Isolation and molecular identification of Leishmania ( Viannia ) peruviana from naturally infected Lutzomyia peruensis (Diptera: Psychodidae) in the Peruvian Andes

Abstract

Leishmania (Viannia) peruviana was isolated from 1/75 Lutzomyia peruensis captured during May 2006 in an endemic cutaneous leishmaniasis region of the Peruvian Andes (Chaute, Huarochiri, Lima, Peru) . Sand fly gut with promastigotes was inoculated into a hamster and the remaining body was fixed in ethanol. L. (Viannia) sp. was determined by polymerase chain reaction (PCR), and Leishmania species through molecular genotyping by PCR-restriction fragment length polymorphism analyses targeting the genes cpb and hsp70, resulting L. (V.) peruviana. The infected sand fly appeared 15 days after the rains finished, time expected and useful real time data for interventions when transmission is occurring.Key words: Leishmania peruviana -Lutzomyia peruensis -polymerase chain reaction-restriction fragment length polymorphism -Peru An important step for the incrimmination of Leishmania vectors is the report of naturally infected sand fly species (Killick-Kendrick & Ward 1981). The proportion of sand flies infected with Leishmania in places frequented by humans is taken into account. Several methods have been applied for this requirement, being the sand fly gut dissection first used, although it is the less productive one because of the low Leishmania infection rate in sand flies (0.001-2.6) (Perez et al. 1994), the requirement of living sand flies and well trained personnel for sand fly gut dissection. To date this technique is still being used with the purpose of isolating the parasite from an individual sand fly. Sand flies can also be cryopreserved in liquid nitrogen to be dissected months later. Young et al. (1987) dissected 18,463 sand flies, obtaining 11 (0.059%) with Leishmania promastigotes in Colombia. In another technique, the whole sand fly body is homogenized in pools of up to 24 individuals and then inoculated into hamsters, where Leishmania infection produces lesions in at least six weeks. Finally, the parasites are isolated by aspiration of the lesions and transferred to culture medium. Using this technique the number of infected sand fly individuals is unknown and it is possible to have a mixture of Leishmania strains in a single pool. Sentinel hamsters have also been used to obtain Leishmania strains from sand flies (Herrer 1982a). The animals are exposed to sand fly bites in endemic areas and then aspirates of different parts of the hamster body are taken and inoculated into culture medium to isolate the parasites. Here indeed, the infected sand fly species are unknown. Detection of Leishmania DNA is now a commonly used technique. Sand flies can be preserved dried or frozen and large number of sand flies can be processed individually or in pools. By means of polymerase chain reaction (PCR) it is possible to amplify and detect DNA of less than one Leishmania parasite . Leishmania species identification is now done by using specific primers designed for different Leishmania genes currently used as targets for molecular genotyping (Garcia et al. 2004(Garcia et al. , 2005.The search for Leishmania vectors in Per...

Natural infections of Pintomyia verrucarum and Pintomyia maranonensis by Leishmania (Viannia) peruviana in the Eastern Andes of northern Peru

“…ayacuchensis and Lu. peruensis, which distribute in western valleys of the Central Andes and Northern and Central Andes, respectively, were identified as vectors of L. (V.) peruviana, the primary etiological agent of CL in the Peruvian Andes [13][14][15][16]. On the other hand, the vector responsible for the transmission of CL remains to be elucidated in endemic areas of the Eastern Andes.…”

“…The vector species of L. (V.) peruviana was identified as Lutzomyia ayacuchensis in the western valley of central Andes [13] and Lu. peruensis in northern and central Andes [14][15][16][17]. Pintomyia verrucarum, a widely distributing species in Andean highlands, was reported to have the capacity to transmit L. (V.) peruviana under experimental conditions [18], and the natural infection of Pi.…”

Epidemiological survey of Lutzomyia longipalpis infected by Leishmania infantum in an endemic area of Brazil

“…The classic method used to identify the parasite in the sandflies' gut is the dissection of the digestive tract, followed by direct microscopic examination. However, a limitation of this technique is the difficulty associated with the processing of a large number of specimens (PEREZ et al, 2007). The advantage of dissection should also be mentioned, i.e., the detection of developmental infectious stages with recording of metacyclic forms in the foregut (SADLOVA et al, 2011).…”