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

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

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

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

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

Finfish and aquatic invertebrate pathology resources for now and the future

Utilization of finfish and aquatic invertebrates in biomedical research and as environmental sentinels has grown dramatically in recent decades. Likewise the aquaculture of finfish and invertebrates has expanded rapidly worldwide as populations of some aquatic food species and threatened or endangered aquatic species have plummeted due to overharvesting or habitat degradation. This increasing intensive culture and use of aquatic species has heightened the importance of maintaining a sophisticated understanding of pathology of various organ systems of these diverse species. Yet, except for selected species long cultivated in aquaculture, pathology databases and the workforce of highly trained pathologists lag behind those available for most laboratory animals and domestic mammalian and avian species. Several factors must change to maximize the use, understanding, and protection of important aquatic species: 1) improvements in databases of abnormalities across species; 2) standardization of diagnostic criteria for proliferative and nonproliferative lesions; and 3) more uniform and rigorous training in aquatic morphologic pathology

Finfish and aquatic invertebrate pathology resources for now and the future

Utilization of finfish and aquatic invertebrates in biomedical research and as environmental sentinels has grown dramatically in recent decades. Likewise the aquaculture of finfish and invertebrates has expanded rapidly worldwide as populations of some aquatic food species and threatened or endangered aquatic species have plummeted due to overharvesting or habitat degradation. This increasing intensive culture and use of aquatic species has heightened the importance of maintaining a sophisticated understanding of pathology of various organ systems of these diverse species. Yet, except for selected species long cultivated in aquaculture, pathology databases and the workforce of highly trained pathologists lag behind those available for most laboratory animals and domestic mammalian and avian species. Several factors must change to maximize the use, understanding, and protection of important aquatic species: 1) improvements in databases of abnormalities across species; 2) standardization of diagnostic criteria for proliferative and nonproliferative lesions; and 3) more uniform and rigorous training in aquatic morphologic pathology