Jpn. J. Infect. Dis., 60, 245-249, 2007
Original Article
Natural Vertical Transmission of Dengue Virus in Aedes albopictus (Diptera: Culicidae) in Kerala, a Southern Indian State Velayutham Thenmozhi*, Joghee Gowder Hiriyan, Satish Chandra Tewari, Paulraj Philip Samuel, Rajaiah Paramasivan, Rathinasamy Rajendran, Tiruchengode Ramasamy Mani and Brij Kishore Tyagi Centre for Research in Medical Entomology, (Indian Council of Medical Research), Madurai, India (Received October 3, 2006. Accepted May 7, 2007) SUMMARY: The natural occurrence of vertical transmission of dengue viruses in Aedes albopictus (Skuse) mosquitoes was examined in the state of Kerala in southern India. Adults and larvae of Ae. albopictus collected from Kerala were screened for dengue viruses by using enzyme-linked immunosorbent assay with dengue-specific monoclonal antibodies. The possibility of the vertical transmission of dengue virus in Ae. albopictus was further evidenced by the detection of the virus in field-collected adult males as well as females emerged from fieldcollected larvae. Two pools, one pool from the adult males and one pool from the emerged females derived from field-collected larvae, were collected in the relatively hot months of June and March, respectively, and found to be positive for dengue virus antigen. Dengue serotype 2 virus was isolated from field-collected male adults in Kerala. These findings suggest that dengue virus is maintained in Ae. albopictus mosquitoes during the dry season by vertical transmission in nature.
INTRODUCTION
MATERIALS AND METHODS
Dengue fever (DF) is a vector-borne disease that has a major impact on public health in many tropical areas worldwide (1). The principal vector of dengue virus in urban areas is the highly domesticated Aedes aegypti (Linn.) mosquito, whereas Aedes albopictus (Skuse) is an important vector in rural areas. Ae. albopictus is a dengue vector in Asia and the Pacific Islands (2,3). In Southeast Asian countries, dengue epidemic usually occurs during the rainy season and is correlated with an increase in breeding habitats and vector populations (4). In vertical transmission by mosquitoes, the virus is transmitted by the progeny through an infected female mosquito, whereas in venereal transmission, the male transmits the virus to the female during copulation (5). Although several studies have demonstrated the vertical transmission of dengue virus in the laboratory (6-10), few studies have reported the vertical transmission in nature, and still fewer have reported vertical transmission involving Ae. albopictus. In Jodhpur, Rajasthan, western India, Joshi et al. (11,12) confirmed the transovarial cycle of dengue virus in Ae. albopictus reared from viable eggs retrieved from the soil of tree holes. In Peninsular Malaysia, Ahmad et al. (13) observed the occurrence of vertical transmission of dengue virus in Ae. aegypti and Ae. albopictus from fieldcollected larvae. Similarly, in Singapore, Kow et al. (14) have shown the presence of dengue virus in field-collected, adult Ae. aegypti and Ae. albopictus male mosquitoes. In the present paper, we report the occurrence of natural vertical transmission of dengue virus in Ae. albopictus in the state of Kerala in southern India.
Study site: Kerala is an Indian state with a total area of 38,863 km2 and population of 31,838,619. The latitude and longitude are 8°18´N to 12°48´N and 74°52´E to 77°25´E, respectively. Frequent outbreaks of DF have recently been documented in Kerala, southern India (15). Field studies were carried out in Kottayam (9°15´N latitude and 77°25´E longitude), Alleppey (9°30´N latitude and 76°19´E longitude) and Ernakulam (9°58´N latitude and 76°15´E longitude), besides the coastal Thrissur and Thiruvananthapuram districts; all of which have had dengue epidemics during the past few years (Fig. 1). This region is fairly humid and warm throughout the year, with the relative humidity and temperature varying between 70 - 90% and 22 - 35.4°C, respectively. Details of the monthly rain fall and temperature are given in Fig. 2.
*Corresponding author: Mailing address: Centre for Research in Medical Entomology, (Indian Council of Medical Research), 4 Sarojini Street, Chinna Chokkikulam, Madurai 625 002, Tamil Nadu, India. Tel: +91-452-2525131, Fax: +91-452-2530660, Email:
[email protected]
Fig. 1. Map of Peninsular India showing the study sites in Kerala.
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Fig. 2. Average monthly maximum temperature and rainfall from January 2002 to December 2003 in Kerala.
Mosquito collection: Larval survey: In each survey, each area was searched both inside and outside houses for breeding places of Aedes using a single larval technique (16), in which a single larva was picked up from each larval breeding container and reared individually. The emerged adults were identified and recorded. Aedes individuals in immature stages were collected monthly from breeding habitats such as latex collecting cups, cocoa pods, coconut shells, tree holes, plant stumps, mud pots, flower pots, grinding stones, tires, cement tanks and discarded containers systematically. Sylvan environments, particularly rubber plantations in some of the districts in Kerala, offer a unique habitat for Aedes mosquitoes. The collected immatures were reared to adulthood at the Centre for Research in Medical Entomology (CRME) in Madurai, India. The emerged adults were identified as described previously (17), separated by sex and pooled (maximum 20 specimens in a pool). They were stored at –80°C until tested. Adult survey: In the outdoor settings, adult males were collected while resting or landing (wild adults). Mosquitoes resting inside houses were collected in the morning for 15 min per house using a mouth aspirator and flashlight. Two insect collectors spent 2 h each, in each area per survey (4 man-hours per village), and the average number of adults per man-hour (PMH) was estimated. Resting collections were also carried out using the drop net method (18,19). In this method, a large mosquito bed net (200 × 125 cm) is spread over the bushes and vegetation and held tightly in position by four insect collectors, while one person disturbs the vegetation and the other person start collecting mosquitoes resting inside the net using a battery operated mechanical aspirator. Landing collections of mosquitoes were carried out while landing on human volunteer (from whom informed consent was obtained) in the morning and late afternoon for 30 min per volunteer. Adult mosquitoes were morphologically identified in the field and pooled (maximum 20 mosquitoes per pool). Detection of dengue virus: Antigen capture enzyme-linked immunosorbent assay (ELISA): Ae. albopictus pools were screened for the presence of dengue viruses by antigen capture ELISA (20,21) using the dengue virus specific monoclonal antibody (MAB) D3-5C9-1 (broadly reactive against all four serotypes of dengue viruses) as the capture antibody and detector MAB 6B6C-1 (broadly reactive against flaviviruses) conjugated with horse radish peroxidase, which was supplied
by the Centers for Disease Control and Prevention (CDC), USA. Each plate contained known DENV-infected larval or suckling mouse brain homogenate as a positive control and the homogenate of an uninfected adult mosquito pool as a negative control and a substrate blank. Insect-bioassay (Toxo-IFA): The suspension of dengue virus antigen-positive ELISA pools was intracerebrally inoculated into 50 early third instar Toxorhynchites splendens (Widemann) larvae and incubated for 14 days at 32°C (2224). Head squash preparations were examined by indirect immunofluorescent assay (IFA) using dengue virus serotypespecific MABs dengue type 1 (D2-IF1-3), dengue type 2 (3H5-1-21), dengue type 3 (D6-8A1-12) and dengue type 4 (1H10-6-7) also supplied by the CDC. Data analysis: The proportion of infected mosquitoes was estimated as minimum infection rate (MIR) using the formula described by Gajanana et al. (25). RESULTS Sixty-eight percent of the total individuals gathered from the sample sites in Kerala were identified as Ae. albopictus by entomological investigation (Fig. 3). The latex collecting cups (Fig. 6) were found to be the main breeding sites (79.7%) followed by grounded coconut shells (Fig. 4). Over a period of 3 years (March 2002 to June 2005), a total of 1,445 fieldcollected Ae. albopictus male mosquitoes in 101 pools and 1,817 field-collected Ae. albopictus female mosquitoes in 160 pools were screened for dengue virus antigen by ELISA. Among them, 1 and 4 of the male and female field-collected adult pools were found to be positive for dengue virus antigen, respectively. From March 2002 to August 2003, out of 77 pools (1,472 specimens) of reared males and 76 pools (1,485 specimens) of reared females screened, only 1 pool was found to be positive for dengue virus antigen; this was a pool of female Ae. albopictus collected in March 2002 from Mundakayam in the Kottayam district (Table 1). In total, 6 pools of Ae. albopictus that was positive for dengue virus by ELISA--i.e., 1 positive pool of field-collected male adults, 4 positive pools of field-collected female adults and 1 positive pool of female adults emerged from field-collected larvae-were further processed by Toxo-IFA for serotyping. Out of the 6 positive pools, dengue type 2 virus was isolated from 3 246
Fig. 3. Species composition of mosquitoes in Kerala. Others* include Malaya genurostris, Cx. pallidothorax, Ae. vittatus, Cx. minutissimus, Cx. brevipalpis, Ae. niveus, Tx. splendens and Ae. krombeini.
Fig. 4. Breeding habitats of Aedes albopictus in Kerala.
Table 1. Detection of dengue virus antigen in Aedes albopictus collected in Kerala, India
Month
No. of positive pools
Mar ’02 Jun Aug Sep Nov Dec Feb ’03 May Jun Jul Aug Jan ’04 Apr May Jun Jul Aug Sep Oct Nov Dec Jan ’05 Mar Apr May Jun
– 0 – – 0 0 0 0 0 – – – 0 0 0 0 0 0 0 0 0 0 0 0 0 1
Total
1
[MIR/1,000]
Wild adults1) Male No. of Total no. of No. of pools mosquitoes positive tested tested pools –
– – – 16 14 6 3 2 4 3 8 4 – 1 9 3 14
– 42 – – 11 15 15 52 132 – – – 183 236 76 25 6 63 7 136 15 – 1 170 23 237
– 0 – – 0 0 0 0 0 – – 0 0 2 0 0 0 0 0 0 0 – 0 0 1 1
101
1,445
4
2 – – 1 1 1 2 7
[0.07]
Female No. of Total no. of pools mosquitoes tested tested
No. of positive pools
Reared adults 2) Male No. of Total no. of No. of pools mosquitoes positive tested tested pools
Female No. of Total no. of pools mosquitoes tested tested
– 2 – – 2 4 2 3 5 – – 2 29 33 9 3 3 3 3 19 12 – 1 5 3 17
– 40 – – 20 78 49 57 84 – – 5 259 173 76 28 9 22 8 329 211 – 3 51 36 279
0 0 0 0 0 0 0 0 0 – 0
5 17 1 1 13 16 4 11 6 – 3
100 335 16 10 255 320 61 207 113 – 55
1 0 0 0 0 0 0 0 0 0 0
5 18 1 1 13 13 5 10 6 1 3
100 360 23 25 256 260 108 182 135 6 30
– – – – – – – – – – – – – –
– – – – – – – – – – – – – –
– – – – – – – – – – – – – –
– – – – – – – – – – – – – –
– – – – – – – – – – – – – –
– – – – – – – – – – – – – –
160
1,817
0
77
1,472
1
76
1,485
[2.20]
[0]
[0.07]
1)
: Field-collected adult mosquitoes. : Adult mosquitoes emerged from field-collected immature stage mosquitoes. MIR/1,000 = Minimum infection rate. The estimated number of postive mosquitoes per 1,000 mosquitoes tested. 2)
pools (2 pools were field-collected female adults collected in May 2004 and 1 pool was field-collected male adults collected in June 2005). The serotyping could not be accom-
plished in 3 positive pools (2 pools of field-collected female adults collected in May 2005 and June 2005 and 1 pool of reared adult females collected in March 2002). Toxo-IFA 247
DISCUSSION In India, Ae. albopictus has often been incriminated as a dengue vector in urban environments (26), and also occasionally in rural settings (27). However, recently Ae. albopictus has been incriminated as the vector in Kerala by isolating dengue type 2 virus, even in the absence of Ae. aegypti which carries enormous epidemiological significances (28). This supports Gubler’s (29) hypothesis that, at some point in the past, probably with the clearing of the forests and development of human settlements, dengue viruses moved out of the jungles and entered into a rural environment where they were (and continued to be) transmitted to humans by peri-domestic mosquitoes such as Ae. albopictus. In this study, dengue viruses were detected in Ae. albopictus collected during relatively hot months (March, May and June). During the study period, dengue type 2 virus was isolated from field-collected Ae. albopictus males in Kerala in June 2005 and two isolates of dengue type 2 viruses were recorded in wild caught females of Ae. albopictus in Kerala in May 2004 (Table 1). In Nagercoil in the Kanyakumari district (adjacent to Kerala), during the dengue outbreak in July 2003, 1 pool of Ae. albopictus males derived from field-collected larvae was found to be positive for dengue virus (27), which also suggested the natural transovarial transmission of dengue viruses in Ae. albopictus. In this study, the MIR values of wild adult male and female Ae. albopictus in Kerala were 0.07 and 2.20, respectively. In general, although the MIRs were relatively low in field-collected males, if the mechanism of infection in these infected males was vertical transmission from an infected maternal parent, and equal numbers of male and female offspring was found to be infected (8), then it is likely that a similar proportion of females in the field were also infected via the same mode of vertical transmission. Also, through a venereal transmission mechanism (5), the infected males (Ae. albopictus) could pass the virus to the females, and the latter could in turn pass it to their offspring vertically. The MIR of reared female Ae. albopictus in our study was 0.07 in the Thiruvananthapuram district (Table 1). Thus our results indicate that Ae. albopictus played a significant role in the maintenance of dengue viruses acquired through vertical transmission in nature, possibly indicating a mechanism for the persistence of dengue virus within an urban environment. It is also important to evaluate the competence of local dengue vectors in each epidemic-prone area.
Fig. 5. Results of Toxorhynchites splendens-immunofluorescent assay (Toxo-IFA).
ACKNOWLEDGMENTS We are grateful to the Director General, Indian Council of Medical Research, New Delhi, for providing facilities and encouragement. All monoclonal antibody preparations used in this study were kindly provided by Dr. D.J. Gubler and Dr. Barbara W. Johnson, Centers for Disease Control and Prevention, Fort Collins, Colorado, USA. Sincere thanks are due to all the laboratory and field supporting staff of CRME, Madurai involved in this study. We appreciate the excellent help rendered by Mr. A. Venkatesh, CRME in preparation of this manuscript, particularly in DTP work.
Fig. 6. Latex collecting cup--breeding habitat of Aedes albopictus in Kerala.
negative and positive pictures are shown in Fig. 5. All of the total 6 ELISA-positive pools were negative for dengue type 1, 3 and 4 viruses in Toxo-IFA. Five pools of field-collected Ae. aegypti male mosquitoes (7 specimens), and 2 pools of reared adult males from fieldcollected larvae (25 specimens) and 2 pools of reared adult females (28 specimens) of Ae. aegypti collected during the study were negative for dengue virus antigens. In addition, 11 pools (64 specimens) of Armigeres subalbatus and 1 pool (12 specimens) of Aedes vittatus were also tested by ELISA for dengue virus antigen and all were negative.
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