Detection of Leishmania (Leishmania) infantum RNA in fleas and ticks collected from naturally infected dogs.

The occurrence of the insect vector (sand flies) with low rates of Leishmania infection, as well as autochthonous transmission in the absence of the natural vector in dogs, have been reported. These unexpected data suggest a hypothesis of other arthropods as a possible way of Leishmania transmission. The prevalence of Leishmania (Leishmania) infantum in fleas and ticks collected from dogs with canine visceral leishmaniasis (CVL), as well as parasite viability, were evaluated herein. The presence of L. (L.) infantum was assayed by PCR and ELISA in ectoparasites and biological samples from 73 dogs living in a Brazilian endemic area. As the occurrence of Leishmania DNA in ticks and fleas is expected given their blood-feeding habits, we next investigated whether parasites can remain viable inside ticks. PCR and ELISA confirmed that 83% of the dogs had CVL. Fleas and ticks (nymphs, male and female adults) were collected in 55% and 63% of the 73 dogs, respectively. Out of the 60 dogs with CVL, 80% harbored ectoparasites infected with L. (L.) infantum. The infection rates of the ectoparasites were 23% and 50% for fleas and ticks, respectively. The RNA analysis of the extract from ticks left in laboratory conditions during 7 to 10 days after removal from CVL dogs showed that parasites were alive. In addition, live parasites were also detected inside adult ticks recently molted in laboratory conditions. These findings indicate a higher infection rate of L. (L.) infantum in ticks and fleas, but they do not conclusively demonstrate whether these ticks can act as vectors of CVL, despite the fact that their rates were higher than those previously described in Lutzomyia longipalpis. The presence of viable L. (L.) infantum in ticks suggests the possible importance of dog ectoparasites in CVL dissemination.

A comparison of the immune parameters of dogs infected with visceral leishmaniasis using Western blot and neutralization techniques.

The Western blot technique was used to demonstrate the presence of antibodies in the blood of dogs that presented canine visceral leishmaniasis. This technique was used against some specific molecules present in the lysate of the promastigote form of Leshmania chagasi. Through the association of the results of the Western blot technique with the morphological alterations seen as a result of the serum neutralization technique performed in McCoy cells (which mimetizes the macrophage) it was possible to observe the role of some molecules of great relevance in determining the disease in symptomatic dogs as well as that of some other molecules associated with asymptomatic infected dogs that may become transmitters as well as differentiating them as asymptomatic resistant dogs. In the sera analyses carried out during the immunobloting a variation of 9 to 27 immunoreacting bands was observed, which were then compared using Dice's similarity coefficient. In the dendrogram constructed on the basis of the coefficient, 50% similarity was observed among the total number of reagent bands with the promastigote lysate, thus creating five groups. The main difference observed related to the clinical condition of the dogs: symptomatic and asymptomatic dogs were found in separate groups. The asymptomatic group of dogs was distributed in two different places in the dendrogram because they presented two different behavior patterns regarding the cellular morphology in the serum neutralization reaction: the presence or absence of cellular lysis. According to this analysis it is possible to evaluate the immune status and associate it with specific markers observed in the reaction found in the Western blot strips.

A comparison of the immune parameters of dogs infected with visceral leishmaniasis using Western blot and neutralization techniques

The Western blot technique was used to demonstrate the presence of antibodies in the blood of dogs that presented canine visceral leishmaniasis. This technique was used against some specific molecules present in the lysate of the promastigote form of Leshmania chagasi.Through the association of the results of the Western blot technique with the morphological alterations seen as a result of the serum neutralization technique performed in McCoy cells (which mimetizes the macrophage) it was possible to observe the role of some molecules of great relevance in determining the disease in symptomatic dogs as well as that of some other molecules associated with asymptomatic infected dogs that may become transmitters as well as differentiating them as asymptomatic resistant dogs. In the sera analyses carried out during the immunobloting a variation of 9 to 27 immunoreacting bands was observed, which were then compared using Dice's similarity coefficient. In the dendrogram constructed on the basis of the coefficient, 50 percent similarity was observed among the total number of reagent bands with the promastigote lysate, thus creating five groups. The main difference observed related to the clinical condition of the dogs: symptomatic and asymptomatic dogs were found in separate groups. The asymptomatic group of dogs was distributed in two different places in the dendrogram because they presented two different behavior patterns regarding the cellular morphology in the serum neutralization reaction: the presence or absence of cellular lysis. According to this analysis it is possible to evaluate the immune status and associate it with specific markers observed in the reaction found in the Western blot strips.

A técnica de Western blot foi utilizada para demonstrar a presença de anticorpos do soro de cães, que apresentavam leishmaniose visceral canina, contra algumas moléculas específicas no lisado da forma promastigota de Leshmania chagasi.Através da associação da técnica de Western blot com as alterações morfológicas observadas como resultado da técnica de soro-neutralização em células McCoy (que mimetizam o macrófago) foi possível observar o papel de algumas moléculas de maior relevância para a determinação da doença em cães sintomáticos bem como o papel de outras moléculas na predição de cães infectados assintomáticos com o potencial de serem transmissores e ainda diferenciá-los como cães assintomáticos resistentes. Na análise dos soros durante a reação de immunoblotting observou-se uma variação de 9 a 27 bandas imunorreagentes, que foram comparadas utilizando-se o coeficiente de similaridade de Dice. No dendrograma construído com base no coeficiente, observou-se 50 por cento de similaridade entre as bandas totais reagentes com o lisado de promastigota formando cinco agrupamentos. A principal diferença foi observada com respeito à condição clínica, ou seja, cães sintomáticos e assintomáticos ficaram em grupos separados. Os soros dos cães assintomáticos distribuídos em dois grupos diferentes do dendrograma apresentaram padrões de comportamento diferentes, quanto à morfologia celular na reação de soro-neutralização, ou seja, a presença ou ausência de lise celular. De acordo com esta análise foi possível avaliar o status imunitário e associá-lo com determinados marcadores específicos observados na reação encontrada nas fitas de Western blot.

Flebotomíneos de várzea do rio Aguapeí, região noroeste do Estado de São Paulo, Brasil / Sand flies in the Aguapeí river floodplain, northwest area of State of São Paulo, Brazil

OBJETIVO: Investigar a sazonalidade de flebotomíneos de acordo com sua ocorrência e densidade. MÉTODOS: A pesquisa for realizada em área de várzea do rio Aguapeí, do município de Mirandópolis, Estado de São Paulo. Os flebotomíneos foram capturados mensalmente com armadilhas automáticas luminosas, instaladas das 18:00 às 7:00 horas, durante um ano (2004-2005), em dois locais: varanda de um rancho de pesca e mata. Utilizou-se a média de Williams para o cálculo da sazonalidade dos flebotomíneos e teste de qui-quadrado para comparação. RESULTADOS: Foram capturados 35.995 flebotomíneos. Cinco espécimes eram Brumptomyia avellari, um Psathyromyia (Xiphomyia) hermanlenti e os demais Nyssomyia neivai, que apresentou freqüência mais elevada no inverno. É o primeiro registro de Ps. hermanlenti no Estado. CONCLUSÕES: A alta densidade de Nyssomyia neivai, um dos vetores suspeitos de transmitir a leishmaniose tegumentar americana, aponta o risco de transmissão dessa doença no local, principalmente nos períodos mais secos do ano.

OBJECTIVE: To investigate the seasonal variation of sand flies regarding their occurrences and densities. METHODS: The study was conducted in the Aguapeí river floodplain in the state of São Paulo, southeastern Brazil. Sand flies were monthly captured with automatic light traps set up between 18:00 and 7:00 hours, over a period of one year (2004-2005), at two sites: veranda of a domicile and in a forest on the banks of Aguapeí River. The Williams' average was used for estimating the seasonal variation and the Chi-square test for comparison. RESULTS: A total of 35,995 specimens were captured: five Brumptomyia avellari, one Psathyromyia (Xiphomyia) hermanlenti and the rest Nyssomyia neivai, which had the highest frequency during the winter. Ps. hermanlenti is first recorded in state of São Paulo. CONCLUSIONS: The high density of Nyssomyia neivai, a suspected vector of cutaneous leishmaniasis, points out to the risk of transmission of this disease in this setting, especially during drier periods of the year.

[Sand flies in the Aguapeí river floodplain, northwest area of State of São Paulo, Brazil]. / Flebotomíneos de várzea do rio Aguapeí, região noroeste do Estado de São Paulo, Brasil.

OBJECTIVE: To investigate the seasonal variation of sand flies regarding their occurrences and densities. METHODS: The study was conducted in the Aguapeí river floodplain in the state of São Paulo, southeastern Brazil. Sand flies were monthly captured with automatic light traps set up between 18:00 and 7:00 hours, over a period of one year (2004-2005), at two sites: veranda of a domicile and in a forest on the banks of Aguapeí River. The Williams' average was used for estimating the seasonal variation and the Chi-square test for comparison. RESULTS: A total of 35,995 specimens were captured: five Brumptomyia avellari, one Psathyromyia (Xiphomyia) hermanlenti and the rest Nyssomyia neivai, which had the highest frequency during the winter. Ps. hermanlenti is first recorded in state of São Paulo. CONCLUSIONS: The high density of Nyssomyia neivai, a suspected vector of cutaneous leishmaniasis, points out to the risk of transmission of this disease in this setting, especially during drier periods of the year.