Advanced Perkinsus marinus infections in Crassostrea ariakensis maintained under laboratory conditions.ABSTRACT The Suminoe oyster, Crassostrea ariakensis, has been under investigation since the early 1990s for potential use in restoring the commercial harvest or for aquaculture aquaculture, the raising and harvesting of fresh- and saltwater plants and animals. The most economically important form of aquaculture is fish farming, an industry that accounts for an ever increasing share of world fisheries production. of oysters in the Chesapeake Bay, USA. Initial studies focusing on C. ariakensis documented a significant level of tolerance to the protozoan protozoan (prō'təzō`ən), informal term for the unicellular heterotrophs of the kingdom Protista. Protozoans comprise a large, diverse assortment of microscopic or near-microscopic organisms that live as single cells or in simple parasite Perkinsus marinus, a pathogen found in almost all reaches of the Bay and widely acknowledged as one of the main reasons for the decline in the eastern oyster, Crassostrea virginica, harvest since the late 1980s. Crassostrea ariakensis was demonstrated to acquire P. marinus, however infection intensities, as measured using Ray's thioglycollate medium assay indices, generally were found to be light. As part of a series of experiments to study potential impacts on the Chesapeake Bay region of pathogens found in C. ariakensis in Asia, a challenge experiment was conducted to study the pathogenicity of Perkinsus olseni to C. ariakensis. During this study, we observed the acquisition of moderate and heavy infection intensities of P. marinus in triploid triploid /trip·loid/ (trip´loid) having triple the haploid number of chromosomes (3n).
Having three times the haploid number of chromosomes in the cell nucleus.
n. C. ariakensis oysters being maintained in the laboratory. Results suggest that there may be some risk of mortality from P. marinus if C. ariakensis is held under stressful conditions at least in hatchery hatchery
a commercial establishment dedicated to the hatching of bird eggs to provide day old chicks and poults to the poultry industry.
the contents of unfertilized eggs. Used in petfood manufacture. or laboratory settings.
KEY WORDS: Perkinsus marinus, Crassostrea ariakensis, PCR PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (PCR) , RFTM, oyster
The eastern oyster, Crassostrea virginica (Gmelin, 1791), an integral part of the economy and ecology of the Chesapeake Bay, has been in a severe state of decline in recent decades. Two pathogens, Haplosporidium nelsoni (Haskin, Stauber & Mackin, 1966) and Perkinsus marinus (Mackin, Owen & Collier, 1950), the parasites responsible for the diseases known as MSX MSX - Microsoft Extended and dermo respectively, have contributed significantly to the decimation DECIMATION. The punishment of every tenth soldier by lot, was, among the Romans, called decimation. of the oyster populations in Chesapeake Bay since the 1950s (Sindermann 1990).
The decline in oyster production in Virginia led to the formation of a number of panels in the early 1990s to make recommendations on restoring oyster populations. One option currently being seriously considered is the introduction and use of a nonnative oyster in Chesapeake Bay. A Virginia Institute of Marine Science (VIMS VIMS Virginia Institute of Marine Science
VIMS Visible and Infrared Mapping Spectrometer
VIMS Visual Information Management System(s)
VIMS Vehicle Information Management System
VIMS Virtual Incident Management System ) study conducted in 1996 focusing on the Pacific oyster, Crassostrea gigas (Thunberg, 1793), documented lower disease susceptibility in C. gigas than in the native eastern oyster, C. virginica; however, growth rates of the Pacifc oyster were equal to or inferior to the native oyster in the Chesapeake Bay (Calvo et al. 1999). A 1998 field-based study on another Asian oyster, the Suminoe oyster, Crassostrea ariakensis (Fujita, 1913), documented rapid growth and survival in that species, as compared with C. virginica, even when endemic diseases were prevalent (Calvo et al. 2001). Although initial baseline samples of C. ariakensis in that study revealed a 12% prevalence of P. marinus, all subsequent samples collected after field deployment showed C. virginica with higher P. marinus prevalence than C. ariakensis. Additionally, Ray fluid thioglycollate medium (RFTM) (Ray 1952) diagnoses showed several heavy P. marinus infections in C. virginica, however, only light infections were observed in C. ariakensis (Calvo et al. 2001).
In this report we describe the first account of moderate to heavy P. marinus infections in C. ariakensis. The oysters in this study were held in laboratory aquaria a·quar·i·a
A plural of aquarium. at VIMS during a challenge experiment conducted to examine the potential pathogenicity of Perkinsus olseni Lester and Davis, 1981, to C. ariakensis, because P. olseni was found during a recent survey of C. ariakensis populations in Asia (Moss & Reece 2005). Prior to the start of the challenge experiment, C. ariakensis were initially determined to be Perkinsus sp.-free, based on molecular diagnostics using PCR (Casas et al. 2002). The oysters acquired detectable levels of the parasite, however, after being held for about 8 weeks in the laboratory aquaria. We used the infected oysters in our challenge study, despite baseline P. marinus infection prevalence, to examine the potential for C. ariakensis oysters to become coinfected with multiple Perkinsus species. We report here our observations regarding the P. marinus infection levels observed in C. ariakensis during this challenge experiment.
MATERIALS AND METHODS
On January 24, 2005, 120 market sized (~75 mm shell height) triploid C. ariakensis were received from the Aquaculture Genetics and Breeding Technology Center hatchery at VIMS. The animals were held for 4 days inside a plastic mesh bag in a holding tank prior to bringing them into the laboratory aquaria. The holding tank was not covered and there was flow-through of nonfiltered York River water of approximately 9[degrees]C and 16 ppt ppt
1. parts per thousand
2. parts per trillion salinity. On removal from the holding tank, 20 C. ariakensis were immediately sacrificed, and gill and mantle tissues were excised aseptically from each animal for DNA extraction. Genomic DNA of each oyster was used in a PCR-based molecular diagnostic assay (Casas et al. 2002) to examine the animals for the presence of DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. from Perkinsus spp. parasites. The animals were subsequently held for 59 days in 10-gallon glass aquaria that were maintained at 20[degrees]C and 25 ppt salinity at a density of approximately 25 oysters per tank and were fed 0.1 g oyster-1 algal algal
pertaining to or caused by algae.
is very rare but systemic and udder infections are recorded. See protothecosis.
the algae Prototheca trispora and P. food daily (Reed Mariculture mariculture
marine aquaculture. , San Jose, CA). At the end of 59 days, on March 24, 2005, five C. ariakensis were sacrificed and gill and mantle were excised aseptically from each animal to be used in Perkinsus spp. screening by PCR assays as described later.
On March 28, 2005, the remaining C. ariakensis (n = 60) were used in a challenge experiment designed to evaluate the pathogenicity of P. olseni to C. ariakensis. Twenty C. ariakensis received single pallial pal·li·al
Of or relating to the cerebral cortex. cavity injections of 100 [micro]L of 25 ppt sterile artificial seawater (SASW SASW Spectral Analysis of Surface Waves
SASW Singapore Association of Social Workers
SASW Smart And Secure Way (security applications)
SASW Standard Application Software ) through a notch in the shell, and 40 animals were inoculated with [10.sup.5] log phase cultured P. olseni cells per gram body weight in a 100-[micro]L volume. The clonal P. olseni culture isolated from a Japanese Venerupis philippinarum (Adams and Reeve, 1850) clam was obtained from Mr. Chris Dungan, Maryland DNR See dynamic noise reduction and domain name resolver. Cooperative Oxford Laboratory, Oxford Maryland, (ATCC ATCC American Type Culture Collection, see there PRA-181). Thirty additional untreated, control C. ariakensis were neither notched nor inoculated, but held in separate aquaria and otherwise maintained and fed in the same manner as the experimental animals for the duration of the experiment. Those C. ariakensis inoculated with 25 ppt SASW were maintained in one 10-gallon aquarium, and those inoculated with P. olseni were split evenly between two aquaria. All aquaria were identical and each tank was covered with a plexiglass lid.
All aquaria environments were maintained at 20[degrees]C to 22[degrees]C and contained 25 ppt aerated aer·ate
tr.v. aer·at·ed, aer·at·ing, aer·ates
1. To supply with air or expose to the circulation of air: aerate soil.
2. , 1-[micro]m filtered York River water, two thirds of which was removed each week and replaced with an equal volume of clean, 25 ppt, 1-[micro]m filtered York River water. Animals were fed daily a single dose of approximately 0.1 g oyster algal feed (Reed Mariculture).
Aquaria were checked daily for oyster mortalities, and moribund animals were removed. The cumulative mortality was calculated for each sample as the sum of the mortalities that occurred during the course of the experiment, which started 59 days after the C. ariakensis were brought into the aquaria, divided by the number of live animals at the start of the experiment. When moribund animals were discovered, if adequate undegraded tissues remained they were processed for analysis. Gill and mantle tissues were removed with a portion preserved in 95% ethanol for DNA extraction and molecular diagnostics and gill, mantle and rectal tissues were processed for Ray fluid thioglycollate medium (RFTM) assay (Ray 1952). After 27[degrees]C incubation for 5-6 days in RFTM, tissues were removed from the culture tubes, macerated on microscope slides and stained with Lugol iodine. Stained, cover-slipped tissue preparations were examined under a light microscope and Perkinsus sp. tissue burdens were enumerated This term is often used in law as equivalent to mentioned specifically, designated, or expressly named or granted; as in speaking of enumerated governmental powers, items of property, or articles in a tariff schedule. on a scale from rare (R) to very heavy (VH) based on the categories of Ray (1952, 1954). Visceral mass tissue sections were preserved in Davidson's solution for histological analysis (Shaw & Battle 1957).
Because of space constraints in the laboratory, the untreated C. ariakensis that were remaining after 37 days were sacrificed and tissues were taken for DNA, RFTM and histological analysis of disease status as described earlier. For those tanks in which C. ariakensis were inoculated with either 25 ppt SASW (one tank) or with P. olseni (two tanks), two randomly chosen oysters from each tank were removed and sacrificed for disease diagnosis on days 21, 44 and 59 postinoculation. The challenge experiment was terminated on day 72 and all remaining animals were sacrificed and tissues preserved as mentioned earlier for DNA, RFTM and histological analysis.
Nucleic Acid Extraction
Genomic DNA was extracted from the excised mantle and gill tissue snips, using a DNeasy Tissue Kit (Qiagen Inc., Valencia, CA), following manufacturer's protocols, except that DNA was eluted in a single 200-[micro]L volume of elution elution /elu·tion/ (e-loo´shun) in chemistry, separation of material by washing; the process of pulverizing substances and mixing them with water in order to separate the heavier constituents, which settle out in solution, from the buffer after a 10 min incubation at room temperature (approximately 20[degrees]C).
SSU SSU Small Subunit
SSU Sonoma State University
SSU Savannah State University (Savannah, Georgia)
SSU Shawnee State University (Ohio)
SSU Salisbury State University Genes
To assure that PCR amplifiable DNA was present in all extracted samples, genomic DNAs were tested using universal small subunit (SSU) ribosomal RNA gene primers 16S-A S-A
sinoatrial. (5' CCG CCG Chicago
CCG Collectible Card Game
CCG Canadian Coast Guard
CCG Country Commercial Guide
CCG Children's Cancer Group
CCG Commission Canadienne des Grains (Canadian Grain Commission) AAT Alpha-1-antitrypsin (AAT)
A blood component that breaks down infection-fighting enzymes such as elastase.
Mentioned in: Chronic Obstructive Lung Disease TCG (Trusted Computing Group, Beaverton, OR, www.trustedcomputinggroup.org) The successor to the Trusted Computer Platform Alliance (TCPA), announced in 2003 by founding members AMD, HP, IBM, Intel and Microsoft. TCG ACA ACA - Application Control Architecture ACC See adaptive cruise control. TGG TGG The Great Gatsby (novel F. Scott Fitzgerald; movie)
TGG Kuala Terengganu, Malaysia - Sultan Mahmood (Airport Code)
TGG Temporary Geographic Grid
TGG Third Generation Gyro
TGG Triple Graph Grammar TTG tTG Tissue Transglutaminase
TTG Telltale Games (website)
TTG TiVo To Go
TTG Tea Tree Gully (South Australia)
TTG Tom Tom Go ATC ATC Air Traffic Control
ATC Average Total Cost
ATC Certified Athletic Trainer
ATC At the Center (Hartford, Maine retreat center)
ATC Applied Technology Council
ATC All Things Considered CTG CTG Cartridge
CTG Center for Technology in Government (SUNY, Albany, New York)
CTG Center for Technology in Government
CTG Computer Task Group (IT consulting company; Buffalo, NY, USA) CCA (1) (Common Cryptographic Architecture) Cryptography software from IBM for MVS and DOS applications.
(2) (Compatible Communications A GT 3') and 16S-B S-B Stoer-Bulirsch (sampling algorithm) (5' GGA GGA Generalized Gradient Approximation
GGA Good Game All
ggA Geschützte Geographische Angabe (German: Protected Geographical Indication)
GGA Global Gecko Association
GGA Georgia Geocachers Association TCC TCC The Car Connection (web site)
TCC Tidewater Community College
TCC Tallahassee Community College
TCC Temporary Continuation of Coverage
TCC Tucson Convention Center (Tucson, AZ, USA) AAG AAG Association of American Geographers (Washington, DC)
AAG Assistant Attorney General
AAG Asociación Argentina de Golf
AAG Anti-Aircraft Gun
AAG Assistant Adjutant General
AAG Australian Association of Gerontology CTT CTT Correios (Portuguese Postal Service)
CTT Certified Technical Trainer
CTT Charity Technology Trust
CTT Cholesterol Treatment Trialists' (collaboration)
CTT Common Task Training GAT CCT CCT Circuit
CCT Commission Canadienne du Tourisme (Canadian Tourism Commission)
CCT Correlated Color Temperature
CCT Common Customs Tariff (EU)
CCT Certificate of Completion of Training TCT TCT The Capital Times (Madison, WI newspaper)
TCT Transcatheter Cardiovascular Therapeutics
TCT The Coroner's Toolkit
TCT Trans Canada Trail
TCT Tcl Core Team
TCT Tsukuba College of Technology (Japan) GCA GCA, ground-controlled approach: see instrument-landing system. GGT GGT
GGT Gammaglutamyltransferase, see there TCA TCA
1. trichloroacetic acid.
2. tricarboxylic acid cycle (Krebs cycle).
TCA Tricyclic antidepressant, see there CCT AC 3') (modified from Medlin et al. 1988) with an expected amplification product of approximately 1,800 bp. Each 25-[micro]L reaction contained 20 mM Tris-HCl (pH8.4), 50 mM KCl, 0.75 mM Mg[Cl.sub.2], 0.1 mM of each dNTP, 0.5 [micro]M each primer, 0.0125 U [micro][L.sup.-t] Taq polymerase, 0.2 mg [mL.sup.-1] BSA 1. BSA - Business Software Alliance.
2. BSA - Bidouilleurs Sans Argent. and 0.5 [micro]L genomic DNA (~10-50 ng total). Amplifications were performed with an initial denaturation denaturation, term used to describe the loss of native, higher-order structure of protein molecules in solution. Most globular proteins exhibit complicated three-dimensional folding described as secondary, tertiary, and quarternary structures. of 94[degrees]C for 4 min, followed by 35 cycles at 94[degrees]C for 30 sec, 45[degrees]C for 30 sec, 65[degrees]C for 2 min, with a final elongation of 65[degrees]C for 2 min. After amplification, 3 [micro]L of PCR product were analyzed by agarose gel electrophoresis Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA, RNA, or protein molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). (2%), stained with ethidium bromide and visualized under UV light. Images were recorded with an Alpha Innotech FlourChem (SanLeandro, CA) imaging system.
Genus-specific Perkinsus sp. PCR Assay
Screening for Perkinsus sp. DNA was performed using Perkinsus genus-specific primers, PerkITS-85 (5' CCG CTT TGT TGT Target
TGT Ticket Granting Ticket (Windows 2000 Kerberos security)
TGT Target Corp (stock symbol)
TGT Turbine Gas Temperature
TGT TDRSS Ground Terminal
TGT Tank Gunnery Trainer
TGT Target Tracker TTG GAT CCC CCC
A very speculative grade assigned to a debt obligation by a rating agency. Such a rating indicates default or considerable doubt that interest will be paid or principal repaid. Also called Caa. 3') and PerkITS-750 (5' ACA TCA GGC GGC Girl Guides of Canada
GGC Greenwood Genetic Center (South Carolina)
GGC Gwasanaeth Gwaed Cymru (Welsh Blood Service)
GGC Generalized Goppa Code
GGC Grosvenor Gallery Company CTC CTC - Cornell Theory Center TAA TAA - Track Average Amplitude TGA See TARGA.
TGA - Targa Graphics Adaptor TG 3') (Casas et al. 2002) that target the internal transcribed spacer ITS (for internal transcribed spacer) refers to a piece of non-functional RNA situated between structural ribosomal RNAs (rRNA) on a common precursor transcript. Read from 5' to 3', this polycistronic rRNA precursor transcript contains the 5' external transcribed sequence (5' ETS), (ITS) region of the ribosomal RNA gene complex. Each PCR reaction contained the following: PCR buffer at a concentration of 20 mM Tris-HCl (pH8.4), 50 mM KCl, 1.5 mM Mg[C.sub.l2], 0.2 mM of each dNTP each primer at 0.1 [micro]M, 0.025 U [micro][L.sup.-1] Taq polymerase, 0.05 mg [mL.sup.-1] BSA and 0.5 [micro]L genomic DNA (~10-50 ng total). Amplifications were performed with an initial denaturation of 95[degrees]C for 4 min followed by 40 cycles of 95[degrees]C for l min, 53[degrees]C for 1 min, 65[degrees]C for 3 min, with a final elongation of 65[degrees]C for 5 min. After amplification, 4 [micro]L of PCR product were analyzed as described earlier.
Perkinsus Species-specific Assays
Identification of the Perkinsus species that was infecting animals shown to have positive amplification products with the Perkinsus genus-specific assay was accomplished through the use of P. marinus and P. olseni species-specific PCR assays. Perkinsus marinus-specific primers PmarITS-70F (5' CCT TTG YTW YTW Yield To Worst GAG WGT WGT Weight
WGT World Games Tour
WGT World Gamemaster Tournament TGC TGC The Golf Channel
TGC The Game Creators (forum)
TGC Trading Card Game
TGC Time-Gain Compensation
TGC The Gungan Council
TGC The Golden Compass (Phillip Pullman book)
TGC Take Good Care CAG CAG 1 Chronic atrophic gastritis 2 Coronary angiography, see there ATG ATG antithymocyte globulin.
lymphocyte immune globulin (antithymocyte globulin equine, ATG, ATG equine, LIG)
Pharmacologic class: Immunoglobulin
Therapeutic class: Immunosuppressant 3') and PmarITS-600R (5' CGA (Color/Graphics Adapter) The first video display standard for the IBM PC. This low-resolution system was superseded by EGA and then VGA. CGA required a digital RGB Color Display monitor. See PC display modes.
CGA - Color Graphics Adapter GTT GTT,
n See test, glucose tolerance.
GTT Glucose tolerance test, see there TGC GAG TAC 1. TAC - Translator Assembler-Compiler. For Philco 2000.
2. TAC - Terminal Access Controller. CTC KAG KAG Kapitalanlagegesellschaft (German: Capital Investment Company)
KAG Klingon Assault Group (Star Trek fan club)
KAG Kiln-formed Architectural Glass
KAG Kick Ass Girls AG 3') (Audemard et al. 2004) and P. olseni-specific primers designed for this study Pols-140F (5' GAC GAC Great American Country
GAC Global Assembly Cache (Microsoft .NET)
GAC Global Assembly Cache
GAC Granular Activated Carbon
GAC Gustavus Adolphus College (St. CGC CGC Canine Good Citizen (AKC Dog Title)
CGC Commission Géologique du Canada (Geological Survey of Canada)
CGC Confédération Générale des Cadres (French labor union) CTT AAC (Advanced Audio Coding) An audio compression technology that is part of the MPEG-2 and MPEG-4 standards. AAC, especially MPEG-4 AAC, provides greater compression and better sound quality than MP3, which also came out of the MPEG standard. GGG GGG German Goo Girls (pornography website)
GGG Giggle (email, USENET, chat slang)
GGG Gadolinium Gallium Garnet
GGG Gimme Gimme Gimme (TV show) CCG TGT T 3') and PolsITS-600R (5' GGR GGR Geography
GGR Golden Gate Region
GGR Graham Goode Racing
GGR Gesellschaft der Germanisten Rumäniens
GGR God Given Right
GGR Global Greek Radio (Greek online radio station)
GGR German Galaxy Rangers CTT GCG GCG Genetics Computer Group
GCG Good Corporate Governance
GCG Global Consumer Group
GCG Global Church of God
GCG Generalized Conjugate Gradient
GCG Global Change Game
GCG Geological Curators' Group
GCG Giant-Cell Granuloma AGC AGC Automatic Gain Control
AGC Automotive Glass Cartridge (fuse)
AGC Associated General Contractors
AGC Associated General Contractors of America
AGC Atypical Glandular Cells
AGC Attorney-General's Chambers ATC CAA Caa
See CCC. AG 3') were used in separate 25-[micro]L reactions. PCR reactions for the P. marinus ITS region contained the following: PCR buffer at a concentration of 20 mM Tris-HCl (pH8.4), 50 mM KCl, 1.5 mM Mg[Cl.sub.2], 0.2 mM each dNTP, each primer at 0.1 [micro]M, 0.025 U [micro][L.sup.-1] Taq polymerase, 0.05 mg [mL.sup.-1] BSA and 0.5 [micro]L genomic DNA (~10-50 ng). Amplifications were performed with an initial denaturation of 95[degrees]C for 4 min followed by 40 cycles of 94[degrees]C for 1 min, 57[degrees]C for 1 min, 65[degrees]C for 3 min, with a final elongation of 65[degrees]C for 10 min. PCR reactions for the P. olseni ITS region contained the following: PCR buffer at a concentration of 20 mM Tris-HC1 (pH8.4), 50 mM KCl, 1.5 mM Mg[Cl.sub.2], 0.2 mM each dNTP, each primer at 0.1 [micro]M, 0.025 U [micro][L.sup.-1] Taq polymerase, 0.05 mg [mL.sup.-1] BSA and 0.5 [micro]L genomic DNA (~10-50 ng). Thermocycling parameters were as follows: an initial denaturation of 95[degrees]C for 4 min followed by 40 cycles of: 94[degrees]C for 1 min, 62[degrees]C for 1 min, 65[degrees]C for 3 min, all followed by a final elongation step of 65[degrees]C for 10 min. After amplification, for each species-specific reaction, 4 [micro]L of PCR product were analyzed as described earlier.
Specificity of P. olseni primers was tested against P. marinus and Perkinsus chesapeaki McLaughlin, Tall, Shaheen, Elsayed and Faisal 2000, DNAs. In addition, amplification products from positive P. olseni-specific reactions were sequenced. PCR products were cloned into the plasmid pCR4-TOPO and transformed into E. coli using a TOPO TOPO Tri-N-Octylphosphine Oxide
TOPO Trioctyl-Phosphine Oxide
ToPo Torposten (German Military Gate Post)
TOPO Tunable Optical Parametric Oscillator TA Cloning Kit (Invitrogen, Carlsbad, CA) following the manufacturer's protocols. Transformed bacterial colonies were picked from agar plates using a sterile wooden toothpick toothpick,
n a wood sliver used to cleanse the interdental space.
toothpick, balsa wood,
n a triangular wedge of balsa wood used to clean the teeth interproximally and stimulate the interdental gingival tissues. and were inoculated into 10 [micro]L of sterile water in 200 [micro]L plastic strip tubes. Inoculated water samples were boiled for 4 min at 94[degrees]C and 0.5 [micro]L of the boiled preparation was used in an M13 PCR reaction using the forward and reverse primers supplied in the cloning kit. Each 25 [micro]L reaction contained the following: 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM Mg[Cl.sub.2], 0.2 mM each dNTP each primer at 1 [micro]M, 0.25 U [micro][L.sup.-1] Taq polymerase, and 0.2 mg [mL.sup.-1] BSA. Thermocycling parameters were as follows: an initial denaturation at 94[degrees]C for 2 min, followed by 30 cycles of 94[degrees]C for 30 sec, 54[degrees]C for 30 sec and 72[degrees]C for 1 min, followed by a final elongation at 72[degrees]C for 5 min. Following amplification using the M13 primer pairs, 3 [micro]L of each PCR product were analyzed as mentioned earlier. Prior to sequencing, PCR products from clones containing the correct insert size were treated with shrimp alkaline phosphatase (SAP) and exonuclease exonuclease /exo·nu·cle·ase/ (ek?so-noo´kle-as) any nuclease specifically catalyzing the hydrolysis of terminal bonds of deoxyribonucleotide or ribonucleotide chains, releasing mononucleotides. I (Exo I) (Amersham Biosciences, Piscataway, NJ), to degrade nucleotides and singlestranded DNA (primers) remaining after PCR. Five micro liters of the M13 PCR product were combined with 0.5 units of SAP and 5.0 units of Exo I, and incubated at 37[degrees]C for 30 min, 80[degrees]C for 15 min, and 15[degrees]C for at least 5 s. Clean PCR products from plasmid inserts were sequenced bidirectionally using the Big Dye Terminator kit (Applied Biosystems, Norwalk, CT) with MI3 sequencing primers, and using 5-[micro]L reactions with 0.125 times the concentration of Big Dye reagent specified in the manufacturer's protocols. Each 5 [micro]L reaction contained 0.0625 [micro]L of Big Dye, 0.96875 [micro]L of 5x buffer, 1.6 pmol of each primer, and 10 ng of clean PCR product. Thermocycling parameters were as follow: 25 cycles of 96[degrees]C for 1 min, 96[degrees]C for 10 sec, 50[degrees]C for 5 sec, 60[degrees]C for 4 min, followed by a final incubation at 4[degrees]C until the sequencing reaction products were precipitated using the ethanol/sodium acetate precipitation method (ABI Abi (ā`bī) [short for Abijah], in the Bible, King Hezekiah's mother.
(Application Binary Interface) A specification for a specific hardware platform combined with the operating system. User Bulletin, April 11, 2002). Precipitated sequencing reaction products were resuspended in 20 [micro]L of Hi-Di formamide (Applied Biosystems) and 10 [micro]L of each were electrophoretically separated on an ABI 3100 Prism Genetic Analyzer.
restriction fragment length polymorphism
restriction fragment length polymorphism.
For further confirmation of the presence of P. marinus or P. olseni DNA, and to further verify the specificity of the Perkinsus species-specific primer sets, genus-specific PerkITS-85/750 amplification products were digested with Mbo I restriction endonuclease (New England Biolabs New England Biolabs (NEB) produces and supplies reagents for the life science industry. NEB offers a large selection of recombinant and native enzymes for genomic research. It also offers products in the areas related to proteomics and drug discovery. , Beverly, MA). Based on analyzing published and unpublished sequence data of all known Perkinsus species, this enzyme will cut the approximately 760 bp ITS fragment of P. marinus DNA that is generated in the genus-specific assay to yield a 3-fragment digestion profile unique to this species. The fragments should be approximately 17,226 and 264 bp in length, For P. olseni, Mbo I should cut the ITS region fragment to yield a 4-fragment digestion profile with fragment lengths of approximately 64, 85, 251 and 262 bp in length. Each 10-[micro]L-digestion reaction contained 5 [micro]L of PCR product, 8.2 [micro]L of sterile distilled water, 1.5 [micro]L of 10X NEB buffer 3, and 0.3 [micro]L of restriction enzyme. As controls, separate oyster genomic DNA samples, determined previously to be infected with P. olseni and P. marinus, and plasmid-purified P. chesapeaki ITS DNA was first amplified with the Perkinsus genus-specific primers, Perk-ITS85/ 750, as described above and then digested with Mbo I restriction endonuclease. We chose to use only P. marinus, P. olseni and P. chesapeaki DNAs as controls, because these were the Perkinsus species, that we expected might be present in our samples. Perkinsus olseni was being used in the experiment and P. marinus and P. chesapeaki are common to Chesapeake Bay and potentially their DNAs could have contaminated the 1-p~m filtered York River water, although this was not likely. PCR products were digested at 37[degrees]C for 6 h and 10 [micro]L of the digested PCR product was electrophoresed on a 3% agarose gel that was 1.5% agarose and 1.5% low melt agarose (Fisher Scientific, Raleigh, NC), stained with ethidium bromide and visualized under UV light. Images were recorded with an Alpha Innotech FlourChem (SanLeandro, CA) imaging system.
Tissues preserved for histological analysis in Davidson's solution (Shaw & Battle 1957) were dehydrated de·hy·drate
v. de·hy·drat·ed, de·hy·drat·ing, de·hy·drates
1. To remove water from; make anhydrous.
2. To preserve by removing water from (vegetables, for example). in a series of ethanol baths, infiltrated with paraffin and embedded in paraffin blocks prior to sectioning. Sections of 5-[micro]m thickness were stained with Harris-hematoxylin and eosin eosin /eo·sin/ (e´o-sin) any of a class of rose-colored stains or dyes, all being bromine derivatives of fluorescein; eosin Y, the sodium salt of tetrabromofluorescein, is much used in histologic and laboratory procedures. . Histological sections of infected oysters were examined using light microscopy to visualize Perkinsus sp. parasite cells in situ.
In-situ Hybridization hybridization /hy·brid·iza·tion/ (hi?brid-i-za´shun)
1. crossbreeding; the act or process of producing hybrids.
2. molecular hybridization
Representative tissue sections from a few of the C. ariakensis oysters determined to have P. marinus or mixed infections of both P. marinus and P. olseni by species-specific PCR were evaluated by in situ hybridization in situ hybridization A method for localizing a sequence of DNA, mRNA, or protein in a cell or tissue; the use of a DNA or RNA probe to detect a cDNA sequence in chromosome spreads or in interphase nuclei or an RNA sequence of cloned bacterial or cultured (ISH ISH In Situ Hybridization
ISH Isolated Systolic Hypertension
ISH Irish Sport Horse
ISH Intermediate System Hello
ISH International Society of Hypnosis
ISH Information Super Highway
ISH International Superhits (Green Day album) ). A genus-specific 5' end digoxigenin-labeled Perkinsus probe (Elston et al. 2004) was used to specifically target SSU rRNA sequences. Perkinsus species-specific probes, PmarLSU-181DIG (5'-GACAACGGCGAACGACTC-3'), specific to P. marinus, and PolsLSU-464DIG (5'-CTCACAAGTGCCAAACAACTG-3'), specific to P. olseni, were designed by locating unique regions in aligned available Perkinsus species LSU LSU Louisiana State University
LSU Large Subunit
LSU La Salle University (Philadelphia, PA)
LSU La Sierra University
LSU Link State Update (OSPF)
LSU Learning Support Unit rRNA gene sequences. Digoxigenin-labeled oligos were obtained from Operon Biotechnologies, Inc. (Huntsville, AL). The protocol followed for ISH was that previously published (Stokes & Burreson, 1995) with the modifications published by Elston et al. (2004). Pronase at a final concentration of 125 [micro]g [mL.sup.-1] was used for permeabilization during a 30-min incubation, and a probe concentration of 7 ng [micro][L.sup.-1] was used for hybridization. The species-specific probes were tested for specificity with numerous Perkinsus sp.-infected reference tissues, including P. marinus in C. virginica, P. chesapeaki in Mya arenaria Linnaeus, 1758 and P. olseni in Haliotis laevigata Donovan, 1808. Controls for each Perkinsus species-specific probe were tested identically except that they received hybridization buffer lacking probe during the hybridization step.
Genus-specific PCR and RFTM Assay Results
All tissue samples used for DNA extraction yielded high quality genomic DNA as indicated by strong 1,800 bp amplification products that were clearly visible by UV illumination of agarose gels following PCR with the SSU rRNA gene universal primers. Perkinsus genus-specific PCR-based diagnostic screening of an initial baseline subset of 20 C. ariakensis sacrificed on January 24, 2005 showed that all animals were free of Perkinsus DNA. After being held in the laboratory aquaria for 59 days, prior to inoculation, five C. ariakensis were sacrificed and genus-specific PCR screening indicated a 100% prevalence of Perkinsus sp. DNA.
RFTM and Perkinsus genus-specific PCR screening results for the C. ariakensis that were inoculated with SASW or P. olseni cells are shown in Table 1. RFTM data were not collected during the first day of subsampling For the signal processing technique, see .
In computer graphics, subsampling (or "downsampling") is the process of reducing an image to a smaller size. It is a type of image scaling, usually used to alter the appearance of an image or reduce the quantity of information required (Day 21), however the PCR screening indicated the presence of Perkinsus sp. DNA in both oysters sampled from those inoculated with SASW and in three of the four oysters sampled from the P. olseni injections. There were no Perkinsus cells observed by RFTM assays in the day 44 SASW sample, although Perkinsus sp. DNA was found by the PCR assay in one of the two oysters sampled. Very light-light RFTM rankings were found for two of the four P. olseni-inoculated oysters sampled on day 44 and three of the four were positive in the Perkinsus genus-specific PCR assay. On day 59 both of the SASW-inoculated oysters sampled had moderate-moderate/heavy Perkinsus sp. tissue burdens and Perkinsus sp. DNA was found in both by the PCR assay. Three of the four P. olseni-inoculated oysters that were sampled on day 59 had light-moderate infections as indicated by the RFTM assay and Perkinsus DNA was detected in all four of the oysters. On day 72, when the experiment was terminated, six SASW- and 15 P. olseni-treated oysters remained and were sacrificed. Perkinsus DNA was found by the PCR assay in all of the oysters remaining from the two treatment groups (Table 1). Perkinsus sp. cells were not detected, however, by the RFTM assay in three of the SASW-inoculated or in one of the P. olseni-inoculated oysters. The other three remaining SASW-inoculated oysters had rare RFTM rankings. Rare-light/moderate tissue burdens were found in nine P. olseni-inoculated oysters at the end of the experiment and four P. olseni-inoculated oysters had heavy-very heavy infections as indicated by the RFTM assay.
RFTM and P. marinus PCR assay results for the 29 untreated C. ariakensis that were still alive and were sacrificed on day 37 of the challenge experiment are shown in Table 2. Eighteen of these oysters were ranked as having rare to light Perkinsus sp. tissue burdens, two were ranked as light/moderate and two as moderate-moderate/heavy tissue burdens. Seven had no observable Perkinsus sp. cells in the RFTM assay, however five of these were positive in the PCR assay. Perkinsus marinus DNA, as indicated by the PCR assay, was found in 23 of these 29 untreated oysters (Table 2), with no DNA detected in three of the oysters with a rare RFTM ranking and in one with a light infection as indicated by the RFTM assay.
Mortality was observed in untreated oysters and in those injected with either SASW or P. olseni (Table 3). For the untreated C. ariakensis, two died during the 37 day period that they were held in the aquaria. For the SASW treatment and the P. olseni treatment, cumulative mortality after 72 days was 40.0% and 46.2%, respectively. Of the 27 dead C. ariakensis removed from the untreated tank and the experimental aquaria during the course of the experiment, it was possible to conduct RFTM analysis on 11 oysters and PCR analysis on 22. All tissues taken from dead C. ariakensis were PCR positive for Perkinsus sp. DNA. Of these 11 oysters examined by RFTM, 2 had none, or rarely observable Perkinsus sp. cells, 2 had very light or light tissue burdens, 1 had a light/moderate tissue burden and 6 had moderate to heavy tissue burdens of Perkinsus sp. cells.
Species-specific PCR Screening for Perkinsus marinus and Perkinsus olseni
DNAs from all oysters shown to be PCR positive with the Perkinsus genus-specific assay were analyzed using both P. marinus-specific and P. olseni-specific primers. Because the P. olseni primers were new for this study, specificity was tested against P. marinus and P. chesapeaki DNAs. The P. olseni primers did not amplify DNA from these other Perkinsus species. Sequencing of amplification products further confirmed the specificity as sequences of amplification products from all positive P. olseni-specific reactions matched those of GenBank deposited P. olseni (P. atlanticus) sequences. The five C. ariakensis taken as a baseline sample immediately prior to the start of the study, and all untreated oysters had only P. marinus DNA. In addition, all but two of the C. ariakensis that were SASW- or P. olseni-inoculated and that were Perkinsus sp. positive with the genus-specific primers, were positive for only P. marinus DNA. Two oysters that were inoculated with P. olseni at the start of the challenge and were sampled on days 21 and 72 were positive for both P. marinus and for P. olseni DNA. Tissue from all dead oysters collected from either the experimental or untreated aquaria were found to be PCR-positive for P. marinus only.
The P. olseni and P. marinus positive control DNA isolated from cultured cells, and plasmid-purified Perkinsus chesapeaki ITS region DNA, were amplified in the Perkinsus genus-specific assay and were digested with Mbo I restriction endonuclease. Results confirmed that P. marinus ITS region DNA has a unique digestion profile when digested with Mbo I and the observed fragment sizes of approximately 17 bp, 226 bp and 264 bp were consistent with the fragment sizes expected for P. marinus.
Ten animals shown by PCR to have Perkinsus sp. DNA, and whose tissues showed light to very heavy RFTM rankings were chosen for further histological examination. A tissue section from one animal with a very heavy RFTM ranking that had died during the experiment showed gross lesions (Fig. 1A) and observable parasite cells when stained with Harris-hemotoxylin and eosin (Fig. 1B). Parasite cells were numerous and observed systemically throughout the visceral mass. Obvious lesions caused by Perkinsus sp. infection were not observed by histology in tissue sections taken from animals with light or moderate Perkinsus sp. tissue burdens as determined by the RFTM assays.
[FIGURE 1 OMITTED]
ISH Assay Results
In-situ hybridization probes designed to target the LSU rRNA genes of P. marinus or P. olseni exhibited specificity for the targeted Perkinsus species. In the specificity tests, the P. marinus probe hybridized only to the Perkinsus cells in the sample of P. marinus in C. virginica and the P. olseni probe hybridized to Perkinsus sp. cells in the control sample of P. olseni in Haliotis laevigata. The probes did not cross-react with nontargeted Perkinsus species or host tissues in other control samples. Figure 2A to D shows four consecutive sections from the heavily infected oyster shown in Figure 1. Figure 2A is an H&E-stained section. Figure 2B shows positive in-situ hybridization with the PmarDIGLSU-181 probe to Perkinsus sp. cells indicating that the lesions were caused by P. marinus. The P. marinus-specific probe bound to cells throughout the digestive epithelium, gonads and gonoducts. In situ hybridization reactions conducted without probe (Fig. 2C), or with the PolsDIGLSU-464 probe (Fig. 2D), produced no signal in host tissues of this oyster. The PolsDIGLSU-464 probe was also tested against two individuals that were inoculated with P. olseni and found to harbor both P. marinus and P. olseni DNA by the PCR assays. The P. marinus probe hybridized to Perkinsus sp. cells in the dually infected oysters, however, no binding of the P. olseni probe was observed, suggesting that infections by P. olseni were extremely light, or that only P. olseni DNA was present and not viable cells.
[FIGURE 2 OMITTED]
Initial screening using a PCR-based diagnostic method on a sample of the triploid C. ariakensis oysters that were obtained for the challenge study suggested that they did not harbor P. marinus. A small sample (n = 5) of these oysters screened 59 days later, however, had 100% P. marinus DNA prevalence as indicated by the P. marinus- specific PCR assay. We suggest that the C. ariakensis oysters either acquired a small number of P. marinus cells that were not numerous enough to be detected by our initial PCR assay of gill/mantle tissue, while in the VIMS hatchery before collection for this experiment, or while being held in unfiltered Please wikify (format) this article or section as suggested in the Guide to layout and the Manual of Style.
Remove this template after wikifying. This article has been tagged since York River water for 4 days prior to being brought into the aquaria where they were subsequently held only in filtered York River water. The P. marinus proliferated to PCR detectable levels after 59 days in aquaria with disease developing and mortality subsequently occurring in not only those oyster groups that had been subjected to notching and inoculation with either SASW or P. olseni for the challenge study, but also in the control group of unnotched and untreated oysters that were simply held for an additional 37 days during the challenge experiment.
RFTM and PCR assay results for the C. ariakensis initially inoculated with either 25 ppt SASW or cultured P. olseni cells for the challenge study revealed a progression of P. marinus infection in the oyster tissues. Sampling and subsequent PCR-based screening of both the 25 SASW and P. olseni-inoculated oysters on Days 21 and 44 indicated that P. marinus DNA was found in 83.3% and 66.7% of the oysters, respectively. We believe that the apparent decrease in P. marinus prevalence on day 44 reflects sampling error related to the small sample sizes (n = 6) assayed to determine PCR-based Perkinsus sp. prevalence at each time point. Although P. olseni DNA was found in two of the P. olseni-inoculated oysters (one each on days 21 and 72), there was an increase in the observed PCR prevalence of P. marinus DNA to 100% in samples of all treated oysters collected on days 59 and 72. Likewise, as determined by RFTM assays, the tissue burdens for the two positive individuals were light in the P. olseni-treated oysters collected on day 44 of the experiment. However, among samples of these same oyster groups that were collected on days 59 and 72, not only light infections, but also moderate to very heavy tissue burdens were observed. Interestingly, disease progression in those C. ariakensis not notched or inoculated, mimicked that seen in the manipulated experimental oysters based on the RFTM-based tissue burdens. By day 37 when the control oysters were sacrificed, the observed Perkinsus sp. tissue burdens ranged from light to moderate/heavy.
During the course of the experiment, there were many instances where dead oysters were discovered in the various treatment groups, with little or no tissue available for either PCR or RFTM analysis. It is important to note, however, that of the 1 dead oysters with available tissue, more than half (55%) had moderate to heavy tissue burdens.
Prevalence and intensity of P. marinus infections in C. virginica oysters are closely linked to temperature and salinity, and the rate of infection in the natural environment is believed to be proportional to the number of waterborne infective cells (Andrews & Hewatt 1957, Chu et al. 1994). Warm water temperatures, 20[degrees]C to 25[degrees]C, and high salinity >15 ppt, have been shown to correlate with times of maximal proliferation of parasite cells, as well as highest incidences of infection in oysters (Andrews & Hewatt 1957, Chu et al. 1994). Mortality of infected oyster hosts depends on the level of infection, with an apparent dose of between 10 and 100 cells adequate to establish infection (Chu 1996). Because of the effects of salinity and temperature on the parasite, the infections caused by P. marinus are seasonal in the Chesapeake Bay, with maximum parasite prevalence recorded in late summer and minimum prevalence observed during the winter months (Burreson & Ragone Calvo 1996). York River water conditions during the time that the oysters were in the holding tank were approximately 9[degrees]C and 16 ppt salinity. Environmental conditions of the York River were not likely favorable for P. marinus proliferation at the start of these experiments; however, it has been shown that parasite cells can remain viable in over wintering conditions of temperatures as low as 4[degrees]C and a salinity of 4 ppt (Chu & Greene 1989, Ragone Calvo & Burreson 1994, Chu 1996). Therefore, we cannot discount the possible presence of parasite cells in the York River water during the holding period, when the oysters were in unfiltered water. The experimental conditions under which the oysters were held, 20[degrees]C and 25 ppt salinity, would have favored the proliferation of P. marinus cells present in the oysters, accounting for the increase in infection prevalence observed after the baseline sampling.
Cumulative mortalities were highest in those treatments where oysters were subjected to the extra stress of notching and injection, and the highest Perkinsus sp. tissue burdens were seen in dead C. ariakensis. We may have observed higher cumulative mortalities in the untreated control oysters, as well, had they been held for a longer time period. In C. virginica, digestive gland epithelia ep·i·the·li·a
A plural of epithelium. and the stomach are often heavily colonized Colonized
This occurs when a microorganism is found on or in a person without causing a disease.
Mentioned in: Isolation with P. marinus and damaged. As parasite proliferation increases to lethal levels, massive tissue sloughing occurs, which eventually contributes to the death of the animals (Mackin, 1951). In this study, tissue sections from a moribund C. ariakensis with a very heavy RFTM ranking showed dense, systemic P. marinus infection and the ISH assays confirmed that all of the observable Perkinsus sp. cells were P. marinus. Collectively, the results presented here strongly suggest that P. marinus was an important contributing factor to the death of these oysters.
Because P. olseni was found during a recent survey of C. ariakensis populations in Asia (Moss & Reece 2005), this study was initiated to examine the pathogenicity of P. olseni to C. ariakensis. At the termination of this experiment, however, PCR based diagnostics suggested that a relatively low number of the P. olseni-inoculated C. ariakensis (n = 2) harbored both P. marinus and P. olseni DNA. This result may indicate that C. ariakensis are not readily susceptible to P. olseni, or that virulence attenuation Loss of signal power in a transmission.
The reduction in level of a transmitted quantity as a function of a parameter, usually distance. It is applied mainly to acoustic or electromagnetic waves and is expressed as the ratio of power densities. of the parasite may have occurred during the culturing period prior to the use of the parasite as inoculum inoculum /in·oc·u·lum/ (-ok´u-lum) pl. inoc´ula material used in inoculation.
n. pl. . Virulence attenuation with in-vitro cultured P. marinus cells has been documented (Ford et al. 2002). Recent studies, however, have demonstrated that cultured cell virulence is enhanced by supplementing media with host oyster homogenate homogenate /ho·mog·e·nate/ (ho-moj´in-at) material obtained by homogenization.
material obtained by homogenization. (Macintyre et al. 2003, Earnhart et al. 2004) and therefore, we plan to do homogenate supplementation of media for Perkinsus sp. isolates used in future challenge studies.
Although little data on P. olseni pathogenicity was obtained during this experiment, it provided valuable information regarding the potential for advanced P. marinus infections to occur in C. ariakensis. Prior field studies conducted in Chesapeake Bay have indicated that although C. ariakensis is capable of acquiring P. marinus infections (Calvo et al. 2001), there was no evidence that C. ariakensis was susceptible to the advanced parasite infections known to occur in C. virginica. We contend that the experimental environmental conditions, under which the oysters were held for a total of 5 months, combined with the stress of the unnatural aquaria environment and experimental manipulation, may have promoted the development of the intense P. marinus infections in C. ariakensis that were observed here. This study provides valuable information on potentially problematic disease issues including parasite proliferation that can arise if P. marinus-infected C. ariakensis encounter stress challenges in the wild or aquaculture environment, or if they are held in hatcheries or laboratories under stressful conditions.
The authors thank Dr. S.K. Allen, Jr. of the Aquaculture and Genetics Breeding Center at VIMS for providing Crassostrea ariakensis oysters; Georgeta Constantin and Alanna Alanna may refer to:
An abbreviation for "no meaningful figure". You'll often see this when comparing financial data among companies where a certain ratio or figure isn't applicable.
For example, if company has negative earnings, it cannot have a P/E ratio. 4570431. VIMS contribution number 2718.
Andrews, J. D. & W. G. Hewatt. 1957. Oyster mortality studies in Virginia. ? II. The fungus disease caused by Dermocystidium marinum in oysters of the Chesapeake Bay. Ecol. Monogr. 27:1-26.
Audemard, C., K. S. Reece & E. M. Burreson. 2004. Real-time PCR for the detection and quantification of the protistan pro·tist
Any of the eukaryotic, unicellular organisms of the former kingdom Protista, which includes protozoans, slime molds, and certain algae. parasite Perkinsus marinus in environmental waters. Appl. Environ. Micro. 70:6611-5618.
Burreson, E. M. & L. M. Ragone Calvo. 1996. Epizootiology of Perkinsus marinus disease of oysters in Chesapeake Bay, with emphasis on data since 1985. J. Shellfish Res. 15:17-34.
Calvo, G. W., M. W. Luckenbach & E. M. Burreson. 1999. A comparative field study of Crassostrea gigas (Thunberg, 1973) and Crassostrea virginica (Gmelin, 1791) in relation to salinity in Virginia. J. Shellfish Res. 18(2):459-464.
Calvo, G. W., M. W. Luckenbach, S. K. Allen, Jr. & E. M. Burreson. 2001. A comparative field study of Crassostrea ariakensis (Fujita 1913) and Crassostrea virginica (Gmelin) in relation to salinity in Virginia. J. Shellfish Res. 20:221-229.
Casas, S. M., A. Villalba & K. S. Reece. 2002. Study of perkinsosis in the carpet shell clam Tapes decussates in Galacia (NW Spain). Identification of the aetiological AE`ti`o`log´ic`al
a. 1. Pertaining to ætiology; assigning a cause.
Adj. 1. aetiological - of or relating to the philosophical study of causation
aetiologic, etiologic, etiological
2. agent and in vitro modulation of zoosporulation by temperature and salinity. Dis. Aquat. Org. 50:51-65.
Chu, F.-L. E. 1996. Laboratory investigations of susceptibility, infectivity, and transmission of Perkinsus marinus in oysters. J. Shellfish Res. 15(1):57-66.
Chu, F.-L. E. & K. H. Green. 1989. Effect of temperature and salinity on in vitro culture of the oyster pathogen, Perkinsus marinus, (Apicomplexa: Perkinsea). J. Invertebr. Pathol. 53:260-268.
Chu, F.-L. E., A. K. Volety & G. Constantin. 1994. Synergistic effects of temperature and salinity on the response of oysters (Crassostrea virginica) and Pacific (Crassostrea gigas) oysters: temperature and salinity effects. J. Shellfish Res. 12(1):21-27.
Earnhart, C. G., M. A. Vogelbein, G. B. Brown, K. S. Reece & S. L. Kaattari. 2004. Supplementation of Perkinsus marinus cultures with host plasma or tissue homogenate enhances their infectivity. Appl. Environ. Microbiol. 70(1):421-431.
Elston, R. A., C. F. Dungan, T. R. Meyers & K. S. Reece. 2004. Perkinsus sp. infection risk for Manila clams, Venerupis philippinarum (A. Adams and Reeve, 1850) on the Pacific coast of North and Central America. J. Shellfish Res. 23(1):101-105.
Ford, S. E., M. M. Chintala & D. Bushek. 2002. Comparison of in-vitro-cultured and wild-type Perkinsus marinus. 1. Pathogen virulence. Dis. Aquat. Org. 51(4):187-201.
MacIntyre, E. A., C. G. Earnhart & S. L. Kaattari. 2003. Host oyster tissue extracts modulate in vitro protease protease /pro·te·ase/ (pro´te-as) endopeptidase.
Any of various enzymes, including the proteinases and peptidases, that catalyze the hydrolytic breakdown of proteins. expression and cellular differentiation in the protozoan parasite, Perkinsus marinus. Parasitology Parasitology
The scientific study of parasites and of parasitism. Parasitism is a subdivision of symbiosis and is defined as an intimate association between an organism (parasite) and another, larger species of organism (host) upon which the parasite is 126: 293-302.
Mackin, J. G. 1951. Histopathology his·to·pa·thol·o·gy
The science concerned with the cytologic and histologic structure of abnormal or diseased tissue.
The study of diseased tissues at a minute (microscopic) level. of infection of Crassostrea virginica (Gmelin) by Dermocystidium marinum Mackin. Owen and Collier. Bull. Mar. Sci. Gulf and Caribbean 1:72-87.
Medlin, L., H. J. Elwood, S. Stickel & M. L. Sogin. 1988. The characterization of enzymatically amplified eukaryoticl6S-like rRNA-coding proteins. Gene 71:491-499.
Moss, J. A. & K. S. Reece. 2005. Pathogens of the Asian oyster, Crassostrea ariakensis, in its native range: current findings and work in progress. J. Shellfish Res. 24(2):668.
Ragone Calvo, L. M. & E. M. Burreson. 1994. Characterization of over-wintering infections of Perkinsus marinus (Apicomplexa) in Chesapeake Bay oysters. J. Shellfish Res. 13:123-130.
Ray, S. M. 1952. A culture method for the diagnosis of infections with Dermocystidium marinum Mackin, Owen and Collier in oysters. Science 116:360-361.
Ray, S. M. 1954. Biological studies of Dermocystidium marinum, a fungus parasite of oysters. Rice Institute Pamphlet. 114 pp.
Shaw, B. L. & H. I. Battle. 1957. The gross microscopic anatomy of the digestive tract of the oyster Crassostrea virginica (Gmelin). Can. J. Zool. 35:325-357.
Sindermann, C. J. 1990. Principal diseases of marine fish and shellfish. Vol. 2. San Diego, CA: Academic Press, Inc. 516 pp.
Stokes, N. A. & E. M. Burreson. 1995. A sensitive and specific DNA probe for the oyster pathogen Haplosporidium nelsoni. J. Eukaryor Microbiol. 42:350-357.
JESSICA A. MOSS, EUGENE M. BURRESON AND KIMBERLY S. REECE *
Virginia Institute of Marine Science, The College of William and Mary Noun 1. William and Mary - joint monarchs of England; William III and Mary II , Gloucester Point, VA 23062
* Corresponding author. E-mail: firstname.lastname@example.org
TABLE 1. RFTM ranking and PCR-based Perkinsus genus-specific assay screening results of the challenge study oysters that were notched and inoculated with either SASW or cultured P. olseni cells and sampled from the experimental aquaria on days 44, 59 and 72. Day 44 (n = 2) Day 44 (n = 4) SASW P. olseni # # PCR # # PCR RFTM Ranking RFTM Pos RFTM Pos None (N) 2 1 2 1 Rare (R) Very Light (VL) 1 1 Light (L) 1 1 Light/Moderate (LM) Moderate (M) Moderate/Heavy (MH) Heavy (H) Very Heavy (VH) Day 59 (n = 2) Day 59 (n = 4) SASW P. olseni # # PCR # # PCR RFTM Ranking RFTM Pos RFTM Pos None (N) 1 1 Rare (R) Very Light (VL) Light (L) 2 2 Light/Moderate (LM) Moderate (M) 1 1 1 1 Moderate/Heavy (MH) 1 1 Heavy (H) Very Heavy (VH) Day 72 (n = 6) Day 72 (n = 15 SASW P. olseni # # PCR # # PCR RFTM Ranking RFTM Pos RFTM Pos None (N) 3 3 1 1 Rare (R) 3 3 1 1 Very Light (VL) 2 * 2 Light (L) 6 6 Light/Moderate (LM) 1 1 Moderate (M) Moderate/Heavy (MH) Heavy (H) 1 1 Very Heavy (VH) 3 3 * Species-specific PCR assays indicated that one of these individuals, as well as one P. olseni-inoculated individual collected on day 21 (data not shown), contained DNA from both P. marinus and P. olseni. Only P. marinus DNA was detected in all other individuals. TABLE 2. RFTM ranking and PCR-based P. marinus assay screening results of control untreated C. ariakensis (unnotched and uninoculated) on day 37 of the challenge study. Day 37 (n = 29) RFTM Ranking # Individuals # P. marinus PCR+ None (N) 7 5 Rare (R) 7 4 Very Light (VL) 3 3 Light (L) 8 7 Light/Moderate (LM) 2 2 Moderate (M) 1 1 Moderate/Heavy (MH) 1 1 Heavy (H) Very Heavy (VH) (Note: P. olseni DNA was not detected in any of these oysters.) TABLE 3. Daily observed C. ariakensis mortalities during the course of the challenge experiment. Day Untreated 25 ppt SASW Perkinsus olseni 2 1 11 1 37 1 43 na * 1 49 na 4 l 53 na 1 55 na 4 58 na 1 1 59 na 1 64 na 1 2 66 na 1 72 na 6 * na = not applicable because the oysters from the untreated tank were sacrificed on day 37. Column headings indicate the sample treatments.