Surveillance for ebola virus in wildlife, Thailand.
Although EBOV has historically been viewed as a virus from Africa, recent studies found that bat populations in Bangladesh and China contain antibodies against EBOV and Reston virus recombinant proteins, which suggests that EBOVs are widely distributed throughout Asia (5,6). Thus, an outbreak in Asian countries free of EBOV diseases may not only be caused by importation of infected humans and/or wildlife from Africa but may arise from in-country filovirus-infected wildlife. Serologic and molecular evidence for filoviruses suggests that members of the order Chiroptera (bats) may be their natural reservoir (7).
As part of a proactive biosurveillance program, we conducted a cross-sectional study for EBOV infection in bats and macaques in Thailand. We screened 500 Pteropus lylei bats collected from 10 roosting sites during March-June 2014 (online Technical Appendix, http://wwwnc.cdc. gov/EID/article/20/12/15-0860-Techapp1.pdf) for antibodies against EBOV antigen by using an ELISA validated by the Centers for Disease Control and Prevention (Atlanta, GA, USA) (8).
Bats and macaques were captured with permission from the Department of National Parks, Wildlife and Plant Conservation. The Institutional Animal Care and Use Committee at the University of California, Davis (protocol #16048) approved the capture and sample collection protocols.
To further screen a wide range of wildlife species in Thailand for active EBOV infection, we sampled and tested 699 healthy bats, representing 26 species, and 50 long-tailed macaques (Macaca fascicularis). Additional bat species were randomly captured ([greater than or equal to] 50/site) in 6 provinces in Thailand during 2011-2013 and identified by morphologic traits. Macaques were captured and sampled in March 2013 from 1 site at Khao Chakan, Sa Kaeo Province, and released at the same site. Blood, saliva, urine, and feces were collected from anesthetized macaques or nonanesthetized bats. All animals were released after sample collection. Details on species screened, sample sizes, and trapping localities are provided in the Table.
All nonblood specimens were collected in nucleic acid extraction buffer (lysis buffer) and transported on ice to the World Health Organization Collaborating Centre for Research and Training on Viral Zoonoses laboratory (Bangkok, Thailand) for storage and testing. Three types of specimen (saliva, urine, and serum) were collected from individual animals and pooled.
Nucleic acid was then extracted with NucliSENS easyMAG (bioMerieux, Boxtel, the Netherlands) and analyzed by reverse transcription PCR (RT-PCR). A consensus RTPCR was used to screen for all known species of Ebola virus and Marburg virus, including EBOV (9). In total, 5 RTPCRs were performed on each specimen, a regimen that included 4 sets of primers specific to known filoviruses and 1 degenerate primer set to detect novel viruses in this family. The sensitivity of RT-PCR on synthetic standard was 50-500 copies/reaction (9). We ran 3,745 PCRs, covering a range of assays, to increase detection sensitivity. All specimens examined were negative for filoviruses by EBOV ELISA and PCR (Table). For P lylei ELISA screening, optical density values for all 500 bats ranged from 0.000 to 0.095, well below the potential positive cutoff value of 0.2.
Assuming a population size of [approximately equal to]5,000 bats/roost and a sample size of 50 bats/site, we have 95% confidence that if >6% of the population had antibodies against EBOV antigen, we would have detected it. If we assume that all 500 animals are part of 1 large panmictic population, and we have 95% confidence that if EBOV were circulating in >0.5% of the population, we would have detected it. Therefore, although we cannot rule out infection of this species with 100% confidence, P. lylei bats, the most abundant species of large pteropid bats in Thailand, are highly unlikely to be reservoirs for EBOV
Our sample sizes for PCR screening of other bat species in this study were much smaller, and we had no supported serologic data, but these negative results could add to the knowledge of filovirus infection in nontissue specimens from healthy bats. Previous studies have detected Ebola virus-like filovirus RNA in lung tissue of healthy Rousettus leschenaultia bats in China (10) and from organs and throat and rectal swab specimens from a die-off of Miniopterus schreibersii bats in Spain (4). In our study, which included 22 M. schreibersii and 132 M. magnate bats, none of the bats tested positive for filoviruses. One limitation of the cross-sectional sampling strategy used here, however, is that PCR-negative findings do not necessarily mean that the bats were not infected in the past. Although we found no evidence of filovirus infection in wildlife species tested in Thailand, we believe that continuing targeted surveillance in wildlife should enable early detection and preparedness to preempt emerging zoonoses.
This study was supported by a research grant from Department of National Parks, Wildlife and Plant Conservation, the Thailand Research Fund (RDG5420089), the Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University (RES560530148HR), Health and Biomedical Science Research Program by National Research Council of Thailand and Health System Research Institute, the Research Chair Grant, the National Science and Technology Development Agency, Thailand, and the Naval Health Research Center (BAA-10-93) under the Cooperative Agreement no. W911NF-11-2-0041, and the United States Agency for International Development Emerging Pandemic Threats PREDICT project.
(1.) Gire SK, Goba A, Andersen KG, Sealfon RSG, Park DJ, Kanneh L, et al. Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak. Science. 2014; 345:136972. http://dx.doi.org/10.1126/science.1259657
(2.) Peterson AT, Bauer JT, Mills JN. Ecologic and geographic distribution of filovirus disease. Emerg Infect Dis. 2004; 10:40-7. http://dx.doi.org/10.3201/eid1001.030125
(3.) Miranda MEG, Miranda NLJ. Reston ebolavirus in humans and animals in the Philippines: a review. J Infect Dis. 2011; 204(Suppl 3):S757-60. http://dx.doi.org/10.1093/infdis/jir296
(4.) Negredo A, Palacios G, Vazquez-Moron S, Gonzalez F, Dopazo H, Molero F, et al. Discovery of an ebolavirus-like filovirus in Europe. PLoS Pathog. 2011; 7:e1002304. http://dx.doi.org/10.1371/ journal.ppat.1002304
(5.) Olival KJ, Islam A, Yu M, Anthony SJ, Epstein JH, Khan SA, et al. Ebola virus antibodies in fruit bats, Bangladesh. Emerg Infect Dis. 2013; 19:270-3. http://dx.doi.org/10.3201/eid1902.120524
(6.) Yuan JF, Zhang YJ, Li JL, Zhang YZ, Wang LF, Shi ZL. Serological evidence of ebolavirus infection in bats, China. Virol J. 2012; 9:236. http://dx.doi.org/10.1186/1743-422X-9-236
(7.) Olival KJ, Hayman DTS. Filoviruses in bats: current knowledge and future directions. Viruses. 2014; 6:1759-88. http://dx.doi.org/ 10.3390/v6041759
(8.) Rollin P, Nichol S, Zaki S, Ksiazek T. Arenaviruses and filoviruses. In: Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D, editors. Manual of clinical microbiology. Washington (DC): ASM Press; 2011. p. 1514-29.
(9.) Zhai J, Palacios G, Towner JS, Jabado O, Kapoor V, Venter M, et al. Rapid molecular strategy for filovirus detection and characterization. J Clin Microbiol. 2007; 45:224-6. http://dx.doi.org/ 10.1128/JCM.01893-06
(10.) He B, Feng Y, Zhang H, Xu Lin, Yang W, Zhang Y, et al. Filovirus RNA in fruit bats, China [letter]. Emerg Infect Dis. 2015; 21:167577. http://dx.doi.org/10.3201/eid2109.150260
Supaporn Wacharapluesadee, Kevin J. Olival, Budsabong Kanchanasaka, Prateep Duengkae, Supakarn Kaewchot, Phimchanok Srongmongkol, Gittiyaporn Ieamsaard, Patarapol Maneeorn, Nuntaporn Sittidetboripat, Thongchai Kaewpom, Sininat Petcharat, Sangchai Yingsakmongkon, Pierre E. Rollin, Jonathan S. Towner, Thiravat Hemachudha
Author affiliations: King Chulalongkorn Memorial Hospital and Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (S. Wacharaplluesadee, N. Sittidetboripa, T Kaewpom, S. Petcharat, S. Yingsakmongkon, T Hemachudha); EcoHealth Alliance, New York, New York, USA (K.J. Olival); Department of National Parks, Bangkok (B. Kanchanasaka, S. Kaewchot, P. Srongmongkol, G. Ieamsaard, P. Maneeorn); Kasetsart University Faculty of Forestry, Bangkok (P. Duengkae); Centers for Disease Control and Prevention, Atlanta, Georgia, USA (P.E. Rollin, J.S. Towner)
Address for correspondence: Supaporn Wacharapluesadee, World Health Organization Collaborating Centre for Research and Training on Viral Zoonoses, King Chulalongkorn Memorial Hospital, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand; email: email@example.com
Table. Overview of bats and macaques tested by Ebola virus IgG ELISA or PCR for filoviruses, Thailand, 2011-2014 Species Host family No. tested (no. positive) Chiroptera Pteropus lylei Pteropodidae 500 (0) Cynopterus brachyotis Pteropodidae 10 (0) C. sphinx Pteropodidae 4 (0) Eonycteris spelaea Pteropodidae 12 (0) Macroglossus sobrinus Pteropodidae 2 (0) Megaerops niphanae Pteropodidae 1 (0) Rousettus amplexicaudatus Pteropodidae 3 (0) Hlpposlderos armlger Hipposideridae 113 (0) H. clneraceus Hipposideridae 4 (0) H. larvatus Hipposideridae 33 (0) H. lekagull Hipposideridae 158 (0) Megaderma lyra Megadermatidae 1 (0) Miniopterus magnate Vespertilionidae 132 (0) M. pusillus Vespertilionidae 1 (0) M. schreibersii Vespertilionidae 22 (0) Myotis horsfieldi Vespertilionidae 6 (0) M. muricola Vespertilionidae 1 (0) Rhinolophus shameli Rhinolophidae 44 (0) R. coelophyllus Rhinolophidae 7 (0) R. luctus Rhinolophidae 1 (0) R. malayanus Rhinolophidae 4 (0) R. microglobosus Rhinolophidae 1 (0) R. pusillus Rhinolophidae 1 (0) Scotophllus kuhlll Vespertilionidae 1 (0) Taphozous longimanus Emballonuridae 27 (0) T. melanopogon Emballonuridae 110 (0) Total 699 (0) Primate Macaca fascicularis Cercopithecidae 50 (0) Species Test method * Specimen type ([dagger]) Chiroptera Pteropus lylei ELISA Serum Cynopterus brachyotis PCR Pooled C. sphinx PCR Pooled Eonycteris spelaea PCR Pooled Macroglossus sobrinus PCR Pooled Megaerops niphanae PCR Pooled Rousettus amplexicaudatus PCR Pooled Hlpposlderos armlger PCR Pooled H. clneraceus PCR Pooled H. larvatus PCR Pooled H. lekagull PCR Pooled Megaderma lyra PCR Pooled Miniopterus magnate PCR Pooled M. pusillus PCR Pooled M. schreibersii PCR Pooled Myotis horsfieldi PCR Pooled M. muricola PCR Pooled Rhinolophus shameli PCR Pooled R. coelophyllus PCR Pooled R. luctus PCR Pooled R. malayanus PCR Pooled R. microglobosus PCR Pooled R. pusillus PCR Pooled Scotophllus kuhlll PCR Pooled Taphozous longimanus PCR Pooled T. melanopogon PCR Pooled Total Primate Macaca fascicularis PCR Pooled Species Location ([double dagger]) Chiroptera Pteropus lylei a Cynopterus brachyotis b C. sphinx b Eonycteris spelaea b Macroglossus sobrinus b Megaerops niphanae b Rousettus amplexicaudatus b Hlpposlderos armlger b H. clneraceus b H. larvatus b, c H. lekagull b Megaderma lyra b Miniopterus magnate b, c M. pusillus b M. schreibersii b Myotis horsfieldi b M. muricola b Rhinolophus shameli b R. coelophyllus c R. luctus b R. malayanus c R. microglobosus b R. pusillus b Scotophllus kuhlll b Taphozous longimanus b T. melanopogon b Total Primate Macaca fascicularis d * ELISA for IgG against Ebola virus. ([dagger]) Nucleic acid extraction from Pooled saliva, serum, and urine. ([double dagger]) a, Central Thailand; b, Eastern Thailand; c, Chaing Mai Province; d, Kao Chakani, Sa Kaeo Province.
|Printer friendly Cite/link Email Feedback|
|Author:||Wacharapluesadee, Supaporn; Olival, Kevin J.; Kanchanasaka, Budsabong; Duengkae, Prateep; Kaewchot,|
|Publication:||Emerging Infectious Diseases|
|Article Type:||Letter to the editor|
|Date:||Dec 1, 2015|
|Previous Article:||Genetic characterization of highly pathogenic avian influenza a(H5N6) virus, Guangdong, China.|
|Next Article:||Probability of spirochete borrelia miyamotoi transmission from ticks to humans.|