Application of a multiplex PCR for the detection of protozoan pathogens of the eastern oyster Crassostrea virginica in field samples (original) (raw)
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Diseases of Aquatic Organisms, 2005
We have developed a PCR-assay for the diagnosis of juvenile oyster disease (JOD) based on the detection of Roseovarius crassostreae directly from affected oysters. Species-specific primers are used to amplify the 16S-23S rDNA internal transcribed spacer (ITS) of R. crassostreae, and confirmation of product identity is accomplished by restriction enzyme analysis. No false positives were obtained with either closely related bacterial species or from other DNAs present in oyster samples. The assay has the potential to detect as few as 10 cells of R. crassostreae per oyster when samples are taken from the inner valve surfaces of the animal. Inclusion of material from soft body surfaces is not necessary, and may reduce sensitivity approximately 10-fold. In a JOD-affected population, a positive PCR result was obtained from all oysters from which these bacteria were subsequently cultured. The assay also detected the presence of R. crassostreae in 2 oysters from which no R. crassostreae isolates were recovered. No R. crassostreae was detected by either PCR or bacteriology in oysters from a population that was not exhibiting JOD-signs. This assay is expected to advance regional disease management efforts and provide valuable insights into the disease process and epizootiology of JOD.
Diseases of Aquatic Organisms, 2007
With the drastic decline of eastern oyster Crassostrea virginica populations in the Chesapeake Bay due to over-fishing, diseases and habitat destruction, there is interest in Maryland and Virginia in utilizing the non-native oyster species Crassostrea ariakensis for aquaculture, fishery resource enhancement, and ecological restoration. The International Council for the Exploration of the Sea (ICES) recommends that non-native species be examined for ecological, genetic and disease relationships in the native range prior to a deliberate introduction to a new region. Therefore, a pathogen survey of C. ariakensis and other sympatric oyster species was conducted on samples collected in the PR China, Japan and Korea using molecular diagnostics and histopathology. Molecular assays focused on 2 types of pathogens: protistan parasites in the genus Perkinsus and herpesviruses, both with known impacts on commercially important molluscan species around the world, including Asia. PCR amplification and DNA sequence data from the internal transcribed spacer region of the rRNA gene complex revealed the presence of 2 Perkinsus species not currently found in USA waters: P. olseni and an undescribed species. In addition, 3 genetic strains of molluscan herpesviruses were detected in oysters from several potential C. ariakensis broodstock acquisition sites in Asia. Viral gametocytic hypertrophy, Chlamydia-like organisms, a Steinhausia-like microsporidian, Perkinsus sp., Nematopsis sp., ciliates, and cestodes were also detected by histopathology.
A Quantitative Competitive Polymerase Chain Reaction Assay for the Oyster Pathogen Perkinsus Marinus
Journal of Parasitology, 2000
A quantitative competitive polymerase chain reaction (QCPCR) assay was developed for the oyster parasite Perkinsus marinus. PCR primers for the rRNA gene region of P. marinus amplified DNA isolated from P. marinus but not from Perkinsus atlanticus, Crassostrea virginica, or the dinoflagellates Peridinium sp., Gymnodinium sp., or Amphidinium sp. A mutagenic primer was used to create a competitor plasmid molecule identical to the P. marinus target DNA sequence except for a 13-bp deletion. Both P. marinus and competitor DNA amplified with equivalent efficiencies. Each of 25 oysters was processed by 5 P. marinus diagnostic methods-Ray's fluid thioglycollate medium (FTM) tissue assay, FTM hemolymph assay, whole oyster body burden assay, QCPCR of combined gill and mantle (gill/mantle) tissue, and QCPCR of hemolymph. The QCPCR assay enabled detection of 0.01 fg of P. marinus DNA in 1.0 g of oyster tissue. QCPCR of gill/mantle tissue or hemolymph as well as the body burden assay detected infections in 24 of 25 oysters. Ray's FTM tissue assay detected only 19 infections. The FTM hemolymph assay detected only 22 infections. Regression analysis of QCPCR results and FTM results indicated that the QCPCR assays were effective in quantitating P. marinus infections in oyster tissues.
This study was undertaken to develop a quantitative polymerase chain reaction assay that would improve the utility of PCR for detecting Haplosporidium nelsoni (MSX), a serious parasite of the eastern oyster Crassostrea virginica. A competitive PCR sequence was generated from the H. nelsoni small subunit ribosomal DNA fragment, originally described by Stokes and colleagues, that was amplified by the same PCR primers and had similar amplification performance. Assays performed using competitor dilutions ranging from 0.05 to 500 pg/µl DNA were used to test oyster samples designated using histological techniques as having " light " or " heavy " MSX infections. Visual diagnoses were confirmed equally well with three methods: densitometry of ethidium-bromide-stained agarose, densitometry of SYBRGreen-stained polyacrylamide gels, and analysis by GeneScan 3.0 of fluorescent products detected in ultrathin gels. Oysters diagnosed as negative for MSX tested as negative or light by PCR. Oysters with light MSX infections generally had less than 5 pg/µl infectious DNA. Oysters with heavy infections generally corresponded to 5 pg/µl or greater competitor dilutions.