Detection of Perkinsus marinus Extracellular Proteins in Tissues of the Eastern Oyster Crassostrea virginica: Potential Use in Diagnostic Assays

Abstract

Perkinsus marinus, the cause of serious losses of the eastern oyster Crassostrea virginica, secretes extracellular proteins (ECP) in culture (in vitro) including serine proteases. The production of similar ECP in the eastern oyster (in vivo) and their role in pathogenicity, however, remain to be elucidated. The induction and dissemination of these proteins within infected eastern oysters could be critical to understanding the pathologic mechanisms employed as well as to providing the means for rapid and specific diagnosis. To quantify and examine the production of these proteins in the host, polyclonal antibodies were produced against ECP produced during the in vitro culture of P. marinus. By the use of a Western blot technique, these antibodies were shown to recognize a 62-kDa protein antigen expressed in lightly infected oysters, whereas three antigens (62, 38, and 28 kDa) were recognized in heavily infected oysters. Additionally, there was an antigen that is expressed in vitro (40 kDa) that was not detected in vivo and two that were detected in vivo (120 and 32 kDa) but not in vitro. An enzyme-linked immunosorbent assay (ELISA) employing these antibodies indicated that the concentration of secreted antigens is significantly related (P < 0.05) to the number of cells of P. marinus in infected eastern oyster tissues, as determined by the standard Ray's fluid thioglycollate medium (RFTM) assay. Significant differences (P < 0.025) between the ELISA and RFTM assays were only observed when oysters possessed less than one hypnospore per gram of tissue. Thus this ELISA proved to be an excellent diagnostic tool, generating values that correlated with the number of protozoal cells present within infected tissues.