Phlebotomine sandfly (Diptera: Psychodidae) diversity and their Leishmania DNA in a hot spot of American Cutaneous Leishmaniasis human cases along the Brazilian border with Peru and Bolivia

Abstract

In this study, we identified the phlebotomine sandfly vectors involved in the transmission of American Cutaneous Leishmaniasis (ACL) in Assis Brasil, Acre, Brazil, which is located on the Brazil-Peru-Bolivia frontier. The genotyping of Leishmania in phlebotomines was performed using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism. A total of 6,850 sandflies comprising 67 species were captured by using CDC light traps in rural areas of the municipality. Three sandfly species were found in the state of Acre for the first time: Lutzomyia georgii, Lu. complexa and Lu. evangelistai. The predominant species was Lu. auraensis/Lu. ruifreitasi and Lu. davisi (total 59.27%). 32 of 368 pools were positive for the presence of Leishmania DNA (16 pools corresponding to Lu. davisi, and 16 corresponding to Lu. auraensis/Lu. ruifreitasi), with a minimal infection prevalence of 1.85% in Lu. davisi and 2.05% in Lu. auraensis/Lu. ruifreitasi. The Leishmania species found showed maximum identity with L. (Viannia) guyanensis and L. (V.) braziliensis in both phlebotomine species. Based on these results and similar scenarios previously described along the Brazil/Peru/Bolivia tri-border, the studied area must take into consideration the possibility of Lu. davisi and Lu. auraensis/Lu. ruifreitasi as probable vectors of ACL in this municipality.

Fig. 1. : Map of Brazil highlighting the state of Acre, location of the district headquarters of Assis Brasil municipality and points of sandfly capture (legends 1, 2, 3 and 4). Irecê road: point 1 (10º53’40.55”S/ 69º36’2.53”W); point 2 (10º53’3.35”S/ 69º35’25.95”W); point 3 (10º52’46.11”S/ 69º35’12.81”W). São Francisco road: point 1 (10º56’49.5”S/ 69º38’17.4”W); point 2 (10º55’49.40”S/ 69º38’49.96”W); point 3 (10º55’52.4”S/ 69º38’38.6”W); point 4 (10º55’22.6”S/ 69º38’36.0”W). Museu road: point 1 (10º56’0,95”S/ 69º31’33,54”W); point 2 (10º56’4.49”S/ 69º30’45.71”W); point 3 (10º55’46.97”S/ 69º29’47.54”W); point 4 (10º55’58.30”S/ 69º29’11.07”W). Riparian Forest: point 1 (10º58’09.4”S/ 69º43’07.8”W); point 2 (10º56’4.49”S/ 69º30’45.71”W); point 3 (10º55’46.97”S/ 69º29’47.54”W); point 4 (10º55’58.30”S/ 69º29’11.07”W).

Fig. 2. : landscape aspect of the sandfly collection areas, Assis Brasil Municipality, Acre state, Brazil. The satellite images were obtained through Google Earth (May 2010).

Fig. 3. : mkDNA gene polymerase chain reaction (PCR) products and SL RNA Multiplex PCR resulting from sandfly pools. 2% agarose gel stained with GelRed. (A) mkDNA gene PCR. Lines 1-3: samples from Lutzomyia davisi pools; Line 4: Lu. auraensis/Lu. ruifreitasi pool; Line 5: Leishmania (Leishmania) amazonensis reference strain and Line 6: males of Lutzomyia sp. (B) Multiplex PCR for the Leishmania complex. Line 1: males of Lutzomyia sp.; Lines 2, 5: samples from Lu. davisi pools; Lines 3, 4: samples from Lu. auraensis/Lu. ruifreitasi pools; Lines 6-8 Leishmania spp. reference strains; Line 6: L. (Viannia) braziliensis; 7: L. (L.) amazonensis; 8: L. (L.) infantum chagasi. M: 100 bp size marker (Invitrogen).

Fig. 4. : polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for identification of Leishmania species in sandflies. The hsp70 PCR products (234 bp) amplified within pools were digested by the enzymes HaeIII and BstuI. 12% silver-stained polyacrylamide gel. Lines 1, 11: Negative control of male Lutzomyia spp.; Lines 2, 12: Lu. auraensis/Lu. ruifreitasi pool I; Lines 3, 13: Lu. davisi pool II; Lines 4, 14: Lu. auraensis/Lu. ruifreitasi pool III; Lines 5, 15: Lu. auraensis/Lu. ruifreitasi pool IV; Lines 6, 16: Lu. davisi pool V; Lines 7, 17: positive control of L. (Viannia) braziliensis; Lines 8, 18: positive control of L. (V.) guyanensis; Lines 9, 19: positive control of L. (L.) amazonensis. M: 100 bp size marker (Invitrogen).