Subcutaneous cysticercosis due to Taenia crassiceps (Cestoda: Taeniidae) in an imported steppe lemming in Japan.

This study describes a subcutaneous proliferative cysticercosis in a pet steppe lemming, Lagurus lagurus (Rodentia: Cricetidae), bred and imported from Czech Republic into Japan. Numerous metacestodes were collected from the subcutaneous cystic lesion of the left medial thigh. Four surgical removals were coupled with anthelmintic treatment but ended with recurrence. Based on morphological features and mitochondrial DNA sequences, the metacestodes were identified as the larval stage of Taenia crassiceps (Zeder, 1800). This is the first case of infection with larval T. crassiceps in rodents of the genus Lagurus, and becomes the third case of the parasite detected from imported animals in Japan. Related public health concerns are discussed.

African pygmy hedgehog adenovirus: Virus replication, virus-induced cytopathogenesis and activation of mitogen-activated protein kinase signaling pathways in infected MDCK cells.

We examined several aspects of African hedgehog adenovirus (AhAdv-1) that was isolated from an African pygmy hedgehog, including: replication kinetics of, virus-induced cytopathic effect (CPE), activation status of mitogen-activated protein kinase (MAPK) signaling pathways, and possible roles of these signaling pathways in virus replication and virus-induced CPE in MDCK cells. AhAdv-1 efficiently replicated and induced CPE in infected cells and caused accumulation of cleaved caspase-3 at 24 h post-infection (p.i.), suggesting apoptosis induction. Analysis of several intracellular signal transduction pathways, which are involved in apoptosis, showed activation of p38 MAPK, Akt and ERK1/2 pathways at 3 h p.i., and upregulation of phosphorylated SAPK/JNK at 24 h p.i. Although p38 MAPK inhibitor and SAPK/JNK inhibitor suppressed activation of the respective pathways in infected cells, they did not inhibit virus-induced CPE. Treatment of infected cells with inhibitor of the Akt pathway, the p38 pathway, the SAPK/JNK pathway or the ERK pathway revealed that inhibitors of p38 pathway inhibited viral replication by real-time PCR and TCID50 assay in infected MDCK cells, suggesting that AhAdv-1 uses p38 pathway for multiplication in infected cells.

New canine parvovirus 2a infection in an imported Asian small-clawed otter (Aonyx cinereus) in Japan.

Post-import from the Republic of Indonesia to Japan in 2017, two juvenile, captive bred Asian small-clawed otters (Aonyx cinereus) exhibited gastrointestinal symptoms, including vomiting, diarrhea, and hematemesis, and died. One of them was examined postmortem. Microscopically, the small intestinal mucosa was necrotic with crypts lined by regenerating large epithelial cells. A gastric cardiac mucosal ulcerative lesion containing fungal yeasts and pseudohyphae morphologically indicated Candida spp. The lymph nodes exhibited marked lymphoid depletion. Canine parvovirus 2 (CPV-2) was isolated from an oral swab, and virus protein 2 (VP2) gene sequencing revealed new CPV-2a. To our knowledge, this is the first new CPV-2a infection report in Asian small-clawed otters. This infection should be considered in gastrointestinal symptom-related cases in this species.

Molecular evidence for vaccine-induced canine distemper virus and canine adenovirus 2 coinfection in a fennec fox.

A 61-d-old fennec fox (Vulpes zerda), 11 d after receiving a multivalent, modified-live virus vaccine containing canine distemper virus (CDV), canine adenovirus 2 (CAdV-2), parainfluenza virus, parvovirus, and canine coronavirus, developed oculonasal discharge, and subsequently convulsions, and hemoptysis, and died. Microscopic changes in the cerebrum were evident, including neuronal degeneration and necrosis; intracytoplasmic eosinophilic inclusion bodies were observed in astrocytes. CDV was detected in the brain tissue by immunohistochemistry. Pulmonary lesions of multifocal necrotizing bronchopneumonia had Cowdry type A intranuclear inclusions in the bronchial epithelial cells. Electron microscopy revealed crystalline arrays of adenovirus-like particles within the intranuclear inclusions. Additionally, the hemagglutinin gene of CDV and the CAdV-2 DNA polymerase gene were detected in the fennec fox; sequence analysis showed 100% identity with those of the vaccine strain viruses. To our knowledge, vaccine-induced CDV and CAdV-2 coinfections using molecular analysis have not been reported previously. Therefore, vaccine strains should be considered prior to CDV vaccination in nondomestic carnivores.

Molecular characterization of feline paramyxovirus in Japanese cat populations.

Feline paramyxovirus (FPaV) is a member of the family Paramyxoviridae that has been reported only in Germany and the United Kingdom. We detected FPaV for the first time in Japan by transcriptome sequencing of cat urine samples. We determined the genome structure of FPaV and conducted a phylogenetic analysis. It was found that FPaV belongs to the genus Jeilongvirus and forms a clade with Mount Mabu Lophuromys virus 1 (MMLV-1). FPaV lacks a small hydrophobic (SH) gene that is found in members of the genus Jeilongvirus; however, some jeilongviruses also do not have this gene. These results provide information about the diversity and evolution of paramyxoviruses.

Complete Genome Sequence of an Adenovirus-1 Isolate from an African Pygmy Hedgehog (Atelerix albiventris) Exhibiting Respiratory Symptoms in Japan.

This study reports the complete genome sequence of an African pygmy hedgehog adenovirus-1 isolate from an African pygmy hedgehog which displayed respiratory symptoms that included nasal discharge, sniffling, coughing, and respiratory distress. The viral genome is 31,764 bp long and shows four deletion sites compared to that of skunk adenovirus-1.

Epidemic nodular facial myxomatous dermatitis in juvenile Cranwell's horned frogs Ceratophrys cranwelli.

In 2017, approximately 40 out of 100 captive Cranwell's horned frogs Ceratophrys cranwelli from several facilities in Japan exhibited protruding facial lesions. Histopathological examination was performed on 6 specimens with such lesions randomly selected from 2 facilities. Lesions consisted of scattered stellate to spindle-shaped cells without atypia in an abundant myxoid matrix and occasional lymphocytic infiltrates. Maxillary bone was resorbed. No etiological organisms were detected using light microscopy or metagenomic analysis of the lesions. Macroscopic and histological assessments indicate that the lesions are associated with nodular facial myxomatous dermatitis, which has never been reported in amphibians.

Molecular and histopathological features of Cryptosporidium ubiquitum infection in imported chinchillas Chinchilla lanigera in Japan.

Long-tailed chinchillas Chinchilla lanigera are popular rodent species kept both in households, where they are hand-raised as pets, and in zoological facilities. From January 2016 to February 2017, 13 juvenile chinchillas from five facilities in Japan were diagnosed with cryptosporidiosis at the animal hospital. Eight of the cases were fatal. All of the animals were imported from the Czech Republic by the same vendor. Histopathological and multilocus sequence analyses using 18S ribosomal RNA, actin, 70-kDa heat shock protein, and 60-kDa glycoprotein genes confirmed Cryptosporidium ubiquitum of subtype XIId as the etiological agent. Multilocus analysis demonstrated the presence of two new sequence types closely related to the C. ubiquitum Xlld strain isolated from a human in the USA. This study indicated that potentially zoonotic Cryptosporidium is widespread and may have caused a high number of deaths among imported juvenile chinchillas.

Morphological and molecular characterization of Cystoisospora sp. from Asian small-clawed otters Aonyx cinereus.

In this study, we provide the first description of Cystoisospora infection in Asian small-clawed otters (Aonyx cinereus). In July 2017, three juvenile otters recently imported from the Republic of Indonesia showed severe diarrhea and were diagnosed with coccidial infection; two of them eventually died. Fecal examination revealed the presence of numerous oocysts. Sporulated oocysts showed typical Cystoisospora features, measuring 24.6 ±â€¯1.6 (22.0-27.0) × 21.8 ±â€¯1.4 (19.0-25.0) µm, with an oocyst length/width ratio of 1.1 ±â€¯0.1 (1.0-1.3). Each sporocyst contained four sporozoites in a head-to-tail arrangement. The Stieda body was absent, and the sporocyst residuum was present. These morphological characteristics differentiated this species from the other valid Cystoisospora species described from mustelids. Molecular analysis was conducted at two loci: the nuclear 18S ribosomal RNA and mitochondrial cytochrome c oxidase subunit I genes. The 18S sequence showed high similarity with canine Cystoisispora ohioensis (1-bp difference, 1422/1423 [99.9%]). At the cytochrome c oxidase subunit I gene locus, the sequence from otters was identical to that of feline Cystoisospora rivolta (847/847 [100%]). Phylogenetic analyses using concatenated data demonstrated that Cystoisospora sp. from otters and C. rivolta grouped together in the same Cystoisospora clade. Based on these data, we concluded that Cystoisospora sp. detected from otters appeared to be highly similar to C. rivolta.

First report of fatal disseminated microsporidiosis in two inland bearded dragons Pogona vitticeps in Japan.

Introduction.Encephalitozoon pogonae is a newly described pathogen belonging to the phylum Microsporidia. In Austria and the USA, this species has been isolated from fatal and disseminated cases of captive-bred inland bearded dragons. Here, we report the case of fatal disseminated microsporidiosis caused by E. pogonae in two bearded dragons in Japan. Case Presentation. The two lizards from different private households in Tokyo, Japan, had been brought to an animal hospital for examination. In both cases, the animal presented with a history of weight loss for several weeks. There were no improvements in clinical symptoms and the lizards deteriorated and finally died. Histopathological examination demonstrated necrotizing granulomatous inflammation attributed to disseminated microsporidian infection. Nucleotide sequencing of the nuclear ribosomal internal transcribed spacer region identified the microsporidian as E. pogonae with sequence identity of 100 %. Conclusion. We report the first case, to our knowledge, of disseminated microsporidiosis caused by E. pogonae in inland bearded dragons in Japan. Although it is difficult to diagnose prenatally since the signs are nonspecific, the disease should be considered in the differential diagnosis of chronic infections that do not respond to antibiotics.

Establishment of serological test to detect antibody against ferret coronavirus.

Since there is no available serological methods to detect antibodies to ferret coronavirus (FRCoV), an enzyme-linked immunosorbent assay (ELISA) using recombinant partial nucleocapsid (N) proteins of the ferret coronavirus (FRCoV) Yamaguchi-1 strain was developed to establish a serological method for detection of FRCoV infection. Many serum samples collected from ferrets recognized both a.a. 1-179 and a.a. 180-374 of the N protein, but two serum samples did not a.a. 180-374 of the N protein. This different reactivity was also confirmed by immunoblot analysis using the serum from the ferret.Therefore, the a.a. 1-179 of the N protein was used as an ELISA antigen. Serological test was carried out using sera or plasma of ferrets in Japan. Surprisingly, 89% ferrets in Japan had been infected with FRCoV. These results indicated that our established ELISA using a.a. 1-179 of the N protein is useful for detection of antibody to FRCoV for diagnosis and seroepidemiology of FRCoV infection.

Ranavirus in an outbreak of dermatophilosis in captive inland bearded dragons (Pogona vitticeps).

BACKGROUND: Various pathogens cause skin diseases in lizards. An outbreak of skin disease occurred in a population of 100 inland bearded dragons at a breeding facility in Japan; 50 developed skin lesions and 15 mortalities were reported. HYPOTHESIS/OBJECTIVES: To identify the pathogens responsible for the skin lesions and to determine the cause of death. ANIMALS: Eight specimens (three dead, two euthanized, three living) were randomly selected from a group of inland bearded dragons with skin lesions consisting of multifocal superficial dermatitis. METHODS: Biopsy samples were taken from skin lesions and healthy skin of the live specimens. Postmortem examinations were performed on the dead and euthanized specimens. Skin samples were processed for microbiological culture, 16S ribosomal DNA (rDNA) sequencing for bacteria, ranavirus major capsid protein (MCP) gene sequencing and histopathological examination. Histopathological examinations were also performed on postmortem tissue samples. RESULTS: Both Austwickia chelonae and ranavirus (DNA) were detected in the skin lesions. Pathological examination revealed no significant visceral lesions caused by A. chelonae or ranavirus infection in dead specimens. CONCLUSIONS AND CLINICAL IMPORTANCE: To the best of the authors' knowledge this is the first report of dermatophilosis due to A. chelonae infection in lizards and the first description of a concurrent infection with a ranavirus. The combined infection associated with the skin lesions may have been the primary cause of death. Co-infection with a ranavirus should be considered as a possible factor in cases of reptilian dermatophilosis.

Spontaneous oral chytridiomycosis in wild bullfrog tadpoles in Japan.

Batrachochytrium dendrobatidis (Bd) infects Anuran larvae (tadpole) mouthparts and causes oral chytridiomycosis, which can be diagnosed in tadpoles by detecting mouthparts deformities. However, oral chytridiomycosis may or may not be observable, depending on species, tadpole stage and season, and has never been reported in Japan. We aimed to observe oral chytridiomycosis characteristics in bullfrog (Lithobates catesbeiana) tadpoles, determine associated pathologic features and investigate the usability of bullfrog tadpoles in Japanese Bd field surveys. Wild-captured bullfrog tadpole mouthparts were examined macroscopically, histopathologically and by molecular biological examination. Macroscopic lesions were observed in 21 of 59 tadpole mouthparts. Lesions were most frequently located in the lower jaw sheaths and were mainly recognized by partial depigmentation (11 tadpoles; some were completely depigmented) and thinning of the pigmented layer (10 tadpoles). Partial defects of the tips and blunt cutting edges of the jaw sheaths were observed with severe jaw sheath depigmentation. Whitened tooth rows were observed in 7 tadpoles. Histologically, the stratified epithelium (pigmented epithelium) showed partial or diffuse hypopigmentation or pigment loss. Irregular stratified epithelium thickening with hyperkeratosis or parakeratosis was observed in the jaw sheaths. Bd infection was confirmed in 20 of 21 tadpoles presenting jaw sheath deformities, by histopathological examination and/or nested polymerase chain reaction. Depigmentation and thinning of the pigmented layers of jaw sheaths were associated with Bd infection. Thus, diagnosis of Bd infection by macroscopic observation of bullfrog tadpole mouthparts is feasible. This is the first report of oral chytridiomycosis in wild bullfrog tadpoles in Japan.

Molecular evidence of Sarcocystis species in captive snakes in Japan.

Sarcocystis nesbitti, using snakes as the definitive host, is a causative agent of acute human muscular sarcocystosis in Malaysia. Therefore, it is important to explore the distribution and prevalence of S. nesbitti in snakes. Nevertheless, epizootiological information of S. nesbitti in snakes remains insufficient because few surveys have assessed Sarcocystis infection in snakes in endemic countries. In Japan, snakes are popular exotic pet animals that are imported from overseas, but the degree of Sarcocystis infection in them remains unclear. The possibility exists that muscular sarcocystosis by S. nesbitti occurs in contact with captive snakes in non-endemic countries. For a total of 125 snake faecal samples from 67 snake species collected at animal hospitals, pet shops and a zoo, this study investigated the presence of Sarcocystis using polymerase chain reaction (PCR) for the 18S ribosomal RNA gene (18S rDNA). Four (3.2%) faecal samples were positive by PCR. Phylogenetic analysis of the 18S rDNA sequences obtained from four amplification products revealed one isolate from a beauty snake (Elaphe taeniura), Sarcocystis zuoi, which uses rat snakes as the definitive host. The isolate from a Macklot's python (Liasis mackloti) was closely related with unidentified Sarcocystis sp. from reticulated pythons in Malaysia. The remaining two isolates from tree boas (Corallus spp.) were closely related with Sarcocystis lacertae, Sarcocystis gallotiae and unidentified Sarcocystis sp. from smooth snakes, Tenerife lizards and European shrews, respectively. This report is the first of a study examining the distribution of Sarcocystis species in captive snakes in Japan.

First report of Veronaea botryosa as a causal agent of chromomycosis in frogs.

A dematiaceous hyphomycete, isolated from frogs, was determined as the possible etiologic agent of a case of systemic chromomycosis this cold-blooded animal. The fungus was identified as Veronaea botryosa on the basis of morphological features observed in histopathological examination and molecular phylogenetic evidence. Although V. botryosa is known to be distributed widely in litter and as a human pathogen, this is the first confirmed report of its involvement in a lethal infection in a cold-blooded animal, including an anuran.

Batrachochytrium dendrobatidis prevalence and haplotypes in domestic and imported pet amphibians in Japan.

The international trade in amphibians is believed to have increased the spread of Batrachochytrium dendrobatidis (Bd), the fungal pathogen responsible for chytridiomycosis, which has caused a rapid decline in amphibian populations worldwide. We surveyed amphibians imported into Japan and those held in captivity for a long period or bred in Japan to clarify the Bd infection status. Samples were taken from 820 individuals of 109 amphibian species between 2008 and 2011 and were analyzed by a nested-PCR assay. Bd prevalence in imported amphibians was 10.3% (58/561), while it was 6.9% (18/259) in those in private collections and commercially bred amphibians in Japan. We identified the genotypes of this fungus using partial DNA sequences of the internal transcribed spacer (ITS) region. Sequencing of PCR products of all 76 Bd-positive samples revealed 11 haplotypes of the Bd ITS region. Haplotype A (DNA Data Bank of Japan accession number AB435211) was found in 90% (52/58) of imported amphibians. The results show that Bd is currently entering Japan via the international trade in exotic amphibians as pets, suggesting that the trade has indeed played a major role in the spread of Bd.

Eradication of the chytrid fungus Batrachochytrium dendrobatidis in the Japanese giant salamander Andrias japonicus.

The purpose of this study was to establish a method for eradicating a chytrid fungus (Batrachochytrium dendrobatidis; Bd) from the Japanese giant salamander Andrias japonicus. The emerging agent (Bd) has a high rate of detection in this endangered amphibian species, which is designated as a special natural monument in Japan. Four Japanese giant salamanders with Bd confirmed by PCR assay were bathed in 0.01% itraconazole for 5 min d-1 over 10 successive days. PCR assays were conducted prior to treatment, on Days 5 and 10 of treatment, and on Days 7 and 14 post-treatment. By treatment Day 5, all individuals tested negative for Bd and remained negative until the end of the experiment. No side effects associated with itraconazole were observed. The present method appears to be a safe and effective approach for Bd eradication and may contribute to reducing the threat and spread of Bd among endangered amphibians. Notably, this study represents the first reported Bd eradication experiment involving Japanese giant salamanders.

Plasma biochemical reference values in clinically healthy captive bearded dragons (Pogona vitticeps) and the effects of sex and season.

BACKGROUND: Bearded dragons are one of the most popular pet lizard species, and biochemical reference values are useful for health management of these reptiles. OBJECTIVES: The objectives of this study were to measure plasma biochemical values in healthy captive bearded dragons, determine reference values, and evaluate the effects of sex and season on the results. METHODS: Blood samples were collected from 100 captive healthy bearded dragons in Tokyo during the summer and winter. Plasma biochemical measurements were performed using a dry-slide automated biochemical analyzer. The data were then compared based on sex and season using 2-way ANOVA. RESULTS: Globulin, cholesterol, and calcium concentrations of females were higher in both summer and winter compared with the values obtained for males. Both males and females had higher uric acid concentrations in winter than in summer. When compared with males, females had a higher chloride concentration in summer and a higher total protein concentration and aspartate aminotransferase activity in winter. Potassium concentration in males was lower in winter than in summer, whereas in females cholesterol concentration was lower in winter than in summer. CONCLUSIONS: Biochemical values that differed based on sex and season in bearded dragons were similar to those in other lizards. These differences reflect physiologic differences in reproductive status in females and seasonal changes in temperature and hydration status. Plasma biochemical values established for bearded dragons in this study will be useful in the diagnostic assessment of captive animals.

Treatment of spontaneous chytridiomycosis in captive amphibians using itraconazole.

We performed this study in order to establish an effective, simple and safe treatment for chytridiomycosis. The subjects were 12 amphibians (11 anurans of 4 different species and 1 urodela) diagnosed with chytridiomycosis by clinical signs and a PCR test. A 0.01% aqueous solution of the antifungal agent itraconazole was used to treat the subjects, and we evaluated the efficacy of treatment by 3 methods: clinical signs, direct microscopy and a nested PCR test. A 10-min immersion in a 0.01% aqueous solution of itraconazole every other day for a total of 7 treatments resulted in an improvement of clinical signs in 11 of the 12 cases. Specifically, we observed an abatement of increased sloughing and disappearance of zoosporangia by direct microscopy. DNA fragments of Batrachochytrium dendrobatidis were not detected by a PCR test at the end of treatment, nor were they detected after treatment (20-57 days following treatment; average, 34.4 days). No recurrence was observed 12 months after the end of treatment, nor did we observe any obvious side effects from itraconazole. Therefore, we recommend this as a treatment method for chytridiomycosis and as an elimination technique for use in captive amphibians.

First report of spontaneous chytridiomycosis in frogs in Asia.

This is the first report of amphibian chytridiomycosis in Asia. We discovered a lethal outbreak in Japan, among 45 exotic frogs from 18 species kept for breeding by a private owner. Of these 45 frogs, 16 died and another 7 were found to be infected by chytrid fungus Batrachochytrium dendrobatidis (Bd) but survived after treatment. Bd was detected in frogs from 9 species (Lepidobatrachus laevis, Ceratophrys cornuta, C. cranwelli, C. ornata, C. calcarata, Chacophrys pierotti, Occidozyga lima, Leptodactylus pentadactylus and Plethodontohyla tuberata).