantenidos en cultivos axénicos puros y mixtos / Quantitative approach to the analysis of the growth and morphometric change in populations of Leishmania chagasi and Trypanosoma cruzi maintained in pure and mixed axenic cultures

Las relaciones que se establecen entre géneros de la familia trypanosomatidae en condiciones de coexistencia en el mismo medioambiente pueden estar vinculadas a respuestas compensatorias inter-poblacionales que incluyen cambios morfológicos (diferentes estadios) y morfométricos (diferencias mensurables). El análisis cuantitativo de tales respuestas en cultivos axénicos puros de Leishmania chagasi y trypanosoma cruzi, así como en isomezclas axénicas de L. chagasi-T. cruzi mantenidas in vitro, no ha sido abordado, desconociéndose por lo tanto, particularidades biológicas. Muestras interdiarias de cultivo se fijaron, colorearon, observaron, digitalizaron y procesaron cuantitativamente. Además de cuantificar las densidades poblacionales, se registraron las magnitudes numéricas de variables morfométricas que, posteriormente, se analizaron con herramientas estadísticas. Los resultados indicaron cambios específicos en las variables investigadas, así como heterogeneidad morfométrica entre los mismos morfotipos de los mismos géneros al ser mantenidos en cultivos puros o mixtos. Los modelos de cambio morfométrico de L. chagasi y T. cruzi en cultivos puros difieren de los modelos de cambio morfométrico en los cultivos mixtos (L. chagasi-T. cruzi). Las metodologías biométricas discriminan, en términos morfométricos, poblaciones del mismo estadio (morfotipo) en ambientes diferentes.

The relations established among genera of the Trypanosomatidae family in coexisting conditions in the same environment may be linked to inter-population compensatory answers that include morphological (differences among stages) and morphometrical (measurable difference) changes. The quantitative analysis of these answers in Leishmania chagasi and Trypanosoma cruzi pure axenic cultures, as well as in L. chagasi - T. cruzi axenic iso-mixtures in vitro maintained has not been approached, and consequently, potentially useful biological particularities in the control of these important human parasites are unknown. Every other day culture samples were fixed, stained, observed, digitalized and quantitatively processed. In addition to quantify, the population densities and the appearance-disappearance stage (morphotypes) dynamics, the numeric magnitudes of the morphometric variables were recorded and later analyzed with multivariate statistical techniques. The results indicate specific changes in the investigated variables, as well as morphometric heterogeneity between the same morphotypes of the same genera when maintained in pure or mixed cultivation. The morphometric change models for L. chagasi and T. cruzi in pure culture differ from the models of morphometric change in mixed cultivation (L. chagasi-T. cruzi). The biometric methodologies discriminate in morphometric terms populations of the same stage (morfotype) in different environments.

Kinetics of growth of Leishmania (Leishmania) chagasi cycle in McCoy cell culture

The kinetics of growth of Leishmania performed in vitro after internalization of the promastigote form in the cell and the occurrence of the transformation of the parasite into the amastigote form have been described by several authors. They used explants of macrophages in hamster spleen cell culture or in a human macrophage lineage cell, the U937. Using microscopy, the description of morphologic inter-relationship and the analysis of the production of specific molecules, it has been possible to define some of the peculiarities of the biology of the parasite. The present study shows the growth cycle of Leishmania chagasi during the observation of kinetic analysis undertaken with a McCoy cell lineage that lasted for a period of 144 hours. During the process, the morphologic transformation was revealed by indirect immunofluorescence (IF) and the molecules liberated in the extra cellular medium were observed by SDS-PAGE at 24-hour intervals during the whole 144-hour period. It was observed that in the first 72 hours the promastigote form of L. chagasi adhered to the cell membranes and assumed a rounded (amastigote-like) form. At 96 hours the infected cells showed morphologic alterations; at 120 hours the cells had liberated soluble fluorescent antigens into the extra cellular medium. At 144 hours, new elongated forms of the parasites, similar to promastigotes, were observed. In the SDS-PAGE, specific molecular weight proteins were observed at each point of the kinetic analysis showing that the McCoy cell imitates the macrophage and may be considered a useful model for the study of the infection of the Leishmania/cell binomial.

Cinéticas de crescimento de Leishmania realizadas in vitro após a internalização da forma promastigota na célula e a ocorrência da transformação do parasito na forma amastigota foram descritas por vários autores, seja com a utilização de explantes de macrófagos em células de baço de hamster ou atualmente da célula de linhagem de macrófago humano U937. Aliando a microscopia à descrição das inter-relações morfológicas e à síntese de moléculas específicas foi possível esclarecer pontos sobre a biologia do parasito. O presente estudo mostra o acompanhamento do ciclo de crescimento da Leishmania chagasi em uma cinética realizada com células de linhagem McCoy, no período de 144 horas. Durante o processo, as transformações morfológicas foram reveladas pela reação de imunofluorescência indireta (RIFI) e as moléculas liberadas no meio extracelular foram observadas pelo método de SDS-PAGE, em intervalos de 24 horas no período de 144 horas. Observou-se que nas primeiras 72 horas, a forma promastigota da L. chagasi fica aderida à membrana das células com aspecto arredondado (amastigota-like). Em 96 horas as células infectadas apresentaram alterações morfológicas; em 120 horas, as células liberaram, para o meio extracelular, antígenos fluorescentes solúveis; e em 144 horas foram observadas novas formas alongadas dos parasitos como se fossem promastigotas. No SDS-PAGE, proteínas com pesos moleculares específicos são observadas em cada ponto da cinética, mostrando que a célula McCoy parece mimetizar o macrófago e que pode ser um modelo útil para o estudo da infecção do binômio leishmânia/célula.

Reproduction of Leishmania (Leishmania ) infantum chagasi in conditioned cell culture growth medium

Leishmânias podem ser produzidas em meio de crescimento condicionado, após o cultivo de células McCoy (MCC). Promastigotas crescidas em meio semi-sólido NNN foram inoculadas em MCC, inicialmente, 100 parasitos por poço com 2,5 mL de McCoyMCC, em placas com 24 poços, sua multiplicação foi observada por uma cinética de 120 horas. Após este tempo, o meio estava saturado de promastigotas. A reprodução das leishmânias foi acompanhada a cada 24 horas, com contagem do número de parasitos por campo fotomicrografado. Como vantagem da técnica do crescimento da leishmânia em MCC tem-se o seu baixo custo, com pequena quantidade de parasitos pode-se obter o aumento da densidade de promastigotas em tempo reduzido. Com o emprego dessa técnica pode-se estudar o comportamento e a multiplicação das leishmânias nos vertebrados e invertebrados, assim como, obter antígenos, tanto brutos (leishmânia) como solúveis, produzidos pelos parasitos, que poderão ser úteis para se desenvolver kits de diagnósticos.

Experimental infection of Leishmania L. chagasi in a cell line derived from Lutzomyia longipalpis (Diptera: Psychodidae)

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6 percent) and on day 4 in the J774 cells (51 percent). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.

Study of factors affecting growth of leishmania in cultures

The rate of growth of Leishmania major and L. infantum in El-ON's culture media supplemented with human, dog, rat and avian blood was studied in vitro. Rabbit blood was used as a control. The effect of culture with these types of blood on the infectivity of both Leishmania strains to albino mice was also studied. The results showed that a good yield of both L. major and L. infantum parasites can be obtained in culture by using avian blood as substitute for rabbit blood in El-ON's medium. In addition, rat blood gave good results with L. infantum. The morphological forms of L. major and L. infantum on all types of blood supplemented media [elongated promastigotes, spindle promastigotes, promastigotes and amastigoes] were present all through the culture period with variable percentages. The infectivity of experimental animals was not affected by culture of both Leishmania strains on rabbit, human, rat, dogs as well as avian blood supplemented media

Vector-host parasite inter-relation in leishmaniasis-III-impact of blood meal from natural vertebrate hosts on the survival and the development of L. infantum and L. major in phlebotomus langeroni [Diptera: Psychodidae]

Phlebotomus langeroni collected from a leishmaniasis endemic focus at El-Agamy, Alexandria, Egypt, were found to fed on blood from man, dogs [Canis familiaris] and rats [Rattus rattus]. The effect of the kind of blood meal on the development and the life cycle of L. infantum and L. major in laboratory reared P. langeroni was investigated. A membrane feeding technique was used to infect sand flies. Gut smears of infected females were examined immediately after feeding and daily for 16 days. Nectomonads and short promastigote forms of L. infantum or L. major were detected in females fed on human, dog and rat blood at all intervals. Paramastigotes [infective stage] were present only in females fed on dog blood containing L. infantum or L. major and in those fed on rat blood containing L. major