Associations of Environmental Conditions and Vibrio parahaemolyticus Genetic Markers in Washington State Pacific Oysters

Abstract

Vibrio parahaemolyticus is a naturally occurring bacterium in estuarine waters and is a major cause of seafood-borne illness. The bacterium has been consistently identified in Pacific Northwest waters and elevated illness rates of vibriosis in Washington State have raised concerns among growers, risk managers, and consumers of Pacific oysters (Crassostrea gigas). In order to better understand pre-harvest variation of V. parahaemolyticus in the region, abundance of total and potentially pathogenic strains of the bacterium in a large number of Washington State Pacific oyster samples were compared with environmental conditions at the time of sampling. The Washington Department of Health regularly sampled oysters between June and September at over 21 locations from 2014 to 2018, resulting in over 946 samples. V. parahaemolyticus strains carrying three genetic markers, tlh, trh, and tdh, were enumerated in oyster tissue using a most probable number-PCR analysis. Tobit regressions and seemingly unrelated estimations were used to formally assess relationships between environmental measures and genetic markers. All genetic markers were found to be positively associated with temperature, independent of the abundance of other genetic markers. Surface water temperature displayed a non-linear relationship, with no association observed between any genetic marker in the warmest waters. There were also stark differences between surface and shore water temperature models. Salinity was not found to be substantially associated with any of the genetic variables. The relative abundance of tdh+ strains given total V. parahaemolyticus abundance (pathogenic ratio tdh:tlh) was negatively associated with water temperature in colder waters and decreased exponentially as total V. parahaemolyticus abundance increased. Strains carrying the trh gene had a pronounced positive association with strains carrying the tdh gene but was also negatively associated with the tdh:tlh pathogenic ratio. These results suggest that there are ecological relationships of competition, growth, and survival for V. parahaemolyticus strains in the oyster tissue matrix. This work also improves the overall understanding of environmental associations with V. parahaemolyticus in Washington State Pacific oysters, laying the groundwork for future risk mitigation efforts in the region.

Nested spatial and temporal modeling of environmental conditions associated with genetic markers of Vibrio parahaemolyticus in Washington state Pacific oysters

“…This non-linear relationship supports previous findings of a negative relationship between salinity and V. parahaemolyticus at high (greater than 23 ppt) salinity levels (Davis et al, 2017). Although previous studies in Washington did not observe an association with salinity (Flynn et al, 2019; Paranjpye et al, 2015), complex non-linear relationships with salinity and V. parahaemolyticus have been found in other bodies of water (DePaola et al, 2003; USFDA, 2005; Johnson, 2015; Davis et al, 2017; Martinez-Urtaza et al, 2016). Further, the previous PNW studies only considered salinity measurements at the time of sampling, whereas statistically significant relationships in this study were identified using a time-lagged measure of salinity.…”

“…This suggests consistency in this relationship across these ecologically diverse waters which was possibly due to random intercepts accounting for baseline differences in genetic targets. The inconsistency between strains of V. parahaemolyticus containing the tdh marker compared to tlh or trh in relation to water temperature had not previously been observed (Flynn et al, 2019).…”

“…The genetic ratios were not correlated with temperature, salinity, or genetic variables ( Figures S5 ). As previously noted in Flynn et al, 2019, we checked for shore and surface collinearity, which revealed a high VIF, and so the former was excluded from our models ( Figure S6 ).…”

“…Whole oyster tissue within a sample was homogenized and V. parahaemolyticus genetic markers enumerated using a most-probable-number per gram (MPN/g) based real-time PCR assay. Details of this assay have been described previously (Flynn et al, 2019; Glover II, 2015), and the MPN-PCR analytical results have been validated. Briefly, hemolysin genetic markers tdh and tlh were targeted across all sampling years, with trh also being targeted beginning in 2014.…”

“…It is difficult, however, to identify broadly applicable relationships between environmental determinants and abundance of the bacterium as many of these associations vary significantly across geographic regions, time periods, and by the genetic marker of interest (DePaola et al, 2003; USFDA, 2005; Turner et al, 2013; Johnson, 2015; Paranjpye et al, 2015; Davis et al, 2017). Previous findings in the PNW have laid the groundwork to characterize space–time residual dependencies for the relationships between environmental measures and V. parahaemolyticus abundance in the region (Flynn et al, 2019; Paranjpye et al, 2015). Statistical assessments accounting for spatial heterogeneity and autocorrelation is also important to consider across the varied aquatic “zones” found in Washington state (Davis et al, 2020).…”