Molecular Detection and Genetic Diversity of Blastocystis in Korean Dogs.

Blastocystis is a genus of unicellular heterokont parasites belonging to a group of organisms known as Stramenopiles, which includes algae, diatoms, and water molds. Blastocystis includes several species that habitat in the gastrointestinal tracts of organisms as diverse as humans, farm animals, birds, rodents, reptiles, amphibians, fish, and cockroaches. It is important to public health and distributed globally, but its prevalence in dogs in Korea has not been reported to date. Here, we collected 787 canine fecal samples and assessed Blastocystis infection by age, sex, region, season, and diarrhea symptoms. We determined Blastocystis subtypes using phylogenetic analyses based on 18S rRNA gene sequences. We identified, 10 Blastocystis positive samples (1.3%). A higher proportion of infected dogs was asymptomatic; however, infection rates did not significantly differ according to region, age, sex, and season. Phylogenetic analysis showed that the Blastocystis sp. identified belonged to 4 subtypes (STs), ST1, ST5, ST10, and ST14, thus revealed the genetic diversity of Blastocystis sp. in dogs Korean. This is first report on the presence of Blastocystis sp. in dogs Korean. This study revealed a lower infection rate than expected and differed from previous studies in STs. Further studies are warranted to observe the national infection status of Blastocystis in dogs and the genetic characteristics of this genus.

A case of active incomplete biliary cirrhosis in an aged female Japanese macaque (Macaca fuscata).

We describe the first case of biliary cirrhosis in Japanese macaque. Clinical signs had not been detected. The liver was nodular. Histopathologically, portal-to-portal pattern of fibrosis might have indicated chronic cholestasis. Fibrotic septa were infiltrated with inflammatory cells. Therefore, this case could be diagnosed as active incomplete biliary cirrhosis.

An outbreak of toxoplasmosis in squirrel monkeys (Saimiri sciureus) in South Korea.

BACKGROUND: Toxoplasma gondii (T. gondii) is an intracellular protozoan parasite that can infect warm-blooded animals including humans. New World monkeys, such as squirrel monkeys, are more susceptible to T. gondii than Old World monkeys, often developing fatal disease. METHODS: In this study, seven of thirteen dead squirrel monkeys at Seoul Grand Park were tested to find the cause of sudden death. RESULTS: The main histopathological findings included interstitial pneumonia, necrotizing hepatitis, and splenitis. Periodic acid-Schiff staining of liver, spleen, and lung revealed cyst structures consistent with bradyzoites. Amplification of the B1 gene was detected in the liver or spleen of all monkeys. Additionally, a restriction fragment length polymorphism assay and phylogenetic analysis of the GRA6 amplicon revealed a consistent clustering with the type II strain of T. gondii. CONCLUSIONS: This study is the first report of T. gondii infection of squirrel monkeys in Korea, and the first report of type II T. gondii based on GRA6 analysis in Korea.

Seroprevalence and risk factors of Besnoitia besnoiti infection in Korean cattle - short communication.

Besnoitia besnoiti is an obligate intracellular parasite that is transmitted by direct contact or via mechanical transmission by flies as vectors. Besnoitiosis causes economic losses in the cattle industry and is regarded as a re-emerging disease in Europe. This study evaluated the seroprevalence of B. besnoiti in Korean cattle using a commercial ELISA kit. Among 558 serum samples, 19 (3.4%) tested seropositive for B. besnoiti. The statistically significant risk factors included age (≥ 2 years), sex (castrated males), and region (lower latitudes) (P < 0.05). The overall seroprevalence suggested a wide distribution of B. besnoiti infection in cattle reared in Korea. Thus, the practice of intensive cattle husbandry and the regionally different seroprevalence of B. besnoiti infection in cattle in Korea warrant routine monitoring and vector control to reduce economical losses due to bovine besnoitiosis in the country.

Severe whipworm (Trichuris spp.) infection in the dromedary (Camelus dromedarius).

One adult (13-yr-old) and two young (3-4-yr-old) male dromedary camels (Camelus dromedarius) from the Seoul Zoo displayed anorexia and chronic diarrhea for 2 wk. Direct fecal smear examination revealed Trichuris spp. infection. After confirmation of the infection, fenbendazole was orally administered as a suspension; this was repeated two times at 3-wk intervals. A high initial dose (20 mg/kg) was followed by administration at the recommended dose (10 mg/kg). Starting on the day following the first treatment, a large number of adult whipworms were discharged with the feces over a 3-day period. Two young male dromedary camels gradually recovered. However, the adult male dromedary camel developed continuous bloody mucoid diarrhea and died 2 days after treatment. Postmortem examination revealed that numerous whipworms were attached to the mucosa throughout the large intestine.

Diversity of fecal parasitomes of wild carnivores inhabiting Korea, including zoonotic parasites and parasites of their prey animals, as revealed by 18S rRNA gene sequencing.

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.

Combining vertebrate mitochondrial 12S rRNA gene sequencing and shotgun metagenomic sequencing to investigate the diet of the leopard cat (Prionailurus bengalensis) in Korea.

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.

Tick Populations and Molecular Analysis of Anaplasma Species in Ticks from the Republic of Korea.

The present study was performed to survey the dominant tick populations and molecularly determine the pathogenic agents of anaplasmosis in ticks from Gyeongsang, Republic of Korea. A total of 3825 questing ticks were collected by the flagging method from 12 sites near animal farms in Gyeongsang from March to October 2021. A molecular genomic study was performed with ticks stored in 70% ethanol to detect Anaplasma genes by the previously described method. The monthly incidence of ticks varied by developmental stages, i.e., nymphs, adults, and larvae, and each of their populations peaked in May, March, and October, respectively. The predominant tick species were Haemaphysalis longicornis, Haemaphysalis sp., Haemaphysalis flava, Ixodes nipponensis, and Amblyomma testudinarium in order. To determine the Anaplasma infection rate, collected ticks were pooled into 395 groups. The minimum infection rate (MIR) of Anaplasma was 0.7% (27 pools). That of A. phagocytophilum was highest (23 pools, MIR 0.6%), followed by A. phagocytophilum-like Anaplasma spp. clade B (2 pools, MIR 0.1%), A. bovis (1 pool, MIR 0.1%), and A. capra (1 pool, MIR 0.1%), respectively. In this study, five species of ticks, including unidentified Haemaphysalis species, were collected in 12 survey sites in Gyeongsang, but their prevalence was somewhat different according to the tick species and survey sites. Further, the incidence rate (6.8%) of 4 Anaplasma spp. was not as high in tick pools. However, the results of this study may offer a basis for future epidemiological research and risk assessment of tick-borne diseases.

Conservation implications of elucidating the Korean wolf taxonomic ambiguity through whole-genome sequencing.

The taxonomic status of the now likely extirpated Korean Peninsula wolf has been extensively debated, with some arguing it represents an independent wolf lineage, Canis coreanus. To investigate the Korean wolf's genetic affiliations and taxonomic status, we sequenced and analysed the genomes of a Korean wolf dated to the beginning of the 20th century, and a captive wolf originally from the Pyongyang Central Zoo. Our results indicated that the Korean wolf bears similar genetic ancestry to other regional East Asian populations, therefore suggesting it is not a distinct taxonomic lineage. We identified regional patterns of wolf population structure and admixture in East Asia with potential conservation consequences in the Korean Peninsula and on a regional scale. We find that the Korean wolf has similar genomic diversity and inbreeding to other East Asian wolves. Finally, we show that, in contrast to the historical sample, the captive wolf is genetically more similar to wolves from the Tibetan Plateau; hence, Korean wolf conservation programmes might not benefit from the inclusion of this specimen.

Detection of gastrointestinal parasites in raccoon dogs (Nyctereutes procyonoides) in the Seosan reclaimed lands, Korea.

This study examined gastrointestinal parasites of raccoon dogs in the Seosan reclaimed lands, Korea. We collected 882 raccoon dog fecal samples and examined them using a fecal flotation technique following standard procedures. Identification was based on the morphology and size of the parasite eggs and oocysts. Nine different helminth eggs and oocysts of Isospora spp. were identified. In total, 314 (35.5%) fecal samples were positive for gastrointestinal helminths or Isospora spp. Double (6%), triple (1%), and quadruple (0.3%) infections were observed. The rates of parasite infection in spring, summer, fall, and winter were 29.8, 20.7, 39.0, and 42.9%, respectively. These results indicate that raccoon dogs host diverse helminths and Isospora spp., posing a potential risk of transmitting parasites to domestic pets and residents in the area. Active epidemiological surveillance with modern molecular techniques and ancillary epidemiological tools is recommended.

Using DNA metabarcoding and a novel canid-specific blocking oligonucleotide to investigate the composition of animal diets of raccoon dogs (Nyctereutes procyonoides) inhabiting the waterside area in Korea.

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 host-specific resistome in environmental feces of Eurasian otters (Lutra lutra) and leopard cats (Prionailurus bengalensis) revealed by metagenomic sequencing.

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.

DNA-based detection of Leptospira wolffii, Giardia intestinalis and Toxoplasma gondii in environmental feces of wild animals in Korea.

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.

Ovarian adenocarcinoma with metastases in a white rhinoceros.

A 36-y-old white rhinoceros (Ceratotherium simum) was presented with respiratory distress, sanguineous vaginal exudate, and anorexia. The clinical signs progressed over 40 d, and the rhinoceros died. Autopsy revealed significant ascites; a unilateral, 12.5-cm diameter, polypoid mass in the left ovary; a white, firm transmural mass in the right uterine horn; a white, friable mass in the lung; and white-to-tan, friable small nodules in the diaphragm. Histologic examination revealed similar neoplastic cells in the masses in all 4 locations, composed predominantly of epithelial cells proliferating in a tubulopapillary pattern with significant nuclear atypia and numerous atypical mitotic figures (18-42 per 2.37 mm2). Immunohistochemistry for CK7 (cytokeratin 7) and CK20 (cytokeratin 20) suggest that the ovarian, pulmonary, and diaphragmatic lesions were of ovarian origin and that the ovary was the primary tumor site.

Skin lesions associated with Demodex sp. in a llama (Lama glama). [corrected]

A 2.5-yr-old female llama (Lama glama) [corrected] with skin lesions was presented to the Animal Health Center in Seoul Grand Park Zoo, Korea. Mites of the genus Demodex in the absence of other mites or fungi were identified from the lesions by skin scrapings. The bodies were elongated, tapered, and 200-280 1m in length; four pairs of stumpy legs were present at the front of the body, and the striated opisthosoma constituted about half of the body length. Histologic examination of the skin biopsy showed typical folliculitis, hyperkeratinization of epidermis, and infiltration of inflammatory cells, consisting mainly of eosinophils and monocytes, in dermis. Although mites were undetected in the dilated hair follicles, the histologic features are consistent with descriptions of infestation by Demodex spp. This is believed to be the first case of skin lesions associated with Demodex sp. in L. glama. [corrected] Incidental findings were previously described in a different species of llamas (Lama glama) without any recognized symptoms. Treatment with amitraz (0.025%) eliminated the mites and resolved the clinical signs.

DNA metabarcoding-based diet survey for the Eurasian otter (Lutra lutra): Development of a Eurasian otter-specific blocking oligonucleotide for 12S rRNA gene sequencing for vertebrates.

The Eurasian otter (Lutra lutra) is an endangered species for which diet analyses are needed as part of its conservation efforts. Eurasian otters feed on vertebrates, such as fishes, and invertebrates, such as crustaceans, but their detailed taxonomies are not fully understood in part due to limited resolving power of traditional morphological identification methods. Here, we used high-throughput sequencing (HTS)-based DNA metabarcoding approaches to analyze diet profiles of Eurasian otters inhabiting a marshy estuary area in Korea. We investigated their diet profiles based on spraint sampling followed by DNA metabarcoding analyses targeting 12S rRNA gene region for vertebrates, 16S rRNA gene region for invertebrates, and cytochrome c oxidase 1 (COI) gene region for fishes. For the vertebrate analysis, a blocking oligonucleotide (OBS1) was designed to suppress amplification of DNA fragments derived from the otters. The 12S rRNA gene sequencing assay detected species belonging to fishes (95%) and amphibians (3.3%). Fishes detected by 12S rRNA gene sequencing included crucian carp (Carassius auratus), mullets (Mugil spp.), bluegill (Lepomis macrochirus), and northern snakehead (Channa argus), which were also detected by COI gene sequencing. Among invertebrates, mud flat crabs (Helicana spp.) and shrimps (Palaemon spp.) were abundant. The designed blocking oligonucleotide OBS1 effectively inhibited amplification of the otter's DNA, with only up to 0.21% of vertebrate sequence reads assigned to the otter. This study demonstrated that HTS-based DNA metabarcoding methods were useful to provide in-depth information regarding diet profiles of the otters at our sampling site. By using HTS-based DNA metabarcoding approaches, future research will explore detailed taxonomies of their diets across locations and seasons.

Severe whipworm (Trichuris spp.) infection in the hamadryas baboon (Papio hamadryas).

A 3-year-old male hamadryas baboon (Papio hamadryas) at the Seoul Zoo, Korea, died without any previous symptoms. Necropsy revealed severe whipworm infection in the large intestine. The animal weighed 2.6 kg and had a blood clot at the anus. Numerous whipworms were found attached to the intestinal wall, with their anterior ends embedded in the mucosa. Fecal microscopy revealed typical barrel-shaped, brown eggs of Trichuris spp., with hyaline polar plugs at each end. Histopathological examination revealed the thin anterior part of Trichuris spp. embedded in the mucosal layer and the thick posterior part at the mucosal surface or hanging freely in the intestinal lumen. This case emphasizes the importance of parasitic infection management in zoo animals.

First record of Ginkgo-toothed beaked whale (Mesoplodon ginkgodens) stranded in Korea.

Two stranded whales were found dead on the coast of Jeju, South Korea. Based on the outer appearance and autopsy findings, one was determined to be an adult and the other a calf. The carcasses were dissected for species identification and pathological examination. A genetic analysis was performed, and the morphological characteristics of the skull observed. Then, 448 bp of the 5' half of the mitochondrial (mt) DNA control region and 413 bp of the mtDNA cytochrome b gene were sequenced. A BLAST search revealed that the whales were ginkgo-toothed beaked whales (Mesoplodon ginkgodens). Morphological comparison of the adult skull with the holotype specimen confirmed the result. This is the first record of a stranded ginkgo-toothed beaked whale in Korea.

Sarcoptic mange in wild raccoon dogs (Nyctereutes procyonoides) in Korea.

Infestation with Sarcoptes scabiei was diagnosed from four wild raccoon dogs (Nyctereutes procyonoides) accidentally captured and presented to the Animal Health Center in Seoul Grand Park Zoo, Korea. Diagnosis was done by microscopic and histologic examination from skin lesions. Sarcoptes scabiei was the only species detected from the lesions and characterized by dorsoventrally flattened and round bodies, sucker-like pulvilli borne on long nonjointed pretarsi, triangular scales and spinelike setae on the dorsum, and three epimeres that are chitinous extensions of the coxae of the legs. In addition, infiltration of mast cells in the dermis was associated with infestation of the burrowing mite. This is the first report of sarcoptic mange in raccoon dogs in Korea. Because heavy infestation with S. scabiei was found in all of the captured wild raccoon dogs, further work is necessary to develop prophylactic interventions to prevent the spread of sarcoptic mange in free-living raccoon dogs in Korea.

Comparison of fecal microbiota of three captive carnivore species inhabiting Korea.

This study aimed at characterizing fecal microbiota of three captive carnivore species of leopard cats Prionailurus bengalensis, Eurasian otters Lutra lutra and raccoon dogs Nyctereutes procyonoides. We used DNA barcoding sequencing to analyze 16S rRNA genes of uncultured bacteria in the feces collected in the Seoul Zoo. The sequencing analyses revealed that: 1) Firmicutes was the most dominant phylum for all three animals; 2) bacterial genus-rank compositions were distinct across species of the animals; and 3) bacterial community memberships were different across species of the studied animals. We expect such baseline information is useful for better understanding of these endangered species and future management of their health in zoos.