Abalone Withering Syndrome Disease Dynamics: Infectious Dose and Temporal Stability in Seawater

Withering syndrome (WS) is a chronic bacterial disease that affects numerous northeastern Pacific abalone Haliotis spp. The causative agent of WS is an obligate intracellular Rickettsiales-like bacterium (WS-RLO) that remains unculturable, thereby limiting our understanding of WS disease dynamics. The objectives of our study were to (1) determine the temporal stability of WS-RLO DNA outside of its abalone host in 14 degrees C and 18 degrees C seawater, (2) develop a standardized protocol for exposing abalones to known concentrations of WS-RLO DNA, and (3) calculate the dose of WS-RLO DNA required to generate 50% infection prevalence (ID50) in the highly cultured red abalone Haliotis rufescens. The WS-RLO stability trials were conducted in October 2016, February 2017, and June 2017. A quantitative PCR (qPCR) analysis was used to quantify bacterial DNA for 7 d in seawater collected at an abalone farm in southern California, where the pathogen is now endemic. For all trials and temperature treatments, WS-RLO DNA was unstable in seawater for longer than 2 d. To determine an ID50, groups of uninfected juvenile red abalone were subjected to 3-h bath exposures with four concentrations of WS-RLO at 0, 10(3), 10(4), and 10(5) DNA copies/mL. Abalone feces were tested biweekly for the presence of WS-RLO DNA, and abalone tissues were sampled 9 weeks postinfection for histological and qPCR analyses. The ID50 results indicated that our protocol was successful in generating WS-RLO infections; a pathogen dose of 2.3 x 10(3) DNA copies/mL was required to generate a 50% infection prevalence in red abalone tissue. These findings are critical components of disease dynamics that will help assess WS transmission risk within and among abalone populations and facilitate appropriate management and restoration strategies for both wild and cultured abalone species in WS-endemic areas.