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Dengue virus detection in Aedes aegypti larvae from southeastern Brazil Samyra Giarola Cecílio1*, Willer Ferreira Silva Júnior1, Antônio Helvécio Tótola2, Cíntia Lopes de Brito Magalhães3, Jaqueline Maria Siqueira Ferreira4, and José Carlos de Magalhães1 ¹Laboratório de Microbiologia Geral e Enzimologia, [email protected] Laboratório de Bioquímica e Imunologia Celular e Molecular, Departamento de Química, Biotecnologia e Engenharia de Bioprocessos (DQBIO/Campus Alto Paraopeba/UFSJ), Ouro Branco, MG, Brazil 3 Laboratório de Biologia e Tecnologia de Micro-organismos, Departamento de Ciências Biológicas, (DCB/UFOP), Ouro Preto, MG, Brazil 4 Laboratório de Microbiologia (Campus Centro Oeste Dona Lindu/UFSJ), Divinópolis, MG, Brazil 2
Received 11 August 2014; Accepted 3 December 2015 ABSTRACT: The transmission of dengue, the most important arthropod-borne viral disease in Brazil, has been intensified over the past decades, along with the accompanying expansion and adaptation of its Aedes vectors. In the present study, we mapped dengue vectors in Ouro Preto and Ouro Branco, Minas Gerais, by installing ovitraps in 32 public schools. The traps were examined monthly between September, 2011 through July, 2012 and November, 2012 to April, 2013. The larvae were reared until the fourth stadium and identified according to species. The presence of dengue virus was detected by real time PCR and agarose gel electrophoresis. A total of 1,945 eggs was collected during the 17 months of the study. The Ovitrap Positivity Index (OPI) ranged from 0 to 28.13% and the Eggs Density Index (EDI) ranged from 0 to 59.9. The predominant species was Aedes aegypti, with 84.9% of the hatched larvae. Although the collection was low when compared to other ovitraps studies, vertical transmission could be detected. Of the 54 pools, dengue virus was detected in four Ae. aegypti pools. Journal of Vector Ecology 40 (1): 71-74. 2015. Keyword Index: Aedes, dengue virus, ovitraps, transovarial transmission, oviposition, mosquitoes.
INTRODUCTION Dengue virus (DENV) has been the most important arboviral disease in the world, responsible for significant morbidity and mortality, especially in tropical countries (Bhattacharya et al. 2013, Khan et al. 2013, Restrepo et al. 2014). Dengue is caused by the four antigenically distinct serotypes of the virus (DENV 1-4), which belong to the genus Flavivirus, family Flaviviridae, and is transmitted by infected Aedes mosquitoes (Añez and Rios 2013, Restrepo et al. 2014). Aedes aegypti is the primary vector, followed by Aedes albopictus, which also transmit the viruses causing West Nile, yellow fever, and chikungunya fevers (Regis et al. 2008, Primavesi 2013, Restrepo et al. 2014). The predilection of females for human blood enhance disease transmission among humans (Carrington and Simmons 2014). Illness with any of the four serotypes results in wide range of symptoms, from a milder dengue fever (DF) to the serious and often fatal dengue hemorrhagic fever (DHF) and hemorrhagic shock syndrome (DSS) (Gubler 1998). The disease brings considerable health, social, and economic problems to endemic areas. With the ongoing search for an efficient vaccine and an antiviral drug, prevention is still the best way to control the disease, which is possible through vector control in several forms (Bäck and Lundkvist 2013, Resende et al. 2012, Resende et al. 2013). Various trap models to capture and measure the density of the vector have been designed, and their use in many countries has shown them to be a better strategy than classical surveillance methods for monitoring (Regis et al. 2008, Regis et al. 2013). Ovitraps, or mosquito egg traps, exhibit a great sensitivity even at low vector densities (Santos et al. 2003). Moreover, they can detect transovarial transmission of the virus, which
has epidemiological importance in disease maintenance by the possibility of establishing new outbreaks in non-epidemic periods (Vélez et al. 1998, Kow et al. 2001). Thus, the present study was designed to assess the circulation of Ae. aegypti and Ae. albopictus vectors with the Ovitrap Positivity Index (OPI) and Eggs Density Index (EDI), and detect dengue virus transovarial transmission in Ouro Branco and Ouro Preto, Brazil. MATERIALS AND METHODS Study area and experimental design Ouro Branco and Ouro Preto are located in southeastern Brazil in Minas Gerais state, with estimated populations of 36,000 and 70,000, respectively. Sixteen public schools from each district, in partnership with local health surveillance departments, were chosen to compose the study, totaling 32 collection points. The ovitrap installation was conducted from September, 2011 to July, 2012 and from November, 2012 until May, 2013, totaling 17 months of collection. The traps consisted of a black plastic container (15 x 10 cm). In each one, three wooden paddles were fixed with clips for egg deposition. In order to improve the attractiveness, the ovitraps were filled with 500 ml of 10% grass infusion (Panicum maximum, Jacq), prepared according to Reiter et al. (1991). One trap was installed monthly per school, remaining at the institution for seven days to exclude the possibility of becoming a source of adults. Eggs were counted, hatched, and larvae were identified to species (Ae. aegypti or Ae. albopictus) at the 4th instar. They were separated into pools up to 40 larvae according to the month and site of collection and stored at -80° C. The entomological indicators calculated were
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OPI (positive traps/traps installed x 100) and EDI (eggs collected/ traps installed). RNA extraction and RT-PCR Pools were macerated with the UNIQUE® ultrasonic tissue macerator. Total RNA was extracted using the RNeasy QIAGEN Kit® according to the manufacturer’s protocol. As a positive control, RNA extracted from the supernatant taken from DENV2 infected C6/36 cells was used. The presence of viral RNA was detected using reverse transcription-polymerase chain reaction (RT-PCR). For the synthesis of complementary DNA (cDNA), the reaction mixture contained 5.0 µl of each extracted RNA, 1.0 μl of the reverse primer (10 pmol) (Chien et al. 2006), 0.5 μl of RiboLock RNase Inhibitor (40 U/μl) Fermentas® and 7.25 μl of nuclease-free water Fermentas®. It was incubated for 5 min at 70° C and 5 min at 4° C. Subsequently, it was added to a mixture of 1.5 μl of dNTPs (10 mM) Fermentas®, 0.75 μl of M-MLV reverse transcriptase (200 U/μl) - Fermentas® and 4 μl of M-MLV Reaction Buffer 5X – Fermentas®. The incubation was carried out for 60 min at 42° C. The reaction was performed in Life-BIOER Express®. The reaction was carried out on StepOne™, Applied Biosystems®. The reaction mixture contained 2 μl of cDNA, 3 μl of each primer (2 pmols) (Chien et al. 2006), 10 μl of Power SYBRPCR Master Mix-Applied Biosystems®, and 2 μl of nucleasefree water Fermentas®, totaling 20 μl per sample. The reaction followed the steps of an initial denaturation at 95° C for 10 min, 40 amplification cycles of 15 s at 95° C, and 1 min at 60° C. cDNA from infected C6/36 cells was used as a positive control and sterile water as a negative control. PCR products were analyzed by electrophoresis in 1.5% agarose gels to confirm the size of amplicons. Ten μl were electrophoresed at 80 V for 1 h in Tris-acetate-EDTA (TAE) pH 8.3. GeneRuler 50 bp DNA Ladder Fermentas® was used as a molecular weight marker. The gel was stained with 0.1 μl/ml ethidium bromide solution and photographed under ultraviolet light. RESULTS Ovitrap installation During the 17 months of trap operation, 1,945 eggs were collected: 812 in Ouro Branco and 1,133 in Ouro Preto. Values of OPI ranged from 0 to 28.1%, with the highest values in January to April, 2013. EDI values ranged from 0 to 59.9%, with the highest values found in December, 2012 and January, 2013. Both cities presented larger numbers of Ae. aegypti. Of the 1,113 hatched eggs (57.2%), 945 (84.9%) were identified as Ae. aegypti and 168 (15.1%) as Ae. albopictus. The larvae were distributed in 54 pools according to the point of collection/date and species. Real-time PCR and agarose gel electrophoresis Of the 54 pools, four of Ae. aegypti showed amplification after the 24th- 30th cycle in real time PCR. Samples were electrophoresed to confirm the size of amplicons. Primers designed by Chien et al. (2006) amplify a segment of 146 bp. These bands could be visualized in agarose gels, corresponding to DENV, confirming transovarial transmission. Of the four positive pools, three were
Figure 1. Agarose gel electrophoresis of the amplified products by real-time PCR. Lane 1: standard molecular weight of 50 bp; lane 2: dengue virus 2 control; lanes 3-9: cDNA samples from eggs collected at different schools. Identification of 146 bp fragments in lanes 2, 3, 4, 5, 6, and 9 positive for dengue virus. There are also fragments of ± 50 bp, relative to dimers of primers. from O. Branco and one was from O. Preto schools. The pools were from eggs collected in January to April, 2013. As shown in Figure 1, the DNA samples analyzed by electrophoresis on agarose gel stained with ethidium bromide, with bands of 146 bp corresponding to DENV. Some lightly stained nonspecific bands can also be observed which are fragments of ± 50 bp, relative to dimers of primers. DISCUSSION The present study aimed to monitor Aedes by ovitrap installation. Devices that detect an increase in population of these vectors are valuable for health surveillance departments to better plan their control actions, find areas with higher incidence, and prevent disease outbreaks. Due to vertical transmission and resistance of eggs to long periods of dessication, it is important to focus the prevention of dengue on Aedes eggs. Oviposition traps have been used in several studies to monitor vector populations (Almeida et al. 2006, Fantinatti et al. 2007, Costa et al. 2008, Miyazaki et al. 2009, Carvalho-Leandro et al. 2010, Resende et al. 2013). Braga et al. (2000) and Cardoso et al. (1997) found higher sensitivity from oviposition traps than from larval surveys to detect Ae. aegypti. This methodology was more efficient than adult traps (Gama et al. 2007, Honorio et al. 2009). In the present study, although egg collection had been low (higher OPI 28.13 – April, 2013 and EDI 59.9 – January, 2013) when compared with other studies with ovitraps (Resende et al. 2013, Zeidler et al. 2008, Nunes et al. 2011), data are consistent with historical cases in both cities, neither of which prepare epidemic registers and are cold and mountainous. Moreover, there were peaks in the numbers of eggs collected in the warmer months of the year, while the other months had much lower or even null rates. This could allow the detection of fluctuations in vector density, suggesting that warmer months have an increased circulation of these arthropods, with a higher risk of disease transmission. Vector-borne infectious diseases are generally seasonal. Indeed, in Brazil, dengue has a seasonal pattern, with a higher incidence of cases in the first five months of the year that are
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warmer and wetter (Reiter 2001, Zell 2004). According to Morin et al. 2013, temperature is an essential element for dengue epidemics. This variable affects the proliferation of both the vector and virus. With the increase of daily minimum and maximum temperatures, there is a faster development of larval populations and the rate of pathogen replication within the insect (shorter extrinsic incubation period) (Russell 1998, Gubler et al. 2001, Reiter 2001). Transovarial transmission of dengue virus occurs both in the laboratory and in the field, playing an important role in the dissemination of the disease. Vertical transmission in Ae. aegypti and Ae. albopictus larvae was detected in studies by Cecilio et al. (2009) and Pessanha et al. (2011). According to Joshi et al. (2002), vertical transmission in nature does not occur in more than 20% of the progeny. In this work, using real time PCR, DENV amplification could be detected in four of the 54 pools of Ae. aegypti larvae hatched in the laboratory. Considering this small rate of vertical transmission occurrence, the positivity of pools (7.41%) was high and the results obtained are of great importance. Results were passed on to local health surveillance departments that have committed to plan a greater intervention in these locations. REFERENCES CITED Almeida, P.S., A.D. Ferreira, V.L Pereira, M.G Fernandes, and W.D. Fernandes. 2006. Distribuição espacial de Aedes albopictus na região sul do Estado de Mato Grosso do Sul. Rev. Saúde Públ. 40: 1094-1100. Añez, G. and M. Rios. 2013. Dengue in the United States of America: A worsening scenario? Biomed. Res. Int. 2013: 1-13. Bӓck, A.T. and A. Lundkvist. 2013. Dengue viruses: an overview. Infect. Ecol. Epidemiol. 3: 1-21. Bhattacharya, M.K., S. Maitra, A. Ganguly, A. Bhattacharya, and A. Sinha. 2013. Dengue: A growing menace - a snapshot of recent facts, figures and remedies. Int. J. Biomed. Sci. 9: 61-67. Braga, I.A., A.C. Gomes, M. Nelson, R.C.B. Melo, D.P. Bergamaschi, and J.M.P. Souza. 2000. Comparação entre pesquisa larvária e armadilha de oviposição para detecção de Aedes aegypti. Rev. Soc. Bras. Med. Trop. 33: 347-353. Cardoso Jr., R.P., S.A.S. Scandar, N.V. Mello, S. Ernandes, M.V. Botti, and E.M.M. Nascimento. 1997. Detecção de Aedes aegypti e Aedes albopictus na zona urbana do município de Catanduva-SP, após controle de epidemia de dengue. Rev. Soc. Bras. Med. Trop. 30: 37-40. Carrington, L.B. and C.P. Simmons. 2014. Human to mosquito transmission of dengue viruses. Front. Immunol. 5: 1-8. Carvalho-Leandro, D., A.L.M. Ribeiro, J.S.V. Rodrigues, C.M.R. Albuquerque, A.M. Acel, F.A. Leal-Santos, D.P. Leite Jr., and R.D. Miyazaki. 2010. Temporal distribution of Aedes aegypti Linnaeus (Diptera, Culicidae), in a hospital in Cuiabá, State of Mato Grosso, Brazil. Rev. Bras. Entomol. 54: 701–706. Cecílio, A.B., E.S. Campanelli, K.P. Souza, L.B. Figueiredo, and M.C. Resende. 2009. Natural vertical transmission by Stegomyia albopicta as dengue vector in Brazil. Braz. J. Biol. 69: 123-127. Chien, L.J., T.L. Liao, P.Y. Shu, J.H. Huang, D.J. Gubler, and G.J. Chang. 2006. Development of Real-time reverse transcriptase PCR assays to detect and serotype dengue viruses. J. Clin. Microbiol. 44: 1295–1304.
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Costa, F.S., J.J. Silva, C.M. Souza, and J. Mendes. 2008. Dinâmica populacional de Aedes aegypti (L) em área urbana de alta incidência de dengue. Rev. Soc. Bras. Med. Trop. 41: 309-312. Fantinatti, E.C.S., J.E.L. Duque, A.M. Silva, and M.A. NavarroSilva. 2007. Abundância e Agregação de Ovos de Aedes aegypti L. e Aedes albopictus (Skuse) (Diptera: Culicidae) no Norte e Noroeste do Paraná. Neotrop. Entomol. 36: 960-965. Gama, R.A., E.M. Silva, I. M. Silva, M.C. Resende, and A.E. Eiras. 2007. Evaluation of the Sticky MosquiTRAPTM for detecting Aedes (Stegomyia) aegypti (L.) (Diptera: Culicidae) during the dry season in Belo Horizonte, Minas Gerais, Brazil. Neotrop. Entomol. 36: 294-302. Gubler, D.J. 1998. Dengue and dengue hemorrhagic fever. Clin. Microbiol. Rev. 3: 48–96. Gubler, D.J., P. Reiter, K.L Ebi, W. Yap, R. Nasci, and J.A. Patz. 2001. Climate variability and change in the United States: potential impacts on vector- and rodent-borne diseases. Environ. Hlth .Perspect. 2: 222-233. Honório, N.A., C.T. Codeço, F.C. Alves, M.A.F.M. Magalhães, and R. Lourenço-De-Oliveira. 2009. Temporal distribution of Aedes aegypti in different districts of Rio de Janeiro, Brazil, measured by two types of traps. J. Med. Entomol. 46: 1001– 1014. Joshi, V., D.T. Mourya, and R.C. Sharma. 2002. Persistence of dengue-3 virus through transovarial transmission passage in successive generations of Aedes aegypti mosquitoes. Am. J. Trop. Med. Hyg. 67: 158-161. Khan, M.I.H., E. Anwar, A. Agha, N.S.M. Hassanien, E. Ullah, I.A. Syed, and A. Raja. 2013. Factors predicting severe dengue in patients with dengue fever. Medit. J. Hematol. Infect. Dis. 5: e2013014. Kow, C.Y., L.L. Koon, and P.F. Yin. 2001. Detection of dengue viruses in field caught male Aedes aegypti and Aedes albopictus (Diptera:Culicidae) in Singapore by type–specific PCR. J. Med. Entomol. 38: 475-479. Miyazaki, R.D., A.L.M. Ribeiro, M.G. Pignatti, and J.H. Campelo Jr. 2009. Monitoring of Aedes aegypti mosquitoes (Linnaeus, 1762) (Diptera: Culicidae) by means of ovitraps at the Universidade Federal de Mato Grosso Campus, Cuiabá, State of Mato Grosso. Rev. Soc. Bras. Med. Trop. 42: 392-397. Morin, C.W., A.C. Comrie, and K. Ernst. 2013. Climate and dengue transmission: evidence and implications. Environ. Hlth. Perspect. 121: 1264-1272. Nunes, L.S., R.B.R. Trindade, and R.N.P. Souto. 2011. Avaliação da atratividade de ovitrampas a Aedes (Stegomyia) aegypti Linneus (Diptera: Culicidae) no bairro Hospitalidade, Santana, Amapá. Bio. Amaz. 1: 26-31. Pessanha, J.E.M., W.T. Caiaffa, A.B. Cecilio, F.C.M. Iani, S.C. Araujo, J.C. Nascimento, E.G. Kroon, F.A. Proietti, and J.R Arias. 2011. Cocirculation of two dengue virus serotypes in individual and pooled samples of Aedes aegypti and Aedes albopictus larvae. Rev. Soc. Bras. Med. Trop. 44: 103-105. Primavesi, R. 2013. Wedding fever: fever in the returning traveler. Diagnosis: Dengue fever. Can. Fam. Phys. 59: 742-744. Regis, L.N., R.V Acioli, J.C.S. Júnior, M.A.V. Melo-Santos, W.V. Souza, C.M. Ribeiro, J.C. da Silva, A.M. Monteiro, C.M. Oliveira, R.M. Barbosa, C. Braga, M.A. Rodrigues, M.G. Silva, P.J. Ribeiro, W.H. Bonat Jr., L.C. de Castro Medeiros,
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M.S. Carvalho, and A.F. Furtado. 2013. Sustained reduction of the dengue vector population resulting from an integrated control strategy applied in two Brazilian cities. PlosOne 8: e67682. Regis, L., A.M. Monteiro, M.A.V Melo-Santos, J.C. Silveira Jr., A.F. Furtado, R.V. Acioli, G.M. Santos, M. Nakazawa, M.S. Carvalho, P.J. Ribeiro Jr., and W.V. Souza. 2008. Developing new approaches for detecting and preventing Aedes aegypti population outbreaks: basis for surveillance, alert and control system. Mem. Inst. Oswaldo Cruz 3: 50-59. Reiter, P. 2001. Climate change and mosquito-borne disease. Environ. Hlth. Perspect. 109: 141-161. Reiter, P., M.A. Amador, and C. Nelson. 1991. Enhancement of the CDC ovitrap with hay infusions for daily monitoring of Aedes aegypti populations. J. Am. Mosq. Contr. Assoc. 7: 52-55. Resende, M.C., T.M. Azara, I.O. Costa, L.C. Heringer, M.R. Andrade, J.L. Acebal, and A.E. Eiras. 2012. Field optimisation of MosquiTRAP sampling for monitoring Aedes aegypti Linnaeus (Diptera: Culicidae). Mem. Inst. Oswaldo Cruz 107: 294-302. Resende, M.C., I.M. Silva, B. Ellis, and A.E. Eiras. 2013. A comparison of larval, ovitrap and MosquiTRAP surveillance for Aedes (Stegomyia) aegypti. Mem. Inst. Oswaldo Cruz 108: 1024-1030.
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Restrepo, B.N., M.E. Beatty, Y. Goez, R.E. Ramirez, G.W. Letson, F.J. Diaz, L.D. Piedrahita, and J.E. Osorio. 2014. Frequency and clinical manifestations of dengue in urban Medellin, Colombia. J. Trop. Med. 2014: 1-8. Russell, R.C. 1998. Mosquito-borne arboviruses in Australia: the current scene and implications of climate change for human health. Int. J. Parasitol. 28: 955-969. Santos, S.R.A., M.A.V. Melo-Santos, L. Regis, and C.M.R. Albuquerque. 2003. Field evaluation of ovitraps consociated with grass infusion and Bacillus thuringiensis var. israelensis to determine oviposition rates of Aedes aegypti. Dengue Bull. 27: 156-162. Vélez, I.D., M.L. Quiñones, M. Suárez, V. Olano, L.M. Murcia, E. Correa, C. Arévalo, L. Pérez, H. Brochero, and A. Morales. 1998. A presencia de Aedes albopictus en Leticia, Amazonas, Colombia. Biomédica 18: 192-198. Zeidler, J.D., P.O.A. Acosta, P.P. Barrêto, and J.S. Cordeiro. 2008. Vírus dengue em larvas de Aedes aegypti e sua dinâmica de infestação, Roraima, Brasil. Rev. Saúde Públ. 42: 986-991. Zell, R. 2004. Global climate change and the emergence/reemergence of infectious diseases. Int. J. Med. Microbiol. 293: 16-26.
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Dengue virus detection in Aedes aegypti larvae from southeastern Brazil Samyra Giarola Cecílio1*, Willer Ferreira Silva Júnior1, Antônio Helvécio Tótola2, Cíntia Lopes de Brito Magalhães3, Jaqueline Maria Siqueira Ferreira4, and José Carlos de Magalhães1 ¹Laboratório de Microbiologia Geral e Enzimologia, [email protected] Laboratório de Bioquímica e Imunologia Celular e Molecular, Departamento de Química, Biotecnologia e Engenharia de Bioprocessos (DQBIO/Campus Alto Paraopeba/UFSJ), Ouro Branco, MG, Brazil 3 Laboratório de Biologia e Tecnologia de Micro-organismos, Departamento de Ciências Biológicas, (DCB/UFOP), Ouro Preto, MG, Brazil 4 Laboratório de Microbiologia (Campus Centro Oeste Dona Lindu/UFSJ), Divinópolis, MG, Brazil 2
Received 11 August 2014; Accepted 3 December 2015 ABSTRACT: The transmission of dengue, the most important arthropod-borne viral disease in Brazil, has been intensified over the past decades, along with the accompanying expansion and adaptation of its Aedes vectors. In the present study, we mapped dengue vectors in Ouro Preto and Ouro Branco, Minas Gerais, by installing ovitraps in 32 public schools. The traps were examined monthly between September, 2011 through July, 2012 and November, 2012 to April, 2013. The larvae were reared until the fourth stadium and identified according to species. The presence of dengue virus was detected by real time PCR and agarose gel electrophoresis. A total of 1,945 eggs was collected during the 17 months of the study. The Ovitrap Positivity Index (OPI) ranged from 0 to 28.13% and the Eggs Density Index (EDI) ranged from 0 to 59.9. The predominant species was Aedes aegypti, with 84.9% of the hatched larvae. Although the collection was low when compared to other ovitraps studies, vertical transmission could be detected. Of the 54 pools, dengue virus was detected in four Ae. aegypti pools. Journal of Vector Ecology 40 (1): 71-74. 2015. Keyword Index: Aedes, dengue virus, ovitraps, transovarial transmission, oviposition, mosquitoes.
INTRODUCTION Dengue virus (DENV) has been the most important arboviral disease in the world, responsible for significant morbidity and mortality, especially in tropical countries (Bhattacharya et al. 2013, Khan et al. 2013, Restrepo et al. 2014). Dengue is caused by the four antigenically distinct serotypes of the virus (DENV 1-4), which belong to the genus Flavivirus, family Flaviviridae, and is transmitted by infected Aedes mosquitoes (Añez and Rios 2013, Restrepo et al. 2014). Aedes aegypti is the primary vector, followed by Aedes albopictus, which also transmit the viruses causing West Nile, yellow fever, and chikungunya fevers (Regis et al. 2008, Primavesi 2013, Restrepo et al. 2014). The predilection of females for human blood enhance disease transmission among humans (Carrington and Simmons 2014). Illness with any of the four serotypes results in wide range of symptoms, from a milder dengue fever (DF) to the serious and often fatal dengue hemorrhagic fever (DHF) and hemorrhagic shock syndrome (DSS) (Gubler 1998). The disease brings considerable health, social, and economic problems to endemic areas. With the ongoing search for an efficient vaccine and an antiviral drug, prevention is still the best way to control the disease, which is possible through vector control in several forms (Bäck and Lundkvist 2013, Resende et al. 2012, Resende et al. 2013). Various trap models to capture and measure the density of the vector have been designed, and their use in many countries has shown them to be a better strategy than classical surveillance methods for monitoring (Regis et al. 2008, Regis et al. 2013). Ovitraps, or mosquito egg traps, exhibit a great sensitivity even at low vector densities (Santos et al. 2003). Moreover, they can detect transovarial transmission of the virus, which
has epidemiological importance in disease maintenance by the possibility of establishing new outbreaks in non-epidemic periods (Vélez et al. 1998, Kow et al. 2001). Thus, the present study was designed to assess the circulation of Ae. aegypti and Ae. albopictus vectors with the Ovitrap Positivity Index (OPI) and Eggs Density Index (EDI), and detect dengue virus transovarial transmission in Ouro Branco and Ouro Preto, Brazil. MATERIALS AND METHODS Study area and experimental design Ouro Branco and Ouro Preto are located in southeastern Brazil in Minas Gerais state, with estimated populations of 36,000 and 70,000, respectively. Sixteen public schools from each district, in partnership with local health surveillance departments, were chosen to compose the study, totaling 32 collection points. The ovitrap installation was conducted from September, 2011 to July, 2012 and from November, 2012 until May, 2013, totaling 17 months of collection. The traps consisted of a black plastic container (15 x 10 cm). In each one, three wooden paddles were fixed with clips for egg deposition. In order to improve the attractiveness, the ovitraps were filled with 500 ml of 10% grass infusion (Panicum maximum, Jacq), prepared according to Reiter et al. (1991). One trap was installed monthly per school, remaining at the institution for seven days to exclude the possibility of becoming a source of adults. Eggs were counted, hatched, and larvae were identified to species (Ae. aegypti or Ae. albopictus) at the 4th instar. They were separated into pools up to 40 larvae according to the month and site of collection and stored at -80° C. The entomological indicators calculated were
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OPI (positive traps/traps installed x 100) and EDI (eggs collected/ traps installed). RNA extraction and RT-PCR Pools were macerated with the UNIQUE® ultrasonic tissue macerator. Total RNA was extracted using the RNeasy QIAGEN Kit® according to the manufacturer’s protocol. As a positive control, RNA extracted from the supernatant taken from DENV2 infected C6/36 cells was used. The presence of viral RNA was detected using reverse transcription-polymerase chain reaction (RT-PCR). For the synthesis of complementary DNA (cDNA), the reaction mixture contained 5.0 µl of each extracted RNA, 1.0 μl of the reverse primer (10 pmol) (Chien et al. 2006), 0.5 μl of RiboLock RNase Inhibitor (40 U/μl) Fermentas® and 7.25 μl of nuclease-free water Fermentas®. It was incubated for 5 min at 70° C and 5 min at 4° C. Subsequently, it was added to a mixture of 1.5 μl of dNTPs (10 mM) Fermentas®, 0.75 μl of M-MLV reverse transcriptase (200 U/μl) - Fermentas® and 4 μl of M-MLV Reaction Buffer 5X – Fermentas®. The incubation was carried out for 60 min at 42° C. The reaction was performed in Life-BIOER Express®. The reaction was carried out on StepOne™, Applied Biosystems®. The reaction mixture contained 2 μl of cDNA, 3 μl of each primer (2 pmols) (Chien et al. 2006), 10 μl of Power SYBRPCR Master Mix-Applied Biosystems®, and 2 μl of nucleasefree water Fermentas®, totaling 20 μl per sample. The reaction followed the steps of an initial denaturation at 95° C for 10 min, 40 amplification cycles of 15 s at 95° C, and 1 min at 60° C. cDNA from infected C6/36 cells was used as a positive control and sterile water as a negative control. PCR products were analyzed by electrophoresis in 1.5% agarose gels to confirm the size of amplicons. Ten μl were electrophoresed at 80 V for 1 h in Tris-acetate-EDTA (TAE) pH 8.3. GeneRuler 50 bp DNA Ladder Fermentas® was used as a molecular weight marker. The gel was stained with 0.1 μl/ml ethidium bromide solution and photographed under ultraviolet light. RESULTS Ovitrap installation During the 17 months of trap operation, 1,945 eggs were collected: 812 in Ouro Branco and 1,133 in Ouro Preto. Values of OPI ranged from 0 to 28.1%, with the highest values in January to April, 2013. EDI values ranged from 0 to 59.9%, with the highest values found in December, 2012 and January, 2013. Both cities presented larger numbers of Ae. aegypti. Of the 1,113 hatched eggs (57.2%), 945 (84.9%) were identified as Ae. aegypti and 168 (15.1%) as Ae. albopictus. The larvae were distributed in 54 pools according to the point of collection/date and species. Real-time PCR and agarose gel electrophoresis Of the 54 pools, four of Ae. aegypti showed amplification after the 24th- 30th cycle in real time PCR. Samples were electrophoresed to confirm the size of amplicons. Primers designed by Chien et al. (2006) amplify a segment of 146 bp. These bands could be visualized in agarose gels, corresponding to DENV, confirming transovarial transmission. Of the four positive pools, three were
Figure 1. Agarose gel electrophoresis of the amplified products by real-time PCR. Lane 1: standard molecular weight of 50 bp; lane 2: dengue virus 2 control; lanes 3-9: cDNA samples from eggs collected at different schools. Identification of 146 bp fragments in lanes 2, 3, 4, 5, 6, and 9 positive for dengue virus. There are also fragments of ± 50 bp, relative to dimers of primers. from O. Branco and one was from O. Preto schools. The pools were from eggs collected in January to April, 2013. As shown in Figure 1, the DNA samples analyzed by electrophoresis on agarose gel stained with ethidium bromide, with bands of 146 bp corresponding to DENV. Some lightly stained nonspecific bands can also be observed which are fragments of ± 50 bp, relative to dimers of primers. DISCUSSION The present study aimed to monitor Aedes by ovitrap installation. Devices that detect an increase in population of these vectors are valuable for health surveillance departments to better plan their control actions, find areas with higher incidence, and prevent disease outbreaks. Due to vertical transmission and resistance of eggs to long periods of dessication, it is important to focus the prevention of dengue on Aedes eggs. Oviposition traps have been used in several studies to monitor vector populations (Almeida et al. 2006, Fantinatti et al. 2007, Costa et al. 2008, Miyazaki et al. 2009, Carvalho-Leandro et al. 2010, Resende et al. 2013). Braga et al. (2000) and Cardoso et al. (1997) found higher sensitivity from oviposition traps than from larval surveys to detect Ae. aegypti. This methodology was more efficient than adult traps (Gama et al. 2007, Honorio et al. 2009). In the present study, although egg collection had been low (higher OPI 28.13 – April, 2013 and EDI 59.9 – January, 2013) when compared with other studies with ovitraps (Resende et al. 2013, Zeidler et al. 2008, Nunes et al. 2011), data are consistent with historical cases in both cities, neither of which prepare epidemic registers and are cold and mountainous. Moreover, there were peaks in the numbers of eggs collected in the warmer months of the year, while the other months had much lower or even null rates. This could allow the detection of fluctuations in vector density, suggesting that warmer months have an increased circulation of these arthropods, with a higher risk of disease transmission. Vector-borne infectious diseases are generally seasonal. Indeed, in Brazil, dengue has a seasonal pattern, with a higher incidence of cases in the first five months of the year that are
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warmer and wetter (Reiter 2001, Zell 2004). According to Morin et al. 2013, temperature is an essential element for dengue epidemics. This variable affects the proliferation of both the vector and virus. With the increase of daily minimum and maximum temperatures, there is a faster development of larval populations and the rate of pathogen replication within the insect (shorter extrinsic incubation period) (Russell 1998, Gubler et al. 2001, Reiter 2001). Transovarial transmission of dengue virus occurs both in the laboratory and in the field, playing an important role in the dissemination of the disease. Vertical transmission in Ae. aegypti and Ae. albopictus larvae was detected in studies by Cecilio et al. (2009) and Pessanha et al. (2011). According to Joshi et al. (2002), vertical transmission in nature does not occur in more than 20% of the progeny. In this work, using real time PCR, DENV amplification could be detected in four of the 54 pools of Ae. aegypti larvae hatched in the laboratory. Considering this small rate of vertical transmission occurrence, the positivity of pools (7.41%) was high and the results obtained are of great importance. Results were passed on to local health surveillance departments that have committed to plan a greater intervention in these locations. REFERENCES CITED Almeida, P.S., A.D. Ferreira, V.L Pereira, M.G Fernandes, and W.D. Fernandes. 2006. Distribuição espacial de Aedes albopictus na região sul do Estado de Mato Grosso do Sul. Rev. Saúde Públ. 40: 1094-1100. Añez, G. and M. Rios. 2013. Dengue in the United States of America: A worsening scenario? Biomed. Res. Int. 2013: 1-13. Bӓck, A.T. and A. Lundkvist. 2013. Dengue viruses: an overview. Infect. Ecol. Epidemiol. 3: 1-21. Bhattacharya, M.K., S. Maitra, A. Ganguly, A. Bhattacharya, and A. Sinha. 2013. Dengue: A growing menace - a snapshot of recent facts, figures and remedies. Int. J. Biomed. Sci. 9: 61-67. Braga, I.A., A.C. Gomes, M. Nelson, R.C.B. Melo, D.P. Bergamaschi, and J.M.P. Souza. 2000. Comparação entre pesquisa larvária e armadilha de oviposição para detecção de Aedes aegypti. Rev. Soc. Bras. Med. Trop. 33: 347-353. Cardoso Jr., R.P., S.A.S. Scandar, N.V. Mello, S. Ernandes, M.V. Botti, and E.M.M. Nascimento. 1997. Detecção de Aedes aegypti e Aedes albopictus na zona urbana do município de Catanduva-SP, após controle de epidemia de dengue. Rev. Soc. Bras. Med. Trop. 30: 37-40. Carrington, L.B. and C.P. Simmons. 2014. Human to mosquito transmission of dengue viruses. Front. Immunol. 5: 1-8. Carvalho-Leandro, D., A.L.M. Ribeiro, J.S.V. Rodrigues, C.M.R. Albuquerque, A.M. Acel, F.A. Leal-Santos, D.P. Leite Jr., and R.D. Miyazaki. 2010. Temporal distribution of Aedes aegypti Linnaeus (Diptera, Culicidae), in a hospital in Cuiabá, State of Mato Grosso, Brazil. Rev. Bras. Entomol. 54: 701–706. Cecílio, A.B., E.S. Campanelli, K.P. Souza, L.B. Figueiredo, and M.C. Resende. 2009. Natural vertical transmission by Stegomyia albopicta as dengue vector in Brazil. Braz. J. Biol. 69: 123-127. Chien, L.J., T.L. Liao, P.Y. Shu, J.H. Huang, D.J. Gubler, and G.J. Chang. 2006. Development of Real-time reverse transcriptase PCR assays to detect and serotype dengue viruses. J. Clin. Microbiol. 44: 1295–1304.
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