An improved procedure to count Perkinsus marinus in eastern oyster hemolymph

摘要

Perkinsus marinas infection intensity in Crassostrea virginica can be quantified without killing of oysters by determining parasite density in hemolymph samples incubated in fluid thioglycollate medium (FTM). The goal of this study was to improve existing protocols for counting of P. marinas in oyster hemolymph. Specifically, the objectives were to examine the effects on parasite number and diameter of. 1) adding supplements to FTM such as lipid and oyster extract; 2) incubating with various FTM preparations with and without agar or beef extract; 3) incubating with various hemocyte densities (10(5), 10(6), and 10(7) hemocytes/mL of FTM) in a constant FTM volume; 4) incubating with different volumes of FTM (0.2 mL, 1.0 mL, 5.0 mL, and 25.0 mL); and 5) sodium hydroxide digestion of cellular debris. From these results, an improved hemolymph protocol was developed. The diameters and numbers of enlarged parasites or hypnospores in hemolymph of 20 oysters measured by the improved protocol and the standard FIM hemolymph assay of Gauthier and Fisher were compared. Finally, the standard and improved protocols were compared with the FTM body burden assay. The diameter of hypnospores from samples processed with the improved protocol (26 +/- 13 mum) was significantly greater than the diameters from samples processed with the standard protocol (10 +/- 4 mum). The number of hypnospores in samples processed with the improved protocol (8.6 x 10(3) +/- 3.3 x 10(3)) was significantly greater than the numbers in samples processed with the standard protocol (1.9 x 10(3) +/- 3.4 x 10(3)). Results of the body burden assay were significantly correlated with results of the standard hemolymph assay and with results of the improved hemolymph assay. The coefficient of determination (r(2) = 0.7602) and slope (0.91189) of the regression of the FTM body burden assay against the improved FTM hemolymph assay was improved from the coefficient of determination (0.5543) and slope (0.61257) of the regression of the FTM body burden assay against of the standard FTM hemolymph assay.