Detection of Leishmania braziliensis in naturally infected individual sandflies by the polymerase chain reaction

Abstract

The natural infection of sandflies by Leishmania in wild-caught specimens was studied, using the polymerase chain reaction (PCR)-hybridization technique. The PCR was carried out using 2 oligonucleotides (primers 3J1 and 3J2) derived from a repetitive nuclear DNA sequence. The primers support the enzymatic amplification of a fragment of approximately 500 bp, present in the nuclear DNA of Leishmania braziliensis. The expected band was observed in 5 of 65 sandflies containing flagellates. After hybridization with a species-specific probe, we confirmed natural infection by L. braziliensis. The technique allowed the identification of Lutzomyia gomezi and Lu. panamensis as vectors of L. braziliensis in an endemic area of cutaneous leishmaniasis in Urama, Puerto Cabello district in Venezuela. As far as we are aware, this work constitutes the first report of natural infection of Lu. panamensis with L. braziliensis in the study area. We also demonstrate that PCR-hybridization is a suitable approach to establish the Leishmania-sandfly relationship and will be useful in epidemiological studies of leishmaniasis in endemic areas.

Study on phlebotomine sand fly (Diptera: Psychodidae) fauna in Belo Horizonte, state of Minas Gerais, Brazil

“…Based on the results of the current work, the use of Shannon traps allowed us to evaluate the presence of some species that were not collected (Passos et al 1993) and it has been shown to be highly sensitive in detecting protozoa parasites into phlebotomine sand flies (Rodriguez et al 1999, Miranda et al 2002, Michalsky et al 2002. Nevertheless, previous reports have shown that phlebotomine infectivity rates in nature are low, 0.2 to 2%, making the parasite finding difficult (Rodriguez et al 1999, Miranda et al 2002.…”

Isolation ofLeishmania (Viannia) braziliensisfromLutzomyia spinicrassa(species groupVerrucarum) Morales Osorno Mesa, Osorno & Hoyos 1969, in the Venezuelan Andean Region

“…mexicana DNA sample or any other negative controls previously mentioned. PCR and hybridization are very useful techniques for species specific identification of parasites in naturally infected sandflies as was previously demonstrated by Rodriguez et al (1999) mainly when parasites in the gut of the specimen are not very abundant. Here we report the utility of primers and probe derived from the nuclear DNA (Rodriguez et al, 1997) in the specific identification of Le.…”

“…Leishmania identification was carried out according to Rodriguez et al (1999). The samples were heated for one hour at 56 o C, and the DNA was extracted with The DNA probe was labelled using the digoxigenin-anti digoxigenin kit (Boehringer-Mannhein, Germany) according to the instructions of the manufacturer.…”

“…The samples were heated for one hour at 56 o C, and the DNA was extracted with The DNA probe was labelled using the digoxigenin-anti digoxigenin kit (Boehringer-Mannhein, Germany) according to the instructions of the manufacturer. Labelling and detection of the hybridized product was carried out as previously described (Rodriguez et al, 1999).…”

Definición de las condiciones de temperatura y almacenamiento adecuadas en la detección de ADN de Leishmania por PCR en flebotominos.

“…Actualmente, la detección de parásitos del género Leishmania en los insectos incluye métodos que van desde la búsqueda directa de los flagelados en el tracto digestivo del insecto hasta técnicas moleculares que usan principalmente K ADN (12,(26)(27)(28)(29)(30). La técnica de reacción en cadena de la polimerasa (PCR), por su alta sensibilidad y especificidad, ha facilitado adelantar estudios ecoepidemiológicos en áreas de alta endemicidad, suministrando datos más precisos en periodos de tiempo más cortos (17,29).…”