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Foot-and-mouth disease in Asiatic black bears (Ursus thibetanus) Kirsty Officer, Nguyen Thi Lan, Leanne Wicker, Nguyen Thi Hoa, Annemarie Weegenaar, Jill Robinson, Yamaguchi Ryoji and Panayiotis Loukopoulos J VET Diagn Invest published online 18 August 2014 DOI: 10.1177/1040638714547256 The online version of this article can be found at: http://vdi.sagepub.com/content/early/2014/08/17/1040638714547256

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547256 research-article2014

VDIXXX10.1177/1040638714547256Foot-and-mouth disease in bears in VietnamOfficer et al.

Brief Communication

Foot-and-mouth disease in Asiatic black bears (Ursus thibetanus)

Journal of Veterinary Diagnostic Investigation 1­–9 © 2014 The Author(s) Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/1040638714547256 jvdi.sagepub.com

Kirsty Officer,1 Nguyen Thi Lan, Leanne Wicker, Nguyen Thi Hoa, Annemarie Weegenaar, Jill Robinson, Yamaguchi Ryoji, Panayiotis Loukopoulos

Abstract. Foot-and-mouth disease (FMD) is a highly contagious, debilitating, and globally significant viral disease typically affecting cloven-hoofed hosts. The diagnosis of FMD in bears in Vietnam is described. The current study describes a confirmed case of FMD in a bear species, and the clinical signs compatible with FMD in a Malayan sun bear. Thirteen Asiatic black bears (Ursus thibetanus) and 1 Malayan sun bear (Helarctos malayanus) were apparently affected. In August 2011, an adult bear became lethargic, and developed footpad vesicles. Over 15 days, 14 out of 17 bears developed similar signs; the remaining 3 co-housed bears and another 57 resident bears did not. All affected bears developed vesicles on all footpads, and most were lethargic for 24–48 hr. Nasal and oral lesions were noted in 6 and 3 cases, respectively. Within 1 month, all looked normal. Foot-and-mouth disease virus (FMDV) was detected by reverse transcription polymerase chain reaction, classified as serotype O, and isolated by virus isolation techniques. Phylogenetic analysis demonstrated clustering of 3 bear isolates, in a branch distinct from other FMDV type O isolates. The outbreak likely occurred due to indirect contact with livestock, and was facilitated by the high density of captive bears. It showed that Asiatic black bears are capable of contracting FMDV and developing clinical disease, and that the virus spreads easily between bears in close contact. Key words: Asiatic black bears; foot-and-mouth disease; Malayan sun bears; outbreak; viral disease. Foot-and-mouth disease is a highly contagious acute febrile viral disease typically affecting cloven-hoofed livestock and characterized by vesicular lesions in the mouth and on the feet. Foot-and-mouth disease virus (FMDV; order Picornavirales, family Picornaviridae, genus Aphthovirus) has a wide host range, high excretion levels, a rapid rate of replication, a low minimum infective dose, antigenic variation, environmental stability, multiple transmission modes, and the ability to persist subclinically in some ruminants, making it one of the most contagious and difficult to control infectious diseases of animals.1,2 These features, its debilitating nature, and its real and potential impact on international trade, make FMD the most economically significant veterinary disease in the world.11 While all members of the order Artiodactyla are thought to be susceptible to FMD,22 domestic cloven-hoofed livestock species, including cattle, pigs, sheep, and goats are considered the most significant hosts due to their role in the epidemiology of the disease.1 African buffalo (Syncerus caffer) play an important role as persistent carriers, and other African wildlife species including greater kudu (Tragelaphus strepsiceros) and impala (Aepyceros melampus) are thought to play a role in maintaining the disease.23 A range of other wildlife belonging to order Artiodactyla are known to be naturally susceptible, but are not considered epidemiologically significant hosts under natural conditions.1,5,10,22,24–26

There are reports of natural infection with FMDV in several non–cloven-hoofed wildlife species including Asiatic elephant (Elephas maximus) and African savannah elephant (Loxodonta africana),1,10 European hedgehog (Erinaceus europaeus),10 eastern gray kangaroo (Macropus giganteus),6 Brazilian tapir (Tapirus terrestris) and Asiatic tapir (Tapirus indicus),25 and brown bear (Ursus arctos).9,17 With the exception of free-ranging hedgehogs infected with FMDV in the vicinity of an outbreak in cattle,10 all other cases were small numbers of captive animals. A range of non–cloven-hoofed species have been experimentally infected with FMDV, From the Vietnam Bear Rescue Centre, Animals Asia Foundation, Tam Dao National Park, Vinh Phuc, Vietnam (Officer, Weegenaar); Key Laboratory of Veterinary Biotechnology and Department of Veterinary Pathology, Faculty of Veterinary Medicine, Vietnam National University of Agriculture, Ha Noi, Vietnam (Nguyen Thi Lan, Nguyen Thi Hoa); PREDICT Program, Wildlife Conservation Society, Ha Noi, Vietnam (Wicker); Animals Asia Foundation, Sheung Wan, Hong Kong (Robinson); Department of Veterinary Pathology, University of Miyazaki, Japan (Ryoji); Laboratory of Pathology, Faculty of Veterinary Medicine, Aristotle University, Thessalonica, Greece (Loukopoulos); and California Animal Health and Food Safety Laboratory, School of Veterinary Medicine, University of California–Davis, San Bernardino, CA (Loukopoulos). 1

Corresponding Author: Kirsty Officer, Animals Asia Foundation, 97 Tran Quoc Toan Street, Hoan Kiem, Ha Noi, Vietnam. [email protected]

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including rodents, rabbits, moles, armadillo, hedgehogs, squirrels, marsupials, monotremes, reptiles, primates, birds, cats, and dogs.1,8,10,22,25 There is a previous report of suspected FMD in Asiatic black bears (Ursus thibetanus),16 where the diagnosis was based on clinical signs and was not confirmed. The authors were unable to find any reports of FMD in the Malayan sun bear (Helarctos malayanus). Foot-and-mouth disease is endemic in Vietnam,14,15 and outbreaks regularly occur in cattle, pigs, and buffalo (Vietnam Ministry of Agriculture and Rural Development Department of Animal Health Disease Information, available at: http://www.cucthuy.gov.vn. In Vietnamese). Serotypes O, A, and Asia-1 are known to be co-circulating in Vietnam,14,15 with serotype O being the most prevalent.15 The current study describes an outbreak of FMD in August and September 2011 in a group of bears housed at a rescue center in Tam Dao National Park, northern Vietnam, and the subsequent isolation and identification of the virus. Sixteen Asiatic black bears (Ursus thibetanus) and 1 Malayan sun bear (Helarctos malayanus) were housed in the affected bear house, which consisted of indoor dens only. An additional 57 bears were residents at the center, in separate housing and outdoor enclosures. The 17 bears (2 cubs, 7 juveniles, and 8 adults of unknown age) in the affected house were transferred to the center over a number of years after confiscation by, or voluntary handover to, the government of Vietnam from private owners, illegal captors, or illegal bear bile extraction facilities; in Vietnam, bear bile is used both as a traditional medical treatment, and as an additive in alcohol and commercial products. The bears were housed in various group sizes, ranging from single den occupancy, to the largest group comprising 6 bears. Neighboring individuals or groups of bears had contact through bars. There was another bear house 3.5 m from the affected one, and both houses had open bars occupying part of their outer walls. Daily husbandry was provided by bear-keeping staff who live in the surrounding rural area. All staff changed their boots on arrival to work, and walked through 2% bleach footbaths at all bear house thresholds. Keeping staff did not work between bear houses, but had close contact with each other at break times, and food buckets and enrichment toys were shared between houses. Bear food, which included daily fresh fruit, vegetables, and browse, and weekly raw beef bones, was sourced from local markets and plantations, and delivered by vehicle. Wheels were sprayed with 1% potassium peroxymonosulfatea on entry. Bones were frozen for at least 2 weeks at –20°C before being thawed and fed to bears. On August 28, 2011 (day 1) an adult male Asiatic black bear was reluctant to walk or eat, and had swellings on his footpads, which developed into vesicles the next day. Four days later (day 5), an adult female living in the adjacent den developed similar signs, and her female den mate subsequently developed signs on day 7. On day 6, a juvenile male housed in a den across the corridor from the first 2 cases developed similar signs, and over the next 2 days, his 5 den

mates (4 juveniles and 1 adult) developed signs. Two cubs, neighboring the juvenile group, developed foot lesions on day 9. On day 11, a juvenile sun bear housed next to the cubs developed severe foot lesions. The final 2 cases (days 15 and 16) were 2 adult female Asiatic black bears sharing a den across the corridor from the sun bear. Fourteen out of 17 bears in the house showed signs. The remaining 2 adult female and 1 juvenile male Asiatic black bears did not develop clinical signs despite having contact through bars with affected animals. On day 6, the house was placed under quarantine. A clothing and boot change was instigated for all staff entering and leaving the house, any nonwaste items leaving the house were soaked in 1% potassium peroxymonosulfate, and waste was disposed of separately to limit potential spread to other areas of the center. None of the 57 bears in other houses and enclosures developed clinical signs. All affected bears developed vesicles on all of their footpads (Figs. 1–3). The vesicles on the pads of younger bears were notably more severe. The adult bears, with thicker, tougher pads, developed moist cracks in their pads, and the vesicles tended to be smaller and peripheral (Fig. 2B). Lesions on the nasal planum were seen in 6 cases, ranging from small discrete well-demarcated erythematous areas, to generalized crusting of the mucocutaneous junction of the nares (Fig. 1A). Subtle oral mucosal lesions were noted on close inspection under anesthetic of the mouths of 3 bears, and ranged from a few small (1–4 mm in diameter), poorly defined, occasionally coalescing areas of hyperemia and/or mild erosion to several very small well-demarcated mildly raised circular vesicles of 1–3 mm diameter on the buccal mucosa (Fig. 1B); in 1 case, a 3-mm mild erosion focus was observed on the hard palate. No tongue lesions or ptyalism were noticed in any of the cases. Affected bears were lethargic and reluctant to eat for 24– 48 hr from the onset of signs, apart from the 2 cubs, which continued to eat and play as normal. The most severely affected bears showed difficulty walking for up to 4 days, but would eat if food was placed in front of them. The vesicles persisted for 2–4 days before they deflated and began to slough, exposing ulcerated pad tissue (Fig. 2C, 2D). At this time, affected bears were more comfortable walking, and their activity levels and appetites returned to normal. The outer vesicle layer disintegrated and fully sloughed within 14 days. The exception was the sun bear, where the entire outer surface of both hind footpads blistered and subsequently sloughed (Fig. 3). Secondary vesicles and bullae formed on the exposed underlying tissue, making it extremely difficult for her to walk, and she took 12 days to return to normal activity. One month later, the footpads of all bears were clinically normal. All affected bears were treated with oral meloxicamb at 0.2 mg/kg for 3 days, then 0.1 mg/kg for 11 days, and oral cephalexinc at 20 mg/kg for 14 days. On day 8, a young adult female Asiatic black bear (B1) that had been showing symptoms for less than 24 hr was

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Foot-and-mouth disease in bears in Vietnam

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Figure 1.  Macroscopic lesions in Asiatic black bears (Ursus thibetanus) in Vietnam with foot-and-mouth disease. A, lesions on the nose of a juvenile bear 4 days after first showing clinical signs; B, lesions on the oral mucosa of a young adult bear (B1) on the first day of showing clinical signs; C, vesicles on the hind foot of B1 on the first day of showing clinical signs; D, vesicles on fore foot of B1 on the first day of showing clinical signs.

Figure 2.  Macroscopic lesions in Asiatic black bears (Ursus thibetanus) in Vietnam with foot-and-mouth disease. A, vesicles on the fore foot of a juvenile bear on the first day of showing clinical signs; B, cracks and a vesicle on the hind foot of an adult bear 2 days after first showing clinical signs; C, fore foot of a juvenile bear 7 days after first showing clinical signs; D, fore foot of a young adult bear (B1) 10 days after first showing clinical signs.

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Figure 3.  Macroscopic lesions in a juvenile Malayan sun bear (Helarctos malayanus; B3) in Vietnam showing signs compatible with foot-and-mouth disease. A, sloughing of the entire outer surface of the hind footpads 5 days after the bear first showed clinical signs; B and C, lesions on underlying raw pad surface of hind foot 8 days after first showing clinical signs and 3 days after sloughing of outer pad.

anesthetized. Anesthesia was induced with zolazepam–tiletamined reconstituted with medetomidinee given at a dose of 1.25 mg/kg tiletamine–zolazepam and 0.0125 mg/kg medetomidine via a syringe pole. After intubation, anesthesia was maintained with isofluranef and oxygen. Samples collected included oral and rectal swabs, vesicle fluid, vesicle epithelial tissue, urine, and blood. On day 9, a juvenile female Asiatic black bear (B2) that had developed signs the previous day was similarly anesthetized and the same set of samples collected. On day 16, an adult female Asiatic black bear (B4) that developed signs that morning was anesthetized and the same set of samples collected, as well as feces. Feces were collected from the den floor of the affected sun bear (B3) on day 12 and frozen. Vesicle fluid samples were collected aseptically, and stored in viral transport medium (VTM) and RNA

stabilization tubes.g Urine was collected via sterile cystocentesis into plain cryogenic vials. Oral and rectal samples were collected using sterile polyester swabs and stored in VTM. Blood was collected aseptically from the jugular vein and whole blood, and separated sera were frozen. All samples were stored at –80°C. Vesicle epithelium was stored in VTM and 10% buffered formalin. Another affected Asiatic black bear (B5) was anesthetized for abdominal surgery 10 days after first developing vesicular lesions on her footpads. Rectal and oropharyngeal swabs and urine were collected. At this time, her foot lesions were almost fully healed. Asiatic black bears B1, B2, and B4 were re-anesthetized 30, 42, and 23 days, respectively, after initial sampling. Rectal and oropharyngeal swabs were collected, stored in VTM, and frozen. The affected bears were fed bovine bones 5 days prior to day 1. Swabs were taken from 6

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Foot-and-mouth disease in bears in Vietnam bones from the same batch, stored in VTM, and frozen. Formalin-fixed samples were trimmed, paraffin embedded, and processed routinely for histologic examination at the Centre for Pathology, Bach Mai Hospital, Ha Noi. Two vesicle fluid samples and 1 epithelial sample from Asiatic black bear B1 were submitted on the day of collection to the National Centre for Veterinary Diagnosis (NCVD), Ha Noi, for routine enzyme-linked immunosorbent assay (ELISA) for FMD viral antigen detection and serotyping (data not shown). All subsequent sample testing was performed at the Key Laboratory of Veterinary Biotechnology, Vietnam National University of Agriculture (VNUA) and is described herein. Viral RNA was extracted using a commercial kitg according to the manufacturer’s instructions, and stored at –20°C until use. Reverse transcription polymerase chain reaction (RT-PCR) was performed in duplicate on the extracted RNA using a one-step RT-PCR kit,g following the manufacturer’s instructions and standard procedures.27 Reverse transcription PCR was performed using the primer set 1F 5′-GCCTGGTCTTTCCAGGTCT-3′ and 1R 5′-CCAGTCCCCTTCTCAGATC-3′ to amplify the 5′-untranslated region (UTR) of the FMDV genome, recognized as suitable for primary diagnosis of FMDV serotypes O, A, C, and Asia-1.19 A serotype O Manisa vaccine strainh was used as the control.17 The RT-PCR reaction mixture contained 5 µl of the extracted RNA solution, 12.5 µl of 2× reaction mix, 0.5 µl of forward primer, 0.5 µl of reverse primer, 0.5 µl of Taq polymerase mix,i and 6 µl of deionized water, to a 25-μl final reaction volume. Complementary DNA was synthesized using a thermocycler j The reaction mixture was incubated at 50°C for 30 min, 95°C for 5 min, followed by 35 cycles with an initial denaturation step at 95°C for 15 sec, annealing at 55°C for 60 sec and elongation at 72°C for 60 sec, and a final elongation step at 72°C for 5 min. Subsequently, a one-step multiplex RT-PCR method was used to confirm the serotype. A universal primer (VN-VP1R) from the conserved 2B region, and 3 serotype-specific primers (VN-OF, VN-As1F, VN-AF) from the hypervariable regions of the VP1 coding gene, designed to differentiate between FMDV serotypes O, A, and Asia-1 based on the expected length of the PCR products,13 were used. Confirmed FMDV serotype strains isolated previously at VNUA were used as positive controls. Briefly, the master mixture contained 5 µl of the extracted RNA solution, 0.5 µl of each primer, 1 µl of deoxyribonucleotide triphosphate, 1 µl of polymerase, 5 µl of buffer, and 11 µl of deionized water. Complementary DNA was synthesized, and the mixture was incubated at 50°C for 30 min, 95°C for 15 min, followed by 35 cycles with an initial denaturation step at 95°C for 30 sec, annealing at 55°C for 30 sec and elongation at 72°C for 60 sec, and incubation at 72°C for 10 min. The PCR products were subjected to agarose gel electrophoresis and analyzed for expected band sizes. Identical results were obtained in all duplicate runs.

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Table 1.  Outcome of bear samples submitted for Foot-andmouth disease virus testing.* Animal ID/ Collection date† B1   Day 2   Day 2   Day 2   Day 2   Day 32   Day 32 B2   Day 2   Day 44   Day 44 B3   Day 2 B4   Day 1   Day 1   Day 1   Day 1   Day 23   Day 23 B5   Day 10   Day 10   Day 10   Day 10  

Sample

RTPCR ELISA

Vesicle fluid Vesicle epithelium Vesicle fluid Vesicle fluid Oral swab Rectal swab

+‡ +‡ +‡ + – –

+‡ – +‡ ND ND ND

Vesicle fluid Oral swab Rectal swab

+ – –

ND ND ND

Feces

–

ND

Vesicle fluid Blood Urine Feces Oral swab Rectal swab

+ + + + – –

ND ND ND ND ND ND

Oral swab Rectal swab Blood Urine Bovine bone swabs

– – – – –

ND ND ND ND ND

Virus isolation   ND ND ND + ND ND   – ND ND   ND   + ND ND ND ND ND   ND ND ND ND ND

* RT-PCR = reverse transcription polymerase chain reaction; ELISA = enzyme-linked immunosorbent assay; ND = not done; + = positive for Foot-and-mouth disease virus (FMDV) or antibodies; – = negative for FMDV or antibodies. † Denotes number of days after first clinical signs were seen in that animal. ‡ Tested at the National Centre for Veterinary Diagnosis, Ha Noi, Vietnam; all other samples tested at Vietnam National University of Agriculture, Ha Noi, Vietnam.

Baby hamster kidney (BHK-21) cells were cultured in Dulbecco modified Eagle medium (DMEM)i supplemented with 10% heat-inactivated fetal bovine serumi and penicillin + streptomycini at 37°C with 5% CO2. Cells were infected with the samples suspected to contain FMDV (Table 1). After 1 hr of absorption at 37°C, cells were washed for 1 min with 0.1 ml of phosphate buffer (pH 6.0), and washed again extensively with DMEM. The infection was allowed to proceed in DMEM, and cells were observed daily for the presence of cytopathic effects (CPE) for 7 days. Cells were harvested when 95% CPE were observed, frozen at –80°C, thawed, gently centrifuged, and the supernatant removed and subjected to RT-PCR to detect FMDV as above. Following electrophoresis, PCR products were purifiedg and subjected to direct sequencingk BLAST software3 was used to examine the homology of the acquired sequences

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Figure 4.  Phylogenetic tree showing the phylogenetic relationship between the 3 bear Foot-and-mouth disease virus (FMDV) isolates from the current outbreak, and representative FMDV serotype O isolates from Japan, China, Taiwan, and the United Kingdom accessed from the GenBank database, based on analysis of the 5′-untranslated region of the genome.

with those of publicly available FMDV sequences. Sequence analysis softwarel was used to compare the resultant nucleotide sequences to each other, and to the corresponding nucleotide sequences of the FMDV vaccine strain and a porcine FMDV isolate. The resultant gene sequences were assembled and aligned pairwise using the ClustalW sequence analysis package.12 The alignments were visually checked and manually corrected. A consensus phylogenetic tree was generated for the bear FMDV sequences and for selected FMDV sequences deposited in the GenBank database using the maximum likelihood method based on the Tamura–Nei model with 1,000 bootstrap replications. A diagnosis of FMD was established on the basis of the clinical, epidemiologic, gross, and histologic findings combined with the RT-PCR, ELISA, and virus isolation results (Table 1). Two vesicle fluid samples and a vesicle epithelium sample from Asiatic black bear B1 tested at NCVD were positive for FMDV on RT-PCR. The 2 vesicle fluid samples were positive for FMDV serotype O on ELISA. On the basis of these initial tests, additional samples were tested at VNUA

for further confirmation of the diagnosis. Vesicle fluid samples from Asiatic black bears B1, B2, and B4 were positive for FMDV on RT-PCR, as were blood, urine, and fecal samples tested from B4. Additionally, FMDV was isolated from vesicular fluid of 2 Asiatic black bears (B1, B4). Samples collected from Asiatic black bear B5 10 days after first showing clinical signs, and samples from B1, B2, and B4 after their recovery were negative for FMDV. Samples from bovine bones stored with those fed to affected bears 5 days prior to the outbreak were negative for FMDV. All positive samples were classified as serotype O, based on the results of the multiplex RT-PCR method and detection of the expected length of PCR product. Comparison of the 5′-UTR and VP1 genome sequences obtained from this outbreak showed 100% homology between the bear FMDV isolates. Comparison of the VP1 sequences obtained with a Vietnamese porcine isolate used as the control showed 98.1% homology. On phylogenetic analysis of the 5′-UTR sequences (Fig. 4), the 3 bear FMDV isolates were clustered together on a sub-branch, distinct but close to other branches containing

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Foot-and-mouth disease in bears in Vietnam FMDV type O isolates from other geographical regions. The 3 bear FMDV isolate sequences and the Vietnamese porcine isolate sequence used were deposited in GenBank (accession nos. AB933316, AB933317, and AB933318 for the bear isolates and AB933319 for the porcine isolate). In histological sections of the superficial foot pad lesions examined, the stratum corneum showed marked keratinocyte ballooning degeneration and focally extensive keratinocyte dissociation, and moderate numbers of neutrophilic infiltrates. Variably sized, often coalescing, roundish or irregularly shaped cavities were also present in the stratum corneum. These contained amorphous and sparse fibrillar lightly eosinophilic material (edema), large numbers of clusters or individual sloughed degenerate or apoptotic keratinocytes, moderate numbers of dead and viable neutrophils, small numbers of lymphocytes and plasma cells, and few erythrocytes. Many of these cavities opened to the epidermal surface. Viral inclusion bodies were not observed. Observation of the characteristic gross and histological lesions displayed, along with the results of combined laboratory tests (ELISA, RT-PCR, and virus isolation), confirms that the clinical signs seen in the 14 affected bears were attributable to FMDV infection. The present study describes confirmed FMDV infection in bears, one of very few FMD outbreaks reported in bears of any species, and, to the authors’ knowledge, the first report of FMD in bears in Asia, which is home to 5 of the 8 extant species of bear. A fecal sample from the affected sun bear (B3) was negative. However given that it was not a fresh sample and that feces contain only small amounts of virus,18 the authors presume based on the clinical signs shown, the expression of the clinical signs within the timeline of the confirmed FMDV outbreak, and the proximity of this bear to Asiatic black bears definitively diagnosed with FMDV, that the sun bear was also suffering from FMD. There are previous reports of clinical signs in bears after natural infection during presumed FMD outbreaks in zoos in Stuttgart16 and Buenos Aires.9 In the case of the Stuttgart Zoo, 3 Asiatic black bears were affected, and the clinical description is very similar to what was observed in the current outbreak. However, all previously reported cases in bears were diagnosed on clinical signs only, with none confirmed by laboratory testing. It is somewhat surprising that, to date, there are no published reports of FMD symptoms in sun bears or in any bears in Asia given the high numbers of captive bears in a number of FMD endemic countries in Asia, including Vietnam and China where bears are intensively “farmed” in bile extraction facilities where high concentrations of bears are kept in small, closely aligned cages, often in proximity to livestock species. The route of entry of the virus into the rescue center is unknown. The most recent bear to enter the affected house was the asymptomatic juvenile, which arrived 1 day prior to day 1, having completed 45 days isolated in a separate quarantine area of the center. There was no other possible direct contact with infected live animals. It is known that aerosol

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transmission is favored over smooth surfaces such as water,1 making this method of transmission less likely given the forested mountainous topography that separates the center from the nearby rural zone. People in contact with infected animals are exposed to massive amounts of FMDV and carry it on their clothes, skin, and in their hair, and have been proven to act as mechanical vectors of the virus.4 At the time of the outbreak, bear-keeping staff did not shower or change their clothes on arrival to work, and therefore were a possible means of passive transfer. Other possible fomite sources include food and edible browse, and delivery vehicles. The bears in the affected house were fed previously frozen bovine bones 5 days before day 1. Foot-and-mouth disease virus survives postmortem in bone marrow, and freezing bones at –20°C is unlikely to kill the virus, if present.20 Testing of in-contact bones did not reveal FMDV, but this does not rule out this potential source. The close homology between the bear FMDV isolates and a Vietnamese porcine FMDV isolate, combined with frequency of FMD outbreaks in rural Vietnam, and the large quantities of virus excreted by infected pigs and cattle,2 makes indirect transmission from local livestock a likely source of the outbreak. There were no official reports of FMD outbreaks around the same time in the local district or the province (http://www.cucthuy.gov.vn). However, anecdotal evidence suggests not all outbreaks are reported to the relevant authorities, and ineffectual enforcement of regulations, including reporting, is a regional issue of concern.7 Stocking density and housing conditions are known to influence FMDV dose and spread,1 and, in the current case, the high density of bears (17 in 144 m2) housed indoors in close contact may account for the rapid spread of the disease through the affected bear house. However, given the highly contagious nature of the disease, its ability to spread, and that peak infectivity can be prior to lesion development,2 it is surprising that cases did not develop in any other bear houses or enclosures during the first 5 days before strict quarantine was imposed. Three bears were apparently unaffected, although it is possible they were subclinically infected, as described in other species,21 or that they had immunity from previous exposure. Recovery from natural infection in other species has been shown to protect against the development of clinical FMD.2 Clinical records at the center contain 2 previous reports of vesicles on footpads of bears, both observed in adult bears 7 months prior to the current outbreak. The lesions were relatively mild, no diagnostic tests were performed, and the bears both spontaneously recovered within 5 days. Further work is needed to assess serological titers and levels of previous exposure in this bear population, and antibody responses in the clinically affected bears. Foot-and-mouth disease is characterized by high morbidity and low mortality, and acute fatal myocarditis is sometimes seen in young animals, particularly very young calves,

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lambs, and piglets.2 High morbidity, including 2 cubs, was seen in this outbreak, but no mortality occurred. Lesions on the feet and in and around the mouth are predominant clinical signs of FMD in cattle and sheep.1 In the current outbreak of FMD in bears, footpad vesicles were the primary lesions observed in all cases, as similarly described in the suspected previous cases in Asiatic black bears16 and a kangaroo.6 The predominance of foot lesions with a lack of conspicuous mouth lesions follows the clinical progression in pigs.5 Nonfoot lesions were restricted to relatively mild crusting of the nasal planum in some cases, and very mild oral mucosal ulcerations that were only seen on close examination under general anesthesia. Given that vesicles tend to develop at areas of physical stress or mechanical trauma,22 the concrete floors could account for the severity of the foot lesions, particularly in the sun bear, which paced frequently. Foot lesions were most severe in young bears, presumably because of their increased levels of activity and thinner pad tissue. The reluctance to eat seen in most cases was likely due to the presumed initial febrile condition and painful foot lesions restricting movement, with no indication that it was due to mouth or tongue lesions. Virus was found in urine from 1 bear, collected directly from the bladder via aseptic cystocentesis confirming that it was not infected via voiding past preputial lesions, as suggested in other species.2 The negative RT-PCR result on rectal and oropharyngeal swabs from Asiatic black bears B1, B2, and B4 collected 32, 44, and 23 days after their clinical signs were first noticed suggests that these animals did not continue to shed virus in feces or saliva beyond the clinical course of the disease. It has been previously reported that FMDV can infect, both naturally and experimentally, a large range of animals outside the commonly infected cloven-hoofed livestock host species.1,5,10,22,25 The current outbreak demonstrated that FMDV is capable of infecting and causing disease in 2 species of bear, and spreading between them. The results of the present study confirmed that FMDV was responsible for the clinical signs seen. Further work is needed to elucidate the genetic relationship of the FMDV in the bear samples and FMDV isolates from livestock in Vietnam. While this diagnosis adds to evidence that the virus can naturally infect non–cloven-hoofed species, and expands the species range reported, it should not be assumed that unexpected hosts such as bears play a significant role in the natural epidemiology of the disease.1,25 It is likely the outbreak occurred due to the high density of bears in captivity and the indirect contact the bears had with livestock in a FMD endemic area. While it does not follow that wild bears could be implicated in the natural spread of the disease, the findings are nonetheless significant for captive bear facilities around the world, and add to the overall understanding of the disease process in an uncommon host species. This confirmed outbreak has proven that bears are capable of contracting FMDV and showing clinical disease, and that the virus spreads easily between bears in close contact.

Acknowledgements The authors wish to thank Dr. Dang Van Duong for the histology preparations and his insightful comments, and Dr. Nguyen Tung at the National Centre for Veterinary Diagnosis for his assistance with the initial diagnosis. The authors would also like to thank Tuan Bendixsen, Caroline Nelson, Rae Joy (dec.), Falk Wicker, Pham Thi Hao, Tran Van Long, and the bear-keeping team for their assistance during the diagnostic workup and management of the outbreak. The authors are grateful to Tim Hyndman for his comments on part of the article.

Sources and manufacturers a. Virkon, Antec International Ltd, Sudbury, Suffolk, United Kingdom. b. Curemed Healthcare Pvt Ltd, New Delhi, India. c. Hau Giang Pharmaceutical, Can Tho City, Vietnam. d. Zoletil, Virbac, Carros, France. e. Domitor, Orion Corp., Espoo, Finland. f. Aerrane, Baxter Healthcare Corp., Deerfield, IL. g. RNAlater TissueProtect tubes, QIAamp viral RNA mini kit, QIAquick PCR purification; Qiagen GmbH, Hilden, Germany. h. Aftopur, Merial SAS, Lyon, France. i. Invitrogen RT/Platinum Taq polymerase mix, Gibco DMEM + GlutaMAX-I, Gibco FBS, GIBCO PenStrep; Life Technologies, Grand Island, NY. j. Mastercycler ep gradient S system, Eppendorf AG, Hamburg, Germany. k. CEQ 8000 genetic analysis system DNA, Beckman Coulter Inc., Brea, CA. l. GENETYX, Genetyx Corp., Tokyo, Japan.

Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.

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