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Abstracts
Session: The State of Oyster Disease
Molecular Technologies: Applications to Dermo Disease
Presented By:
Gerardo R. Vasta (vasta@umbi.umd.edu), Jose A. F. Robledo, Eric J. Schott, Wolf T. Pecher, Hafiz Ahmed, and Keiko Saito
Center of Marine Biotechnology, University of Maryland Biotechnology Institute
Center of Marine Biotechnology, University of Maryland Biotechnology Institute
Introduction: During the past years, continued studies on the morphology, physiology, and biochemistry of Perkinsus marinus, and the effects of P. marinus on the oyster host, have resulted in substantial progress towards understanding the parasite's virulence and the environmental conditions that lead to epizootic outbreaks. The application of molecular approaches to Dermo disease in recent years, however, has resulted not only in the development of specific and sensitive diagnostic tools for Perkinsus spp, but also in a deeper understanding of various aspects of the parasite's biology and its interactions with the bivalve hosts. Although Fong et al. (Mar. Biol. Biotechnol., 2:346, 1993) amplified Perkinsus marinus rRNA genes by applying PCR to infected oyster hemocytes as early as 1993, the breakthrough that facilitated the comprehensive application of molecular approaches to the study of Dermo disease was the development by several research groups of in vitro culture techniques for Perkinsus spp. that enabled the production of large biomass of genetically homogeneous cells for isolation of nucleic acids and proteins. The unrestricted availability of Perkinsus spp. clonal cultures to the scientific community from a recognized public repository (ATCC, http://www.atcc.org) made it possible for several laboratories to apply molecular technologies to most of the species currently described.
Molecular diagnostics: The first molecular method for diagnosis of P. marinus was a PCR-based assay targeted to the intergenic spacer (IGS) of the ribosomal RNA gene. This method was adapted into a semiquantitative format, and is sensitive enough to detect a single trophozoite in 30 mg of oyster tissue. This was later followed by a similar PCR-based method targeted to the internal transcribed spacer (ITS). The IGS proved to be a useful target for the development of strain-specific primers for P. marinus (types I and II), and more recently, for PCR-based diagnosis of P. andrewsi and P. atlanticus. Further, the high conservation of IGS sequences close to the 5'end of the SSU rRNA in all Perkinsus spp. characterized at present enabled the design of primers that amplify all species currently available as holotype (hapantotype) cultures from ATCC (P. marinus, P. andrewsi, and P. atlanticus) and based on the sequence information available would also amplify P. olseni. This Perkinsus "genus-specific" PCR-based assay complements the species- and strain-specific assays developed earlier, and strengthen the detection of yet un-described Perkinsus spp. or those for which specific detection assays are not currently available. Quantitative PCR-based assay formats were later developed, and include competitive PCR (QC-PCR), real-time PCR (Taqman), and ELISA-PCR. The relevance of the species-/strain-specific molecular diagnostic methods over standard diagnostic methods (FTM, histology, etc) resides in that in some areas, such as Chesapeake Bay, multiple Perkinsus spp and strains are sympatric, and can be present simultaneously in the same individual oyster or clam.
Description of new species/strains: The characterization of rRNA genes from Perkinsus isolates from various host species, complemented with ultrastructural and gross morphology studies, led to the description of new Perkinsus spp. including P. andrewsi and P. chesapeaki. However, for P. qugwadi, a species that had been described as such based on morphological criteria, the later application of molecular approaches contributed to raise concerns about its true taxonomic placement within the genus Perkinsus. [JF1]The application of molecular techniques has recently enabled the assessment of the genetic variability among isolates of Perkinsus spp. from various regions along the Atlantic and Gulf coasts of US.
Phylogenetic analysis: The phylogenetic affinities of P. marinus within the Alveolata has been controversial, and continues to generate great interest. Initially classified as a fungus, it was later placed within the Apicomplexa based on ultrastructure of the flagellated life stage. Molecular studies, however, suggested a closer affiliation to the dinoflagellates. The recent description of Parvilucifera infectans, a close relative of Perkinsus spp, lead to the establishment of the phylum Perkinsozoa, which would include both genera. An additional genus (Cryptophagus) has been recently added. The most recent phylogeny, based on tubulin, actin, and rRNA sequences, places the perkinsids as one of the earliest diverging group of the lineage leading to dinoflagellates. This phylogenetic position, however, has been inferred from limited available sequence information, and additional genetic characterization of species from both clades, Dinozoa and Apicomplexa, will be required to support it.
Identification and structural/functional characterization of selected genes and their products: The availability of RNA and DNA from Perkinsus spp. cultures, and the use of PCR primers or hybridization probes based on consensus sequences from taxonomic related species, enabled the isolation and characterization of genes of interest for phylogenetic analysis, such as actins and tubulins, and for use in studies related to virulence and intracellular survival, such as superoxide dismutases (SODs) and iron transporters (Nramp). The application of molecular techniques has "subverted" the classical approach to structure/function studies, by avoiding the typical requirement of large quantities of the purified authentic protein. The bacterial expression of recombinant P. marinus SOD1 and SOD2 enabled the crystallization and resolution of both structures without prior purification of the authentic proteins. Gene complementation in heterologous systems (defective yeast mutants) led to the characterization of subcellular compartmentalization and biological function of the SOD gene products. Finally, this information facilitated both molecular and biochemical/physiological studies on the effect of stressors, such as reactive oxygen species and iron depletion, on gene expression, and cell viability. This has resulted in the identification of pathways the parasite uses to abrogate or avoid intracellular oxidative attack by the oyster hemocytes. For biomolecules synthesized by complex enzymatic pathways, such as carbohydrates and lipids, the implementation of classical biochemical approaches has been equally successful. The structural/functional characterization of Perkinsus proteins that may have critical roles as virulence factors, or in the parasite's metabolism, may lead to the rational design of chemotherapeutic agents for use in aquaculture settings.
Genomics: From the above it becomes clear that the knowledge of the complete gene repertoire of P. marinus could lead to the identification of additional genes required for parasite's virulence and, at the same time, suitable as targets for intervention. Therefore, it has become of great interest to elucidate the complete sequence of the P. marinus genome, and to examine gene expression under selected conditions by "expressed sequence tags" (ESTs). These comprehensive gene discovery programs in P. marinus are currently supported by NOAA/Sea Grant and NSF-USDA, and the information generated will be made available to the scientific community via public databases. ESTs generated from P. marinus propagated under various environmental conditions or exposed to host factors will result in unique gene expression profiles. Research on transcriptome differences among Perkinsus spp. will provide insight into their distinct virulence or responses to the host environment. These powerful approaches may be conceptualized as "ecogenomics " and "comparative genomics". Similar genomic studies are currently being carried out on the oyster elsewhere, and complementary technologies, including the construction of BAC libraries and microarrays, proteomics, and novel mathematical techniques, will contribute to examine oyster disease with a comprehensive, multidisciplinary approach.
The future: Implementation of molecular technologies such as those described above will rapidly increase the depth of knowledge of the parasite's biology, and facilitate future hypothesis-driven research on how environmental, host, and genetic factors modulate virulence in this ecologically and economically important pathogen. Furthermore, the vast information yielded by the application of genomic approaches, and the hypotheses-driven studies deriving from these, will make Perkinsus spp. excellent models for basic studies on fundamental processes that are common not only to parasites of mollusks and other invertebrates, but also parasites of medical and veterinary relevance. (Supported by awards from Sea Grant/NOAA, NRAC, NSF and USDA).
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