Introduction. Medical and Veterinary Entomology ( 1998) 12,

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1 Medical and Veterinary Entomology ( 1998) 12, Abundance of Lutzomyia ovallesi but not Lu. gomezi (Diptera: Psychodidae) correlated with cutaneous e i s h man i as i s i n c i den ce i n n o rt h -ce n t ra Ven ezu e a 1 M. DORA FELCANGEL and JORGE RABNOVCH Facultad de Ciencias de la Salud, Universidad de Carabobo, and Escuela de Malariologia Dr. Arnold Gabaldh, Ministerio de Sanidad y Asistencia Social, Maracay, Venezuela and *Departamento de nvestigaci6n, Universidad de Belgrano, Buenos Aires, Argentina Abstract. n north-central Venezuela Lutzomyia gomezi and Lu. ovallesi are the main endophiliclanthropophilic species of phlebotomine sandflies implicated as vectors of cutaneous leishmaniasis (CL). Lutzomyia ovallesi has been found infected with Leishmania braziliensis (1.2%) and less often with Le. mexicana (.7%), while Le. braziliensis infections have also been found in Lu. gomezi (.47%). We investigated population densities of these sandflies using two sampling mcthods with four series of collections between January 1991 and March 1995 at El ngenio, Miranda State. All-night outdoor collections from a Shannon trap were correlated with indoor collections from CDC light-traps by linear regression, which proved to be statistically significant for both species. Estimated numbers of female sandflies per house per night were found to be proportional to monthly precipitation (i.e. rainfall), with a lag time of seven months for Lu. ovallesi and of six months for Lu. gomezi. Predominance of Lu. ovallesi over Lu. gomezi (c. 1 : ) was observed throughout the year, with the number of infected females estimated as.43 f_.47 Lu. ovallesi and.85?.124 Lu. gomezi per CDC trap per house per night (ratio - 5:l). The mean rate of new CL cases per house per year and sandfly abundance were correlated by linear regression, showing a statistically significant relationship for Lu. ovallesi but not for Lu. gomezi. The negative intercept indicated that, on average, the CDC trap density exceeds 8 Lu. ovallesi femaleshouselyear before new CL cases occur at El ngenio. Key words. Leishmania braziliensis, Le. mexicana, Lutzomyia ovallesi, Lu. gomezi, Psychodidae, sandflies, cutaneous leishmaniasis, leishmaniasis vectors, population ecology, intradomiciliary transmission, Venezuela. ntroduction The widespread Neotropical sandflies Lurzomyia gornezi (Nitzulescu) and Lu. ovallesi (Ortiz) (Diptera: Psychodidae: Phlebotominae), occur sympatrically in Colombia (Porter & De Foliart, 1981). Panama (Chaniotis et al., 1971) and Venezuela (Feliciangeli, 1987). Bionomics of these species have been described by Williams (197), Chaniotis et al. (1971), (1972). Correspondence: Dr M. Dora Feliciangeli, Universidad de Carabobo, Apdo 4873, Maracay, Venezuela. mdora@telcel.net.ve Porter & De Foliart (1981), Travi etal. (1988), Alexander & Young (1992), Alexander etal. (1992) and Aiiez et al. (1994). The anthropophilic habits and natural infection of Lu. ovallesi with Leishmania promastigotes have been reported from Belize and Panama (Williams, 197), Colombia (Young el al., 1987), Guatemala (Rowton et al., 1991, 1992) and Venezuela (Feliciangeli etal., 1988, 1991, 1994; Bonfante Garrido et al., 1991). Rowton ef al. (1991, 1992) implicated Lu. ovallesjas vector of Le. braziliensis Vianna in Guatemala whereas, from isoenzyme profiles in Venezuela, Bonfante Garrido et al. (1991) interpreted the parasites as hybrid Leishmania braziliensis X guyanensis (Floch). Natural infections with promastigotes in Lu. gomezi have been found in Panama by Johnson.etal Blackwell Science Ltd 121

2 122 M. D. Feliciangeli and J. Rabinovich TSHANNON TRAP Fig. 1. Sketch Map of El ngenio village, showing houses (W) used in this study, and its location in Miranda State, Venezuela. Table 1. Survey details of four series of collections of sandflies at El ngenio, January 1991-March See Fig. 1 for locations of numbered houses used in S1, S2 and S3. For S4, CDC light traps were operated in about half (29/55 = 53%) of the houses on 12 nights per month. Survey No. Type of collection No. nights Collections Dates per month Times (hourshight) s1 2 collectors with Jan X2=6 illuminated Shannon trap Feb 1992 (2 collectors) c. 9 m from house No. 4 s2 CDC light-trap house Nos. 1-5 Nov Feb (5 houses) 12 X 5 = 6 s3 2 collectors with Nov-Feb X 2 = 24 illuminated Shannon trap (2 collectors) c. 9 m from house No. 4 s4 CDC light-trap June X 12 = houses March 1995 (29 houses)

3 Lutzomyia ovallesi and cutaneous leishmaniasis incidence in Venezuela 123 Table 2. Lutzomyia ovallesi female abundance at El ngenio, Miranda State, Venezuela: 4 surveysr methods, Numbers in square brackets [n] represent the number of nights per month of operation of the Shannon traps. Numbers in curved brackets (n) represent the size of the sample, i.e. no. houses X no. nights (to the left of the slash) and the number of nights of CDC trap operation (number to the right of the slash). Survey 1 (S1) Survey 3 (S3) 2 collectors with Survey 2 (S2) 2 collectors with Survey 4 (S4) illuminated Shannon trap CDC light trap illuminated Shannon trap CDC light-trap Month/Year c. 9 m from house No. 4 house Nos. 1-5 c. 9 from house No houses Jan [2] Feb [] Mar [] Apr [2] May [2] Jun [2] Jul [3] Aug [2] Sept [2] Oct [2] Nov [2] 25 [ 11 Dec [] Jan [2] Feb [2] 647 [] Nov 92 Dec 92 Jan 93 Feb 93 Mar [3] 41 [l] [4] [l] 15.2 [7] Nov 93 8 (15/3) [3] Dec (2/4) [4] Jan (1614) [4] Feb (2/5) 12.1 [5] Jun 94 5 (15/3) Jul (44/6) Aug (56/6) Sept (56/6) Oct (6/6) Nov (28/1) Dec (188/7) Jan (134/5) Feb (191/7) Mar (167/6) (1963), in Colombia by Young eral. (1987) and in Ecuador by Gomez & Hashiguchi (1987). Miranda State in north-central Venezuela is considered to be a mesoendemic area for cutaneous leishmaniasis (CL), with an average of 212 new cases per year giving a prevalence of 1.7 per 1OOOOO people in About 34% of the cases are clustered in El ngenio (Feliciangeli etal., 1994) where CL incidence during was 122 cases among 265 people inhabiting this village (O Daly et al., 1995). Lutzomyia ovallesi seems to be the main vector of CL in this area, with Lu. gomezi as a subsidiary vector. Using PCR and DNA-probes, Lu. ovallesi was found infected predominantly with Le. braziliensis (1.2%) and occasionally with Le. mexicana (.7%), whereas Lu. gomezi was only found infected with Le. braziliensis (.47%; Bamos et al., 1994; Feliciangeli et al., 1994). For several species of Lutzomyia, a correlation between precipitation and sandfly abundance has been recognized (Barretto, 1943; Pifano etal., 196; Scorza et al., 1968; Chaniotis etal., 1971; Christensen etal., 1972; Porter & De Foliart, 1981; Feliciangeli, 1987; Travi et al., 1988; de Queiroz et al., 1991; Alexander et al., 1992; Villaseca et al., 1993; Comer et al., 1994; Momson et al., 1995a,b). Villaseca et al. (1993) correlated the mean intradomiciliary abundance of Lu. peruensis (Shannon) with human cases of uta (Leishmania peruviana Velez) in Peru, pointing out the usefulness of this kind of analysis for distinguishing the differential vector roles of sympatric species of Lutzomyia and for finding the major sites of transmission to humans. They also stressed the importance of such information for vector control. n hying to establish more precisely the relationship between rainfall and abundance of Lu. ovallesi and Lu. gomezi indoors, for a better understanding of their relative vectorial importance, this paper analyses data for precipitation and sandflies collected at El ngenio by two sampling methods intermittently over four years, u

4 124 M. D. Feliciangeli and J. Rabinovich Table 3. Lutzomyia gomezi female abundance at El ngenio, Miranda State, Venezuela, See Table 2 for interpretation of details. Survey 1 (Sl) Survey 3 (S3) 2 collectors 2 collectors with with illuminated Survey 2 (S2) illuminated Shannon trap CDC light Shannon trap Survey 4 (S4) c. 9 m from trap house c. 9 from house CDC light-trap MonthNear house No. 4 Nos. 1-5 No houses Jan 91 Feb 91 Mar 91 Apr 91 May 91 Jun 91 Jul 91 Aug 91 Sept 91 Oct 91 Nov 91 Dec 91 Jan 92 Feb 92 Nov 92 Dec 92 Jan 93 Feb 93 Mar 93 Nov 93 Dec 93 Jan 94 Feb 94 Jun 94 Jul 94 Aug 94 Sept 94 Oct 94 Nov 94 Dec 94 Jan 95 Feb 95 Mar [2] 17.9 [l] 13.6 [l] 11.9 [2] 7.7 [2] 24.7 [2] 1.2 [3] 1.7 [2] 11.9 [2] 2.6 [2] 23.8 [2] 34.9 [ [2] [2].2 (15/3) 3.5 (2/4).4 (1614). (2/5) 23.5 [l] 27 r [3] 15 [ [4] 37. [l] 4.6 [7] 4. [3] 14.9 [4] 11.3 [4] 2.7 [5] (15/3).16 (44/6).2 (56/6).7 (56/6).18 (6/6).29 (2811).29 (188/7).42 (134/5).24 (191/7).13 (167/6) Materials and Methods Study area The study area is located at the El ngenio village (alt. 4 m; lat. 1"31'N, long. 66'34'W) in the Cordillera de la Costa foothills, 3.5 km from the Guatire city and 3 km east of Caracas City (Fig. 1). This region belongs to the hemontane Dry Forest (Ewe1 & Madriz, 1968) or Basic Tropophile Montane Forest (Huber & Alarch, 1988). During Guatire had an average annual precipitation of 853 mm and a mean temperature of 2 C. The climate is markedly seasonal, with five dry months (December-April) in which evapotranspiration exceeds precipitation. According to a census in 199 (OCE, 1991). the village consisted of 55 houses, ten of which were inhabited or under construction at the initiation of this study, with a population of 254 people (145 men and 19 women). Phlebofomine sandflies Between January 1991 and March 1995 we made four surveys (designated S1, S2, S3 and S4) of sandflies at El ngenio, using inconsistent sampling design and methods. For S1 and S3, a fluorescent illuminated Shannon trap (Shannon, 1939) was located c. 9 m from house numbers 4 (Fig. 1). For S2 and S4, a CDC light-trap was employed overnight in the sleeping quarters of each house (Table 1). The taxonomic identification of Lu. ovallesi and Lu. gomezi followed Feliciangeli (198). Details of the collection, handling and parasitological procedures are described in Feliciangeli ef al. (1994). -, Data analysis Cross-correlation analyses were applied to female Lu. ovallesi and Lu. gomezi abundance and monthly precipitation 1998 Blackwell Science Ltd, Medical and Veterinaly Entomology, 12,

5 Lutzomyia ovallesi and cutaneous leishmaniasis incidence in Venezuela 125 Average female Lu. ovallesilnightlcollector in Shannon traps Fig. 2. Lurzomyia ovallesi linear regression of Shannon trap collections outdoors against CDC light-trap collections indoors (standardized for 12 hour night period) j 4 a Average female Lu. gornezilnightlcollector in Shannon traps Fig. 3. LurzomYia gomezi linear regression of cokctions outdoors against CDC light-trap collections indoors (standardized for 12 hour night period). " Q :i:,. j /.. ii Fig. 4. Monthly rainfall and Lu. ovallesi and Lu. gomezi femaleshouselnight. time series. To obtain homogeneous data for sandfly abundance, results of the various surveys were converted to the number of female sandflies per house per night, i.e. standardized as if for CDC light-trap collections hours. For the S1 survey, as collections were undertaken for only three hours per night (18.3CL21.3 hours), allowances were made for the distinctive patterns of sandfly activity through the course of the night, as interpreted from survey S3 when only C 6.5% of all Lu. ovallesi and C 5.3% Lu. gomezi were collected in the first three hours of the night collections. Thus, the number of female sandflies captured per man-hour per night in S1 was divided by.276 and.547 to convert the 3-

6 126 M. D. Feliciangeli and J. Rabinovich Lag Corr First variable: Lu. ovallesi abundance Lagged variable: Precipitation Fig. 5. Cross-correlation between monthly rainfall and Lu. ovallesi femaleshousehight. hour estimates to 12-hour estimates for Lu. ovallesi and Lu. gomezi, respectively. Shannon trap collection data (Sl, S3) were converted to CDC trap equivalents by linear regression through the origin, based on the CDC all-night data (S2, S4). Values were averaged for months with multiple data points, i.e. November 91 and February 92 for surveys S1 and S3; November 93, December 93, January 94 and February 94 for surveys S2 and S4). As cross-correlation analysis requires continuous and equally spaced observations, data gaps for the period March-October 1992, April-October 1993 and March-May 1994 (corresponding with lowest sandfly abundance) were filled by inference from values obtained during survey S1 (the only survey with the necessary monthly information). Monthly rainfall data for the Guatire climatological station (3.5 km east of El ngenio) for years were provided by the Venezuelan Ministry of Environment and Renewable Natural Resources. Time series for precipitation and standardized abundance of Lu. ovallesi and Lu. gomezi females indoors were tested for significance of cross-correlation and lagged regression. Leishmania infections All sandflies collected by Shannon trap during the 14 month period from January 1991 to February 1992 (Feliciangeli, et al., 1994), and blood-fed sandflies collected indoors during June 1994-March 1995, were dissected and Leishmania infections were identified using PCR and DNA probes. Results Monthly sandfly collections from Shannon traps (surveys S 1, S3) and from CDC traps (surveys S2, S4) are summarized in Table 2 for Lu. ovallesi and Table 3 for Lu. gomezi, expressed as the average number of sandflies per night. Conversion of the Shannon collections (1 2 Wnightloutdoors) to equivalent CDC (12 Wnightlindoors) figures by linear regression, proved to be statistically significant for Lu. ovallesi (CDC =.1428?.197 Shannon; F1,15 = , P <.3; Fig. 2) and for Lu. gomezi (CDC =.29147?.4847 Shannon; F1,15 = , P <.24; Fig. 3). The overall time series of monthly rainfall and sandflied househight showed an apparent lag between these two variables (Fig. 4). Cross-correlation analysis proved to be statistically significant for Lu. ovallesi abundance following precipitation with lags of 5, 6 and especially 7 months (r =.4716; SE =.158; Fig. 5). For Lu. gomezi the lag wzs statistically significant for only 6 (r =.528, SE =.1491; Fig. 6) and 7 months after precipitation. Linear regression analyses of sandfly abundance on precipitation were significant for precipitation 5-7 months

7 Lutzomyia ovdlesi and cutaneous leishmaniasis incidence in Venezuela 127 Lag Corr First variable: Lu. gomezi abundance Lagged variable: Precipitation ~~.5 1. Fig. 6. Cross-correlation between monthly rainfall and Lu. gomezi femaledhousehight. Y=O.O35871'X 2.4 : 1 Y=O.O4428^X ' Precipitation (rnrnlrnonth) Fig. 7. Linear regression of Lu. ovallesi femaleshousehight and precipitation, lagged by seven months. a 1 Lu gornezf lags precipfiation by 6 months previously, with the highest correlation coefficient for Lu. ovallesi being a lag of seven months (r =.6947, F1,44 = 4.113, P <.1; Fig. 7), and a lag of six months for Lu. gomezi (r =.1423, F1,- = , P <.1; Fig. 8). Lutzomyia ovallesi outnumbered Lu. gomezi by 8-1% during most months; the lowest proportion of Lu. ovallesi was nearly 65% in December 1993 (Fig. 9). The average sandfly abundance per house per night (based on 51 months of data) converted to 12 h CDC trap equivalent was for Lu. ovallesi and.42?.48 Lu. gomezi. Dominance of Lu. ovallesi over Lu. gomezi was a ratio of c. 1 : 1 overall. Both species fluctuated in relative synchrony closely following the

8 M. D. Feliciangeli and J. Rahinovich Q 26 o c c 1.%.- C.- CL e Fig. 9. Monthly rainfall and percentage of Lu. ovallesi femaleshousehight with Lu. gornezi femaleshousehight at El ngenio, January March precipitation pattern. Cross-correlation between the percentage of Lu. ovallesi and precipitation was statistically significant, with a lag of six months (r =.4955, S.E. =.1491). During January 1991 to February 1992, Leishmania braziliensis infections were found in only Lu. gomezi: one in November 1991, the other in February n Lu. ovallesi the infection rate fluctuated with sandfly abundance, and also apparently followed precipitation with a lag of 5-6 months (data unsuitable for cross-correlation analysis with such a short time series). Monthly time series of sandfly abundance, infection rates and precipitation are shown in Fig. 1. The overall infection rate in Lu. ovallesi during the period January 91-February 92, was = 1.19%. n the period June 94- March 95, no infection was detected among 166 fed Lu. ovallesi and six fed Lu. gomezi collected indoors. When sandfly abundance is multiplied by infection rate, the mean numbers of positive sandfly femaleshousehight were estimated to be.43?.47 Lu. ovallesi and Lu. gomezi, resulting in a ratio of 5 : 1 in favour of Lu. ovallesi as the most important vector. The monthly relationship of sandfly abundance and CL incidence could not be analysed because monthly data of the new CL cases were not available. However, O Daly efal. (1995) reported annual incidence at El ngenio as 14, 15, 28 and 35 new cases for 1991, 1992, 1993 and 1994, respectively, from which we calculated the mean rate of new cases per house per year. We then carried out linear regressions with annual sandfly abundance (femaleshouse/year) as the independent variable and number of new caseshouselyear as the dependent variable. For Lu. gomezi the regression was not statistically significant. The regression for Lu. ovallesi was highly significant (Fig. ll), with a negative intercept of c. 82 indicating that, on average, the density of Lu. ovallesi females exceeds 8 per CDC traphouse/year before new CL cases occur at El ngenio. Discussion Lutzomyia ovallesi is considered to be a dry habitat species in Panama (Chaniotis et al., 1971; Christensen et al., 1972) and in Venezuela (Feliciangeli, 1987) where climatic conditions are comparable. n Colombia, Travi et al. (1988) observed that Lu gomezi was prevalent in the dry season, although no clear correlation was found with the monthly rainfall, n Guatemala, Rowton et al. (1991) reported population peaks of Lu. ovallesi in March and in July at the beginning of the rainy season. The power of time series analyses to assess temporal associations between sandfly abundance and metereological factors was first demonstrated by Momson etal. (1995a) for 1998 Blackwell Science Ltd, Medical and Vererinary Entomology, 12,

9 Lutzomyia ovallesi and cutaneous leishmaniasis incidence in Venezuela Fig. 1. Monthly rainfall, density of Lu. ovallesi females and Leishmania infection rates at El ngenio, January 1991-February z ll Q P r z Q u) P g.2 Y= 'X., Lu. ovallesi fernales/house/year in CDC traps Fig. 11. Linear regression of Lu. ovallesi females/house/year and the rate of new cases/house/year at El ngenio, Lu. longipalpis. Our application of this method to Lu. ovallesi and Lu. gomezi indicated a statistically significant positive association between rainfall and indoor abundance of sandfly females, with a time lag of about 6 months. Both species occur as adult populations throughout the year. The unimodal seasonal pattern of adult sandfly population density suggests that breeding site conditions of both species are most productive during the period immediately before and during the dry season. This interpretation agrees with observations made in Panama by Rutledge & Mosser (1972) who pointed out that protection of sandfly breeding sites from flooding, caused by rainfall or soil run-off, was the main environmental factor affecting sandfly population abundance and species composition in tree-base habitats. Lutzomyia gomezi populations were higher in the dry January-March period of the year (Rutledge & Ellenwood, 1975). Quiescence and dispause of sandfly immature stages under adverse conditions have been invoked by several authors to explain the virtual absence of adult sandflies, or their very low densities, during some months of the year (Ward & Killick-Kendrick, 1974; Morrison et al., 1995a). This explanation could account for the paucity of Lu. ovallesi and Lu. gomezi adults during the wet season. Evaluation of man-vector contact and identification of the sites where leishmaniasis occurs may help towards personal protection as a component of control strategies. Cutaneous leishmaniasis in the New World is traditionally considered as a sylvatic disease, with people becoming infected when they visit forested areas (WHO, 199). However, epidemiological observations have begun to indicate more domiciliation of vector species (Scorza et al., 1985; Comes ei al., 1986), leading to increasing CL incidence. Contrasts between two well-studied foci in north-central Venezuela, viz San Esteban - a focus of low endemicity (Feliciangeli, 1987) and El ngenio - a hyperendemic focus, are attributed to the way that in El ngenio the same two anthropophilic vector species, Lu. ovallesi and 1998 Blackwell Science Ltd, Medical and Veterinary Enfomdogv, 12,

10 13 M. D. Feliciangeli and J. Rabinovich Lu. gomezi, both exhibit more endophilic behaviour. Data on blood-feeding sources of Lu. ovallesi caught by CDC traps inside houses in El ngenio showed that 45% of the freshly engorged females had taken human blood (Gomez et al., 1995). Our entomological findings, coupled with observations of CL cases in very young children who stay mainly at home (O Daly et al., 1995), led us to consider that CL transmission in El ngenio may be intradomiciliary in most cases. The positive correlation between Lu. ovallesi density inside houses and CL incidence during constitutes additional evidence pointing to domestic transmission. The lack of a similar correlation for Lu. gomezi, coupled with its much lower density and infection rate, indicates the limited epidemiological importance of this species as a local endophilic vector of CL, although it may be a subsidiary exophilic vector. We showed that outdoor and indoor sandfly population densities are strongly correlated, and we recognize the probabilities of leishmaniasis transmission occurring in both situations. Whereas the relative proportions of sandflies biting and resting outdoorslindoors are unknown, we do have data on the densities trapped indoors and CL incidence among villagers. Making the extreme assumption that all new CL cases were transmitted indoors, we estimated that c. 82 Lu. ovallesi females per CDC traplhouselyear are needed to produce a new human case of leishmaniasis. f even a small fraction of the new CL cases were acquired outdoors, the minimum number of Lu. ovallesi needed indoors for transmission to occur would have to be even larger. We are therefore encouraged to investigate the impact of domestic vector control in this hyperendemic focus of CL in north-central Venezuela. Acknowledgements This work was funded by the Commission of the European Community (Contract T*S3-CT93-247) and the University of Carabobo (Project FCS-91-44) and it was sponsored by PAHO (Caracas, Venezuela). The technical assistance of Mr A. Bravo and Mr Freddy Arias is also gratefully acknowledged. References Alexander, B., Ferro, C., Young, D.G., Morales, A. & Tesh, R.B. (1992) Ecology of plebotomine sand flies (Diptera: Psychodidae) in a focus of Leishmania (Viannia) brasiliensis in northeastern Colombia. Memorias do nstituto Oswaldo Cruz, 87, Alexander, B. & Young, D.G. (1992) Dispersal of phlebotomine sandflies (Diptera: Psychodidae in a Colombian focus of Leishmania (Viannia) braziliensis. Memorias do nstituto Oswaldo Cruz, 87, ABez, N., Nieves, E., Cazorla, D., Oviedo, M., Lug de Yarbuh, A. & Valera, M. (1994) Epidemiology of cutaneous leishmaniasis in MCrida, Venezuela Altitudinal distribution, age, structure, matural infection and feeding behaviour of sandflies and their relation to the risk of transmission. Annals of Tropical Medicine and Parasitology, 88, Barretto, M.P. (1943) Observacoes sobre a biologia, em condigoes naturais, dos flebdtomos do Estado de Sao Paulo (Diptera, Psychodidae). Sao Paulo, Faculdade de Medicina da Universidade de Sao Paulo, PhD Thesis. Banios, N.A., Rodriguez, N., Feliciangeli, M.D., Ulrich, M., Telles, S., Pinardi M.E.J. & Convit, J. (1994) Coexistence of two species of Leishmania in the digestive tract of the vector Lutzomyia ovallesi. American Journal of Tropical Medicine and Hygiene, 51, Bonfante-Ganido, R., Spinetti, H., Cupillo, E., Momen, H. & Grimaldi, G., Jr (1991). Lutzomyia ovallesi (Diptera, Psychodidae) as a vector of cutaneous leishmaniasis in Venezuela. 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