Schuster, C.J., M.L. Kent, J. Peterson, and J.L. Sanders. 2022. Multi-state occupancy model estimates probability of detection of an aquatic parasite using environmental DNA: Pseudoloma neurophilia in zebrafish aquaria. Journal of Parasitology https://doi.org/10.1101/2022.02.16.480730
Pseudoloma neurophilia is the most common pathogen reported in zebrafish (Danio rerio) research facilities. Assays for prevalent zebrafish pathogens are continually expanding, however for P. neurophilia, the only diagnostic methods available are through lethal sampling, often requiring euthanasia of the entire population for accurate estimates of prevalence in small populations. Thus, there is a need to develop non-lethal screening methods that are both sensitive and informative. Here, we describe a nonlethal assay to detect Pseudoloma neurophilia in tank water using a previously developed qPCR assay that was adapted to the droplet digital PCR (ddPCR) platform. The assay detected concentrations as low as 77.5 spores/L and was specific for P. neurophilia. The assay was then validated by testing water from five tanks with infected zebrafish over a three-month period. Each tank was tested using three different water sampling methods: water from flowing tanks, static water, or water from static spawning events. Prevalence data and occupancy modeling revealed that samples collected in static conditions were more informative than samples from flow-through conditions, with prevalence of positive samples at 80% or higher compared to 47%, respectively. Prevalence of positive water samples correlated with prevalence of infection in fish, determined by qPCR.