The takeover of Vibrio coralliilyticus

Vibrio tubiashii is a known bacterial pathogen of the larval Eastern Oyster (Crassostrea virginica) and pacific oysters (Crassostrea gigas) and has been responsible for causing major shortages in oysters for commercial shellfish producers.  Whilst V.tubiashii is a known bacterial pathogen of shellfish, Vibrio coralliilyticus until recently was known as a coral-specific pathogen, but recent mortality in fish and shellfish has been linked to this pathogenic bacteria. Until recently, V.coralliilyticus isolates (ATCC 19105 and Pacific isolates RE11 and RE98) were misidentified as strains of V.tubiashii, suggesting that these two bacterial pathogens are closely related or play similar ecological roles. It has since been found that V.tubiashii actually plays a less significant role in the pathogenicity of shellfish than originally thought, with V.coralliilyticus being the major contributor to shellfish pathogenicity.

This discovery lead to the whole-genome sequencing of RE98, a highly pathogenic strain previously identified as V.tubiashii derived from an Oregon shellfish hatchery. As expected – the results showed that the RE98 strain was in fact a V.coralliilyticus strain (Richards et al., 2014). The similarities of these two Vibrios' is highlighted by the dramatic losses of Oysters from Willipa Bay, west coast USA, as a pathogen wreaking havoc along the most productive estuaries is yet to be distinguished as either V.tubiashii of V.coralliilyticus, but their involvement is undoubted.

The bacterial cultures used for V.coralliilyticus were ones previously identified as V.tubiashii (ATCC BAA-450;ATCC 19105 and  RE22). All isolates were routinely grown in Luria-Bertoni broth or agar containing 2% added NaCl. Incubations were carried out at 26°C and broths were incubated with shaking at 250rpm. All larva was soured from Shellfish farms in Quilcene, WA, where they were shipped (along with algae and seawater) to the laboratory used for this experiment. Larvae were maintained at 26 °C and oysters were fed on larvae sourced from the same collection point as themselves. Larvae were enumerated under a stereo zoom microscope and counts were recorded. Small portions of V.coralliilyticus or V.tubiashii colonies were incubated overnight at the same temperature and rpm as the larvae, and were used to reinoculate fresh tubes of LB broth on the morning of the assay; followed by incubation at 26 °C and the same rpm treatment until optical density (600nm) was attained. Vibrio-negative controls (uninoculated wells), a second set of medium controls, and the diluted culture-containing wells (1µl either of either V.coralliilyticus or V.tubiashii, undiluted or at various dilutions) were used.

In Eastern Oyster larvae all four V.coralliilyticus strains produced 100% mortality at concentrations between 2.9x10^4. The 50% lethal dose of V.coralliilyticus isolates ranged from 1.1x10^4 CFU/ml to 3.0x10^4 CFU/ml. This is believed to be the first time that this Vibrio has been shown to cause mortality in this species of Oyster. The Pacific oyster larvae, in contrast, experienced 100% mortality when exposed to 2.7x10^4 to 6.8x10^4 CFU/ml of V.coralliilyticus (excluding one duplicate assay for ATCC strain BAA-450, where the highest dilution test of 4.6x10^4 CFU/ml gave only 56.5& mortality). The 50% lethal dose for Pacific oysters ranged from1.2x10^4 to 4.0x10^4 ( a larger range than for the Eastern Oyster). This data revealed that different strains of V.coralliilyticus had different powers of infections on different larvae (.g strain BAA-450 was x2 infections to Eastern Oysters compared with Pacific) , and the lowest 50% lethal dose for both Oyster species was obtained with V.coralliilyticus RE98, suggesting this particular strain is the most pathogenic among the tested isolates.

V.tubiashii strains gave more varied results…With Eastern oysters treated with strain 19106, had a  50% lethal dose of 3.8x10^3 CFU/ml, making it the most pathogenic out of the 6 strains tested on European oysters. In Pacific Oysters, neither of the V.tubiashii strains showed significant pathogenicity, and the overall results indicate that V.coralliilyticus is pathogenic to both Oyster larvae, whilst V.tubiashii strains are predominantly infectious in the Eastern Oyster larvae.

This paper does highlight that different results would be obtained under varying environmental conditions (e.g temperature, salinity…) as Vibrio pathogenicity of both species seemed highest at high salinities, and they suggest further investigations into the salinity of fishery hatcheries to help explain the pathogenic patterns in aquaculture. Acidification of waters has also been linked to a reduction in shellfish immunity, negatively impacting shell formation, and noted that west coast shellfish populations being negatively affected by upwelling of water from ‘dead-zones’. It is clear from this paper that stress is likely to alter the health status of oyster larvae, consequently affecting the 50% lethal dosage of pathogens. It is also clear from this paper (as well as a multitude of others (Tout et al., 2015)) that environmental conditions play major roles in the health of our Oceans, and vibrios simply exploit conditions which boost their population, and thus virulence. In order to try and reduce the negative impact of Vibrio strains in shellfish and aquaculture, it is not simply a question of creating defense mechanisms for shellfish (e.g phage therapy) from these potential opportunistic Vibrios, but also to mitigate and control the environmental conditions which seem to optimize Vibrio populations.

Richards, G.P., Watson, M.A., Needleman, D.S., Church, K.M. and Häse, C.C., 2015. Mortalities of Eastern and Pacific oyster larvae caused by the pathogens Vibrio coralliilyticus and Vibrio tubiashii. Applied and environmental microbiology, 81(1), pp.292-297.

Richards, G.P., Bono, J.L., Watson, M.A. and Needleman, D.S., 2014. Complete genome sequence for the shellfish pathogen Vibrio coralliilyticus RE98 isolated from a shellfish hatchery. Genome announcements, 2(6), pp.e01253-14.

Tout, J., Siboni, N., Messer, L.F., Garren, M., Stocker, R., Webster, N.S., Ralph, P.J. and Seymour, J.R., 2015. Increased seawater temperature increases the abundance and alters the structure of natural Vibrio populations associated with the coral Pocillopora damicornis. Frontiers in microbiology, 6, p.432.

This paper has cited Tout et al.,2015, and focuses on the interactions between phytoplankton and Vibrio species : 

Main, C.R., Salvitti, L.R., Whereat, E.B. and Coyne, K.J., 2015. Community-level and species-specific associations between phytoplankton and particle-associated Vibrio species in Delaware's inland bays. Applied and environmental microbiology, 81(17), pp.5703-5713.