in Taiwan - Journal of Wildlife Diseases

DOI: 10.7589/2015-04-090

Journal of Wildlife Diseases, 51(4), 2015, pp. 923–928 # Wildlife Disease Association 2015

Rabies Virus Infection in Ferret Badgers (Melogale moschata subaurantiaca) in Taiwan: A Retrospective Study Jen-Chieh Chang,1,3,5 Kuo-Jung Tsai,1,5 Wei-Cheng Hsu,1 Yang-Chang Tu,1 Wei-Chieh Chuang,1 ChiaYi Chang,1 Shih-Wei Chang,2 Te-En Lin,2 Kuo-Yun Fang,2 Yung-Fu Chang,4 Hsiang-Jung Tsai,1 and ShuHwae Lee1,6,7 1Animal Health Research Institute, 376 Zhongzheng Rd., Danshui Dist., New Taipei City 25158, Taiwan; 2Endemic Species Research Institute, 1 Ming-shen East Road, Chichi Township, Nantou County 552, Taiwan; 3Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, 250 Kuo Kuang Rd., Taichung 402, Taiwan; 4Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-5786, USA; 5These authors contributed equally to this article; 6Current address: Animal Drugs Inspection Branch, Animal Health Research Institute, No. 21, Qiding, Zhunan Township, Miaoli County 35054, Taiwan; 7Corresponding author (email: [email protected])

2012). In Taiwan, domestic rabies cases have not been detected since 1961; along with Japan and Singapore, Taiwan had been considered rabies-free in recent decades. Nevertheless, an outbreak was detected among ferret badgers (Melogale moschata subaurantiaca) in 2013 (Chiou et al. 2014). Following the three confirmed index cases, more than 400 additional rabid animal cases had been identified by December 2014. Most of these cases were diseased and dead ferret badgers except for one house shrew (Suncus murinus), one domestic dog (Canis lupus familiaris), and five masked palm civets (Paguma larvata). The outbreak was spread over nine cities and counties ranging from central to southern and eastern Taiwan (Fig. 1). The Council of Agriculture and the Centers for Disease Control (Taiwan) applied measures to control wildlife rabies and to prevent human exposures (Wu et al. 2014). Based on preliminary analyses, nucleotide sequences were found to be similar between Taiwanese rabies virus isolates and mainland Chinese isolates. It was hypothesized that the virus was introduced several years ago and has since diverged evolutionarily. To test this hypothesis, we screened for rabies viral antigens and RNA in frozen ferret badger specimens. Fifteen frozen stored ferret badgers collected from 2010 to 2013 (Table 1) were shipped to the Animal Health Research Institute, New Taipei City, Taiwan.

Fifteen ferret badgers (Melogale moschata subaurantiaca), collected 2010–13 and stored frozen, were submitted for rabies diagnosis by direct fluorescent antibody test and reverse transcription PCR. We detected seven positive animal samples, including some from 2010, which indicated that the ferret badger population in Taiwan had been affected by rabies prior to 2010. Key words: Ferret badger, rabies, retrospective study, Taiwan.

ABSTRACT:

The rabies virus is a member of the genus Lyssavirus within the family Rhabdoviridae. Its genome is a negative-sense, single-stranded RNA of approximately 12,000 bases. The genome consists of five genes that encode the nucleoprotein (N), glycoprotein (G), matrix protein (M), RNA polymerase (L), and phosphoprotein (P) (Yousaf et al. 2012). Rabies is prevalent in most Asian countries. For example, in the Philippines 200 to 300 human cases are reported annually, with virus populations thought to have evolved independently on different islands (Saito et al. 2013). Rabies is thought to have been introduced into the Philippines around the early 19th (Gong et al. 2010) or early 20th (Meng et al. 2011) century. In mainland China, approximately 2,000 persons per year die of rabies and mortality rates have been increasing since 2000. The coverage of rabies vaccination in dogs is reported to be over 50% in metropolitan areas but fewer than 3% in rural areas (Yang and Dong 923

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JOURNAL OF WILDLIFE DISEASES, VOL. 51, NO. 4, OCTOBER 2015

FIGURE 1. Distribution of rabies-affected counties and cities in Taiwan. Shaded area includes the counties and cities where rabid ferret badgers were tested during the 2013 epidemic. Counties and cities where the ferret badgers were sampled in this study are indicated with their names.

Brain tissues of the ferret badgers were obtained through necropsy and subjected to direct fluorescent antibody test (FAT) for detecting rabies virus antigens as well as to nucleic acid extraction followed by reverse transcription PCR (RT-PCR) for rabies viral RNA. The FAT protocol was based on the World Organisation for Animal Health (OIE) Terrestrial Manual (OIE 2014) and the standard protocol for rabies diagnosis in the US (see “Protocol for Postmortem Diagnosis of Rabies in Animals by Direct

Fluorescent Antibody Testing” [www.cdc. gov/rabies/pdf/rabiesdfaspv2.pdf]). Briefly, smears of brain tissues were fixed to microscope slides with acetone and airdried. Smears were stained with fluorescein isothiocyanate–labeled monoclonal antirabies antibody (Fujirebio Diagnostics, Malvern, Pennsylvania, USA), washed with phosphate-buffered saline, and examined. The aggregates of rabies virus antigens showed brilliant apple green fluorescence (Fig. 2) detected by fluorescent microscopy. The stained smears were scanned at a magnification of 2003 for apple green inclusions. Morphologic examination to indicate rabies was performed at a 4003 magnification. Samples were rated rabies positive or negative based on a numerical scoring system of +1 to +4. A positive is at least +3 to +4 intensity and has a +2 to +4 distribution of antigen in slides made from brain stem, cerebellum, or hippocampus. A negative finding for rabies would be made by examination of at least the brain stem and cerebellum. A piece of the brain stem was homogenized and the RNA was extracted using TRIzol Reagent (Life Technologies, Grand Island, New York, USA). The N gene was amplified by RT-PCR using primer NF1 (59-AGAAGAAGCAGACAATGTCATCT-39) and NR3 (59-GCTCTGATTGCACTCGGATTGA-39). The RT-PCR was carried out in a reaction mixture containing 4.5 U AMV reverse transcriptase (Promega Co., Madison, Wisconsin, USA), 0.5 U Taq polymerase (Bertec Enterprise Co., New Taipei City, Taiwan), 6.25 nmole dNTP (Bertec Enterprise Co.), and 20 pmole of both primers. The reaction was performed with a G-Storm thermal cycler (G-Storm, Somerton, Somerset, UK) under the following conditions: 40 min at 42 C, 2 min at 95 C, 35 cycles at 95 C for 60 s, 50 C for 80 s, and 72 C for 60 s followed by a final extension of 7 min at 72 C. The product was analyzed on a 2% agarose gel and subjected to nucleotide sequencing by

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TABLE 1.

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Ferret badgers sampled 2010–13 in Taiwan and used for rabies diagnosis.

Sample no.

Date of sampling

Location

Rabies diagnosisa

2702 2703 2704 2705 2706 2707 2710 2711 2712 2713 2715 2717 2719 2014-3145 2014-3146

21 July 2010 20 March 2010 17 July 2010 10 November 2010 30 August 2012 23 October 2012 3 January 2013 20 November 2012 November 2012 19 November 2012 31 January 2013 17 March 2013 8 July 2013 5 August 2011 11 August 2011

Jhushan, Nantou County Jiji, Nantou County Guoshing, Nantou County Sanyi, Miaoli County Lugu, Nantou County Hengshan, Hsinchu County Jhushan, Nantou County Lugu, Nantou County Heping, Taichung City Douliou, Yunlin County Jhushan, Nantou County Jhongpu, Chiayi County Fushing, Taoyuan County Caotun, Nantou County Guoshing, Nantou County

Positive Negative Positive Negative Positive Negative Positive Negative Negative Negative Negative Positive Negative Positive Positive

a

The diagnosis was based on the results of a fluorescent antibody test and further supported by reverse transcription PCR.

commercial sequencing services (Mission Biotech, Taipei City, Taiwan). Nucleotide sequences were analyzed using Lasergene version 7 (DNASTAR, Inc., Madison, Wisconsin, USA). Sequences from 25 rabid ferret badgers diagnosed in July and August 2013, including the first three cases (OIE 2013), were used for comparing sequence similarity. Of the 19 frozen ferret badgers submitted, 15 provided quality brain tissues, and seven of the 15 were positive by FAT.

FIGURE 2. The aggregates of rabies virus antigens showed brilliant apple green fluorescence by the fluorescent antibody test. The stained smears of brain stem were scanned at a magnification of 2003. Bar550 mm.

The positive signals were detected in brain stem, cerebellum, hippocampus, or cerebrum. Of the seven positive ferret badgers, six were sampled in Nantou and one in Chiayi (Table 1). The RT-PCR and nucleotide sequencing confirmed that the RNA extracted from the seven FATpositive tissues was rabies virus–specific. The nucleotide sequences of the N gene from the seven positive ferret badgers were analyzed with the 25 sequences obtained from infected ferret badgers. The sequences from ferret badgers in Nantou County (Nt2704, 2706, 2710, 2014-3145, 2014-3146) were 98.7– 100.0% identical to those from Taichung and Nantou. The sequence from ferret badger Cy2717, sampled in Chiayi, was 97.3–99.4% identical to those from Southern Taiwan (Yunlin, Chiayi, Tainan, and Kaohsiung). Our retrospective study demonstrates that the 2013 epidemic of ferret badgers in Taiwan did not result from a newly introduced virus. Although the number of ferret badgers we examined was limited, ferret badger no. 2704, which was the earliest specimen among the positive cases, showed that rabies virus was present by 2010 (Table 1). These findings imply

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TABLE 2. N gene sequences of rabies viruses from ferret badgers in Taiwan used in the study.

Ferret badger no.

TW-1680 Th2408 Th2484 TW-1682 Nt1938 Nt1983 Nt2169 Nt2702 Nt2704 Nt2706 Nt2710 2014-3145 2014-3146 Cy2263 Cy2717 TW-1685 Tn1766 Tn1832 TW1694 Kh1830 Kh1879 Kh2261 YL2167 Pt2485 Pt2946 TW-1614 Tt2329 Tt2514 HL1956 R2012-26a R2012-88a R2013-01a a

Location (county or city)

Sequence accession no.

Taichung Taichung Taichung Nantou Nantou Nantou Nantou Nantou Nantou Nantou Nantou Nantou Nantou Chiayi Chiayi Tainan Tainan Tainan Kaohsiung Kaohsiung Kaohsiung Kaohsiung Yunlin Pingtung Pingtung Taitung Taitung Taitung Hualien Nantou Yunlin Nantou

KF501181 KP860167 KP860168 KF501182 KP860149 KP860150 KP860151 KP860155 KP860156 KP860157 KP860158 KP892527 KP892528 KP860138 KP860139 KF501184 KP860171 KP860172 KF501185 KP860142 KP860143 KP860144 KP860184 KP860160 KP860161 KF501180 KP860178 KP860179 KP860140 KF620487 KF620488 KF620489

Rabies viruses of the first three rabid ferret badgers in Taiwan according to official report on the World Organisation for Animal Health (OIE) database.

that this epidemic in ferret badgers began earlier but was not recognized until 2013. The phylogenetic tree based on the N gene indicated that five retrospective rabies viruses were clustered with current rabies viruses in ferret badgers, including the first three cases (Fig. 3). Comparison of the N genes in the seven positive ferret badgers to those in the 25 current rabies cases (Table 2) showed .91% identity. Consistently, the viruses isolated from ferret badgers in Fujiang and Jiangxi, two southeastern provinces in mainland China, gave 98.7–99.1% nucleotide identity in full-length N protein

genes (Liu et al. 2010), also revealing a close genetic relationship among ferret badger rabies virus in the same area. In contrast, in southeastern China a rabies virus isolated from a ferret badger in Zhejiang Province had only 89% nucleotide identity to those isolated from dogs in the same province (Zhang et al. 2009). The differences suggested that the virus circulates within a single-species wildlife population, such as the case of the ferret badger, indicating a limited host range virus population that has retained genetic stability. Interchanges of viral gene pools among host species in the same ecosystem could be significantly limited in Taiwan, at least for the time being. Ferret badgers collected from northwestern Taiwan (Miaoli, Hsinchu, and Taoyuan) were negative, consistent with conclusions that the current epidemic was a localized event. Geographic isolation by rivers and higher urbanization in northern Taiwan may contribute to the lack of rabies spread to northern Taiwan. The other domestic animals diagnosed with rabies by May 2015 were one juvenile domestic dog, one house shrew, and five masked palm civets. These results should be noted by veterinarians or wildlife conservation workers in order to protect themselves when in contact with suspected animals. Although few spillover (cross-species transmission) cases from these ferret badger populations were detected under the surveillance program, ferret badgers and other potential wildlife reservoir populations still constitute a health risk. Wildlife rabies awareness should be altered because potential rabies transmission from ferret badgers to humans, dogs, and wildlife cannot be excluded (Liu et al. 2010). The Council of Agriculture (COA), Taiwan, has already implemented strategies for wildlife rabies control including health education, animal surveillance, companion animal management and vaccination, a diagnostic laboratory, study and research for ferret badger rabies virus, vaccination of ferret badgers, and epidemiology. The COA provided animal vaccines to reinforce the

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FIGURE 3. Phylogenetic analysis of rabies viruses from ferret badgers in Taiwan. The evolutionary tree was inferred based on the nucleoprotein gene using the neighbor-joining method and was rooted as CTN181(GenBank accession no. EF564174). The percentage (.70) of replicate trees in which the associated viruses clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. Within the tree, seven retrospective samples (Nt2702, Nt2704, Nt2706, Nt2710, Cy2717, 2014-3145, 2014-3146) are presented as closed circles and the first three rabid ferret badgers (R2012-26, R2012-88, R2013-01) are presented as triangles. The tree indicated that seven retrospective samples were clustered with rabies viruses of the current epidemic, including the first three cases in ferret badgers.

vaccination of dogs and cats in hot spots (mountainous enzootic area). In addition, the Rabies Control Central Epidemic Command Center established on 1 August 2013 has provided effective interministerial collaboration to achieve the goal of “no human infection cases” (Inoue and Fei 2014). The use of pre- and postexposure prophylaxis in humans had also been recommended and would be provided when humans are bitten by wildlife or pet animals in enzootic areas. We thank Fan Lee for assistance in drafting this manuscript; our collaborators in Endemic Species Research Institute, and Wei-Lien Chi (Department of Veterinary Medicine, National Pingtung University of Science and Technology) for

submitting specimens. Financial support for this research was provided by grant 102AS-10.1.1-HI-H2 from the Council of Agriculture, Executive Yuan, Taiwan. LITERATURE CITED Chiou HY, Hsieh CH, Jeng CR, Chan FT, Wang HY, Pang VF. 2014. Molecular characterization of cryptically circulating rabies virus from ferret badgers, Taiwan. Emerg Infect Dis 20:790–798. Gong W, Jiang Y, Za Y, Zeng Z, Shao M. 2010. Temporal and spatial dynamics of rabies viruses in China and Southeast Asia. Virus Res 150:111–118. Inoue S, Fei CY. 2014. Epidemiology of rabies in Taiwan and the capacity building of rabies diagnosis in Japan. J Vet Epidemiol 18:11–17. Liu Y, Zhang S, Wu X, Zhao J, Hou Y, Zhang F, Velasco-Villa A, Rupprecht CE, Hu R. 2010. Ferret badger rabies origin and its revisited

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