Observations of a Protistan Disease Similar to QPX in Mercenaria mercenaria (Hard Clams) from the Coast of Massachusetts

During the summer and fall of 1995, in clam aquaculture leases at two locations on the coast of Massachusetts, significant mortalities were observed to occur primarily in 112 role= presentation style= font-size: 0.9px; display: inline-block; position: relative; \u3e- to 2-year old hard clams (Mercenaria mercenaria,quahog) planted in the leases. Examination of hard clams from those leases suggested a parasite similar to QPX (Quahog Parasite Unknown), as described by Whyte, S. K., Cawthorn, R. J., and McGladdery, S. E. (1994,Can. Dis. Ag. Org.19, 129–136), was responsible for the poor condition of affected clams and the resulting high mortality. In clam tissues, the QPX-like parasite formed thalli surrounded by a mucofilamentous net. Larger thalli underwent endosporulation producing sporangia that contained approximately 40 endospores. Rupture of the sporangium released the endospores into the surrounding tissue. Inflammatory response by the clam to infection was chronic, active, and granulomatous. The occurrence both of phagocytic multinucleated giant inflammatory cells and inflammatory encapsulation of parasites were commonly identified as part of the response. Moderately to severely infected clams showed significantly reduced growth and a poorer condition index as compared to uninfected or mildly infected clams

Development and validation of a real-time quantitative PCR assay for the detection and quantification of Perkinsus marinus in the Eastern oyster, Crassostrea virginica

Author Posting. © National Shellfisheries Association, 2009. This article is posted here by permission of National Shellfisheries Association for personal use, not for redistribution. The definitive version was published in Journal of Shellfish Research 28 (2009): 459-464, doi:10.2983/035.028.0306.Perkinus marinus causes a devastating disease, known as Dermo, in the Eastern oyster Crassostrea virginica. Routine detection of the disease is traditionally accomplished by the use of the Ray/Makin assay, using Fluid Thioglycollate Medium (RFTM). A simple real-time quantitative PCR assay was developed as a diagnostic tool to detect and quantify P. marinus, to complement and serve as an alternate to the RFTM method. Using a dual-labeled probe approach, a sensitive assay was designed to accurately detect a range of one to several thousand P. marinus organisms present in oyster tissues. A simple extraction method was used to increase throughput of the assay. Cultured P. marinus cells were quantified prior to DNA extraction, generating a standard curve and allowing cell counts to be derived from PCR cycle threshold values. Direct comparison of the RFTM and real-time PCR methods was accomplished by using tissue samples from the same oyster for both tests. Plotting cycle threshold values against the known Mackin index value generated a standard curve with a coefficient of regression of 0.9. Our results indicate that correlations could be made between this molecular based approach and traditional methods, allowing results generated from the PCR assay to be easily translated into the understood Mackin scale

Combined effects of a parasite, QPX, and the harmful-alga, Prorocentrum minimum on northern quahogs, Mercenaria mercenaria

Northern quahogs, Mercenaria mercenaria (L.), frequently are infected with the parasite Quahog Parasite Unknown (QPX, Labyrintohomorpha, Thraustochytriales), which can cause morbidity and mortality of the quahogs. Possible interactions between this parasitic disease and exposure to the harmful dinoflagellate Prorocentrum minimum in M. mercenaria were studied experimentally. Quahogs from Massachusetts with variable intensity of QPX infection were exposed, under controlled laboratory conditions, to cultured P. minimum added to the natural plankton at a cell density equivalent to a natural bloom. After 5 days of exposure, individual clams were diagnosed histologically to assess prevalence and intensity of parasitic infection, as well as other pathological conditions. Further, cellular defense status of clams was evaluated by analyzing hemocyte parameters (morphological and functional) using flow-cytometry. Exposure of quahogs to P. minimum resulted in: a lower percentage of phagocytic hemocytes, higher production of reactive oxygen species (ROS), larger hemocyte size, more-numerous hemocytic aggregates, and increased numbers of hemocytes in gills accompanied by vacuolation and hyperplasia of the water-tubular epithelial cells of the gills. Quahogs had a low prevalence of QPX; by chance, the parasite was present only in quahogs exposed to P. minimum. Thus, the effect of QPX alone on the hemocyte parameters of quahogs could not be assessed in this experiment, but it was possible to assess different responses of infected versus non-infected quahogs to P. minimum. QPX-infected quahogs exposed to P. minimum had repressed percentage of phagocytic hemocytes, consistent with immuno-modulating effect of P. minimum upon several molluscan species, as well as smaller hemocytes and increased hemocyte infiltration throughout the soft tissues. This experiment demonstrates the importance of considering interactive effects of different factors on the immunology and histopathology of bivalve shellfish, and highlights the importance of considering the presence of parasites when bivalves are subjected to harmful-algal blooms

Chemical impacts in fish and shellfish from Cape Cod and Massachusetts Bays

This paper is not subject to U.S. copyright. The definitive version was published in Environment Cape Cod 1, no. 3 (1998): 68-85.Mununichogs, soft shell clams, and blue mussels from some or all of 10 sites in Boston Harbor and Massachusetts and Cape Cod Bays were examined histologically: a suite of pathological changes previously known to be associated with chemical contamination were found in animals from the more contaminated sites. In particular, liver tumors were evident in 14% of the adult mununichogs from the Island End River, a tributary of the Mystic River in Boston Harbor. Additionally, a number of pathologies previously shown to be associated with chemical exposure were seen in the two bivalve species at a number of contaminated sites. Induction of cytochrome P45() IA (CYPIA) was also seen in muntntichogs from the more contaminated sites: CYPIA induction is a biochemical change associated with exposure to dioxin and other planar halogenated and aromatic hydrocarbons. These findings suggest that there are measurable biochemical and pathological changes in intertidal fish and shellfish from the more contaminated parts of the Massachusetts Bays system. These types of changes were less evident in the two reference sites in Cape Cod Bay

Host susceptibility hypothesis for shell disease in American lobsters

Author Posting. © American Fisheries Society, 2007. This article is posted here by permission of American Fisheries Society for personal use, not for redistribution. The definitive version was published in Journal of Aquatic Animal Health 19 (2007): 215-225, doi:10.1577/H06-014.1.Epizootic shell disease (ESD) in American lobsters Homarus americanus is the bacterial degradation of the carapace resulting in extensive irregular, deep erosions. The disease is having a major impact on the health and mortality of some American lobster populations, and its effects are being transferred to the economics of the fishery. While the onset and progression of ESD in American lobsters is undoubtedly multifactorial, there is little understanding of the direct causality of this disease. The host susceptibility hypothesis developed here states that although numerous environmental and pathological factors may vary around a lobster, it is eventually the lobster's internal state that is permissive to or shields it from the final onset of the diseased state. To support the host susceptibility hypothesis, we conceptualized a model of shell disease onset and severity to allow further research on shell disease to progress from a structured model. The model states that shell disease onset will occur when the net cuticle degradation (bacterial degradation, decrease of host immune response to bacteria, natural wear, and resorption) is greater than the net deposition (growth, maintenance, and inflammatory response) of the shell. Furthermore, lesion severity depends on the extent to which cuticle degradation exceeds deposition. This model is consistent with natural observations of shell disease in American lobster

Evidence That Qpx (Quahog Parasite Unknown) Is Not Present In Hatchery-Produced Hard Clam Seed

A protistan parasite known as QPX (Quahog Parasite Unknown) has been recently associated with disease and mortality of adult hard clams, Mercenaria mercenaria, from Canada to Virginia. There is concern that the organism may be transported in hatchery-reared seed. Tissue sections of 2,203 seed clams (\u3c1-20 mm) from 13 different hatcheries in six states, collected from 1995 to 1997 and examined by pathologists in three laboratories, failed to show QPX or QPX-like organisms. Further, QPX was not detected in a total of 756 hatchery-produced clams examined during their first year of field growout. From this, we conclude that hatchery-produced seed clams are an unlikely source of QPX organisms

Lethal marine snow : pathogen of bivalve mollusc concealed in marine aggregates

Author Posting. © The Authors, 2005. This is the author's version of the work. It is posted here by permission of American Society of Limnology and Oceanography for personal use, not for redistribution. The definitive version was published in Limnology and Oceanography 50 (2005): 1983-1988, doi:10.4319/lo.2005.50.6.1983.We evaluated marine aggregates as environmental reservoirs for a thraustochytrid pathogen, Quahog Parasite Unknown (QPX), of the northern quahog or hard clam, Mercenaria mercenaria. Positive results from in situ hybridization and denaturing gradient gel electrophoresis confirm the presence of QPX in marine aggregates collected from coastal embayments in Cape Cod, Massachusetts, where QPX outbreaks have occurred. In laboratory experiments, aggregates were observed and recorded by entering a quahog’s pallial cavity, thereby delivering embedded particles from the water column to its benthic bivalve host. The occurrence of pathogen-laden aggregates in coastal areas experiencing repeated disease outbreaks suggests a means for the spread and survival of pathogens between epidemics and provides a specific target for environmental monitoring of those pathogens.This work was funded by an NSF grant as part of the joint NSF-NIH Ecology of Infectious Disease program, by the Woods Hole Oceanographic Institution (WHOI) Sea Grant Program, under a grant from the National Oceanic and Atmospheric Administration, U.S. Department of Commerce, and a National Science Foundation Graduate Fellowship to M. Lyons

Development of a multiplex qPCR for the quantification of three protozoan parasites of the eastern oyster Crassostrea virginica

A multiplex quantitative PCR (qPCR) assay for the simultaneous detection of 3 eastern oyster Crassostrea virginica parasites, Perkinsus marinus, Haplosporidium nelsoni, and H. costale, was developed using 3 different fluorescently labeled hydrolysis probes. The primers and probe from a previously validated singleplex qPCR for P. marinus detection were combined with newly designed primers and probes specific for H. nelsoni and H. costale. The functionality of the multiplex assay was demonstrated on 2 different platforms by the linear relationship of the standard curves and similar cycle threshold (CT) values between parasites. Efficiency of the multiplex qPCR assay on the Roche and BioRad platforms ranged between 93 and 101%. The sensitivity of detection ranged between 10 and 100 copies of plasmid DNA for P. marinus and Haplosporidium spp., respectively. The concordance between the Roche and BioRad platforms in the identification of the parasites P. marinus, H. nelsoni, and H. costale was 91, 97, and 97%, respectively, with a 10-fold increase in the sensitivity of detection of Haplosporidium spp. on the BioRad thermocycler. The concordance between multiplex qPCR and histology for P. marinus, H. nelsoni, and H. costale was 54, 57, and 87%, respectively. Discordances between detection methods were largely related to localized or low levels of infections in oyster tissues, and qPCR was the more sensitive diagnostic. The multiplex qPCR developed here is a sensitive diagnostic tool for the quantification and surveillance of single and mixed infections in the eastern oyster

Sea Star Wasting Disease in \u3cem\u3eAsterias forbesi\u3c/em\u3e along the Atlantic Coast of North America

As keystone species, sea stars serve to maintain biodiversity and species distribution through trophic level interactions in marine ecosystems. Recently, Sea Star Wasting Disease (SSWD) has caused widespread mass mortality in several sea star species from the Pacific Coast of the United States of America (USA) and Asterias forbesi on the Atlantic Coast. A densovirus, named Sea Star associated Densovirus (SSaDV), has been associated with the wasting disease in Pacific Coast sea stars, and limited samples of A. forbesi. The goal of this research is to examine the pathogenesis of SSWD in A. forbesi on the Atlantic Coast of the USA and to determine if SSaDV is associated with the wasting disease in this species. Histological examination of A. forbesi tissues affected with SSWD showed cuticle loss, vacuolation and necrosis of epidermal cells, and oedema of the dermis, but no consistent evidence indicating the cause of the lesions. Challenge experiments by cohabitation and immersion in infected water suggest that the cause of SSWD is viral in nature, as filtration (0.22 μm) of water from tanks with sea stars exhibiting SSWD did not prevent the transmission and progression of the disease. Death of challenged sea stars occurred 7–10 d after exposure to infected water or sea stars, and the infectivity crossed species (A. forbesi and Pateria miniata) with equal penetrance. Of the 48 stars tested by quantitative real time PCR, 29 (60%) were positive for the SSaDV VP1 gene. These stars represent field-collected sea stars from all geographical regions (South Carolina to Maine) in 2012–2015, as well as stars exposed to infected stars or water from affected tanks. However, a clear association between the presence of SSaDV and SSWD signs in experimental and field-collected A. forbesi was not found in this study

Probiotic strains for disease management in hatchery larviculture of the eastern oyster Crassostrea virginica

Bacterial pathogens are a major cause of mortality in bivalve hatcheries, and outbreaks can result in shortages of seed supply to the grow-out industry. The use of probiotic bacteria is a potential preventative measure to limit the impact of bacterial diseases. Previous research showed that the marine bacteria Phaeobacter inhibens S4 (S4) and Bacillus pumilus RI06—95 (RI) protect larval eastern oysters (Crassostrea virginica) when challenged with the pathogens Vibrio tubiashii RE22 (now Vibrio coralliilyticus RE22) and Roseovarius crassostreae CV919-312T. In this study, these probiotic bacteria were tested under hatchery conditions. Daily addition of S4 and RI (104 colony forming units (CFU)/ml) to 100-l culture tanks resulted in a significant decrease in the levels of total Vibrios in water and tank surfaces (P < 0.05), but not in oysters. Larval growth and survival was unaffected by the probiotic treatments. Larvae treated with probiotics in the hatchery showed significantly less mortality than larvae from control tanks when exposed to 105 CFU/ml of V. coralliilyticus RE22 for 24 h in a laboratory challenge. These results suggest that S4 and RI are safe and potentially effective tools to limit disease outbreaks in oyster hatcheries