Asian Pac J Trop Dis 2012; 2(4): 268-272 268

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Document heading doi: 10.1016/S2222-1808(12)60059-0 2012 by the Asian Pacific Journal of Tropical Disease. All rights reserved. 襃 distribution and West Nile virus infection in zoos and in important sites of migratory and resident birds, Thailand

* Tanasak Changbunjong1 , Thekhawet Weluwanarak1, Namaoy Toawan1, Parut Suksai1, Poonyapat Sedwisai1, Tatiyanuch Chamsai1, Charoonluk Jirapattharasate1, Sivapong Sungpradit1, Yudthana Samung2, Parntep Ratanakorn1

1Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic , Faculty of Veterinary Science, Mahidol University, Nakorn Pathom, Thailand

2Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

ARTICLE INFO ABSTRACT

Objective: Article history: To investigate the distribution of mosquito species in the zoos and in important sites ( ) Received 15 March 2012 Methods:of migratory and resident birds and evaluate West Nile virus WNV infection in mosquito species. 2009 Received in revised form 27 April 2012 Mosquitoes distribution investigation was carried out bimonthly from January Accepted 28 May 2012 to December 2010 in five areas of birds, Thailand by using Centers for Disease Control, light Available online 28 August 2012 traps, and gravid traps. Mosquitoes were identified, pooled into groups of up to 50 mosquitoes by species, places and time of collection and tResults:ested for WNV infection by viral isolation and reverse transcriptase polymerase chain reaction. A total of 66 597 mosquitoes comprising 26 8 sCulexpecie stritaeniorhynchus in genera wer e collectCulexed. T hvishnuie five most abCulexundan sitienst mosquito sCulexpecie squinquefasciatus collected were Keywords: (79 3 ) (8 2 ) (6 ) Anopheles peditaeniatus. % , . % , % , (3.3%) and (1.1%). All 1 736 mosquito pools were negative for viral isolation Mosquito Conclusions: and reverse transcriptase polymerase chain reaction. This study provides new West Nile virus information on number of mosquito species present and their relative abundance. Although Vector our study found no evidence of WNV in the avifaunal sources of Thailand, mosquito active Surveillance surveillance should be continuously conducted. The cooperation between related organizations Zoo is needed for early detection of WNV disease and development of effective veterinary and public Migratory bird health policies in this region. Resident bird

[4] 1. Introduction suggested that this virus can be classified in seven lineages , molecular phylogenetic studies have shown that isolates of the virus can be just divided into two lineages. WNV lineages W N (WNV) I eFlavivirusst ile virus is a mosquito-borne virus in the strains are widely distributed in most continents, whereas genus of the family Flaviviridae that affects mostly lineages II have mostly been found in Africa[5,6]. T T WNV birds but also other species and humans. his virus ransmission cycle of Culex involves birds serving as the belongs to the Japanese encephalitis virus (JEV) serogroup natural reservoir hosts and species mosquitoes serving viruses. The other members of these serogroup are Japanese as the enzootic and/or epizootic vectors[7]. The infection of S L M V encephalitis virus, t. ouis encephalitis virus, urray alley most wild bird species is ae.gsymptomatic, while some species of encephalitis virus and Kunjin virus (subtype of WNV)[1]. WNV birds, especially corvids ( . crows), can be severely affected was first isolated from the blood of a sick woman in the West and have high mortality rates[8,9]. There are among 64 mosquito Nile District of Uganda in 1937[2]. WNV is widely distributed in species and 326 bird species found with WNV positive[10,11]. several countries of Africa, Europe, North America, Australia, The viruses can infect humans and domestic animals, such as and Asia (especially in India)[3]. Although it has been horses, which are generally thought to be incidental hosts that can develop diseases from moderate flu-like symptoms to *Corresponding author: Tanasak Changbunjong, The Monitoring and Surveillance [12] Center for Zoonotic Diseases in Wildlife and Exotic Animals, Faculty of Veterinary fatal encephalitis . Science, Mahidol University, 999 Putthamontol-4 Road, Salaya, Nakhon Pathom 73170, Zoo, migratory and resident birds are potential sources for Thailand. WNV[13-16] I T Tel/Fax: 66 (0) 2441 5238 monitoring . n hailand, there are many species E-mail: [email protected] of captive and resident birds in the zoos. Especially at Dusit zoo, Bangkok, there is a large number of crows (Corvus Tanasak Changbunjong et al./Asian Pac J Trop Dis 2012; 2(4): 268-272 269

) H P J B J [17] macrorhynchos , species that may be used as sentinels for eron, heasant-tailed acana and ronze-winged ˚aca΄na ΄΄. WNV outbreak in Thailand. Furthermore, several places of Bu˚ng΄chaw΄΄ak zoo, Suphan Buri Province (N14 54 42.5 , Thailand such as Bung Boraphet, Nakorn Sawan Province are E100 02 51.1 ) exhibits a wide variety of animal species. This [17] 45 a common for migratory and residents birds that may be zoo is abundant in2 bird species, possessing more than a potential source of WNV. Every year many species of birds species in 8 000 m of aviary, especially Pheasant, Peacock, escape the cold weather by migrating into Thailand[18], and Painted Stork, Lesser Whistling-Duck, Parrot groups. these birds may play an important role for WNV. Few data Moreover, outside the cages, there are many species of resident are available on the current WNV situation in Thailand. In a birds flying around the area. [15] recent study, antibodies to WNV were found in horses , but D˚usi΄t zoo,΄΄ locate˚d a΄t Kha΄΄o Din Park in the Bangkok city ( 13 46 29 23 100 31 56 63 ) WNV-specific RNA was not detected. Therefore, there is no N . , E . , is the oldest and mos2t popular evidence in the past indicating the presence of indigenous zoo in Thailand[24]. It covers a total area of 0.189 km and has WNV infections to our knowledge. more than 1 600 animal species. This zoo is classified as a full- Centers for Disease Control (CDC) published guidelines for function animal park completed with facilities like an animal monitoring WNV and other arboviruses in the U.S. The goal hospital, a zoo museum, an educational centre, a cafeteria, of these comprehensive surveillance programs is to identify and a relaxation area. Besides the zoo animals, there are huge WNV activity in birds, mosquitoes and horses as sentinels for numbers of large-billed crows (Corvus macrorhynchos) that potential outbreaks in humans. Mosquito surveillance is one of live and within the zoo. ˚ ΄ ΄΄ [19-22] ( 13 31 04 2 programs widely used in several countries . The important B˚an΄gpu,΄΄ Samut Prakarn Province N . , goals of mosquito surveillance are to identify potential E100 39 22.0 ) has a high diversity in shore and water bird mosquito vectors in a particular area, monitoring population species that visit its mangrove swamps in winter[25]. Example densities of those vectors, and determining infection rates[23]. of these birds are Brown-headed Gull, Asian Dowitcher, Little Our study was conducted to investigate the distribution of Heron, Terns, Ruddy Shelduck, Black-winged Stilt, Lesser mosquito species in zoos, important areas of migratory and Sand-Plover, Whimbrel, Spotted Redshank, Curlew Sandpiper, resident birds and to evaluate WNV infection in these mosquito and Ruddy Turnstone. T species. he information from this study will be useful for N future research on epidemiological studies, detection and Phitsanulok prevention of WNV in Thailand.

A1 2. Materials and methods

2.1. Study areas Nakhon Sawan A2 Five localities including zoos and sources of migratory and resident birds in Thailand were used in this mosquito distribution (Figure 1). B W P B R D an ang et is located˚ in΄ the ΄΄ang a˚ka΄m i΄΄strict of A3 Phitsanulok Province (N16 41 58.2 , E100 11 02.1 ). This location is the large source of egrets in Thailand. Birds are Suphan Buri found near the Yom River, which has an abun2dant food supply for birds: wild fish in the area over 0.128 km . Bird species at Bangkok the site include Cattle Egret, Little Egret, Intermediate Egret, A4 Samut Prakarn Great Egret, Chinese Egret, Black Crowned Night Heron, Little A5 Cormorant, Oriental Darter and Asian Open billed Stork. Bung Borap˚het΄ Wat΄΄er Bird ˚Pa΄rk, l΄΄ocated in Nakorn Sawan ( 15 43 21 8 100 17 38 6 ) Province N . , E 2 . , is the largest wetland in Thailand with about 212.38 km in freshwater. This location has a large collection of migratory birds during the winter season (November to March) and is an important area for Figure 1. several resident bird species. The total number of found bird Map of mosquito collected sites in Thailand. A1 B W P A2 B B A3 B A4 species at the site is approximately of 250. At the site water : an ang et; : ung oraphet; : ungchawak zoo; : Dusit zoo; A5: Bangpu. A birds, shore birds and accipiters birds can be observed. mong 2.2. Mosquito collection often sighted migratory birds are Grey Heron, Purple Heron, Spot-billed Pelican, Black-headed Ibis, As’ian Open bill Stork, Lesser Whistling-Duck, Garganey, Baer s Pochard and Mosquitoes were collected on various occasions bimonthly Northern Pintail. Whereas the resident birds include Cotton by using CO2-baited CDC light traps which dry ice was used Pygmy-goose, Little Cormorant, Common Coot, Great Egret as a source of CO2 to attract mosquitoes and CDC gravid traps Watercock, Purple Swamphen, Little Grebe, Chinese Pond with fermented hay (John W. Hock Company, Gainesville, USA) Tanasak Changbunjong et al./Asian Pac J Trop Dis 2012; 2(4): 268-272 270

2009 2010 from January to December . Each five traps operated 3. Result from 6 p.m. until 6 a.m. on each study day. The mosquitoes were transported alive to laboratory for species ideetn tialfication by using description and keys of Rattanarithikul [26-29]. There were 66 597 mosquitoes collected. They were They were pooled by species, place, and time of collection. The subdivided into 61 534 mosquitoes for CDC light and 5 063 1 50 T CDC (T 1) A number of mosquitoes per pool ranged from to . hey were mosquitoes for gravid traps able . totAedeomyia,al of eight 80 (26 ) stored at - 曟 until tested for virus. gAedes,enera Anopheles, species Armigeres,of mosquito eCoquillettidia,s were collecte dCulex,: 2.3. Viral isolation Mansonia T and .Culex he stritaeniorhynchusix most abundant mosqCulexuito svishnuipecies (79 3 ) collecteCulexd wer esitiens . % , P 1 000 L (8 2%) (6 0%) (3 3%) ools of mosquitoes were homogenized in 毺 of Anopheles. , peditaeniatus . , . and minimum essential medium (MEM 10暳, Penicillin G, (1.1%). Mosquito species distribution S F ) 1 5 L T 2 T treptomycin and ungizone in . m appendorf tube by using for the five collected areas is shown in able . he spCulexecies T plastic pestle. he homogenated mosquito was centrifuged dtritaeniorhynchusistribution was markedCulexly diffevishnuirent am ong study areas. at 4 000 rpm for 10 min and supernatant was passed 0.45 毺m and were the predominant T 200 L B C B B B W syring filter. he 毺 of samples were inoculated into baby species at unCulexg haquinquefasciatuswak zoo, ung oCulexraphet gelidusand an ang (BHK 21) 37 5% P hamster kidney - cells and incubated at 曟 in a et, whereas Culex sitiens Culex gelidus, were at CO2 incubator for 2 hours. 150 毺L of the samples of BHK-21 Dusit zoo, and and at Bangpo. When cells were discarded and added 500 毺L of maintenance the number of mosquito densities was compared among the medium (2% fetal bovine serum in MEM) and then incubated at five areas, Ban Wang Pet had the highest mosquito densities 37 曟 in 5% CO2. We checked the presence of cytopathic effect (41.8%) followed by Bung Boraphet (26.7%), Bung Chawak zoo (CPE) daily for 3 days. Positive CPE was confirmed by reverse (22.3%), Bangpo (6.2%) and Dusit zoo (3.0%), respectively (Table transcription polymerase chain reaction (RT-PCR). 2). T 1 736 2.4. RNA extraction and RT-PCR he total mosquito pools were negative isolation for WNV. These results were confirmed by using RT-PCR and all of them were negative for WNV. Viral RNA was extracted from mosquitoes by using a viral Table 1 nucleic acid extraction kit (Geneaid Biotech Ltd., Taiwan). T T The RT-PCR was performed using a one-step RT-PCR kit otal number of mosquito species collected at five areas in hailand, 2009-2010. (QIAGEN Ltd., Germany) for the screening both WNV and JEV Number collected infection. The reaction mixture contained 0.125 毺L of forward ′ Mosquito species Light traps Gravid traps Total Percent primer JE/WN-OF 5 GRA ARM GDG ARG ACA TYT GGT GTG Aedeomyia catasticta ′ ′ 2 0 2 <1.0 G 3 , 0.125 毺L of reverse primer JE/WN-OR 5 CGG GGT CTC ′ 21 8 29 <1.0 CTC TAA CCT CTA GTC C 3 , 2 毺L of template DNA, 5 毺L of Aedes mediolineatus 1 0 1 <1.0 5X OneStep RT-PCR buffer (QIAGEN), 1 毺L of 10 mM dNTP Aedes vexans 6 0 6 <1.0 mix (QIAGEN), 1 毺L of OneStep RT-PCR enzyme (QIAGEN) Anopheles argyropus 3 0 3 <1.0 and RNase-free water was added to a total volume of 25 毺 Anopheles barbirostris 12 0 12 <1.0 L PCR 1 50 曟 Anopheles campestris . cycling conditions were as follows: cycle at for 2 0 2 <1.0 30 1 95 15 35 94 Anopheles nigerrimus minutes; cycle at 曟 for minutes; cycles at 曟 57 0 57 <1.0 45 70 45 72 90 Anopheles peditaeniatus for seconds followed by 曟 for seconds, 曟 for 748 1 749 1.1 72 10 P Anopheles sinensis seconds and final extension at 曟 for min. ositive and 2 0 2 <1.0 Anopheles sundaicus negative controls were included in each run. Positive controls 40 0 40 <1.0 Anopheles vagus for WNV were obtained from the United States Geological 1 8 9 <1.0 Anopheles tessellatus Survey while positive controls for JEV were obtain from JE 5 0 5 <1.0 Armigeres subalbatus vaccine strain Beijing-1. Negative controls consisted of master 4 29 33 <1.0 Coquillettidia crassipes mix minus RNA templates. PCR product were separated by gel 6 0 6 <1.0 Culex bitaeniorhynchus electrophoresis and visualized under UV light. The specific 19 3 22 <1.0 Culex fascocephala size of PCR product for WNV and JEV was 580 and 591 base 4 0 4 <1.0 Culex gelidus pairs, respectively. If the RT-PCR positive specimens will be 516 31 547 <1.0 Culex pseudovishnui confirmed for WNV by using specific primer including WN233 186 126 312 <1.0 ’ ’ ’ Culex quinquefasciatus (5 TTG TGT TGG CTC TCT TGG CGT TCTT 3 ) and WN640c (5 978 1 226 2 204 3.3 ’ Culex sitiens CAG CCG ACA GCA CTG GAC ATT CATA 3 ). The RT-PCR 3 965 5 3 970 6.0 Culex tritaeniorhynchus cycling conditions will be used 1 cycle at 50 曟 for 30 minutes; 50 193 2 613 52 806 79.3 Culex vishnui 1 cycle at 95 曟 for 15 minutes; 35 cycles at 94 曟 for 45 seconds 4 547 928 5 475 8.2 Lutzia fuscana followed by 55 曟 for 45 seconds, 72 曟 for 30 seconds and the 0 83 83 <1.0 Mansonia indiana final extension at 72 曟 for 10 minutes. The specific size for 49 2 51 <1.0 Mansonia uniformis WNV was 408 base pairs. 167 0 167 <1.0 Total 61 534 5 063 66 597 100.0 Tanasak Changbunjong et al./Asian Pac J Trop Dis 2012; 2(4): 268-272 271 Table 2 Species distribution of mosquitoes collected at five areas in Thailand, 2009-2010. Mosquito species Number collected Dusit zoo Bung chawak Bang pu Bung Boraphet Ban Wang Pet Total Aedeomyia catasticta 0 0 0 2 0 2 Aedes aegypti 27 1 0 0 1 29 Aedes mediolineatus 0 0 0 1 0 1 Aedes vexans 0 0 0 6 0 6 Anopheles argyropus 0 0 0 1 2 3 Anopheles barbirostris 0 1 0 0 11 12 Anopheles campestris 0 0 0 2 0 2 Anopheles nigerrimus 0 2 0 55 0 57 Anopheles peditaeniatus 1 71 0 333 344 749 Anopheles sinensis 0 0 0 2 0 2 Anopheles sundaicus 0 0 40 0 0 40 Anopheles vagus 1 8 0 0 0 9 Anopheles tessellatus 0 5 0 0 0 5 Armigeres subalbatus 6 0 0 0 27 33 Coquillettidia crassipes 1 0 0 5 0 6 Culex bitaeniorhynchus 0 3 0 18 1 22 Culex fascocephala 0 0 0 0 4 4 Culex gelidus 169 49 125 11 193 547 Culex pseudovishnui 0 91 0 40 181 312 Culex quinquefasciatus 1 661 318 104 10 111 2 204 Culex sitiens 0 10 3 875 65 20 3 970 Culex tritaeniorhynchus 93 12 575 0 14 788 25 350 52 806 Culex vishnui 34 1 681 0 2 241 1 519 5 475 Lutzia fuscana 18 4 0 30 31 83 Mansonia indiana 2 3 0 22 24 51 Mansonia uniformis 1 0 1 156 9 167 Total 2 014 (3.0%) 14 822 (22.3%) 4 145 (6.2%) 17 788 (26.7%) 27 828 (41.8%) 66 597 (100%)

4. Discussion The relative proportion of mosquito species varied according to collection methods and study area[22]. In this study, CDC light traps allowed us to collect a higher number and species Active mosquito surveillance was carried out in five of mosquitoes than etC DalC gravid traps, in agreement with the important bird sites in Thailand. This survey provides new study of Williams [30]. For example, gravid traps were information on the number of mosquito species present and not as efficient as CDC Mansonialight traps to coAnophelesllect several species their relative abundance at different sites. More than 60 000 Coquillettidiaof mosquito such as sp., sp., and mosquitoes were collected and included 26 different species. sp. Although CDC gravid traps collected fewer They are the mosquito species that occur in Thailand[27]. The individuals and fewer species than light traps, the infection relative species distribution in these five study areas was rate of mosquitoes infete calted was higher in the gravid traps clearly different and was in agreement with the differences in presented by Williams [30]. habitat characteristics in each study site. BungChawak zoo and Culex In th equinquefasciatus present study, we fouCulexnd tw ovishnui species of mosquitoes Bung Boraphet, Ban Wang Pet are found in areas near rivers, ( and ) that could act WNV T wetlands, and are surrounded by rice fiCulexelds. I tritaeniorhynchusn these two sites, as potential vectors of in hailand as they have Culexbeen WNV A [3,31] F the predominCulexant mo vishnuisquito speCulexcies w vishnuias tritaeniorhynchusisolated the strai ns ofCulex bitaeniorhynchus in sia . urthermore, followed by . is a common and and presented in this a widespread species frequently found in association with study could also act as potential vectors of WNV in Thailand Culex tritaeniorhynchus[28] Culex quinquefasciatus . waCulexs the since these species reported from experiment studies in I [3] geliduspredominant species of mosquito at Dusit zoo followed by ndia . A WNV . Both these two species Culexare co mquinquefasciatusmon species ass ociated lthough our study found no evidence of in the with humans and animals[28]. occurs avifaunal sources of Thailand, the mosquito active surveillance abundantly in houses and in practically all types of human should be continuously conducted. The first project surveying and animal shelters in urban communities. Breeding sites of WNV in Thailand was during 2005-2006[15] and also found both these two mosquito species have a wide range of habitats WNV negative in mosquitoes collected at bird areas including including clear, turbid or polluted fresh to blackish water in Bung Boraphet; Nakonsawan province, Wat phailom, Pathum T N H L S N P Culexground quinquefasciatus pools, ditches, pits, welCulexls, dra igelidusns and containers for hani province and ong an ake, akon akon rovince. ; while can be found Our study also confirms the situation of WNV infection in in pounds, swamps, marshy depression, ditches, pits, wells, mosquitoes at Bung Boraphet area. I T WNV stream margins, seepage pCulexools, rsitiensice fields, wheel tracks, and n hailand, could be introduced in near future under footprints[28]. At Bangpo, was the predominant the current circumstances of many people and materials mosquito speciesCulex that wgelidusas mo stly found in the brackish water entering and leaving the country within short periods of time, or coastal areas, is also found in this area. the development of transportations, the importation of illegal Tanasak Changbunjong et al./Asian Pac J Trop Dis 2012; 2(4): 268-272 272 67 – birds and other domestic pets, unintentional introduction of (1): 67 75. virus-infected mosquitoes or other vector species. Moreover, [14] R eed KD, Meece JK, Henkel JS, Shukla SK. Birds, migration some bird species such as crows, which are the major WNV and emerging zoonoses: west nilCline vi rMedus, lResyme disea1se, 2003 (1) amplifier and other birds especially ardeid birds, important in–fluenza A and enteropathogens. ; : vertebrate host of WNV in Asia[3] are presented in Thailand. 5 12. 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