Prevalence and distribution of QPX, Quahog Parasite Unknown, in hard clams Mercenaria mercenaria in Virginia, USA.

In July 1996, the Virginia Institute of Marine Science initiated a sampling program to examine wild and cultured hard clams Mercenaria mercenaria for QPX, Quahog Parasite Unknown, a protistan parasite associated with severe mortalities of hard clams in localized areas in maritime Canada and Massachusetts, USA. The sampling program set out to seasonally monitor wild clams from one site, James River, Virginia, and cultured clams from 2 sites, Chincoteague Bay and Mattawoman Creek, Virginia. Histological examination of initial samples revealed 8% prevalence of the parasite in 1-2 yr old cultured clams in Chincoteague Bay. This is the first documentation of QPX in Virginia. To ascertain the distribution of the parasite in Virginia, the survey was expanded between August 1996 and July 1997 to include 16 additional sites. A total of 1305 wild and cultured clams was sampled from Chesapeake Bay tributaries and coastal areas where harvest and culture occur. QPX was not found in Chesapeake Bay, but was present in cultured clams from 3 coastal embayments--the original Chincoteague Bay site, Burton Bay and Quinby Inlet. The parasite was found in Chincoteague Bay at each sample period at prevalences ranging from 8 to 48%. Infections were generally light to moderate intensity and were most often observed in mantle and gill tissues. The maximum prevalence was observed in May 1997 and coincided with notable clam mortalities. QPX prevalences at the other sites were low, ranging from 4 to 15%. To date QPX has not had a significant impact on Virginia's hard clam fishery and aquaculture industry; however, the presence of the pathogen in 3 of the state's most productive hard clam growout areas warrants continued monitoring and research.

Molecular diagnostics, field validation, and phylogenetic analysis of Quahog Parasite Unknown (QPX), a pathogen of the hard clam Mercenaria mercenaria.

Quahog Parasite Unknown (QPX) is a protistan parasite that causes disease and mortality in the hard clam Mercenaria mercenaria. PCR primers and DNA oligonucleotide probes were designed and evaluated for sensitivity and specificity for the QPX organism specifically and for the phylum Labyrinthulomycota in general. The best performing QPX-specific primer pair amplified a 665 bp region of the QPX small-subunit ribosomal DNA (SSU rDNA) and detected as little as 1 fg cloned QPX SSU rDNA and 20 fg QPX genomic DNA. The primers did not amplify DNA of uninfected hard clams M. mercenaria or of the thraustochytrids Schizochytrium aggregatum, Thraustochytrium aureum, and T. striatum. The general labyrinthulomycete primers, which were designed to offer broader specificity than the QPX primers, amplified a 435 bp region of SSU rDNA from QPX, and a 436 to 437 bp region of SSU rDNA from S. aggregatum, T. aureum, and T. striatum, but did not amplify that of the clam M. mercenaria. Field validation of the QPX-specific primer pair, through comparative sampling of 224 clams collected over a 16 mo period from a QPX endemic site in Virginia, USA, indicated that the PCR assay is equivalent to histological diagnosis if initially negative PCR products are reamplified. Oligonucleotide DNA probes specific for QPX and the phylum Labyrinthulomycota were evaluated for in situ hybridization assays of cell smears or paraffin-embedded tissues. Two DNA probes for QPX offered limited sensitivity when used independently; however, when used together as a probe cocktail, sensitivity was greatly enhanced. The probe cocktail hybridized to putative QPX organisms in tissues of hard clams collected from Virginia, New Jersey, Massachusetts and Canada, suggesting that the QPX organisms in these areas are either very closely related or the same species. The QPX probe cocktail did not hybridize with clam tissue or with the thraustochytrids S. aggregatum, T. aureum, and T. striatum. The labyrinthulomycete DNA probe hybridized with QPX and the 3 thraustochytrids, with no background hybridization to clam tissue. SSU rDNA sequences were obtained for the putative QPX organisms from geographically distinct sites. Phylogenetic analyses based on the QPX and Labyrinthulomycota sequences confirmed earlier reports that QPX is a member of this phylum, but could not definitively demonstrate that all of the QPX organisms were the same species.

Perkinsus marinus tissue distribution and seasonal variation in oysters Crassostrea virginica from Florida, Virginia and New York.

Perkinsus marinus infection intensity was measured in eastern oysters Crassostrea virginica collected in October and December 1993, and March, May, and July 1994 from 3 U.S. sites: Apalachicola Bay (FL), Chesapeake Bay (VA), and Oyster Bay (NY). Gill, mantle, digestive gland, adductor muscle, hemolymph, and remaining tissue (including gonadal material and rectum) were dissected from 20 oysters from each site at each collection time. Samples were separately diagnosed for P. marinus infections by incubation in Ray's Fluid Thioglycollate Medium (RFTM) and subsequent microscopic quantification of purified enlarged hypnospores. At all sampling times and sites, average P. marinus infection intensity (g wet wt tissue-1 or ml hemolymph-1) was lowest in hemolymph samples, and generally highest in the digestive gland. Perkinsus marinus prevalence was 100% at both FL and NY sites for each of the 5 collection times, and, for the VA site, was less than 100% in only 1 month (May 1994). Seasonal intensity patterns and mean total body burdens differed among the sites. Average body burden was highest in VA during October and progressively declined to a minimum in May. This decline was probably due to mortality of heavily infected oysters and diminution of parasite activity associated with colder temperatures and reduced salinities. Intensities varied little during the months of October and December at both the FL and NY sites. Minimum average intensities were observed in March in FL oysters and May in NY oysters. Relatively high P. marinus infection levels that persisted throughout the winter in NY oysters compared with VA oysters could reflect constant high salinity in Long Island Sound which favors parasite activity, and also rapid decline in temperature in the fall that may have prevented epizootic oyster mortalities.

Real-time PCR investigation of parasite ecology: in situ determination of oyster parasite Perkinsus marinus transmission dynamics in lower Chesapeake Bay.

Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica on the East Coast of the United States. Transmission dynamics of this parasite were investigated in situ for 2 consecutive years (May through October) at 2 lower Chesapeake Bay sites. Compared to previous studies where seasonal infection patterns in oysters were measured, this study also provided parasite water column abundance data measured using real-time PCR. As previously observed, salinity and temperature modulated parasite transmission dynamics. Using regression analysis, parasite prevalence, oyster mortalities and parasite water column abundance were significantly positively related to salinity. Perkinsus marinus weighted prevalence in wild oysters and parasite water column abundance both were significantly related to temperature, but the responses lagged 1 month behind temperature. Parasite water column abundance was the highest during August (up to 1,200 cells/l) and was significantly related to P. marinus weighted prevalence in wild oysters, and to wild oyster mortality suggesting that parasites are released in the environment via both moribund and live hosts (i.e. through feces). Incidence was not significantly related to parasite water column abundance, which seems to indicate the absence of a linear relationship or that infection acquisition is controlled by a more complex set of parameters.

Dementia associated with the antiphospholipid syndrome: clinical and radiological characteristics of 30 patients.

OBJECTIVE: To analyse the clinical and radiological characteristics of patients with dementia associated with the antiphospholipid syndrome (APS). METHODS: Twenty-five patients were identified by a computer-assisted (MEDLINE, National Library of Medicine, Bethesda, MD) search of the literature to locate all cases of dementia associated with APS published in English, Spanish and French from 1983 to 2003. Additionally, we included five patients from our clinics. RESULTS: There were 21 (70%) females and 9 (30%) males. The mean age of patients was 49+/-15 yr (range 16-79 yr). Fourteen (47%) of the patients suffered from primary APS, 9 (30%) had systemic lupus erythematosus and 7 (23%) had 'lupus-like' syndrome. Ten (33%) patients had Sneddon's syndrome and 2 (7%) had cerebral lesions described as Binswanger's disease. Other APS-related manifestations included thrombocytopenia in 12 (40%) patients, cerebrovascular accidents in 11 (37%), heart valve lesions in 8 (27%), deep vein thrombosis in 7 (28%), migraine in 7 (23%), seizures in 4 (13%); five of the 21 (24%) female patients had nine spontaneous abortions. Lupus anticoagulant was present in 21/29 (72%) patients and anticardiolipin antibodies were present in 24/29 (83%) patients. Cortical infarcts were found in 19 (63%) patients, subcortical infarcts in 9 (30%), basal ganglia infarcts in 7 (23%) and signs of cerebral atrophy in 11 (37%). Anticoagulation was used in 14/25 (56%) patients, steroids in 12/25 (48%), aspirin in 6/25 (24%) and dypiridamole in 5/25 (20%). CONCLUSIONS: Dementia is an unusual manifestation of APS but one which has a high disability impact in a patient's daily life. In order to prevent these consequences, an echocardiographic and cerebral CT or MRI evaluation are recommended in all patients with APS. Furthermore, ruling out APS should be recommended in the clinical approach to dementia, especially in young patients.

Acute osmotic tolerance of cultured cells of the oyster pathogen Perkinsus marinus (Apicomplexa:Perkinsida).

Cultured Perkinsus marinus cells were exposed for 24 hr to salinities of 0, 3, 6, 9, 12 and 22 ppt at temperatures of 1, 5, 10, 15 and 28 degrees C in artificial seawater (ASW) and to the same salinities at 28 degrees C in ASW with the osmotic concentration adjusted with sucrose to the equivalent of 22 ppt. At 28 degrees C mortality increased as salinity decreased below 22 ppt. Mortality was greater than 99% at 0 ppt and greater than 90% at 3 ppt. Mortality was 70% at 6 ppt, 43% at 9 ppt and 20% at 12 ppt. Mortality was low (< 5%) and equal to that at 22 ppt in all treatments where osmotic concentration was maintained with sucrose. Mortality occurred rapidly, within 5 min of exposure to experimental conditions. In the region where mortality was most sensitive to salinity changes (6-12 ppt), lower temperature caused an increase in mortality, but the temperature effect was significant only at 9 ppt.

Effect of in vitro exposure to tributyltin on generation of oxygen metabolites by oyster hemocytes.

Mollusks depend chiefly on hemocyte-mediated cytotoxic mechanisms such as reactive oxygen species (ROS) to defend against pathogenic microorganisms. The effect of in vitro tributyltin chloride (TBT) exposure on ROS generation by oyster (Crassostrea virginica) blood phagocytes is quantified in this study. Luminol-augmented chemiluminescence (LCL) was used to measure ROS activity of resting and zymosan-stimulated cells after 1 or 20 hr TBT exposure. LCL is thought to measure primarily the activity of the myeloperoxidase/hydrogen peroxide/ halide antimicrobial pathway. Hemocytes in TBT-free medium (controls) produced low level LCL, which was markedly stimulated by the addition of zymosan particles. Both resting and zymosan-stimulated LCL values were significantly inhibited by > or = 80 ppb TBT after either 1 or 20 hr of exposure. Exposure to < or = 2 ppb TBT concentrations for 20 hr produced slightly enhanced LCL activity, suggesting a hormesis-like effect. Partial reversibility of TBT suppression of LCL took place when previously exposed cells were put in TBT-free medium. The TBT concentrations used in these studies were not cytolethal in vitro and were considerably less than oyster tissue levels recorded after chronic, sublethal in vitro exposures. The data suggest that the common aquatic contaminant TBT can interact rapidly with C. virginica hemocytes to produce a partially reversible immunotoxicological lesion. Xenobiotic-induced suppression of ROS production by hemocytes may increase host susceptibility to infectious diseases.