There are many surveillance and monitoring programs underway around the world that are aimed at specific diseases of concern. In Europe several countries monitor for specified diseases under the European Council Directive 91/67/EEC and associated amendments, as well as the European Commission Decision 96/240/EC. Likewise, Australia has an extensive surveillance programme for specific reportable diseases under their AquaPlan (AquaPlan 2000). Most recently, Canada had to implement a surveillance programme to address the first time detection of an OIE listed molluscan pathogen, Haplosporidium nelsoni, the causative agent of MSX disease. Coincidentally, this outbreak occurred at the same time as the Expert Consultation in Rome in October, 2002, and necessitated the early departure of Dr. McGladdery who was in charge of coordinating the contingency plan response. Since much of the Expert Consultation discussions up to that point were brought immediately to bear in addressing this disease emergency, it was considered appropriate to include the outbreak history and controls based on the surveillance that was implemented (and is ongoing) as a case-study (Atlantic Canadian oyster disease surveillance) for this report.
Historic reference data
Traditionally, Canadas east coast native oyster species, Crassostrea virginica, has been considered to be free of the diseases that have impacted the same species along the mid-eastern Atlantic coast of the United States. In the late 1980s a shellfish health programme was initiated with the objective of compiling a disease database that could be used to assess the risk of moving oysters between the four Atlantic Provinces and three hydrographic water bodies. The database comprised of histopathology information and a reference slide collection.
Between 1988 and 2002, over 8 000 oysters were examined histologically as part of disease and aquaculture development research projects (variable sample sizes), health checks for licensed stock transfers (for depuration relay and seed/broodstock for culture purposes) (sample size n = 60), and investigation of reports of abnormal mortalities, growth or spawning events (variable sample sizes). During this period, no OIE listed pathogens of oysters were detected, and annual reports to OIE were based on these observations.
In 1998, the data generated from this general surveillance was used to establish zones within Atlantic Canada for use by government introductions and transfers committees, as well as for teaching purposes. Based on like-to-like health profiles, all oysters in the southern Gulf of St. Lawrence were considered to be homogenous, while those in Cape Breton were considered to be distinct, based on experimental demonstration of ongoing susceptibility to Malpeque Bay Disease, which is present at subclinical levels in oysters throughout the southern Gulf of St. Lawrence and southwestern Nova Scotia.
Detection of suspect OIE listed disease
Suspicious observations were found by the shellfish health laboratory in October 2002 in a sample of oysters submitted from Cape Breton due to heavy mortalities. The histology was sent to the OIE reference laboratory for molluscan diseases at the Virginia Institute of Marine Sciences. Duplicate samples from the suspicious case were process for scanning and transmission electron microscopy, as well as for PCR using OIE published primer for MSX and SSO (seaside organism) parasites.
At the same time, additional samples were received from Cape Breton independently from other stakeholders. Protocols for enhanced record-keeping and sign-off were initiated for laboratory personnel to track samples being sent for second opinion; for electron microscopy; and for PCR analysis in Moncton and Virginia (PCR done blind on all samples).
A contingency plan was developed with two stages:
(i) Actions required on Presumptive diagnosis of a significant infectious agent; and
(ii) Actions required on Confirmation of a significant infectious agent.
Stage (i) involved alerting local and national authorities of suspicion of a serious infection, as well as the oyster growers who submitted the samples and the provincial authorities. A communications plan was also developed with scenarios to cover something new killing oysters in Cape Breton, as well as first time detection of an OIE reportable pathogen.
Confirmation of MSX disease in Canada
On 18 October 2002, confirmation of the infection being Haplosporidium nelsoni, the parasite responsible for MSX disease in Crassostrea virginica, was received from the OIE reference laboratory. The Chief Veterinary Officer for Canada was notified immediately and he notified the OIE General Secretariat. Canada was no longer considered to be an MSX-free country.
The contingency plan prepared for confirmation of MSX was implemented and a meeting held in Cape Breton on 21 October 2002 with affected leaseholders and First Nations food fishery stakeholders, along with federal and provincial authorities and fish health veterinary services. This meeting was used to:
(a) Identify priority sites for sampling to map out extent of MSX infections in Atlantic Canada. Sites selected were those reporting mortalities, at neighbouring sites, with direct oyster transfers from the positive site over the preceding 18 months, with indirect links to the hydrographic area containing the infected site, and with no links to the affected site.
(b) Establish interim control measures to prevent spread while samples were collected, analysed and results produced for feedback. This included voluntary cessation of the native food fishery, closure of leases (harvest fishery closed for conservation reasons), and development of harvest protocols for lease-holders to help get product out of the water and to market live with no intermediate washing or resoaking that could spread MSX.
Surveillance to determine the geographic extent of MSX infection of Atlantic Canadian oyster populations
The sampling protocols were developed in consultation with provincial extension officers familiar with all oyster industry stakeholders in the area. One site per day was visited and two teams of two personnel were deployed. Disinfection of clothing and equipment was undertaken pre- and post- each site visit and oysters were collected by the biologists, using farm equipment and wet-gear. The approach used was to start at the sites furthest removed from the known positive area and work inwards from there.
At the same time samples were received from the two neighbouring provinces. These were from sites that had reported observations of mortalities over the summer, but did not consider them abnormal at the time. They were also sites with indirect links to Nova Scotia oyster processors with live-holding capability.
On receipt of the oysters at the laboratory, they were logged into a special log with code numbers which were used for blind samples for PCR and cross-checking histology readings. All initial samples received were processed using sterile flaming between oysters. This was to ensure that any PCR results received could be tracked back to individual oysters.
Sample sizes were 60. Thirty of the oysters were processed immediately for histology and tissues preserved for PCR. The remaining 30 were fixed, but not processed further for immediate histology, and tissues fixed for PCR examination, as required.
Protocols for reading slides included a sweep for obvious clinical infections - noting whether or not there was spore development. As soon as a positive slide was confirmed, the entire sample was recorded as positive and examination moved to the next sample. It was considered more important at this stage of surveillance to map presence/absence than prevalence. Negative samples following the sweep and high power search and rescue examination of the entire tissue section were flagged for processing the remaining 30 animals. Any suspicious but inconclusive histological observations were flagged for PCR examination.
Subsamples of tissue sections from each sample of 30 were flagged for analysis by a second slide-reader. This included slides with anything suspicious but inconclusive.
Mussels from the positive area were also submitted for examination due concerns over proposed transfers to mussel growing sites outside the area. These were processed for histology, but also analysed using MSX-PCR.
The surveillance period (November-December) is not recommended for detection of subacute infections by MSX, however heavy infections were found in oysters from the Bras dOr Lakes area of Cape Breton with direct transfer links to the affected site. Other sites outside the Bras dOr Lakes system showed no clear evidence of MSX or related pathology. However, a couple of oysters from the southern Gulf of St. Lawrence contained low numbers of plasmodia with no evidence of proliferation or pathology. These were subsequently identified as SSO, a related parasite, previously thought to be absent from Canadian oysters. The salinity range in the southern Gulf is not considered normal for SSO, and the patchy, light, and subclinical infections detected subsequently indicated a diffuse, ubiquitous distribution, rather than the point-source heavy infection profile found for MSX within Bras dOr Lakes.
Follow-up sampling to address establishment questions
The direct transfer links to the positive site raised the question of how long the parasite may have been present in Cape Breton oysters and if it had established infections in surrounding wild populations. Additional samples were collected in late November-December that indicated that the disease had spread to oysters on neighbouring beds that were not subject to handling or seed transfers.
Preliminary zonation for mapping
Bras dOr Lakes as a whole were zoned as positive for MSX, despite the presence of some sites that showed no evidence of infection. This was because they could not be isolated from positive sites hydrographically or from routine human activities within Bras dOr Lakes.
A preliminary Buffer Zone was delineated around the outer coast of Cape Breton where results showed no evidence of MSX and despite there being seed transfers from the affected area of Bras dOr Lakes in early summer 2002.
The remaining east coast oysters were designated as being in MSX-negative areas, although analysis of these negative samples was continued over the winter.
Zonation for management strategies
The presence of spores, indicative of advanced infections and potential for release of MSX sporoplasm into the water, along with detection of plasmodia in neighbouring wild oyster samples, indicated the strong possibility that MSX had established an infection process in affected waters. Although observations suggested the disease outbreak was recent, this may have had an incubation period dating back to the previous fall to permit infections of neighbouring oysters. These observations, along with the fact that MSX cannot be transmitted directly between oysters in the laboratory, indicated that some reservoir, other than oysters, had become involved in MSX transmission in the Bras dOr Lakes ecosystem.
All these facts suggested that any attempt to remove oysters in order to eradicate MSX from the system would likely be futile. Thus, emphasis was placed on defining zones within which MSX could effectively be contained. Results from ongoing surveillance in 2003 continue to indicate that MSX is confined within Bras dOr Lakes and all transfer controls are based on this remaining a positive zone.