Test selection and sampling requirements for aquatic animal imports

Information for foreign authorities

The purpose of these tables is to advise foreign authorities which diagnostic tests must be used when exporting susceptible aquatic species to Canada where testing or surveillance is a requirement and indicated on the approved zoosanitary export certificate.

These test selection and sampling requirements apply to all end uses that require testing for export or surveillance with the exception of food service, retail use, and further processing for human consumption.

Trade partners must select either the World Organisation for Animal Health (WOAH; founded as Office International des Épizooties (OIE)) or Canadian test indicated below. For diseases not listed by the WOAH, countries are expected to use the identified "Canadian test" below. Test selection or sample size may only be changed in situations where Canada has performed a country evaluation or assessed a country's aquatic animal health regulatory controls as part of the negotiated requirements for export to Canada.

All sampling must be completed using a screening test on apparently healthy populations. Tissues and sample size requirements are also indicated in the table below.

Pooling is not allowed; except for oyster seed and post-larval shrimp when conducting PCR testing on individual animals is not possible.

Please note

The WOAH tests listed below are based on the WOAH Manual of Diagnostic Tests for Aquatic Animals disease chapters sections 5 "Rating of tests against purpose of use". Generally, screening tests with an a) or b) in the "Targeted surveillance" column of the WOAH manual are the recommended tests for import.

Sample size has been determined based on either 1 or 2% prevalence and 95% confidence and takes into account disease clustering.

This document is subject to change based on new scientific evidence and development of more advanced protocols.

Finfish diseases

Table 1: The test type, sample size and sample type required by disease for the import of susceptible species of finfish into Canada
Diseases	WOAH test	Canadian test	Sample size	CFIA types of tissue samples, and/or lifestage
Infectious salmon anaemia virus (ISAv) pathogenic strains and non-pathogenic strains (HPR0)	Screening test: Real time RT-PCR (Snow et al method) Confirmatory test: cRT-PCR (segment 6) and sequencing and virus isolation (HPR deleted strains)	Screening test: qRT-PCR^{Table Note 1} (segment 8) Confirmatory test: cRT-PCR (segment 6) and sequencing and virus isolation (for HPR deleted strains)	Originating from a Country or zone that is free, to all zones^{Table Note 2} in Canada: 175 for pathogenic ISA and 350 for HPR0 Originating from a Country or zone that is not free or has an unknown health status to a zone^{Table Note 2} in Canada that is free, provisionally free or a buffer zone: Refuse entry^{Table Note 3} Originating from a Country or zone that is not free or has an unknown health status to a zone^{Table Note 2} in Canada that is infected: 175 for HPR deleted ISA and 350 for HPR0 ISA	Lethal testing-Pathogenic strains: Mid- kidney and heart HPR0: gill^{Table Note 4} Nonlethal broodstock testing: Pathogenic strains: ovarian fluid, milt, HPR0: gill^{Table Note 4}
Viral haemorrhagic septicemia virus	Screening test: Virus isolation Confirmatory test: PCR with sequencing to determine strain	Screening test: qRT-PCR Confirmatory test: cRT-PCR and sequencing and virus isolation	175	Lethal testing -Anterior kidney and brain; spleen and heart can be added if larger sample required for virus isolation Non-lethal broodstock testing: ovarian fluid, milt
Infectious haematopoietic necrosis virus	Screening test: Virus isolation Confirmatory test: PCR	Screening test: qRT-PCR Confirmatory test: cRT-PCR and sequencing and virus isolation	175	Lethal testing -Anterior kidney and brain; spleen and heart can be added if larger sample required for virus isolation Non-lethal broodstock testing-eggs, ovarian fluid, milt
Infectious pancreatic necrosis virus^{Table Note 5}	N/A	Screening test: qPCR Confirmatory test: cPCR and sequencing and virus isolation	Originating from a Country or zone that is free to all zones^{Table Note 2} in Canada: 175 Originating from a Country or zone that is not free or has an unknown health status to a zone^{Table Note 2} in Canada that is free, provisionally free or a buffer zone: 350 Originating from a Country or zone that is not free or has an unknown health status to a zone^{Table Note 2} in Canada that is infected: 175	Lethal testing - spleen and kidney Non-lethal broodstock testing- ovarian fluid, milt^{Table Note 6}
Epizootic haematopoietic necrosis virus	Screening test: Virus isolation Confirmatory test: PCR	Screening test: qPCR Confirmatory test: cPCR and sequencing and virus isolation	175	Lethal testing – anterior-mid kidney and spleen
*Oncorhynchus masou virus disease (Oncorhynchus masou virus)*	Screening test: Virus isolation Confirmatory test: PCR confirmation As per the WOAH Manual of Diagnostic Tests for Aquatic Animals	Screening test: qPCR Confirmatory test: cPCR and sequencing and virus isolation	175	Lethal testing -anterior – mid kidney, skin lesions and spleen Non-lethal broodstock testing- ovarian fluid
*Gyrodactylosis (Gyrodactylus salaris)*	Screening test: Visual inspection and wet mount Confirmatory test: PCR and sequencing	Screening test: cPCR and morphometry Confirmatory test: cPCR and sequencing	175	Whole fish and/or fins
Koi herpes virus	Screening test: PCR Confirmatory test: PCR and sequencing	Screening test: qPCR Confirmatory test: cPCR (that targets a different segment of the genome) and sequencing	175	Anterior kidney, spleen, brain, gill and intestine
Spring viraemia of carp virus	Screening test: Virus isolation	Screening test: qRT-PCR Confirmatory test: cPCR and sequencing and virus isolation	175	Kidney spleen, gill and brain
Red sea bream iridovirus	Screening test: PCR with sequencing confirmation	Screening test: qPCR Confirmatory test: cPCR and sequencing and virus isolation	175	Anterior - mid Kidney and spleen
*Ceratomyxosis (Ceratomyxa shasta)^{Table Note 5}*	N/A	Screening test: Visualization of parasite on gills or in gut smear Confirmatory test: PCR	175	Gills, pancreas, pyloric cecae and adjacent intestine
*Whirling disease (Myxobolus cerebralis)^{Table Note 5}*	N/A	Screening Test: Pepsin Trypsin digest Confirmatory test: cPCR and sequencing and visualization of spores	175	Head including gill arches and operculum
Salmonid alphavirus	Screening test: Real time PCR Confirmatory test: cPCR sequencing confirmation	Screening test: qPCR Confirmatory test: cPCR and sequencing and serology	175	Heart and kidney

Table Notes

Table Note 1

Canada requires testing for ISAV HPR0 using gill as the sample tissue and using the method described in Snow et al. 2006 New Diagnostic Technology: Applications in Animal Health and Biologics Controls Dev Biol 126:133-135.

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Table Note 2

Finfish Reportable Diseases-Declarations by Province and Marine Area health status in Canada

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Table Note 3

While diagnostic test methods are available for ISA HPR0, due to the transient expression and seasonal nature of this strain, and the difficulty in detecting infection when the prevalence is low, it is not possible at this time through testing alone to reduce the importation risk of ISA HPR0 to negligible.

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Table Note 4

Gill must be run independently of other tissues sampled.

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Table Note 5

This disease is listed in the Canadian Reportable Disease Regulations

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Table Note 6

Smail DA¹, Munro ES. Isolation and quantification of infectious pancreatic necrosis virus from ovarian and seminal fluids of Atlantic salmon, Salmo salar L. J Fish Dis. 2008 Jan;31(1):49-58.

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Mollusc diseases

For diseases of molluscs that are considered as foreign animal diseases in Canada, histology alone does not have sufficient sensitivity for screening of apparently healthy populations.

Table 2: The test type, sample size and sample type required by disease for the import of susceptible species of molluscs into Canada.
Disease	WOAH test	Canadian test	Sample size	CFIA types of tissue samples, and/or lifestage
Abalone viral mortality (abalone herpes-like virus)	Screening test: PCR Confirmatory test: in situ hybridization or PCR and sequencing	Screening test: qPCR Confirmatory test: cPCR and sequencing and In situ hybridization	175	Adult animals: Mouth and the neural tissue of the cerebral commissure, pleuropedal and buccal ganglia (juveniles and adults). Larvae, spat use whole animals (qPCR only).
*Bonamia exitiosa*	Screening test: PCR Confirmatory test: PCR with sequencing for confirmation	Screening test: qPCR Confirmatory test: cPCR and sequencing and histology	175	Adult animals: 3-5mm section through gill and mantle, gonad and digestive gland. Larvae, spat use whole animals.
*Bonamia ostreae*	Screening test: real-time PCR Confirmatory test: PCR with sequencing for confirmation	Screening test: qPCR or histology Confirmatory test: cPCR and sequencing and histology	175	Adult animals: Gill, mantle, gonad and heart for PCR; Sagittal section through gill, digestive gland for histology. pools of 2-3 ml for spat.
Haplosporidium nelsoni^{Table Note 7}	N/A	Screening test: qPCR or histology Confirmatory test: cPCR and sequencing and histology	Originating from a Country or zone that is free, to all zones^{Table Note 8} in Canada: All susceptible species: 175 except C. gigas: 350 Originating from a Country or zone that is not free or has an unknown health status to a zone^{Table Note 8} in Canada that is free, provisionally free or a buffer zone: Refuse entry Originating from a Country or zone that is not free or has an unknown health status to a zone^{Table Note 8} in Canada that is infected: 175 all species except C. gigas: 350	Adult animals: Gill, mantle, digestive gland for PCR; Sagittal section through gill and mantle, heart and digestive gland for histology. 2-3 ml pools for spat.
*Marteilia refringens*	Screening test: PCR Confirmatory test: PCR and sequencing or in situ hybridization	Screening test: qPCR Confirmatory test: cPCR and sequencing and histology	175	Adult animals: 3-5mm section through gill, mantle, gonad and digestive gland. Larvae, spat use whole animals.
Marteiliodes chungmuensis^{Table Note 9}	N/A	Screening test: Histology Confirmatory test: cPCR and sequencing	175	Adult animals ( female only): 3 to 5mm section through gills, mantle, ovarian tissue, and digestive gland
Mikrocytos mackini^{Table Note 7}	N/A	Screening test: qPCR or histology Confirmatory test: cPCR and sequencing and histology	175	Adult animals: Sagittal section through base of gills, digestive gland, gonad, mantle and gills for PCR and histology.
Ostreid herpesvirus 1	Screening test: qPCR Confirmatory test: cPCR and sequencing (WOAH Manual of Diagnostic Tests for Aquatic Animals)	Screening test: qPCR Confirmatory test: cPCR and sequencing and In situ hybridization	175	Adult animals: 3 to 5mm section through gill, mantle, digestive gland and adductor muscle. Larvae, spat use whole animals.
*Perkinsus marinus*	Screening test: PCR Confirmatory test: In situ hybridization or PCR with sequencing	Screening test: qPCR Confirmatory test: cPCR and sequencing and histology	175	Adult animals: 3-5mm section through gill, mantle, gonad and digestive gland.
*Perkinsus olseni*	Screening test: PCR Confirmatory test: In situ hybridization or PCR with sequencing	Screening test: qPCR Confirmatory test: cPCR and sequencing and histology	175	Adult animals: 3-5mm section through gill mantle, gonad and digestive gland.
*Withering syndrome of abalone (Xenohaliotis californiensis)*	Screening test: PCR Confirmatory test: PCR and sequencing	Screening test: PCR Confirmatory test: cPCR and sequencing and In situ hybridization	175	Adult animals: Posterior oesophagus and digestive gland.

Table Notes

Table Note 7

This disease is listed in the Canadian Reportable Disease Regulations

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Table Note 8

Mollusc Reportable Diseases-Declarations by Province and Marine Area

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Table Note 9

This disease is listed in the Canadian Health of Animals Regulations Schedule VII

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Crustacean Diseases

Histopathology alone cannot be used for confirmation of screening test in apparently healthy populations because histopathology does not have sufficient specificity.

Table 3: The test type, sample size and sample type required by disease for the import of susceptible species of crustaceans into Canada.
Diseases	WOAH test	Canadian test^{Table Note 10}	Sample size	CFIA types of Tissue samples, and/or lifestage
Taura syndrome virus	Screening test: RT-qPCR Confirmatory test: In situ DNA probes hybridization	Screening test: qPCR Confirmatory test: cPCR and sequencing and In situ hybridization	175	Whole post-larvae or antennal gland, lymphoid organ, cuticular epithelium of gills, exoskeleton, appendages and anterior foregut for lethal sampling; hemolymph, gills and pleopod for non-lethal sampling
White spot syndrome virus	Screening test: RT-qPCR Confirmatory test: In situ hybridization	Screening test: qPCR^{Table Note 11} Confirmatory test: cPCR and sequencing and In situ hybridization	175	Whole post-larvae or antennal gland, hematopoietic organ, cuticular epithelium of gills, exoskeleton, appendages and anterior foregut for lethal sampling; hemolymph, gills and pleopod for non-lethal sampling
Yellowhead virus	Screening test: RT-qPCR Confirmatory test: In situ hybridization	Screening test: qPCR Confirmatory test: cPCR and sequencing and In situ hybridization	175	Whole post-larvae or lymphoid organ and cuticular epithelium of gills, exoskeleton, appendages and anterior foregut for lethal sampling; hemolymph, pleopods and gills for non-lethal sampling
*Crayfish plague (Aphanomyces astaci)*	Screening test: RT-qPCR Confirmatory test: cPCR and sequencing	Screening test: qPCR^{Table Note 11} Confirmatory test: cPCR and sequencing and histology (microscopic examination)	175	Soft abdominal cuticle, uropod and telson
Infectious hypodermal and hematopoietic necrosis virus	Screening test: RT-qPCR Confirmatory test: In situ hybridization	Screening test: qPCR Confirmatory test: cPCR and sequencing and In situ hybridization	350	Whole post-larvae or antennal gland, hematopoietic organ, lymphoid organ, cuticular epithelium of gills, exoskeleton, appendages and anterior foregut for lethal sampling; hemolymph, gills and pleopod for non-lethal sampling
Infectious myonecrosis virus	Screening test: RT-qPCR Confirmatory test: In situ hybridization	Screening test: qPCR Confirmatory test: cPCR and sequencing and In situ hybridization	175	Whole post-larvae or white muscle, and lymphoid organ for lethal sampling; hemolymph and pleopod for non-lethal sampling
White tail disease	Screening test: RT-qPCR Confirmatory test: In situ hybridization	Screening test: qPCR Confirmatory test: cPCR and sequencing and In situ hybridization	175	Whole post-larvae or striated muscle from cephalothorax, abdomen and tail. for lethal sampling; pleopod for non-lethal sampling
Necrotizing hepatopancreatitis	Screening test: RT-qPCR Confirmatory test: In situ hybridization	Screening test: cPCR^{Table Note 11} Confirmatory test: cPCR and sequencing and In situ hybridization	175	Whole post-larvae or hepatopancreas
Acute hepatopancreatic necrosis disease (AHPND)	Screening test: real time PCR (Preliminary enrichment culture for detection of VpAHPND) Confirmatory test: histopathology or bioassay	Screening test: qPCR^{Table Note 11} Confirmatory test: cPCR and sequencing and In situ hybridization	175	Whole post-larvae or hepatopancreas, stomach

Table Notes

Table Note 10

Molecular assays are described in: Arseneau, J.R. and Laflamme, M. 2016. Development of RT-qPCR methodologies for the detection of viral pathogens in crustaceans of commercial importance. Can. Tech. Rep. Fish. Aquat. Sci. 3134: iii+21pp.

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Table Note 11

Method is published in the WOAH Manual of Diagnostic Tests for Aquatic Animals.

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