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.
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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 150m 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.
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CDV is a morbillivirus, which is known to cause systemic diseases in canids, mustelids (ferrets, mink), raccoons, collared peccaries, seals, and felids.3,22 It has emerged as a potentially devastating pathogen in certain wild felid populations, including lions, tigers, lynx, and bobcat.23,24Although CDV has only one serotype, it has multiple strains with di®erent virulence and neurotrop-ism. In Taiwan, serum antibodies to CDV had been detected in a captive leopard cats25; and CDV infection had been diagnosed in 2 wild TWFBs by histopathology and RT-PCR.26However, no CDV sequences of TWFBs were submitted to the GenBank database. In the present study, CDV infection was detected only in MPCs, in which distinct eosinophilic intranuclear and intracyto-plasmic inclusion bodies could be easily detected in the epithelium of airway and syncytial cells, but not in the brain tissue. CDV infection can be easily di®erentiated from rabid TWFBs and MPCs, in which intracyto-plasmic Negri bodies could be easily found in neurons. In Taiwan, it is believed that CDV-infected dogs and/or wild carnivores are the most likely source of infection for the susceptible wild-ranging carnivores.25
S. hyicus is a Gram-positive coccus knowing to cause a fatal generalized exudative epidermitis in newborn piglets.27,28It has also been isolated from animals with septic polyarthritis and from bovine mastitis.29,30 Two cases of human infection, including a donkey bite-induced wound infection31 and a bacteremia in a farmer,32have been reported. To our knowledge, this is the ¯rst case of S. hyicus-associated pneumonia. The source of the infection and whether it requires any predisposing factor for the pneumonia development are not known.
Parasitism is a common ¯nding in free-living wildlife, including zoo animals. Although parasitism-associated severe illness may be occasionally encountered in wild-life, most of the natural cases display limited or more often no clinical e®ects as an incidental ¯nding. In the present study, parasitism was also the most common
¯nding; however, many of the parasites cannot be identi¯ed simply based on their morphological char-acteristics seen in tissue sections. Lungworms are nem-atode of the order Strongylida that infest the lungs of vertebrates. The most common lungworms belong to the superfamily either Trichostrongyloidea or Metas-trongyloidea, but not all the species in these super-families are lungworms. Lungworms infest a wide range of mammals and have a direct or indirect and complex life cycle. For those lungworms with indirect life cycle, snail, slug, or earthworm plays the role of intermediate host for those susceptible terrestrial mammals.33,34 Owing to the conserved nature of ribosomal RNA
(rRNA) and its potential use for providing informative characters for comparative analysis with other nematode taxa, the nuclear DNA locus has been used for phylogenetic analysis in nematode, including Metas-trongyloide.33Based on the facts of host site speci¯city, parasite morphology, and snail and earthworm being the preferred food for TWFBs in the ¯eld, A. cantonesis was initially considered as a potential candidate for the worms found in the lungs of TWFBs. PCR amplicons with the expected size of 1134 bp were obtained by using the reported primers designed based on the 18S rRNA gene of A. cantonesis19; surprisingly, further sequencing and phylogenetic analysis of the 18S rRNA gene revealed that the lungworm seen in TWFBs was genet-ically more closely related to P. decorus, a lungworm of sea mammals, instead of A. cantonesis. In badger spe-cies, several di®erent lung nematodes have been reported in European badgers (Melesmeles), including A. falci-formis, A. vasorum, A. pridhami, Crenosoma sp., and Capillaria aerphil.35 For most Metastrongyloidea spe-cies, gastropods such as slugs and snails act as the in-termediate hosts. However, nongastropod inin-termediate hosts also exist, such as ¯shes and earthworms in the life cycle of P. decorus in pinnipeds and Metastrongylus spp.
in suids, respectively.33For A. cantonesis, the larvae in the permissive host such as rats will complete the pul-monary migration from the pulpul-monary artery to the terminal bronchioles and pharynx. Whereas in nonper-missive hosts such as mice, guinea pigs, rabbits, rhesus monkeys, and humans, the larvae generally fail to complete the pulmonary migration and remain in the central nervous system until death.34TWFBs are spec-ulated to be a permissive host for the lungworm based on its complete life cycle. To our knowledge, this is also the
¯rst report of lungworm infestation in TWFBs; however, whether this parasite is truly a new species closely re-lated to P. decorus requires further elucidation.
Pearsonema plica and P. feliscati, known as bladder worms, are nematode parasites that inhabit in the uri-nary bladder and rarely in the ureters and renal pelvis of various domestic and wild carnivores. This parasite has a worldwide distribution and is frequently seen in dogs and foxes. Its life cycle is indirect and involves earth-worm as an intermediate host.36 The species of the bladder worm found in CEMs could not be determined simply based on their morphological characteristics histologically. If the bladder worm that we found belongs to one of the two species of Pearsonema men-tioned above, this parasite should be found in TWFBs more easily instead of in CEMs because earthworm is the known intermediate host. Whether the bladder worm found in the study is a new species or there are other 82 H.-Y. Chiou et al.
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intermediate hosts aside from earthworm requires fur-ther elucidation.
Sarcoptic mange is a worldwide, highly contagious, parasitic skin disease of mammals. The etiologic agent is the burrowing mite Sarcoptes spp., which also causes scabies in humans. Many wild animal species, including red fox (Vulpes vulpes), develop extensive skin lesions and eventually die.37 In Taiwan, there were only two cases of wild Formosan serows (Capricornis swinhoei) infested with Sarcoptes scabiei and Chorioptic texanus reported in 2007.38
In the present study, lingual capillariasis due to Trichinelloidea (Capillariiadae) infestation was diag-nosed in four TWFBs. Trichinelloidea infestation was also observed in the skin of one TWFB and one SCV. To our knowledge, the nematode Capillaria xenopodis (Pseudocapillaroides xenopi) which causes cutaneous and/or epidermal capillariasis has only been reported in South African clawed frogs (Xenopus laevis).39,40 An unusual zoonotic trichuroid nematode, Anatrichosoma sp.,41 has been identi¯ed to cause ulcerative podo-dermatitis in cats.41,42 This parasite has also been identi¯ed occasionally in human mouth lesions.43 How-ever, no information of cutaneous and/or lingual capil-lariasis (Trichinelloidea) is available in wild carnivores.
The ¯ndings of the present study have clearly dem-onstrated that there are many unknown diseases cryp-tically circulating in the wildlife sharing the same habitat with us, and some of which may even have se-rious impacts on our and other animal's health, wildlife conservation, and economy. These ¯ndings also reinforce the importance of an e®ective wildlife disease surveil-lance. The information accumulated from years of sys-temic survey may allow us to predict future emergence of known and unknown pathogens.4
Acknowledgments
The study was supported in part by Grants 101AS-10.1.2-BQ-B1(1), 102AS-10.1.1-101AS-10.1.2-BQ-B1(1), 102AS-10.1.1-BQ-B1 (3), 103AS-10.1.1-BQ-B1(1), 104AS-10.1.2-BQ-B1(1), and MOST 103-3114-Y-518-001 from the Bureau of Ani-mal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, Taiwan.
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