Spontaneous oral chytridiomycosis in wild bullfrog tadpoles in Japan

Abstract

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.

Comparison of methods for detection of chytrid fungus (<i>Batrachochytrium dendrobatidis</i>) in bullfrog tadpole mouthparts

“…We previously confirmed that the wild bullfrog Lithobates catesbeiana tadpole is a suitable animal model for investigating Bd distribution in Japan [ 5 ]. Thus, in the present study, we compared the sensitivity of two detection methods for Bd: nested polymerase chain reaction (PCR) for Bd genomic DNA from (1) swab-scraped mouthpart samples (hereon referred to as “swab nested PCR”) and (2) resected mouthpart samples (hereon referred to as “tissue nested PCR”).…”

“…The detection rates of swab nested PCR and tissue nested PCR were investigated using samples from 120 wild bullfrog tadpoles. The tadpoles were collected from two sites: 60 from Saihaku county in Tottori prefecture on May 13, 2012, and 60 from Seihi town, Saikai city of Nagasaki prefecture on February 17, 2013; these collection times corresponded to the time at which the Bd infection rate is reportedly high [ 5 , 10 ], in conformity with the Invasive Alien Species Act [ 7 ]. The collected bullfrog tadpoles were anesthetized and then euthanized, as described previously [ 5 ], for measuring body length and body weight.…”

“…The tadpoles were collected from two sites: 60 from Saihaku county in Tottori prefecture on May 13, 2012, and 60 from Seihi town, Saikai city of Nagasaki prefecture on February 17, 2013; these collection times corresponded to the time at which the Bd infection rate is reportedly high [ 5 , 10 ], in conformity with the Invasive Alien Species Act [ 7 ]. The collected bullfrog tadpoles were anesthetized and then euthanized, as described previously [ 5 ], for measuring body length and body weight. The bullfrog tadpoles collected from the two sites were at developmental stages 25 to 41 [ 3 ]; the average length and body weight of tadpoles collected in Tottori and Nagasaki were 9.7 cm (6.2–11.6 cm) and 14.1 g (4.1–22.6 g), and 8.8 cm (5.3–11.0 cm) and 9.0 g (3.6–14.3 g), respectively.…”

“…All mouthparts of tadpoles in Group 1 were swabbed with sterile cotton swabs because Bd was previously reported to occur on both the jaw sheaths (the tip and the basement) and tooth rows [ 5 ], and the mouthparts were then resected. The swabs and resected mouthparts were stored frozen in a sterile microtube at −20°C for nested PCR.…”

“…The swabs and resected mouthparts were stored frozen in a sterile microtube at −20°C for nested PCR. Bd DNA extraction from the swab samples was conducted using the NucleoSpin® Tissue kit (Macherey-Nagel, Duren, Germany) in accordance with a previous method [ 5 ]. Bd DNA was extracted from resected mouthparts using the same method after homogenization with a disposable homogenizer (BioMasher®, Nippi Inc., Tokyo, Japan).…”