DISEASES OF AQUATIC ORGANISMS Vol. 40: 137-146.2000 Published March 14 Dis Aquat Org ~ Molecular detection of Marteilia sydneyi, pathogen of Sydney rock oysters Sarah N. ~leeman'l*,Robert D. ~dlard~ '~epartmentof Parasitology, University of Queensland, Brisbane, Queensland 4072, Australia '~rotozoaSection, Queensland Museum, PO Box 3300, South Brisbane, Queensland 4101, Australia 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 pos- sible 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 equiva- lent 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 amplifica- tion 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 sensitiv- ity 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 ~nfectivestages. In situ hybrid- sati ion conducted on paraffin sections will determine the location of the parasite within the host for n~orphologicalcharacterisation. KEY WORDS: Marteilia sydneyi . PCR primers . DNA probe . In sjtu hybridisation . Saccostrea com- mercialis . rDNA gene sequence INTRODUCTION limits and prevent further geographic spread of the disease, potential control is hindered by the incom- Marteilia sydneyi, the aetiological agent of QX dis- plete understanding of the life cycle of this pathogen. ease, is recognised as the most pathogenic parasite of Development and ultrastructure of presporulating and the Sydney rock oyster Saccostrea commercialis in sporulating stages in the oyster host are described culture on the east coast of Australia, where stock mor- (Perkins & Wolf 1976); however, the stage infective to talities in excess of 90% have been recorded. Prior to the oyster and its origin are unknown. Failure to infect 1994, QX infections were recorded from the Great oysters experimentally with mature spores (Lester 1986) Sandy Strait (25'30's) south to the Macleay River and recent evidence which suggests a limited period (31" S),after which time its range had extended further of viability of spores in seawater (Wesche et al. 1999) south to include the Georges River estuary (34"s) have implicated the involvement of an intermediate (Adlard & Ernst 1995).Although current management host. strategies exist to keep stock losses within manageable Traditionally, detection of QX in oyster tissue in- volved microscopical examination of histological sec- tions. Wet smears of hepatopancreas, in which refrac- tile bodies in sporonts are characteristic of infection, O Inter-Research 2000 Resale of full article not permitted 138 Dis Aquat Org 40: 137-146, 2000 provided a simple and rapid diagnosis when sporonts for 6 to 24 h in 10% buffered formalin, dehydrated in were present. Current diagnosis is based on the exam- alcohol and embedded in paraffin. Histological sec- ination of Hemacolor (Merck)-stained imprints of he- tions (6 pm) were stained in haematoxylin and eosin patopancreds which enable rapid detection of all de- (H&E) to confirm presence or absence of M. sydneyi velopmental stages, including presporulating stages, in adjacent sections. undetectable in wet preps, within a few days of initial DNA extraction and purification. Genomic DNA was infection. These techniques cannot identify unambigu- extracted from purified sporonts, Marteilia sydneyi in- ously stages of Marteilia sydneyi of unknown morpho- fected Saccostrea commercialis digestive gland and logy that may occur in alternate hosts, highlighting the sperm of uninfected oysters by digestion in a solution need for an appropriate diagnostic tool. An indirect of extraction buffer (100 mM Tris, pH 8.0, 100 mM fluorescent antibody test (IFAT)was developed (Roubal EDTA, pH 8.0, 100 mM NaCl), 10% SDS and Pro- & Lester 1987) but was considered an unreliable guide teinase K (20 mg ml-') at 56°C for 4 to 24 h. Remaining to infection as it did not react to the contents of the proteins and polysaccharides were removed by phenol/ spore (Roubal et al. 1989), the most likely stage to be chloroform/isoamyl alcohol extraction and nucleic present in an alternate host. acids recovered by ethanol precipitation (see Sam- DNA techniques display levels of sensitivity and brook et al. 1989).DNA concentration and purity were specificity, irrespective of life cycle stage, required for estimated by measuring the 260/280 optical density such a test. PCR and in situ hybridisation assays have ratio of a solution following RNase treatment (0.2 pg proven to be usefu! toc!s fer diszasc detection in RNase per pi oi yenomic DNA). aquatic animals (Adlard & Lester 1995, Chang et al. Primer synthesis. A reverse primer, based on the 1996, Durand et al. 1996, Lo et al. 1997, Wang et al. putative first internal transcribed spacer (ITS1) se- 1997) and are considered suitable tools for life cycle quence given by Anderson et al. (1995),was designed studies (Fong et al. 1993, Stokes & Burreson 1995, and designated PR02. It was synthesised from the re- Stokes et al. 1995). Polymerase chain reaction (PCR) verse complement of PRO1 (Anderson et al. 1995) such primers developed by Anderson et al. (1995) were that PR02 = 5'-TCA AGG GAC ATC CAA CGG TC-3'. found to be specific to Marteilia sydneyi. However, This primer was then used in a PCR amplification with their location in the rDNA gene cluster was not con- a forward primer designated RA2 (5'-GTC CCT GCC firmed and subsequent analysis revealed low levels of CTT TGT ACA CA-3') with annealing site in a con- sensitivity in PCR (Adlard unpubl. data). served region of the small subunit (SSU), 180 nucleo- In this paper we describe the development and opti- tide bases upstream of the boundary between the SSU misation of 2 diagnostic assays based on PCR and in and ITS 1. situ hybridisation, utilising PCR primers that amplify This PCR produced an amplified fragment of 430 bp specific DNA regions within the ribosomal DNA clus- when compared on an agarose gel with a molecular ter of Marteilia sydneyi, which can then be applied to weight standard (100 bp ladder, GibcoBRL). The nu- investigate the role of possible alternate hosts in the cleotide sequence was determined for this fragment life cycle of this important pathogen. using the same primers as sequencing primers, then compared for homology with known SSU sequences (Cryptocaryon irntans, and compared using Blast rou- METHODS tine on GenBank) to confirm its location in the rDNA cluster. To increase the species specificity for hybridis- Specimen preparation. Oysters were tested for the ation of a PCR-generated probe, a second forward presence of Marteilia sydneyi by microscopical exami- primer designated LEG 1,5'-CGATCT GTG TAG TCG nation of Hemacolor (Merck)-stainedimprints of oyster GAT TCC GA-3', was designed from this sequence digestive gland. Infected oysters were classified as within the original fragment, but positioned 48 nucleo- having: (1) presporulation stage of infection if plas- tide bases downstream of the SSU/ITS1 boundary. moaia and secondary cells were identified i.n imprints; PCR. PCR was performed under the following reac- and (2) progressi.ve stage of infection if all develop- tion parameters unless otherwise stated, expressed as mental stages (plasmodia, secondary cells, immature final concentrations: MgC12 2 mM; buffer 67 mM Tris- and mature sporonts) were present. Sporonts of M. HCl, 16.6 mM (NH,)SO,, 0.45% (v/v) Triton X-100; sydneyi were purified from homogenised digestive dNTP's 200 mM; primers 10 pm01 each; Taq polymer- gland of infected oysters by centrifugation on discon- ase (Boehringer, Mannheim) 3 U; DNA 50 ng, ultra- tinuous sucrose and Percoll gradients as described pure water to 50 p1 total. Thermal cycling parameters by Mialhe et al. (1985). Tissue samples of uninfected were as follows: denaturation at 95OC for 60 s; primer oysters, oysters with presporulation stages of QX and annealing at 55'C for 30 S; chain extension at 72°C for oysters with progressive stages of infection were fixed 60 S, repeated for 30 cycles with a final cycle incorpo- Kleeman & Adlard: Molecular detect~onof Marteilia sydneyi 139 rating a 7 rnin extension. Electrophoresis of amplified Mannheim) in a spot test on nylon membranes accord- products was conducted through submarine agarose ing to the methodology in the application manual. gels (1.2% [w/v] agarose, 1.2 p1 ethidium bromide DIG-labelled DNA probe sensitivity. Sensitivity of [l0 mg ml-' w/v] in 20 m1 total, for 30 rnin at 100 V per the probe was determined in dot-blot hybridisations. 20 mA) and examined and photographed under ultra- Serial dilutions from 10 ng to 1 pg of Marteilia sydneyi violet light. A molecular weight standard (100 bp PCR cloned DNA were heat-denatured and spotted ladder, Gibco BRL) was used to estimate the size of onto nylon membranes then fixed by UV crosslinking products.