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.
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.
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.
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.