title: Molecular detection of Marteilia sydneyi, pathogen of Sydney rock oysters.
abstract: The life cycle of Marteilia sydneyi, the aetiological agent of QX disease in the Sydney rock oyster Saccostrea commercialis, is not known. We have developed and optimised 2 diagnostic assays, the polymerase chain reaction (PCR) and in situ hybridisation, for use in investigating the role of possible alternative hosts in the life cycle of this pathogen. PCR primers, designed within the ITS1 rDNA of M. sydneyi, amplified a 195 bp fragment. Sensitivity of the PCR assay was assessed using DNA extracted from known numbers of sporonts purified from infected oyster digestive gland. DNA equivalent to 0.01 sporonts was detectable following agarose gel electrophoresis. The potential inhibitory effect of the presence of host DNA on the PCR assay was tested by the addition of oyster genomic DNA during amplification. Concentrations of host DNA in excess of 50 ng per 20 microliters reaction reduced the sensitivity of the test. Environmental validation of the PCR assay was demonstrated by the amplification of M. sydneyi DNA from 50 ng of genomic DNA extracted from QX-infected oysters. A DNA probe was constructed using the M. sydneyi unique primers and was able to detect 10 pg of M. sydneyi PCR amplified DNA in dot-blot hybridisations. The probe hybridised with presporulating and sporulating M. sydneyi stages in paraffin sections of oyster digestive gland. No non-specific binding was observed. Hybridisation consistency and signal intensity decreased as sporonts matured. While the high sensitivity and specificity of the PCR test will allow rapid screening of large numbers of potential alternative hosts for the presence of parasite DNA, it does not actually identify infective stages. In situ hybridisation conducted on paraffin sections will determine the location of the parasite within the host for morphological characterisation.
authors.affiliation: Department of Parasitology, University of Queensland, Brisbane, Australia. s.kleeman@mailbox.uq.edu.au
authors.forename: S N
authors.initials: SN
authors.lastname: Kleeman
authors.forename: R D
authors.initials: RD
authors.lastname: Adlard
mesh_terms: D000818:Animals; D016054:DNA, Protozoan / Q000737:chemistry; D004275:DNA, Ribosomal / Q000032:analysis; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D056890:Eukaryota / Q000235:genetics / Q000302:isolation & purification*; D017403:In Situ Hybridization / Q000662:veterinary; D008018:Life Cycle Stages; D010125:Ostreidae / Q000469:parasitology*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D017422:Sequence Analysis, DNA
chemical_list: D016054:DNA, Protozoan; D004275:DNA, Ribosomal