American Visceral Leishmaniasis Dissociated from Lutzomyia Longipalpis (Diptera, Psychodidae) in the State of Espírito Santo, Brazil

ARTIGO ARTICLE 365

American visceral leishmaniasis dissociated from Lutzomyia longipalpis (Diptera, Psychodidae) in the State of Espírito Santo, Brazil

Leishmaniose visceral americana dissociada da presença de Lutzomyia longipalpis (Diptera, Psychodidae) no Estado do Espírito Santo, Brasil

Israel de Souza Pinto 1 Claudiney Biral dos Santos 1 Gabriel Grimaldi Jr. 2 Adelson Luiz Ferreira 1 Aloísio Falqueto 1

Abstract Introduction

1 Centro de Ciências da The occurrence of American visceral leishmania- American visceral leishmaniasis is a zoonotic dis- Saúde, Universidade Federal do Espírito Santo, Vitória, sis in the State of Espírito Santo, Brazil has always ease caused by the protozoan Leishmania infan- Brasil. been associated with the presence of the Lutzomy- tum chagasi (Cunha & Chagas, 1937) (Kinetoplas- 2 Instituto Oswaldo Cruz, ia longipalpis vector. The geographic distribution tida, Trypanosomatidae). Its transmission occurs Fundação Oswaldo Cruz, Rio de Janeiro, Brasil. of this vector in this state is related to the pres- as a result of the biting of female phlebotomine ence of specific geoclimatic characteristics, such sand flies infected with the protozoan 1. Correspondence as a dry climate, low elevations (< 450m), steep Classic epidemiological studies indicate a I. S. Pinto Unidade de Medicina slopes and rocky outcrops. The occurrence of hu- strong association between L. i. chagasi and the Tropical, Centro de Ciências man autochthonous cases of American visceral sand fly Lutzomyia longipalpis (Lutz & Neiva, da Saúde, Universidade leishmaniasis in municipalities without these 1912) (Diptera, Psychodidae) which is pointed out Federal do Espírito Santo. Av. Marechal Campos 1468, geoclimatic characteristics justifies the present as the main vector of the disease. This correlation Vitória, ES study and our main goal was to test the associa- is based on the following factors: the occurrence 29043-900, Brasil. tion between the occurrence of American visceral of human and canine American visceral leish- [email protected] leishmaniasis and the presence of the Lu. longi- maniasis cases associated with the geographic palpis. Sand flies were captured monthly from and ecological distribution of this sand fly spe- July 2006 to August 2007 using Shannon and cies; finding of Lu. longipalpis naturally infected CDC traps in two municipalities with records of with L. i. chagasi; experimental infection of Lu. autochthonous American visceral leishmaniasis longipalpis with L. i. chagasi; attraction of Lu. lon- and one with no record. We captured 13,112 sand gipalpis to L. i. chagasi reservoirs (dogs and foxes, flies, but no Lu. longipalpis was found. The ab- mainly) and experimental transmission of L. i. sence of Lu. longipalpis and the possible role of chagasi by infected Lu. longipalpis 1,2,3,4,5,6,7,8,9. another American visceral leishmaniasis vector Some authors support the participation in these localities were discussed. of other sand fly species in the transmission of American visceral leishmaniasis 10, based on their Psychodidae; Vector Ecology; Leishmaniasis coincidental distribution with the disease. There are also those authors that defend the participation of other arthropods in American visceral leishmaniasis transmission, based on findings of natural infection in fleas 11 and ticks 12. However, none of these authors confirmed the capacity of

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these arthropods to transmit the disease through pattern found in endemic areas of the Rio Doce biting. valley, in the Central-North region of the state 17. In Brazil, the first American visceral leish- Moreover, the Southern region of Espírito Santo maniasis cases were registered by Penna 13 who is isolated from the endemic areas of American found Leishmanias in 41 of 47,000 post-mortem visceral leishmaniasis in the Rio Doce valley by viscerotomy exams. This author registered dis- geographical and climatic barriers that limit ease cases in the states of Alagoas, Bahia, Ceará, vector dispersion. Yet, the municipalities in the Pará, Pernambuco, Piauí, Rio de Janeiro, Rio northern part of Rio de Janeiro state that border Grande do Norte and Sergipe. At present, around the municipalities in the South of Espírito Santo 3,000 new cases of American visceral leishmania- state did not register the occurrence of American sis are registered annually in Brazil, the majority visceral leishmaniasis cases nor the presence of associated with rural areas. However, urbaniza- Lu. longipalpis (unpublished data). tion processes of both the disease and the Lu. Considering that Lu. longipalpis was never longipalpis vector in cities such as Belo Hori- found in collections carried out in the Southern zonte (Minas Gerais) and Campo Grande (Mato portion of Espírito Santo state, the occurrence Grosso do Sul) 14,15 have been occurring in the of American visceral leishmaniasis cases in this last years. region justified the systematic studies of the sand In Espírito Santo state, American visceral fly fauna for vector identification. In this sense, leishmaniasis was first registered by Martins et the objective of this study was to test the asso- al. 16, in the municipalities of Colatina and Baixo ciation between the occurrence of American vis- Guandu. At present, it is distributed in 10 mu- ceral leishmaniasis and the presence of Lu. lon- nicipalities, all located in the Center-North of the gipalpis in the new localities that have registered state. In all endemic areas, disease transmission the disease. was always associated with the presence of Lu. longipalpis. Using the geoprocessing tool within the Geo- Material and methods graphical Information System, Feitoza et al. 17 studied the geographic and climatic variables The study was performed in the Central-South of endemic areas in Espírito Santo. The authors region of Espírito Santo state in the localities of concluded that potential areas for the occur- Belém district (20°47’29” S; 40°00’09” W) and rence of American visceral leishmaniasis and Santo Antônio district (20°50’40” S; 40°56’21” W) Lu. longipalpis present characteristics such as that belong, respectively, to the municipalities of sloping relief, elevation up to 450 meters above Vargem Alta and Rio Novo do Sul, where autoch- sea level, dry climate and rocky outcrops. Those thonous human American visceral leishmaniasis characteristics always appear in the areas with cases had occurred. For the sand fly fauna com- registers of autochthonous American visceral parison, Joebinha district (20°41’56” S; 40°47’41” leishmaniasis human cases, which also coincide W) in the municipality of Alfredo Chaves was also with the presence of Lu. longipalpis. It is impor- included in the study as it had registered autoch- tant to note that, until recently, the disease used thonous American tegumentary leishmaniasis to be registered only in the municipalities local- cases. ized in the Rio Doce valley, in the Central-North The three locations present similar geo-cli- region of Espírito Santo state. American visceral matic characteristics, as follows: humid climate leishmaniasis had never been registered in mu- influenced by the air currents coming from the nicipalities localized in the South portion of the Atlantic Ocean; annual pluviometer indices state. higher than 1,200mm; and sloping relief, with In the last five years, however, three autoch- rare rocky outcrops 17. The region contains small thonous disease cases were registered in the mu- forest patches that are the remains of the Atlantic nicipalities of Rio Novo do Sul and Vargem Alta, Rain Forest which was devastated to give room to in the southern region of Espírito Santo state. coffee crops, and latter, in part, to banana crops Using the Polymerase Chain Reaction and isoen- and extensive cattle rearing. zymes techniques, the etiologic agent isolated In the period between August 2006 and July from two patients was identified as L. i. chagasi. 2007, monthly sand fly collections were per- The epidemiological investigation showed no formed exploring: the modified environment, evidence of the introduction by immigrants, and represented by the domiciles in which leishmani- also no evidence of the migration of ill people, asis cases had occurred, including their annexes or their relatives, to known endemic areas. From and domestic animal shelters; and the preserved a geographic and climatic perspective, the area environment, represented by secondary forests where new cases occurred is different from the next to the domiciles.

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The captures were performed during the Mi. schreiberi was the predominant species in the three initial nocturnal hours using active search modified environment and Ny. intermedia the in Shannon traps with the help of the Castro suc- predominant species in the preserved environ- tion manual capturer in the modified environment. In Joebinha district (Alfredo Chaves), Ny. ment and two CDC light traps installed in the intermedia was the predominant species in the preserved environment. two environment types, followed by Pi. fischeri. The collected specimens were placed in tubes The specific richness (S) and the diversity in- containing ethanol (70%) and taken to the labo- dex (H) were higher in Belém district, both in the ratory, where they were assembled according to modified (S = 13; H = 0.51) and in the preserved the technique proposed by Barreto & Coutinho 18 environments (S = 7; H = 1.23), when compared and identified according to the taxonomic cri- with the modified (S = 2; H = 0.52) and preserved teria proposed by Galati 19,20. The sand flies’ ge- (S = 2; H = 0.50) environments in Santo Antônio neric names were abbreviated as proposed by district and with the modified (S = 4; H = 0.21) Marcondes 21. and preserved (S = 6; H = 1.03) environments in Using the PAST program (http://folk.uio.no/ Joebinha district. When the two environment ohammer/past/) 22, the following parameters types were compared, the specific richness was were estimated in each environment: the specif- higher for the modified environment in Belém ic richness (S), the equitability index (J) and the district and for the preserved environment in Jo- Shannon diversity index (H). ebinha district. In Santo Antônio district, the specific richness was the same for both environment types (Table 1). The diversity index was more Results than twice the value in the preserved environment compared with the modified environment A total of 13,112 sand fly specimens belonging to in Belém district and more than four times the 15 species (H = 0.59) were collected (Table 1). No value in Joebinha district, while in Santo Antônio instance of Lu. longipalpis, the main American district they were similar (Table 1). In Joebinha visceral leishmaniasis vector in the New World, district and in Belém district the equitability in- was captured in the three locations under study. dex was higher for the preserved environment The only species captured in all three locali- when compared with the modified environment, ties were Nyssomyia intermedia (Lutz & Neiva, while in Santo Antônio district the index was sim- 1912) and Micropygomyia schreiberi (Martins, ilar in both environment types (Table 1). Falcão & Silva, 1975). Other species appeared in Belém district and Joebinha district including: Lutzomyia alencari (Martins, Souza & Falcão, Discussion 1962), Micropygomyia quinquefer (Dyar, 1929), Migonemyia migonei (França, 1920) and Pin- The sand flies fauna found in the two American tomyia fischeri (Pinto, 1926). Yet some species visceral leishmaniasis foci in the Central-South were captured in just one locality: Brumptomyia portion of Espírito Santo state presents a specific nitzulescui (Costa Lima, 1932), Evandromyia ed- composition very different from that found in wardsi (Mangabeira, 1941), Evandromyia termi- other transmission areas in Espírito Santo and in tophila (Martins, Falcão & Silva, 1964), Nyssomyia Brazil 15,23. However, this fauna is very similar to whitmani (Antunes & Coutinho, 1939), Pintomy- that found in typical areas of American tegumen- ia bianchigalatiae (Andrade-Filho, Aguiar, Dias & tary leishmaniasis transmission, in which the Falcão, 1999), Pintomyia monticola (Costa Lima, species Ny. intermedia, Mg. migonei and Pi. fis- 1932), Pintomyia serrana (Damasceno & Arouck, cheri predominate, like in the locality of Joebinha 1949) and Psathyromyia shannoni (Dyar,1929) in (Alfredo Chaves) and in other regions in Espírito Belém district and Sciopemyia sordellii (Shannon Santo and in Brazil 24,25,26,27. & Del Ponte, 1927) in Joebinha district. The absence of Lu. longipalpis in two areas In Belém district (Vargem Alta), Ny. interme- in the South portion of Espírito Santo that reg- dia was the predominant species both in the pre- istered autochthonous American visceral leish- served and in the modified environment. In the maniasis human cases suggests a dissociation of modified ambient, it was followed by Mg. migo- the disease occurrence from the presence of this nei and Pi. fischeri. In the preserved environment insect in the state. Although in agreement with though, it was followed by Mi. schreiberi and Pi. the geo processing model proposed by Feitoza fischeri. In Santo Antônio district (Rio Novo do et al. 17, this absence is unexpected due to the Sul), individuals belonging to only two species fact that Lu. longipalpis is considered the only were captured in both environment types: Mi. vector of the disease in the Southeastern region schreiberi and Ny. intermedia. In this location, of Brazil 23.

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Table 1

Sand ﬂ ies collected in modiﬁ ed and preserved environments in the municipalities of Vargem Alta, Rio Novo do Sul and Alfredo Chaves, Espírito Santo State, Brazil, between August 2006 and July 2007.

Species Vargem Alta (Belém district) Rio Novo do Sul (Santo Antônio district) Modified Preserved Modified Preserved environment environment environment environment ♂ ♀ Subtotal ♂ ♀ Subtotal ♂ ♀ Subtotal ♂ ♀ Subtotal

Brumptomyia nitzulescui 1 - 1 ------Brumptomyia sp. - - - - 2 2 ------Evandromyia edwardsi 1 2 3 1 3 4 ------Evandromyia termitophila 1 - 1 ------Lutzomyia alencari - 1 1 ------Micropygomyia quinquefer 1 - 1 1 2 3 ------Micropygomyia schreiberi 8 7 15 8 12 20 33 2 35 1 - 1 Migonemyia migonei 1,032 147 1,179 - 2 2 ------Nyssomyia intermedia 7,799 2,170 9,969 32 21 53 7 2 9 1 3 4 Nyssomyia whitmani 1 - 1 ------Pintomyia bianchigalatiae - 3 3 ------Pintomyia fischeri 346 443 789 - 5 5 ------Pintomyia monticola - 1 1 ------Pintomyia serrana - 1 1 ------Psathyromyia shannoni 1 1 2 ------Sciopemyia sordellii - 1 1 ------Total 9,193 2,774 11,967 42 47 89 40 4 44 2 3 5 Specific richness (S) 13 7 2 2 Equitability index (J) 0.21 0.63 0.73 0.72 Diversity index (H) 0.57 1.23 0.52 0.50

(continues)

In an area of American visceral leishmania- logic agent of American tegumentary leishma- sis in Pernambuco state in the Brazilian North- niasis, including the finding of females naturally eastern region, Carvalho et al. 10 pointed to Mg. infected with this protozoan 29,30. migonei as a possible vector of L. i. chagasi, in The finding of Lu. alencari in the locality of spite of not having found females infected with Belém district, although in low density, deserves L. i. chagasi. For identification of this insect as more detailed studies to verify its possible par- a vector, the authors took into account mainly ticipation in the American visceral leishmaniasis their abundance in the intra and peri domicili- epidemiology in that locality, especially if one ary environments of residences in which human takes into account its phylogenetic proximity cases had occurred. Yet, Mg. migonei presents an with Lu. longipalpis. attraction for dogs 28, which can act as L. i. chaga- In Santo Antônio district, the transmission of si reservoirs. In this sense, the finding of this sand the disease might not have occurred due to the fly species in Belém district could explain the oc- sand flies biting since the two species found, Mi. currence of American visceral leishmaniasis in schreiberi and Ny. intermedia, are not considered this locality, but it would exclude Santo Antônio disease transmitters among human beings. The district, where the insect was not captured. Fi- first species, Mi. schreiberi, possibly feeds on rep- nally, Mg. migonei is found in many municipali- tiles and it was never found biting human beings ties next to the American visceral leishmaniasis to get fed. The second species, Ny. intermedia, endemic areas, even within Espírito Santo state, was never found naturally infected with L. i. cha- where the disease was never registered. However, gasi, in spite of being anthropophilic, attracted in the Brazilian Southeastern and Northeastern by a domiciliary reservoir such as dogs and able regions, Mg. migonei has been identified as a sec- to be experimentally infected. On the other hand, ondary vector of Leishmania braziliensis, an etio- the evidence of the natural infection with L. bra-

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Table 1 (continued)

Sand ﬂ ies collected in modiﬁ ed and preserved environments in the municipalities of Vargem Alta, Rio Novo do Sul and Alfre- do Chaves, Espírito Santo State, Brazil, between August 2006 and July 2007.

Species Alfredo Chaves (Joebinha district) Total Modified Preserved environment environment ♂ ♀ Subtotal ♂ ♀ Subtotal ♂ ♀ Subtotal

Brumptomyia nitzulescui ------1 - 1 Brumptomyia sp. ------2 2 Evandromyia edwardsi ------2 5 7 Evandromyia termitophila ------1 - 1 Lutzomyia alencari - 1 1 1 1 2 1 3 4 Micropygomyia quinquefer - - - 3 3 6 5 5 10 Micropygomyia schreiberi - - - 1 - 1 51 21 72 Migonemyia migonei 1 - 1 - - - 1,033 149 1,182 Nyssomyia intermedia 872 30 902 24 13 37 8,735 2,239 10,974 Nyssomyia whitmani ------1 - 1 Pintomyia bianchigalatiae ------3 3 Pintomyia fischeri 40 9 49 4 3 7 390 460 850 Pintomyia monticola ------1 1 Pintomyia serrana ------1 1 Psathyromyia shannoni ------1 1 2 Sciopemyia sordellii ------1 1 Total 913 40 953 33 21 54 10,221 2,891 13,112 Specific richness (S) 4 6 15 Equitability index (J) 0.15 0,71 0.21 Diversity index (H) 0.21 1.03 0.59

ziliensis together with the insect anthropoph- maniasis transmission in the region. Yet, the find- ily, the coincidental distribution with American ing of Pi. fischeri in the localities of Belém district tegumentary leishmaniasis and the attraction for and Joebinha district supports the fact that the reservoir animals, leads us to believe that this spe- fauna has a pattern typical of that found in an cies could be considered the main disease trans- American tegumentary leishmaniasis transmis- mitter in the Southeastern region of Brazil 25,27,28. sion area, since the species, like Mg. migonei, is It is possible that fleas can participate in the incriminated as a secondary vector of the dis- transmission of American visceral leishmaniasis ease. in Santo Antônio district since infection by L. i. The fact that the species richness and diver- chagasi was detected in females of Ctenocepha- sity index are higher in Belém district may be re- lides felis felis (Bouché, 1835) by means of kDNA- lated to the size of the forest fragment and the PCR (unpublished data). However, the sensibility composition of the vegetation around the frag- of the molecular technique can allow the detec- ment in the preserved environment. In this location of the parasite DNA in the blood ingested by tion, the forest fragments are bigger and located the fleas of an infected dog and therefore it does next to banana trees, unlike in the other locations not prove that these parasites can install them- that contain smaller fragments that are next to selves in the flea’s digestive tract to be transmit- cattle rearing areas. On the other hand, the higher ted to other dogs or to human beings. species richness for the modified environment in The fauna found both in Belém district and Belém district can be related with the proximity in Santo Antônio district were similar to those of residences to the forest and with the presence found in Joebinha district. The predominance of of food sources for chicken and pigs. The diver- Ny. intermedia shows the potential for American sity index and the equitability index, as expected, tegumentary leishmaniasis transmission in these were higher in the preserved environment in areas, but does not prove American visceral leish- Belém and Joebinha districts. Possibly, the forest

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represents a system in equilibrium, with smaller visceral leishmaniasis in these locations. Con- variations of temperature and humidity, favoring sidering the absence of sand flies suspected of the development of sand flies. transmitting the American visceral leishmaniasis In conclusion, the present study dissociates in Santo Antônio district and the low frequency the occurrence of autochthonous American vis- of the two species collected in these locales, it ceral leishmaniasis human cases from the pres- is possible that other vectors or other transmis- ence of Lu. longipalpis, as this insect is not found sion mechanisms occur in the region. However, in two foci of the disease in the southern part of additional studies that reinforce this hypothesis Espírito Santo state. Moreover, the study con- must be developed, including the investigation tributes to the sand flies fauna knowledge in the of natural infection in sand flies and the inves- region and points out the possibility of the par- tigation of possible L. i. chagasi vertebrate res- ticipation of other sand fly species, apart from ervoirs in these localities in both domestic and Lu. longipalpis, in the transmission of American sylvatic environments.

Resumo

A ocorrência de leishmaniose visceral americana no neos foram capturados mensalmente de julho de 2006 Estado do Espírito Santo, Brasil, sempre foi associada a agosto de 2007 com uso de armadilhas de Shannon à presença do vetor Lutzomyia longipalpis. A distribui- e CDC em dois municípios com registros de leishma- ção geográfica desse vetor no estado está relacionada à niose visceral americana autóctone e um sem registros. ocorrência de características geoclimáticas específicas, Foram capturados 13.112 flebotomíneos, mas nenhum tais como clima seco, baixas altitudes, relevo acidenta- Lu. longipalpis foi encontrado. A ausência de Lu. lon- do e afloramentos rochosos A ocorrência de casos hu- gipalpis e a possível participação de outro vetor de manos autóctones de leishmaniose visceral americana leishmaniose visceral americana nessas localidades em municípios sem essas características geoclimáticas foram discutidas. justificou o presente estudo, com objetivo de testar a associação entre a ocorrência de leishmaniose visceral Psychodidae; Ecologia de Vetores; Leishmaniose americana e a presença de Lu. longipalpis. Flebotomí-

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Contributors Acknowledgments

I. S. Pinto participated in the collection of the sand flies, Our thanks to the Núcleo de Entomologia e Malacologia species identification, data analysis and interpretation at the Espírito Santo State Ministry for Health and the and the writing up of the article. C. B. Santos and A. L. Tropical Medicine Unit at the Federal University of Espi- Ferreira participated in the sand fly collection, species rito Santo for their support in carrying out this project. identification and writing up the article. G. Grimaldi Jr. To the Coordenação e Aperfeiçoamento de Pessoal de writing up the article and was responsible for the leish- Nível Superior (CAPES) for a grant for I. S. Pinto. And to mania species identification. A. Falqueto participated Drs. Yuri L. R. Leite, Marcelo T. Tavares and Reginaldo P. in the sand fly collection, writing up the article and was Brazil for their comments on earlier drafts. To L. G. S. R. responsible for coordinating the study. Bauzer for translated the manuscript.

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