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 optirnised 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 genornic DNA during amplification. Concentrations of host DNA in excess of 50 ng per 20 p1 reaction reduced the sensitivity of the test. Environmental validation of the PCR assay was demonstrated by the amplification of M. sydneyl 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 ~nfective stages. In situ hybridsati ion conducted on paraffin sections will determine the location of the parasite within the host for n~orphological characterisation.

Marteiliosis in molluscs: A review

“…While we still have little knowledge about the biology of M. refringens, the life-cycle of other paramyxeans is even less understood although recent advance in the development of molecular detection tools should, in the near future, enable investigators to go forward on this topic (Kleeman and Adlard 2000;Itoh et al 2003).…”

“…In numerous papers published on this paramyxean parasite and on M. sydneyi from Australia, the existence of a complex lifecycle had been postulated, although its transmission routes and life-cycle was unknown and until now remains uncertain (Balouet 1979;Balouet et al 1979;Lauckner 1983;Grizel 1985;Figueras and Montes 1988;Berthe et al 1998;Kleeman and Adlard 2000;Audemard et al 2000Audemard et al , 2002.…”

“…To date, gene probes used in the diagnosis of marteiliosis in cultured molluscs have been developed for detection of the most economically important paramyxeans, namely Marteilia refringens (Lubat 1990;Le Roux et al 1999, M. sydneyi (Anderson et al 1995;Kleeman and Adlard 2000) and M. chungmuensis (Itoh et al 2003). These techniques are slowly moving from development in specialized laboratories for research purposes, to routine application and are expected to find an increasing use in routine marteiliosis monitoring programs in mollusc aquaculture and in efforts to prevent the spread of paramyxeans.…”

“…in each sporangium (rather than four in M. refringens), and the presence of a heavy layer of concentric membranes surrounding mature M. sydneyi spores. In situ hybridisation has been proposed to detect infections and confirm the identity of paramyxean parasites in a histological section (Le Roux et al 1999;Kleeman and Adlard 2000;Itoh et al 2003). Probes have different levels of specificity, depending on the region of the genes where they have been designed (Kleeman et al 2002b).…”

“…Often, however, when DNA sequences have been determined by different laboratories they do not correspond to the same gene or region of a gene, impeding taxonomic studies and sequence comparisons for the development of diagnostics. Efforts were made to obtain the same regions of sequences from related organisms to optimize the chance of developing probes and PCR primers with the desired specificity and in the case of paramyxeans, efforts have focused on the small-subunit ribosomal RNA gene (Le Roux et al 1999Berthe et al 2000;Kleeman and Adlard 2000;Itoh et al 2003). These rRNA genes are useful targets for diagnostic tests because there are many copies in the genome, which can help to ensure good sensitivity, and they offer a mosaic of conserved and variable regions which allow analyses at various levels of resolution.…”