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NO54 - 4

Identification of the Epitope Recognized by the Anti-Red Sea Bream Iridovirus (RSIV) Monoclonal Antibody M10 Using a Phage Display RSIV Peptide Library

Tomokazu Takano1*, Tomomasa Matsuyama1, Yasuhiko Kawato1, Takamitsu Sakai2, Jun Kurita2, Yuta Matsuura1, Sachiko Terashima1, Kazuhiro Nakajima3 and Chihaya Nakayasu1

1 Nansei Main Station, National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, Mie 519-0193, Japan
2 Tamaki Laboratory, National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, Mie 519-0423, Japan
3 National Research Institute of Fisheries and Environment of Inland Sea, Japan Fisheries Research and Education Agency, Hiroshima 739-0452, Japan

(Received June 17, 2019)

ABSTRACT--Red sea bream iridoviral disease (RSIVD) spreads readily throughout the marine environment because of its wide variety of host species. Use of a rapid diagnostic method, such as the immunofluorescence antibody test, helps to control the disease, so the anti-RSIV monoclonal antibody M10 (mAb M10) has been developed in Japan. In the present study, we carried out epitope mapping using the phage display method to identify the antigen of mAb M10. A phage display RSIV peptide library was constructed to cover the entire genome of RSIV, then phage clones recognized by the antibody were selected by biopanning. The selected clones harbored partial fragments of the laminin-type epidermal growth factor-like domain (LEGFD) gene. N-terminal and C-terminal deletion peptides were then prepared from the amino acid sequence deduced from the smallest fragment to precisely determine the epitope. Finally, seven amino acids, EYDCPEY, located in the extracellular domain of the LEGFD protein were determined to be the epitope. Identical residues of the epitope were also identified from the LEGFD protein in other megalocytiviruses including the infectious spleen and kidney necrosis virus and turbot reddish body iridovirus. mAb M10 is considered to be widely available for the diagnostics of megalocytivirus infections.

Key words: mAb M10, epitope, Megalocytivirus, phage display library, red sea bream iridovirus, RSIV

Bucephalid Metacercarial Infection in Wild Larval and Juvenile Ayu Plecoglossus altivelis

Sho Shirakashi1*, Tsukasa Waki2 and Kazuo Ogawa3

1 Aquaculture Research Institute, Kindai University, Shirahama 3153, Wakayama 649-2211, Japan
2 Graduate School of Science, Toho University, Funabashi Miyama 2-2-1, Chiba 274-8510, Japan
3 Meguro Parasitological Museum, Shimo-Meguro 4-1-1, Meguro, Tokyo 153-0064, Japan

(Received September 3, 2019)

ABSTRACT--Ayu Plecoglossus altivelis is a native amphidromous fish in Japan and an important target for farming and recreational fishing. We have found high infection of hitherto unknown digenean metacercariae in wild ayu larvae and juveniles captured from the sea and a river in Wakayama Prefecture. The infection prevalence was 95% and 100% in larvae and juvenile ayu, respectively with variable intensities up to 566 metacercariae per fish. The metacercariae were located primarily around fins and inside caudal fins, but appeared to cause no serious pathological features to the fish. The phylogenetic analysis based on the internal transcribed spacer 2 and 28S rDNA sequences revealed its identity as a member of Bucephalinae (Bucephalidae) with no identical genetic information on the database. Morphological characteristics of the larval trematode resembled those of the genus Prosorhynchoides. Genetically identical metacercariae were also found from Scomber japonicus and Engraulis japonicus captured at the same location as P. altivelis with variable infection levels, which suggested that it was a marine species. Experimental infection challenge of the metacercariae to several marine fish via oral intubation failed to obtain adult specimen and thus the precise identification of the parasite remains unknown.

Key words: Plecoglossus altivelis, Bucephalidae, Bucephalinae, Scomber japonicus, Engraulis japonicus, 28S rDNA, ITS2, metacercariae

Application of Hydrogen Peroxide-Melanin Bleaching and Fluorescent Nuclear Staining for Whole-Body Clearing and Imaging in Fish

Kunihiko Futami*, Oto Furukawa, Masashi Maita and Takayuki Katagiri

Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan

(Received July 5, 2019)

ABSTRACT--Recently, our lab reported that CUBIC, a tissue-clearing technique, could help reveal the initial route of infection, its spread, and the localization of pathogens in infected goldfish Carassius auratus. However, this technique, in its original form, failed to clear melanin pigmented parts of the fish. We show here that bleaching with H2O2 clears melanin pigments of Black Moor goldfish without causing severe histological damage. Furthermore, nuclear staining with PI helps visualize the internal structures of cleared fish in a dark field. Since bleaching does not significantly quench the fluorescence of GFP, it may be applied to the in vivo imaging of GFP.

Key words: CUBIC, melanin bleaching, hydrogen peroxide, propidium iodide, GFP