A Spirochaete is suggested as the causative agent of Akoya oyster disease by metagenomic analysis

<div><p>Mass mortality that is acompanied by reddish browning of the soft tissues has been occurring in cultured pearl oyster, <i>Pinctada fucata martensii</i>. The disease is called Akoya oyster disease (AOD). Although spreading pattern of the disease and transmission experiments suggest that the disease is infectious, the causative agent has not yet been identified. We used shotgun and 16S rRNA-based metagenomic analysis to identify genes that are present specifically in affected oysters. The genes found only in diseased oysters were mostly bacterial origin, suggesting that the causative agent was a bacterial pathogen. This hypothesis was supported by the inhibition of AOD development in naïve oysters injected with the hemolymph of diseased animals followed immediately with penicillin bath-administration. Further analyses of the hemolymph and mantle specifically and universally detected genes of bacteria that belong to phylum Spirochaetes in diseased pearl oysters but not in healthy oysters. By <i>in situ</i> hybridization or immunostaining, a <i>Brachyspira</i>-like bacterium was observed in the smears of hemolymph from affected oysters, but not from healthy oysters. Phylogenetic analysis using 16S rRNA sequences showed that the presumptive causative bacterium was outside of but most closely related to family Brachyspiraceae. We propose ‘Candidatus <i>Maribrachyspira akoyae</i>’ gen. nov, sp nov., for this bacterium.</p></div

Strategy for analysis of shotgun metagenomic data.

<p>Strategy for analysis of shotgun metagenomic data.</p

Effect of penicillin administration on cumulative mortality and a-value of the survivors.

<p>The a-value of the survivors is represented with bars and cumulative mortality is plotted with open circles. Asterisks indicate significant differences between negative control groups (Wilcoxon signed-rank test, <i>p</i>-value<0.05).</p

Normalized abundance distribution of each taxon at the phylum level from healthy and diseased pearl oyster in (A) hemolymph and (B) mantle.

<p>Percentage of total sequence reads in samples from healthy and diseased pearl oyster of hemolymph and mantle is presented. The error bars show calculated standard deviation of the mean. Asterisks indicate significant difference between relative abundance for healthy and diseased oysters (Wilcoxon signed-rank test, <i>P</i>-value<0.05).</p

Sites of Akoya pearl oyster collection (open circles).

<p>Sites of Akoya pearl oyster collection (open circles).</p

Bacterial diversity of the healthy and diseased pearl oysters.

<p>Shannon alpha (A, B) and Bray-Curtis beta (C, D) diversity of the hemolymph (A, C) and mantle (B, D) microbes were evaluated using OTUs at the phylum level. Box plots show median, 25th, and 75th percentiles, and whiskers represent minimum and maximum values. Student’s t test; *p<0.05; **P<0.01.</p

Survivor adductor muscle a-value (bars) and cumulative mortality (plotted points) are shown for each group.

<p>The groups are color coded as follows: positive control, black; negative control, white; sediment-inoculated groups, orange; and supernatant-inoculated groups, blue. Letters indicate statistically significant differences: a, cumulative mortality significantly different from the negative control (Fisher's exact test, p<0.05); b, cumulative mortality significantly different from the supernatant-injected group (Fisher's exact test, p<0.05); c, mean a-value significantly different from the supernatant control (Wilcoxon signed-rank test, p<0.05); and d, mean a-value significantly different from negative control (Wilcoxon signed-rank test, p<0.05).</p

Phylogenetic tree for 16S rRNA gene sequences of putative causative agent and those of representative type species of phylum Spirochaetes.

<p>Strain names and accession numbers of the 16S rRNA gene sequences follow the species name. The tree was constructed using the maximum-likelihood algorithm. Numbers at nodes represent the confidence limits estimated based on 1000 bootstrap replicates. The 16S rRNA gene sequence of <i>Fusobacterium nucleatum subsp</i>. <i>nucleatum</i> ATCC25586^T was used to root the tree. Bar, 0.05 <i>K</i>nuc in nucleotide sequences.</p

Fluorescence micrographs showing specific detection of bacteria cells from diseased pearl oyster hemolymph after (A) <i>in situ</i> hybridization and (B) fluorescence immunostaining.

<p>Fluorescence micrographs showing specific detection of bacteria cells from diseased pearl oyster hemolymph after (A) <i>in situ</i> hybridization and (B) fluorescence immunostaining.</p

Summary of sample characteristics for metagenomic analysis.

<p>Summary of sample characteristics for metagenomic analysis.</p