Preliminary investigation of Culicidae species in South Pantanal, Brazil and their potential importance in arbovirus transmission

Investigação preliminar das espécies de culicídeos do Pantanal Sul brasileiro e sua potencial importância na transmissão de arbovírus

VIROLOGY

Preliminary investigation of Culicidae species in South Pantanal, Brazil and their potential importance in arbovirus transmission^*

Investigação preliminar das espécies de culicídeos do Pantanal Sul brasileiro e sua potencial importância na transmissão de arbovírus

Alex Pauvolid-Corrêa^I,1,**; Fernando Neto Tavares^I,2; Jeronimo Alencar^II; Julia dos Santos Silva^II,3; Michele Murta^I; Nicolau Maués Serra-Freire^III,4; Aiesca Oliveira Pellegrin^IV; Hélcio Gil-Santana^II; Anthony Érico Guimarães^II; Edson Elias da Silva^I

^ILaboratório de Enterovirus, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Ministério da Saúde, Avenida Brasil 4365, Manguinhos, 21045-900 Rio de Janeiro, RJ, Brasil

^IILaboratório de Díptera, Setor de Culicídeos, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Ministério da Saúde, Avenida Brasil 4365, Manguinhos, 21045-900 Rio de Janeiro, RJ, Brasil

^IIILaboratório de Ixodides, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Ministério da Saúde, Avenida Brasil 4365, Manguinhos, 21045-900 Rio de Janeiro, RJ, Brasil

^IVEmbrapa Pantanal, Ministério da Agricultura Pecuária e Abastecimento, Rua 21 de Setembro 1880, Nossa Senhora de Fátima, 79320-900 Corumbá, Mato Grosso do Sul, Brasil

^{Correspondence to}

SUMMARY

In view of the high circulation of migratory birds and the environmental and climatic conditions which favor the proliferation of arthropods, the Brazilian Pantanal is susceptible to circulation of arboviruses. However, the amount of data concerning arbovirus vectors in this area is scarce; therefore the aim of this study was to conduct a preliminary investigation of Culicidae species in the Nhecolândia Sub-region of South Pantanal, Brazil and their potential importance in the arbovirus transmission. A total of 3684 specimens of mosquitoes were captured, 1689 of which caught in the rainy season of 2007, were divided into 78 pools and submitted to viral isolation, Semi-Nested RT-PCR and Nested RT-PCR, with a view to identifying the most important arboviruses in Brazil. Simultaneously, 70 specimens of ticks found blood-feeding on horses were also submitted to the same virological assays. No virus was isolated and viral nucleic-acid detection by RT-PCR was also negative. Nevertheless, a total of 22 Culicidae species were identified, ten of which had previously been reported as vectors of important arboviruses. The diversity of species found blood-feeding on human and horse hosts together with the arboviruses circulation previously reported suggest that the Nhecolândia Sub-region of South Pantanal is an important area for arbovirus surveillance in Brazil.

Keywords: Arbovirus; Pantanal; Culicidae; Ticks; Nhecolândia.

RESUMO

Regiões como o Pantanal brasileiro, que apresentam fatores como riqueza de fauna silvestre incluindo circulação de aves migratórias e condições ambientais e climáticas favoráveis à proliferação de artrópodes estão potencialmente sujeitas à circulação de arbovírus. Entretanto, poucos trabalhos foram realizados acerca da presença de arbovírus em potenciais vetores no Pantanal. Neste sentido o principal objetivo deste trabalho foi conduzir uma investigação preliminar para presença de arbovírus em amostragens de culicídeos capturados na Sub-região da Nhecolândia no Pantanal Sul. Um total de 3684 mosquitos foi capturado, dos quais 78 grupos compondo uma amostragem de 1789 espécimes foram submetidos às técnicas de isolamento viral e RT-PCR para os mais importantes arbovírus no Brasil. Simultaneamente, 70 espécimes de carrapatos capturados durante hematofagia em cavalos também foram submetidos à pesquisa viral. Não houve isolamento viral em nenhuma amostra analisada e os resultados de detecção de ácido nucléico viral foram também negativos. Entretanto, foram identificadas 22 espécies de culicídeos, dez das quais previamente reportadas como vetores de importantes arbovírus. A competência vetorial de espécies capturadas durante hematofagia em humanos e cavalos aliada ao relato prévio de circulação de arbovírus sugerem a Sub-região da Nhecolândia como uma importante área de vigilância para arbovírus no Centro-Oeste do Brasil.

INTRODUCTION

Arboviruses are maintained in nature due to their biological transmission from an infected vertebrate host to another through hematophagous arthropods, mainly by mosquitoes, ticks, sand flies and biting midges⁵⁶. The Pantanal is a vast sedimentary plain of approximately 140,000 km² located in South America covering part of Brazilian, Bolivian and Paraguayan territories²⁶. However, about 85% of the area is located in Brazil, where 65.5% is situated in Mato Grosso do Sul State (MS) and 35.5% in the Mato Grosso State (MT), respectively recognized as South and North Pantanal^4,36. The region is a large floodplain whose dynamics are regulated primarily by the flood-pulse, whereby periods of flooding and drought alternate and constitute the primary factor governing the ecology of the plains³³. The considerable annual and multi-annual variability affects the biota with different intensities and on different time scales^8,32,39,31. The Pantanal wetland is classified into sub-regions that differ in their vegetation, flooding and physiognomy^1,2. The Nhecolândia Sub-region in MS, which occupies 19.48% of the Pantanal area, is one of the largest of these floodplains⁴⁸. Like other periodically flooded plains, the Brazilian Pantanal presents a set of factors that could be important for the circulation and maintenance of arboviruses. Among these factors, the richness of wildlife including the presence of migratory birds³⁸ and climatic conditions⁴⁹ that favor the proliferation of arthropods, sets the region a promising area for arbovirus surveillance in Brazil, in particular for the early detection of unrecognized or not previously reported species in Brazil, which may be circulating silently in the country through enzootic cycles in the Brazilian Pantanal⁴².

The circulation of Maguari virus (MAGV), Ilheus virus (ILHV), Tacaiuma virus (TCMV), Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV) was demonstrated, through arbovirus surveillance in horses from the Nhecolândia Sub-region in the 1990's²⁵. Despite this, data concerning arbovirus vectors in the area are scarce, with the exception of a few reports such as an interesting study about the feeding habits of Culicidae species by means of the precipitin technique conducted in North Pantanal³ and the description of Culicidae fauna that can be potential vectors of infectious diseases in an impacted area of MS, outside South Pantanal²³. Although ticks have not commonly been detected as arbovirus vectors in Brazil⁵⁴, these arthropods have been reported as the most important vectors of disease-causing pathogens in domestic and wild animals¹⁵ and their potential competence as arbovirus transmitters in Brazil has still to be considered. Some tick studies had been conducted in Pantanal, although most have focused on ecological aspects and wild fauna^9,32,11,12. The aim of this study was to conduct a preliminary investigation into Culicidae species and their potential importance as arbovirus transmitters mainly to human and horse hosts during the rainy and dry seasons in the Nhecolândia Sub-region of South Pantanal, Brazil, and the detection of the presence of arboviruses in the Culicidae and Ixodidae specimens sampled in the area.

MATERIAL AND METHODS

Area of study and collections - A sampling of mosquitoes authorized by the Brazilian Institute of Environment and Natural Resources (Licence Number IBAMA 002/2007) was conducted, during the rainy season (February) and dry season (November) of 2007, on the Nhumirim Ranch (18º 58' S and 56º 37' W), a property of Embrapa Pantanal (Brazilian Agricultural Research Company). The ranch presents a mosaic of vegetation areas including forest, savanna, floodable grassland areas and shallow lakes that display varying degrees of salinity and coalescence with the system in flood located in the Nhecolândia Sub-region of South Pantanal, approximately 150 km east of Corumbá city, a municipality of MS, Brazil. Specimens of ticks detected blood-feeding on horses of the property were also collected, but only in February. Adult mosquitoes were collected from several sites randomly selected using CDC automatic light traps and Shannon light traps, both without bait, and suction tubes while landing to blood-feed on horses and on research team members, as usually described^35,52.

Culicidae and Ixodidae sampling on equines - Three Culicidae collections were undertaken for one hour at different times (5:00 pm, 7:00 pm and 8:00 pm) in February, and three manual collections of Ixodidae were made on different days in the same month from 6:00 am to 9:00 am. In the dry season, the only catch of Culicidae species was undertaken from 6:00 pm to 7:00 pm.

Culicidae sampling on team members- In February, six one-hour collections were made starting at different times (10:00 am, 3:00 pm, 7:00 pm and 8:00 pm). In November, three collections of approximately two hours each were made starting at 8:00 am, 9:00 am and 1:00 pm.

Culicidae capture using CDC light trap - In the rainy season, only one capture was performed, from 6:00 pm to 6:00 am. In the dry season, four catches were conducted in the same period.

Culicidae capture using Shannon light trap - In February, one capture was undertaken, from 9:00 pm to 10:00 pm. During the dry season, only one capture was performed, from 6:00 pm to 7:30 pm.

Identification of arthropods - Specimens were placed in coolers and transported to the field station where they were immobilized by chilling and identified through direct observation of the morphological characters. The arthropod identification was carried out at the Oswaldo Cruz Institute (IOC), by the Diptera Laboratory according to the dichotomous keys for South American mosquitoes^{27,28,24,17,14,20} and by the Ixodides Laboratory according to the dichotomous key for Brazilian ixodological fauna⁷. After identification, 1689 specimens of mosquitoes and 70 specimens of ticks captured during the rainy season were separated by trap, date and point of capture and up to 100 specimens were pooled by species. The pools were placed in sterile cryovials and transported in liquid nitrogen to the Enterovirus Laboratory of IOC for further processing. The arthropods were stored at -70ºC in a freezer until trituration, RNA extraction and RT-PCR. Mosquitoes sampled in the dry season were identified but were not investigated for arboviruses.

Processing and virus isolation - In the laboratory, pools from one up to 50 mosquito specimens were placed in polypropylene capped culture tubes with four copper-clad steel beads (BBs) and 2mL of modified BA-1diluent (5% heat-inactivated fetal bovine serum in Medium 199 with glutamine, NaHCO3, penicillin [100U/mL], streptomycin [100mg/mL], fungizone 1000x and TRIS 0.5M). Because of the anatomical characteristics of the ticks, their trituration was carried out with sterile mortar and pestle in the presence of 2mL of modified BA-1. The suspensions were vortex and clarified by centrifugation at 6000 RPM for 15 minutes at 25 ºC. Aliquots of 200µL of each pool were inoculated into VERO cell monolayer tubes [5 x 10⁵ cells/mL]. The tubes were kept at 36ºC for one hour with gentle motion at each 15 minutes to optimize virus adsorption. At the end of this period 1800µL of Medium 199 containing 2% of bovine fetal serum, was added followed by incubation at the same temperature for seven days. Tubes were observed daily under an inverted microscope for evidence of cytopathic effects (CPE). The non-observation of CPE after three consecutive passages of seven days each was considered as demonstrating the absence of cytopathogenic viruses in a given pool.

Duplex RT-PCR followed by Semi-Nested PCR - RNA was extracted by using a commercial kit (QIAamp© Viral RNA Mini Kit QIAGEN), in accordance with the manufacturer's instructions. Reverse transcription was conducted in extracted RNA with Superscript II (Invitrogen) system and random primers [500µg/mL] (Random Primers PROMEGA). Aliquots of 10µL of cDNA served as templates in subsequent Duplex RT-PCR followed by Semi-Nested PCR assay for Venezuelan equine encephalitis virus (VEEV), Western equine encephalitis virus (WEEV), Mayaro virus (MAYV), Ilheus virus (ILHV), Rocio virus (ROCV), Yellow fever virus (YFV) and Saint Louis encephalitis virus (SLEV) detection, as previously described¹⁰.

Nested RT-PCR for flaviviruses - Five microliter aliquots of each cDNA underwent PCR amplification using a set of degenerate Flavivirus specific primers. The first round was conducted with external primers FlagR2 (5`tgt cca cts ccc ctt tgr tct 3`) and FlagF1 (5`aca tga tgg gra aam gwg aga 3`) for 40 cycles of 20 sec at 94ºC, 45 sec at 50ºC and one min at 68ºC. A second round was conducted with 5µL of the first amplicon using flavivirus-specific inner primers FlagR1 (5`tcc cai ccg gck gtg tca tc 3`) and FlagF2 (5`gcc atw tgg twc atg tgg 3`). These primer sets were engineered to match sequences of the NS5 gene of SLEV, YFV, Japanese encephalitis virus (JEV), Dengue virus (DENV) and West Nile virus (WNV) genome fragments. The same cycling parameters were used as in the first reaction.

RESULTS

A total of 3684 Culicidae specimens captured were identified as belonging to 22 species and 70 Ixodidae specimens were identified as Amblyomma cajennense and Anocentor nitens. Culicidae specimens of 18 species were identified blood-feeding on research team members and at least six species on horses. Of that total of mosquitoes captured, 17 species were identified among the 2139 specimens captured during the rainy season and nine among the 1545 specimens captured during the dry season (Table 1). Ps. albigenu was the most abundant Culicidae species in the rainy season (55.91%) of which 96.91% were caught blood-feeding on research team members indicating anthropophily. Ma. titillans was the most abundant species in the dry season (78%) of which the majority (60.5%) were caught in CDC automatic light traps.

Interestingly, the light traps show higher abundance during the dry season than in the rainy season. Ninety-four mosquitoes were caught per hour of capture (mq/hr) in the rainy season and 299 mq/hr in the dry season with the Shannon light trap while the CDC automatic light trap caught 10 mq/hr in the rainy season and 20 mq/hr in the dry season. On the other hand, the suction tubes used to capture host-seeking female mosquitoes on horses and research team members were more efficient during the rainy season. The captures on horses were 126 mq/hr in February and 53 mq/hr in November, while on research team members the numbers were, respectively, 262 mq/h and 24 mq/h.

Viral detection and isolation - No virus was isolated and viral nucleic-acid detection by RT-PCR was also negative in 78 pools containing a total of 1689 specimens of 17 mosquito species and in 18 pools containing a total of 70 specimens of two tick species.

DISCUSSION

Beyond the previously reported Culicidae species in the two most recent studies^3,23, we here report the presence of thirteen additional unidentified species in North Pantanal and six species in MS. Ma. titillans was the most abundant species found in this present study. These data are in agreement with those of a previous study undertaken in North Pantanal³. However, despite the high prevalence of this species in the dry season (78%), Ps. albigenu accounted for more than half mosquitoes captured in the rainy season (55.91%). In spite of this and interestingly, Ps. albigenu was not found during the sampling performed in November. This absence suggests an ecological complexity of the sub-region and this should be taken into consideration by entomologists in arbovirus studies, since EEEV has already been detected in this species in the Peruvian Amazon⁵¹.

The Ixodidae species identified in this study have commonly been reported for equine blood-feeding^21,19,47. Despite a report on the experimental arbovirus transmission of Amblyomma cajennense⁶, these species have not usually been reported in natural arbovirus cycles in Brazil⁵⁴. Regarding the epidemiological and epizootiological relevance of the Culicidae species identified in the present study, at least ten out of the 22 had already been reported in the transmission of important arboviruses. In Brazil, Ps. ferox was found infected by MAGV, ILHV and MAYV and related to ROCV transmission during a serious outbreak which occurred in São Paulo State (SP) in 1975^30,46. This Culicidae species was also reported as a vector in ILHV and VEEV cycles in the Peruvian Amazon and Central America, respectively^16,51.

Oc. argyrothorax was found naturally infected by ILHV and specimens of Sa. chloropterus were related to the transmission of ILHV and YFV in Guatemala and Panama in the 1950's and were also found infected by YFV in Brazil^44,45,46. In the same decade the transmission of VEEV in Ecuador and the Peruvian Amazon by Ma. titillans was reported²⁹. Specimens of Ps. albigenu were reported as EEEV vector in the Peruvian Amazon⁵¹ and An. triannullatus was found naturally infected by TCMV as well as Oc. scapularis by ILHV, MAGV and the Mucambo virus (MUCV)⁴⁶. Oc. scapularis further demonstrated its ability to transmit ROCV when experimentally infected and is considered to be one of the main species involved in this arbovirus cycle in nature³⁴. Cx. quinquefasciatus and Cx. declarator have been described in SLEV transmission^50,22,53,46 and Cx. quinquefasciatus and An. albitarsis have been reported in the transmission of WEEV in the US and Argentina respectively⁴³. Cx. quinquefasciatus has further been found naturally infected by EEEV and the Oropouche virus (OROV)⁴⁶.

As for the absence of cytopathogenic viruses in the arthropod samples, a set of factors involving the capture of the samples and the way in which they have been analyzed should be examined. Factors such as the non epizootic periods of collection and possible loss of virus infectivity as a result of environmental conditions in the field, such as the high temperature-humidity indices in the rainy season, may have influenced the results. Besides, despite the existence of reports of equine encephalitis cases in the region in the 1990's²⁵, collections and captures performed in February 2007 were conducted in the absence of recent official reports of epidemics, epizootics or even isolated cases of symptomatic infection by arboviruses. Epidemiological surveillance involving arthropods has shown that even during human or animal arbovirus outbreaks, the viral isolation in cell culture from mosquito samples is not easy.

Since 1999, the epidemiological surveillance for WNV in USA has reported reduced rates of viral isolation from Culicidae samples even in epidemic areas^37,5. In a study accomplished to isolate viruses in the Peruvian Amazon, the presence of arboviruses was detected only in 1.09% of the more than half a million mosquitoes analyzed⁵¹. In the tropical forests the life expectancy of mosquitoes during the rainy season can be very low, which can greatly reduce the possibility of the isolation of arboviruses¹³. In the 1990's, after an equine epizooty of encephalomyelitis in the Paraná State (PR) in Brazil, the attempt to isolate the agent in mice, from about 1800 mosquitoes, was unsuccessful¹⁸.

The non-detection of viral RNA in the arthropod pools can also be attributed to the same factors considered for the results of viral isolation, such as the non-epizootic periods of collection and environmental conditions in the field. The results of several previous studies have demonstrated low levels of positive results even during epidemics. In 2000, during the mosquito surveillance for WNV in the USA, the presence of virus RNA was detected by RT-PCR in 3.6% of 9952 mosquito pools evaluated⁵⁵.

Another important issues are the environmental conditions prevailing during identification of mosquito species. Although carried out as described and recommended by several authors, the optimization of this step could minimize the reduction in the viral titer that may occur by virtue of abrupt changes in temperature and humidity. The use of triethylamine should be considered as an alternative to chilling for the immobilization of mosquitoes. In an arbovirus study in the Peruvian Amazon, the use of triethylamine showed some advantages such as immobilization with lesser humidity, considered an important factor in the reduction of the virus titer^41,40.

A total of 22 Culicidae species were identified, ten of which had been previously reported naturally infected with or as vectors of important human arboviruses. The diversity of species found blood-feeding on horses and mainly on human beings infers the potential susceptibility of these hosts to many arbovirus infections, and this must be taken into account in the epidemiological and epizootiological surveillance of arboviruses in Brazil. In this scenario, the advancement of environmental degradation in the region can lead not only to ecological but also health impact. For over two centuries, the Brazilian Pantanal has remained preserved, mainly because of the secular economic activity of the local population based on extensive beef cattle breeding that occupied large areas of native pastures with relatively low impact in the wild fauna and flora. However, the recent advancement of plantations of sugarcane around the floodplain and the arrival of cattle ranchers from other regions of Brazil, the management of Pantanal has been suffering drastic changes, as the deforestation for planting of exotic pastures, which may be compromising the fragile balance of flooding and drought in the floodplain. These changes can directly impact the fluctuations of vector populations, which could lead to the favoring of certain species of higher capacity of transmission of arboviruses resulting in outbreaks of arboviruses, as has been reported in the Amazon region⁵³. Finally, the detection of arbovirus vectors associated with previous reports of the equine circulation of EEEV, WEEV, TCMV, MAGV and ILHV²⁵ suggests that the Nhecolândia Sub-region of Southern Pantanal is a key area for arbovirus surveillance.

ACKNOWLEDGEMENTS

The authors thank Dr. Roger Nasci of the CDC, United States of America (USA) who kindly supplied the protocols for mosquito trituration, the Brazilian Government, through CNPq and IOC/FIOCRUZ for their financial support and Embrapa Pantanal staff for its technical support at the field station.

Received: 08 August 2009

Accepted: 19 January 2010

Presented in part at the XIX National Meeting of Virology, Caxambú, Minas Gerais, November 16-19th, 2008.

• 1. Adamoli I. O Pantanal e suas relações fitogeográficas com os cerrados. Discussão sobre o conceito "Complexo Pantanal". In: 32ş Congresso Nacional de Botânica, Teresina; 1982. p. 109-19.
• 2. Adamoli J. Fisiografia do Pantanal. In: Allem AC, Valls JFM. Recursos forrageiros nativos do Pantanal Mato-Grossense. (Documentos, 8). Brasília: Embrapa Cenargen; 1987.
• 3. Alencar J, Lorosa ES, Silva JS, Lopes CM, Guimarães AE. Observações sobre padrões alimentares de mosquitos (Diptera: Culicidae) no Pantanal Mato-Grossense. Neotrop Entomol. 2005;34:681-7.
• 4. Alho CJR, Lacher TE, Conçales HC. Environmental degradation in the Pantanal ecosystem. Bioscience. 1988;38:164-71.
• 5. Andreadis TG, Anderson JF, Vossbrinck CR. Mosquito surveillance for West Nile virus in Connecticut, 2000: isolation from Culex pipiens, Cx. restuans, Cx. salinarius, and Culiseta melanura Emerg Infect Dis. 2001;7:670-4.
• 6. Aragão HB. Ixodidas brasileiros e de alguns paízes limítrophes. Mem Inst Oswaldo Cruz. 1936;31:759-843.
• 7. Aragão HB, Fonseca F. Notas de ixodologia. VIII: Lista e chave para os representantes da fauna ixodológica brasileira. Mem Inst Oswaldo Cruz. 1961;59: 115-30.
• 8. Barros ATM. Seasonality and relative abundance of Tabanidae (Diptera) captured on horses in the Pantanal, Brazil. Mem Inst Oswaldo Cruz. 2001;96:917-23.
• 9. Bechara GH, Szabó MP, Duarte JM, Matushima ER, Pereira MC, Rechav Y, et al. Ticks associated with wild animals in the Nhecolândia Pantanal, Brazil. Ann N Y Acad Sci. 2000;916:289-97.
• 10. Bronzoni RVM, Baleotti FG, Nogueira RMR, Nunes M, Figueiredo LTM. Duplex reverse transcription-PCR followed by nested PCR assays for detection and identification of Brazilian alphaviruses and flaviviruses. J Clin Microbiol. 2005;43:696-702.
• 11. Cançado PH, Piranda EM, Mourão GM, Faccini JL. Spatial distribution and impact of cattle-raising on ticks in the Pantanal region of Brazil by using the CO₂ tick trap. Parasitol Res. 2008;103:371-7.
• 12. Cançado PH, Zucco CA, Piranda EM, Faccini JL, Mourão GM. Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) as a parasite of pampas deer (Ozoctoceros bezoarticus) and cattle in Brazil's Central Pantanal. Rev Bras Parasitol Vet. 2009;18:42-6.
• 13. Causey OR, Causey CE, Maroja OM, Macedo DG. The isolation of arthropod-borne viruses, including members of two hitherto undescribed serological groups, in the Amazon region of Brazil. Am J Trop Med Hyg. 1961;10:227-49.
• 14. Consoli RAGB, Lourenço-de-Oliveira R. Principais mosquitos de importância sanitária no Brasil. Rio de Janeiro: Editora Fiocruz; 1994.
• 15. De La Fuente J, Estrada-Pena A, Venzal JM, Kocan KM, Sonenshine DE. Overview: ticks as vectors of pathogens that cause disease in humans and animals. Front Biosci. 2008;13:6938-46.
• 16. Downs WG, Spence L, Aitken THG. Studies on the virus of Venezuelan Equine Ecephalomyelitis in Trinidad, W.I. III. Reisolation of virus. Am J Trop Med Hyg. 1962;11:841-3.
• 17. Faran ME, Linthicum KJ. A handbook of the Amazonian species of Anopheles (Nyssorhynchus) (Diptera, Culicidae). Mosq System. 1981;13:1-81.
• 18. Fernández Z, Richartz R, Travassos da Rosa A, Soccol VT. Identificação do vírus causador da encefalomielite eqüina, Paraná, Brasil. Rev Saude Publica. 2000;34:232-5.
• 19. Flechtmann CHW. Ácaros de importância médico-veterinária. 3Ş ed. São Paulo: Nobel; 1985.
• 20. Forattini OP. Culicidologia médica: identificação, biologia, epidemiologia. v. 2. São Paulo: Editora da Universidade de São Paulo; 2002.
• 21. Freitas MG, Costa HMA, Costa JO, Iide P. Entomologia e acarologia médica e veterinária. 6Ş ed. Belo Horizonte: Precisa Ed.Gráfica; 1984.
• 22. Goés P, Bruno-Lobo M. Estudos sobre arbovírus: síntese do problema e plano inicial de trabalho: Epidemiologia. An Microbiol. 1961;9(A): 79-111.
• 23. Gomes AC, Natal D, Paula MB, Urbinatti PR, Mucci LF, Bitencourt MD. Riqueza e abundância de Culicidae (Diptera) em área impactada, Mato Grosso do Sul, Brasil. Rev Saude Publica. 2007;41:661-4.
• 24. Gorham JR, Stojanovich CJ, Scott HG. Clave ilustrada para los mosquitos anofelinos de Sudamerica Oriental. Atlanta: U.S. Departament of Health, Education and Welfare, Public Health Service; 1967.
• 25. Iversson LB, Silva RAMS, Travassos da Rosa APA, Barros VLRS. Circulation of eastern equine encephalitis, western equine encephalitis, Ilheus, Maguari and Tacaiuma viruses in equines of the Brazilian Pantanal, South America. Rev Inst Med Trop Sao Paulo. 1993;35:355-9.
• 26. Junk WJ, Cunha CN. Pantanal: a large South American wetland at a crossroads. Ecol Eng. 2005;24:391-401.
• 27. Lane J, Cerqueira NL. Os sabetíneos da América. Arch Zool Est Sao Paulo. 1942;3:473-849.
• 28. Lane J. Neotropical Culicidae. v 1 e 2. São Paulo: Editora Universidade de São Paulo; 1953.
• 29. Levi-Castillo R. The problem of human and equine encephalomyelitis in Ecuador. Acta Trop. 1952;9:77-80.
• 30. Lopes OS, Sacchetta LA, Francy DB, Jakob WL, Calisher CH. Emergence of a new arbovirus disease in Brazil: isolation of Rocio virus from Psorophora ferox (Humboldt, 1819). Am J Epidemiol. 1981;113:122-5.
• 31. Marques MI, Adis J, Santos GB, Battirola LD. Terrestrial arthropods from tree canopies in the Pantanal of Mato Grosso, Brazil. Rev Bras Entomol. 2006;50:257-67.
• 32. Martins JR, Medri IM, Oliveira CM, Guglielmone A. Ocorrência de carrapatos em tamanduá-bandeira (Myrmecophaga tridactyla) e tamanduá-mirim (Tamandua tetradactyla) na região do Pantanal Sul Mato-Grossense, Brasil. Ciênc Rural. 2004;34:293-5.
• 33. Medina-Júnior PB, Rietzler AC. Limnological study of a pantanal saline lake. Braz J Biol. 2005;65:651-9.
• 34. Mitchell CJ, Forattini OP. Experimental transmission of Rocio encephalitis virus by Aedes scapularis (Diptera: Culicidae) from the epidemic zone in Brazil. J Med Entomol. 1984;21:34-7.
• 35. Mitchell CJ, Monath TP, Sabattini MS, Cropp CB, Daffner JF, Calisher CH, et al. Arbovirus investigations in Argentina, 1977-1980. II: Arthropod collections and virus isolations from Argentine mosquitoes. Am J Trop Med Hyg. 1985;34:945-55.
• 36. Moraes AS, Resende EK, Rodrigues CAG, Mauro RA, Galdino S, Oliveira MD, et al. Embrapa Pantanal: 25 anos de pesquisas em prol da conservação do pantanal, os desafios do novo milênio. In: III Simpósio sobre Recursos Naturais e Sócio-Econômicos do Pantanal. Corumbá: Embrapa Pantanal; 2000.
• 37. Nasci RS, White DJ, Stirling H, Oliver JA, Daniels TJ, Falco RC, et al.West Nile virus isolates from mosquitoes in New York and New Jersey, 1999. Emerg Infect Dis. 2001;7:626-30.
• 38. Nunes AP, Tomás WM. Aves migratórias ocorrentes no Pantanal: caracterização e conservação. (Documentos, 62). Corumbá: Embrapa Pantanal; 2004.
• 39. Nunes da Cunha C, Junk WJ. Year-to-year changes in water level drive the invasion of Vochysia divergens in Pantanal grasslands. Appl Veg Sci. 2004;7:103-10.
• 40. O'Guinn ML, Lee JS, Kondig JP, Fernandez R, Carbajal F. Field detection of Eastern equine encephalitis virus in the Amazon basin region of Peru using reverse transcription-polymerase chain reaction adapted for field identification of arthropod-borne pathogens. Am J Trop Med Hyg. 2004;70:164-71.
• 41. O'Guinn ML, Turell MJ. Effect of triethylamine on the recovery of selected South American alphaviruses, flaviviruses, and bunyaviruses from mosquito (Diptera: Culicidae) pools. J Med Entomol. 2002;39:806-8.
• 42. Pauvolid-Corrêa A, Varella RB. Aspectos epidemiológicos da febre do oeste do Nilo. Rev Bras Epidemiol. 2008;11:463-72.
• 43. Reisen WK, Monath TP. Western equine encephalomyelitis. In: Monath TP, editor.The Arboviruses: epidemiology and ecology. v 3. Florida: CRC Press; 1988.
• 44. Rodaniche E, Galindo P. Isolation of Ilhéus virus from Sabethes chloropterus captured in Guatemala in 1956. Am J Trop Med Hyg. 1957;6:686-7.
• 45. Rodaniche E, Galindo P, Johnson CM. Isolation of yellow fever virus from Haemagogus lucifer, H. equinus, H. spegazzinii falco, Sabethes chloropterus and Anopheles neivai captured in Panama in the fall of 1956. Am J Trop Med Hyg. 1957;6:681-5.
• 46. Segura MNO, Castro FC. Atlas de Culicídeos na Amazônia Brasileira: características específicas de insetos hematófagos da família Culicidae. Belém: Agência Brasileira; 2007.
• 47. Serra-Freire NM. Ácaros (carrapatos e outros). In: Marcondes CB, editor. Entomologia médica e veterinária. São Paulo: Editora Atheneu; 2001.
• 48. Silva JV, Abdon MM. Delimitação do Pantanal brasileiro e suas sub-regiões. Pesq Agrop Bras. 1998;33:1703-11.
• 49. Soriano BMA, Galdino S. Análise das condições climáticas em 2000 na Sub-Região da Nhecolândia, Pantanal, Mato Grosso do Sul, Brasil. (Boletim de Pesquisa e Desenvolvimento, 30). Corumbá: Embrapa Pantanal; 2002.
• 50. Tsai TF, Mitchell CJ. St. Louis encephalitis. In: Monath TP, The Arboviruses: epidemiology and ecology. v 4. Florida: CRC Press; 1988.
• 51. Turell MJ, O'Guinn ML, Jones JW, Sardelis MR, Dohm DJ, Watts DM, et al. Isolation of viruses from mosquitoes (Diptera:Culicidae) collected in the Amazon basin region of Peru. J Med Entomol. 2005;42:891-8.
• 52. Vaidyanathan R, Edman JD. Sampling with light traps and human bait in epidemic foci for eastern equine encephalomyelitis virus in southeastern Massachusetts. J Am Mosq Control Assoc. 1997;13:348-55.
• 53. Vasconcelos PFC, Travassos da Rosa APA, Rodrigues SG, Travassos da Rosa ES, Dégallier N, Travassos da Rosa JFS. Gestão imprópria do ecossistema natural na Amazônia brasileira resulta na emergência e reemergência de arbovírus. Cad. Saude Publica. 2001;17:155-64.
• 54. Vieira, AML, Souza CE, Labruna MB, Mayo RC, Souza SSL, Camargo-Neves VLF. Manual de vigilância acarológica. Superintendência de controle de endemias, Secretaria de estado da saúde, Governo do Estado de São Paulo. São Paulo: Imprensa Oficial; 2004.
• 55. White DJ, Kramer LD, Backenson PB, Lukacik G, Johnson G, Oliver J, et al. Mosquito surveillance and polymerase chain reaction detection of West Nile virus, New York State. Emerg Infect Dis. 2001;7:643-9.
• 56. World Health Organization (WHO). Virosis transmitidas por artrópodos y roedores (Informes Técnicos 719). Geneva: Graficas Reunidas; 1985.

*

This work is part of Alex Pauvolid-Correa's MSc thesis, defended at the Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Ministério da Saúde, Rio de Janeiro, Brazil, in 2008.

**

Present address: Laboratório de Flavivírus, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Ministério da Saúde, Avenida Brasil 4365, Manguinhos, 21045-900 Rio de Janeiro, RJ, Brasil;

1

Scholarship of CNPq in the Doctor of Tropical Medicine Program at the Fundação Oswaldo Cruz (FIOCRUZ);

2

Scholarship of IOC/FIOCRUZ in the Doctor of Parasitic Biology Program at the Fundação Oswaldo Cruz (FIOCRUZ);

3

Scholarship of CNPq in the Doctor of Zoology Program at the Universidade Federal do Rio de Janeiro (UFRJ);

4

Scholarship in Research Productivity 1B of CNPq.

Publication Dates

• Publication in this collection
  12 Mar 2010
• Date of issue
  Feb 2010

History

• Received
  08 Aug 2009
• Accepted
  19 Jan 2010