Studying and monitoring the ecology of Leishmania parasites is essential to understanding and controlling the epidemiology of the diseases they cause. In French Guiana, cutaneous leishmaniases (CLs) are endemic over the entire territory and the annual incidence was estimated to be approximately 2 cases per 1,000 inhabitants between 1979 and 2000.1,2 Since 2002, the number of cases has substantially increased, mainly in Brazilian immigrants working in gold mining placers situated in the inland dense forests. In the Department of Parasitology at Cayenne General Hospital, 348 new cases of CL were recorded in 2004 (Rotureau B and others, unpublished data). At least five Leishmania species are known to be sympatrically transmitted in the sylvatic ecotopes of the entire French Guianan territory3: Leishmania (Viannia) guyanensis (in more than 90% of the cases), L. (Leishmania) amazonensis, L. (V.) braziliensis, L. (V.) naiffi and L. (V.) lainsoni.1,4
Since the investigations of Dedet and others,5 there has been no study on the Leishmania reservoir hosts’ ecology in the Guyanas region.3 In addition, there is an absence of studies of Leishmania parasites in South American bats. Bats constitute approximately 48% (range = 43–55%) of all mammalian species recorded in Amazonian lowland rainforests,6 and are undoubtedly the most common mammals, in terms of population’s size, in neotropical forests. In French Guiana, bats are found in high numbers in all forested and non-forested landscapes, as well as in human-made clearings and agricultural and urbanized habitats.7 Bats, which are known to harbor several trypanosomatid parasites, are thus likely to be in contact with a large panel of potential vectors and reservoir hosts. Nevertheless, only a few studies have investigated their potential involvement in Leishmania life cycles.8–11 Thus, their possible role as reservoir host for Leishmania was investigated in three distinct forested ecotopes of French Guiana where numerous human CL cases have been reported. In 2003 and 2004, mammals were sampled with different trapping methods in both dry and rainy seasons to obtain the most representative panel of animals. We emphasized trapping common Chiroptera species living in anthropized ecotopes. In parallel, we collected non-flying mammals to monitor their actual involvement in the ecology of Leishmania parasites.
Chiroptera were collected in lightweight 50-denier mist nets set at ground level and maintained for a few hours in individual tissue bags until examination. Rodents and marsupials were caught alive in large live traps (14 × 14 × 41 cm; Tomahawk Live Trap Co., Tomahawk, WI) and small live traps (8 × 9 × 23 cm; Sherman Traps, Tallahassee, FL) set at ground level, as well as in live wire-mesh traps (10 × 11 × 33 cm; Besançon Technologie Services, Besançon, France) set in trees and lianas 1.5–2.5 meters above ground. Two or three traps were grouped at stations and distributed every 20 meters along two 500-meter lines. They were baited with pieces of various local fruits (mangos, papaws, and bananas) or peanut butter–coated hazelnuts. A 100-meter line of pitfalls (11 buckets) was also used. A list of vouchered specimens for morphologic examination is available upon request. Field collecting and sampling permits were provided by the Direction de l’Environnement de la Guyane Française (Cayenne, French Guiana) to one of the authors (FC).
Collections were made at three locations in the northern part of French Guiana where human CL cases have been reported in high numbers by the neighboring health centers. A total of 153 mammals were caught in a primary forest growing on white sands in Angoulème (53°40′W, 5°20′N) in September–October 2003 and May 2004, 79 in a 15–30-year-old secondary forest near the Petit-Saut hydroelectric dam (53°05′W, 5°05′N) in September 2003, and 36 in a slightly degraded secondary flooded forest in Kaw (52°05′W, 4°30′N) in April 2004. Ten additional samples were collected in peri-urban sites in 2003 and 2004 (around Cayenne, Macouria and Sinnamary) by trapping by the local population or by wildlife veterinary rescue centers. In Angoulème, the number of captures tripled between the dry season (September–October 2003) and the following wet season (May 2004), with an average of 4 and 11 captures/day, respectively. The same tendency was observed in the species diversity. This difference could be explained by seasonal variations in French Guiana, especially hygrometric variations, and consequent fluctuations in alimentary resource availability.
Animals were examined directly for skin lesions. Biopsy specimens of skin (141), liver (137), wing membrane (132), and nasal mucosa (1) were sterilely obtained and cultured in vitro at 24°C for one month in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 20% fetal calf serum, 2 mM L-glutamine, 25 mM HEPES, pH 7.4, 1% non-essential and essential amino-acids in minimal essential medium, and 50 IU/mL penicillin/streptomycin. After one month, DNA was extracted from the culture media containing crushed tissue biopsies by a simplified protein salting-out method12 and screened by a Leishmania-specific polymerase chain reaction (PCR) routinely used in our laboratory for diagnosis of human cutaneous punch biopsy specimens. Sequences located between the end of the small subunit and the 5.8S region of the ribosomal RNA genes were amplified with the primers SSU-12103-D (5′-GGGAATATCCTCAGCACGT-3′) and 5.8S-13333-R (5′-CGACACTGAGAATATGGCATG-3′) as described elsewhere.13
A total of 411 biopsy specimens from 278 mammals (47 species in 36 genera, Tables 1 and 2) were collected over a two-year period. We focused our sampling on bats with 216 captures for 29 species. No flagellate was found throughout our sampling by either culture or specific PCR (0 of 278 specimens). Although reporting negative results—as in this study—is not very appealing, we nevertheless present these data which i) complete those by others (see below) and ii) will contribute to a better assessment of prevalence of Leishmania infection in French Guianan mammals.
Culture in RPMI 1640 medium is routinely used in our laboratory for the diagnosis of human CL and has been useful in many field studies. The simplified protein salting-out DNA extraction procedure12 and the Leishmania-specific PCR amplification of the internal transcribed spacer 1 region of the ribosomal RNA genes13 have also been useful in detecting parasites. Leishmania transmission cycles likely occur in the three studied areas because 15, 74, and 95 new human CL cases were recorded in the Iracoubo Health Center near Angoulème, the Saint-Elie Health Center near Petit-Saut, and the Régina and Cacao Health Centers near the forest of Kaw, respectively, in 2003 and 2004.
In French Guiana, two distinct cycles of leishmaniases were described in the late 1980s.14 Both occur in the rain forest, but at different altitudes. There is a L. (V.) guyanensis cycle in the canopy with the arboreal sand fly Lutozomyia umbratilis as the vector and at least one mammal of the canopy, the two-toed sloth Choloepus didactylus, as a reservoir. There is also a L. (L.) amazonensis cycle at ground level with Lu. flaviscutellata as the vector and Proechimys cuvieri as the main reservoir host.3,14 Human infections in French Guiana were shown to result from incursions into the forest throughout the territory, mostly for professional activities.14 However, many points remain unclear regarding the ecology of some clinically important parasites such as L. (V.) braziliensis, which could be responsible for mucocutaneous leishmaniases, especially concerning its wild Amazonian reservoir hosts. In addition, new reservoir hosts are frequently found infected with parasites such as L. (L.) amazonensis.
The absence of parasites in the non-volant mammal group can be mainly explained by the small numbers studied (62 specimens). There were 18 different species represented in 62 captures and 16 of those species were represented by less than 10 individuals. In a previous study in French Guiana over a seven-year period, Dedet and others5 found parasites in 2 (1.6%) of 122 Didelphis marsupialis and 5 (5.6%) of 89 Proechimys (5.6%). Philander opossum, Metachirus nudicaudatus, and Oryzomys megacephalus were also reported to harbor Leishmania in Amazonia (studies reviewed by Rotureau3). Thus, additional field studies on a larger scale are required to verify these preliminary results and to monitor the actual ecology of Leishmania reservoir hosts.
Concerning chiropters, we have collected 216 specimens belonging to 4 families, with emphasis on sampling common and abundant species. However, the largest sample size of any species caught was 54 (Carollia perspicillata), and 24 of the 29 species were represented by less than 10 individuals. According to Post and Millest15 and assuming that each of the bat species is equal in its susceptibility and maintenance of the infection, the statistical minimum prevalence detectable in the 216 bats examined is 1.4% (95% confidence). Thus, the maximum likely frequency of infected specimens in our 216 negative samples is 1.4%. On the basis of the Carollia perspicillata population sampled (54 specimens), the minimum prevalence detectable is 5.4%. Similarly, the respective maximum likely frequencies of infected specimens are 8.4% for Molossus molossus (34) and 11.3% for Artibeus planirostris (25). Since bats constitute 48% of all mammalian species recorded in the lowland rain forest and are the most common mammal in terms of population size,6 a prevalence of less than 1.4% would still provide a significant maintenance host population. Thus, although we obtained negative results, they should be carefully interpreted because of the limited set of populations studied. Moreover, one can assume that a host sample taken in one year is not necessarily as representative of the parasite dynamics as a host sample taken in another year in the same area, regardless of the uniformity of the host trapping technique.
Carollia perspicillata, M. molossus, and A. planirostris (synonymous with A. jamaicensis16) are known to colonize a wide range of habitats. In French Guiana, they have been recorded in primary and secondary forests, savannas, costal swamps, and mangroves, as well as in peri-urban and urban environments.7 By living in anthropized ecotopes, these species are in contact with sand flies such as Lu. flaviscutellata, the main vector of L. (L.) amazonensis. Moreover, trypanosomatid parasites transmission from bats to sand flies was observed, because Lu. vespertilionis was shown to be preferentially attracted to Emballonuridae and able to transmit chiropteran trypanosomes.17,18 In Venezuela, Lu. longipalpis, the primary vector of L. (L.) infantum, was shown to feed on at least four different species of bats,11 and bat wings were routinely used to feed sand flies in a Brazilian laboratory.19
In neotropical forests, bats are undoubtedly the most common mammals.20,21 Are they not involved in the Leishmania parasitic cycles in the Guyanas as our results suggest? Evaluating the role that wildlife play in the maintenance of an infectious agent is quite difficult. It is possible that Chiroptera are not suitable as hosts for Leishmania; several mammalian orders, such as Insectivora and Lagomorpha, also appear to be unsuitable hosts. However, this issue may be resolved either by additional sampling of a larger panel of species from different ecotopes, and/or by direct experimental infection of bats with Leishmania parasites.
Non-volant mammals studied
Flying mammals (Order: Chiroptera) collected
Address correspondence to Brice Rotureau, Laboratoire Hospitalo-Unversitaire de Parasitologie et Mycologie Médicale, Equipe EA 3593, Unité de Formation et de Recherche en Médecine de l’Université des Antilles et de la Guyane, Campus Saint-Denis, BP 718, 97336 Cayenne, French Guiana. E-mail:
Authors’ addresses: Brice Rotureau and Bernard Carme, Laboratoire Hospitalo-Universitaire de Parasitologie et Mycologie Médicale, Equipe EA 3593, Unité de Formation et de Recherche en Médecine de l’Université des Antilles et de la Guyane, Campus Saint-Denis, BP 718, 97336 Cayenne, French Guiana, Telephone: 33-594-28-72-60, Fax: 33-594-28-72-63, E-mail:
Acknowledgment: We thank Jacques Cuisin for his friendly and efficient help during the trapping sessions.
Financial support: This work was supported by the University of the French West Indies and the French Guiana (Cayenne, French Guiana), the French Contrat Plan Etat-Région No. 2365, and the Institut National de la Santé et de la Recherche Médicale (Paris, France). Field-collecting activities were supported by a grant from the Ministère de l’Outre-Mer (Paris, France) to Francois Catzeflis.
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