Mixed Domestic Infestation by Rhodnius Prolixus Sta¨ L, 1859

MIXED DOMESTIC INFESTATION BY RHODNIUS PROLIXUS STA¨ L, 1859 AND PANSTRONGYLUS GENICULATUS LATREILLE, 1811, VECTOR INCRIMINATION, AND SEROPREVALENCE FOR TRYPANOSOMA CRUZI AMONG INHABITANTS IN EL GUAMITO, LARA STATE, VENEZUELA

M. DORA FELICIANGELI, HERNA´ N CARRASCO, JAMES S. PATTERSON, BENNY SUAREZ, CLARA MARTIŃEZ, AND MEHUDY MEDINA Facultad de Ciencias de la Salud, BIOMED, Universidad de Carabobo, Maracay, Venezuela; Facultad de Medicina, Instituto de Medicina Tropical, Universidad Central de Venezuela, Caracas, Venezuela; London School of Hygiene and Tropical Medicine, London, United Kingdom; Laboratorio de Chagas, Direccioń General de Salud Ambiental y Contralorıá Sanitaria, Ministry of Health and Social Development, Maracay, Venezuela

Abstract. Mixed infestation of nymphs and adults of Rhodnius prolixus Sta¨l, 1859 and Panstrongylus geniculatus Latreille, 1811 was detected in 3 (15%) of 20 dwellings in El Guamito, an endemic focus of Chagas disease in Lara State, Venezuela. In one of the houses, both species were positive for Trypanosoma cruzi: 14.3% (R. prolixus) and 20% (P. geniculatus ). The overall infection rate in 143 of 352 R. prolixus was 16.1%. Parasites isolated from R. prolixus were identified as T. cruzi I by random amplified polymorphic DNA analysis. Dot−enzyme-linked immunosorbent assays of 36 R. prolixus showed that 58.3% of the R. prolixus had fed on humans. The gut contents of one fifth-instar nymph of P. geniculatus that was positive for T. cruzi also reacted with anti-human serum. A questionnaire was used to gather data on the demographic and socioeconomic characteristics of the population. An indirect immunofluorescent test, an indirect hemaglutination test, and an ELISA were used to detect the presence of antibodies against T. cruzi in 84 of 86 inhabitants and in 15.5% of people more than 20 years old. The relative risk (RR) of infection was greater in men than Of the people more than 15 years old, 36.6% had no .(0.54−4.80 ס confidence interval 95% ,1.61 ס in women (RR formal education. All respondents recognized triatomine bugs, but they did not relate them to Chagas disease transmission. A total of 85.7% of the houses were “ranchos” suitable for the colonization of triatomine bugs. The possible domiciliation of P. geniculatus and the implications of competition with R. prolixus for resources are discussed. Since there is no clear separation of food sources, abiotic factors such as microclimatic variation within houses may be critical to predict the outcome of the process of competition and potential domestication of this generally sylvatic species.

INTRODUCTION sylvatic bugs often show high infection rates5 and may estab- Human Chagas disease is caused by the parasitic protozoan lish new domestic cycles of T. cruzi. Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) that At present, T. cruzi is divided into T. cruzi I and T. cruzi II6, which broadly correspond to the principal zymodemes is mainly transmitted through fecal contamination by triatom- 7 ine bugs (Hemiptera: Reduviidae). It is endemic in all Latin (Z1 and Z2); a third zymodeme group Z3, also known as American countries (20°N−45°S), where approximately T. cruzi IIa, is considered by some to be more closely related to T. cruzi I.8 These three zymodeme groups have been as- 80−100 million people are at risk and 11−12 million people 9 are estimated to be infected.1 sociated with different vectors and reservoirs. The identifi- In Venezuela, a national Chagas Disease Control Program cation of the parasite strain circulated by sylvatic bugs enter- was undertaken in the 1960s with the aim of interrupting ing houses is therefore of extreme interest in terms of acquir- ing a better knowledge of the etiology and epidemiology of intradomestic transmission by vector control using residual 7 spraying of dieldrin indoors and hexachlorocyclohexane out- Chagas disease. Miles and others, using isoenzyme analysis, doors and replacing the mud-walled, palm-roofed huts with reported the presence of T. cruzi Z1 and Z3 in Venezuela. However, there is little new information available on this cement block dwellings with zinc roofs on a large scale (Na- 10 tional Rural Housing Program) to deprive the domestic matter, although a phenotypic and genotypic characteriza- Rhodnius prolixus of resting and breeding sites. During the tion of a number of isolates will be published elsewhere 1980s, diendrin was replaced with fenitrothion in most states (Carrasco H and others, unpublished data). and the routine screening of all public hospital blood banks In this paper, we report mixed domestic infestation by for Trypanosoma cruzi was instituted using enzyme-linked R. prolixus and Panstrongylus geniculatus. The latter species immunosorbent assays (ELISAs). Efforts to improve health appears to be in the process of domiciliation in El Guamito, education were also implemented. These interventions have Lara Sate, Venezuela. The food sources of the bugs, the iden- reduced the annual incidence of T. cruzi infection from ap- tification of T. cruzi isolated from both species, and a sero- proximately 10 per 1,000 inhabitants in the 1950s to 1 per logic survey among the inhabitants are also reported. 1,000 in the 1980s. This has decreased a huge burden of disease among the rural poor. However, analysis of epidemio- MATERIAL AND METHODS logic indicators of the last 10 years has shown that transmission has not been interrupted and may now be increasing.2 Study area. The research was carried out in El Guamito Although the presence of R. prolixus in human dwellings (09°47Ј00ЉN, 69°20Ј14ЉW) in the Municipality of Iribarren, remains the primary risk factor for maintenance of the do- Lara State, Venezuela, a wet pre-mountain forest with an mestic transmission, invasion by wild triatomine species has average annual precipitation between 1,200 and 2,200 mm recently been documented in Venezuela, indicating possible and an annual temperature range of 18–24°C.11 El Guamito is domestic adaptation.3,4 These findings deserve attention since a small village of 25 houses12 scattered from 429–1043 m, sur- 501 502 FELICIANGELI AND OTHERS rounded by a large variety of subsistence agriculture (coffee, against humans, mice, and some domestic animals (dog, banana, avocado, corn, beans) (75%), with secondary forest chicken, pig, cow, and horse) (Sigma, St. Louis, MO). Serum (10%) and primary forest (15%). Most of the houses are dilutions in (phosphate-buffered saline [PBS] 1:100) from se- made of a structure of sticks plastered by a mixture of mud lected hosts were used as positive controls. Triatomine blood and straw, which is called “bahareque” in the colloquial lan- meal samples on filter paper were eluted individually in 100 guage, with thatched or zinc roofs. Very few were constructed ␮L of PBS (pH 7.4) at overnight at 4°C. After 24 hours, 5 ␮l of sun-baked mud bricks, which are called “adobe” houses. of each eluate was transferred to small piece of nitrocellulose Entomologic study. Searching for triatomine bugs and natu- membrane in flexible polyvinyl chloride plates and incubated ral infection with trypanosomatids. In response to the re- for one hour at 37°C. The plates were blocked with 150 ␮Lof ported presence of Chagas disease vectors, an initial visit to El PBS, 1% bovine serum albumin for 30 minutes at room tem- Guamito was carried out in November 2000. House searches perature and then washed three times with PBS, 0.05% were conducted using torches and fine forceps in cracks in Tween 20. The anti-IgG host-specific peroxidase conjugate walls, pictures on the walls, beds, and household goods in 10 was diluted in PBS, 0.05% Tween 20 according to previously houses. The finding in two houses of mixed infestations by determined ratios, mixed with 50 ␮L of each heterologus se- R. prolixus and P. geniculatus led to subsequent visits for rum to reduce cross-reactivity and to enhance specificity, in- more extensive collections. The triatomine bugs were trans- cubated for one hour at 37°C, and washed three times with ported to the laboratory, where feces or gut contents were PBS, Tween 20. Peroxidase substrate (4-cloro-naphthol) was examined for the presence of trypanosomatids. The first added and incubated in the dark for 30 minutes. The reaction group of positive fecal samples were fixed in methanol for five was stopped by washing with distilled water. The samples minutes, dried, and stained with 10% Giemsa. Additionally, a were considered positive if defined blue-purple spots devel- small amount of each sample was kept in lysis buffer (10 mM oped on an antigen dot. Tris, pH 8, 10 mM EDTA, 1 ␮g/␮L of proteinase K) for a Epidemiologic study. With the aim of evaluating the preva- polymerase chain reaction (PCR). Inoculations and cultures lence of antibodies to T. cruzi among the population at risk, were attempted as described later in this report. in May 2001 families of 21 houses gave informed consent for Identification of T. cruzi. Parasites from R. prolixus were inclusion in a study according to a protocol reviewed and obtained by pulling out the digestive tract, mixing in two or approved by the Committee of Bioethics of the Instituto de three drops of physiologic saline, inoculating intraperitoneal- Altos Estudios Dr. Arnoldo Gabaldon of the Ministry of ly into laboratory mice and subsequent blood culture, and/or Health and Social Development. A questionnaire on demo- direct culture of bug feces in diphasic blood agar medium. graphic data (age, sex, occupation, education, time in the lo- After isolation, the parasites were grown at 26°CinRPMI cality, knowledge of Chagas disease) structure of the houses, 1640 medium (GIBCO-BRL, Paisley, Scotland) supple- and peridomestic habitat was completed with the help of the mented according to the method of Miles.13 Genomic DNA householders. Finger prick blood samples were collected on was isolated and purified using the Nucleon DNA Extraction Whatman (Clifton, NJ) no. 1 filter paper to screen for anti- Kit (Amersham Pharmacia Biotech, Ltd., Bucks, United bodies to T. cruzi. Samples were kept dry in plastic bags and Kingdom) following the manufacturer’s instructions. The re- transported to the Laboratorio de Chagas, Direccio´ n de Salud sulting DNA was suspended in 100 ␮L of TE buffer (10mM Ambiental y Contralorı´a Sanitaria, Ministry of Health and Tris-HCl, pH 7.2, 1mM EDTA). The isolates were identified Social Development (Maracay, Venezuela). and typed to T. cruzi group using the random amplified poly- Three serologic tests were used for detecting antibodies to morphic DNA (RAPD) technique as reported by Carrasco T. cruzi as routinely applied at national level: 1) an indirect and others.14 Alternatively, parasites were detected after ex- immunofluorescent test (IFAT),17 2) an indirect hema- traction of genomic DNA directly from P. geniculatus feces glutination test (IHT),18 and 3) an ELISA (Code UM 2014; using the Wizard Genomic DNA Purification System (Promega, UMELISA Chagas-SUMA, Havana, Cuba). The IHT and Madison, WI), followed by amplification of a 280-basepair ELISA antigens were soluble protein extracts of T. cruzi pro- product, with specific primers derived from the sequence of duced at the Instituto de Medicina Tropical, Universidad a 480-bp fragment obtained by RAPD analysis with primer Central de Venezuela (Caracas, Venezuela) according to the A2. This sequence has been shown to be species specific for method of Maekelt.19 Cut-off points for each test were IFAT: T. cruzi; no amplification product is obtained with DNA of 1:32, IHT: 1:32, and ELISA: 0.030 optical density values. different Kinetoplastidae, including T. rangeli and Leishma- Samples were considered positive when a positive reaction nia spp. The sequences of the 480-bp T. cruzi species-specific was obtained in two of the three tests. fragment and the amplification primers will be published elsewhere (Carrasco H and others, unpublished data). Parasite RESULTS DNA was amplified in an MJ thermal reactor (PTC200; MJ Research, Inc., Waltham, MA). Amplified DNA products Entomologic and parasitologic results. Among 20 houses were analyzed by electrophoresis on agarose gels, stained inspected for triatomine bugs, 14 were found positive for Table 1 shows the .(70% ס with ethidium bromide, and photographed on an ultraviolet R. prolixus (infestation index transilluminator. number of specimens collected, instars, and results of exami- Blood meal identification by dot-ELISA. Blood meal iden- nation for T. cruzi. A total of 352 R. prolixus were collected, tification was carried out on triatomine bugs positive for of which 143 were examined. Twenty-three were found A total of .(16.08% ס and on a random sample of negative bugs positive for T. cruzi (infection index (17 ס T. cruzi (n .were collected in one house (No (281 ס The technique used was previously standardized 79.8% of the bugs (n .(20 ס n) were (15 ס for identification of blood meals in phlebotomine sand 2). Ninety-nine were examined and 15.15% (n flies.15,16 We used IgG peroxidase antiserum conjugates infected. DOMESTIC INFESTATION OF R. PROLIXUS AND P. GENICULATUS 503

TABLE 1 Rhodnius prolixus collected, examined and positive for Trypanosoma cruzi in 20 houses in El Guamito, Lara State, Venezuela

No. (%) No. (%) No. (%) positive for collected examined T. cruzi Males 74 (21.0) 29 (39.2) 5 (17.2) Females 78 (22.2) 42 (53.8) 8 (19.1) Instar V 66 (18.8) 26 (39.4) 0 (0.0) IV 62 (17.5) 19 (30.6) 3 (15.8) III 57 (16.2) 18 (31.6) 7 (38.9) II 14 (3.97) 8 (57.4) 0 (0.0) I 1 (0.28) 1 (100) 0 (0.0) Total 352 143 (40.6) 23 (16.08)

Table 2 shows the results of triatomine bugs catches in the three houses where a mixed infestation of two triatomine species was found and their examination for T. cruzi. A total of 36 R. prolixus and 11 P. geniculatus were collected (3.3:1.0). FIGURE 1. Polymerase chain reaction amplification with primers Tc83F and Tc83R of a 280-basepair (bp) DNA fragment from feces of Only one P. geniculatus was positive for T. cruzi.(11.1%), a triatomine bugs. Lane 1, Rhodnius prolixus from house 11; lane 2, fifth-instar nymph. Panstrongylus geniculatus from house 11; lane 3, R. prolixus from Identification of T. cruzi. The PCR amplification of a 280- house 1; lane 4, degraded product; lane 5, purified DNA of Trypa- bp nuclear DNA fragment in feces from P. geniculatus indi- nosoma cruzi obtained from R. prolixus from house 11. cated the presence of T. cruzi in the intestine of this triatomine bug, as shown in Figure 1. The pattern of bands obtained by RAPD of the parasites isolated from R. prolixus corre- able for colonization by triatomine bugs. At the time of the sponded to T. cruzi I when compared with the TCI (Z1) study, 51 men and 35 women lived there, of whom 38.4% reference strain WA250 cl10,14 as shown in Figure 2. It is were less than 15 years old. A total of 36.6% of the people noteworthy that both species of triatomine bugs were found had no formal education. The most frequent occupations in the same house. Blood meal identification. The sample obtained from the stomach of the fifth-instar P. geniculatus that was positive for T. cruzi reacted with the human antiserum. Seventy-five per- were (27 ס cent of the samples obtained from R. prolixus (n not reactive. Twenty-one blood (9 ס reactive and 25% (n meals reacted only with human antiserum (58.3%), one with human and dog antisera (2.8%), one with human and mouse antisera (2.8%), and four with chicken antisera (11.1%). Among 15 R. prolixus positive for T. cruzi, 13 (86.7%) had fed on humans. Demographic and serologic results. The demographic and socioeconomic indicators showed high levels of poverty in El Guamito where 85.7% of the dwellings were “ranchos” suit-

TABLE 2 Mixed infestation by Rhodnius prolixus (Rp)andPanstrongylus geniculatus (Pg) in 3 of 20 houses in El Guamito, Lara State, Venezuela*

House no.

51112

Rp Pg Rp Pg Rp Pg Males 5 (2) 3 (3) Females 2 (0) 1 (1) 3 (3) Instar V 1 (0) 7 (5) 3 (2) 2 (2) 2 (2) IV 1 (0) 2 (0) 2 (2) FIGURE 2. Random amplified polymorphic DNA profiles ob- III 2 (1) 3 (3) 2 (2) tained with primer A2 of Trypanosoma cruzi stocks isolated from the II 3 (2) 1 (1) 2 (2) intestine of Rhodnius prolixus. Lane M, 1-kb molecular mass marker; I lane 1, R. prolixus from house 11; lane 2, R. prolixus from house 1; reference strain ס reference strain CAN II; lane Z1 ס Total 10 2 21 (3+) 5 (1+) 5 4 lane Z3 ס no. of bugs positive for WA250 cl10B; lane Z2 reference strain Esmeraldo cl3. Values on ס + .Values in parentheses are number of bugs dissected * Trypanosoma cruzi. the left are in basepairs. 504 FELICIANGELI AND OTHERS were domestic activities (29.1%), farmers (37.2%), and stu- it repeatedly attacked people. Although no human infections dents (8.1%). were detected, T. cruzi I (Z1) was isolated from bugs, pigs, All but two men were screened for antibodies to T. cruzi. and opossums collected in the area. This indicates the need The overall seroprevalence was 15.5% (nine men and four for a program to control Chagas disease in the Amazon Basin. women) and all were more than 30 years old. No significant In northcentral Venezuela, 20 specimens of P. geniculatus of difference by sex was found. However, the relative risk (RR) different stages were found in a house in Hoyo de la Puerta, Miranda State, associated with Rattus rattus living in a cavity ,1.61 ס of infection was greater in men than in women (RR -All infected women inside the house.3 However, no investigation for T. cruzi in .(0.54−4.80 ס confidence interval 95% were housekeepers and all of the men were farmers. fection was done. In both of these cases, no other triatomine All of the heads of households said that they recognized species was found associated with P. geniculatus nymphal triatomine bugs; 14 (82.4%) said they had seen them inside stages, indicating that P. geniculatus successfully colonized houses. Additionally, 10 (58.8%) had seen them in the peri- the houses. domestic habitats. However, 78.9% said they knew nothing of The present work in El Guamito, Lara State, Venezuela Chagas disease, although 89.5% recognized the bugs as a seems to be the first report of mixed infestation of R. prolixus problem for their family. and P. geniculatus adults and nymphs. Mathematical models, A total of 61.1% of the families had lived in the houses they such as the Lotka-Volterra equations,24,25 have been used to occupied for more than 10 years. However, the rest were chil- hypothesize what would happen when two species live to- dren of older inhabitants who had married and constructed gether in the same habitat. It is accepted that competition for their own houses in the same village. Eight houses had bugs food and space would occur, which might give rise to transient and seropositive occupants, six houses had bugs but no sero- coexistence with eventual competitive exclusion to the point positive occupants, three houses had no bugs but seropositive of the replacement of one species by the other. The logistic occupants, while three houses had no bugs and no seroposi- model predicts that competing species can co-exist only if tive occupants. Fisher’s exact one-tailed test showed no sig- interspecific competition is relatively weak compared with intraspecific competition. The competitive exclusion principle .(0.5743 ס nificant difference among the groups (P (CEP), as derived by Gause,26 states that two species that occupy the same habitat cannot also occupy the same ecologic DISCUSSION niche. Any two species that occupy the same niche will compete with each other to the detriment of one of the species, The village of El Guamito in Lara State is a long estab- which will thus be excluded. The CEP assumes a number of lished focus of Chagas disease in Venezuela, as demonstrated conditions that must be met for it to be obtained. Among the by the distribution of the presence of antibodies to T. cruzi most important, 1) the environment must be spatially uni- among its inhabitants, which were detected only in adults, form, 2) the environment must be temporally constant, 3) the majority of whom had lived all their life in El Guamito. time must be sufficient to allow exclusion, and 4) species must The bias in relative risk towards men could be interpreted as have the opportunity to compete. the result of outdoor transmission, probably due to contami- An example of transient co-existence of T. dimidiata and nation of men with the urine or feces of Didelphis marsupia- R. ecuadoriensis in Ecuador has been reported (Abad-Franch lis,20 natural reservoir of T. cruzi, which are commonly F, unpublished data). The ability of Triatoma infestans to re- hunted for food. However, because of the maintenance of place T. sordida in a laboratory experiment27 is supported by poor dwelling conditions, which are highly favorable for the observations of the spreading of T. infestans in domestic ec- presence of R. prolixus, and because of the presence of T. cruzi otopes in Bolivia,28 and is evidence of the successful replace- in these vectors feeding on humans and dogs, recent infec- ment of one triatomine species by another better adapted tions cannot be excluded. The additional presence of P. ge- species after a period of stability of the human popula- niculatus also infected with T. cruzi in a pre-imaginal stage tion, followed by significant migration. What is the case for and the fact that it had fed on humans confirms that not only R. prolixus and P. geniculatus? Theoretically, it could be ar- R. prolixus but also P. geniculatus is involved in an active gued that they could coexist indefinitely if there is a distinc- domestic cycle. tion between their niches, i.e., P. geniculatus exploiting the The RAPD analysis showed that the triatomine bugs were humid dusty burrow-like areas in the house (perhaps feeding infected with T. cruzi I (Z1). These results are in agreement only on domestic animals, e.g., dogs and/or chickens or pigs), with those obtained with many other isolates found circulat- leaving R. prolixus to occupy the drier parts and feeding on ing in domestic and sylvatic cycles in different localities in humans. However, the identification of the blood meal in one Portuguesa State in west-central Venezuela, which were also P. geniculatus collected in El Guamito indicates that this identified as T. cruzi I. would be not the situation. Nevertheless, P. geniculatus and Panstrongylus geniculatus is a sylvatic triatomine bug wide- R. prolixus have different relative humidity requirements, spread in 16 Latin American countries,5 which is commonly which are higher for P. geniculatus. This factor may be critical associated with the armadillo Dasypus novemcinctus and only in modulating the outcome of the process of competition be- occasionally enters houses when attracted by light.21 It has tween these two species and the domestication of this sylvatic been frequently reported to be infected with T. cruzi, and in species. In El Guamito, it was observed that the three houses Brazil mainly with T. cruzi Z3.22 The potential for domicili- where P. geniculatus was found shared similar features: all ation of P. geniculatus was suggested by Valente and others23 were made of adobe bricks rather than “bahareque”, and in the Amazon Basin of Brazil, where hundreds of specimens piles of adobe were found inside and outside the houses. were found infesting pig sties adjacent to human dwellings In conclusion, the follow-up of invasion of houses by P. and specimens were collected from 10 houses, in one of which geniculatus in the field, the study of the association of its DOMESTIC INFESTATION OF R. PROLIXUS AND P. GENICULATUS 505 presence with the features of the niches occupied, and experi- 9. Gaunt M, Miles MA, 2000. The ecotopes and evolution of tri- mental simulation in the laboratory are necessary to confirm atomine bugs (Triatominae) and their associated trypano- and predict if incipient domiciliation of P. geniculatus may somes. Mem Inst Oswaldo Cruz 95: 557–565. 10. Feliciangeli MD, Dujardin JP, Bastrenta B, Mazzarri M, Villegas give rise to successful domestic colonization. J, Flores M, Mun˜ oz M, 2002. Is Rhodnius robustus (Hemiptera: Reduviidae) responsible for Chagas disease transmission in Received August 18, 2003. Accepted for publication February 6, western Venezuela? Trop Med Int Health 7: 180–187. 2004. 11. Ewel JJ, Madriz A, 1968. Zonas de Vida de Venezuela. Memoria Explicativa Sobre el Mapa Ecolo´gico. Caracas: Editorial Sucre. Acknowledgments: We thank Carlos Mendez, Roseliano Visval, and 12. OCEI, 1990. XII Censo General de la Poblacioń y Vivienda. No- Annhy Torrellas for their technical assistance. menclador de Centros Poblados. Caracas: OCEI Financial support: This work was supported by the Wellcome Trust 13. Miles MA, 1993. Culturing and biological cloning of Trypano- (Project no. 062984/Z/00/Z). The Chagas Disease Intervention Ac- soma cruzi. Hyde JE, ed. Methods in Molecular Biology, Pro- tivities – European Community, the MCT-FONACIT, S1-98000388, tocols in Molecular Parasitology. Totowa, NJ: Humana Press and the CDCH-UCV-09-34-4097-01. Inc., 15−28. 14. Carrasco HJ, Frame IA, Valente SA, Miles MA. Genetic ex- Authors’ addresses: M. Dora Feliciangeli and Benny Suarez, Facultad change as possible source of genomic diversity in sylvatic de Ciencias de la Salud, BIOMED, Universidad de Carabobo, Nu´ - populations of Trypanosoma cruzi. Am J Trop Med Hyg 54: cleo Aragua, Apartado 4873, Maracay, Venezuela. Hernań Carrasco 418–424. and Clara Martıńez, Facultad de Medicina, Instituto de Medicina 15. Gomez B, Sanchez E, Feliciangeli MD, 1998. Man-vector contact Tropical, Universidad Central de Venezuela, Caracas, Venezuela. of phlebotomine sandflies (Diptera: Psychodidae) in north- James S. Patterson, London School of Hygiene and Tropical Medi- central Venezuela, as assessed by bloodmeal identification us- cine, London WC1E 7HT, United Kingdom. Mehudy Medina, Labo- ing a dot-ELISA. J Am Mosq Control Assoc. 14: 28–32. ratorio de Chagas, Direccio´ n General de Salud Ambiental y Con- 16. Agrela I, Sańchez E, Gomez B, Feliciangeli MD, 2002. Feeding tralorıá Sanitaria, Ministry of Health and Social Development, Ma- behavior of Lutzomyia pseudolongipalpis (Diptera: Psycho- racay, Venezuela. didae), a putative vector of visceral leishmaniasis in Venezu- Reprint requests: M. Dora Feliciangeli, Facultad de Ciencias de la ela. J Med Entomol 39: 440–445. Salud, BIOMED, Universidad de Carabobo, Nu´ cleo Aragua, Apar- 17. Camargo ME, 1966. Fluorescent antibody test for the diagnosis of tado 4873, Maracay, Venezuela, Telephone : 58-243-233-5822, Fax: American trypanosomiasis. Technical modification employing 58-243-242-5333; E-mail: [email protected]. preserved culture forms of Trypanosoma cruzi in a slide test. Rev Inst Med Trop Sao Paulo 8: 227–234. REFERENCES 18. Kagan IG. 1970. Serologic diagnosis of parasitic diseases. N Engl J Med 282: 685−686. 1. Dias JC, 2000. Chagas disease control and the natural history of 19. Maekelt GA, 1960. Die komplement bindungs reaction der Cha- human Chagas disease: a possible interaction? Mem Inst Os- gas krankheit. Z Trop Med Parasitol 21: 39–44. waldo Cruz 95 (Suppl II): 14–22. 20. Deane MP, Kenzi HL, Jansen AM, 1986. Double development 2. Feliciangeli MD, Campbell-Lendrum D, Martinez C, Gonzalez cycle of Trypanosoma cruzi in the opossum. Parasitol Today 2: D, Coleman P, Davies C, 2003. Chagas disease control in Ven- 146–147. ezuela: lessons for the Andean region and beyond. Trends 21. Lent H, Wygodzinsky P, 1979. Revision of the Triatominae Parasitol 19: 44–49. (Hemiptera, Reduviidae), and their significance as vectors of 3. Reyes-Lugo M, Rodriguez-Acosta A, 2000. Domiciliation of Chagas’disease. Bull Am Mus Nat Hist 163: 123–520. the sylvatic Chagas disease vector Panstrongylus geniculatus 22. Povoa MM, de Souza AA, Naif RD, Arias JR, Naif MF, Bian- Latreille, 1811 (Triatominae: Reduviidae) in Venezuela. Trans cardi CB, Miles MA, 1984. Chagas disease in the Amazon R Soc Trop Med Hyg 94: 508. Basin. IV. Host records of Trypanosoma cruzi zymodemes in 4. Soto Vivas AA, Barazarte H, Molina de Fernańdez D, 2001. the States of Amazonia and Rondonia. Brazil. Ann Trop Med Primer registro de Eratyrus mucronatus (Hemiptera: Reduvi- Parasitol 78: 479–487. idae) en el ambiente domiciliario en Venezuela. Entomotro- 23. Valente VC, Valente SAS, Noireau F, Carrasco HJ, Miles MA, pica 16: 215–217. 1998. Chagas disease in the Amazon basin: association of Pan- 5. Schofield CJ, 1994. Triatominae: Biolog´ıa y Control. West Sussex, strongylus geniculatus (Hemiptera: Reduviidae) with domestic United Kingdom: Eurocommunica Publications. pigs. J Med Entomol 35: 99−103. 6. Anonymous, 1999. Recommendations from a Satellite Meeting. 24. Lotka AJ, 1925. Elements of Physical Biology. Baltimore: Wil- International Symposium to Commemorate the 90th Anniver- liams & Wilkins. sary of the Discovery of Chagas Disease. April 11−16, 1999, 25. Volterra V, 1926. Variations and fluctuations of the number of Rio de Janeiro, Brazil. MemInstOswaldoCruz94: 429−432. individuals in animal species living together. Chapman RN, ed. 7. Miles MA, Povoa MM, Prata A, Cedillos RA, de Souza AA, Animal Ecology. New York: McGraw-Hill, 409−448. Macedo V, 1981. Do radically dissimilar Trypanosoma cruzi 26. Gause GF, 1934. The Struggle for Existence. New York: Hafner. strains (zymodemes) cause Venezuelan and Brazilian forms of 27. Bar ME, Oscherov EB, Dambrovsky MP, Porcel EA, Alvarez Chagas’ disease? Lancet i: 1338−1340. BM, 1994. Interaction between populations of Triatoma in- 8. Mendonça MBA, Neheme NS, Santos SS, Cupolillo E, Vargas N, festans and Triatoma sordida. Rev Saude Publica 28: 59–68. Junqueira A, Naiff RD, Barrett TV, Coura JR, Zingales B, 28. Noireau F, Brenie`re SF, Cardozo L, Bosseno MF, Vargas F, Fernandes O, 2002. Two main clusters within Trypanosoma Peredo C, Medinacelli M, 1996. Current spread of Triatoma cruzi zymodeme 3 are defined by distinct regions of the ribo- infestans at the expense of Triatoma sordida in Bolivia. Mem somal RNA cistron. Parasitology 124: 177–184. Inst Oswaldo Cruz 91: 271–272.