\ ‘Conway Ti HOSTS OF SANDFLY VECTORS OF LEISHMANIA BRAZILIENSIS GUYANENSIS IN THE CENTRAL AMAZON OF BRAZIL* HOWARD A. CHRISTENSEN, JORGE R_ ARIAS, ANA MARIA ok VASQUEZ, ‘AN RULA. BE PREIT: Gorgas Memorial Laboratory, APO Miami 34002 or Apartado 0981, Panama 5, Republic of Panema, ‘and Instituto Nacional de Pesquisas de Amazonia, Coiza Postal $78 Monans 69000, Amazonas, Brasil Abstract The blood meals of 2,569 phlebotomine sandflies from areas endemic for cu- taneous leishinaniasis in the central Amazon of Brazil were tested by the microcapillary precipitin method to determine their vertebrate hosts. The two-toed sloth, Choloepus didac- 4ylus, was the predominant host of two incriminated vectors of Leishmania brasiliensis guy- anensis inthe region, Lutzomyia wmbratilis and La. anduzei (64.0% and 63.65%, respectively) The Lu. “skannoni” group, a complex of several species in which femal are indistinguish- able, also fed predominantly on sloths (73.0%). Species comprising the Lu. “shannoni” group, have not been implicated as vectors of leishmaniasis; however, their foeding patterns in the study area illustrate their potential involvement in the transmission of the parasites to two- toed sloths, which are the principal reservoir hosts of L. byacliensis in Panama, Rodents, and particularly por nes, were the second most frequently fed-on mammal by Lu. um brats (11.6%) and the Lu. “shannoni” group (8.5%). Lutsomyia anduzei (Rozeboom, 1942) was first reported to be a possible vector of cutaneous leish- maniasis by Floch and Abonnenc in French Guy~ ana," based on epidemiological dara. Wijers and Linger, working in Surinam, provided more sub- stantive eviddence of this sandy’s involvement in the transmission of leishmaniasis in their report? in that 12 of 1,209 Lu. anduzei collected in an endemic area were infected with promastigote fla- tellates. The flagellates from two of the fies were inoculated into the skin on the back of a hamster; hhowever, parasites were not demonstrated on his- ‘ological examination after 1 month. Itis probable that some of the sandflies reported by these in- vestigators'~* were Lu. wmbratlés rather than Li. andusei, since Ward and Killick-Kendick diseov- fered that Lu, andusei in Pard State, Brazil, was Accepted 16 Sepember 1981 * This investigation received financial support fom the UNDP/World Bank WHO Special Programme for [Research and Training in Tropical Diseases, the Con- selho Nacional de Desenvolvimento Cientifico » Tee- nologico grandes Endemias Grant PDE. 1005.01, and Trslitto Nacional de Pesquisas da Amazonia Project - Inattato Nacional de Pesquisas di Amazonia, Caixa Pstal 478, Manaus 69000, Amazonas, Brasil actually a complex comprised of several closely ‘elated taxa," one of which was later desctibed by Ward and Fraiha as a new species, Lu. umbva~ tilis.* Promastigotes found by Lainson et al. in 4 of 55 Lu. umbratlis (reported as Lu. andusei) {rom northern Brazil produced nodular lesions at the site of inoculation in hamsters, and am sotes were demonstrable on stained smears from the nodules. Arias and Freitas isolated leishmanial Dpromastigotes (infective to hamsters) from three Lu. andusei and 12 Lu, umbvatils in the Manaus ‘area of central Brazil. ‘The subject of the present paper concerns the results of precipitin tests on the blood meals of 2,574 sandifies, collected from tree trunks during the latter study, to delineate ther vertebrate hosts. ‘The object was to determine the potential reser- voirs of leishmaniasis by the elucidation of the feeding profiles of incriminated vectors MATERIALS AND METHODS Study sites ‘The study sites comprised four terra firma forest localities along the Torquato Tapajos Highway {AM-010), an area known to be endemic for Leishmania braziliensis guyanensis, the etiologi cal agent for “pian-bois.” These areas were: 1) Cidade Nova, 2} Colonia Santo Antonio, 3) Ducke Forest Reserve, and 4) CEPLAC Field Station 239240 Cidade Nova and Colonia Santo Antonio are two ‘esidential areas about 5 km from the city of Man: ‘aus, 2 km before the entrance to the airport, Ci- dade Nova isa recently deforested area where the government is constructing 15,000 low-cost houses, ‘There have been several clinical cases of cuta- neous leishmaniasis from this area during the time of this study. The entrance to Colonia Santo An= tonio is only several hundred meters down the highway, and is an area which has been colonized for many years. Ducke Forest Reserve is a bio- logical reserve 26 km northeast ofthe city of Man- aus on the AM.O10 highway; this area has been the site of many sandily studies and has been char acterized by Penny and Arias.* CEPLAC Field Station is 30 kto northeast of Manaus, 4 km from the Ducke Forest Reserve. Leishmania b. guy: danensis has been isolated from naturally infected Lu, umbratifis and Lu. anducei collected in the CEPLAC Field Station and the Ducke Forest Re- Sandjiy collections All collections were from sandy resting sites on large trees. Only blood-engorged females were collected, and these were placed individually in eotton-stoppered glass tubes, Sandiies were dissected in a drop of physiolog- ical saline and the blood meals were transferred to numbered sections of No. 1 Whatmann filter paper. All samples were refrigerated and subse- quently weapped in aluminum foil and mailed to Gorgas Memorial Laboratory in Panama where they were analyzed Processing of blood meats Filter papers containing sandAy blood-meal ‘smears were placed in 12 > 7S-mam test tubes con- taining 0.3 ml of 0.85% buffered saline; the ma- tela was then refrigerated overnight at 5°C. The next day each blood meal was gently expressed from the filter paper with a wooden applicator stick, The filter paper was removed from each tube and the eluate tested with a Hemastix to de- termine if the material contained sufficient blood to process by the microcapillary precipitin tes, ‘The antigens in the eluate were then screened by class, order, and family-specific antisera. Prepa- ration of the antisera has been reported previ- ously." (CHRISTENSEN HT AL, Tame Vertebrate hosts, a5 determined by the microcapilary Drecpitn test, of Lutzoryia umbratilisfrom Manaus, Bros! Mammalia nidentied) esa Perissodactsla(unidemtied) mH Edentata Bradypodidae (soths) oe 640 Unidentsed St32 Myrmecophagiae (anteater) © 192.0, Rodentia Brethizonidae(porcupines) 1151 Unidentified wna Heteromyidae (piny pocket mice) 10.1 Mutidae oo rats) 1 on Primates Unidentited sos Cebidae onkeys) 6 06 Lezomorphe Leporidae rabbits) wus Morsipiali Didelphidae (opossums) i oo Carnivora Procyonidae foals eaccoons, te} Unidentified 2 02 Artiodaesla ‘Suid (ig) 1 Total ors 100 RESULTS A total of 2,569 blood meals from the following sanelly species was tested by the precipitin meth od: Lu. umbvatilis, Lu, “shannoni” group, La. andusei, Lu. tuberculata and Lu. furcata, The vertebrate hosts of 1,955 (76.1%) of these sandtlies were identified, in the majority of cases to the family level of specificity. Lutsomyia wmbratilis, the principal vector species in the study areas, was the most prevalent sandfly collected from the tree trunks, Edentates ‘were the predominant hosts of Lat. wmbratlis; sloths comprised 64.0% of all feedings (Table 1). (OF the 624 females which had fed on sloths, 207 tested with Choloepus genus-specific antisera were postive indicating that the majority, if not all, of sloth feedings were derived from C. didactylus, the twoetoed sloth. A single double feeding on Bradypodidae and Cebidae also was detectedHOSTS OF Lurzostyia SP Tame? 4s determined by the microcapillary ‘of Lutzomyia “shannon” group from Veriebrate hosts, ‘breciitn test, ‘Manaus, Bras ves (unidentified 1 on Marmalia (anidentifed) 8 Perissodactyla(unidentised) Sos Bdensate Bradypodidae (othe) os 0 Unidentiied 19 "20 “Myrmecophanidae (anteater) re Rodentia Erethizontidae (porcupine) s&s Unidentied os Dasyproctidac agouti) oz Carnivora Procyoniae (oats, raccoons, ete.) 22.2 Unidentife 408 Felidae (cas) Ae fall Primates Cedidae (monkess) R43 Unidentibed 2 02 Calithricdae tmarmasets) 1 on Lagomorpha “Lepore (rabbit) sos Marsupiatia Didelphidae posums) 308 Total ost 100 Blooc-engorged females of the Lu. “shannon” group may’ have included Lu, shannon, Lar den- rophila, and Lu. abonnenci in the collection since females are indistinguishable. This group represented the second most common taxa en= countered in the study. Although species of this ‘group are not considered to be of importance as Leishmania vectors with respect to clinical cases in the Manaus region,” their feeding habits are very similar to that of Lu. mbratils in that sloths accounted for 73.0% and porcupines 8.5% of the 951 hosts identified (Table 2). Similarly, all 291 of the 694 Bradypodidae tested with Choloepus- specific antisera were positive. In addition to the single feelings, one specimen had fed on members of three families, Bradypodidae, Didelphidae and Dasypodidae. ‘Twenty-two Lu, anduzei, another proven Leishmania vector species in the region,” had fed (on the following hosts: Bradypodidae 14 (63.6%) 2a Erethizontidae three (13,6%), Myrmecophagidac ‘two (9.1%), and one each (4.6%) on Cebidae, an unidentified edentate and an unidentified mam- ‘mal. The blood meals of five Lu. fuberculata were identified as from two unidentified mammals, and fone each from Bradypodidae, Didelphidae, and Leporidae. Two Lu. furcata blood meals tested ‘were from unidentified rodents Tt should be noted that the small amount of blood and/or blood meals older than about 24 hhouts in specimens tested precluded the identii- cation of some hosts below the class or order level of specificity. Antisera titers decrease towards the seneric level due to necessary absorption with sera from cross-reacting hosts. Discussion. Precipitin tests, performed by Dr. P. F. L. Boreham, on Lu. umbratilis collected in north Brazil from widely separate trees and reported by Lainson and Shaw" showed that 35 females from fone batch had fed on the following animals: pri ‘mates 20 51.3%), rodents 11 (28%), and anteaters ' (18%), Analysis of 36 additional blood meals of Lu. wmbratilis from three adjacent trees showed ‘that 35 had fed on primates and one on a porcu- pine. Lainson and Shaw emphasized the fact that samples of sandily vectors from highly localized habitats can be misleading in attempts to elucidate the vertebrate source of their leishmaniasis, ifthe sandily species concerned is an opportunistic feed- er." These authors agreed, however, that the re- sults of blood-meal tests on a given sandy species collected from numerous widely dispersed areas, cover a long period must eventually afford some indication of the most likely reservoins) of Leish- mania, especially if they are related to the detec tion of the parasite in dissected sandfis from the same area at the same time, Tnasmuch as our study involved numerous collecting sites over an area extending for 30 km during the period from June 1978 to October 1980, and since during the “study £. b. guyanensis was isolated from La, um- bratilis and Lu. anducei, the preponderant num- ber of feelings by Lu. umbratilis on C, didactylus incriminates this edentate as a natural reservoir host of L. 6. guyanensis in the terra firma forest ‘of the Manaus area. Our findings, and the recent report by Lainson etal." of isolating Lb. guya- nnensis from C. didactylus captured in Jari, Para State, Brazil, implicate two-toed sloths as one of the principal reservoirs of the disease in the north=242 (CHRISTENSEN ET AL ‘em Amazon region. Another species of two-toed sloth, Choloepus haffmanni, previously had been incriminated as the principal reservoir of 1. bra- tiliensis in the Republic of Panama." Two- toed sloths should be considered as potential res- ervoirs of parasites comprising the L. brasiliensis complex throughout the distribution of these ‘edentates from Nicaragua to central Brazil ‘The limited but pertinent data presented on the feeding habits of the second L. brasiliensis vector, Lu, anducei, further emphasizes the significant role of C. didartylus in the epidemiology of the disease in the Brazilian endemic foci studied. Although none of the 128 promastigote flagel- lates isolated from the Lu. “shannon” group by Arias and Freitas infected hamsters,’ the high fre~ queney of sloth feedings by these taxa illustrates their potential involvement in the transmission, cycle of leishmaniasis. If such involvement is es- tablished in this group in the future by isolating Leishmania parasites, the most important role of this complex undoubtedly would be the transmis- sion between sloths, with a very minor role in transmission to man because of their rather min- imal anthropopbagic habits."" ACKNOWLEDGMENTS ‘The authors gratefully acknowledge the assis: tance of Dr. Bustorgio Mendez in the identifica tion of animals from which antisera were pre- pared, and the technical assistance of Salomon Puga, Joao F. Vidal, Roberto Rojas, Raimundo Nonato L. Santos, Celina Gonzalez, Matia Jose Silva and Jose Montenegro |. Foch, H., and Abonpene,E., 1952. Diptres phe Totomus dela Guyane ets Antilles Francais Foune de Linion Francais, No. 14, 207 9 2, Wifes, D. J. Bey and Linger, Ry 1985. Man biting ‘Sandie in Surinam (Duteb Guiana: Phleboto. us andusei asa possible vector of Leishmania Drasilimsis. dnw. Trop. Med. Parasitol 60: ors 4. Ward, RD. and Kilek-Kendrick, R, 1974. Field and laboratory observations on'Psvchodopyaus Iainsoni Pratha & Ward and other sandies (Dip- tera, Phlebotomidae) fom the Transamazonica 18 4 Highway, Paré State, Brazil Bull. Entomol Res, 04: 215-221 Ward, RD, and Fraha, H., 1977. Latzomyio um ‘rai, a’ new species of sand Ay fom Brazil Diptera Psychodidae). J. Med, Entomol. 14 ust Lainson, R., Ward, R, D. and Shaw, J. J. 1976 CCtaneous leishmaniasis in north Bra Lats ‘myia onduzel as a major vector, Trans. R. Soe Trop, Med. Hyg, 70: 171-172, Arias, J. Ry and Freitas, RA. de, 1977. On the ‘ectrs of cutaneous leishmaniasis inthe central Amazon of Brazil. Acta Amazon, 7: 293-298 Arias, J. R., and Freitas, RA. de, 1978. Sabre os ‘vetores de leishmaniose cutanea na Amazonia ‘central do Braail. 2 ineidencin de fagelaos est Fcbotomos sevaticos. deta Amazon. 8: 387-396 Penny, N.D., and Aras, J.B, 108t. fects of the “Amazon Forest. Columbia Caiversty Press Ia press) Christensen, H. A. and Vasques, A. M. de, 198 ost feeding profiles of Rhodnius pllescens (He: miptera: Reduvidae in evra villages of central Panama. Am J. Trop. Med. Hyg. 10: 282-28, Lxinson, Rand Shaw, J. Jo, 1995. The role of ‘animals in the epidemiology’ of South American leishmaniasis Pages 1-116 0 W. H.R Lumsden and D. A, Evans, eds. Biology of the Kineto- Plastida, Volume’? Academic Press, London, ‘New Vork and San Francis, Lainson, Ri, Shaw, J.T and Povoa, M. The ‘importance of elentates (lochs and anteater) 4 primary reservoite of Leishmania brsiien, ‘is gujanensis, causative agent of “pias” ‘in northern Brazil Trans, R. Soc. Twp. Med, Hyg ln pres) Herter A.. and Telford, 8. R., Je, 1969, Leioke ‘mania Brasliensis jolted font shoths in Pane ima, Science, 104: 1419-1820. Herrer, Ay Cheistensen, HLA, and Beumer, R. J 1973. Reservoir hosts of cutaneous leishmaniasis among Panamanian forest mammals. dm. J Trop. Med. Hyg, 22: 585-501 Herre, A. and Christensen, HL A., 1976, Epide- tmilogial patterns of cutaneous leishmaniasis in Panama. II. Endemic persistence ofthe disease Am J. Trop. Med. Hyp. 25" S488 Christensen, H. A. and Hrrer, A. 1979. Suscep- tilt of Sand fies (Diptera aychodida) to Tey Danosomatidae from two-toed slths (Edentat Hradypodidae. J. Med. Entomol, 16: 424-427 Herter A., and Christensen, HUA, 1980, Leoh ‘mania brasiliensis in the Panamanian two-toed Sloth, Choloepus Retfwanni. dm. J. Trop. Med. Hyg, 20° 1196-1200, Avia JU R., and Freitas, R.A. de, 1977. Flebo- {omos da Amazonia central do Brasil, I. Restl- tados obtios das capturas fas com iscae hu mana ¢ equina (Diptera: Psychodidae) Aeta Amazon, 72 507-527