台灣野生食肉目動物疾病監控調查：鼬獾狂犬病毒鑑定與特性分析首例

國立臺灣大學獸醫專業學院獸醫學研究所 博士論文

Graduate Institute of Veterinary Medicine School of Veterinary Medicine

National Taiwan University Doctoral Dissertation

台灣野生食肉目動物疾病監控調查：鼬獾狂犬病毒鑑定 與特性分析首例

Disease Surveillance and Monitoring in Wild-Ranging Carnivores in Taiwan: The First Identification and Characterization of Rabies Virus in

Ferret Badgers

邱慧英

Hue-Ying Chiou

指導教授：龐 飛 博士 鄭謙仁 博士 Advisors: Dr. Victor Fei Pang

Dr. Chian-Ren Jeng

中華民國 104 年 6 月

June 2015

謹以此論文

獻給

我的師長、家人、朋友

以及直接或間接參與台灣野生動物救傷、疾病監測和

狂犬病研究的夥伴們

目錄 Contents

摘 要

I

Abstract III

Chapter I

General introduction

1

Chapter II

Disease Surveillance in Rescued and Road-killed Wild- Ranging Carnivores in Taiwan

Taiwan Veterinary Journal 2015, 41:1-12

17

Chapter III

Pathological Characterization and Molecular Detection of Rabies Virus in the Rabid Ferret Badgers of a Recent Outbreak in Taiwan

Journal of Wildlife Diseases, 2015, accepted

30

Chapter IV

Molecular Characterization of Cryptically Circulating Rabies Virus from Ferret Badgers, Taiwan

Emerging Infectious Diseases, 2014, 20:790-798

64

Chapter V

Conclusions

80

I

摘要

本研究的目的是調查台灣野生食肉目動物死亡與潛在病因，並針對發現的 重要或有趣的疾病進行深入研究。在 2011 年 8 月至 2015 年 1 月期間共收集 51 例救傷死亡或路死的食肉目動物屍體，經由詳細解剖與組織病理學檢查、分子 與免疫學分析、微生物學及寄生蟲學檢測等進行病因分析。這些案例包括 31 例 台灣鼬獾(TWFBs) (Melogale moschata subaurantiaca)、12 例白鼻心(MPCs) (Paguma larvate taivana)、5 例麝香貓(SCCs) (Viverricula indica pallida)及 3 例食蟹獴(CEMs) (Herpestes urva)。人畜共通狂犬病與致死性犬瘟熱分別於 4 例 台灣鼬獾與 3 例白鼻心被確診。

台灣鼬獾狂犬病的特徵性病理變化為非化膿性腦膜腦脊髓炎、神經節炎及 形成典型的細胞質內包涵體- Negri bodies，受影響的中樞神經系統以腦幹病變 最為嚴重。此外，大腦皮質部、丘腦與腦幹的神經元與神經氈具有不同程度的 海綿狀變性。在非神經組織的代表性病變包括腎上腺壞死與間質淋巴球性唾液 腺炎。經由免疫組織化學染色法(IHC)與螢光抗體試驗(FAT)，病毒抗原可在神 經組織的神經元細胞與軸突/樹突等區域，以及全身不同組織間的巨噬細胞中被 偵測到。結果顯示，腦幹、大腦皮質部、海馬角、丘腦與下視丘是台灣鼬獾狂 犬病分子診斷的理想的採樣區。我們利用不同單位提供的 2004-2012 年間福馬 林固定、石蠟包埋的舊有台灣鼬獾腦組織，進行免疫組織化學染色回溯性研 究，發現狂犬病陽性案例最早可追溯至 2004 年。

為了分析台灣鼬獾狂犬病病毒的源起，我們完成三株狂犬病病毒全基因體 定序分析，並由公告的幾株狂犬病毒(RABV)核蛋白(N)與醣蛋白(G)序列進行親 緣地理學分析顯示，台灣鼬獾狂犬病病毒（RABV-TWFB）來自亞洲譜系並已 獨立演化，其近緣病毒株 China I (包括中國鼬獾狂犬病病毒株; RABV-CNFB) 和菲律賓狂犬病病毒的分化年代在 158-210 年前，而台灣鼬獾狂犬病病毒株的

II

最近共祖起源年代約在 91-113 年前。我們的研究顯示，此次地方性台灣鼬獾狂 犬病病毒基因分析的古老親緣結果說明了台灣鼬獾的狂犬病病毒株可能隱晦的 潛藏在環境中長期的慢慢傳播，而致一直未被檢出，此病毒與宿主間的交互作 用及其潛存的機制值得進一步研究。

III

Abstract

The objective of this study was to investigate the causes of death and potential diseases carried by the wild-ranging carnivores in Taiwan. For those interesting and essential diseases, further studies in depths were performed. A total of 51 carcasses from rescued but dead or road-killed carnivores, collected during the period of August 2011 to January 2015, were necropsied for histopathology, molecular and

immunological assays, microbiology, and parasitology. The cases included 31 Taiwan ferret badgers (TWFBs) (Melogale moschata subaurantiaca), 12 masked palm civets (MPCs) (Paguma larvate taivana), 5 small Chinese civets (SCCs) (Viverricula

indica pallida), and 3 crab-eating mongooses (CEMs) (Herpestes urva). Zoonotic

rabies and fatal canine distemper were diagnosed in 4 TWFBs and 3 MPCs, respectively.

The characteristic pathological changes of rabid TWFBs were nonsuppurative meningoencephalomyelitis, ganglionitis, and formation of typical intracytoplasmic Negri bodies with brain stem affected the most. Additionally, variable spongiform degeneration, primarily in the perikaryon of neurons and neuropil, was observed in the cerebral cortex, thalamus, and brain stem. In the non-nervous tissue,

representative lesions included adrenal necrosis and lymphocytic interstitial

sialoadenitis. By immunohistochemical (IHC) staining as well as fluorescent antibody test (FAT), positive viral antigens were detected in the perikaryon of the neurons and axonal and/or dendritic processes in the nervous tissue and in the macrophages scattered in various tissues throughout the body. The findings suggest that brain stem, cerebral cortex, hippocampus, thalamus and hypothalamus are ideal sampling regions for molecular diagnosis of RABV in TWFBs. Retrospective study using archived

IV

formalin-fixed and paraffin-embedded tissues of TWFBs revealed the earliest IHC- positive rabid TWFB case in 2004.

To examine the origin of this viral strain, we sequenced three complete genomes and acquired multiple rabies virus (RABV) nucleoprotein (N) and glycoprotein (G) sequences. Phylogeographic analyses demonstrated that the RABV of TWFB (RABV- TWFB) is a distinct lineage within the Asian group, and has been differentiated from its closest lineages, China I (including Chinese ferret badger isolates; RABV-CNFB) and Philippines, 158-210 years before present. The most recent common ancestor of RABV-TWFB was originated 91-113 years ago. The ancient origin of the endemic RABV-TWFB illustrates that this RABV variant could be cryptically circulated in the environment without being recognized for a long period of time. The underlying mechanism is worthy of further study and may shed light on the complex interaction between RABV and its host.

Chapter I

General Introduction

1.1 Background

Zoonoses are a growing concern. The concept of “One World, One Health” has recently been repeatedly stressed, indicating a worldwide tight link between animal diseases and public health. It is known that approximately 60% of known human pathogens and over 75% of the emerging and re-emerging human diseases in the past two decades originate from domestic or wild animals; moreover, approximately 80%

of the pathogens that could potentially be used in bioterrorism are of animal origin as well (Dehove, 2010). Additionally, wild animals often act as sentinels for animal diseases, which could serve as an effective management and control of these diseases in domestic animals. Therefore, surveillance of wildlife diseases must be considered as equally important as surveillance and control of diseases in domestic animals (Dehove, 2010). However, owing to the fact that symptoms and signs of disease in wildlife are not as readily observed as in domestic animals and specimens for

laboratory analysis are more difficult to collect, the implementation of early detection of and response to disease outbreaks thus usually relatively slow.

The new and emerging wildlife diseases may pose a serious risk to animal welfare, human health, wildlife conservation, and economic productivity. Ebola virus infection is a good example, which has caused 26 outbreaks mainly in Central and West Africa since its first discovery in 1976, and the most recent outbreak in West Africa has resulted in 10,460 human deaths by the end of March 2015

(http://apps.who.int/ebola/current-situation/ebola-situation-report-1-april-2015-0).

Current evidence has strongly implicated that bats, including fruit bats and

insectivorous bats, are the reservoir hosts for Ebola viruses. It has been suggested that

uptake of partially eaten fruits and pulp dropped by Ebola virus-infected fruit bats by land mammals such as gorillas and duikers forms a possible indirect transmission chain from the natural host to animal populations; the outbreaks in human are

acquired initially via close contact with these infected bats or land mammals followed by close contact among people (Leroy et al., 2004).

Although dogs are considered the principal host of rabies, rabies virus (RABV) infection is also dispersed in many species of wild Carnivora and Chiroptera, which have become the major source of human infection in many countries in Europe and North America where dog vaccination programs are well established (Barbosa et al., 2008). It was estimated that rabies caused over 60,000 human deaths in the world in 2010, primarily in Africa and Asia (World Health, 2013). Avian influenza (AI) is an infectious viral disease of birds, which often causes no apparent signs of illness in wild water fowl such as ducks and geese. AI viruses can sometimes spread to

domestic poultry and cause large-scale outbreaks of serious disease leading to severe economic impact such as H5N1, H5N8, H7N1, H7N9 (Sartore et al., 2010), some of which have also been reported to cross the species barrier and cause disease in

humans and other mammals such as H5N1, H7N9 (Cowling et al., 2013; Leong et al., 2008; Li et al., 2014).

Disease surveillance in animals is the key for early detection of the underlying, new or even emerging diseases (Cox-Witton et al., 2014). Wildlife disease

surveillance has become an integral component in the identification and control of emerging animal and zoonotic diseases potentially hazard to human and domestic animal welfare (Daszak et al., 2000). Both passive and active surveillance strategies have been used in wildlife disease investigation (Stallknecht, 2007). Government-

supported active and passive wildlife disease survey programs have long been carried in Taiwan; however, zoo animals were often the major target simply because the majority of these exhibited animals were imported and the varieties and population of native wild-ranging wild animals are very limited. Owing to the more and more important role of wildlife on the emerging and re-emerging disease development in and between animals and humans, the target in the wildlife disease surveillance program has been readjusted and started to switch gradually from admixture of zoo and native wild-ranging wildlife to native wild-ranging wildlife only since 2011.

Through this program, carcasses of native wild-ranging wildlife have been routinely submitted either to the National Taiwan University (NTU) or to the National Pingtung University of Science and Technology for disease surveillance and monitoring.

1.2 Rabies

Rabies is possibly one of the oldest zoonotic disease. It is caused by the rabies virus (RABV), a neurotropic virus belonging to the Lyssavirus of Rhabdoviridae (Warrell and Warrell, 2004). Rabies is a worldwide disease and only a small number of countries and regions are free from this disease. The RABV infects nearly all warm-blooded animals causing progressive, severe neurological signs and death invariably (Jackson and Fu, 2013). According to World Health Organization (WHO), there were over 60,000 rabies-associated human deaths worldwide in 2010, primarily in Africa and Asia (WHO, 2013). The virus cannot penetrate intact skin; thus, bitten by a rabid animal is the most important route of transmission in humans. Dogs are considered as the principal host of the disease in developing countries. In developed countries such as the United States, the important sources of transmission include bats and wild carnivores; animal exposures in other countries; and rare cases of infection

via inhalation or organs transplant. Interestingly, only 40% to 50% of victims of bites from rabid animals will develop rabies (Kauffman and Goldmann, 1986).

1.3 Genome of rabies virus

The RABV is enveloped and contains a single-stranded, negative-sense

ribonucleic acid (RNA) genome of approximately 12 kb (Holmes et al., 2002; Yousaf

et al., 2012). The genome of RABV encodes five genes whose order is highly

conserved. These genes code for nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L), are arranged in a strictly conserved order (3’-N-P-M-G-L-5’) (Dietzschold et al., 1983;

Nagaraja et al., 2008; Wunner and Conzelmann, 2013). All rhabdoviruses have two major structural components, a helical ribonucleoprotein (RNP) core and the

surrounding envelop. The N protein forms the RNP core with the P and L proteins and plays a critical role in transcription and replication (Kuzmin et al., 2012; Nagaraja et

al., 2008). The remaining two structural proteins of the rabies virion, the G and M, are

associated with the lipid-bilayer envelope that surrounds the RNP core. N protein is involved in the encapsidation of the genome RNA and protection of the RNA from endogenous ribonucleases activity (Wunner and Conzelmann, 2013) as well as the modulation of viral RNA transcription and replication. The P protein is capable of oligomerization or binding to the nucleoprotein-RNA template (Gigant et al., 2000); it interacts with the cytoplasmic dynein light chain protein, which helps the axoplasmic transport of viral nucleocapsid; it also acts as a chaperone of soluble nascent N protein (Raux et al., 2000; Yang et al., 1999). The M protein is involved in the down

regulation of viral RNA transcription, condensation of helical nucleocapsid cores into tight coils, association with membrane bilayers, and cytopathogenesis of infected cells

(Wunner and Conzelmann, 2013). It confers the characteristic bullet shape to the virion. The G protein forms approximately 400 trimeric spikes which are tightly arranged on the surface of the virus; it plays important roles in the attachment of RABV to the host-cell surface as well as in the pathogenicity and neurovirulence of RABV (Morimoto et al., 1999; Tuffereau et al., 1989). The L protein is the

polymerase of the RNP core and is involved in the majority of enzymatic activities of the polymerase complex in viral RNA transcription and replication (Morimoto et al., 1999; Nagaraja et al., 2008).

1.4 Rabies in wildlife

Wildlife has become an important source of RABV infection in many developed countries. Prior to 1951, domestic dogs and cats were the major source of rabies in the United States. More than 8,000 cases were reported among dogs in 1946. However, with the institution of vaccination programs for domestic animals carried on

throughout the United States in the 1940s, the prevalence shifted from dogs and cats to wildlife. Rabies in wild animals, particularly bats, raccoons, foxes, and skunks, has accounted for almost 85% of all reported cases in the United States since 1976

(Kauffman and Goldmann, 1986; Krebs et al., 2003a). The disease is generally regarded as having a single-species reservoir with spillover to other dead-end hosts (Krebs et al., 2003a; Krebs et al., 2003b). It is known that mustelids, including the genera Melogale, Meles, and Mellivora of the weasel family Mustelidae, are also susceptible to rabies (Barnard, 1979; Wandeler et al., 1974a; Wandeler et al., 1974b;

Wandeler et al., 1974c). The Chinese ferret badger (CNFB) (Melogale moschata

moschata) has been considered as a primary rabies host and source of human rabies in

southeast China (Liu et al., 2010; Zhang et al., 2009; Zhenyu et al., 2007).

In natural or experimental RABV infection, non-suppurative

meningoencephalomyelitis and ganglionitis are the characteristic histopathological findings (Abreu et al., 2014; Balachandran and Charlton, 1994; Bundza and Charlton, 1988; Charlton et al., 1983; Stein et al., 2010). However, the severity and distribution of the lesion and the amount and distribution of viral antigens are variable, depending on the host, viral strain, and clinical course (Balachandran and Charlton, 1994;

Charlton, 1984; Charlton et al., 1987a; Charlton et al., 1987b; Hamir et al., 1992;

Hamir et al., 2011).

1.5 History of rabies in Taiwan

The earliest written record regarding human rabies case in Taiwan could be traced back to the early nineteenth century when Taiwan was still under the colonial rule of Japanese. Starting from 1930, the Japanese began to control rabies in Taiwan by producing inactivated vaccine for prevention and treatment of human rabies and for canine use along with instituting strict dog registration and poisoning stray dogs.

Prior to 1948 when rabies re-emerged in Taiwan, there had been no record of rabies for more than ten years. Following the ending of the World War II, traffics between Taiwan and Shanghai, Hongkong or Hainan, where rabies existed, became heavy. On June 17, 1948, a human rabies case originated from Shanghai was diagnosed at the Taiwan University Hospital. Owing to the lack of infectious disease specialist during the period of 1948 to 1958, dog-bite human cases were treated simply by surgeons without prevention and/or post-exposure vaccination and immunoglobulin treatment.

A total of 782 deaths occurred during the outbreak with the highest number of 238 deaths in 1951 followed by102 deaths in 1952. In 1956, the Joint Commission on

Rural Reconstruction and the Taiwan Provincial Health Department instituted rabies

control measures, including vaccinating dogs with vaccines imported from the United States and culling stray dogs to control animal reservoir. Following the successful control measures, human death from rabies ended in 1958 and the last rabid dog case was reported in 1961 (Liu, 2013). Since then, Taiwan declared to have eradicated rabies and listed as rabies-free by the World Organisation for Animal Health (OIE)

(Wu et al., 2014) until 2013. Prior to the re-emerging of rabies in 2013, there were 3

imported dog-bite human rabies cases, in which 2 cases were from China occurring in 2002 and 2012 and 1 case was from the Philippines in 2013; all these three patients died. The Bureau of Animal and Plant Inspection and Quarantine (BAPIQ) of the Council of Agriculture has contracted National Taiwan University to conduct disease surveillance of wild animals since 2011. In 2013, rabies was added to the disease list under surveillance and in the same year rabies was diagnosed in Taiwan ferret- badgers (TWFBs) (M. moschata subaurantiaca) (Chiou et al., 2014). The rabid TWFBs showed severe encephalopathy, but repeated testing for etiologies causing encephalopathy, including canine distemper and measles, failed to identify the cause of death; however, the results of RT-PCR for RABV turned out to be positive in June 2013. Following reporting the findings to BAPIQ on June 24, the specimens were then submitted to the Animal Health Research Institute, Council of Agriculture, on June 26 for confirmation, After the Council of Agriculture convened a rabies expert meeting on July 16, the diagnosis of rabies was confirmed and the incident was reported to OIE on July 17. May 23 of 2012, the date of the first rabid TWFB submitted for examination, became the onset date of the current endemic.

1.6 Objectives of study

Disease surveillance in rescued and road-killed wild-ranging carnivores in

Taiwan (Chapter II)

Recent highly pathogenic avian influenza and rabies events have shown just how important disease surveillance of free-ranged wildlife can be in dealing with zoonotic diseases. Controlling the pathogen at its source in animals could help to avoid

subsequent public health problems. This study was part of a continual disease surveillance and monitoring program in captive and free-range wildlife. Routine clinical observation and pathological examination for wildlife in Taiwan were performed. The gross and histopathological examination for each animal was

recorded. In addition, bacteriological, parasitic and PCR examination were included.

Potential zoonotic and important infectious diseases were monitored in order to prevent their spreading to humans and domestic animals. Results obtained from the study can provide information regarding the current status of wildlife diseases in Taiwan to the government, which will be beneficial to animal welfare, human health, wildlife conservation, and economic productivity.

Pathological characterization and viral detection in rabid Taiwan ferret badgers (Chapter III)

Wildlife has become an important source of RABV infection in many developed countries due to well established canine vaccination programs. The Chinese ferret badger (CNFB) (Melogale moschata moschata) is known to be a primary rabies host and source of human rabies in southeast China. It is known that the severity and distribution of rabies-associated pathological changes and the amount and distribution of viral antigens vary among different hosts, viral strains, and

durations of the clinical disease. Although rabies-associated lesions have been reported in various animal species and in human beings, the associated pathological

changes and the distribution of viral antigens have not yet been fully described in ferret badgers (FBs). However, the information is essential for establishing more accurate sampling and diagnosis modalities for FB-associated rabies, especially when composite sampling is not feasible. The present study was to characterize the

pathological changes and the pattern of viral antigen distribution regarding the recent outbreak of rabies in TWFBs.

Genomic organization and characterization of the rabies virus of Taiwan ferret badger-associated rabies (Chapter IV)

The recent outbreak of rabies in TWFBs has caused widespread panic among public. As there has been no reported rabid case in dogs or humans for more than 50 years in Taiwan, it is important to understand whether the current outbreak is an

emerging, a re-emerging or actually a cryptically circulated disease, its relations with

the other RABV lineages. The present study tried to clarify whether the current

outbreak of the Taiwanese ferret badger-associated rabies (TWFB-AR) is an emerging, a re-emerging or actually a cryptically circulated disease. The possible origin of this outbreak and its relations with the CNFB-associated rabies (CNFB-AR) in mainland China via the genomic organization and characterization as well as the analysis of genetic diversity and phylogeographic origin of RABV-TWFB were investigated. In addition, a possible mechanism contributing to the limited host range of RABV-TWFB is also discussed.

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Chapter II

Disease Surveillance in Rescued and Road-killed Wild-Ranging

Carnivores in Taiwan

Reprinted from Taiwan Veterinary Journal, 2015, Authors: Hue-Ying Chiou,

Kuang-Sheng Yeh, Chian-Ren Jeng, Hui-Wen Chang, Li-Jen Chang, Ying-Hui

Wu, Fang-Tse Chan, Victor Fei Pang, Title of article: Disease Surveillance in

Rescued and Road-killed Wild-Ranging Carnivores in Taiwan, 41(2):73-84,

copyright (2015) with permission from Chinese Society of Veterinary Science and

World Scientific Publishing Co. Pte. Ltd.

DISEASE SURVEILLANCE IN RESCUED AND ROAD-KILLED WILD-RANGING

CARNIVORES IN TAIWAN

Hue-Ying Chiou

^*

, Kuang-Sheng Yeh

^*

, Chian-Ren Jeng

^*,†

, Hui-Wen Chang

^*,†

, Li-Jen Chang

^‡

, Ying-Hui Wu

^‡

,

Fang-Tse Chan

^§

and Victor Fei Pang

^*,†,¶

*Graduate Institute of Veterinary Medicine School of Veterinary Medicine

National Taiwan University Taipei 10617, Taiwan, ROC

†Graduate Institute of Molecular and Comparative Pathobiology School of Veterinary Medicine, National Taiwan University

Taipei 10617, Taiwan, ROC

‡Veterinary O±ce, Taipei Zoo, Taipei 11656, Taiwan, ROC

§Endemic Species Research Institute, Council of Agriculture Executive Yuan, Nantou 55244, Taiwan, ROC

Received 9 April 2015 Accepted 17 April 2015 Published 12 June 2015

ABSTRACT

The objective of this study was to investigate the causes of death and potential diseases carried by the wild-ranging carnivores in Taiwan through a government-supported disease survey program. During the period of August 2011 to January 2015, a total of 51 carcasses from rescued but dead or road-killed carnivores were necropsied for histopathology, molecular and immunological assays, microbiology, and parasitology. The cases included 31 Taiwan ferret badgers (TWFBs) (Melogale moschata subaurantiaca), 12 masked palm civets (MPCs) (Paguma larvate taivana), 5 small Chinese civets (SCCs) (Viverricula indica pallida), and 3 crab-eating mongooses (CEMs) (Herpestes urva). Zoonotic rabies and fatal canine distemper were diagnosed in four TWFBs and three MPCs, respectively. A high prevalence rate of lungworm infestation (23/31; 74.2%) was observed in TWFBs. In addition, a unique fatal Staphylococcus hyicus pneumonia and a fatal heavy systemic sarcopic mange infestation were diagnosed in a TWFB and a suckling MPC kid, respectively. Road tra±c accidents and stray dog-associated killing were the most common etiologies for the death of wild-ranging carnivores.

Keywords: Carnivores; Taiwan ferret badger; Masked palm civet; Small Chinese civet; Crab- eating mongoose; Rabies; Canine distemper.

¶Corresponding author: Victor Fei Pang, Graduate Institute of Molecular and Comparative Pathobiology, School of Veterinary Medicine, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan, ROC. Tel:þ886-2-33663867; Fax:

þ886-2-23621965; E-mail: [email protected] Taiwan Veterinary Journal, Vol. 41, No. 2 (2015) 73–84 DOI:10.1142/S1682648515500067

73 Taiwan Vet J 2015.41:73-84. Downloaded from www.worldscientific.com by NATIONAL TAIWAN UNIVERSITY on 06/28/15. For personal use only.

18

INTRODUCTION

Wildlife has become widely recognized to play an im- portant role in the epidemiology of emerging and zoo- notic diseases.^1–4The new and emerging wildlife diseases may pose a serious risk to animal welfare, human health, wildlife conservation, and economic productivity. Ebola virus infection is a good example, which has caused 26 outbreaks mainly in Central and West Africa since its

¯rst discovery in 1976, and the most recent outbreak in West Africa has resulted in 10,460 human deaths by the end of March 2015 (http://apps.who.int/ebola/current- situation/ ebola-situation-report-1-april-2015-0). Cur- rent evidence has strongly implicated that bats, includ- ing fruit bats and insectivorous bats, are the reservoir hosts for Ebola viruses. It has been suggested that uptake of partially eaten fruits and pulp dropped by Ebola virus- infected fruit bats by land mammals such as gorillas and duikers forms a possible indirect transmission chain from the natural host to animal populations⁵; the outbreaks in human are acquired initially via close contact with these infected bats or land mammals followed by close contact among people.

Although dogs are considered the principal host of rabies, rabies virus (RABV) infection is also dispersed in many species of wild Carnivora and Chiroptera, which have become the major source of human infection in many countries in Europe and North America where dog vaccination programs are well established.^6,7It was es- timated that rabies caused over 60,000 human deaths in the world in 2010, primarily in Africa and Asia.⁸Avian in°uenza (AI) is an infectious viral disease of birds, which often causes no apparent signs of illness in wild water fowl such as ducks and geese. AI viruses can sometimes spread to domestic poultry and cause large- scale outbreaks of serious disease leading to severe eco- nomic impact such as H5N1, H5N8, H7N1, H7N9,⁹some of which have also been reported to cross the species barrier and cause disease in humans and other mammals such as H5N1, H7N9.^10–12

Disease surveillance in animals is the key for early de- tection of the underlying or even emerging diseases.¹³ Wildlife disease surveillance has become an integral com- ponent in the identi¯cation and control of emerging ani- mal and zoonotic diseases potentially hazardous to human and domestic animal welfare. Both passive and active surveillance strategies have been used in wildlife disease investigation.¹⁴There are numerous situations in which it is important to determine whether a particular disease of interest is present in a free-ranging wildlife population.

However, adequate disease surveillance can be labor- intensive and expensive and thus there is substantial

motivation to conduct it as e±ciently as possible. Sur- veillance is often based on the assumption of a simple random sample, but this can almost always be improved upon if there is auxiliary information available about disease risk factors.¹⁵Owing to the fact that conducting an unbiased surveillance in free-ranging mammal popula- tions is often more challenging, the passive opportunistic case identi¯cation is, thus, generally more often used for the detection of disease events in wild animals.¹⁶

Government-supported active and passive wildlife disease survey programs have long been carried in Taiwan, however, zoo animals were often the major target simply because the majority of these exhibited animals were imported and the varieties and population of native wild-ranging wild animals are very limited.

Owing to the well-known role of wildlife on the emerging and re-emerging disease development in and between animals and humans, the target in the wildlife disease surveillance program has been readjusted and switched to the native wild-ranging wildlife since 2011. Through this program, carcasses of native wild-ranging wildlife have been routinely submitted either to the National Taiwan University (NTU) or to the National Pingtung University of Science and Technology for disease sur- veillance and monitoring. We report herein the ¯ndings of the causes of death and potential diseases carried by the native wild-ranging carnivores in Taiwan at NTU through the wildlife disease surveillance program during the period of August 2011 to January 2015.

MATERIALS AND METHODS Animals

During the period of August 2011 to January 2015, 51 carcasses of free-ranging carnivores from wildlife ¯rst aid station, and the rescue center, animal disease control center and/or animal protection and health inspection o±ce of di®erent regions of Taiwan were submitted to the School of Veterinary Medicine at NTU for routine disease surveillance (Table 1). These cases included 31 Taiwan ferret badgers (TWFBs) (Melogale moschata subaurantiaca), 12 masked palm civets (MPCs) (Paguma larvate taivana), 5 small Chinese civets (SCCs) (Viver- ricula indica pallida), and 3 crab-eating mongooses (CEMs) (Herpestes urva).

Sample Collection

Full necropsy was performed and representative tissue samples, including cerebrum, cerebellum, brain stem, 74 H.-Y. Chiou et al.

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liver, spleen, lungs, heart, tongue, esophagus, gastroin- testinal (GI) tract, pancreas, kidney, adrenal gland, urinary bladder, testicle, ovary, uterus, skeletal muscle, and haired skin, were collected. The tissue samples were

¯xed in 10% neutral bu®ered formalin and processed routinely for histopathological examination. Fresh tis- sues, especially brain, lung, and liver, were collected from each animal and stored at
80^C for subsequent nucleic acid extraction.

Histopathological Examination

All tissue blocks were sectioned at 5
m, stained with hematoxylin and eosin (H&E), and examined by light microscopy.

Microbiological Analysis

Where gross lesions suggestive of a bacterial infection were seen, tissue specimens were inoculated onto 5%

sheep blood agar (CMP^TMÞ and incubated at 37^C for overnight. Suspected colonies were further identi¯ed using a Vitek 2 Compact automated system (Biom
erieux, Marcy-I'Etoile, France) to the species level.

Immunohistochemical Staining

The IHC staining was performed using the advanced Super Sensitive^TMPolymer-HRP IHC Detection System (BioGenex Lab., Fremont, CA, USA) according to the manufacturer's instructions with primary mouse anti- Rabies virus (RABV) glycoprotein IgG2a (Abcam Inc., Boston, MA, USA) and anti-Canine distemper virus (CDV) nucleoprotein IgG2b (Santa Cruz Biotechnology Inc., Dallas, Texas, USA) monoclonal antibodies.

Depara±nized slides were treated with 100
g/mL pro- teinase K (AppliChem Inc, St. Louis, MO, USA) for 15 min, and then incubated in 3% H₂O₂ for 10 min to quench the activity of endogenous peroxidase. Following

incubation with PowerBlock reagent (BioGenex uni- versal blocking reagent) for 10 min and wash in Tris- bu®ered saline Tween-20 (TBST) bu®er, the slides were incubated with primary antibody for 60 min, Super- Enhancer (BioGenex HRP kit) for 30 min, and Polymer- HRP reagent (BioGenex HRP kit) for 40 min with gentle wash in TBST bu®er for 5 min after each step. Following treatment with the chromogen-DAB (BioGenex HRP kit) for 3 min, the slides were rinsed with TBST bu®er, drained, and counterstained with Mayer's hematoxylin for 1 min. Positive and negative controls were run par- allel in each assay.

Fluorescent Antibody Test (FAT)

For the detection of RABV antigens, brain smears were prepared either with a frozen ground composite brain tissue suspension of cerebrum, hippocampus, thalamus, and hypothalamus left from the preparation for RNA extraction or with impression smears made from cerebrum, cerebellum, and brain stem from each necropsied animal. The smears were then air-dried at room temperature, ¯xed in acetone (Merck) at
20^C for 20 min, washed 3 times with PBS, and stained with either direct (DFA) or indirect (IFA) immuno°uores- cence assay. For the DFA, the air-dried and acetone-

¯xed brain smear slides were stained with a commer- cially available FDI °uorescein isothiocyanate (FITC)-conjugated anti-rabies monoclonal globulin (Fujirebio Diagnostics, Inc., Malvern, PA, USA), incu- bated at 37^C for 30 min, and washed in PBS for 3 times.

For the IFA, the air-dried and acetone-¯xed brain smear slides were incubated with the primary anti-RABV glycoprotein IgG2a monoclonal antibody (Abcam) at a 1:500 dilution at room temperature for 60 min, washed in PBS for 3 times, stained with an FITC-conjugated goat anti-mouse IgG antibody (Bethyl Lab., Inc., Montgomery, TX, USA) at room temperature for 60 min, and washed in PBS for 3 times. The viral anti- gens targeted by the FITC-labeled antibody appeared as apple-green °uorescent ¯nely to clumped granular aggregates under the °uorescent microscope (Optiphoto II, Nikon, Tokyo, Japan).

Reverse Transcription Polymerase Chain Reaction and Sequence Analysis

Fresh brain and lung specimens were screened for RABV and CDV by reverse transcription polymerase chain reaction (RT-PCR). Representative tissue specimen from each animal, approximately 25 mg each, was mixed Table 1. The Number of Dead Wild-Ranging

Carnivores Submitted for Necropsy During the Period of August 2011 to January 2015.

Year

Species 2011 2012 2013 2014 2015

TWFB 1 3 7 12 8

MPC 1 3 4 4

SCC





1 3 1

CEM





1


2

Disease Surveillance in Rescued and Road-Killed Wild-Ranging Carnivores 75

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with 1 mL of TRIzol reagent (Invitrogen, CA, USA) and homogenized. Following the addition of 0.2 mL of chloroform, the samples were centrifuged at 12; 000  g for 15 min. The upper aqueous phase was collected for total RNA extraction by using the RNeasy Mini Kit (QIAGEN Inc, Hilden, Germany) followed by cDNA synthesis by using the Transcriptor ¯rst strand cDNA synthesis kit (Roche Diagnostics, IN, USA) according to the manufacturer's instructions. Details of the RT-PCR primers¹⁷used and associated references are summarized in Table2.^17,18 The amplicons were further sequenced (Tri-IBiotech Inc, Taipei, Taiwan) and compared with those viral sequences deposited in GenBank/EMBL/

DDBJ using the NCBI's BLAST program (http://www.

ncbi.nlm.nih.gov/BLAST).

Polymerase Chain Reaction and Sequence Analysis

The total DNA was extracted from lung tissue emulsion and/or larvae obtained from lung wash using DNeasy^r blood & tissue kit (QIAGEN Inc, Hilden, Germany) according to the manufacturer's instructions. Owing to the fact that Angiostrongylus cantonesis is a common lungworm in wild rats in Taiwan and the natural feed habitat of TWFBs for snails and earthworms, which are the common intermediate hosts of A. cantonesis, polymerase chain reaction (PCR) for the detection of A. cantonesis was performed. Details of the PCR pri- mers used and the associated Ref. 19 are listed in Table 2. The amplicons were further sequenced (Tri- IBiotech Inc, Taipei, Taiwan) and compared with the sequences of various related parasites deposited in GenBank/EMBL/DDBJ using the NCBI's BLAST program (http://www.ncbi.nlm.nih.gov/BLAST).

Phylogenetic Analysis

Multiple sequence alignments were constructed by using Clustal W²⁰ and MEGA6.06 software programs (www.

megasoftware.net). The phylogenetic tree was generated using the maximum-likelihood method with 1000 boot- strap replicates. Scale bar indicates nucleotide sub- stitutions per site.

Parasitological Examination

Parasites were preserved in 70% ethanol or 10% formalin and examined by dissecting microscopy.

RESULTS

General Condition

Many of the carcasses received had su®ered variable degrees of traumatic injuries such as punctured wounds, diaphragmatic hernia, inguinal hernia and/or bone fractures along with external and internal hemorrhages as well as tears and/or ruptures of internal organs.

Death related to road tra±c accident or bites from other animals were diagnosed in 77.4% of TWFBs (24/31), 75% of MPCs (9/12), 60% of SCCs (3/5), and 100% of CEMs (3/3) (Table 3). Owing to no large carnivores present in Taiwan, stray dogs are highly speculated to be responsible for the killing.

Viral Diseases

The most signi¯cant viral diseases detected in the study were rabies in four TWFBs and canine distemper in three MPCs. Aside from emaciation and rough hair coat, Table 2. Oligonucleotides Used in the Study.

Primer Pair Sequence 5⁰–3⁰ Target Amplicon Size (bp) References

Rabies virus

RV-N-F ACGCTTAACAACAAAACCATAGAAG N gene 1300 17

RV-N-R CGGATTGACGAAGATCTTGCTCAT

RV-G-F CATCCCTCAAAAGACTTAAGGAAAG G gene 500

RV-G-R CCGAGGAGATGAGGTCTTCGGGAC

Canine distemper virus

RHF AACAATGCTCTCCTACCAAGA H gene 1800 18

RHF4 AATGCTAGAGATGGTTTAATT

Angiostrongylus cantonensis

AngioF1 ATCATAAACCTTTTTTCGAGTATCCA 18S rRNA 1134 19

AngioR1 TCTCGAGACAGCTCAGTCCCGG

76 H.-Y. Chiou et al.

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no apparent gross lesions were seen in the four rabid TWFBs. Nonsuppurative meningoencephalomyelitis and ganglionitis with formation of typical intracytoplasmic Negri bodies were the characteristic histopathological

¯ndings [Figs.1(A) and1(B)]. The lesions were widely distributed in the brain with brain stem a®ected the most. Additionally, variable spongiform degeneration, primarily in the perikaryon of neurons and neuropil, was observed in the cerebral cortex, thalamus, and brain stem. In the non-nervous tissue, representative lesions included adrenal necrosis and lymphocytic interstitial sialoadenitis. The FAT and IHC staining [Fig. 1(C)]

demonstrated a widely distributed pattern of RABV antigens in the neurons and nerve processes. The results

of RT-PCR and subsequent sequence analysis of the amplicons further con¯rmed that the virus belonged to the classic RABV.²¹

Two rescued adult MPCs displaying neurologic signs of severe ataxia and seizures prior to death were submitted. No apparent gross lesions were found. Mi- croscopically, both had nonsuppurative meningoence- phalomyelitis [Fig.2(A)] with gliosis, areas of necrosis, accumulation of gitter cells and hypertrophied (gemis- tocytic) astrocytes, and occasional syncytial cell forma- tion. No distinct intranuclear or intracytoplasmic inclusion bodies were identi¯ed in the brain tissue. Both animals had no pneumonia but mild bronchiolitis. CDV antigens were detected in the hypertrophied astrocytes Table 3. The Incidence of Various Categories of Disease Conditions Detected in the

Necropsied Wild-Ranging Carnivores.

Animal Species

TWFB MPC SCC CEM

Disease Condition n ¼ 31 n ¼ 12 n ¼ 5 n ¼ 3

Trauma 24^a 9 3 3

Virus 4 3 0 0

Bacteria 1 0 0 0

Fungus 0 1 1 0

Parasitism 25 5 3 2

Lung

Lungworms 23 2 0 0

Liver

Nematode larvae 9 0 0 0

Tongue

Trichinelloidea (Capillariiadae) 4 0 0 0

Sarcocystis 1 0 0 0

Esophagus

Unidenti¯ed nematodes 4 0 0 0

GI tract

Unidenti¯ed nematodes 16 2 3 1

Trichostrongyloidea 2 0 3 0

Cestodes 3 0 0 1

Ascaridida 1 0 2 0

Trematodes 0 2 0 0

Pancreas

Trematodes 1 1 0 0

Urinary bladder

Bladder worm (Pearsonema spp. formerly Capillaria) 0 0 0 1 Skin

Trichinelloidea 1 0 1 0

Demodex mites 1 0 0 0

Sarcoptic mange 0 1 0 0

Unknown^b 2 1 1 0

aThe number of animal with the speci¯c disease condition.

bNo signi¯cant gross and/or microscopic lesions.

Note: n = Total number of animal examined.

Disease Surveillance in Rescued and Road-Killed Wild-Ranging Carnivores 77

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(A)

(B)

(C)

Fig. 1 Rabies, TWFB (Melogale moschata subaurantiaca).

(A) Cerebrum. Nonsuppurative encephalitis, characterized by lymphoplasmacytic perivascular cu±ng (right up corner) with formation of varying numbers of intracytoplasmic eosinophilic Negri bodies in the neurons (arrows). H&E stain. (B) Peri- renal ganglion. Ganglionitis, characterized by the in¯ltration of lymphocytes, plasma cells, and macrophages as well as degen- eration and necrosis of ganglion cells (arrowheads) with for- mation of intracytoplasmic eosinophilic Negri bodies (arrows).

H&E stain. (C) Brain stem. Strong rabies virus antigen-posi- tive signals widely distributed in the neurons and nerve pro- cesses. Immunohistochemical (IHC) staining, polymer-HRP method, AEC substrate, Mayer's hematoxylin counterstain.

(A)

(B)

(C)

Fig. 2 CDV infection, MPC (Paguma larvate taivana). (A) Cerebrum. Nonsuppurative encephalitis, characterized by the presence of lymphoplasmacytic cu±ng, areas of necrosis (arrows), and gliosis. H&E stain. (B) Cerebrum. Strong CDV antigen-positive signals widely distributed in the neurons and astrocytes. IHC staining, polymer-HRP method, DAB sub- strate, Mayer's hematoxylin counterstain. (C) Lung. Intersti- tial pneumonia, characterized by the in¯ltration of lymphocytes, plasma cells and macrophages and proliferation of type II pneumocytes with formation of syncytial cells (long arrows) and eosinophilic, intranuclear (short arrows) and intracytoplasmic (arrowheads) inclusion bodies in the alveolar septa. H&E stain.

78 H.-Y. Chiou et al.

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and neurons [Fig. 2(B)]. The results of RT-PCR and subsequent sequence analysis of the amplicons further con¯rmed CDV infection (data not shown). In another female MPC kid, weighing approximately 400 gm, the lung lobes were bilaterally pale pink and rubbery to ¯rm when palpated. The trachea was ¯lled with a moderate amount of serosanguineous °uid and some ¯brin strands.

A small amount of similar exudate and some ¯brin strands oozed from the small airways and lung paren- chyma. Microscopically, di®use bronchointerstitial pneumonia along with proliferation of bronchiolar epi- thelial cells and alveolar type II pneumocytes and syn- cytial cell formation was revealed [Fig. 2(C)].

Intranuclear and intracytoplasmic eosinophilic inclusion bodies were found in the epithelium of the airway and alveoli. Syncytial cells with 3–5 nuclei were randomly distributed and some of them also contained intracyto- plasmic and/or intranuclear inclusions [Fig.2(C)]. CDV infection was further con¯rmed by IHC staining, RT- PCR, and sequence analysis of the amplicons (data not shown).

Bacterial and Fungal Diseases

The only bacterial disease identi¯ed in the study was a fatal staphylococcal pneumonia found in a TWFB.

The lesion involved the entire apical and cardiac lobes bilaterally. The a®ected lobes were di®usely dark red to mottled red white with some ¯brin loosely attached to the anterio-ventral portion and adjacent thoracic wall. There was a large amount of translucent, ser- osanguinous pleural e®usion [Fig.3(A)]. The hilar lymph nodes were enlarged. Microscopically, the lung lesion was characterized as a bilateral ¯brinosuppurative nec- rotizing bronchopneumonia. There were areas of marked mucosal epithelial necrosis and transmural suppurative in°ammation in the bronchi [Fig. 3(B)]. Necrosis along with variable edema, ¯brin deposition, hemorrhage, and accumulation of degenerate neutrophils and macro- phages was apparent in the lung parenchyma [Fig.3(C)].

Large numbers of bacterial colonies were present on the surface of the necrotic bronchial mucosa and widely distributed in the lung parenchyma [Figs. 3(B) and 3(C)]. The lobular septa were markedly dilated by edema, ¯brin deposition, and in¯ltration of in°ammatory cells. Areas of thrombosis were also noted [Fig. 3(C)].

Bacterial culture using the lung tissue and pleural e®u- sion successfully isolated a pure nonhemolytic bacterium forming white colonies on the blood agar. The bacterium was further identi¯ed as Staphylococcus hyicus via the Vitek 2 Compact automated system.

(A)

(B)

(C)

Fig. 3 S. hyicus pneumonia, TWFB (Melogale moschata sub- aurantiaca). (A) Opened thoracic cavity. The apical and cardiac lung lobes are non-collapsed, ¯rm, and dark red to mottled red white with deposition of some ¯brin in the anterio-ventral portion; the pleural cavity is ¯lled with a large amount of translucent, serosanguinous e®usion. (B) Bronchus. Necrotizing bronchitis, characterized by extensive mucosal necrosis and transmural in°ammation along with formation of large numbers of bacterial colonies (arrows). H&E stain. (C) Lung. Necrotizing pneumonia, characterized by the presence of extensive necrosis and in¯ltration of neutrophils and macrophages with edema, ¯- brin deposition, congestion, hemorrhage, and formation of thrombus (arrows) and large numbers of bacterial colonies (arrowheads). H&E stain.

Disease Surveillance in Rescued and Road-Killed Wild-Ranging Carnivores 79

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The only fungal disease detected was the Candida- induced glossitis in a SCC and a suckling MPC kid, which had su®ered severe lethal sarcoptic mange infestation.

Parasitism (Table 3)

Parasitism was the most common disease condition seen in the wild-ranging carnivores in the present study, in which lungworm had the highest prevalence rate, espe- cially in TWFBs and MPCs.

Lungs Lungworm infestation was recorded in 74.2%

of TWFBs (23/31) and 16.7% of MPCs (2/12), but no case was seen in SCCs and CEMs. Grossly, the lungs with lungworm infestation displayed areas of consoli- dation and variable congestion. Multiple pale yellow to white spots, ranging from 0.1 to 0.2 cm in diameter, were randomly distributed on the surface and in the paren- chyma of the lungs. Microscopic examination revealed areas of variable subacute to chronic verminous pneu- monia. Multiple aggregates of varying numbers of sections of a nematode, including adults and larvae, were randomly distributed in the lung parenchyma [Fig. 4(A)]. The adult worms and larvae were present primarily in the alveolar spaces, bronchioles, and/or bronchi. The cross sections of the adult worms were approximately 200–300
m in diameter and character- ized by having a thin cuticle, a pseudocoelom lined by coelomyarian-polymyarian musculature, an intestinal tract lined by a few multi-nucleated cells, ovaries, and uterus ¯lled with oocytes, embryos, and developing lar- vae [Fig. 4(A)]. The larvae were 10–20
m in width, deeply basophilic, and occasionally coiled. Around the parasites, there was variable granulomatous in°amma- tion, characterized by the in¯ltration of epitheloid macrophages, lymphocytes, plasma cells mixed with some eosinophils, neutrophils, and multinucleated giant cells [Fig.4(B)]. By using the pair of primers AngioF1/

AngioR1, PCR amplicons with the expected size of 1134 bp were obtained from the DNA extracted from lung tissue emulsion and/or larvae. However, the results of further sequencing and phylogenetic analysis at the 18S rRNA gene suggested that the lungworm found in TWFBs is more closely related to Para¯laroides dec- orus, a lungworm of sea mammals, rather than to A. cantonesis (Fig.5).

Alimentary system In the alimentary system, parasitism was a quite common incidental ¯nding in all wild-ranging carnivores and could be seen in various parts of the system. The incidental alimentary parasite infestation found in TWFBs included parasite migration

in hepatic parenchyma (9/31), characterized by the presence of scattered necrotic migration tract surrounded by varying numbers of eosinophils and mononuclear in-

°ammatory cells; Trichinelloidea (Capillariadae) in the lingual mucosal epithelium (4/31), characterized by having a thick cuticle in the adult worm and striated shell and bipolar plugs in the eggs; Sarcocystis in the muscle of tongue (1/31); unidenti¯ed nematodes in the esophageal mucosa (4/31); unidenti¯ed nematodes in the lower GI tract (16/31); Trichostrongyloidea (2/31), cestodes (3/

(A)

(B)

Fig. 4 Lungworm infestation, TWFB (Melogale moschata subaurantiaca). (A) Lung. Verminous pneumonia, character- ized by the presence of multiple cross sections of a nematode in the alveolar spaces and in¯ltration of mixed macrophages, lymphocytes, plasma cells, and neutrophils in the alveolar septa; the adult nematode (arrows) having a thin cuticle, a pseudocoelom lined by coelomyarian-polymyarian muscula- ture, an intestinal tract lined by a few multi-nucleated cells, ovaries, and uterus ¯lled with larvae (arrowheads). H&E stain.

(B) Lung. Verminous pneumonia, characterized by the accu- mulation of large numbers of epitheloid macrophages mixed with some lymphocytes, plasma cells, eosinophils, and neu- trophils around a few larvae (arrows) in the lung parenchyma.

H&E stain.

80 H.-Y. Chiou et al.

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31) and Ascaridida (1/31) in the small intestine; and trematodes in the pancreatic duct (1/31). In MPCs, there were unidenti¯ed nematodes (2/12) and trematodes (2/

12) in the GI tract, and trematodes (1/12) in the pan- creatic duct. In SCCs, unidenti¯ed nematodes (3/5), Trichostrongyloidea (3/5), and Ascaridida (2/5) were detected in the small intestine. In CEMs, unidenti¯ed nematodes (1/3) and cestodes (1/3) were found in the small intestine.

Skin Heavy sarcoptic mange infestation-associated death was diagnosed in a suckling MPC kid. Grossly, the skin lesion was characterized by formation of large areas of thick crusts with no alopecia. Microscopically, the a®ected regions showed severe epidermal hyperplasia, hyperkeratosis, and a large number of mites and eggs distributed in the upper epidermis along with mixed in°ammatory cell in¯ltration in the epidermis and upper dermis. Other incidental cutaneous parasitic infestation included Trichinelloidea (suspected Anatrichosoma and Capillariiadae) in a SCC (1/5) and a TWFB (1/31) co- infected with Demodex.

Urinary tract Bladder worm infestation was diag- nosed in a CEM (1/3), in which there were a moderate number of adult Pearsonema sp. (formerly Capillaria) with eggs in the mucosal epithelium along with mild in°ammation. The eggs had a typical oval shape and a thick capsule with striated shell and bipolar plugs.

Skeletal muscle Infestation with Sarcocystis sp. was found in 1 TWFB (1/31). Microscopically, there were many protozoal cysts, ranging from 30 to 150
m in the

myo¯bers of the tongue. The cysts had a thin hyalinized wall and contained numerous crescent bradyzoites.

Minimal muscular degeneration and ¯brosis with in¯l- tration of a few mixed in°ammatory cells were also noted.

DISCUSSION

Rabies is one of the most important and possibly the oldest zoonotic diseases. Owing to the well-established canine vaccination programs, wildlife has become an important source of RABV infection in many developed countries. Through the government-supported disease surveillance program of dead wild-ranging native wild carnivores, rabies was diagnosed in TWFBs in mid-June of 2013. Since the ¯rst discovery of rabid TWFBs in 2013, rabies has been diagnosed in additional 452 TWFBs, 4 MPCs, 1 shrew, and 1 puppy bitten by rabid- TWFB by March 26, 2015. (https://www.baphiq.gov.

tw/news list.php?menu¼1924&typeid¼1948). Prior to the most recent outbreak, Taiwan had been considered as a rabies-free region for more than 50 years. Phylo- geographic analyses indicated that the TWFB-associ- ated RABV is a distinct lineage among the Asian isolates. This particular lineage might have been di- verged from its closest lineages, China I and Philippines, more than 150 years.²¹The discovery of this re-emerging important zoonotic disease further emphasizes the es- sentiality of a systemic disease surveillance in wild- ranging wildlife.

Fig. 5 Phylogenetic analysis of the lungworm detected in TWFB (Melogale moschata subaurantiaca) and nematodes of the order Strongylida using 18S rRNA. Multiple sequence alignments were constructed by using Clustal W and MEGA6.06 software programs. The phylogenetic tree was generated using the maximum-likelihood method with 1000 bootstrap replicates. Scale bar indicates nucleotide substitutions per site.

Disease Surveillance in Rescued and Road-Killed Wild-Ranging Carnivores 81

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