Molecular Diagnostic Screening Enables Simultaneous Detection of the Shellfish Parasites Bonamia ostreae and Bonamia exitiosa

As a member of the World Trade Organization, Canada is required to ensure that imports and exports of fish and seafood products are free of infectious aquatic pathogens (disease-causing agents). In Canada, this responsibility falls to the National Aquatic Animal Health Program (NAAHP), which is co-delivered by Fisheries and Oceans Canada (DFO) and the Canadian Food Inspection Agency (CFIA).

Central to protecting Canada’s reputation for producing high-quality fish and seafood is the ability to deliver accurate, reliable, and consistent diagnostic testing for aquatic pathogens, which is carried out by three of the four laboratories in the DFO National Aquatic Animal Health Laboratory System (NAAHLS). The labs also conduct targeted research and provide scientific advice in support of the NAAHP.

At the Pacific Biological Station in Nanaimo, British Columbia, Fisheries and Oceans Canada lead molecular biology technician Geoff Lowe (back) and lead shellfish health technician Gary Meyer (front) use traditional light microscopy to screen for the presence of federally regulated Bonamia parasites in oyster tissues.

Photo: Cathryn Abbott (DFO)

For the past several years, NAAHP surveillance for shellfish pathogens in British Columbia has relied on a long-standing traditional method of diagnostic screening—called histopathology—that involves staining tissue sections from host species and examining them under a microscope. While this approach is effective, it is not efficient for screening a large number of individuals.

Molecular diagnostic methods

“Recently, NAAHP has been moving toward enabling the use of molecular diagnostic methods,” says Fisheries and Oceans Canada research scientist Dr. Cathryn Abbott of the Pacific Biological Station Aquatic Animal Health Laboratory in Nanaimo, B.C. One such technique, quantitative polymerase chain reaction (qPCR), enables very rapid and sensitive screening to detect the genetic material (i.e., DNA) of a pathogen or other target organism.

“A qPCR test has very high sensitivity, which means it can detect a very small amount of the target, which is important for minimizing false negative test results,” says Abbott.

Bonamia ostreae and Bonamia exitiosa

In 2015–2016, Abbott and her research team conducted experiments aimed at enabling NAAHLS member laboratories to use a molecular diagnostic method of screening for the presence of two closely related aquatic parasites—Bonamia ostreae and Bonamia exitiosa. Both are listed by and notifiable to the World Organisation for Animal Health (OIE). In Canada, known or suspected cases of infection by either parasite must be reported to the Canadian Food Inspection Agency.

The parasites can cause lethal infections in several species of oysters, including European Flat Oyster (Ostrea edulis). Signs of infection may include a yellowish discolouration and extensive lesions in some internal organs, although many oysters show no such signs before they die. In 2004, B. ostreae caused high mortality among cultured stocks of European Flat Oysters in British Columbia, resulting in the demise of this aquaculture species on the West Coast. Neither species of Bonamia parasites has ever been detected in populations of O. edulis in Eastern Canada. The closely related parasite B. exitiosa has never been reported in Canada. However, this native of the southern hemisphere has caused widespread mortality among New Zealand Dredge Oysters.

Given that oysters rank as the second most valuable shellfish aquaculture species in Canada based on total farm gate value of almost $30 million in 2014, surveillance for B. ostreae and preventing B. exitiosa from entering Canada is a high priority for the National Aquatic Animal Health Program.

Adapting and validating a qPCR test

“Of the published molecular tests for detecting these two species of Bonamia parasites, none have been completely validated to international standards as defined by the OIE. Our goal was to have a qPCR test for use in our laboratories that meets our specific regulatory need to screen a large number of shellfish for both parasites simultaneously. This is much more time and cost efficient and typically more accurate than traditional light microscopy,” says Abbott. “We needed a species-level molecular diagnostic screening approach and associated data that clearly measure how well the test performs in the context of what we need it to do.”

After adapting an existing test to make it suitable to meet the laboratories’ needs, the research team evaluated its analytical performance including the risk of false positive or false negative results, and the consistency of its performance within a lab (repeatability) and between labs (reproducibility). The research team also adopted a procedure that uses DNA sequencing—determining the order of the chemical building blocks of a DNA molecule—to confirm whether samples that tested positive using the screening qPCR test are, in fact, positive.

Expanding the National Aquatic Animal Health Program’s diagnostic screening repertoire

The results of this research will increase the scope of NAAHLS laboratories to screen for CFIA-regulated parasites in shellfish by enabling them to implement an analytically validated qPCR-based test for detecting B. ostreae and B. exitiosa.

In February 2016, the aquatic animal health lab at the Pacific Biological Station achieved international accreditation, which means it meets the standards for diagnostic laboratories established by the International Organization for Standardization (ISO).

For more information, see:Fisheries and Oceans Canada’s National Aquatic Animal Health Laboratories Attain International Accreditation.”

This work was funded through the Centre for Aquatic Animal Health Research and Diagnostics.

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