Leishmaniasis in Brazil: XIII. Isolation of Leishmania from Armadillos

239

TRANSACTIONSOF THE ROVAL SOCIETV OF TROFICAL MEDICINE AND HVGIENE, VOL. 73, No. 2, 1979

leishmaniasis in Brazil: XIII. Isolation of Leishmania from armadillos (Dasypus novemcinctus) , and observations on the epidemiology of cutaneous leishmaniasis in north Pará State

R. LAINSON,J. J. SHAW,R. D. WARD,P. D. READYAND R. D. NAIFF Instituto Evandro Chagas,Fundação Serviços de Salíde Pública,Caixa Postal 3, Belém,Pará, Brasil

Iso/ation 01 Leishmania Irom armadi//os: north Pará State (1".54'S : 48".47'W). Leishmania Armadillos have long been suspected as a source was isolated from three of lhe armadillos (21.4%), of Leishmania infecting man in South America, with positive cultures obtained from lhe heart- principally due to early literature (much of it blood, spleen and liver of lhe first, blood and liver unscientific in nature), which has indicated a marked of lhe second, and lhe spleen only of lhe third: association of phlebotomine sandflies with these no isolations were made from lhe skin. The flagel- animaIs. lates were in each case infective to hamsters, in lhe The examination of armadillo burrows often skin of which they produced small, nodular lesions reveals numbers of sandflies resting there, some- containing scanty amastigotes (Fig. 1). Finally, times engorged with blood, but they are usually laboratory-reared sandflies, Lutzomyia longipalpis non-anthropophilic species (e.g. Brumptomyia spp.) (Lutz & Neiva), were fed on promastigotes through unassociated with human leishmaniasis. Unsub- chick-skin membranes, in lhe manner described by stantiated accounts are also common of veritable clouds of sandflies pursuing armadillos in the forest, at night. The indian (Tupi) name for the armadillo in the States of Pará and north Mato Grosso is "tatu", and that for the sandfly "tatu- quira"-literally, "armadillo-fly" (VON lHERING, 1968). This does suggest, then, some basis for the almost folkloric association of the "tatu" with sandflies and leishmaniasis. As far as we know, however, no evidence has until now been produced to support this belief. Between March and August, 1978, we examined 14 nine-banded armadillos (Dasypus novemcinctus) by culturing heart-blood and pieces of spleen, liver and skin in NNN blood-agar medium. The animaIs showed no skin lesions and appeared to be in perfect health: alI were from forest, near Moju,

Fig 2. Pylorus of a sandfly, Lutzomyia /ongipalpis, experimentally Pig. 1. Leish",ania from an armadillo, Dasypus /1ovemcinclus: infected with the sameparasite: note auached flagellates- a type of intrace\lular amastigotesin a smear of skin-lesion of experimenta\ly development typical of leishmaniae of the L. braziliemis complexo infected hamster. Bouin-fixed, Giemsa stained. Fresh preparation, phase contrasto 240 LEISHMANIASISIN BRAZIL : XIII

WARD et alo (1978). Dissection of these insects, results of this attempt will be given elsewhere, but tive to six days later, showed prolific development of the following observations on Leishmaniainfections the parasite, with attachment of round-stumpy found in a number of the sandflies we dissected are forms to the wall of the hindgut (pylorus and ileum) appropriate here, for the insects were caught very (Fig. 2). Some flies also showed heavy growth of close to where the infected armadillo was shot. free, elongated promastigotes throughout the A total of 129 engorged sandflies were captured midgut and up to the cardia. from tree-trunks, during the dar, and maintained The Leishmania from the armadillos is clearly individually in 3 X 1 inch plastic tubes, in the not a member of the L. mexicanacomplex, parasites manner described by WARD (1972) and modified by of which do not undergo development in the hind- LAINSONet ai. (1977). Throughout the tive days of gut ofthe sandfly (LAINSON& SHAW,1972; LAINsoN capture, they were kept in the forest in a polystyrene et al., 1977). From its behaviour it is close to box, closed at night and open by dar, and containing members of the L. braziliensis group, in which the a wad of cotton wool soaked in water to preserve hindgut development is an obligate feature. Other maximum humidity. The sucrose supply of each characters indicating this relationship are the poor fly was changed daily, and a check made for deaths growth of the parasite in hamster skin (LAINSON& and oviposition. During the capture of the naturally SHAW, 1972) and the small size of the amastigotes engorged sandflies from the tree-trunks, the (SHAW & LAINSON,1976). catchers' hands and arms were constantly attacked by unfed females: 77 of these were caught, and Observations on lhe epidemiology of cutaneous kept under the same conditions for subsequent leishmaniasisin north Brazil dissection. In September, 1978, we continued our previous On return to Belém, alI sandflies dying after investigations on "pian-bois" in our study area oviposition were mounted for identification. Those near Monte Dourado, north Pará State (0°'47'S : 48 which survived egg-Iaying were dissected, 52°'40'W), where we had incriminated the sandfly identified and examined for evidence of promasti- Lutzomyia umbratilis Ward & Fraiha as an important gote infection: the results are given in Table I. vector of Leishmania braziliensis guyanensisto man The natural infections in alI eight infected sand- (LAINSONet al., 1976; WARD & FRAIHA,1977). flies were remarkably heavy, with stumpy attached Armadillos were abundant in the area and one parasites packing the pylorus and scattered, to was shot at night: it is noteworthy that a number of lesser extent down the ileum. Free, elongated sandflies were seen attempting to feed on the dead fiagellates extended throughout the midgut, up to body shortly afterwards. Material from the animal the cardia, and were often seen escaping from the was sent, on ice, to our laboratory in Belém: cut end of the oesophagus.lu each case,intradermal cultures of peripheral blood were not possible, and inoculation of the parasites into hamsters produced those Eram the liver were lost due to fungal con- small, nodular lesions containing amastigotesof the tamination. Cultures Eram the spleen, however, L. braziliensis type: cultures were prepared from yielded good growth of promastigotes which proved these, in NNN medium. infective to hamsters, in which they produced Finally, both men engaged in the capture of the cuntaneous lesions containing amastigotes, similar sandflies subsequently developed cutaneous leish- to those described above. maniasis, with totaIs of six and seven lesions The main objective during these studies was to respectively, on their hands and arms. The parasites obtain blood-fed Lu. umbratilis and establish a were isolated in NNN culture and inoculated intra- laboratory colony of this important sandfly. The dermally into hamsters: the resulting lesions

Table I-Examination of blood-fed sandflies taken from tree-trunks, and others attacking mau, ln an area endemic for cutaneous leishmaniasis ("pian-bois") in north Pará State, Brazil

blood-fed, from tree-trunks, daytime attacking man, daytime No. infected No. infected Species Total No. with Total No. with caught dissected Leishmania caught dissected Leishmania Lu. umbratilis 83 25 o 72 23 1 Lu. whitmani 37* 21* 7* 3 O Lu. dendrophila 4 O O O Lutzomyia sp., shannoni group 2 2 O O O Lu. anduzei 1 O 1 O Lu. damascenoi O 1 O Lu. rorotaensis O O TotaIs: 129 48 7 77 23 1 * Of the total of 37 blood-fed Lu. whitmani caught off trees, 34 were from a single tree, and 6 of them were infected. 11 R. LAINSONet ai. 241 resembled in every way those caused by the Leish- high rate of infection (33' 3 '10)tound in Lu. whit- mania from the sandflies. mani during the present investigations. It may well have been due to chance: this species is scarce, at Discussion ground leveI, in our study area, and has a very We are uncertain as yet, if the Leishmania from patchy distribution. In this respect it is significant the armadillos from Moju and Monte Dourado are that of the 37 blood-fed Lu. whitmani taken from the same; and the role of these edentates in the randomly selected trees, 34 were from a single tree epidemiology of cutaneous leishmaniasis in north and 6 of them were infected. What role, if any, Brazil must remain speculative until comparative this insect may play in the epidemioIogy of "pian- studies are completed on our isolates from arma- bois" has still to be determined: possibly it is dillos, sandflies and mano largely restricted to transmission among the wild In previous studies in the Monte Dourado area, mammalian hosts. seven different samples of sandflies captured at There is little doubt in our minds that of the various periods in November, 1975 (LAINSONet ai., two sandflies found infected, Lu. umbratilis must 1976), and in February, March and July 1976 stiII be regarded as the major vector of L. brazil- (unpublished observations) have revealed infection iensis guyanensisto man in the endemic areas we rates of 7'30;0,5'3%, 1.00;0, I'So;o, 3'00;0, 3'6% have studied, for it is very much more abundant and 1'1 % in Lu. umbratilis: the over-all figure was than Lu. whitmani. af a total of 77 sandflies attack- 20 infected flies out of 1,139 dissected (1'750;0)' ing the collectors in these studies, for example, af these, 13 were very heavy infections, of the type 72 (93.5%) were Lu. umbratilis and only three described above, and alI produced infections in (3'9%) Lu. whitmani. Finally, 23 of these Lu. hamsters. As five of six persons engaged in the umbratilis were dissected and one (4.3%) proved capture of these sandflies subsequently developed to be infected. The fact that the catchers between cutaneous leishmaniasis, it was reasonably con- them developed 13 separate leishmanial lesions, cluded that Lu. umbratilis was the major vector clearly indicates that they were bitten by many transmitting the infection to mano more than one infected Lu. umbratilis: other infected Promastigote infections were seen in three other specimens of this fly must have been included in species of sandflies during these earlier studies, but the 49 which were mounted without dissection, or their nature remained undetermined. Thus, two were among others which escaped capture. of 34 Lu. whitmani (Antunes & Coutinho) showed scanty flagellates associated with a recent bloodmeal, in the midgut, but inoculated hamsters died Acknowledgements a few days later: on another occasion, one of 15Lu. These studies were financed by the Wellcome anduzei (Rozeboom) had a heavy hindgut Trust, London, with additional support from the infection and abundant, free promastigotes in the Superintendência de Desenvolvimento da Ama- bloodmeal remnants, but the flagellates proved zônia (SUDAM) do Brasil. We are much indebted non-infective to hamsters. Finally, unidentified to the Company JARI Florestal e Agropecuária, promastigotes were seenin 13 of 112 Lu. dendrophila Ltda., for facilities provided in Monte Dourado. (Mangabeira): a few flies showed attached hindgut AIso to Srs. Manoel C.M. de Souza, Iorlando da forros, but most had only scanty promastigotes Rocha Barata and José I. de Almeida for their swimming within the recently ingested blood. Six untiring efforts in the field; and to Sras. M. Dantes attempts to infect hamsters with these parasites de Farias and Suêd de N.F. Silva, and Srs. Antonio failed. J. de Oliveira Monteiro and José P.N. Cruz for No promastigote infections were found in the technical assistance. following sandfly species: 202 Psychodopygus maripaensis (Floch & Abonnenc), 22 Ps. davisi (Root), 11 Ps. hirsutus (Mangabeira), 10 Lu. gomezi References (Nitzulescu), 6 Lu. jlaviscutellata (Mangabeira), Arias, J. R. & Freitas, R. A. de (1977). On the 6 Lu. pacae (Floch & Abonnenc), 5 Ps. paraensis vectors of cutaneous leishmaniasis in the central (Costa Lima), 4 Lu. damascenoi (Manga beira), Amazon of Brazil. 1. Preliminary findings. Acta 3 "Phlebotomusde Souvenir" (Floch & Abonnenc: = Amazônica.. 7, 293-294. Lu. yuilli Young & Porter ?), 3 Lu. infraspinosa Ihering, R. von (1968). Dicionârio dos animais do (Manga beira) and 2 Ps. bispinosus Fairchild & Brasil. Editôra Universidade de Brasília: pub- Hertig. af these, Ps. maripaensisis highly anthropo- lished by the Serviço Gráfico da Comp. Mel- philic, but feeds principally at night, when man horamentos de São Paulo, Indústrias de Papel, is not normally working in the forest: it does not Rua Tito 479, São Paulo. seem to be part of the tree-trunk fauna, at least at Lainson, R. & Shaw, J. J. (1972). Leishmaniasis ground leveI. The remaining speciesare also largely of the New World: taxonomic problems. British nocturnal in their feeding habits, and have always Medical Bulletin, 28, 44-48. formed a very minor proportion of the sandflies Lainson, R., Ward, R. D. & Shaw, J. J. (1976). attacking man in the Monte Dourado area. Epi- Cutaneous leishmaniasis in north Brazil: Lutzo- mastigotes of unidentified trypanosomes were myia anduzei as a major vector. Transactions of found in 1 Lu. dendrophila and 7 Lu. umbratilis. the Royal Society of Tropical Medicine and In view of these previous findings, and a recent Hygiene, 7O, 171-172. report of L. braziliensis-like parasites in Lu. umbra- Lainson, R., Ward, R. D. & Shaw, J. J. (1977). tilis from near Manaus, Amazonas State, Brazil Leishmania in phlebotomid sandflies: VI. Im- (ARIAS& FREITAS,1977), we were surprised by the portance of hindgut development in distinguish- 242 LEISHMANIASIS IN BRAZIL : XII

ing between parasites of the Leishmania mexicana Ward, R. D. & Fraiha, H. (1977). Lutzomyia and L. braziliensis complexes. Proceedings of the umbratilis, a new speciesof sand fty from Brazil. Royal Society, London, Series B, 199, 309-320. (Diptera: Psychodidae). Journal of Medica! Shaw, J. J. & Lainson, R. (1976). Leishmaniasis in Entomology,14,313-317. Brazil: XI. Observations on the morphology of Ward, R. D., Lainson, R. & Shaw, J. J. (1978). Leishmania of the braziliensis and mexicana Some methods for membrane feeding of labora- complexes. Journal of Tropical Medicine and tory reared, neotropical sandfties (Diptera: Hygiene, 79, 9-13. Psychodidae). Annals of tropical Medicine and Ward, R. D. (1972). Some observations on the Parasitology, 72, 269-276. biology and morphology of the immature stages of Psychodopygus wellcomei Fraiha, Shaw & Lainson, 1971 (Diptera: Psychodidae). Memorias do Instituto Oswaldo Cruz, 10, 15-28. Acceptedfor publication 30th November,1978.

International HUNDRED YEARS DF MALA RIA RESEARCH

Date: 17th to 19th January, 1980 Venue : lnstitute of Postgraduate Medical Education and Research 244 Acharya J. C. Bose Road, Calcutta 700 020, lndia Further information from : Professor K. P. Sen Gupta, Convenor, lnternational Symposium, Hundred Years of Malaria Research lnstitute of Postgraduate Medical Education and Research, 244 Acharya J. C. Bose Road, Calcutta 700 020 Sponsored by : lndian National Science Academy lndian Council of Medical Research National Academy of Medical Science Clndia) lndian Statistical lnstitute Dept. of Science and Technology, Government of lndia Dept. or Health and Family Welfare, Government of West Bengal Scientific Sessionswill include : Epidemiology of malaria Vector biology, ecology and behaviour Parasitology Genetics of parasite, vector and host lmmunology Drugs Contrai measures