The raccoon dog (Nyctereutesprocyonoides) is a medium-sized omnivore native to Asia. Because they live close to human habitation, and therefore, there is likely bidirectional influence between raccoon dogs and humans, it is important to investigate their potential risks. Here, to identify potential risks of carriage of antimicrobial resistance (AMR) and human pathogens by raccoon dogs, we investigated spatioseasonal patterns of fecal resistome (collection of antimicrobial resistance genes: ARGs), pathogenome (collection of virulence factor genes: VFGs), and microbiome (collection of bacterial species) of raccoon dogs inhabiting an urban forest area and a rural rice paddy area in Korea. Metagenomic sequencing revealed that the compositions of fecal resistome, pathogenome, and microbiome were all patterned by season, and we hypothesize that the observed patterns are due to seasonal changes in the diet of omnivorous raccoon dogs. Furthermore, although less pronounced than the seasonal differences, we also observed the geographical differences in the resistome, with aminoglycoside resistance genes being more prevalent in the rural area, which may reflect the geographical difference in selective pressures for AMR emergence, such as the use of manure that may contain antimicrobials in rice cultivation in agricultural areas. Additionally, our network analysis revealed that specific antimicrobial resistance genes were linked to specific bacterial pathogens, e.g., tetA-P to Clostridium. Overall, our study successfully revealed for the first time that not only the microbiome, but also the resistome and pathogenome of wild animals change spatioseasonally, and that the risk of AMR in bacterial pathogens laden by raccoon dogs is spatioseasonal.

Bats can be phylogenetically classified into three major groups: pteropodids, rhinolophoids, and yangochiropterans. While rhinolophoids and yangochiropterans are capable of laryngeal echolocation, pteropodids lack this ability. Delicate ear movements are essential for echolocation behavior in bats with laryngeal echolocation. Caudal auricular muscles, especially the cervicoauricularis group, play a critical role in such ear movements. Previously, caudal auricular muscles were studied in three species of bats with laryngeal echolocation, but to our knowledge, there have been no studies on non-laryngeal echolocators, the pteropodids. Here, we describe the gross anatomy of the cervicoauricularis muscles and their innervation in Cynopterus sphinx by using diffusible iodine-based contrast-enhanced computed tomography and 3D reconstructions of immunohistochemically stained serial sections. A previous study on bats with laryngeal echolocation reported that rhinolophoids have four cervicoauricularis muscles and yangochiropterans have three. We observed three cervicoauricularis muscles in the pteropodid C. sphinx. The number of cervicoauricularis muscles and their innervation pattern were comparable to those of non-bat boreoeutherian mammals and yangochiropterans, suggesting that pteropodids, and yangochiropterans maintain the general condition of boreoeutherian mammals and that rhinolophoids have a derived condition. The unique nomenclature had been previously applied to the cervicoauricularis muscles of bats with laryngeal echolocation, but given the commonality between non-bat laurasiatherians and bats, with the exception of rhinolophoids, maintaining the conventional nomenclature (i.e., M. cervicoauricularis super ficialis, M. cervicoauricularis medius, and M. cervicoauricularis profundus) is proposed for bats.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a cytokine that binds to fibroblast growth factor-inducible 14 (Fn14). The TWEAK/Fn14 pathway promotes intestinal inflammation, induces C-X-C motif chemokine ligand 10 (CXCL10), and is upregulated in the intestine of humans with inflammatory bowel disease. However, the role of this pathway in canine chronic enteropathy (CE) remains unclear. Therefore, we measured TWEAK, Fn14, and CXCL10 mRNA levels in the intestinal mucosa of dogs with CE. Real-time PCR analysis revealed significantly lower TWEAK and CXCL10 mRNA levels in the duodenal mucosa of dogs with antibiotic-responsive enteropathy (ARE), a type of CE, compared to healthy dogs. The findings suggested the involvement of decreased TWEAK and CXCL10 mRNA levels in the ARE etiology.

The leopard cat ( Prionailurus bengalensis ), an endangered species in South Korea, is a small feline widely distributed in Asia. Here, we investigated the diet of leopard cats in the inland areas of Korea by examining their fecal contents using vertebrate mitochondrial 12S rRNA gene sequencing and shotgun metagenomic sequencing. Shotgun metagenomic sequencing revealed that the feces were rich in DNA not only of vertebrates but also of arthropods and plants, but care should be taken when using shotgun metagenomic sequencing to identify vertebrates at low taxonomic levels (e.g., genus level), as it was often erroneous. Meanwhile, vertebrate mitochondrial 12S rRNA gene sequencing was found to be accurate in the genus-level identification, as the genera identified were consistent with the Korean fauna. We found that small mammals such as murids were their main prey. By using these two sequencing methods in combination, this study demonstrated that accurate information about the overall dietary content and vertebrate prey of leopard cats could be obtained. We expect that the continued community efforts to expand the genome database of wildlife, including vertebrates, will alleviate the problem of erroneous identification of prey at low taxonomic levels by shotgun metagenomic sequencing in the near future.

Consisting of diverse groups of organisms, parasites are among the least studied pathogens despite their enormous impacts on humans, livestock, and wildlife. In particular, little is known about their host specificity and diversity in wildlife. Here, using multiple primer pairs and sequencing 18S rRNA genes of diverse groups of parasites, we aimed to investigate fecal parasitomes of carnivorous wildlife in Korea, namely, the raccoon dog (Nyctereutes procyonoides), the leopard cat (Prionailurus bengalensis), and the Eurasian otter (Lutra lutra). A total of 5 host-specific parasite species were identified, including 2 from raccoon dogs, 2 from leopard cats, and 1 from Eurasian otters. In addition, numerous parasite species of their prey animals were detected in their feces. It was found that the parasitome composition varied between host animals, and it was thought that the difference was attributed to the difference in prey animals, as numerous small mammal parasites were detected from feces of leopard cats inhabiting inland areas and fish parasites from feces of Eurasian otters and raccoon dogs inhabiting waterside areas. Furthermore, 5 zoonotic parasites known to infect humans were identified at the species level. Wildlife-associated zoonoses are expected to increase as the proximity between humans and wildlife increases due to urbanization. Vigilance may be necessary, such as by monitoring parasites in wildlife feces, as was done in this study.

Among bats, rhinolophoids and yangochiropterans, but not pteropodids, exhibit laryngeal echolocation. Although Rousettus has been regarded as the only pteropodid capable of echolocation using tongue clicks, recent evidence suggests that other species of pteropodids are also capable of echolocation using wing clicks. Studies on laryngeal echolocators suggest that delicate ear movements are essential for the echolocation behavior of bats and that the cervicoauricularis muscles play a critical role in such ear movements. In this study, we observed the gross anatomy of cervicoauricularis muscles in three species of pteropodids (Cynopterus sphinx, Eonycteris spelaea, and Rousettus leschenaultii) to examine whether ear muscle anatomy varies among pteropodids with different echolocation types and between pteropodids and laryngeal echolocating bats. We found that M. cervicoauricularis profundus originates from the nuchal crest in tongue-click echolocators (R. leschenaultii) and from the midline in wing-click echolocators (C. sphinx and E. spelaea). In general, tongue-click echolocation using high click rates is considered to be more sophisticated in terms of sonar performance than wing-click echolocation. M. cervicoauricularis profundus originating from the nuchal crest (CPNC) is not common in non-bat laurasiatherian mammals, but can be found in laryngeal echolocating bats. As it pulls the ear pinna caudally in the horizontal plane and increases the access to sound, CPNC found in R. leschenaultii and laryngeal echolocating bats may be a key characteristic of the sophisticated active echolocation behavior of bats.

Background C-C motif chemokine ligand (CCL)5 induces skin inflammation in healthy dogs. In addition, CCL5 is overexpressed in the skin of experimental models of canine atopic dermatitis (cAD). Tumour necrosis factor (TNF)-alpha has been shown to be upregulated in cAD. However, it remains unclear whether TNF-alpha induces CCL5 production in canine keratinocytes.Hypothesis/Objectives To determine the effect of TNF-alpha on CCL5 production in canine keratinocyte culture and investigate possible synergy with interferon (IFN)-gamma and interleukin (IL)-4.Materials and Methods CCL5 protein concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in the culture supernatant of a cell line of canine progenitor epidermal keratinocyte (CPEK) cells stimulated with TNF-alpha with or without inhibitors of the TNF receptor signalling pathway. CCL5 protein concentrations also were measured in CPEK cells stimulated with TNF-alpha in the absence or presence of IFN-gamma, a T-helper (Th)1-type cytokine, and/or IL-4, a Th2-type cytokine.Results TNF-alpha increased CCL5 production in CPEK cells in time- and dose-dependent manners. Inhibitors of the TNF receptor signalling pathway diminished CCL5 production. Although neither IFN-gamma nor IL-4 alone induced CCL5 production in CPEK cells, the combination of TNF-alpha and IFN-gamma, and not IL-4, synergistically enhanced CCL5 production in these cells.Conclusions and Clinical Relevance TNF-alpha may be involved in skin inflammation in dogs by promoting CCL5 production in keratinocytes. Furthermore, the synergistic effect of TNF-alpha and IFN-gamma suggests that the local Th1-type milieu may aggravate skin inflammation. Further studies are required to elucidate the role of TNF-alpha-induced CCL5 production of keratinocytes in the pathogenesis of cAD.

A nine-year-old, castrated male mixed-breed dog presented with a three-month history of sneezing and stertorous breathing. Computed tomography revealed a soft tissue mass in the left nasal cavity with lysis of the cribriform plate. The mass was diagnosed as intranasal sarcoma based on histopathological analysis. The tumor cells were immunohistochemically positive for KIT and platelet-derived growth factor receptor α/β and negative for vascular endothelial growth factor receptor 2 and cyclooxygenase-2. Treatment with toceranib phosphate (TOC) and firocoxib reduced the tumor size, which was defined as partial response (PR). After PR induction, TOC alone mediated survival for 205 days. This case report suggests that the combination of TOC and possibly firocoxib may be a therapeutic option for canine intranasal sarcoma.

Investigation of feces of wildlife, which is considered as reservoirs, melting pots, vectors and secondary sources of antimicrobial resistance genes (ARGs), provides insights into the risks and ecology of ARGs in the environment. Here, we investigated microbiomes, virulence factor genes (VFGs) of bacterial pathogens, and resistomes in environmental feces of Eurasian otters (Lutra lutra) and leopard cats (Prionailurus bengalensis) using shotgun metagenome sequencing. As expected, the taxonomic compositions of bacteria were significantly different between the animals. Importantly, we found that the compositions of ARGs were also significantly different between the animals. We detected ARGs including iri, tetA(P), tetB(P), floR, sulII, strA, strB, tetW and tetY. Some of them were significantly more abundant in either of the host animals, such as strA, strB and tetY in Eurasian otters, and tetA(P), tetW and iri in leopard cats. We also found that some ARGs were selectively correlated to particular VFGs-related bacteria, such as tetA(P) and tetB(P) to Clostridium, and iri to Mycobacterium. We also found that there were positive correlations between Acinetobacter and ARGs of multiple antimicrobial classes. The host-specific resistomes and VFGs-related bacteria may be due to differences in the host's gut microbiome, diet and/or habitat, but further investigation is needed. Overall, this study provided important baseline information about the resistomes of the wildlife in Korea, which may help the conservation of these endangered species and assessment of human health risks posed by ARGs and bacterial pathogens from wildlife.

The raccoon dog (Nyctereutes procyonoides) is known to be an opportunistic generalist who feeds on a wide variety of foods. Historically, their diet has been investigated by morphological observation of undigested remains in feces, requiring specialized knowledge such as osteology, zoology, and phytology. Here, we used DNA metabarcoding of vertebrate 12S rRNA gene and invertebrate 16S rRNA gene to investigate their fecal contents. Additionally, we developed a blocking oligonucleotide that specifically inhibits the amplification of the canid 12S rRNA gene. We confirmed that the blocking oligonucleotide selectively inhibit the amplification of raccoon dog’s DNA without significantly changing the composition of the preys’ DNA. We found that the main foods of raccoon dogs in our study area, the waterside of paddy fields in Korea, were fishes such as Cyprinidae and insects such as mole crickets, which makes sense given the Korean fauna and their well-known opportunistic feeding behaviors. As a method to conveniently and objectively investigate feeding habits of raccoon dogs, this study provided baseline information on DNA metabarcoding. By using DNA metabarcoding, it is expected that the diet habits and ecology of raccoon dogs will be better understood by future research.

The phylogenetic relationships among seven Callosciurus species from the Indochina Peninsula, including the C. honkhoaiensis which is endemic to Hon Khoai Island, were studied using complete mitochondrial cytochrome b gene sequences (1140 bases). We primarily focused on the phylogenetic position of C. pygerythrus, which is distributed in the western part of the peninsula. We identified two main lineages: 1) C. caniceps, C. honkhoaiensis, C. inornatus, C. phayrei and C. pygerythrus, and 2) C. erythraeus and C. finlaysonii. Estimated divergence time between the two lineages was at the junction of the Zanclean and Piacenzian in the Pliocene. Within the first linage, the divergence time of sub-lineages corresponded to the Pliocene-Pleistocene boundary, although phylogenetic relationships were unclear. These two divergence times estimated in the present study correspond to episodes of global cooling, suggesting that climate may have contributed to the divergence of these Callosciurus squirrels.

In this study, we examined the role of the eastern bent-winged bat (Miniopterus fuliginosus) in the dispersion of bat adenovirus and bat alphacoronavirus in east Asia, considering their gene flows and divergence times (based on deep-sequencing data), using bat fecal guano samples. Bats in China moved to Jeju Island and/or Taiwan in the last 20,000 years via the Korean Peninsula and/or Japan. The phylogenies of host mitochondrial D-loop DNA was not significantly congruent with those of bat adenovirus (m2XY = 0.07, p = 0.08), and bat alphacoronavirus (m2XY = 0.48, p = 0.20). We estimate that the first divergence time of bats carrying bat adenovirus in five caves studied (designated as K1, K2, JJ, N2, and F3) occurred approximately 3.17 million years ago. In contrast, the first divergence time of bat adenovirus among bats in the 5 caves was estimated to be approximately 224.32 years ago. The first divergence time of bats in caves CH, JJ, WY, N2, F1, F2, and F3 harboring bat alphacoronavirus was estimated to be 1.59 million years ago. The first divergence time of bat alphacoronavirus among the 7 caves was estimated to be approximately 2,596.92 years ago. The origin of bat adenovirus remains unclear, whereas our findings suggest that bat alphacoronavirus originated in Japan. Surprisingly, bat adenovirus and bat alphacoronavirus appeared to diverge substantially over the last 100 years, even though our gene-flow data indicate that the eastern bent-winged bat serves as an important natural reservoir of both viruses.

The placenta of the Korean water deer was anatomically examined to accumulate basic information regarding its reproductive system. The convex placentomes with five to nine well-developed pedicles were observed in the whole uterine horns, and therefore, the placenta was classified as oligocotyledonary. The evidence indicating the migration of binucleate cells (BNCs) from trophectoderm to the uterine epithelium led to the histological classification of the placenta as synepitheliochorial. The number of fetuses was markedly higher than that in other ruminant species. However, the number of placentomes was found to be similar to the other Cervidae species. Therefore, these results suggest that the Korean water deer may possess special mechanisms or structures at the fetus attachment site to maintain this unusally high number of fetuses.

Morphological variation of the skull was examined in the northern treeshrew (Tupaia belangeri) from various localities across Southeast Asia. Through a multivariate analysis, the treeshrews from South Vietnam exhibited distinct morphological characteristics compared to other populations from Thailand and Laos, and Malaysia. The plots of the specimens of North Vietnam are not randomly mixed with Thailand plots segregation in the scatteregrams of canonical discriminant analysis. Since the skulls of the population from North Vietnam were morphologically similar to those form central Laos and northern and northeastern Thailand, the zoogeographical barrier effect of Mekong River was not clearly confirmed. The population of the Kanchanaburi in western Thailand is clearly smaller in size compared to the other populations. The southern border of the distribution of this species is determined by the Isthmus of Kra or Kangar-Pattani Line. In the northern treeshrew, which is distributed from southern China to Bangladesh and southern Thailand, we have detected osteometrical geographical variation driven by geography. These results indicate that the skull morphology in the Tupaia glis-belangeri complex distinctively differs in South Vietnam, western Thailand, and southern Thailand. The zoogeographical barrier and factor separating these districts are expected to clarify in the future.

A 7-year 6-month-old, castrated male Shiba dog presented with a 1-month history of lethargy, anorexia, vomiting, and frequent watery diarrhea. Weight loss, hypoalbuminemia, anemia, and leukocytosis were detected at the first visit. The dog was diagnosed with non-responsive enteropathy (NRE) based on clinical and histopathological examinations. Since the dog did not respond to the immunosuppressive drugs, fecal microbiota transplantation (FMT) was performed during the treatment with chlorambucil. A single endoscopic FMT into the cecum and colon drastically recovered clinical signs and clinicopathological abnormalities and corrected dysbiosis in the dog. No recurrence or adverse events were observed. The present case report suggests that FMT, possibly together with chlorambucil, might be a treatment option for NRE in Shiba dogs that have poorer prognosis compared with other dog breeds.

Immunohistochemical investigations of the expression of surfactant protein A (SP-A) and surfactant protein D (SP-D) in the uterine and placental tissues of 13 pregnant mares were performed using anti-horse monoclonal primary antibodies. Strong positive reactions for both SP-A and SP-D were observed in the trophoblasts in the microcotyledons of the placentae at 182 to 314 days of gestation; in uterine glandular epithelial cells, faint-to-weak reactions were observed during gestation. This study describes, for the first time, the changes in the SP-A and SP-D expression levels in the endometrium of mares during gestation; the SP-A and SP-D expression levels increased after the second trimester of gestation.

Background Lymphoma in the nasal cavity is the most common tumor of cats' upper respiratory tract. However, the effect of single-agent chlorambucil on nasal or nasopharyngeal lymphoma has not been evaluated in cats. Case description An 8-year-old, castrated male Scottish Fold weighing 3.5 kg presented with an 8-month history of nasal discharge, sneezing, and mild epistaxis. CT and rhinoscopy revealed nasal discharge and slight swelling of the nasopharyngeal mucosa, but no masses and local invasions were detected. Histopathological and immunohistochemical analyses of the nasopharyngeal mucosa demonstrated B-cell lymphoma in the cat. The treatment with chlorambucil led to long-term management of the cat without any side effects. No recurrences of clinical signs have been observed for 754 days. Conclusion The present case report suggests that chlorambucil can be a therapeutic option for feline localized nasopharyngeal B-cell lymphoma without masses and local invasions.

台湾の金門島に生息するユーラシアカワウソ（Lutra lutra chinensis以下，カワウソ）は，比較的長期間にわたり人為的影響を受けなかったことや豊富な人為的水環境の存在により幸運にも絶滅を免れてきた。2018年の糞便痕跡および赤外線カメラの映像に基づく調査から過去の文献同様金門島の西南部はカワウソの利用率が顕著に低いことが判明したが，その他の各エリアの利用頻度について論じるためにはより長期的なデータが必要である。さらに，2017年から２年間実施した赤外線カメラ映像に基づく行動観察から，繁殖や捕食に関する行動に加え，カワウソの人工物利用状況についても明らかになった。またカワウソに脅威を与える存在である野良猫および野良犬については未だ多くがカワウソの生息域内を行動していることが判明した。現在カワウソは台湾内の法律においても厳重に保護されており，人為的影響を殆ど受けることはない。また調査に加え様々な保全の取り組みも行われているが，その保全活動は地元自治体に委ねられており，常に楽観視することができない状態である。地元住民からは経済発展を望む声も多く聞かれ，食害も発生しているため，今後長期的に保全活動を持続するためには地域還元型のカワウソ保全システムを確立する必要がある。そして，将来的な日本の対馬におけるカワウソ保全においては，諸外国の同種の保全活動と課題を参考にし，より効果的な取り組みを行っていくことが肝要である。

Leptospira, Giardia intestinalis and Toxoplasma gondii infections are reported in humans and animals worldwide, but molecular surveillance of these pathogens in Korean wildlife is still limited. Here, we examined the prevalence of these pathogens in environmental feces of Eurasian otters, leopard cats and raccoon dogs using nested PCR followed by DNA sequencing. G. intestinalis was detected in all of three animals, while T. gondii was detected only in leopard cats. Leptospira wolffii was detected in raccoon dog and Eurasian otter. Our results suggest that these animals can act as a reservoir of these zoonotic pathogens. Consistent monitoring of these pathogens in wildlife is needed to prevent from their infections in humans and livestock in Korea.

Background The involvement of interleukin (IL)-33 produced by keratinocytes has been suggested in the pathogenesis of canine atopic dermatitis (cAD). House dust mite (HDM)-derived proteases induce the production of various cytokines and chemokines in keratinocytes via protease-activated receptor-2 (PAR-2); however, their effects on IL-33 mRNA expression in canine keratinocytes have not been determined. Hypothesis/Objective To clarify whether HDM-derived proteases induce IL-33 mRNA expression in canine keratinocytes via PAR-2. Methods and materials Expression of IL-33 mRNA was quantified by real-time PCR in a cell line of canine progenitor epidermal keratinocytes (CPEK) stimulated with Dermatophagoides farinae (Der f) whole body extract, Der f pre-treated with cysteine protease and serine protease inhibitors, and trypsin. Trypsin and Der f-mediated IL-33 mRNA expression also was measured in CPEK cells treated with a PAR-2 antagonist. Results Der f enhanced IL-33 mRNA expression in CPEK cells in incubation time- and dose-dependent manners. Der f pre-treated with a serine protease inhibitor, and not a cysteine protease inhibitor, abrogated an increase in IL-33 mRNA expression in CPEK cells. Trypsin also enhanced IL-33 mRNA expression in CPEK cells. Trypsin-mediated IL-33 mRNA expression was completely abolished by a PAR-2 antagonist, while Der f-mediated IL-33 mRNA expression was partially and significantly diminished by it. Conclusions and clinical relevance Der f-derived serine protease upregulated IL-33 mRNA expression in CPEK cells at least in part via PAR-2. These findings suggest that HDM may be involved in the development of (C)AD by increasing IL-33 mRNA expression in keratinocytes.