Kenichi Tamukai

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).

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.

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.

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.

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.

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 ± 1.6 (22.0–27.0) × 21.8 ± 1.4 (19.0–25.0) μm, with an oocyst length/width ratio of 1.1 ± 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.

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.