Lutzomyia longipalpis naturally infected by Leishmania (L.) chagasi in Várzea Grande, Mato Grosso State, Brazil, an area of intense transmission of visceral leishmaniasis Lutzomyia longipalpis naturalmente infectado por Leishmania (L.) chagasi

The American visceral leishmaniasis (AVL) is caused by parasites belonging to the genus Leishmania (Trypanosomatidae) and is transmitted to humans through the bite of certain species of infected phlebotomine sand flies. In this study, we investigated the natural infection ratio of Lutzomyia longipalpis, the main vector species of AVL in Brazil, in Várzea Grande, Mato Grosso State. Between July 2004 and June 2006, phlebotomine sand flies were captured in peridomestic areas using CDC light-traps. Four hundred and twenty (420) specimens of Lu. longipalpis were captured. 42 pools, containing 10 specimens of Lu. longipalpis each, were used for genomic DNA extraction and PCR (polymerase chain reaction) amplification. Leishmania spp. DNA was detected in three out of the 42 pools tested, resulting in a minimal infection ratio of 0.71%. Restriction fragment length polymorphism (RFLP) analysis indicated that Leishmania (L.) chagasi was the infective agent in the positive pools. Psychodidae; Insect Vectors; Leishmaniasis Introduction American visceral leishmaniasis (AVL) is a public health problem in Brazil. In Várzea Grande, Mato Grosso State, a total of 138 human cases of AVL were reported between 1998 and 2005 1. In 2003, that municipality was considered an area of intense transmission by the Department for Epidemiological Surveillance in the Brazilian Ministry of Health 2. In this context, we carried out the present study in order to determine the natural ratio of Leishmania-infected Lutzomyia longipalpis and the infecting Leishmania species in that area. Materials and methods Várzea Grande (15o32’30”S, 56o17’18”W) is located in the state of Mato Grosso, near to its capital, Cuiabá 3. Phlebotomine captures were carried out for two years during three consecutive days per month, from July 2004 to June 2006. CDC light traps were mounted in peridomestic areas in five houses across three districts 4 of intense AVL transmission (São Matheus and Eldorado – two residences each – and Parque Sabiá – one residence) totaling five traps per day. The selection of areas and residences was based on previous entomological data 4,5, as well as on the prevaNOTA RESEARCH NOTE NATURAL INFECTION OF L. longipalpis BY LEISHMANIA 2415 Cad. Saúde Pública, Rio de Janeiro, 26(12):2414-2419, dez, 2010 lence and incidence of human and canine cases of AVL. Pools containing ten Lu. longipalpis females each were prepared for DNA isolation 6. In order to confirm the extraction of phlebotomine sand fly DNA, these pool samples were amplified by polymerase chain reaction (PCR) in the presence of primers for a constitutive Lutzomyia gene (cacophony) 7,8. The pool samples were also amplified with specific primers for Leishmania spp. 9. In every PCR reaction set, both negative (no DNA) and positive controls (kDNA purified from Leishmania (V.) braziliensis) were included. Product analysis was performed by PAGE (polyacrylamide gel electrophoresis). Since each pool sample comprised ten Lu. longipalpis females, the minimal infection rate was calculated as the number of positive pools times 100 divided by the total number of specimens tested 10. Positive PCR samples were submitted to PCR-RFLP (restriction fragment length polymorphism), aimed to distinguish among the infecting parasite species according to a published protocol 11.