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.

Samples from the field and laboratory exposure to Mikrocytos mackini (a tiny protistan parasite of unknown taxonomic affiliation) confirmed that juvenile Pacific oysters (Crassostrea gigas) are susceptible to infection and the resulting disease. In the laboratory bath exposure experiment, a prevalence of infection approaching 100% and mortalities were observed in the small oysters (about 18mm in shell length). However, in the same laboratory exposure experiment, similar aged geoduck clams (Panope abrupta, about 8mm in shell length) were resistant to infection. The main route of infection in the oysters appeared to be via the digestive tract and possibly the gills where the parasite multiplied within host cells. Other tissues such as the adductor muscle and vesicular connective tissue were subsequently colonized. Although the infection resulted in the mortality of some oysters, others appeared to overcome the disease.

The protistan parasite Mikrocytos mackini, causative agent of Denman Island disease (mikrocytosis), induces mortality and reduces marketability in the Pacific oyster, Crassostrea gigas, in British Columbia, Canada. This parasite is a pathogen of international concern because it infects a range of oyster species, and because its life cycle and mode of transmission are unknown. A digoxigenin-labelled DNA probe in situ hybridisation technique (DIG–ISH) was developed, and its detection sensitivity was compared to standard histological sections stained with haematoxylin and eosin stain (H&E-histo). In H&E-histo preparations, the detection of M. mackini was certain only when the parasite occurred within the vesicular connective tissue of adult oysters. However, the DIG–ISH technique clearly demonstrated the presence of infection in all other host tissues as well as in juvenile oysters with poorly developed vesicular connective tissue. The probe hybridised strongly to M. mackini, did not hybridise to oyster tissues or with the other shellfish parasites tested, and was more sensitive for detecting infections when compared to H&E-histo.

The protistan parasite Mikrocytos mackini, the causative agent of Denman Island disease in the oyster Crassostrea gigas in British Columbia, Canada, is of wide concern because it can infect other oyster species and because its life cycle, mode of transmission, and origins are unknown. PCR and fluorescent in situ hybridization (FISH) assays were developed for M. mackini, the PCR assay was validated against standard histopathological diagnosis, and a preliminary phylogenetic analysis of the M. mackini small-subunit ribosomal RNA gene (SSU rDNA) was undertaken. A PCR designed specifically not to amplify host DNA generated a 544 bp SSU rDNA fragment from M. mackini-infected oysters and enriched M. mackini cell isolates, but not from uninfected control oysters. This fragment was confirmed by FISH to be M. mackini SSU rDNA. A M. mackini-specific PCR was then designed which detected 3 to 4x more M. mackini infections in 1056 wild oysters from Denman Island, British Columbia, than standard histopathology. Mikrocytos mackini prevalence estimates based on both PCR and histopathology increased (PCR from 4.4 to 7.4%, histopathology from 1.2 to 2.1%) when gross lesions were processed in addition to standard samples (i.e. transverse sections for histopathology, left outer palp DNA for PCR). The use of histopathology and tissue imprints plus PCR, and standard samples plus observed gross lesions, represented a 'total evidence' approach that provided the most realistic estimates of the true prevalence of M. mackini. Maximum parsimony and evolutionary distance phylogenetic analyses suggested that M. mackini may be a basal eukaryote, although it is not closely related to other known protistan taxa.

A population study on Chionoecetes tanneri off the west coast of Vancouver Island, British Columbia, Canada revealed the presence of Hematodinium sp. in up to 4% of the captured Tanner crabs. Prior to this encounter, the only reports of Hematodinium sp. in Chionoecetes spp. were from C. bairdi and C. opilio on the coasts of Alaska and Newfoundland.

A eukaryotic parasite of uncertain taxonomic affiliation, with superficial similarity to parasitic dinoflagellates (large plasmodia and numerous trophonts) but a different mechanism of nuclear division and a lack of organelles characteristic of parasitic dinoflagellates, is described from spot prawns (Pandalus platyceros). Up to 20% of the spot prawns examined from Malaspina Strait, British Columbia, were infected. Infections in the majority of the prawns were cryptic (asymptomatic) but of sufficient duration to affect secondary sexual characteristics and castrate the host. Cryptic infections consisted of plasmodia containing numerous nuclei. The plasmodia were observed in the haemal sinuses of all tissues. In mature plasmodia the nuclei stopped dividing and showed a peripheral chromatin ring, an internal chromatin web, and up to three tiny nucleoli. Mature plasmodia divided into numerous uninucleate trophonts, resulting in symptoms of lethargy, orange discoloration, and milky haemolymph caused by a plethora of either spherical or discoid trophonts. Symptomatic infections of the prawns fished with traps rarely exceeded 2%. In 3 of the 156 symptomatic prawns examined, about 30% of the trophonts were in the process of binary fission. During mitosis the nuclear membrane was persistent, but openings (about 0.8 µm in width) at either pole accommodated emergent spindle-pole bodies to which the few chromosomes were attached by microtubules. Attempts to transmit the infection between prawns in the laboratory were unsuccessful.

An ultrastructural study was carried out on Mikrocytos mackini, the cause of Denman Island disease in Pacific oysters Crassostrea gigas in western Canada. Three forms were identified, quiescent cells (QC), vesicular cells (VC) and endosomal cells (EC). QC occurred in the vesicular connective tissue (VCT), haemocytes (hyalinocytes), adductor and heart myocytes, and extracellularly. They had a central round to ovoid nucleus, < 7 cisternae of inactive nuclear membrane-bound Golgi, few vesicles and lysosome-like bodies. VC were rarely extracellular and usually occurred in adductor and heart myocytes, in close association with host cell mitochondria. The contents of the host cell mitochondria appeared to pass through a tubular extension into the cytoplasm of the parasite. Cytoplasmic vesicles resembled the tubular structure in appearance and size. EC occurred in the VCT, in haemocytes and extracellularly. They had a dilated nuclear membrane, sometimes containing a looped membranous structure that appeared to derive from the nucleus, and pass into the cytoplasm. A well-developed anastomosing endoplasmic reticulum connected the nuclear and plasma membranes, and endosomes were present in the cytoplasm. QC and EC cells were frequently observed tightly against, or between, the nuclear membranes of the host cell. Few organelles occurred in all forms of M. mackini, especially QC. The lack of organelles found in most eukaryotic cells, including mitochondria or their equivalents, may be due to obligate parasitism and the utilization of host cell organelles reducing the need for parasite organelles. Alternatively, perhaps M. mackini is a primitive eukaryote. Although phylogenetic affinities could not be determined, it is not a haplosporidian. A developmental cycle is proposed from these findings.

This report describes a simple filtration technique to isolate the parasite Mikrocytos mackini from oyster tissue. The technique is based on successive filtration through filter papers and polycarbonate membrane filters of decreasing mesh using a low vacuum (<8 cm Hg). This technique allows for the recovery of about 1 × 108 parasites (microcells) from about 2 g of heavily infected oyster tissue. About 99% of the particulate material in the final preparation consisted of intact M. mackini.

Japanese scallops Patinopecten yessoensis, introduced into British Columbia, Canada, as a species for aquaculture, proved highly susceptible to an enzootic protistan parasite Perkinsus qugwadi (initially called SPX). Sporadic occurrence of the parasite among cultured scallops sometimes resulted in losses exceeding 90%. Native scallops (Chlamys rubida and Chlamys hastata) were resistant to infection and the pathogenic effects of the parasite. Weathervane scallops Patinopecten caurinus (not amenable to culture but closely related to the Japanese scallop) were not available to test for resistance to infection. Laboratory and field studies showed that first-generation progeny of Japanese scallops that survived an epizootic outbreak of P. qugwadi had a significant increase in resistance to infection and resulting mortalities. Hybrid scallops. resulting from a cross between Japanese scallop females (from the same group of scallops that survived an epizootic outbreak of P. qugwadi) and weathervane scallop males, had similar resistance to P. qugwadi. The identification of scallop stocks that are resistant to P. qugwadi has facilitated the development of a scallop culture industry in British Columbia.

Perkinsus qugwadi was detected in cultured Japanese scallops, Patinopecten yessoensis, held for at least 3 months at 5 of 14 locations along the coast of British Columbia. At four of the locations, infected scallops were observed in over half of the samples examined and the prevalence of infection reached 98%. At one location (Cypress Bay), infected scallops were rarely encountered and when infection was present, the intensity was light. Field studies, laboratory experiments, and commercial grow-out operations indicate that high mortalities among Japanese scallops are associated with P. qugwadi infection. Although some scallops responded to infection with an accumulation of numerous haemocytes in the vicinity of the parasite, other scallops appeared not to respond and were overwhelmed by numerous parasites that multiplied in the connective tissues of all organs. Epizootiology of this pathogen in Japanese scallops suggests that P. qugwadi is enzootic in an unknown host in British Columbia, with the introduced Japanese scallop serving as an aberrant host.

In addition to Crassostrea gigas, Mikrocytos mackini Farley 1988, a pathogenic intracellular protistan of unknown taxonomic affiliations, produces disease and mortalities in other species of economically important oysters (Crassostrea virginica, Ostrea edulis and Ostrea conchaphila). Preliminary evidence suggests that these alternate species may be more susceptible to infection and the resulting disease than the usual host C. gigas. M. mackini isolated from C. virginica and O. edulis were infective for oysters. Warm temperatures (above 15 degrees C) prevented the development of M. mackini in C. gigas, C. virginica and O. edulis.

Denman Island disease, characterized by clinical signs of focal green lesions on the body surface or within the mantle, palps, and adductor muscle of Pacific oysters (Crassostrea gigas), is caused by a protozoan parasite of unknown taxonomic affiliations, Mikrocytos mackini. Detection of M. mackini was more sensitive and rapid by use of tissue imprints than histological sections. Of several isolation procedures investigated, centrifugation of homogenized infected tissues through a 15% sucrose solution enabled the isolation of the highest number of M. mackini with the lowest amount of oyster debris. Experimental transmission showed that oysters exposed to M. mackini by inoculation with isolated parasites had shorter prepatent periods and higher prevalences and intensities of infection than those incubated in homogenates from infected oysters (bath exposure) or those naturally exposed in the field. Experimental transmission was also used to propagate M. mackini in vivo in the laboratory year round. For the development of the disease, exposed oysters required prolonged incubation at low temperatures (about 10 degrees C).

Stained prawn disease (SPD) with clinical signs of black discolouration of the cuticula, especially around the edges of body segments, and black stippling on the surface of the hepatopancreas was caused by a rickettsia-like microorganism with an affinity for fixed phagocytes and haemocytes. This disease was found in prawns from various localities throughout Howe Sound and at one location in the Strait of Georgia, British Columbia, Canada. The distribution of SPD within Howe Sound has not changed since it was first detected in 1989. However, the prevalence has declined to about 4% from a record high of about 15% in July 1990 and March 1991. In areas with these high prevalences, an above-average level of mortality, equated to a decline in survival rates from 57 to 15%, was detected. These mortalities were not attributable to fishing pressure because the affected area has been closed to fishing since November 1988 due to unacceptable levels of dioxin and furan compounds in shellfish tissue samples. Laboratory studies indicated that the SPD agent can be transmitted horizontally by cannibalism and via the water [exposure to screened (1 mm pore size) effluent from infected prawns] and remained infectious for 10 d or more of storage at -10 degrees C. About 50% of the prawns that fed on infected prawns (both fresh and after being frozen) and 25% of the prawns exposed to contaminated water became infected. Most mortalities attributable to SPD occurred between 2 and 4 mo after exposure to the etiological agent in the laboratory.

Preliminary grow-out studies with Patinopecten yessoensis in British Columbia, Canada, revealed the presence of at least three pathogens. At one location, the shell boring polychaete, Polydora websteri, was responsible for 84% mortality. Most surviving scallops were stunted and regrowth of the shell was often abnormal. At another locality, a protozoan of unknown taxonomic affinities (SPX) was observed. Following one year of excellent growth and survival (>90%), about 60% of the surviving scallops died within 3 months (June to August). Creamy-white pustules (up to 5 mm in diameter) were observed in 50% of the scallops. SPX was found in the connective tissues of 84% of the scallops with pustules and in 15% of those without pustules. The third pathogen was also an unidentified protozoan (SPG). However, unlike SPX, SPG was found in 5% to 10% of P. yessoensis from seven localities and infections were always light. In addition to the diseases with recognizable causes, focal lesions of unknown aetiology were associated with scallop mortalities (>65%) in six of seven growout localities during 1989–1990. Visible lesions consisting of pinkish-orange pustules (up to 15 mm in diameter) occurred in up to 30% of P. yessoensis. In addition, 30% to 55% of the scallops had microscopic lesions in the connective tissues of all organs.

The densities and patterns of distribution of the six species of molluscs were determined in the three environments: dry rock surface, "intersection" area, and rock pool, at both midday and midnight low tides during each season. Austrocochlea constricta occurred abundantly within the rock pool area, which was a homogeneous environment at certain times and in certain seasons. The density of Bembicium nanum was inversely related to the presence of water, and this species had a clumped pattern of distribution, except in the rock pool during the winter. Cellana tramoserica occurred in similar densities in all three environments during cool weather, but tended to migrate from the rock surface to the "intersection" environment during hot weather. The densities of Melanerita melanotragus, Montfortula conoidea, and Morula marginalba were greatest in the "intersection" environment, but, because of the generally low densities of these three species on the rock platform, few valid conclusions could be made concerning their abundances and distribution.