A suite of ddPCR assays targeting microbial pathogens for improved management of shellfish aquaculture

ABSTRACT The shellfish aquaculture industry is one of the fastest-growing sectors of global food production, but it is currently facing major challenges stemming from microbial pathogens. This study presents an optimized and validated suite of droplet digital PCR (ddPCR) assays using water samples proximal to oyster farms in North Carolina to quantify pathogens relevant to the aquaculture industry. Two of the molecular assays enable the quantification of the pathogens, Vibrio parahaemolyticus and Perkinsus marinus , that threaten human health and oyster performance, respectively. This work also introduces two ddPCR assays that enable the simultaneous quantification of at least nine ecologically relevant Vibrio spp . using only two sets of primers and probes targeting the glycosyl hydrolase family 18 (GH18) domain of the chiA gene in Vibrio bacteria. The entire suite of assays was applied to single assessments at 12 sites, revealing heterogeneity in microbial pathogen concentrations across the coastal landscape and variability of abundances within individual estuarine river systems. Additionally, a longitudinal study conducted at a demonstration lease elucidated unique temporal trends for all microbial targets. Notably, when concentrations of Vibrio spp . quantified using the two assays targeting the chiA gene reached their maximum, the daily probability of mortality increased, suggesting a role for other ecologically pertinent Vibrio spp . in the progression of mortality that would otherwise be missed. This study highlights the utility of ddPCR for the advancement of shellfish management by offering insights into the spaciotemporal dynamics of microbial pathogens. IMPORTANCE Climate change is drastically altering the environment and changing the abundance and geographical distribution of marine pathogens. These microbial species put additional pressure on the aquaculture industry by acting as sources of disease for animals important to the food industry as well as for humans upon consumption of contaminated food. To address growing concerns, high-resolution monitoring of pathogens can offer insights for effective management in a critical industry. Validated in the field, the suite of molecular droplet digital PCR assays presented here improves upon current methods, enabling the simultaneous quantification of several targets. This technology makes it possible to track pathogens as they move through the environment and reveals changes in abundance that may inform adjustments to farming practices aimed at mitigating negative outcomes. Additionally, this work presents a unique approach to molecular assay design that unveils potential drivers of ecological shifts and emerging etiologies of disease more efficiently.