Perkinsus marinus causes disease of economic importance in Crassostrea virginica. Dead of gaping oysters are the main clinical signs; thin, watery tissue and pale digestive gland are the gross signs. However these signs are not specific to infection with Perkinsus marinus. Crassostrea gigas can be infected but do not develop the disease (Calvo, Luckenbach et al. 1999).
Perkinsus marinus is a pathogenic dinoflagellate of oysters. Recent investigations indicate that this parasite may not belong in the Phylum Apicomplexa where it was initially classified but rather it seems to be more closely related to the Dinoflagellida.
Screening techniques for the pathogen
The standard diagnostic technique for Perkinsus sp. detection in bivalves is the culture of host tissue (usually gills) in the “Ray’s Fluid Thioglycolate Medium” (RFTM). The biggest limitation of the technique is the time it needs to get hypnospores that can be visualised after staining by Lugol’s iodine (usually between 5 and 7 days of incubation in RFTM).
Histology can be used as a screening technique. Sections of tissue should include digestive gland and gills. Positive result is the occurrence of spherical cells about 2-10 µm in diameter (smaller than P. olseni) with a large vacuole and an eccentrically displaced nucleus (see picture below). Cells are often phagocyted by haemocytes.
Picture : Perkinsus marinus cells in the connective tissue of the digestive gland and the gut epithelium of an infected Crassostrea virginica oyster.
In advanced infection only, smears can also be used as a screening technique. Collect haemolymph with a syringe inserted into the adductor muscle, place a drop of haemolymph on a slide and smear. Presence of spherical cells about 2-15 µm in diameter with a large vacuole and an eccentrically displaced nucleus indicates the presence of Perkinsus sp.
Confirmatory techniques for diagnosis
A PCR technique based on the amplification of a part of the rRNA non transcribed spacer (NTS) region has been developed (Marsh A. G., Gauthier et al. 1995; Robledo J. A., Gauthier et al. 1998). A set of primers (PmarITS-70F and PmarITS-600R) for two species specific standard or real-time PCR techniques has also been designed from the intergenic spacer sequence (ITS) (Audemard, Reece K. S. et al. 2004). These last two techniques can detect less than a cell DNA of P. marinus in the reactive medium.
DNA sequencing of the ITS region can be done to identify the species, by comparing the ITS region nucleotide sequences with reference sequences deposited in the GenBank database (http://www.ncbi.nih.gov/entrez/).
A specific DNA probe that targets the LSU of the rRNA gene of P. marinus has been recently developed (Moss et al. 2006). The sensitivity is greater than histology but has not been compared with the RFTM technique.
Comments and recommendations on available diagnostic techniques
The NTS PCR assay has been validated against fluid thioglycollate culture (Robledo J. A., Gauthier et al. 1998). The ITS PCR assay has not been validated against fluid thioglycollate culture. However the ITS primers are recommended over the NTS assay because they are more likely to amplify all Perkinsus marinus strains (Audemard, Reece K. S. et al. 2004).
What should we do for diagnosis at suspicion?
In case of suspicion of perkinsosis due to Perkinsus marinus, oyster tissues including heart, rectum, piece of gill and mantle are placed in Ray’s Fluid Thioglycolate Medium for 5 to 7 days. In parallel piece of tissues should be fixed in ethanol for molecular analysis (PCR/ sequencing).
EU-legislation related to techniques
Perkinsus marinus is listed by the EU.
OIE recommendations related to techniques
Infection with Perkinsus marinus is listed by the OIE Code (2009 version) and by the OIE Manual of Diagnostic Tests for Aquatic Animals (2009 version).
The OIE recommends:
- RFTM culture of tissue for surveillance
- PCR technique for presumptive diagnostic
- ISH for confirmatory diagnostic
OIE Reference Laboratory
: Department of Environmental and Aquatic Animal Health, Virginia Institute of Marine Science, PO Box 1346, Gloucester Point, VA 23062 USA
: Dr. Eugene M. Burreson, e-mail: email@example.com
It is advised to follow the recommendations of the OIE.
Audemard, C., Reece K. S., et al. (2004). Real-time PCR for detection and quantification of the protistan parasite Perkinsus marinus in environmental waters. Appl Environ Microbiol 70(11): 6611-8.
Calvo, G. W., M. W. Luckenbach, et al. (1999). Comparative field study of Crassostrea gigas (Thunberg, 1793) and Crassostrea virginica (Gmelin, 1791) in relation to salinity in Virginia. J Shellfish Res Vol. 12, 18(2): 465-473.
Marsh A. G., J. D. Gauthier, et al. (1995). A semiquantitative PCR assay for assessing Perkinsus marinus infections in the eastern oyster, Crassostrea virginica. J Parasitol 81(4): 577-583.
Moss J.A., Burreson E.M. & Reece K.S. (2006). Advanced Perkinsus marinus infections in Crassostrea ariakensis maintained under laboratory conditions. J. Shellfish Res., 25: 65–72.
ICES (2011). Dermo disease of oysters caused by Perkinsus marinus. Revised and updated by Susan E. Ford. ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish. Leaflet No. 30. 5 pp. see this link.
Robledo J. A., J. D. Gauthier, et al. (1998). Species-specificity and sensitivity of a PCR-based assay for Perkinsus marinus in the eastern oyster, Crassostrea virginica: a comparison with the fluid thioglycollate assay. J Parasitol 84(6): 1237-44.