No abstract

ABSTRACT. In 1989, an unidentified protistan parasite, QPX (Quahaug Parasite X), was found in quahaugs Mercenaria mercenaria (Linnaeus, 1758) from a hatchery on Prince Edward Island, Canada, which was suffering extensive mortalities. The parasite was identical to one reported from mass mortalities of wild populations of quahaugs in the late 1960's. QPX eliclts a massive inflammatory response characterized by extensive infiltration of haemocytes with necrosis of the connective tissues in the digestive gland and within the musculature of the foot. Light microscopy revealed a range of paraslte stages including thick-walled cyst-like stages. The majority of QPX tissue stages were enclosed within a translucent 'halo' indicative of possible host tissue lysls. The same feature was observed in QPX isolated from quahaug tissue cultured on potato dextrose agar. Culture in sterile artificial seawater and on potato dextrose agar resulted in production of a blflagellate stage. Features of all developmental stages observed are described and compared with those of the Thraustochytriales and Labyrinthulales.

Biomass of the protistan parasite QPX (quahaug parasite X) of hard-shell clam Mercenaria mercenaria was enriched from in vitro culture. The nuclear gene encoding the 18S RNA of the small-subunit ribosomal (ssu-rDNA) was recovered using the polymerase chain reaction (PCR) and sequenced. Phylogenetic analysis clearly showed that QPX is a member of phylum Labyrinthulomycota, within which it appears as a specific relative of Thraustochytrium pachydermum. These results confirm the provisional assignment of QPX to the Labyrinthulomycota made previously on the basis of morphological and ultrastructural characters found in some, but not all, geographic isolates.KEY WORDS: QPX, quahaug parasite X · Mercenaria mercenaria · Hard-shell clam · Small-subunit ribosomal DNA · Labyrinthulomycota · Thraustochytrium pachydermum Resale or republication not permitted without written consent of the publisherDis Aquat Org 42: [185][186][187][188][189][190] 2000 trids are found in similar environments, although more typically in association with decomposing plant material or organic detritus than on living plants or algae. A few are of economic importance: Labyrinthula macrocystis has been implicated in the decline of shellfish fisheries along the Atlantic coasts of North America and Europe during the 1930s, due to its (perhaps secondary) role in the 'wasting disease' of the eelgrass Zostera marina (Pokorny 1967), while Labyrinthuloides haliotidis is a pathogen of the abalones Haliotis kamtschatkana and H. rufescens, causing up to 100% mortality in juveniles (Bower 1987a,b, Bower et al.1994.Using the culture method developed by Kleinschuster et al. (1998), we enriched biomass of QPX from infected Mercenaria mercenaria isolated from New Brunswick, and have used the polymerase chain reaction (PCR) to amplify the nuclear gene encoding the small-subunit ribosomal RNA (ssu-rDNA) from this enriched fraction. METHODS AND MATERIALSBiological samples. Infected Mercenaria mercenaria ranging in size from 40 to 87 mm were collected from Sam Orr Pond, St Andrews, New Brunswick, on 16 July 1998. Within 24 h of removal from the water, a dorso-ventral cross-section of tissues was removed from each clam and preserved in 1G4F (Howard & Smith 1983) for histological examination. At the same time, approximately 1 mm 3 samples of gill, mantle, digestive gland, gonad and foot were collected for culture in Minimal Essential Medium (MEM; Sigma M0644).Separation of parasites from tissues. The methods we employed are slight modifications of those used by Bower (1987c) for culture of Labyrinthuloides haliotidis in juvenile abalone (Haliotis rufescens and H. kamtschatkana). The combined tissues were macerated in a 10 ml Potter Elvehjem tissue grinder with 1 ml sterile artificial seawater (SAS). The homogenate was transferred to a 15 ml capped centrifuge tube (Fisher) with an additional 2 ml SAS containing antibiotics (10 000 U penicillin, 10 000 µg streptomycin and 25 µg amphotericin B ml -1 SAS). The homogenate was centrifuged at 1750 × g in a bencht...

As part of a disease resistance experiment, 112 apparently healthy European flat oysters Ostrea edulis L. were exported from Canada (Nova Scotia) into France to test their susceptibility to Bonamia ostreae infection. Twelve oysters died in transit and 17 others died within 2 wk of laboratory quarantine acclimation. All oysters were examined histologically, and the 17 that died during quarantine were assayed for microcells (Bonamia sp. and Mikrocytos mackini) using molecular techniques. A microcell parasite was detected in the connective tissue of 5 of the 112 oysters. Morphological appearance, tissue affinity and molecular characterization through PCR, in situ hybridization (ISH), fluorescence in situ hybridization (FISH) and sequencing revealed a protist related to M. mackini. This is the first report of a parasite of the genus Mikrocytos in a species belonging to the genus Ostrea from the Atlantic Ocean. KEY WORDS: Mikrocytos · Ostrea edulis · In situ hybridization · PCR · Histology Resale or republication not permitted without written consent of the publisherDis Aquat Org 80: [27][28][29][30][31][32][33][34][35] 2008 MATERIALS AND METHODSOysters. The oysters from Nova Scotia were produced in a hatchery and transferred to open water after 4 mo. They were held in suspended culture for the duration of grow out and were ca. 3 yr of age when shipped to France (A. Mallet, pers. comm.). In April 2001, 60 adult Ostrea edulis were collected from this population and histological examination of tissue cross-sections and heart smears to check for the absence of lesions and/or pathogens was done at the Shellfish Health Laboratory of the Fisheries and Oceans Canada, Gulf Fisheries Center (DFO-GFC), Moncton, NB, Canada. Subsequently, 112 cohorts from the same site were shipped in June 2001 and were received after 6 d of transit and maintained in quarantine at the IFREMER laboratory, La Tremblade, France. The quarantine system used chlorine treatment for effluent water, but the incoming water was not treated. Temperature of the water was 14°C.Following the detection of a presumptive Mikrocytos sp. in the oysters at IFREMER, 135 American oysters Crassostrea virginica and 275 Ostrea edulis were collected from several beds in Nova Scotia in late 2001 and in 2002. Of these, 3 samples totaling 189 O. edulis (46%) came from the initial site of sampling (Table 1). A total of 104 O. edulis from this site were examined by histology for pathogens and all of the oysters were examined by PCR for the presence of Mikrocytos sp. and Bonamia ostreae at DFO-GFC.Histology. Oysters were opened and a standard section through the digestive gland, gills, labial palps and mantle was fixed in Davidson's fixative (Moore et al. 1953), paraffin embedded and sections (5 µm thick) stained with haematoxylin and eosin (H&E). A complete examination of the cross-section was performed at 100 × magnification with further examination of any unusual pathology and specifically for the presence of microcells at 1000 × magnification (OIE 2003).Heart ...

In May 1989 a previously unidentified apicomplexan parasite was found in broodstock bay scallops Argopecten irradians from the east coast of Canada. Host tissue responses ranged from focal hemocytic encapsulation to the formation of abscesses containing necrotic hemocytes, parasites and ceroid. Bay scallops held for 6 mo in closed-system, artificial seawater at 22 to 24 "C produced detectable infections much earlier than specimens grown in estuarine waters. Additionally, the lesions in experimentally held bay scallops showed less phagocytosis and encapsulation than those found in fieldsampled bay scallops. Transmission of the parasite appears to be direct, having occurred during the brief exposure of the spat generation to infected broodstock. The parasite was undetectable in bay scallops under 7 to 10 mm in length (i.e. < G mo old) and only appeared in tissue sections after the scallops had been held for 6 wk in warm water. Thioglycollate culture of tissues from infected scallops yielded zoosporangia which stained blue-black with Lugol's iodine. Thls test is diagnostic for Perlunsus species; however, the concentration of zoosporangia in the bay scallop tissues did not attain the levels found in previously described Perkinsus species. The specles herein described is a new species based on both morphological and epidemiological differences from previously described species. The name proposed is Perkinsus karlssoni after John D. Karlsson, who has provided extensive studies of bay scallop reproductive biology as well as parasites. Biflagellate zoospores released from the Lugol-positive zoosporangia when transferred to seawater survived over 6 wk at 4OC, but were unable to tolerate either full-salinity seawater (3.3 %) or salinities under 1.4 %.