Evaluation of methods using ray's fluid thioglycollate medium for diagnosis of Perkinsus marinus infection in the eastern oyster, Crassostrea virginica

Accurate detection and quantification of parasite body burden are critical for understanding many aspects of host-parasite interactions. The standard assay for diagnosing Perkinsus marinus infections in Crassostrea virginica involves incubation of oyster tissue in Ray's fluid thioglycollate medium (RFTM), followed by iodine staining and microscopic examination for parasites. The original RFTM tissue assay is destructive and provides only a ranked level of infection intensity. A recent modification provides a technique to enumerate P. marinus after incubation in RFTM and determines total body burden. Application of this technique to hemolymph samples has provided a nondestructive assay. We provide a critical evaluation of these three assays. Data from previous studies were not adequate to critically evaluate performance of the tissue and hemolymph assays. Sensitivity and accuracy of these assays were therefore compared against weight-standardized body burden in Delaware Bay oysters over the course of a year. Determination of total body burden was significantly more sensitive than the other assays. Neither tissue nor hemolymph assays provided accurate estimates of individual infection intensities and were insensitive at low infection levels. The tissue assay was easier to use and slightly more accurate than the hemolymph assay, and both provided reasonable estimates of average infection level in the population. In summary, total body burden assessment is recommended when highly accurate measures of infection intensity or prevalence are necessary, the tissue assay is recommended for monitoring epizootics because of its simplicity and accuracy at the population level, and the hemolymph assay is only recommended when oysters must be kept alive.