Chytridiomycosis, a disease that has caused amphibian population declines globally and elevated many species of anurans to endangered or threatened status, has recently been declared an internationally notifiable disease. Batrachochytrium dendrobatidis (Bd), the amphibian chytrid fungus causing this disease, has not been previously reported in Korea or on mainland Asia. Thirty-six frog specimens representing 7 species were collected from the wild in South Korea and examined for Bd using standard PCR. Bd was detected in 14 (38.8%) samples from 3 species (Bufo gargarizans, Hyla japonica, and Rana catesbiana). Skin sections from all 14 PCR-positive frogs were examined using 2 staining techniques: haematoxylin and eosin (H&E) and Bd immunoperoxidase (IPX). In histological sections, zoosporangia were found in 6 frogs, with lower sensitivity for H&E (21%) than for IPX (46%). Intensity of infection, based on histopathology, was low in all frogs. These results confirm that Bd is present in South Korea and, hence, on the Asian mainland. Studies are urgently required to determine the impact of chytridiomycosis on Korean amphibians, and to map the distribution of Bd in Korea and other Asian mainland countries.
KEY WORDS: Chytridiomycosis · Batrachochytrium dendrobatidis · Amphibian decline · Korea · Fungus
Resale or republication not permitted without written consent of the publisherDis Aquat Org 86: [9][10][11][12][13] 2009 nerable (Matsui 2004), and Hynobius yangi is classified as endangered (Stuart 2008)
MATERIALS AND METHODSFrogs were collected opportunistically from the northern part of South Korea between June 11 and December 13, 2007 (see Table 1). During nocturnal and occasional diurnal surveys, frogs were captured individually by hand, with a new pair of disposable latex gloves being used for each frog in order to avoid cross-contamination between individuals . The animals were sacrificed by soaking them in a bath of 0.05% aqueous tricaine methane sulfonate (MS-222). We aseptically cut small pieces of skin from the abdomen, inguinal region, and web between toes; we then divided this into 2, and placed 1 in 10% formalin for histological analysis and the other in a 1.5 ml microtube for DNA extraction. Instruments were flamed after dissection of each specimen to prevent cross-contamination. DNA was immediately extracted with Gene Releaser (Bio Ventures). The PCR assay used species-specific primers (Bd1a and Bd2a) located within internal transcribed spacer ITS1 and ITS2 to amplify the 5.8S region of nuclear rDNA (Annis et al. 2004). The PCR products were examined using 1.0% agarose gel and some of the positive bands were cut and sequenced to confirm the Bd sequence. Each sample was tested in triplicate, and was only recorded as positive if all 3 replicates indicated the presence of Bd. For PCR positive amphibians, histological sections of formalin fixed skin were stained using 2 techniques: haematoxylin and eosin (H&E) and Bd immunoperoxidase (IPX), the latter technique using polyclonal antibodies speci...