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NO53 - 2

An Epidemiological Study of Akoya Oyster Disease Using Polymerase Chain Reaction Targeting Spirochaetes Genes

Tomomasa Matsuyama*1, Yuta Matsuura1, Mari Inada1, Tomokazu Takano1, Chihaya Nakayasu2, Takamitsu Sakai1, Sachiko Terashima1, Motoshige Yasuike6, Atushi Fujiwara6, Yoji Nakamura6, Yasushi Tsuchihashi3, Kazushi Odawara4, Syunsuke Iwanaga5 and Tetsuji Masaoka1

1National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, Mie 516-0193, Japan
2Head Quarters, Japan Fisheries Research and Education Agency, Kanagawa 220-6115, Japan
3Mie Prefecture Fisheries Research Institute, Mie 517-6464, Japan
4Ehime Prefecture Fisheries Research Center, Ehime 798-0104, Japan
5Nagasaki Prefectural Institute of Fisheries, Nagasaki 851-2213, Japan
6National Research Institute of Fisheries Science, Japan Fisheries Research and Education Agency, Kanagawa 236-8648, Japan

ABSTRACT―In a previous study, a member of Spirochaeta was presumed to cause Akoya oyster disease (AOD), and Candidatus Maribrachyspira akoyae was proposed as the putative causative agent. With the aim of performing an epidemiological study of AOD, polymerase chain reaction (PCR) assays were developed using the primers designed from shotgun metagenomic sequences identified as having high homology to Spirochaetes genes by a BLAST search. PCR assays with six primer sets produced specific target amplicons (n = 63-67) in pearl oysters collected from five AOD-endemic areas (n = 88), but did not in pearl oysters (n = 36) collected from three AOD-free areas. All individuals (n = 17) with the color index a-value above 3.0, which is an index of AOD development, were positive for all 6 primer sets in PCR. There was a positive correlation between the a-value and the results of PCR. These results show that the previous identification of Spirochaeta as an etiological agent of AOD is reasonable. Sequencing of amplification products confirmed only one nucleotide substitution in the PCR products, suggesting that the pathogenic causative agent of AOD has limited genetic diversity.

Key words: Akoya oyster disease, Candidatus Maribrachyspira akoyae, epidemiology, PCR, pearl oyster, Spirochaeta

Experimental Infection of Ostreid Herpesvirus 1 (OsHV-1) JPType1, a Japanese Variant, in Pacific Oyster Crassostrea gigas Larvae and Spats

Takahiro Nagai* and Misato Nakamori

Fisheries and Marine Technology Center, Hiroshima Prefectural Technology Research Institute, Hiroshima 737-1207, Japan

ABSTRACT―In many locations worldwide, ostreid herpesvirus 1 (OsHV-1) infections are associated with mortalities in Pacific oyster Crassostrea gigas primarily during the summer months. In the present study, experimental infections were performed to investigate the pathogenicity of OsHV-1 JPType1 (a Japanese variant) in hatchery-reared Pacific oyster larvae and spats. The JPType1 virus was prepared from dead oyster spats obtained from a sea cage and was once passed in oyster larvae as the inoculum source of the infection experiments. Compared to pediveliger larvae, D-shaped larvae were more susceptible to infections when the larvae were exposed to serially diluted virus solutions at the concentrations of 3.2 × 106, 3.2 × 105 and 3.2 × 104 virus DNA copies/mL. Real-time PCR analysis revealed 1.6 × 108 virus DNA copies/ng total DNA in a pooled sample of D-shaped larvae. No mortality was observed after 5 days when nine spat groups of differently sizes (mean shell height from 1.8 mm to 6.1 mm) were immersed in seawater containing 2.5 × 106 virus DNA copies/mL; however, 6.3 × 105 virus DNA copies/mg whole weight were detected in a group of spats. Virus concentrations were higher in smaller spat groups than in larger spat groups. The pathogenicity of OsHV-1 JPType1 in larvae and spats was confirmed; however, it declined with the growth of the oyster.

Key words: OsHV-1, herpesvirus, Pacific oyster, pathogenicity, larvae, spat, Japanese virus variant, experimental infection

Francisella halioticida, Identified as the Most Probable Cause of Adductor Muscle Lesions in Yesso scallops Patinopecten yessoensis Cultured in Southern Hokkaido, Japan

Miku Kawahara1, Makoto Kanamori2, Gary R. Meyer3, Tomoyoshi Yoshinaga1 and Naoki Itoh1*

1Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
2Hakodate Fisheries Research Institute, Fisheries Research Department, Hokkaido Research Organization, Hakodate 040-0051, Japan
3Fisheries and Oceans Canada, Pacific Biological Station, BC V9T 6N7, Canada

ABSTRACT―The occurrence of orange/pinkish colored lesions in the adductor muscle of Yesso scallops Patinopecten yessoensis has been known for many years in Japan; however, determination of the causative agent has not been adequately investigated. Histological examination of affected scallops in southern Hokkaido typically revealed intense host responses: hemocyte infiltration, an abundance of necrotic hemocytes, lysis of muscle fibers and in some instances melanin deposits when the lesions occurred adjacent to the shell. Microbiota analysis showed that Francisella halioticida was dominant in the lesions, and in situ hybridization using F. halioticida specific probes also confirmed the presence of this bacterium within the lesions. A F. halioticida specific PCR assay detected this bacterium in the majority of scallop lesions tested. Subsequently, three bacterial isolates were obtained from scallop lesions on modified Eugon agar supplemented with antibiotics, and these bacterial isolates were found to be F. halioticida by 16S rRNA and rpoB gene sequences. These results suggest that infection with F. halioticida is the most likely cause of the adductor muscle lesions observed in Yesso scallops. Field surveys conducted in 2017 of scallops cultured in southern Hokkaido showed that the presence of adductor muscle lesions putatively caused by F. halioticida was significantly related to mortalities and poor growth of scallops.

Key words:Francisella halioticida, lesion, adductor muscle, Patinopecten yessoensis, Yesso scallop, etiology, aquaculture, epidemiology

Aeromonas veronii biovar sobria Associated with Mortalities of Riverine Ayu Plecoglossus altivelis in the Tama River

Hisato Takeuchi1, 2, Aki Namba1, Kazutomo Hori1, Shosaku Kashiwada2 and Nobuhiro Mano1*

1Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University, Kanagawa 252-0880, Japan
2Research Center for Life and Environmental Sciences, Toyo University, Gunma 374-0193, Japan

ABSTRACT―In July 2016, there were mortalities of riverine ayu Plecoglossus altivelis in a tributary of the Tama River, Japan. A Gram-negative, motile and short rod-shaped bacterium was dominantly isolated from all examined dead fish, and identified as Aeromonas veronii biovar sobria. Biochemical characteristics and gyrB sequence of the present strains differed from those of A. veronii strains from ayu in previous years. The present strains also caused higher mortalities to ayu than A. veronii strains previously isolated. These results indicate that the present mortalities of riverine ayu in the Tama River were caused by high pathogenic A. veronii biovar sobria.

Key words: Aeromonas veronii biovar sobria, Plecoglossus altivelis, riverine fish, pathogenicity, the Tama River