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Mem. Inst. Oswaldo Cruz vol.103 no.4 Rio de Janeiro June 2008
Hector De LimaI, +; Noris RodríguezI; Miguel A BarriosI; Ángela ÁvilaI; Israel CañizalesII; Saúl GutiérrezII
IInstituto de Biomedicina, Universidad Central de Venezuela, Apartado 4043, 1010A Caracas, Venezuela
IIGeoclean C.A., Caracas, Venezuela
This report describes the isolation of a Leishmania chagasi strain from a bat (Carollia perspicillata), and its identification using biological methods and molecular characterization. The parasites were isolated in an artificial culture medium from a blood sample extracted from a bat heart. The isolate was then inoculated into the footpads of Balb/c mice, which subsequently developed a typical nodular leishmanial lesion; the parasites were confirmed as Leishmania by smear and histopathology. Molecular characterization of the parasites was performed by polymerase chain reaction with species-specific primers, kDNA restriction pattern following Hae III endonuclease digestion and dot blot hybridization using a kDNA probe. This report demonstrates that bats can be hosts for L. chagasi species and suggests the need for studies to determine whether they may be involved in foci of visceral leishmaniasis.
Key words: Leishmania chagasi - bats - molecular biology - reservoir - Venezuela
Leishmania present a complex life cycle that involves vectors, reservoirs and susceptible hosts that vary according to the ecological conditions where they develop. A great variety of mammals, including more than 100 species, both domestic and wild, have been suggested as potential reservoirs for Leishmania (WHO, 1990). However, it has not been possible to demonstrate the participation of Chiroptera in the Leishmania life cycle, even though they are a relatively large group of mammals present in practically all the ecosystems, including urban and peri-urban environments. There are limited studies investigating the role of bats in the Leishmania life cycle. In the Old World, indirect evidence suggests bats as potential Leishmania reservoirs. Mutinga (1975) demonstrated Leishman-Donovan bodies in 3/104 bats in Kenya, and Morsy et al. (1987) detected anti-Leishmania antibodies in seven bats in Egypt. The frequent presence of phlebotomine sandflies in caves and crevices inhabited by bats suggests that bats may provide an alternative blood support in such niche environments (Killick-Kendrick et al. 1977, Lampo et al. 2000). In Venezuela, Lampo et al. (2000) used bats undergoing feeding trials to demonstrate four species of bats as a blood source for Lutzomyia longipalpis, the main vector of the Leishmania species that causes American visceral leishmaniasis (VL). Rotureau et al. (2006), in a study performed in 216 bats representing 29 species from cutaneous leishmaniasis (CL) foci in French Guiana, found no evidence of Leishmania after a PCR analysis with genus-specific primers and culture of animal samples.
In this report, we isolated Leishmania chagasi from a Carollia perspicillata bat and then identified it using biological characteristics and molecular techniques. The study was performed in and around the SINCOR Biological station facility within the Orinoco Oil Belt. This region is located in the Monagas municipality in the South of the state of Anzoategui, Venezuela (8º18'09"-8º21'48"N and 64º56'51"- 65º01'35"W), 130-164 m a.s.l., located predominantly in the tropical dry forest zone. Annual temperatures range 22-29ºC and the annual precipitation is approximately 1,000 mm.
Bats were captured during one week in August 2006 using two methods, depending on the time of day. Manual direct capture was used during noon hours prior to location of diurnal roosts, and mist nets were used for capture during the night. Taxonomical identification was performed according to Emmons' field guide (1997). The skin of the animals was examined externally for the presence of suspicious lesions. Blood samples (1 ml) were taken from the heart of each animal under ether anesthesia, and the samples were used for fresh microscopic examination, Giemsa stained smears and culture in blood agar base medium (Difco) containing 10% defibrinated rabbit blood and 200 U penicillin. From positive cultures, 106 parasites were inoculated subcutaneously into the ventral pad of the left hind foot of Balb/c mice of the same sex, weight and age (n = 5). Molecular characterization of the parasites consisted of (i) PCR using AJS-3 and DBY species-specific primers that are specific for the Leishmania donovani complex (Lambson et al. 1999, 2000); (ii) dot-blot hybridization using total kDNA probes designed based on the international reference strain L. chagasi; and (iii) kDNA restriction patterns following Hae III endonuclease digestion, as previously described by Rodriguez et al. (2005).
A total of 11 bats were captured, all belonging to one family (Phyllostomidae) but distributed in three genera: Carollia (8), Phylloderma (2) and Rhynophylla (1). None of the bats showed lesions upon external physical examination. All fresh blood microscopic examinations and Giemsa stained blood smears were negative for hemoparasites. A blood culture was positive for Leishmania promastigotes in one animal identified as "M9", a female Carollia perspicillata (MCAR/VE/06/M9). Mice inoculated with promastigotes from the M9 isolate developed a nodular lesion in the inoculated footpad. The lesions were progressive, reaching 5 mm (average size) after one month and the parasites were successfully maintained in mice. The histopathological study revealed a macrophage-rich granuloma laden with amastigotes (Fig. 1).
Gel electrophoresis of the PCR amplicon from the M9 isolate showed the expected diagnostic band of approximately 800 bp (Fig. 2A, line 4), which was also observed in the PCR products from samples of the international reference strains L. infantum and L. chagasi (Fig. 2A, lines 2, 3), although the M9 product also contained additional bands. No product was seen by PCR using primers for Leishmania and the Viannia subgenus (data not shown). Dot blot hybridization of the PCR product with total kDNA from L. chagasi showed a strong hybridization signal with M9 (Fig. 2B, sample 4). Additionally, kDNA analysis with the Hae III restriction enzyme showed strong homology between the M9 restriction pattern and L. chagasi (Fig. 3, lines 4, 3 respectively), which shared products of approximately 850 bp, bands between 650 and 500 bp and one small band of 300 bp.
In this work we report the casual finding of a bat infected with L. chagasi parasites, which suggests a possible role of bats in the leishmaniasis epidemiological cycle. Until now, indirect inconclusive evidence has associated bats with the Leishmania life cycle, although the parasites have never been directly identified in bats (Mutinga 1975, Morsy et al. 1987). This is likely due to outdated techniques and the paucity of studies investigating Leishmania in bats. In contrast, Chiroptera have been found infected with a variety of species of trypanosomes, including Trypanosoma cruzi, the causative agent of Chagas disease (Barnabe et al. 2003). The Leishmania infected bat was captured in an unpopulated area of Monagas in the state of Anzoategui. This may be significant considering that, of the 423 cases of VL reported in Venezuela in the last 10 years (1998-2007), 76 (17.96%) were diagnosed in this state, with no cases being reported in Monagas. Additionally, according to the Venezuelan Ministry of Health, 1146 cases of CL were reported in Anzoategui during the same period, with sporadic cases in Monagas.
This report documents a specific case where a bat harbored a L. chagasi species, possibly due to the coexistence of Lu. longipalpis and bats in the same resting places (Killick-Kendrick et al. 1977, Lampo et al. 2000). Further studies of VL foci are needed to clarify the role of bats as possible reservoirs in the epidemiological chain of this disease.
To Raimundo Rodríguez and Rodrigo Rueda for his technical assistance, Ms. Maria Eugenia Gallinoto and Howard Takiff for the English correction of the manuscript.
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Received: 29 March 2008
Accepted: 20 June 2008
Financial support: Geoclean and Sincrudos de Oriente (SINCOR).