I u/ Am. J. Trop. Med. Hyg.. 61(5), 1999. pp. 846-849 li Copyright 0 1999 by The American Scciety of Tiupical Medicine and Hygiene ,,. LUlZOMYIA NUNEZTOVARI ANGLESI (LE PONT & DESJEUX, 1984) AS A VECTOR OF LEISHMAhXA AMAZONENSIS IN A SUB-ANDEAN &EISHNANIASIS FOCUS OF BOLMA EDDY MARTINEZ, FRANÇOIS,~PONT, MIGUEL TORREZ, JENNY LLERIA, FERNANDO VARGAS, JEAN CLAUDE DUJARDIN, AND JEAN PIEmTUJARDIN Instituto Boliviano de Biología de Altura, Departamento de Enfermedades Tropicales, La Paz, Bolivia; Institur de Rechercke pour le Développement La Paz, La Paz, Bolivia; Prince Léopold Iristitute of Tropical Medicine, Antwerp, Belgium; Génétique Moléculaire des Parasites et des Vecteurs, Institut de Reclterclie pour le D6veloppentertt. Montpellier, France Abstract. Recently, a new Leishnmnia amazonensis focus was described in a sub-Andean region (1,450-2,100 meters above sea level) of Bolivia. In this area, three anthropophilic sandfly species were identified Lutzornyia nuneztovari anglesi Le Pont & Desjeux, 1984, which represented 8699% of the captures, Lu. galatiae Le Pont et al., 1998, and Lu. slmnnoni Dyar 1929. Only Lu. nuneztovari anglesi was found naturally infected by flagellates (16 of 1,715 females). Three Leishmania stocks were isolated and analyzed 6y isoenzyme electrophoresis at 11 loci. No significant isoenzymatic differences were demonstrated between them and 7 stocks isolated from patients from the same area, and previously characterized as L. amazonensis. Moreover, in a simplified protocol, the experimental infection of Lu. nuneztovari anglesi by L. amazonensis was successful in 92% of the surviving specimens. These data are discussed in relation to the Killick-Kendrick criteria. These results strongly suggest that Lu. nunezrovari anglesi is the vector of L. amazorzeizsis at Cajuata, Inquisivi, La Paz, Bolivia. In Bolivia, only two teishnmnia species have been iden- ulation by house-to-house studies. Two hundred inhabitants tified as agents of human cutaneous leishmaniasis: L. (V.) were examined in December 1995 and 215 inhabitants were brazilìensis and L. (L.) amazonensis. The first one is a wide- examined in March 1996. spread parasite in Bolivia,'"' while the second one has been Sandfly collections, dissection, and parasite isolation. reported only Leishmania amazonensis is better From October 1995 to September 1996, except for Novem- known from sylvatic lowlands, especially in Amazonia,8.9 ber 1995 and March 1996, monthly protected human bait where all proven vectors belong to the Lutzonzyia JIaviscu- captures (capturing the sand flies attracted to exposed legs; tellata but the cycle is able to survive in plan- only the authors of this study were subjected to this proce- tation woodland and deforested areas as in Brazil." In the dure) in coffee crops or residual forest were organized on two Venezuelan and Ecuadorian Andes, parasites related to L. consecutive nights each month between 6:OO PM and 1O:OO amazonensis have been described (L. ganhami'2 and L. PM. Female specimens were caught in individual glass tubes, mexicana, l3respectively). Leishmania amazonensis is poten- and immediately dissected in saline solution (0.9%) on glass tially very dangerous and occasionally induces a chronic and slides for microscopic examination. When positive for fla- eventually fatal disease known as cutaneous diffuse leish- gellates, the gut and head content was aspirated into a sy- maniasis (CDL). The first Bolivian case reported by Prado ringe with saline s.olution and subsequently inoculated into Barrientos from the neighboring region of the Yungas was a hamster. Material from hamster lesions developing a gran- . a typical case of CDL probably due to L. amazoneiisis.14 uloma at the inoculation site was aspirated into a syringe Recently, we described an outbreak of cutaneous leishman- containing sterile saline solution and then cultured in tubes iasis in the province of Inquisivi, La Paz,I5where the parasite of diphasic medium (NNN and Schneider's). These were was identified as L. amazonensis on the basis of biologic stored at 24T after changing from diphasic to monophasic and molecular data. The present study provides evidence in- medium (Schneider's). The study was approved by the Sci- dicating that the vector of L amazonensis is Lu. nuneztovari entific Committee of the Instituto Boliviano de Biología de anglesi Le Pont and Desjeux, 1984. Altura. Experimental infection of sand flies. Using local faç$- ties but without temperature and humidity control, 140 wild MATERIALS AND METHODS female Lu. nuneztovari anglesi were blood fed on anesthb- Study area. The L. amazonensis focus is located at Ca- tized hamsters experimentally infected With a patient strain juata and surrounding communities in the province of In- from the same region and previously characterized as L. quisivi in southeastern region (67"15'W, 16942's) of the De- amazonensis by isoenzyme analysis. of these 140 speci- partment of La Paz, Bolivia. The study area is at an altitude mens, 6 were randomly selected 36 hr after the blood meal ranging from 1,450 to 2,100 meters above sea level.15 It is and checked for the presence of promastigotes in the diges- a deforested vaJley with very steep slopes such that the bot- tive gut. After 60 hr, only 13 sand flies had survived, which tom of the valley is shaded early in the afternoon. The hu- were also examined. The other anthropophilic species (LU. man population lives in scattered adobe brick houses with galatiae and Lu. shannoni) were not tested because their corrugated iron roofs. Around the settlements, land is culti- rarity did not pennit us to gather a representative live sample vated with coca plantations, root vegetables, and papaya after 24 or more hours. On the other hand, no member of crops, while residual deciduous forests with xerophytes and these two species was found to be positive for flagellates epiphytes cover the steepest places. The cumulative preva- after field dissection. I lence-of-cutaneous leishmani~asis-wa: determined in the pop- Isoenzyme electrophoresis. Three stocks of parasites iso- 846 I j -__. i -.-_1 ., ..-*-- o 3 LU. NUNEZTOVARI ANGLES1 VECTOR OF L. AMAZONENSIS 847 1 TABLE1 Anthropophilic Lufzomyia sand flies captured by human bite and detection of infection* Lu n anglesi Lu. galariae LIL shannoni Month Fhrh Dissected Infected (‘3%) Flhrlh Dissected Fhrh Dissected . October 28.5 199 2 (1.0) 4.2 34 0.25 2 December 34.5 250 4 (1.6) 1.2 10 0.62 5 January 36.0 138 2 (1.4) 2.0 16 0.62 5 February 6.0 43 o (0.0) 0.4 3 0.25 2 April 81.0 112 2 (1.8) 0.4 3 0.37 3 . May 47.9 243 1 (0.4) 3.5 28 0.00 O June 102.1 303 2 (0.7) 6.4 51 0.25 2 July 66.1 225 1 (0.4) 1.9 15 0.12 1 August 75.7 87 1 (1.1) 2.8 22 0.75 6 September 82.0 115 1 (0.9) 1.7 14 0.37 3 Total 1,715 16 (0.93) 196 29 * Fhlh = females capturedihourhuman; % = percent of infected females. lated from wild LLLnuneztovari anglesi were compared with circumscribed new focus of leishmaniasis with high ende- 7 stocks isolated from human lesions previously identified micity. as L. amaZonensis,’5 as well as with reference strains of L. Sandfly collections. From 86% to 99 % of the female (L.) amazonensis (IFLIVBR/67/PH8), L. (V.) braziliensis sand fies captured on human bait during a one-year period (MHOM/BR/75/M2903), L. (L.) chagasi (MHOM/BlU74/ were Lu. nuneztovari anglesi, the remaining ones were Lu. PP75), L. (L.) mexicana (M”YC/BZ/62/M379), and L. (L.) galatiae and Lu. shannoni, in decreasing order of abundance pifanoi (MHO~VI~W~~/LV135). (Table 1). Cellulose acetate plates (Helena Laboratories, Beaumont, Natural infection of Lu. nuneztovari anglesi. Only Lu. TX) were used. Running conditions and identification tech- nuneztovari anglesi was found infected with flagellates in niques were as described by Dujardin and others.I6 Each the midgut, the pharynx, cibarium, and proboscis. This nat- sample was mixed with a hypotonic enzyme stabilizer, held ural infection was detected each month, except in February. for 30 min on ice, centrifuged for 2 min at 3,500 X g, and Of the 1,715 female Lu. nuneztovari anglesi dissected, 16 immediately subjected to electrophoresis. All aliquots al- (0.93%) were infected with promastigotes (Table 1). lowed the survey of as many as 12 different enzyme sys- Parasite isolation. Four to six weeks after inoculation of tems, including additional analyses for controls or verifica- promastigotes from the gut and head of infected sand flies tions. The following 12 enzyme systems were assayed: acon- into the hind legs of hamsters, three hamsters deve€oped itase (EC 4.2.1.3, ACON), glucose-6-phosphate dehydroge- nodular lesions, without ulceration, which progressively in- nase (EC 1.1.1.49, G6PD), glucose phosphate isomerase (EC creased and developed into metastatic peripheral lesions 5.3.1.9, GPI), a-glycerophosphate dehydrogenase (EC (forelegs, nose, ears, tail, and mucocutaneous zones) after 1.1.1.8, aGPD), isocitrate dehydrogenase (EC 1.1.1.42, 6-8, months. Samples obtained from these lesions showed IDH), malate dehydrogenase (EC 1.1.1.37, MDH), peptidase abundant free parasites as well as many vacuolated histio- 1, substrate-L-leucyl-leucine (EC 3.4.1 1, PEP l), 6-phos- cytes containing parasites. Three stocks, each from a differ- phogluconate dehydrogenase (EC 1.1.1.44, 6PGD), phos- ent hamster, were isolated. Development of parasites in the phoglucomutase (EC 2.7.5.1, PGM), malic enzyme (EC culture medía was observed after 48 hr. 1.1.1.40, ME), mannose phosphate isomerase (EC 5.3.1.8, Experimental infection of sand flies. From the 6 sand MFI), and fructose-1,6 diphosphate (EC 3.1.3.11, FDP).