Genetic detection and discrimination of closely related oyster pathogens for biosecurity and shellfish stock management

Disease outbreaks among shellfish cause significant declines in both wild fisheries and aquaculture production globally. In Aotearoa New Zealand, a long history of flat oyster infections by the haplosporidian parasite Bonamia exitiosa has influenced fishery dynamics in the Foveaux Strait. B. ostreae, which causes high host mortality, was detected in both wild and farmed oysters in the Marlborough Sounds in 2015 and at Stewart Island in 2017, prompting the development of rapid diagnostic tests with high sensitivity and specificity that can be used to simultaneously monitor both Bonamia species. Separate molecular diagnostic assays were developed for B. exitiosa and B. ostreae using droplet digital PCR (ddPCR). Analytical tests indicated both assays were specific and had limits of detection below 0.4 copies µl-1. Estimates of diagnostic performance used latent class analysis conducted on comparisons with previously established test methods. Both ddPCR assays had a diagnostic sensitivity and specificity equal to or greater than tests based on qPCR, end-point PCR, histopathology and heart imprints, validating their use for fishery health assessment and biosecurity monitoring. The new ddPCR assays are preferred over other methods in situations where high throughput and analytic and diagnostic performance are essential, as seen in New Zealand where 2 Bonamia species are sympatric in some regions and may co-infect a single host. Furthermore, the new assays could be adapted for eDNA-based surveillance and combined with other pathogen assays for multiplex assays, enabling further research into Bonamia lifecycles.