Sylvatic American trypanosomiasis in Argentina. Trypanosoma cruzi infection in mammals from the Chaco forest in Santiago del Estero

38 TRA~~SACTIONS OF THE ROYAL SOCIETYOF TROPICALMEDICINEAND HYGIENE(1992) 86, 3841

Sylvatic American trypanosomiasis in Argentina. Trypanosoma mammals from the Chaco forest in Santiago del Ester0

cruziinfection

in

C. Wisnivesky-Coil?, N. J. Schweigmann’, A. Alberti’, S. M. Pietrokovsky’, 0. Conti’, S. Montoya’, A. Riarte’ and C. Rivas* ‘Unidad de Ecologia de Reservorios y Vectores de Parasites, Departamento de Ciencias Biologicas, Facultad de Ciencias Exactas y Naturales, Ciudad Universitaria, Pabellon II, 4” Piso, 1428 BuenosAires, Argentina; ‘Institute National de Diagndstico Paseo Colon 568, Buenos Aires, Argentina

e Investigacidn

de la Enfermedad

de Chagas ‘Dr Mario

Fatala

Chabtn’,

Abstract

Typanosoma

cruzi infection in sylvatic mammalsof the quebracho woodsof the easternpart of Santiagode1

Ester0 province, Argentina, was studied from October 1984to December 1987. 301 mammalsof 20 different specieswere caught. T. cruzi, characterizedbiologically and biochemically, was isolated by xenodiagnosis from 23of 72 (32%) Didelphis albiventris opposums,2136(5.5%) Conepatus chinga skunks,and oneferret (Galictis &a). 53 opossumrefugeswerelocatedand triatominebugswerefound in 2 of them: one male Triatoma infestans, infected with T. cruzi, and 5 uninfected nymphs of T. sordida, had all fed on opossumblood. Electrophoreticzymogrampatternsof the T. cruzi populationsisolatedfrom opossums and skunkswere similarto isoenzymeprofilesalreadydescribedfor populationsisolatedfrom infected humans in Argentina. The smallnumberof triatominesfound in the opossumrefugesseemsinadequateto account for the prevalenceof T. cruzi infection recordedfor thesemammals,soother possiblecontaminativeroutes of infection should be investigated. Introduction Chagasdisease(Americanhumantrypanosomiasis) is a widespreadendemicinfection in Argentina, extending over approximately 2.4 million km (BECKER, 1985). Recentserologicalstudieson 18 yearsold men recorded 5.5% infection bv Trvnanosoma cruzi for the whole country (SEGURA et al; 1985). Although the first studies of Chagas diseasein Argentina involved simultaneousdetection of human casesand studieson enzooticcycles,after the start of the National Control Programmein 1963only epidemiological surveysof the humanpopulationwere carried out. As a result, mostof the existing data on wild hostsof T. cruzi in Argentina were obtainedbefore 1950(MAZZA & ROMAIQA,

1932;

MAZZA

&

SCHREIBER,

timber and coal production. Zone B was essentially similarto A but it had beenuninhabitedduring the last 20 yearsand had becomea hunting area.The northernmost part of zone C was deforestedfor agriculture 10 yearsago.The village of Amamais locatedin the southof zone 6, where only patchesof forest remain. Human dwellinasweresoravedwith deltamethrinbv the National Co&o1 Agency” in September 1985. Zone D was similar to zone A, but the soil wasmore sandywith a forest of higher trees, fewer bushesand more abundant cactaceousplants. Most of the inhabitantsof the settlement (Trinidad) made their living hunting sylvatic animals.The houseswere heavily infestedby Triatoma infestans and had never been treated with insecticides.

1938).

Since 1980,we have conductedseveralstudieson the dynamicsof transmissionof domesticT. cruzi infection in rural areasof the province of Santiagode1Ester0 (WISNIVESKY-COLLI et al., 1985, 1987?1989). These were extended to the enzootic cycle m the forested neighbourhoodof Amama, since an understandingof sylvatic transmissionof T. cruzi and its connectionwith the domesticcycle is relevant to control campaigns.This paperreports on T. cruzi infection in wild mammalsof Amama between 1984and 1987. Materials and Methods Study area

The study area was located in the Department of Moreno, Province of Santiagode1Estero, (27”12’3O”S, 63”02’3O”W).The climate is semiarid, of type BS in Koeppen’s classification (PETTERSEN, 1976). It is markedly seasonal,with a dry seasonbetweenMay and October and a rainy seasonthroughout the rest of the year. Mean annual rainfall ranges between 550 and 600 mm (relative humidity 55-68%) and the mean annualtemperatureis 21-22°C with a meanof 28.5”Cin the warmest month and 5*5”C in the coldest one (ANONYMOUS,1982). Accordine to its bioneonranhical characteristics’,the areabelongs-tothe Chaquezalegion (CABRERA, 1980). The primary forest of hardwood quebracho trees (Schinopsis lorentzii and Aspidospetma quebracho blanco) was intensively exploited for timber production and cattle raisingat the beginningof the 20th century. Most of the herbaceousgrowth was destroyed and replacedby thorn shrubs, resultingin a secondary forest of young trees, shrubs, cacti and bromeliads. Four different forest areas(zonesA-D) were selected astrapping stations. Zone A wasused at that time for

Trapping

and examination

of wild mammals

Mammal trapping wascarried out during 6 surveysof 20 d each,from October 1984to December1987.Three different methodswere employed. (i) Trapping using ‘Tender’ traps (WoodstreamInc.), 35~70x35 cm, and ‘National’ traps, 20x 50x20 cm, for medium sized mammals,and ‘Sherman’traps for rodents. (ii) Manual capture(of all mammalsexcept mice), performedduring the day or at night with the aid of hunting dogs. (iii) Trapping carried out by local hunters using snares. Traps for mediumsizedmammalswere placedonly in forested areas1 to 5 km from houses.Shermantraps were arranged in lines of 10 ‘stations’ (2 traps per station), 10 m apart from eachother, and they werealso placed in cornfields and inside houses. Different baits were tried according to the expected catch. Cow fat mixed with viscera was good for opossums,and they were alsoattracted to baitsmadeof ground mousefood pelletsmixed with fat and banana scent. Shermantraps were baitedwith corn [maize]. All traps were baited at sunset and examined early the following day. Captured mammalswere transported to the field station, allowedto rest for 2 h before examinationand then tranquillized with an intramuscular injection of 2 mgikg of a mixture of 2-acetyl-lo-(3-dimethyl-aminopropyl) ferrothiacine maleate hydrogen (Acedan,@ obtained from Hollidav-Scott) ulus ketamine (20 ma/ kg). Mammalswere weighedand measuredaccordingt’o routine procedures(WHO, 1979). Xenodiagnosiswas carriedout beforekilling the mammals,using4 different triatomine speciesfor e&h animal: Triatoma &jestans, T. sordida and T. platensis (5 third or fourth instar nvmnhs each)and Dipeialogaster maximus(2 first instar nymphs).

39 Triatomines were provided by the Insectary of the National Control Agency, Punilla, Cordoba. Some opossums were marked and released at the site of capture, in order to track them.

refugeswere identified by an agar double diffusion test (WISNIVESKY-COLLI et al., 1987).

Mammal

Three hundred and one mammalsof 20 different specieswerecapturedbetween1984and 1987,including 72 Didelphis albiventris (opossum),4 Marmosa pusilla (mouseopossum),36 Conepatus chinga (skunk), 1 Galictis cuja(ferret), 39 Sylvilagus brasiliensis (tapeti), 15Pediolagus salinicola (mara!,8 Lagostomus maximus (vizcacha), 19 Microcavia australzs (mountain cavy), 72 Cricetidae (voles), 15 Tolipeutes mataco(mataco), 2 Chaetophractus vellerosus (smallarmadillo), 1 C. villosus (hairy armadillo), 5 Felis colocoZo (pampascat), 7 F. geoffroyi (Geoffroy’s cat), 3 F. yagouarondi (yaguarundi), 1 F. catus (domesticcat), and 1 Dusyceon griseus (south American ‘fox’). Most of the opossumsand skunks camefrom forest areas, but 4 opossumsand 2 skunks were caught in peridomesticpremises(Table). Cricetid miceof 3 species were captured and preliminarily identified as Graomys griseoflavus, Akodon sp. andCalomys sp., sincetaxonomic classificationof Argentine cricetid rodentsis at present being revised (REIG, 1986). Only G. griseofravus were caught in both forest and domesticareas,Calomys were found only in cornfields, and Akodon were trapped in both cornfieldsand peridomesticpremises.Cavieswere found near houses. All armadillos were caught by hunting dogs, sincethey never enteredtraps. The four marsupials(M. pusilla) werecollectedby hand, in birds’ nestsand hollow trees. Wild catswere hunted by local inhabitants.

tracking and refuge inspection

In order to get to opossumrefuges and collect associatedtriatomines, theseanimalswere fitted with a spool and line mammaltracking device (MILES et al., 1981) of appropriate weight and range (2500-3000m) before being releasedat sunset. They were always allowed a full night of freedom before being tracked. Refugeswere examinedon the spot, being flushed out when necessarywith tetrametrine (0.2%). Hollow logs lessthan 1.5 m from ground level were examined by flashlight. Contents of cavities in the living treeswere obtained manually and examinedunder direct sunlight on a white fabric placedon the ground. Any triatomines present were collected and put into plastic bagslined with pleated absorbentpaper. and identification of trypanosomes Rectal contentsof bugs usedfor xenodiagnosiswere examined at 30 and 60 d after feeding as previously described(WISNIVESKY-COLLI et al., 1985). To isolate parasites,infected insectswere asepticallydissectedand their intestinesprocessedasdescribedelsewhere(PIETROKOVSKY et al., 1991). To identify isolatedtrypanosomesasT. cruzi, biological as well as biochemicalmethods were used. The biological criteria included (i) ability to infect and multiply in triatomines, (ii) ability to grow in media routinely usedfor T. cruzi culture, (iii) ability to infect laboratory mice, forming intracellular amastigotesin various organs,and (iv) morphologicaland biometrical characteristics(BARRETO, 1965). The first criterion was fulfilled by the isolation of trypanosomesby xenodiagnosis,the secondby culturing infected bug intestines. Trypanosome infectivity to laboratory mice was tested by inoculation of approximately 1.5~ lo5 cultured parasites(7x lo3 metacyclic trypomastigotes)in the back of 8 inbred C3H mice (4-7 daysold). A drop of tail blood wasexaminedmicroscopically at 48 h intervals, beginningon the 7th day after inoculation. Xenodiagnosisusing 5-6 third instar T. infestans nymphswasperformedon infected mice before killing them in the 4th week after inoculation. Most organsaswell aspiecesof skeletalmusclewere removed and fixed in 10% neutral formalin solution. Sections (5-6 pm) were stainedwith haematoxylinand eosinand exammed for amastigotenests. Giemsa-stainedthin blood smearswere also prepared from infected mice. Biochemicalidentification was done by Dr Enrique Montamat at the Facultad de CienciasMedicas, UniversidadNational deCordoba,andinvolved electrophoretic characterizationof the following isoenzymes:aspartate aminotransferase (ASAT, EC 2.6.1. I), glucose-6-phosphate dehydrogenase(G6PD, EC 1.1.1.49), malate dehydrogenase(decarboxylating) (NADP) (malic enzyme, ME, EC 1.1.1.40), glucosephosphateisomerase (GPI, EC 5.3.1.9), phosphoglucomutase(PGM, EC 2.7.5.1) and alcoholdehydrogenase (NADP) (ADH, EC 1.1.1.2) (MONTAMAT et al., 1987). Blood meals of triatomines collected in opossum Isolation

Results Mammal

T. cruzi infection of mammals T. cruzi were isolatedby xenodiagnosis from 23 of 72 (32%) D. albiventris, 2136 (5.5%) C. chinga, and the only ferret captured (G. cuja). All infected opossumswere trapped in forest areas1 km or more awayfrom houses (Table: zonesA, B and D). All animalsfound in zone C were negative by xenodiagnosis.Infected skunks were caught in the settlementof Trinidad (zone D) approximately 200 m from dwellings(Table). No differencein the percentageof infection amongsexeswas found for opossums. T. cruzi isolatesbelonged to 3 different isoenzyme strains(zymodemes).Parasitesisolatedfrom opossnms showedthe sameelectrophoreticzymogrampatternsfor the 6 enzymes tested and were characterized as zymodemeZlO, closely related to zymodemes22 and 212 isolatedfrom chronic and acute patientsin Argentina. T. cruzi isolatesfrom skunksbelongedto 23, which is also characteristic of some human stocks. Parasites isolatedfrom the ferret belongedto zymodemeZ9, which hasalso beenfound in T. infestans (MONTAMAT & DE LUCA

A

Didelphis albiventr$ Conepatus chinga”

“No. infected/no. examined. bIncluding 4 from peridomesticareas. ‘Including 2 from peridomesticareas.

lgl

(35%)

D’ORO,

1990).

of opossum refuges Fifty-three refuges were identified. Fourteen opossumswere releasedand tracked, but only 7 denswere located. Sometimes the threadwasbroken by wandering dogsandpigs,or the opossums lost their tracking device. Forty-six refugeswere found following directions provided by local people. The 53 densfound included 34 Location

Table. Distribution of infected opossums and skunks according Province of Santiago de1 Estero, Argentina (19841987)

Species

trapping

to the capture

area, Department

B

Capture zones C

419(44%) 012

0/6b O/l 1’

of Moreno,

D

Total

8126(31%) 2114(14%)

23/72 (32%) 2136(6%)

40 (64%) small cavities in living trees; 16 (30%) hollow logs; one cavitv in dried cactus (Oauntia auimilok and 2 burrows d earth. There appearid to be no association betweenthe sex of the animal and the type of refuge used. In one case,it wasinferred that the sameanimal visited 2 refugesduring the samenight. Triatomine bugs were found in only 2 (4%) of the examineddens.One T. infestans male, and 2 uninfected nymphsof T. sordida (3rd and4th instars)werecollected from a holein a red quebracho tree (Schinopsis sp.); three 4th instar nymphs of T. sordida were found inside a hollow live cactus. Only the T. infestans male was infectedwith T. cruzi, but all the triatomineshad fed on opossumblood. Discussion

Our finding of T. cruzi infection in opossumsagrees with previous work characterizing Didelphis sp. as primary reservoir hosts of the parasite in America (reviewed by BARRETO, 1979). D. albiventris is widely distributed in the temperate and tropical areasof South America, and in Argentina it is presentfrom the northern border to 40”s (CABRERA, 1958), within the area endemic for Chagasdisease. ReportedT. cruzi prevalencesrangedfrom 25% to 45% in the ChacoRegion of Santa Fe Province (MAZZA & ROMANIA,1932; MAZZA & SCHREIBER, 1938), and 49% (34 of 69) in Santiagode1Ester0(CANAL FEIJOO, 1939). The finding of T. cruzi in the skunk C. chinga hasbeen recently reportedfor the first time (PIETROKOVSKY et al., 1991). Although the observedprevalencein our local skunkswaslow, its epidemiologicalrole deservesfurther investigation. This study was designed to investigate T. cruzi infection in sylvatic ecotopes,soperidomestictrappingof medium sized mammalswas avoided. However, some animals caught near houseswere examined. Large differences were found in the location of infected opossumsand skunks. All infected opossumswere caught in forested areas, at least 1 km away from dwellings, and those trapped in zone C, near the settlementof Amama,were negative. Housesin Amama had beentreatedwith insecticidesin September1985and periodic examinationsperformedup to November 1987 revealed 1141(2.4%) \ , infesteddwellingsin August 1986 and 5141 (12%) in November 1987. However, peridomestic premiseshad not been sprayed, since T. infestans density levelsin them were very low (19 bugs collected in storeroomsand goat pens. 2 of them infected). The 2 infected skunks came from the village of Trinidad where 17 of the 19 houses(95%) harbouredT. infestans, whereasno skunk trapped in the woodswas infected. These facts indicate that skunks probably became infected from domestic sources,as suggestedby the zymogramsof their trypanosomes.A similar origin of infection in opossumpopulationscannotbe ruled out, as they are nomadic animals, able to travel over long distances(HUNSAKER,1977) and prone to approach dwellings,which in our study areaare mostly infested. Two ?actsrule out opossumrefugesassignificantfoci of T. cruzi transmission:the small number that harbouredtriatominesand the presenceof only oneinfected bug of a domiciliary speciesthat had probablyflown from a house.Although a failure in the searchingmethodused for triatominesis nossible,the mevalencerate recorded for local marsupialssuggeststhat (i) opossumsmight actively participatein the domesticcycle and/or(ii) other routes of transmission, different from the classical vectorial one, could be playing a role. The first alternative impliesthat part of the T. cruzi populationwould ‘escape’from the domesticcycle, with opossumsacting as reservoirsof parasitesavailablefor future reintroductions. The secondnossibilitv gained significancewhenlargenumbersof T. &uzi epimas$gotes were found by DEANE et al. (1986) in the lumen of the

analglandsof laboratory-rearedD. marsupialis inoculated with isolatesof opossumorigin. The epimastigotes developedinto metacyclic-liketrypomastigotesinfective for mice and opossums,and the authorssuggestedthat direct transmissionof T. cruzi betweenopossums could explain the high rate of infection found in them in areas wheretriatominesare scarce.However, studiesin Brazil found only low rates of infection with trypanosomesin the anal glands of naturally infected D. marsupialis, ranging from 2.1% (1148) in Amazonas state (NAIFF et al., al.,

1987)to 4.5% (1122)in SantaCatarina(STEIDEL et 1988). Other direct routes, such as vertical transmission,

could not be confirmed by observations on both experimentally (ROMANIA, 1955) and naturally infected opossums (TELFORD & TONN, 1982). It is worth remarking that, as opossums and skunks are hunted and skinned by local people, handling of infected animals involves a potential risk of infection. Acknowledgements

We are grateful to Dr Delmi Canale (Laboratorio de Reservorios y Vectores, Servicio National de Chagas), for providing the triatomines used in xenodiagnosis and to Dr E. Montamat (Universidad National de C6rdoba) for performing the electrophoresis of T. cruzi isolates. Dr Bruno Travi and Dr C. J. Schofield made helpful comments on the manuscript and Dr J. Wright kindly assisted with the English version. Field work was facilitated by the friendly hospitality of Mrs M. Moyano and her family and by local people at Amama and Trinidad who helped us with mammal trapping. This work received financial support from the Secretaria de Ciencia y Tecnica and the University of Buenos Aires, Argentina and the UNDPWorld Bank/WHO Special Programme for Training and Research in Tropical Diseases. References

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Tropical

de S&o Paula,

7, 304315.

Barreto, M. I’. (1979). Epidemiologia. In: Trypanosoma cruzi e Doenca de Chagas, Brener, Z. & Andrade, Z. (editors). Rio de Janeiro: Guanabara Koogan, pp. 89-151. Becker, D. (1985). Politicas sanitarias para el control de la enfermedad de Chagas. In: Factores Biologicosy Ecologicos en la Enfermedad de Chagas, Carcavallo, R. U., Ravinovich, J. E. & Tonn, R. J. (editors). Buenos Aires: Ministerio de Salud y Action Social, pp. 305-308. Cabrera, A. L. (1958). Catalog0 de 10s mamiferos de America de1 Sur. Revista de1 Museo Argentino de Ciencias Naturales ‘Bernardino Rivadavia’,, 4, l-307. Cabrera, A. L. (1980). Bzogeografia de America Latina. Organizacion de Estados Americanos, Programa Regional de Desarrollo Cientifico y Tecnokjgico, monografia no. 13, serie biologia, pp. 69-75. Canal Feijoo, E. J. (1939). Vectores, dep6sitos parasitarios y cases clinicos de la enfermedad de Chaaas en la Provincia de Santiago de1 Estero. Actas y Trabajos ii1 Congreso National de Medicina, Cordoba, Argentina, 1, 97-113. Deane, M. I’., Lenzi, H. L. & Jansen, A. M. (1986). Double developmental cycle of Tqpanosoma cruzi in the opossum. Parasitology Today, 2, 146147. Hunsaker, D. (1977). Ecology of New World marsupials. In: The Biology of Marsupials, Hunsaker, D. (editor). New York: Academic Press, pp. 95-156. Mazza, S. & Romaria, C. (1932). Infeccidn espontanea de la comadreja de1 Chaco santafecino por el Tryjanosoma cruzi. Resumenes VII Reunion de la Sociedad de Patologia Norte, Tucuman, Argentina, pp. 981-989.

Reeional

Mazza, S. & Schreiber, F. (1938). Hallazgo en el Departamento de Obligado, Santa Fe, de otra especie de mustelido naturalmente infectado con Schizotrypanum cruzi, de Triatoma infestans infectados en nidos de comadrejas, de Triatoma platensis infectados en nidos de pcidcidos y de Psammolestes coreodes sin infestation en nidos de dendrocolaptidos. Mision de Estudios de Patologia Regional Argentina, 34, 17-35. Miles, M. A., De Souza, A. A. & Povoa, M. M. (1981). Mammal tracking and nest location in Brazilian forest with an improved spool and line device. Journal of Zoology, London,

195, 331-347.

41 Montamat, E. E. & De Luca d’Oro, G. M. (1990). Polimorfismo enzimatico en Typanosoma cruzi de Argentina. Reunion de la Sociedad Argentina de Protozoologia, Cdrdoba, Argentina, p. 41. Montamat, E. E., Arauzo, S., Cazzulo, J. J. & Subfas, E. (1987). Characterization by electrophoretic zymograms of 19 Typanosoma cruzi clones derived from two chagasic patients. Comparative Biochemical Physiology, 87, 416-421. Naiff, R. D., Naiff, M. F., Barrett, T. V. & Arias,, J. R. (1987). Typanosoma cruzi nas glandulas de Didelphas marsupialis: primeiro registro de infeccoes naturais. Resumos X Congreso .S;fdade Brasileira de Parasitologia, Salvador, Brazil, p. Pettersen, S. (1976). Introduction a la Metereologia. Madrid: Espasa Calpe, pp. 398-412. Pietrokovsky, S. M., Schweigmann, N. J., Riarte, A., Alberti, A., Conti, O., Montoya, S. & Wisnivesky-Colli, C. (1991). The skunk Conepatus chinga as new host of Typanosoma cruzi in Argentina. Journal of Parasitology, 77, 643-645. Reig, 0. (1986). Diversity patterns and differentiation of high Andean rodents. In: High Altitude Tropical Biogeography, Monasterio, M. & Vuilleumier, F. (editors). London: Oxford University Press, pp. 404-439. Romana, C. (1955). Falta de transmision hereditaria de Typanosoma (Schizotypanumj cruzi en Didelphis paraguayense y comentario sobre herencia de1 parasite en otrds mamfferos. Anales de1 Institute de Medicina Regional (Tucumdn), 4, 149-154. Segura, E. L., Perez, A. C., Yanovsky, J. F.,, Andrade, J. & Wynne de Martini, G. J. (1985). Decrease m the prevalence of infection bv Trvaanosoma cruzi (Chapas’ disease) in voune men of Argentina. Bulletin of the pan Americhn &ealtYh Organization, 19, 252-264. Steidel, M., Scholz, A. F., Toma, H. K. & Schlemper, B. R., jr

(1988). Presence of Tppanosoma cruai in the anal glands of naturally infected opossum (Didelphis marsupialis) in the State of Santa Catarina, Brazil. Memorias do Znstituto Oswald0 Cruz, 83, 135-137. Telford, S. R., jr, & Tonn, R. J. (1982). Dinamica de1 Typanosoma cruzi en poblaciones de un reservorio primario Didelphis marsupialis en 10s llanos altos de Venezuela. Boletin de la Oficina Sanitaria Panamericana, 95, 341-364. Wisnivesky-Colli, C., Giirtler, R. E., Solarz, N. D., Lauricella, M. & Segura, E. L. (1985). Epidemiological role of humans, dogs and cats, in the transmission of Typanosoma cruzi, in a central area of Argentina. Revista do Instituto de Medicina Tropical de Sao Paula, 27, 346352. Wisnivesky-Colli, C., Ruiz, A. M., Ledesma, O., Gtirtler, R. E., Lauricella, M., Salomon, D. O., Solarz, N. D. & Segura, E. L. (1987). Ecologia domestica de la tripanosomiasis Americana: perhl alimentario de Triatoma infestans en un area rural de Santiago de1 Estero. Revista de la Sociedade Brasileira de Medicina Tropical, 20, 31-39. Wisnivesky-Colli, C., Ruiz, A. M., Gtirtler, R. E.! Solarz, N. D., Ledesma, O., Bujas, M. A., de Rissio, A. M., Marteleur, A. & Segura, E. L. (1989). Dynamics of transmission of Typanosoma cruzi in a rural area of Argentina. IV. A serological, parasitological and electrocardiographic study of the human population. Medicina (Buenos Aires), 49, 341-350. WHO (1979). Report from the Expert Committee Meeting on Epidemiology of Chagas’ Disease, Brasilia, July 16-19. Geneva: World Health Organization, mimeographed document TDR/EPICHA/79.1. Rev. 1. Received publication

25 July 1989; revised 30 May 1991

29 May

1991;

accepted

Announcement

12th Seminar

on Amebiasis

The 12th Seminar on Amebiasis will be held on 12-14 November 1992 at the National Academy of Medicine, Mexico City. Free communications are invited on all aspects of basic and clinical research on Entamoeba histo2ytica and related Entamoeba. For further details contact Dr Adolf0 Martinez-Palomo, CINVESTAV-IPN, Apartado Postal 14-740, 07000 Mexico, D.F., Mexico, Fax (525) 754-5116, or Dr Louis S. Diamond, Bldg 4, Rm 126, National Institutes of Health, Bethesda, MD 20892, USA, Fax (301)-496-2443.

for