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Survey of natural infection by Leishmania in sand fly species collected in southeastern Brazil
Transactions of the Royal Society of Tropical Medicine and Hygiene 104 (2010) 461–466
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Survey of natural infection by Leishmania in sand fly species collected in southeastern Brazil Leonardo S. Rocha a , Aloísio Falqueto b , Claudiney B. dos Santos c , Adelson L. Ferreira b , Grazielle C. da Grac¸a a , Gabriel Grimaldi Jr. a , Elisa Cupolillo a,∗ a b c
Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brasil Unidade de Medicina Tropical, Universidade Federal do Espírito Santo, UFES, Espírito Santo, Brasil Núcleo de Entomologia, Secretaria Estadual de Saúde e Meio Ambiente do Estado do Espírito Santo, SESA, Espírito Santo, Brasil
a r t i c l e
i n f o
Article history: Received 24 August 2009 Received in revised form 10 February 2010 Accepted 10 February 2010 Available online 26 March 2010 Keywords: Sand flies natural infection Lutzomyia fischeri Lutzomyia ferreirana Leishmaniasis Leishmania braziliensis
a b s t r a c t In this study, we sought to identify sand fly vectors of the Leishmania species that circulate in distinct eco-epidemiological disease-endemic rural areas within the Espírito Santo State in southeastern Brazil. PCR amplification of a conserved region of the minicircle kDNA was used to estimate infection rates in field-captured, peridomestic female sand flies. Only 13 of the 1689 female sand fly specimens (0.77%) actually contained Leishmania DNA. Leishmania braziliensis infections were found in Lutzomyia intermedia and Lu. whitmani, and, for the first time, in Lu. fischeri and Lu. ferreirana. Interestingly, the high rate of genetic polymorphism of the L. braziliensis parasites in one of the disease-endemic areas that were studied may reflect specific transmission cycles involving different sand fly vectors. © 2010 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction Cases of leishmaniasis have been reported in all Brazilian states, with several sand fly species participating in the transmission cycle of the responsible parasites. The cutaneous disease is associated with many Leishmania species,1–3 while the visceral manifestation is only associated with a single Leishmania species, L. infantum (syn. L. chagasi). American cutaneous leishmaniasis (ACL) is endemic and widely distributed throughout Espírito Santo (ES), Brazil. Several ACL cases in both humans and dogs that are associated with L. (Viannia) braziliensis have been reported in this area.4 Disease-endemic foci occur in small, scattered set-
∗ Corresponding author at: Av. Brasil 4365, Pavilhão Leônidas Deane s509, Manguinhos, Rio de Janeiro, RJ, Brazil, 21040-360. Tel.: +55 21 3865 8177; fax: +55 21 2209 4110. E-mail address: ecupoli@ioc.fiocruz.br (E. Cupolillo).
tlements that are found near heavily deforested mountains, in which few remnants of the original vegetation remain. This disease can also be found in old established communities that are located near important cities on the Atlantic Coast. In ES, five human-biting sand fly species are usually collected within ACL-affected areas: Lutzomyia intermedia, Lu. migonei, Lu. whitmani, Lu. fischeri and Lu monticola (A. Falqueto, unpublished data). Although Lu. intermedia is thought to be the main vector of L. braziliensis in ES, other sand fly species, such as Lu. whitmani and Lu. migonei, might act as secondary vectors of the parasite in rural localities.5 In addition, altitude has been shown to influence the level of genetic structuring of Lu. intermedia populations from different regions in the state, particularly in Afonso Cláudio and Viana, where the transmission of L. braziliensis reflects distinct eco-epidemiologic features.6 In contrast, Lutzomyia longipalpis is the only known species that acts as a vector for L. infantum in the ES state. In this state visceral leishmaniasis (VL) occurs in munic-
0035-9203/$ – see front matter © 2010 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2010.02.005
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ipalities in the northwestern region, where the presence of Lu. longipalpis has already been reported. Since the first documented case of VL in 1968, there have been several additional cases associated with L. infantum in humans and dogs.7 In the municipality of Pancas the last report of human VL was in 2000, but there is still a high density of Lu. longipalpis and a high incidence of canine visceral leishmaniasis (CVL).8 Based on the high sensitivity demonstrated for PCR amplification of the conserved region of the minicircle kDNA9–11 , the present study employed this methodology to investigate the rates of L. braziliensis and L. infantum infection in different sand fly species that had been collected from rural areas in ES with documented cases of CL and/or VL. Six different areas were selected, based on the incidence of human disease and some biogeographic characteristics. Furthermore, we postulate that the geographic structuring that has been observed for some L. braziliensis genotypes12,13 may actually reflect specific parasite-sand fly vector interactions. Consistent with this, there was a higher degree of genetic diversity within L. braziliensis isolates from Afonso Cláudio, ES than in isolates from both patients and dogs in Viana.6 This difference is likely to be due to an adaptation of different clones (genotypes) to the host vectors that are specific to each of the two disease-endemic areas. The gene flow estimates for the Lu. intermedia specimens collected at Afonso Cláudio do indicate that these specimens represent a more genetically homogenous population than those from Viana.14 Based upon those data, other sand fly species, such as Lu. whitmani and Lu. migonei, might act as vectors that are involved in the transmission of L. braziliensis within the distinct ecologic region of Afonso Cláudio.
Table 1 Coefficient of incidence of ACL among the rural populations of the municipalities in the Espírito Santo State where sand flies were collected
2. Materials and methods
The insect DNA was extracted individually using the Wizard® SV Genomic DNA Purification System Kit (Promega, Madison, USA) and resuspended in 20 l of hydration solution. The PCR reactions were performed on pooled insect DNA. The insect pools were made with 2 l of individually extracted DNA from 10 different specimens, for a total of 20 l, and are designated as the insect pools. The remaining 18 l of DNA from each female was saved for further analyses, which would be performed after being identified as belonging to a positive pool. Male sand flies were used as negative controls for the extraction. The quality of the extracted DNA was assessed by performing PCR of the cytochrome-b gene16 on the insect pools and the negative control pools. This cytochrome-b amplification was discarded after analysis. For the detection of infected sand flies, extracted DNA was examined by the amplification of a 120 bpconserved region of Leishmania mkDNA, using the primers Forward: 5 -GGGGAGGGGCGTTCTGCGAA-3 and Reverse: 5 -GGCCCACTAT ATTACACCAACCCC-3 .17 Positive controls for the PCR reactions included DNA extracted from promastigotes of the following Leishmania strains: IOC/L566 (MHOM/BR/1975/ M2903) L. braziliensis, IOC/L579 (MHOM/BR1974/PP75) L. infantum, and IOC/L575 (IFLA/BR/1967/PH8) L. amazonensis. These reference strains were derived from the Fiocruz Leishmania collection (Colec¸ão de Leishmania do Instituto Oswaldo Cruz, CLIOC).
2.1. Studied areas The insect specimens were collected between 2004 and 2008 from six distinct municipalities. The incidence of disease in 78 municipalities within ES was calculated in order to select those six areas for this study. Since ACL is considered a typically rural disease in the state, disease incidence was calculated considering only the rural population. Other factors that were considered in the calculations include (A) the average rural population determined for each year between 2004 and 2008 and (B) the average number of ACL cases reported for each year between 2004 and 2008. The formula was ACL incidence = B/A x 100 000 habitants. Within the six selected areas, the incidence of ACL ranged from 1–10 (A); 11–80 (B); and 81–110 (C). These areas were considered herein to be areas of low (A), medium (B) and high (C) disease incidence (Table 1). Some geographical characteristics were also considered for the selection of these areas. Two regions, Afonso Cláudio and Itaguac¸ú, were in the center-west of the state and had a temperate climate, alternating between dry and rainy seasons. Another two, Pancas and Água Doce do Norte, were characterised as hot and dry areas. The remaining two, Iconha and Viana, were in the center-east and characterised as hot and rainy areas (Figure 1).
a b
Municipality
ACL incidencea (2004–2008)
Level of incidenceb
Afonso Cláudio Água D. Norte Iconha Itaguac¸u Pancas Viana
57.40 8.86 2.99 88.49 69.20 108.94
Medium Low Low High Medium High
x 100 000 habitants. Low: 1–10, Medium: 11–80; High: 81–110.
Two of these areas were also selected for the collection of Lu. longipalpis. The municipality of Pancas was selected in light of the presence of the sand fly species along with reported cases of CVL. Afonso Claudio was selected as the other Lu. longipalpis municipality, as it has maintained a presence of Lu. longipalpis despite an absence of VL. 2.2. Capture and identification of the phlebotomines Collections of sand flies were periodically conducted in each geographic area. The sand flies were captured using a manual aspirator in home environments. Female sand flies were identified after dissection in sterile saline, and the last three abdominal segments and head were then transferred to separate 1.5 mL tubes and stored at −70 ◦ C until molecular analysis. Sand flies were identified using the criteria proposed by Young and Duncan.15 2.3. DNA extraction, PCR amplification and electrophoresis
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Figure 1. Map of Espírito Santo, Brazil, depicting the geographic locations where the sand flies were collected. 1. Água Doce do Norte, 2. Pancas, 3. Itaguac¸ú, 4. Afonso Cláudio, 5. Viana and 6. Iconha. BA = Bahia; MG = Minas Gerais; ES = Espírito Santo and RJ = Rio de Janeiro.
An aliquot of 10 l of each PCR reaction was separated electrophoretically on a 6% polyacrylamide gel. The gels were stained using the DNA Silver Staining kit (GE Healthcare - Amersham Biosciences, Piscataway, NJ, USA). A 50 bp DNA Step Ladder provided molecular weight size standards. For the identification of individual infected sand flies within each positive pool, the PCR amplification was performed as described above. 2.4. Sequence analysis Each specimen that was positive by PCR was run on a 2.5% agarose gel, and the 120 bp fragments were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, USA). The purified fragments were then sequenced using the BigDyeTM Terminator v3.0 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and diluted 1:4 in a final volume of 10 l with 20–60 ng of the purified PCR products and 3.2 pmol of the forward and reverse primers. The products were sequenced in duplicate for each primer (two forward and two reverse). 3. Results A total of 1689 female sand flies were collected from the domestic environments of six distinct municipalities from the Espírito Santo state during this study. The distribution of the peridomestic sand fly species has been summarised by study site (Table 2). The most commonly collected sand fly species in all the studied areas was Lu. intermedia. Lu. lenti was very abundant in Água Doce do Norte but not in the other areas. Lu. fischeri, like Lu. intermedia, was found in all municipalities and represented 23% of the specimens collected in Viana and 13.5% of those collected in Afonso Claudio. Although Lu. whitmani was the second most abundant sand fly species collected in Afonso Claudio (23%), it was not found in any other region, except for Itaguac¸ú, which is an area with similar characteristics to those of
Afonso Claudio: a temperate climate, alternating between dry and rainy seasons. The highest numbers of sand fly species were collected in Afonso Claudio, followed by Pancas and Itaguac¸ú with the lowest numbers collected in Água Doce do Norte and Iconha (Table 2). Of the 1689 female sand fly specimens, 13 (0.77%) were found to have Leishmania DNA. Comparative sequence analysis of the PCR fragments (120 bp) with the sequences deposited in the GenBank confirmed the identification of L. braziliensis and its association with at least four sand fly species, namely Lu. intermedia (9/647), Lu. whitmani (1/161), Lu. fischeri (2/166) and Lu. ferreirana (1/9). In some of the wild-caught Lu. fischeri specimens, flagellates other than Leishmania were also detected using molecular analysis (de Souza Rocha et al., unpublished data). Lu. longipalpis was only collected in Afonso Claudio and Pancas, as expected. It was not very abundant in the Afonso Claudio municipality (Table 2). None of the Lu. longipalpis specimens contained any Leishmania DNA. The rate of natural Leishmania infection varied among the municipalities: 0.6% for Afonso Claudio, 3.07% for Itaguac¸ú, 0.4% for Pancas, and 2.6% for Viana. No natural Leishmania infection were detected in sand flies collected in Àgua Doce do Norte and Iconha. Only four species showed natural infection by Leishmania: Lu. intermedia (9/647), Lu. fischeri (2/166), Lu. whitimani (1/161) and Lu. ferreirana (1/9) (Table 2). Dissection of the insects in order to determine the presence of flagellates was not performed. However, when the insects were being prepared for taxonomic identification, flagellates were visualized in the posterior region of the midgut of some specimens that later were identified as Lu. ferreirana. 4. Discussion Approximately 230 different sand fly species are found in Brazil, but only about 8% of these have been implicated
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Table 2 Number and species of Lutzomyia (♀) captured and naturally infected by Leishmania braziliensis by collection site Sand fly species
Lu. intermedia Lu. lenti Lu. fischeri Lu. whitmani Lu. longipalpis Lu. migonei Lu. monticola Lu. quinquefer Lu. schereiberi Lu. pessoai Lu. edwardsi Lu. salesi Lu. ferreirana Lu. firmatoi Lu. chotti Lu. alencari Lu. hirsutus Lu. microps Total
Collection sites (naturally infected) Afonso Cláudio
Agua D. Norte
Iconha
Itaguac¸ú
Pancas
Viana
Total
236 (1) 31 89 (1) 152 (1) 9 43 31 25 6 12 1 9 5 (1) 8 1 658 (4)
20 390 5 415
62 11 3 76
30 (2) 4 9 1 8 9 1 3 65 (2)
149 10 4 (1) 69 1 4 1 1 1 4 1 1 246 (1)
150 (6) 53 23 2 1 229 (6)
647 (9) 431 166 (2) 161 (1) 78 69 33 29 16 13 11 10 9 (1) 8 4 2 1 1 1689(13)
- Species not collected.
as proven or suspected vectors of human L. braziliensis CL in different ecologic and geographic regions.1,18 In the present study, 0.77% (13/1689) of the wild-caught female sand flies were found to be naturally infected with Leishmania. Our findings corroborated the known low leishmanial infection rates found in field-captured peridomestic sand flies in many regions where the disease is endemic.11,19–21 The rates of sand fly infection varied in each area and were in accordance with the transmission of the disease. The highest rates were observed in Itaguac¸ú and Viana, which are areas with the highest ACL incidence. Neither Iconha nor Àgua Doce do Norte, each with a very low ACL incidence, had any infected sand flies. All the identified sand fly infections involved L. braziliensis, with no vector specimen being infected by L. infantum. While this result was expected for Afonso Cláudio since no recent VL cases have been reported in that area, the lack of naturally infected Lu. longipalpis in Pancas was unexpected, since canine LV has active transmission.8 It is likely that the lack of observed Lu. longipalpis infection in this area is a result of the coincidence between the period of collection and the removal of Leishmania-infected dogs from this area as part of the Brazilian Leishmaniasis Control Program. Incrimination of a particular sand fly species as a disease vector is difficult, since the presence of Leishmania in the gut of a wild-caught phlebotomine does not necessarily mean that the insect is able to transmit the parasite to humans.22 Thus, whether some or all of the incriminated sand fly species herein, including Lu. intermedia, Lu. whitmani, Lu. fischeri and Lu. ferreirana, are important vectors or whether some are merely incidental hosts remains unclear. Nevertheless, a close ecologic relationship involved in L. braziliensis infection exists between the parasites, the sand flies and the domestic reservoirs (dogs and possibly equines) that are found in each disease-endemic area.4 Of note, changes were observed in the sand fly densities and/or geographic distribution among vector species
circulating in the study sites (Table 2), which validates our previous observations.5 It is already known that the phlebotomine sand fly Lu. intermedia is dominant in peridomiciliary captures from old, established rural communities.23 This species has been incriminated as the main vector of L. braziliensis in southeast Brazil,24–26 although Lu. whitmani and Lu. migonei also frequently bite humans in the peridomicile. In the present study, Lu. intermedia was the most frequent species found in the studied areas, except in Água Doce do Norte. Although this species was collected in all areas, only three of these areas had confirmed infections by L. braziliensis. To the best of our knowledge, this study is the first to provide evidence of a natural L. braziliensis-Lu. ferreirana combination. One noteworthy fact is that the infected sand fly was captured in a house with a record of a dog with proven L. braziliensis CL. Natural infection by L. braziliensis was also observed in Lu. fischeri, which was collected in two distinct areas. This observation supports the conclusions of Coutinho & Barreto,27 who reported Lu. fischeri as a possible vector of L. braziliensis CL in São Paulo, Brazil. Additional criteria are required to implicate a particular sand fly species as a vector of human leishmaniasis, including the following: contact between the sand fly and humans and/or suspected reservoir hosts, similar distribution of the fly and the parasite, and completion of the parasite’s life cycle in the fly and evidence of bite transmission by the insect.22 Interestingly, Lu. fischeri was also infected with monoxenous lower trypanosomatid parasites (de Souza Rocha et al., unpublished data). Accordingly, flagellates other than Leishmania have also been associated with a number of different sand fly species.28–30 These observations reinforce the importance of applying DNA-based methods that target specific markers for the identification of suspected sand fly vector(s) of a given Leishmania species throughout its geographic range. Pathogens that produce many different genetic variants are more prone to infecting multiple hosts.31 Hence, the
L.S. Rocha et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 104 (2010) 461–466
existence of several sand fly vector species that participate in the transmission cycles in Afonso Claudio would explain the high level of genetic diversity found among L. braziliensis isolates from that disease-endemic area, which is in contrast with the homogeneity of parasites and sand fly species that are found in other regions, like Viana.4,6 Whether or not the genetic variants, representing the parasite genotypes, of this etiologic agent6 interact specifically with several sand fly vector species that circulate within that region5 remains to be determined. If true, these interactions might result from longstanding evolutionary relationships between sand flies and Leishmania, in which the parasites and vectors are connected by unique behavioural32–35 or molecular phenotypes.36 In turn, these relationships could have epidemiological importance,37 with implications for the control or intervention of leishmaniasis. Authors’ contributions: EC, AF and GGJr conceived the study; LSR, EC, AF and GGJr were responsible for the study design; LSR, CBS and ALF carried out the field work; LSR and GCG conducted the PCR assays and sequencing analysis; LSR, EC and GGJr participated in the analysis and interpretation of the results. All the authors prepared the final version of the manuscript, revising it for intellectual content and have seen and approved the final manuscript. EC is the guarantor of the paper Acknowledgements: The Sequencing (PDTIS-Fiocruz).
Genomic
Platform-DNA
Funding: This research was funded by PRONEX/CNPq (National Council for Scientific and Technological Development of the Ministry of Science and Technology), the Carlos Chagas Filho Research Foundation of the State of Rio de Janeiro (FAPERJ) and the European Union (grant INCOCT2005-015407). EC and GG are CNPq fellow researchers; LSR is a Ph.D. student of the Program in Molecular and Cell Biology/FioCruz and is sponsored by CNPq. Conflicts of interest: None declared. Ethical approval: Not required. References 1. Grimaldi Jr G, Tesh RB. Leishmaniasis of the New World: current concepts and implications for future research. Clin Microbiol Rev 1993;6:230–50. 2. Marzochi MC, Marzochi KB. Tegumentary and visceral leishmaniases in Brazil: emerging anthropozoonosis and possibilities for their control. Cad Saude Publica 1994;10:359–75. 3. Silveira FT, Ishikawa EA, De Souza AA, Lainson R. An outbreak of cutaneous leishmaniasis among soldiers in Belem, Para State, Brazil, caused by Leishmania (Viannia) lindenbergi n. sp. A new leishmanial parasite of man in the Amazon region. Parasite 2002;9:43–50. 4. Falqueto A, Sessa PA, Ferreira AL, Vieira VP, Santos CB, Varejão JB, et al. Epidemiological and clinical features of Leishmania (Viannia) braziliensis American cutaneous and mucocutaneous leishmaniasis in the State of Espírito Santo, Brazil. Mem Inst Oswaldo Cruz 2003;98:1003–10. 5. Ferreira AL, Sessa PA, Varejão JB, Falqueto A. Distribution of sand flies (Diptera: Psychodidae) at different altitudes in an endemic region of American cutaneous leishmaniasis in the State of Espírito Santo, Brazil. Mem Inst Oswaldo Cruz 2001;96:1061–7.
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Transactions of the Royal Society of Tropical Medicine and Hygiene journal homepage: http://www.elsevier.com/locate/trstmh
Survey of natural infection by Leishmania in sand fly species collected in southeastern Brazil Leonardo S. Rocha a , Aloísio Falqueto b , Claudiney B. dos Santos c , Adelson L. Ferreira b , Grazielle C. da Grac¸a a , Gabriel Grimaldi Jr. a , Elisa Cupolillo a,∗ a b c
Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brasil Unidade de Medicina Tropical, Universidade Federal do Espírito Santo, UFES, Espírito Santo, Brasil Núcleo de Entomologia, Secretaria Estadual de Saúde e Meio Ambiente do Estado do Espírito Santo, SESA, Espírito Santo, Brasil
a r t i c l e
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Article history: Received 24 August 2009 Received in revised form 10 February 2010 Accepted 10 February 2010 Available online 26 March 2010 Keywords: Sand flies natural infection Lutzomyia fischeri Lutzomyia ferreirana Leishmaniasis Leishmania braziliensis
a b s t r a c t In this study, we sought to identify sand fly vectors of the Leishmania species that circulate in distinct eco-epidemiological disease-endemic rural areas within the Espírito Santo State in southeastern Brazil. PCR amplification of a conserved region of the minicircle kDNA was used to estimate infection rates in field-captured, peridomestic female sand flies. Only 13 of the 1689 female sand fly specimens (0.77%) actually contained Leishmania DNA. Leishmania braziliensis infections were found in Lutzomyia intermedia and Lu. whitmani, and, for the first time, in Lu. fischeri and Lu. ferreirana. Interestingly, the high rate of genetic polymorphism of the L. braziliensis parasites in one of the disease-endemic areas that were studied may reflect specific transmission cycles involving different sand fly vectors. © 2010 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction Cases of leishmaniasis have been reported in all Brazilian states, with several sand fly species participating in the transmission cycle of the responsible parasites. The cutaneous disease is associated with many Leishmania species,1–3 while the visceral manifestation is only associated with a single Leishmania species, L. infantum (syn. L. chagasi). American cutaneous leishmaniasis (ACL) is endemic and widely distributed throughout Espírito Santo (ES), Brazil. Several ACL cases in both humans and dogs that are associated with L. (Viannia) braziliensis have been reported in this area.4 Disease-endemic foci occur in small, scattered set-
∗ Corresponding author at: Av. Brasil 4365, Pavilhão Leônidas Deane s509, Manguinhos, Rio de Janeiro, RJ, Brazil, 21040-360. Tel.: +55 21 3865 8177; fax: +55 21 2209 4110. E-mail address: ecupoli@ioc.fiocruz.br (E. Cupolillo).
tlements that are found near heavily deforested mountains, in which few remnants of the original vegetation remain. This disease can also be found in old established communities that are located near important cities on the Atlantic Coast. In ES, five human-biting sand fly species are usually collected within ACL-affected areas: Lutzomyia intermedia, Lu. migonei, Lu. whitmani, Lu. fischeri and Lu monticola (A. Falqueto, unpublished data). Although Lu. intermedia is thought to be the main vector of L. braziliensis in ES, other sand fly species, such as Lu. whitmani and Lu. migonei, might act as secondary vectors of the parasite in rural localities.5 In addition, altitude has been shown to influence the level of genetic structuring of Lu. intermedia populations from different regions in the state, particularly in Afonso Cláudio and Viana, where the transmission of L. braziliensis reflects distinct eco-epidemiologic features.6 In contrast, Lutzomyia longipalpis is the only known species that acts as a vector for L. infantum in the ES state. In this state visceral leishmaniasis (VL) occurs in munic-
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ipalities in the northwestern region, where the presence of Lu. longipalpis has already been reported. Since the first documented case of VL in 1968, there have been several additional cases associated with L. infantum in humans and dogs.7 In the municipality of Pancas the last report of human VL was in 2000, but there is still a high density of Lu. longipalpis and a high incidence of canine visceral leishmaniasis (CVL).8 Based on the high sensitivity demonstrated for PCR amplification of the conserved region of the minicircle kDNA9–11 , the present study employed this methodology to investigate the rates of L. braziliensis and L. infantum infection in different sand fly species that had been collected from rural areas in ES with documented cases of CL and/or VL. Six different areas were selected, based on the incidence of human disease and some biogeographic characteristics. Furthermore, we postulate that the geographic structuring that has been observed for some L. braziliensis genotypes12,13 may actually reflect specific parasite-sand fly vector interactions. Consistent with this, there was a higher degree of genetic diversity within L. braziliensis isolates from Afonso Cláudio, ES than in isolates from both patients and dogs in Viana.6 This difference is likely to be due to an adaptation of different clones (genotypes) to the host vectors that are specific to each of the two disease-endemic areas. The gene flow estimates for the Lu. intermedia specimens collected at Afonso Cláudio do indicate that these specimens represent a more genetically homogenous population than those from Viana.14 Based upon those data, other sand fly species, such as Lu. whitmani and Lu. migonei, might act as vectors that are involved in the transmission of L. braziliensis within the distinct ecologic region of Afonso Cláudio.
Table 1 Coefficient of incidence of ACL among the rural populations of the municipalities in the Espírito Santo State where sand flies were collected
2. Materials and methods
The insect DNA was extracted individually using the Wizard® SV Genomic DNA Purification System Kit (Promega, Madison, USA) and resuspended in 20 l of hydration solution. The PCR reactions were performed on pooled insect DNA. The insect pools were made with 2 l of individually extracted DNA from 10 different specimens, for a total of 20 l, and are designated as the insect pools. The remaining 18 l of DNA from each female was saved for further analyses, which would be performed after being identified as belonging to a positive pool. Male sand flies were used as negative controls for the extraction. The quality of the extracted DNA was assessed by performing PCR of the cytochrome-b gene16 on the insect pools and the negative control pools. This cytochrome-b amplification was discarded after analysis. For the detection of infected sand flies, extracted DNA was examined by the amplification of a 120 bpconserved region of Leishmania mkDNA, using the primers Forward: 5 -GGGGAGGGGCGTTCTGCGAA-3 and Reverse: 5 -GGCCCACTAT ATTACACCAACCCC-3 .17 Positive controls for the PCR reactions included DNA extracted from promastigotes of the following Leishmania strains: IOC/L566 (MHOM/BR/1975/ M2903) L. braziliensis, IOC/L579 (MHOM/BR1974/PP75) L. infantum, and IOC/L575 (IFLA/BR/1967/PH8) L. amazonensis. These reference strains were derived from the Fiocruz Leishmania collection (Colec¸ão de Leishmania do Instituto Oswaldo Cruz, CLIOC).
2.1. Studied areas The insect specimens were collected between 2004 and 2008 from six distinct municipalities. The incidence of disease in 78 municipalities within ES was calculated in order to select those six areas for this study. Since ACL is considered a typically rural disease in the state, disease incidence was calculated considering only the rural population. Other factors that were considered in the calculations include (A) the average rural population determined for each year between 2004 and 2008 and (B) the average number of ACL cases reported for each year between 2004 and 2008. The formula was ACL incidence = B/A x 100 000 habitants. Within the six selected areas, the incidence of ACL ranged from 1–10 (A); 11–80 (B); and 81–110 (C). These areas were considered herein to be areas of low (A), medium (B) and high (C) disease incidence (Table 1). Some geographical characteristics were also considered for the selection of these areas. Two regions, Afonso Cláudio and Itaguac¸ú, were in the center-west of the state and had a temperate climate, alternating between dry and rainy seasons. Another two, Pancas and Água Doce do Norte, were characterised as hot and dry areas. The remaining two, Iconha and Viana, were in the center-east and characterised as hot and rainy areas (Figure 1).
a b
Municipality
ACL incidencea (2004–2008)
Level of incidenceb
Afonso Cláudio Água D. Norte Iconha Itaguac¸u Pancas Viana
57.40 8.86 2.99 88.49 69.20 108.94
Medium Low Low High Medium High
x 100 000 habitants. Low: 1–10, Medium: 11–80; High: 81–110.
Two of these areas were also selected for the collection of Lu. longipalpis. The municipality of Pancas was selected in light of the presence of the sand fly species along with reported cases of CVL. Afonso Claudio was selected as the other Lu. longipalpis municipality, as it has maintained a presence of Lu. longipalpis despite an absence of VL. 2.2. Capture and identification of the phlebotomines Collections of sand flies were periodically conducted in each geographic area. The sand flies were captured using a manual aspirator in home environments. Female sand flies were identified after dissection in sterile saline, and the last three abdominal segments and head were then transferred to separate 1.5 mL tubes and stored at −70 ◦ C until molecular analysis. Sand flies were identified using the criteria proposed by Young and Duncan.15 2.3. DNA extraction, PCR amplification and electrophoresis
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Figure 1. Map of Espírito Santo, Brazil, depicting the geographic locations where the sand flies were collected. 1. Água Doce do Norte, 2. Pancas, 3. Itaguac¸ú, 4. Afonso Cláudio, 5. Viana and 6. Iconha. BA = Bahia; MG = Minas Gerais; ES = Espírito Santo and RJ = Rio de Janeiro.
An aliquot of 10 l of each PCR reaction was separated electrophoretically on a 6% polyacrylamide gel. The gels were stained using the DNA Silver Staining kit (GE Healthcare - Amersham Biosciences, Piscataway, NJ, USA). A 50 bp DNA Step Ladder provided molecular weight size standards. For the identification of individual infected sand flies within each positive pool, the PCR amplification was performed as described above. 2.4. Sequence analysis Each specimen that was positive by PCR was run on a 2.5% agarose gel, and the 120 bp fragments were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, USA). The purified fragments were then sequenced using the BigDyeTM Terminator v3.0 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and diluted 1:4 in a final volume of 10 l with 20–60 ng of the purified PCR products and 3.2 pmol of the forward and reverse primers. The products were sequenced in duplicate for each primer (two forward and two reverse). 3. Results A total of 1689 female sand flies were collected from the domestic environments of six distinct municipalities from the Espírito Santo state during this study. The distribution of the peridomestic sand fly species has been summarised by study site (Table 2). The most commonly collected sand fly species in all the studied areas was Lu. intermedia. Lu. lenti was very abundant in Água Doce do Norte but not in the other areas. Lu. fischeri, like Lu. intermedia, was found in all municipalities and represented 23% of the specimens collected in Viana and 13.5% of those collected in Afonso Claudio. Although Lu. whitmani was the second most abundant sand fly species collected in Afonso Claudio (23%), it was not found in any other region, except for Itaguac¸ú, which is an area with similar characteristics to those of
Afonso Claudio: a temperate climate, alternating between dry and rainy seasons. The highest numbers of sand fly species were collected in Afonso Claudio, followed by Pancas and Itaguac¸ú with the lowest numbers collected in Água Doce do Norte and Iconha (Table 2). Of the 1689 female sand fly specimens, 13 (0.77%) were found to have Leishmania DNA. Comparative sequence analysis of the PCR fragments (120 bp) with the sequences deposited in the GenBank confirmed the identification of L. braziliensis and its association with at least four sand fly species, namely Lu. intermedia (9/647), Lu. whitmani (1/161), Lu. fischeri (2/166) and Lu. ferreirana (1/9). In some of the wild-caught Lu. fischeri specimens, flagellates other than Leishmania were also detected using molecular analysis (de Souza Rocha et al., unpublished data). Lu. longipalpis was only collected in Afonso Claudio and Pancas, as expected. It was not very abundant in the Afonso Claudio municipality (Table 2). None of the Lu. longipalpis specimens contained any Leishmania DNA. The rate of natural Leishmania infection varied among the municipalities: 0.6% for Afonso Claudio, 3.07% for Itaguac¸ú, 0.4% for Pancas, and 2.6% for Viana. No natural Leishmania infection were detected in sand flies collected in Àgua Doce do Norte and Iconha. Only four species showed natural infection by Leishmania: Lu. intermedia (9/647), Lu. fischeri (2/166), Lu. whitimani (1/161) and Lu. ferreirana (1/9) (Table 2). Dissection of the insects in order to determine the presence of flagellates was not performed. However, when the insects were being prepared for taxonomic identification, flagellates were visualized in the posterior region of the midgut of some specimens that later were identified as Lu. ferreirana. 4. Discussion Approximately 230 different sand fly species are found in Brazil, but only about 8% of these have been implicated
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Table 2 Number and species of Lutzomyia (♀) captured and naturally infected by Leishmania braziliensis by collection site Sand fly species
Lu. intermedia Lu. lenti Lu. fischeri Lu. whitmani Lu. longipalpis Lu. migonei Lu. monticola Lu. quinquefer Lu. schereiberi Lu. pessoai Lu. edwardsi Lu. salesi Lu. ferreirana Lu. firmatoi Lu. chotti Lu. alencari Lu. hirsutus Lu. microps Total
Collection sites (naturally infected) Afonso Cláudio
Agua D. Norte
Iconha
Itaguac¸ú
Pancas
Viana
Total
236 (1) 31 89 (1) 152 (1) 9 43 31 25 6 12 1 9 5 (1) 8 1 658 (4)
20 390 5 415
62 11 3 76
30 (2) 4 9 1 8 9 1 3 65 (2)
149 10 4 (1) 69 1 4 1 1 1 4 1 1 246 (1)
150 (6) 53 23 2 1 229 (6)
647 (9) 431 166 (2) 161 (1) 78 69 33 29 16 13 11 10 9 (1) 8 4 2 1 1 1689(13)
- Species not collected.
as proven or suspected vectors of human L. braziliensis CL in different ecologic and geographic regions.1,18 In the present study, 0.77% (13/1689) of the wild-caught female sand flies were found to be naturally infected with Leishmania. Our findings corroborated the known low leishmanial infection rates found in field-captured peridomestic sand flies in many regions where the disease is endemic.11,19–21 The rates of sand fly infection varied in each area and were in accordance with the transmission of the disease. The highest rates were observed in Itaguac¸ú and Viana, which are areas with the highest ACL incidence. Neither Iconha nor Àgua Doce do Norte, each with a very low ACL incidence, had any infected sand flies. All the identified sand fly infections involved L. braziliensis, with no vector specimen being infected by L. infantum. While this result was expected for Afonso Cláudio since no recent VL cases have been reported in that area, the lack of naturally infected Lu. longipalpis in Pancas was unexpected, since canine LV has active transmission.8 It is likely that the lack of observed Lu. longipalpis infection in this area is a result of the coincidence between the period of collection and the removal of Leishmania-infected dogs from this area as part of the Brazilian Leishmaniasis Control Program. Incrimination of a particular sand fly species as a disease vector is difficult, since the presence of Leishmania in the gut of a wild-caught phlebotomine does not necessarily mean that the insect is able to transmit the parasite to humans.22 Thus, whether some or all of the incriminated sand fly species herein, including Lu. intermedia, Lu. whitmani, Lu. fischeri and Lu. ferreirana, are important vectors or whether some are merely incidental hosts remains unclear. Nevertheless, a close ecologic relationship involved in L. braziliensis infection exists between the parasites, the sand flies and the domestic reservoirs (dogs and possibly equines) that are found in each disease-endemic area.4 Of note, changes were observed in the sand fly densities and/or geographic distribution among vector species
circulating in the study sites (Table 2), which validates our previous observations.5 It is already known that the phlebotomine sand fly Lu. intermedia is dominant in peridomiciliary captures from old, established rural communities.23 This species has been incriminated as the main vector of L. braziliensis in southeast Brazil,24–26 although Lu. whitmani and Lu. migonei also frequently bite humans in the peridomicile. In the present study, Lu. intermedia was the most frequent species found in the studied areas, except in Água Doce do Norte. Although this species was collected in all areas, only three of these areas had confirmed infections by L. braziliensis. To the best of our knowledge, this study is the first to provide evidence of a natural L. braziliensis-Lu. ferreirana combination. One noteworthy fact is that the infected sand fly was captured in a house with a record of a dog with proven L. braziliensis CL. Natural infection by L. braziliensis was also observed in Lu. fischeri, which was collected in two distinct areas. This observation supports the conclusions of Coutinho & Barreto,27 who reported Lu. fischeri as a possible vector of L. braziliensis CL in São Paulo, Brazil. Additional criteria are required to implicate a particular sand fly species as a vector of human leishmaniasis, including the following: contact between the sand fly and humans and/or suspected reservoir hosts, similar distribution of the fly and the parasite, and completion of the parasite’s life cycle in the fly and evidence of bite transmission by the insect.22 Interestingly, Lu. fischeri was also infected with monoxenous lower trypanosomatid parasites (de Souza Rocha et al., unpublished data). Accordingly, flagellates other than Leishmania have also been associated with a number of different sand fly species.28–30 These observations reinforce the importance of applying DNA-based methods that target specific markers for the identification of suspected sand fly vector(s) of a given Leishmania species throughout its geographic range. Pathogens that produce many different genetic variants are more prone to infecting multiple hosts.31 Hence, the
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existence of several sand fly vector species that participate in the transmission cycles in Afonso Claudio would explain the high level of genetic diversity found among L. braziliensis isolates from that disease-endemic area, which is in contrast with the homogeneity of parasites and sand fly species that are found in other regions, like Viana.4,6 Whether or not the genetic variants, representing the parasite genotypes, of this etiologic agent6 interact specifically with several sand fly vector species that circulate within that region5 remains to be determined. If true, these interactions might result from longstanding evolutionary relationships between sand flies and Leishmania, in which the parasites and vectors are connected by unique behavioural32–35 or molecular phenotypes.36 In turn, these relationships could have epidemiological importance,37 with implications for the control or intervention of leishmaniasis. Authors’ contributions: EC, AF and GGJr conceived the study; LSR, EC, AF and GGJr were responsible for the study design; LSR, CBS and ALF carried out the field work; LSR and GCG conducted the PCR assays and sequencing analysis; LSR, EC and GGJr participated in the analysis and interpretation of the results. All the authors prepared the final version of the manuscript, revising it for intellectual content and have seen and approved the final manuscript. EC is the guarantor of the paper Acknowledgements: The Sequencing (PDTIS-Fiocruz).
Genomic
Platform-DNA
Funding: This research was funded by PRONEX/CNPq (National Council for Scientific and Technological Development of the Ministry of Science and Technology), the Carlos Chagas Filho Research Foundation of the State of Rio de Janeiro (FAPERJ) and the European Union (grant INCOCT2005-015407). EC and GG are CNPq fellow researchers; LSR is a Ph.D. student of the Program in Molecular and Cell Biology/FioCruz and is sponsored by CNPq. Conflicts of interest: None declared. Ethical approval: Not required. References 1. Grimaldi Jr G, Tesh RB. Leishmaniasis of the New World: current concepts and implications for future research. Clin Microbiol Rev 1993;6:230–50. 2. Marzochi MC, Marzochi KB. Tegumentary and visceral leishmaniases in Brazil: emerging anthropozoonosis and possibilities for their control. Cad Saude Publica 1994;10:359–75. 3. Silveira FT, Ishikawa EA, De Souza AA, Lainson R. An outbreak of cutaneous leishmaniasis among soldiers in Belem, Para State, Brazil, caused by Leishmania (Viannia) lindenbergi n. sp. A new leishmanial parasite of man in the Amazon region. Parasite 2002;9:43–50. 4. Falqueto A, Sessa PA, Ferreira AL, Vieira VP, Santos CB, Varejão JB, et al. Epidemiological and clinical features of Leishmania (Viannia) braziliensis American cutaneous and mucocutaneous leishmaniasis in the State of Espírito Santo, Brazil. Mem Inst Oswaldo Cruz 2003;98:1003–10. 5. Ferreira AL, Sessa PA, Varejão JB, Falqueto A. Distribution of sand flies (Diptera: Psychodidae) at different altitudes in an endemic region of American cutaneous leishmaniasis in the State of Espírito Santo, Brazil. Mem Inst Oswaldo Cruz 2001;96:1061–7.
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