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Surveillance for ebola virus in wildlife, Thailand.

To the Editor: Active surveillance for zoonotic pathogens in wildlife is particularly critical when the pathogen has the potential to cause a large-scale outbreak. The recent outbreak of Ebola virus (EBOV) disease in West Africa in 2014 was initiated by a single spillover event, followed by human-to-human transmission (1). Projection of filovirus ecologic niches suggests possible areas of distribution in Southeast Asia (2). Reston virus was discovered in macaques exported from the Philippines to the United States in 1989 and in sick domestic pigs in the Philippines in 2008 (with asymptomatic infection in humans) (3). Dead insectivorous bats in Europe were found to be infected by a filovirus, similar to other members of the genus Ebolavirus (4).

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.

DOI: http://dx.doi.org/10.3201/eid2112.150860

References

(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: spwa@hotmail.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.
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Title Annotation:LETTERS
Author:Wacharapluesadee, Supaporn; Olival, Kevin J.; Kanchanasaka, Budsabong; Duengkae, Prateep; Kaewchot,
Publication:Emerging Infectious Diseases
Article Type:Letter to the editor
Geographic Code:9THAI
Date:Dec 1, 2015
Words:1871
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