Fish Health Protection Regulations

As part of the ongoing development of the National Aquatic Animal Health Program (NAAHP), Fisheries and Oceans Canada (DFO) was notified on December 7, 2015 that the Canadian Food Inspection Agency (CFIA) will implement the final portion of NAAHP, the Domestic Movement Control Program (DMCP), effective December 31, 2015. Under DMCP, CFIA will enact new measures, such as zonation and permitting, to support domestic movements of aquatic animals. As of this date, DFO will no longer exercise its authority under the FHPR, including the Manual of Compliance, and intends to move forward with the repeal of the FHPR to remove regulatory overlap with the DMCP.

For more information and questions on CFIA’s Domestic Movement Control Program, please visit the CFIA website at Notice to Industry Change to federal management of aquatic disease in Canada.

Manual of Compliance

Ottawa 1984 (revised 2011)
Minister of Public Works and Government Services Canada 1984 (revised 2011)
reprinted 1989
reprinted 1986
Cat. No. Fs 41-31/31-1984
ISBN 0-662-53060-8
DFO/4281

Correct citation for this publication:

Fisheries and Oceans Canada. 1984 (revised 2011). Fish Health Protection Regulations: Manual of Compliance (Fish. Mar. Serv. Misc. Spec. Publ. 31(Revised): iv+50p.

Contents

Abstract

  1. Introduction
  2. Regulations Respecting the Protection of Fish

Manual of Compliance

  1. Guidelines for Producers
  2. Role of Fish Health Officials
  3. Role of Local Fish Health Officers
  4. Sampling Procedures
  5. Transportation of Samples
  6. Treatment of Samples
  7. Procedures for the Detection of Certain Bacterial Fish Pathogens
  8. Procedures for the Detection of Viruses
  9. Procedures for the Detection of Certain Parasites
  10. Egg Disinfection Procedures

References

Abstract

This revised manual explains the application of the Fish Health Protection Regulations under The Fisheries Act of Canada. The Fish Health Protection Regulations have been amended to reflect a change in authorities for international movement of fish including salmonids. The authority for international movement of fish including salmonids will fall within the domain of the Canadian Food Inspection Agency as of December 2011. Fisheries and Oceans Canada will continue to regulate all interprovincial movement of salmonids after that time until further notification. For more information on the import requirements of the Canadian Food Inspection Agency please visit the CFIA website.

This manual outlines the administrative and inspection procedures to be followed and provides step-by-step procedures for handling fish samples to test for specified bacterial, viral, and myxosporean pathogens of salmonids.

All movements of fish between provinces require a permit. This permit may be issued only to producers who have a Fish Health Protection Regulations Fish Health Certificate that shows that their facility has been inspected according to this Manual of Compliance and indicating the Fish Health Protection Regulations pathogen profile for the facility.

The sampling procedures are based on the probability of detecting an infected specimen in a lot, assuming a certain prevalence of detectable infection. Selection, transportation, and laboratory handling of the samples are described in detail.

The methods provide for the detection of the following pathogens: the redmouth bacterium ( Yersinia ruckeri); the furunculosis bacterium (Aeromonas salmonicida): the protozoans causing whirling disease (Myxobous cerebralis) and ceratomyxosis (Ceratomyxa shasta); the viruses causing viral hemorrhagic septicaemia, infectious hematopoietic necrosis, infectious pancreatic necrosis and infectious salmon anaemia; and other pathogens considered to be notifiable, such as the bacterial kidney disease bacterium (Renibacterium salmoninarum).

I. INTRODUCTION

The future of Canadian fish culture and of recreational and commercial fisheries depends upon healthy fish stocks. International and interprovincial exchange of cultivated species risks the dissemination of serious infectious diseases. Effective programs of prevention and control are necessary to prevent spread of fish disease agents and to alleviate this serious impediment to development of these fisheries. This revised Manual of Compliance explains the application of the Fish Health Protection Regulations under the Fisheries Act of Canada. It presents guidelines for producers, defines the roles of Fish Health Officials and Local Fish Health Officers, and outlines the sampling, handling, and diagnostic procedures that constitute inspections leading to the issuance of a Fish Health Certificate.

The Fish Health Protection Regulations, as published in the Canada Gazette, appear in Section II. They apply to all fish species belonging to the family Salmonidae of the genera listed in Schedule I. The Regulations are designed to minimize the risk of the spread of infectious diseases through inspection of wild and cultured fish stocks and to control the movement of infected fish between provinces/territories. They apply to live and uneviscerated dead cultured fish, eggs (including fertilized eggs or gametes) of cultured and wild fish and products of dead, uneviscerated cultured fish destined to move across provincial boundaries within Canada. In the event of violation of these Regulations, seizure and other powers of the Fisheries Act apply.

Compliance with these Regulations and the issuance of a Fish Health Certificate requires that an existing facility with eggs and/or fish of unknown FHPR pathogen status must have four satisfactory inspections over a period of not less than 18 months before a FHPR Fish Health Certificate may be issued. Inspections must be conducted at intervals of not less than 90 days and not more than 270 days. Note that in the case of egg shipments, only viral testing is required, and schedule II bacterial and parasitic disease agents are not of concern. If eggs or fish are transferred from a source showing disease agent(s) of concern on their Fish Health Certificate, the Fish Health Certificate for the receiving facility will be changed to reflect the FHPR status of the source (if disinfected eggs are transferred; only the status of the Schedule II viruses will be changed for the receiving facility). If a given facility introduces fish or fish eggs from a source that does not hold a valid Fish Health Certificate, the receiving facility will have to re-commence the inspection schedule at inspection one and have four inspections over a minimum of 18 months, with inspections being not less than 90 days or more than 270 days apart, before a Fish Health Certificate can be re-issued.

A new facility using an isolated water supply free of all species of fish and starting with stocks from a source with a valid Fish Health Certificate can obtain a Fish Health Certificate after only one inspection. In this circumstance the Fish Health Certificate must reflect the disease agent profile of the source, plus the results of the one inspection at the receiving facility. For a production facility to retain a Fish Health Certificate, consecutive satisfactory inspections must be conducted as described in section B - REQUIREMENTS TO OBTAIN A FISH HEALTH CERTIFICATE. For all inspections, procedures outlined in this manual are to be followed. Persons wishing to upgrade their facility's Fish Health Certificate, from positive to negative for a Schedule II pathogen, will be required to implement a program to eliminate the specific pathogen(s) and have four consecutive negative (for the specific pathogen(s)) inspections over a minimum period of 18 months with inspections being not less than 90 days or more than 270 days apart.

If further clarification on the interpretation of these regulations is required, contact the Aquatic Animal Health Office, National Registry of Aquatic Animal Health, Fisheries and Oceans Canada, Ottawa, Ontario K1A 0E6; nrfd@dfo-mpo.gc.ca.

II. REGULATIONS RESPECTING THE PROTECTION OF HEALTH OF FISH

This copy of the Fish Health Protection Regulations is provided for ease of use. For all purposes of interpreting and applying the law, users should consult the Regulations as amended, as registered by the Clerk of the Privy Council and published in Part II of the Canada Gazette.

Short Title

  1. These regulations may be cited as the Fish Health Protection Regulations.

Interpretation

  1. In these Regulations,

    "approved" means approved by the Minister (approuvé)

    "cultured fish" means a fish listed in Schedule I that is propagated by man in a fish culture facility and includes the eggs of such fish; (poisson d'élevage);

    "Fish Health Official" means a person approved to inspect fish and fish sources for the purposes of these Regulations; (inspecteur sanitaire des poissons);

    "Local Fish Health Officer" means a person approved as a local fish health officer in charge of the administration and enforcement of these Regulations (agent local de protection de la santé du poisson);

    "Minister" means the Minister of Fisheries and Oceans for Canada; (Ministre)

    "wild fish" means a fish that is listed in Schedule I and includes the eggs of that fish, but does not include a fish that is propagated by man in a fish culture facility; (poisson sauvage).

Prohibition

  1. Subject to subsection (2), a person must not carry cultured or wild fish from one province to any other province without an interprovincial carrying permit.

  2. Subsection (1) does not apply to eviscerated cultured fish.

Permits

  1. Subject to section 5, a Local Fish Health Officer for a province may issue, to a person who applies for one, an interprovincial carrying permit that authorises the person to carry cultured or wild fish into that province.

  2. An interprovincial carrying permit must not be issued unless the person who applies for the permit has obtained a certificate and

    1. the certificate indicates that no disease or disease agent listed in Schedules II to IV was detected; or

    2. the Local Fish Health Officer for a province is satisfied that none of the detected diseases or disease agents indicated on the certificate will be harmful to the conservation and protection of fish in that province.

Certificates

  1. A certificate required pursuant to section 5 is issued by a Fish Health Official and

    1. certifies that the source of the fish was inspected in the approved manner; and

    2. indicates which, if any, of the diseases or disease agents listed in Schedule II to IV were detected during the inspection or inspections, as the case may be.

SCHEDULE I

Fish

All species and hybrids derived from species of fish belonging to the family Salmonidae, including the following genera:

Pacific salmon Oncorhynchus spp.
Danube salmon and Taimens Hucho spp.
Atlantic salmon Salmo spp.
Trout Salmo spp.
Char Salvelinus spp.
Grayling Thymallus spp.
Lenok Brachymystax spp.
Inconnu Stenodus spp.
Whitefish Coregonus spp.
Whitefish Prosopium spp.
Ayu Plecoglossus spp.

SCHEDULE II

Diseases or disease agents found in live fish or their source

  1. Any filterable replicating agent capable of causing cytopathic effects in the cell lines of fish specified by the Minister including, but not limited to:
    1. Viral Hemorrhagic Septicemia (Egtved) (Egtved virus, VHS)
    2. Infectious Hematopoietic Necrosis (IHNV)
    3. Infectious Pancreatic Necrosis (IPNV)
  2. Whirling Disease (Myxobolus cerebralis)
  3. Ceratomyxosis (Ceratomyxa shasta)
  4. Furunculosis (Aeromonas salmonicida)
  5. Enteric Redmouth Disease (Yersinia ruckeri)

SCHEDULE III

Diseases or disease agents found in dead fish or their source

  1. Viral Hemorrhagic Septicemia (Egtved virus, VHSV)
  2. Whirling Disease (Myxobolus cerebralis)

SCHEDULE IV

Diseases or disease agents found in live fish or their source

  1. Myxobacterial infections
  2. Bacterial Kidney Disease (KD bacterium)
  3. Motile Aeromonad Septicemia (motile Aeromonas sp.)
  4. Pseudomonad Septicemia (Pseudomonas spp.)
  5. Vibriosis (Vibrio spp.)

MANUAL OF COMPLIANCE

III. GUIDELINES FOR PRODUCERS

The Fish Health Protection Regulations (FHPR) apply to all facilities from which live and dead cultured fish, fertilised eggs and gametes of cultured and wild fish, and products of uneviscerated dead cultured fish of all species belonging to the family Salmonidae, as designated in Schedule I, which will be shipped from one province/territory to another. "Facilities" include all sites used for the propagation or holding of eggs and/or fish. Persons wishing to transfer fish between provinces/territories within Canada should become familiar with all of the regulations (federal, provincial and municipal) of the region into which they plan to carry fish since requirements may exist in addition to the Fish Health Protection Regulations.

An interprovincial carrying permit is required for each shipment of fish or eggs designated above, except for interprovincial shipments of dead cultured fish within Canada. These interprovincial carrying permits may be issued only for shipments from facilities with a valid Fish Health Certificate indicating that the facility has been inspected by an approved Fish Health Official and has satisfied the inspection requirements detailed in Section B - REQUIREMENTS TO OBTAIN A FISH HEALTH CERTIFICATE below. A list of Fish Health Officials approved for the inspection of facilities may be obtained from the National Registry of Aquatic Animal Health, Fisheries and Oceans Canada, Ottawa.

Anyone wishing to obtain a Fish Health Certificate for their facility within Canada must provide fish at their own expense for inspection. Fish species, other than those listed in Schedule I that are being reared at the facility, are also subject to sampling by the Fish Health Official. The Fish Health Official has flexibility in establishing time and frequency of sampling and selection of fish and must have access to records relating to introductions, losses, disease prevalence and treatments.

Although not specified in the Regulations, surface disinfection of eggs prior to shipment is strongly recommended unless otherwise advised. A suggested procedure for disinfection is given in Section XII of this manual.

A Fish Health Official may wish to alter inspection procedures and/or Fish Health Certificate issuance conditions when circumstances do not allow for a straightforward application of the regulations and/or this Manual of Compliance (e.g. specialized fish growing operations, research facilities, seasonal operations, etc.). In these specialized cases, a note must be made on the Fish Health Certificate.

A fish health diagnostic laboratory may be located in a different province from that in which an inspection is being conducted, requiring the transport of fish samples across provincial boundaries. Such samples should be handled so as to avoid any potential transfer of disease. All persons involved in fisheries research must conform to the requirements and intent of the Regulations and be cognizant of regional conditions.

A. MOVEMENT OF EGGS AND FISH

Before shipment, an interprovincial carrying permit must be obtained from the Local Fish Health Officer of the province/territory to which the shipment is going. Interprovincial carrying permits must accompany all shipments of fish or eggs. Offices at which the Local Fish Health Officers can be reached are listed in Appendix 2.

To acquire an interprovincial carrying permit, producers must provide a copy of their Fish Health Certificate (Appendix 4) with the Source facility DECLARATION portion completed and signed by the owner or manager of the source facility. The Receiving facility INFORMATION portion must also be completed and contain the signature and address of the receiving recipient.

An interprovincial carrying permit allows the transport of fish or eggs from the source facility directly to the receiving facility. The replenishing of water in a truck or other shipment container at any location other than another facility with a Fish Health Certificate of equal or better fish health status or an isolated water source free of all species of fish is the same as an introduction of fish from a source without a valid Fish Health Certificate and invalidates the interprovincial carrying permit.

Movement of Disinfected Eggs of Wild and Cultured Fish

Sources of eggs must be inspected by a Fish Health Official (FHO) and a Fish Health Certificate issued noting the presence or absence of filterable replicating viral agents, including their strains/serotypes if required by the recipient province. Viral agents include, but are not limited to:

  • Viral hemorrhagic septicaemia virus (VHSV)
  • Infectious Hematopoietic Necrosis Virus (IHNV)
  • Infectious Pancreatic Necrosis Virus (IPNV)

The Local Fish Health Officer (LFHO) may, after reviewing the Fish Health Certificate issue an Interprovincial carrying permit if the movement of such eggs will not result in the introduction of a viral agent or strain/serotype of a viral agent listed above and not already known to occur in the receiving province.

In the absence of fish health data indicating the presence of selected pathogens, the LFHOs may designate areas in a province as free of selected pathogens, even though the pathogens have been detected in other parts of the province. LFHOs may reject applications to move eggs from a source that tested positive for a selected pathogen into an area designated free of the specified pathogen. The eggs must be accompanied by an interprovincial carrying permit issued by the LFHO in the receiving province.

Sources of disinfected eggs need only provide information on virus testing when applying for Interprovincial carrying permits. However, when only information on virus testing is provided, eggs must be disinfected at the source and again in the receiving facility. If information on all diseases or disease agents listed in Schedule II of the FHPR are provided (i.e. including bacteria and parasites), and the Source is shown free from those diseases, disinfection of the eggs is not required.

Movement of Live Cultured Fish

Sources of live cultured fish must be inspected by a FHO and a Fish Health Certificate issued noting the presence or absence of diseases/disease agents listed in Schedule II of the FHPR including filterable replicating viral agents, including their strains/serotypes, if required by the receiving province/territory. Viral agents include, but are not limited to:

  • Viral Hemorrhagic Septicemia Virus (VHSV)
  • Infectious Hemaotpoietic Necrosis Virus (IHNV)
  • Infectious Pancreatic Necrosis Virus (IPNV)

Schedule II pathogens also include:

  • Aeromonas salmonicida
  • Yersinia ruckeri
  • Myxobolus cerebralis
  • Ceratomyxa shasta

The LFHO may, after reviewing the Fish Health Certificate, issue an interprovincial carrying permit if the movement of such fish will not result in the introduction of a disease agent or strain/serotype of a disease agent listed above not already known to occur in the receiving province/territory.

In the absence of fish health data indicating the presence of selected pathogens, LFHOs may designate areas in a province as free of selected pathogens, even though the pathogens have been detected in other parts of the province. LFHOs may reject applications to move cultured live fish from a source that has tested positive for a selected pathogen into an area designated free of the specified pathogen.

If the fish at the source are clinically ill with Schedule II disease agent(s), no fish should be transferred until the disease episode is determined by a veterinarian to be controlled. The fish must be accompanied by an interprovincial carrying permit issued by the LFHO in the receiving province.

Receiving a shipment from outside Canada

FHPR facilities wishing to import fish or eggs from outside of Canada, must meet the import requirements as set out by the Canadian Food Inspection Agency. Facilities wishing to maintain their fish health certification under the FHPR will also be required to demonstrate that each shipment meets any additional requirements for diseases listed in the Fish Health Protection Regulations which are not listed in the CFIA import permit. Fish Health testing results covering the diseases not regulated by CFIA, but listed in the FHPR, must be provided for each shipment to the Fish Health Official by the importing facility. Failure to provide fish health testing data for those diseases not covered by the CFIA import permit, to the level as outlined within this manual will result in the invalidation of the FHPR Fish Health Certificate by the FHO.

It is also the responsibility of the importing facility to verify that any other regional or provincial conditions and/or regulations have been met.

Dead, Uneviscerated Cultured Fish

Sources of dead, uneviscerated cultured fish must be inspected by a FHO and a Fish Health Certificate issued noting the presence or absence of disease agents listed in Schedule III, including:

  • Viral Hemorrhagic Septicemia Virus (VHSV)
  • Myxobolus cerebralis

The LFHO may, after reviewing the Fish Health Certificate, issue an interprovincial carrying permit if the movement of such dead, uneviscerated cultured fish will not result in the introduction of a disease agent or strain/serotype of a disease agent listed above and not already known to occur in the receiving province.

In the absence of fish health data indicating the presence of selected pathogens, LFHOs may designate areas in a province or territory as free of selected pathogens, even though the pathogens have been detected in other parts of the province/territory. LFHOs may reject an application to move dead, uneviscerated cultured fish from a source that tested positive for a selected pathogen into an area designated free of the specified pathogen.

FACILITY SAMPLING FOR CERTIFICATION

These guidelines are meant to optimize the detection of infected fish for emerging and reportable pathogens within a facility. Best sampling practices are dependent on several determinants including the type of facility (i.e., open, flow-through, closed, well-source, recirculation), the diseases that are being screened for, and the availability of informative data on those diseases of concerns.

GENERAL GUIDELINES

In order for fish, eggs or sperm to be considered for movement between provinces the product must originate from a FHPR certified facility. Requirements to obtain and maintain FHPR certificates are outlined below. If facility type does not fall under the criteria section B - REQUIREMENTS TO OBTAIN A FISH HEALTH CERTIFICATE, four consecutive satisfactory inspections over a period of not less than 18 months must be conducted. It is recommended, but not mandatory, that two inspections be conducted per calendar year. Under normal circumstances, the four inspections should be conducted at intervals of not less than 90 days and not more than 270 days. Note that in the case of egg shipments, only viral testing is required, and Schedule II bacterial and parasitic disease agents are not of concern. Furthermore, with the exception of the regions where the parasite Ceratomyxa shasta is known to be present, the routine screening of Ceratomyxa shasta (examination of stained smears of intestinal material) is not required, and the testing is only required for fish that shows clinical signs of infection.

Background information for all the lots being sampled should be obtained. This includes data relating to use of vaccines and chemotherapeutants (name, dosage, treatment duration), and the lots sampled (i.e. species, number of fish on hand, age, origin, source documentation including a copy of the valid Fish Health Certificate, and Interprovincial carrying permit if applicable).

The Fish Health Official has flexibility in establishing the time and frequency of sampling and selection of fish which provides the greatest opportunity of detecting any reportable disease agent, and must have access to records relating to introductions, losses, disease prevalence and treatments for the lots being sampled.

Sample selection from multiple cages/tanks/buildings/sites that meet single lot definition for FHPR certification should prioritize, if available: moribund fish, surface swimmers/air gulpers /fish with ectoparasites, gross lesions / exophthalmia / fin erosion / etc, fish at intake/outflow, downstream cage/tank/site, and central cage/tank/site.

Fish samples must be handled rapidly so as to minimize degenerative changes. Samples should be kept cold, but not frozen, and arrive at the laboratory for testing such that testing will be initiated within 72 hours from the time of collection. If samples cannot be brought to the laboratory alive, they should be stored on ice or refrigerated for no longer than 48 hours.

Fish samples should be placed in strong, sealed plastic bags (dead or moribund fish should be kept separately from healthy fish) which are packed in an insulated metal or plastic container with a layer of ice, or ice packs (especially for air shipments), around each bag. When reproductive fluids are being collected, as many of the samples as possible should be from female fish. Male (seminal) and female (ovarian) fluid samples should be shipped in sealed insulated containers on ice. Do not mix seminal and ovarian fluid samples.

Procedures must be adopted to ensure that samples are kept separate from each other, to prevent cross-contamination, tampering, and to eliminate errors in matching results of tests with samples.

The following precautions are recommended to ensure continuity of evidence.
  • Samples should be labelled immediately after they are collected.
  • The labels should be coded in order to track date and time of collection, facility name and, species, size or life stage, facility type or identification number, name of collector and sample number.
  • Documented records should be maintained with date and time of collection, facility name and species, size or life stage, facility type or identification number, name of collector and sample number of all samples collected. Alternatively, the Sample Submission Sheet (Appendix F) can be used as continuity of evidence. Both the producer and the Fish Health Official (or his /her designate) should sign the documentation, or the Sample Submission Sheet, to verify collection of the sample.
  • Samples should be kept in sealed containers between the time of collection and the time of arrival in the laboratory.
  • The samples and record of the samples should be transferred together when samples are passed from one person to another. Both persons must sign the documentation when such transfers take place. An authorized person from the receiving laboratory must sign the documentation when the samples arrive in the laboratory.
  • When possible, the signature of an authorized person shipping the sample should appear on the shipped package such that any tampering with the package will be evident to the receiver. Application of clear wrapping tape over the signature may serve this purpose.
  • Labelling codes in the laboratory should match those assigned to samples collected in the field.
  • If samples are collected from more than one facility, they must be kept separate at all times during transportation and processing in the laboratory. If this condition cannot be met, the sampling of only one facility is allowed.

B. REQUIREMENTS TO OBTAIN A FISH HEALTH CERTIFICATE

Aquaculture Facilities within Canada

i) Land-based facilities using ground water or water supplies free of all species of fish

  • An existing facility, with fish of unknown pathogen status, must have four satisfactory inspections, as outlined in the general guidelines, to receive a Fish Health Certificate.
  • A new facility starting with stocks from a source(s) with a valid Fish Health Certificate(s) can obtain a Fish Health Certificate after one satisfactory inspection. The Fish Health Certificate must reflect the disease agent profile of the source facility plus the results of the one inspection at the receiving facility.
  • Once a facility has received a Fish Health Certificate, one annual satisfactory inspection will be required to maintain the Fish Health Certificate. If the facility has however moved fish in from an uncertified source, it will be required to undergo four consecutive satisfactory inspections, as outlined in the general guidelines, to regain certification.

ii) Land-based Facilities Using Surface Water

  • Four consecutive satisfactory inspections as detailed in the general guidelines are required for the issuance of a Fish Health Certificate.
  • Because the pathogen status of facilities using untreated surface water cannot be maintained with certainty between inspections, such facilities will continue to be inspected ideally twice a year with inspections being no less than 90 days and no more than 270 days apart to maintain their certified status.

iii) Cage Culture Facilities

  • Four consecutive satisfactory inspections, as detailed in the general guidelines, are required for the issuance of a Fish Health Certificate.
  • Because the pathogen status of cage-culture facilities cannot be maintained with certainty between inspections, such facilities will continue to be inspected ideally twice a year with inspections being no less than 90 days and no more than 270 days apart to maintain their certified status.

iv) Eggs of Wild Fish

  • Wild adult broodstock must have a record of two consecutive annual inspections before a Fish Health Certificate may be issued. Where it is not possible to sample the same populations in consecutive years (e.g. Pacific salmon), broodstock from the same area of the river must be tested in the second year.

Upgrading Fish Health Certificates

Facilities wishing to upgrade their Fish Health Certificate from positive to negative for a Schedule II pathogen will be required to implement a programme to eliminate the specific pathogen(s) and have four consecutive negative (for specific pathogen(s)) inspections over a minimum period of 18 months. Inspections must not be less than 90 days or longer than 270 days apart.

C. ADDITIONAL DISEASE TESTING

If there is information indicating that a new strain or serotype of a pathogen listed in Schedules II, III and IV could be introduced into a receiving province with a shipment of eggs or fish, the LFHO in a receiving province/territory may request additional testing in accordance with regional/provincial/territorial policies or regulations to determine the strain and/or nucleic acid profile for a pathogen detected at a source facility.

If additional testing is required by the LFHO, then certain conditions for testing will apply. Additional testing must be coordinated and/or supervised by a FHO or LFHO. Tests conducted shall have a level of sensitivity and specificity comparable with other routine regulatory diagnostic tests, and test procedures must be accessible to both public and private laboratories doing health certification inspections. Additional testing must be conducted by a credible diagnostician, in a timely fashion, and with due regard to provisions for continuity of evidence.

D. APPEAL PROCEDURE

Where the LFHO does not issue an interprovincial carrying permit under the Fish Health Protection Regulations, the LFHO must provide written reasons for rejecting an application. The applicant may request a review of a rejected application by applying to the National Registry of Aquatic Animal Health for a review of the decision. The appeal application should be in the form of a letter to the Assistant Deputy Minister (ADM), Ecosystems and Oceans Science Sector, Fisheries and Oceans Canada and addressed to the attention of the National Registry of Aquatic Animal Health. The letter must be received within 30 days of receipt of the decision by the LFHO not to issue the interprovincial carrying permit.

The application must include a copy of the original application for the interprovincial carrying permit, a copy of the reason(s) given for not issuing the permit, and a list of the reason(s) for requesting a review. Any additional information that the applicant wishes to submit in support of the application must be included with the review request. The applicant must demonstrate that the decision not to issue the interprovincial carrying permit was inconsistent with the Regulations. The National Registry will coordinate the review process, establish a Review Board, participate as an advisor on the Review Board, and hold, assemble and forward documentation to the ADM.

The National Registry will forward a copy of the application to the appropriate DFO Regional Director General and, if necessary, the responsible provincial agencies with a request that the Regional Director General and responsible provincial agencies submit written comments to the Review Board with respect to the application. Written comments should include reasons for either supporting/rejecting the appeal and should be based on supporting scientific data. Peer-reviewed, scientific references should be included with the comments. Comments should be sent to the National Registry within 20 working days of receipt of the application.

The Review Board must be established within 15 working days of receipt of the application and will consist of the National Registry and three persons selected by the National Registry. The role of the National Registry on the Review Board will be advisory. The National Registry will not vote on the recommendation(s) set out in the report prepared by the Review Board.

The role of the Review Board in the review process is advisory. The Review Board provides a report, including its recommendation(s) to the ADM. The ADM makes the final decision whether an interprovincial carrying permit should be issued. The Review Board may request written submissions from anyone who, in the opinion of the Review Board, should be consulted.

The Review Board will prepare a written report to the ADM within 50 working days of the Review Board being established. In exceptional circumstances, the Review Board may request the National Registry for a reasonable extension of time to complete its report. The report will contain the recommendation(s) of the Review Board with respect to the application.

Subject to the Access to Information Act and the Privacy Act as amended from time to time, the Review Board will provide copies of the documentation forwarded to the ADM to the applicant. The documentation will include a copy of the Review Board's report, recommendation(s), the written submissions of the Regional Director General and the responsible provincial agencies, and other written submissions obtained by the Review Board as part of its review. The applicant will have 20 working days from receipt of the documentation to forward a written submission setting out comments. The submission should be in the form of a letter to the ADM addressed to the attention of the National Registry.

The National Registry will forward the documentation to the ADM for review and a final decision. The documentation will include the applicant's application for review (including any supporting documentation filed with the application), the review submissions of the Regional Director General and responsible provincial agencies and any other written submissions obtained by the Review Board in the course of its review, the Review Board's report and recommendation(s), and the applicant's written submission regarding the review board report.

The ADM will make the final decision on whether to issue an interprovincial carrying permit and will provide the decision and the reasons(s) for the decision in writing within 30 working days. The decision will be forwarded to the applicant, copied to the Regional Director General, responsible provincial agencies and the LFHO through the National Registry. If a review relates to a shipment of eggs, any juvenile fish that may hatch from these eggs before the completion of the review procedure will be subject to live fish health requirements of the amended Fish Health Protection Regulations and other related regulations and policies.

Table 1. Summary of review procedure, expressed in working days, for import permit appeals.
Action Expected Elapsed Time After LFHO sends Rejections Letter to Applicant
LFHO sends letter to applicant rejecting application to import eggs or fish Day zero
Applicant sends appeal application to National Registry. National Registry forwards appeal application to RDG and provincial agency if necessary. Day 30
National Registry establishes Review Board Day 45
DFO Regional Director General and responsible provincial agencies submit comments on appeal application to National Registry Day 50
Review Board provides report and recommendations for ADM to the National Registry. National Registry forwards report to the applicant. Day 95
Applicant provides comments to National Registry on Review Board's report and recommendations Day 115
ADM provides written decision on appeal to applicant Day 140

IV. ROLE OF FISH HEALTH OFFICIALS

The Fish Health Official (FHO) must be a qualified specialist in fish disease diagnosis, have access to a laboratory within Canada equipped to undertake the diagnostic procedures outlined in this manual, and have received approval from the Government of Canada (Appendix 3). Those seeking approval are to contact the National Registry of Aquatic Animal Health, providing a copy of their curriculum vitae, three specimens of their signature, and an outline of their laboratory capabilities. All applicants will be required to answer a knowledge questionnaire based on the Fish Health Protection Regulations and the Manual of Compliance, which will be reviewed through the National Registry. Applicants will be advised as to the outcome of their application. The FHO approval will be re-evaluated by the National Registry of Aquatic Animal Health every three years and, if appropriate, renewed for an additional 3 years.

FHOs should avoid and prevent situations that could give rise to a conflict of interest, or the appearance of a conflict of interest to ensure their impartiality and objectivity are maintained when inspecting facilities under the FHPR. This can protect FHOs from conflict of interest allegations and avoid situations of risk. Conflict of interests include situations where a FHO has a monetary or other economic investment or interest in the facility being inspected, or where the facility is owned or operated by the FHO, or by a member of the FHO's family. FHOs should not solicit or accept transfers of economic benefit, beyond the payment for services rendered, which could compromise the integrity of the FHO. When in doubt, FHOs should contact the National Registry.

Evidence that an approved Fish Health Official is not following the intent of the Regulations, i.e., to prevent the spread of infectious disease agents by careful inspection of production sources and control of infected stock movements, by wilfully disregarding either inspection requirements and/or results of inspections, can lead to temporary suspension of approval or permanent removal of approval. Actions will be determined by the National Registry of Aquatic Animal Health, Fisheries and Oceans Canada, Ottawa, Ontario K1A 0E6.

The Fish Health Official conducts an inspection by visiting the production site, viewing all parts of the site, and carrying out the procedures outlined in Sections VI-XI of this manual. The FHO should obtain information from the owner or manager of the site on identification of stocks being inspected and become familiar with records of introductions, losses, disease prevalence and treatment of fish in the facility over the past two years, or from the date of initial entrance to the FHPR inspection program. If all requirements of the Regulations are met and if the inspection schedules outlined earlier have been adhered to, the Fish Health Official may issue a Fish Health Certificate (Appendix 4). A copy of the certificate must be distributed to the owner/manager, a copy of which will accompany each application for an interprovincial carrying permit; the National Registry of Aquatic Animal Health and to the Fish Health Official.

The Fish Health Official must review import permits issued by the CFIA for international imports. The FHO must ensure that disease testing for those diseases not covered by the CFIA certificate is to the standard as outlined in this manual. If disease testing is not to the standard of this manual for those diseases not addressed in the CFIA certificate, the FHO must change the status of the Fish Health Certificate as appropriate and inform the National Registry.

If Fish Health Officials have diagnostic information which they have gathered from sampling outside of the scheduled FHPR inspections or documented diagnostic information from other reliable sources, this information is to be used in determining the facility's FHPR status along with that collected during scheduled FHPR inspections.

If the information indicating a change in the facility's status is obtained before the next scheduled inspection, an amendment to the current Fish Health Certificate must be issued to reflect the new status of the facility. The validity period for a Fish Health Certificate amended to reflect a change in health status is the same as the validity period of the original Fish Health Certificate.

In addition, Fish Health Officials must complete a Fish Health Laboratory Report (Appendix 5) and send a copy of the Lab Report to the National Registry of Aquatic Animal Health, the owner/manager of the facility being inspected to provide evidence of the inspection, and retain a copy for their own files.

If the invalidation of the Fish Health Certificate of a facility is necessary because of failure to meet FHPR requirements, then the following procedures are to be carried out by the Fish Health Official: a letter outlining the reason(s) for invalidation of the Certificate and the steps necessary to regain a Fish Health Certificate are to be sent to the owner/manager of the facility and the National Registry of Aquatic Animal Health.

V. ROLE OF LOCAL FISH HEALTH OFFICERS

Local Fish Health Officers (LFHOs), located within each region of Canada, administer these Regulations for their province/territory. Their responsibilities include a review of Fish Health Certificates and data relevant to a fish or egg source in question, a shipment of fish or eggs in particular, and the health needs of their region. Subsequent to Section 5 of the Regulations, they may issue interprovincial carrying permit to applicants to allow passage of acceptable shipments of live fish or eggs moving between provinces/territories within Canada. An interprovincial carrying permit must accompany each shipment. A standard interprovincial carrying permit is not provided with this manual since provinces or regions have developed interprovincial carrying permits in response to their individual needs.

The notifiable disease agents in Schedule IV are not listed in Schedules II or III, but are considered important by the Fish Health Official. In special cases, just cause may exist for the Local Fish Health Officer to prevent the movement of these notifiable disease agents or other fish pathogens into an area.

Local Fish Health Officers are located at offices listed in Appendix 2 and the National Registry of Aquatic Animal Health can be contacted for current information for any specific province/territory/region.

VI. SAMPLING PROCEDURES

A. CULTURED FISH

Sampling by lot

Unless otherwise noted, sampling of fish shall be according to "lot". Within the FHPR, a production lot is defined as "fish of the same species in an aquaculture establishment originating from the same spawning population and that have shared the same water supply at the source and/or at the receiving site." The same definition applies to broodstock lots. In situations where this lot definition cannot be applied, the Fish Health Official will use his/her discretion to divide the fish into lots, or sample on a facility-based level (as described below). Lot-based sampling is based on the premise that all lots have the same probability of infection (i.e., the probability of infection for the pathogens of concern is assumed equal for each lot). Therefore, all individual lots must be sampled.

Selecting the sample

i) Production fish

Production fish include all sexually immature fish being reared at a facility. A random sample of fish from a unit will yield a certain confidence of obtaining at least one infected specimen if the apparent (detectable) prevalence of infection in the population is at (or above) a specified level. Tables of sample sizes needed in populations of different sizes and different assumed minimal prevalence of infection have been published (Ossiander and Wedemeyer, 1973; Simon and Schill, 1984). However, these tables assume that the diagnostic tests being used to detect disease have perfect (100%) sensitivity (proportion of infected fish that have a positive test result) and specificity (proportion of uninfected fish that have a negative test result). The sample sizes shown in Table 2 incorporate diagnostic error by using the apparent prevalence (as opposed to an assumed minimal prevalence of detectable infection) to detect one or more infected (or diseased) fish in virtually any fish inspection situation. Different parameters are considered so that one can readily vary population size (N), apparent prevalence (AP) (estimated as a measure of the true prevalence and diagnostic accuracy presented in Table 2, and confidence level (CL), to suit his/her own application. For instance, sampling to detect very rare pathogens, or pathogens for which available diagnostic methods lack sensitivity would probably require more restrictive parameters than sampling to detect more common pathogens or pathogens for which sensitive detection methods exist.

It is recommended that a minimum confidence level of 95% and an apparent pre-valence no greater than 5% (n=581 for a > 40,000 population size) be used to establish the sample size (n) per lot needed to detect at least one test-positive fish. Less restrictive parameters may be selected if the selection is supported by relevant scientific data or fish health surveillance or certification data (as it pertains to accuracy of diagnostic test system used and true prevalence estimation, respectively). This must be noted on the Lab Report and/or appended to the Fish Health Certificate. When the sample is being withdrawn from a lot held in a single holding unit (e.g. tank, raceway, or pond), the sample must be selected to contain as many moribund and freshly dead specimens as are available.

Table 2: Sample size (n) required to detect one or more infected specimens in populations (lots) with an assumed minimum prevalence of detectable infection of 5 and 10%. Calculations are based on a 95% level of confidence. For intermediate population sizes, use the sample size for the next larger population listed (Ossiander and Wedemeyer 1973)

Table 2: Number of fish to be sampled when assumed prevalence of detectable infection is 5 or 10%
Population Size
N=
Assumed prevalence of 5%
n=
Assumed prevalence of 10%
n=
50 29 20
100 43 23
250 49 25
500 54 26
1,000 55 27
2,500 56 27
5,000 57 27
10,000 57 27
100,000 57 27
Over 100,000 60 30

ii)Broodstock

Broodstock (sexually mature fish held for reproductive purposes): the same sensitivity in detecting infected individuals must be aimed at. Sampling must be conducted once yearly at spawning time. In those species for which spawning is a terminal event, tissue samples must be collected from all fish involved up to a maximum of 60 fish. For those species that are repeat spawners, 10%2 of all spawners used up to a maximum of 30 fish, must be subjected to lethal sampling. The balance of the samples required to achieve the rate that provides a 95% probability of detecting an infected fish in a lot, assuming the minimum prevalence of detectable infection is 5%, is to be made up of reproduction fluids. Ovarian fluid must account for as many as possible of the reproductive product samples collected.

Samples should not be taken during or immediately after therapeutic treatment. Complete background information for all samples must be obtained. This includes data relating to any recent use of chemotherapeutants, the health history of the facility and the lots from which the samples were taken.

In the event that overt disease signs are noted at the time of sampling, procedures for the detection of notifiable disease agents and other pathogens should be carried out in addition to the procedures for identification of disease agents listed in Schedule II. This includes collecting and preserving additional tissues for confirmation by cell culture and molecular biology at reference laboratories.

When samples of fish or their tissues are processed in pools (rather than individually), as in the virological assays, care must be taken to process those from apparently healthy fish separately from those of freshly dead or moribund fish. For examples on sample sizes from various scenarios please refer to Appendix 1.

Facility-based sampling is meant to allow flexibility in the sampling of fish farms when sampling by this method has shown to be warranted. Facility-based sampling may be used as an alternative to lot-based sampling when both of the following requirements are met:

  1. There are a large number of small lots of valuable fish, or where a facility has a large number of lots of fish; and
  2. The current Fish Health Certificate for the facility is free of Schedule II agents.

When a FHO determines that a fish farm will be sampled using the facility-based sampling method, he/she is required to obtain clearance to do so through an advanced-ruling process coordinated by the National Registry. This clearance must be noted on the Lab Report and/or appended to the Fish Health Certificate. See Appendix 6 for examples on facility based sampling methods.

Sample size for viruses

  1. Production fish (non-broodstock fish): the sample size to be employed is one that gives a 95% probability of detecting an infected specimen in a lot, assuming the minimum prevalence of detectable infection is 5%.
  2. Broodstock (sexually mature fish held for reproductive purposes): the same sensitivity in detecting infected individuals must be aimed at. Sampling must be conducted once yearly at spawning time. In those species for which spawning is a terminal event, tissue samples must be collected from all fish involved up to a maximum of 60 fish. For those species that are repeat spawners, 10% of all spawners used up to a maximum of 30 fish, must be subjected to lethal sampling. The balance of the samples required to achieve the rate that provides a 95% probability of detecting an infected fish in a lot, assuming the minimum prevalence of detectable infection is 5%, is to be made up of reproduction fluids. Ovarian fluid must account for as many as possible of the reproductive product samples collected.

The lethal sampling of only 10% of all the spawners does not conform to the sampling rates set out in Table 2, it is intended to conserve small but valuable populations of spawners that may live to spawn again.

Sample size for bacteria

  1. Production fish must be sampled for bacterial pathogens at a rate that provides a 95% probability of detecting an infected specimen in a lot assuming the minimum prevalence of detectable infection is 5%. For routine purposes, only fish averaging 4 cm or more in fork length need be bacteriologically sampled. Reliable bacteriological sampling of fish smaller than 4 cm in fork length is technically more difficult: such fish need be sampled only when unusual and unexplained mortality rates or disease signs are observed.
  2. Broodstock must be sampled for bacteria in accordance with the sample size already outlined for the lethal sampling of broodstock for virus (see VI A.iib).

Sample size for parasites

  1. Production fish must be sampled for Myxobolus cerebralis and Ceratomyxa shasta at a rate that provides a 95% probability that an infected fish will be detected in a lot assuming the minimum prevalence of detectable infection is 5%.

    M. cerebralis: The fish must be at least 120 days of age for the test to be meaningful because the spores on which the diagnosis is based are slow to develop. In fish grown or held at temperature below 12o C, spore formation may take 9-11 months. (Taylor et al. 1973)

    C. shasta: Routine monitoring of apparently healthy fish for the presence of spores need be performed only with fish averaging at least 120 days of age. Younger fish need to be examined for C. shasta only when unusual and unexplained mortality rates or disease signs are observed.

  2. Broodstock must be sampled in accordance with the sample size already outlined for the lethal sampling of broodstock for virus (see VI A.iib).

Times of sampling and frequency

  1. Production fish: Sampling should be conducted as per section B - REQUIREMENTS TO OBTAIN A FISH HEALTH CERTIFICATE with sampling times and frequency depending on local conditions and the discretion of the Fish Health Official. Because the detection of M. cerebralis and certain viruses is best accomplished using fish of a certain age, spring and fall sampling periods (March-May and September- November) are recommended.
  2. Broodstock: Sampling will be conducted once yearly at spawning time (see VI. A.iib).

B. WILD FISH

Sexually immature fish

All sexually immature fish taken from the wild must be sampled at a rate that will give a 95% probability of detecting an infected specimen in the total catch, assuming the minimum prevalence is 5%. This sampling rate will apply to the types of pathogens (viral, bacterial, and myxosporean) mentioned earlier. The Fish Health Official may use considerable discretion when determining an appropriate sample size for wild fish.

Sexually mature fish

If wild spawners or their fertilized eggs are to be collected, seminal and ovarian fluids must be taken from all the fish involved, up to a maximum of 60 fish. In those species for which spawning is a terminal event, tissue samples must be collected from all fish involved (lethal sampling) up to a maximum of 60 fish. For those species that are repeat spawners, 10% of all spawners used or collected, up to a maximum of 30 fish, must be subjected to lethal sampling. These sampling rates apply to the types of pathogens (viral, bacterial and myxosporean) mentioned earlier. Again, considerable discretion must be used by the Fish Health Official when certifying wild stocks.

C. FERTILIZED EGGS AND GAMETES

The sampling of fertilized eggs or the gametes of fish cannot be relied on to detect disease agents listed in Schedule II. The threat to fish health posed by such eggs or gametes must, therefore, be determined by examining the disease status of the parent fish.

D. NON-SALMONID SPECIES

Other species not belonging to the family Salmonidae occurring at the same facility as salmonid species are subject to sampling by the Fish Health Official for disease agents listed in Schedule II or III.

VII. TRANSPORTATION OF SAMPLES

Fish samples must be handled rapidly in such a way that degenerative changes do not render diagnosis unreliable or impossible. If samples cannot be brought to the laboratory alive, they should be stored on ice or refrigerated for no longer than 48 hours.

A. LIVE FISH

Live fish should be transported in sealed plastic bags that have been partly filled with water and charged with oxygen. The bags should be labelled showing name of hatchery, date of inspection, lot identifier, and number of fish. The bags may then be placed with ice in insulated containers. Under these conditions anaesthetics are not usually required.

B. DEAD FISH

Fish sampled should be placed in sealed plastic bags (dead or moribund fish should be kept separately from healthy fish), labelled as for live fish and packed in an insulated container with a layer of ice around each bag.

C. REPRODUCTIVE FLUIDS

Seminal and ovarian fluid samples should be collected in sterile test tubes and shipped in an insulated container on ice. Do not mix seminal and ovarian fluid samples. For pooling restrictions see X B.4b and X C.

VIII. TREATMENT OF SAMPLES

A. AUTOPSY PROCEDURE

General Comments

The procedure outlined below is designed to facilitate the processing of large numbers of fish for the designated pathogens. Except for very small specimens, the same fish will serve as the source of tissues for the various bacteriological, virological, and myxosporean tests. The bacteriological examination must be performed first. To maximize detection sensitivity, fish must be autopsied within 72 h of sampling and all assay procedures should be initiated within this time.

External examination and sampling

Note all gross abnormalities such as body discoloration, body distension, exophthalmia, ulcers, blebs, inflammation, hemorrhagic areas, clubbed or necrotic gills, and eroded opercula, fins and caudal peduncles. Inoculate the appropriate media and prepare stained smears (as needed) with material from these lesions. Lesion tissue may also be taken for subsequent histology assay.

Internal examination and sampling

Disinfect the surface of the fish and using aseptic techniques expose the kidney. Inoculate the appropriate media and prepare the appropriate smears with kidney material.

Inspect the viscera for abnormalities. Inoculate the appropriate media and prepare smears (as needed) with material from any abnormal organs. Lesion tissue may also be taken for subsequent histology assay. Remove tissues for virological and myxosporean examinations.

B. DISPOSAL OF SAMPLES

The receiving laboratory should handle and dispose of samples and other items likely to be infectious in a manner that precludes the dissemination of disease agents. All materials such as fish carcasses or tissues, transport containers and water, microbial cultures, and contaminated equipment should be autoclaved, incinerated, or otherwise sterilized before being discarded.

IX. PROCEDURES FOR THE DETECTION OF CERTAIN BACTERIAL FISH PATHOGENS

A. SCOPE

The microorganisms covered by these procedures are divided into three categories:

  1. Those pathogens (listed in Schedule II) are considered to be limited in geographical distribution and the absence of which must be verified. These include the following certifiable disease agents:

    • Yersinia ruckeri (Enteric redmouth disease)
    • Aeromonas salmonicida (Furunculosis)
  2. Those pathogens (listed in Schedule IV) that may be ubiquitous, and if detected during a disease outbreak or systematically in the absence of clinical signs in sampled fish, are notifiable. These include:

    • Myxobacteria (e.g. Flexibacter columnaris)
    • Bacterial Kidney Disease (Renibacterium salmoninarum) *
    • Motile Aeromonas sp. (e.g. Aeromonas hydrophila)
    • Pseudomonas spp.(e.g. Pseudomonas fluorescens)
    • Vibrio spp. (e.g. Vibrio anquillarum)
      There is no longer a requirement to make, stain and examine kidney smears for Bacterial Kidney Disease (BKD) from fish collected during a Fish Health Protection Regulation (FHPR) inspection, except where fish show clinical signs of BKD. If there are suspected clinical signs of BKD, prepare and examine smears from the suspect kidney tissue, skin blebs or muscle lesions, attempt growth of R. salmoninarum on a selective BKD growth medium, and take tissue samples for possible histological examination of disease.
      If a client requires kidney smears from all fish to be screened for R. salmoninarum, for reasons over and above those of FHPR certification, this can be undertaken under an agreement between the FHO and the client. The FHO can choose whether or not to provide the diagnostic screening or to prepare slides for examination under an alternate arrangement. Clients should be informed prior to each inspection as to whether or not they require complete sample screening (i.e. examination of kidney smears) for BKD, in order to avoid the need for them to kill more fish for such screening at a later date.
  3. Those bacterial agents not listed in Schedules II and IV, which the Fish Health Official detects and determines to be associated with significant losses and/or disease signs (e.g. Edwardsiella tarda).

B. PROCEDURES

The following procedures represent the minimum requirements in bacteriological testing; they must be applied to all samples taken from lots in which there is an unusually high prevalence of disease signs and/or mortalities. In samples from apparently healthy lots the procedures need only be applied to those fish averaging 4 cm or more in fork length (see VI A. 4a).

  1. Aseptically obtain the following tissues/material and streak on the appropriate medium:
    1. Kidney tissue, preferably from sites that appear abnormal, and external/internal lesion material on Tryptic Soy Agar (TSA);
    2. Gill tissue and/or external lesion material on Shieh's medium (SH agar) or Cytophaga Agar (CA) only if gross pathology suggests a myxobacterial infection.
  2. Prepare Gram-stained smears of kidney tissue and lesion material and examine a minimum of 25 fields (900-1000x magnification). The presence of small gram-positive diplobacilli, frequently present intracellularly, is presumptive evidence for R. salmoninarum (Sanders and Fryer 1980). (Presumptive evidence is strengthened by lack of growth on TSA.)
  3. Incubate the TSA plates at 20o C for five days: examine daily for growth. Incubate the SH or CA plates at 15-20o C for five days: examine daily for growth.
  4. If gross pathology suggests a myxobacterial infection, prepare moist mounts of gill tissue and/or lesion material and examine for the presence of masses of long, thin rods. Select young representative colonies (yellow dry, rhizoid or yellow, moist, spreading) from the SH or CA plates and prepare Gram-stained smears. The presence of long, thin, Gram-negative rods, capable of gliding or creeping motility, constitutes a presumptive diagnosis for myxobacteria.
  5. Select young representative colonies from TSA plates. Differentiate the microorganisms on the basis of the following characteristics:
    1. If the cells are Gram-negative, rod-shaped, oxidase-positive, non-motile, ferment glucose (O.F. test) and usually produce a brown diffusing pigment, the isolate is presumptively A.salmonicida (Griffin et al. 1952). Achromogenic strains of A. salmonicida may occur (Evelyn 1971).
    2. If the cells are Gram-negative, rod-shaped, oxidase, indole and H2S-negative and produce an alkaline/acid (K/A) reaction in Triple Sugar Iron Agar (TSI), the isolate is presumptively Y. ruckeri.
    3. If the isolate differs from 5b.by being indole and H2S-positive and produces both acid and gas in TSI, it is presumptively E. tarda (Amandi et al. 1982).

A flow chart is given for these procedures in which additional features separating motile aeromonads, Pseudomonas spp., and Vibrio spp. are indicated.

Confirmatory testing of presumptively identified certifiable agents must be performed. For a possible exception, see IX C 2g. The serological and / or biochemical and /or molecular tests are to be used for confirmatory testing; serological and biochemical tests to be used are described in IXC 2.

Confirmatory tests for the notifiable agents may be performed at the discretion of the FHO. Identification should be based on whether or not the disease agent is associated with significant losses and/or gross pathology.

Figure 1. Differentation of bacteria isolates on TSA, SH or CA plates (click to enlarge)

C. MATERIALS3 AND METHODS

1. Primary isolation media

  1. Tryptic Soy Agar (Difco)
  2. SH Agar (Shieh 1980)
  • Agar 1.0%
  • Peptone 0.5%
  • Yeast extract 0.05%
  • Magnesium sulphate 0.03%
  • Sodium pyruvate 0.01%
  • Monobasic potassium phosphate 0.01%
  • Dibasic potassium phosphate 0.005%
  • Sodium bicarbonate 0.005%
  • Calcium chloride 0.001%
  • Citric acid 0.001%
  • Sodium acetate 0.001%
  • Barium chloride 0.001%
  • Ferrous sulphate 0.0001%
  • pH 7.0

Note: This is a modification of Shieh's medium. Neomycin (5 μg/mL) and polymyxin B (10 units/mL) can be added to SH agar to facilitate the isolation of myxobacteria by suppressing the growth of other bacteria (Fijan 1969).

  1. Cytophage Agar (Anacker & Ordal 1959)
Tryptone 0.05% Beef extract 0.02%
Yeast extract 0.05% Agar 0.9%
Sodium acetate 0.02% pH 7.2 - 7.4

2. Test Media, Reagents and Methods

  1. Cytochrome oxidase test: : Using a platinum loop, transfer some bacterial growth from an actively growing plate culture to a paper strip impregnated with the appropriate chemicals. After thorough spreading, a positive test is indicated by the development of a bright blue colour within a minute. (MacFaddin 1980)
  2. Motility: Examine log-phase cultures in wet preparations using Tryptic Soy Broth as the suspending medium. If the wet preparation method gives a doubtful result, check the results by stab-inoculating tubes of Motility Test Medium (Difco) or Glucose Motility Deep (GMD) medium (Walters and Plumb 1978).
  3. Differentiation between oxidative and fermentative carbohydrate metabolism: Perform an O.F. test (glucose) as described by MacFaddin (1980). Alternatively, inoculate tubes of GMD medium and interpret the results as described by Walters and Plumb (1978).
  4. Indole production: Perform the test for indole production (MacFaddin 1980)
  5. Triple sugar iron agar: The use of TSI agar and the interpretation of the results of this medium are described by MacFaddin (1980).
  6. Confirmatory slide agglutination test for A. salmonicida (Rabb et al. 1964) and Y. ruckeri: The agglutination test is performed by emulsifying a small amount of bacterial growth in saline (0.9% NaCl) on a clean glass slide. A loopful of antiserum is placed adjacent to the bacterial suspension and the two are mixed by gentle rocking and tilting of the slide. Prepare the appropriate positive and negative controls. A rapid macroscopic clumping of bacterial cells in the test mixture and the positive control (but not in the negative control) constitutes a positive agglutination test. Agglutination in the negative control invalidates the test. Many strains of A. salmonicida autoagglutinate. To prevent autoagglutination place the suspension in boiling water for 15 min prior to performing the slide agglutination test.
  7. Confirmatory tests for R. salmoninarum: If clinical signs or examinations of Gram-stained kidney smears suggest the presence of R. salmoninarum, retain a portion of the suspect kidney tissue and perform one of the following confirmatory tests. If such kidney tissue is unavailable, the presumptive evidence for R. salmoninarum noted earlier (IX.B.2) constitutes a confirmed diagnosis.
    1. Immunodiffusion test (Chen et al. 1974): Prepare immunodiffusion plates by adding 10 mL of medium consisting of Noble agar (1.0%), NaC1 (0.9%), and thimerosal (0.01%) to a 60 mm Petri plate. Punch a pattern of 6mm diameter wells, six of them peripheral to a centre well, so that all wells are 6mm apart. Place 0.1 mL specific antiserum in the centre well. Load the peripheral wells separately with 0.1 mL of saline (negative control), a heavy R. salmoninarum cell suspension in saline (positive control), and a 50% kidney homogenate in saline (test sample). Arrange the loading so test samples are adjacent to positive controls. Incubate the plates in a moist chamber at 15o C for 48 h. A precipitin line of identity or partial identity between a test sample and a positive control constitutes a positive test.
    2. Direct fluorescent antibody test (DFAT) (Bullock et al. 1980): Prepare a kidney smear on a clean glass slide, allow to air dry and fix for 5-8 min in acetone at 20o C. Add 1-2 drops of the recommended optimal dilution of conjugated anti R. salmoninarum serum containing a 1:150-1:200 dilution of rhodamine counterstain (Difco) (optional) to the slide and allow to react for 5-8 min at 20-25o C. Rinse the slide and wash for two min in phosphate buffered saline (pH 7.2) and air dry. Add a drop of mounting fluid (pH 9.0) to the test area, add a coverslip and examine a minimum of 25 fields under oil immersion using a microscope equipped with an ultraviolet light source. A positive control should be prepared and stained in a similar manner. The presence of small fluorescing diplobacilli of typical size and shape constitutes a positive test.
  8. Indirect fluorescent antibody test (IFAT) (Bullock and Stuckey 1975): The indirect fluorescent antibody test can be used in place of the immunodiffusion test and DFAT to confirm the presence of R. salmoninarum. Consult the reference for the proper procedure.
  9. Biochemical confirmation of A. salmonicida and Y. ruckeri:: Confirmatory testing of presumptively identified isolates of A. salmonicida and Y. ruckeri is performed using conventional media (Difco) as described by Edwards and Ewing (1972) or the API-20E miniaturized diagnostic system (bioMérieux SA 69280 Marcy l'Étoile, France) and comparing the results with those obtained for known (positive control) cultures of the disease agent.
    1. Streak bacterial growth on TSA and incubate at 20o C for 24-48 h to obtain a pure culture.
    2. Prepare and inoculate separately the suspect bacterial culture and the known culture as recommended (Difco, API-20E). When confirming presumptively identified isolates of Y. ruckeri, a saline suspension having a final turbidity equivalent to that of a #1 McFarland turbidity standard is recommended as the inoculum.
    3. Incubate the inoculated biochemical test media at 20o C for 24-72 hours. Add the necessary reagents and read the test results as recommended (Difco, API-20E.)
    4. Compare the results obtained for the suspect isolate to those of the known bacterial culture(s). For interpretation of the biochemical profiles for A. salmonicida consult the papers by Paterson (1974) and Paterson et al. (1980); and for Y. ruckeri consult the paper by Stevenson and Daly (1982).
  10. Sensitivity to the vibriostatic agent 0/129 and the antibiotic novobiocin: The test is performed on TSA plates by applying a 0/129 disk and a novobiocin (5μg) disk (Difco) to the medium that has been uniformly surface-seeded with the organism under test. After incubation at 20-22o C for 16-24 h, sensitive organisms show a clear zone around the disk. To prepare 0/129 disks, saturate Whatman antibiotic assay filter paper disks (6mm) with 0.1% 0/129 in acetone. Drain off excess solution and dry the disks at 37o C. The vibriostatic agent 0/129 (2, 4-diamino-6, 7-diisopropylpteridine) can be obtained from Oxoid Inc in Canada. A control disk impregnated only with acetone should be included to preclude the possible inhibitory reaction due to acetone.
  11. Agar block motility test for myxobacteria:
    1. Excise a 5mm square block of agar supporting a suspected myxobacterial colony. Place the block, colony side up, on a glass slide and cover gently with a cover slip.
    2. Examine the margin of the colony under high power for evidence of gliding or creeping motility.
  12. Confirmatory serological tests: Confirmation of identity of bacteria by serological tests such as slide, tube, or micro-well agglutination reactions should be carried out using standard procedures. Sera used should be standardized, preferably absorbed and appropriate for the purpose. Positive control organisms and negative control should be included.
  13. Confirmatory molecular tests: Confirmation of identity of bacteria by molecular tests such as PCR should be carried out using standard procedures. Positive control organisms and negative control should be included.

The approved methodology for detection of bacteria is based upon isolation, followed by Biochemical, serological identification or molecular detection.

X. PROCEDURES FOR THE DETECTION OF VIRUSES

A. SCOPE

  1. Any filterable agent in the fish samples that replicates intracellularly in any of the specified cell lines is certifiable whether or not it can be identified with presently available antisera or molecular techniques, and whether pathogenicity for salmonids exists or is unknown.

    Methodology is dependent upon detection of cytopathic effects (CPE) in susceptible cell cultures.
  2. Any abnormal proliferative lesions (tumours) encountered should be processed by histological methods, and the results of the histopathological evaluation reported.

B. TISSUES TO BE ASSAYED

  1. Sac fry: assay whole. When present, yolk sacs should first be removed and discarded.
  2. Fish averaging 2-4 cm in fork length: remove and discard heads, but retain gills; cut off the tails just posterior to the vents. Mince the remainder of the carcasses and assay.
  3. Fish averaging 4-10 cm in fork length: excise the gills, then eviscerate and assay the combined viscera and gills. After removal of the gills, evisceration is readily accomplished by first cutting off the head, then slitting open the body cavity from the cut end to the vent, and finally cutting and scraping to remove the viscera (including kidney).
    1. Fish averaging 10 cm or more in fork length: assay mixtures of kidney, spleen, pyloric caeca-pancreas, and gills. The appropriate relative volumes of these tissues should be 3:1:1:1, respectively. One part each of anterior, central and posterior kidney must be represented in the sample.
    2. When the fish in this size category are brood fish and reproductive fluids are to be used, as many as possible of the reproductive fluid samples must be from female fish. For maximum sensitivity, assay reproductive fluid samples individually.

C. POOLING

Tissues from a maximum of five fish may be pooled to form one sample. But when preparing pooled samples, apparently healthy fish (or their tissues) must not be pooled with dead and moribund fish (or their tissues). Where possible fish of the same size category should be pooled together.

D. PREPARING INOCULA

Samples must be processed within 72 hours of collection (see VIII A.1). Prior to and during processing, samples must be kept refrigerated or on ice, but not frozen.

  1. Solid tissues: weigh and then homogenize the tissues in a minimal volume of balanced salt solution (BSS) such as Earle's or Hanks' BSS at pH 7.6-7.8. Three methods are available:
    1. Homogenize using a stomacher.
    2. Use a sterile Ten Broeck, or homogenizer designed to allow cooling on ice during homonegization to process small fish or small amounts of tissue. Care should be taken to prevent possible aerosol dissemination of virus.
    3. Use a sterile pre-chilled mortar and pestle to grind the tissues with a small quantity of sterile sand (80-120 mesh silica) until a smooth paste is formed. Equipment used for homogenization of pooled samples within any given lot need not be sterilized between each use.
    4. After tissues have been triturated, dilute each sample extract to a final concentration of 2% tissue suspension in BSS .  Centrifuge the extracts at 2500 x g for 15 min at 4o C and aseptically filter the clarified supernatant through a 0.45 μm pore diameter membrane filter.  To avoid any appreciable loss of virus by absorption on the filter, collect the maximum possible volume of filtrate.
  2. Fluid samples: dilute 1:2 with cold Eagle's minimum essential medium (MEM) at pH 7.2-7.6. Centrifuge at 2500 x g for 15 min at 4o C. Decontaminate as in X D 1.

E. ASSAY

Cell Cultures

For virus assays to detect viruses listed under Schedule II, two of the four recommended continuous cell lines must be used: rainbow trout gonad (RTG-2), Chinook salmon embryo (CHSE-214), epithelioma papulosum cyprini (EPC) or fathead minnow (FHM). The EPC or FHM cell lines must be used in IHN virus enzootic areas. For other filterable replicating agents of concern capable of causing cytopathic effects in the cell lines of fish, cell lines may be used that are scientifically accepted and/or specified in the Manual of Diagnostic Tests for Aquatic Animals of the Office Internationale des Epizooties (OIE). Additional cell lines may be used if requested or required.

Biannually or before each inspection season all stock cell cultures should be tested and found to be free of myoplasma. Each cell type must also be tested for susceptibility to viruses enzootic to the region (i.e., state, province, or watershed). The donor cell cultures used to prepare monolayers for virus detection must be no older than two weeks.

Additional information on fish cell culture and virus information can be found in the OIE Manual of Diagnostic Tests for Aquatic Animals.

Either of the following procedures may be used

  1. Inoculation of preformed monolayers:
    1. Prepare duplicate monolayers of each of two cell lines for each sample to be tested. Plastic multidishes suitable for tissue culture (1.5 to 2.0 cm diameter wells) may be used sealed or unsealed (with the appropriate organic buffer incorporated in the medium).
    2. Use 1.0 mL per well of Eagle's MEM at pH 7.6-7.8 containing Earle's salts, glutamine, and 10% fetal bovine serum (FBS). Alternate antibacterial mixtures for use in the medium are 100 IU penicillin/mL and 100 μm streptomycin/mL, or 50 μg gentamycin/mL. The use of a fungistat (e.g. 25 IU nystatin/mL) is also permitted.
    3. Incubate the cell cultures at 15-20o C; the temperature depends upon when they will be required for the assay. At the time of inoculation the cell monolayers must be 70-90% confluent and not more than as 48 h old.
    4. The growth medium must be removed and the monolayers washed with BSS , which is removed prior to inoculating 0.1 mL of the filter sterilized sample into each well.
    5. Incubate the inoculated cell cultures at 15o C for 60-90 min. Every 20 min gently rock cultures to uniformly spread the inocula. Add one mL of Eagle's MEM containing 2% FBS to each monolayer. Note: the new medium should be of the same composition as in E 2.a ii, except for decreased FBS and the final pH must be 7.6 to 7.8.
    6. Incubate cultures at 15o C.
  2. Simultaneously applied cells and test sample:
    1. Place in tissue culture wells 1.0 mL of medium (see X E 2. a.) containing sufficient cells to produce a 70-90% confluent monolayer on attachment. Duplicate cultures of two cell lines (see X E. 1.) are required per sample.
    2. Immediately add 0.1 mL of the filter sterilized sample to each culture.
    3. Incubate cultures at 15o C.

Controls

For each batch of donor cell cultures, duplicate negative controls must be run. Negative controls must consist of cultures inoculated according to the procedure used for the assays, except that sterile BSS must be used in place of the sample. Positive controls may be used if available.

Procedures to be followed during incubation

  1. Inspect the cultures shortly after inoculation and after 24 h. Thereafter examine cultures at least every day to determine whether CPE has been produced.
  2. A sample is considered negative if there is no CPE in the cultures 14-21 days postinoculation.
  3. If CPE occurs in one or more of the cultures inoculated with the samples, the presence of a filterable, replicating agent must be verified. Filter (0.45 μm pore diameter) the culture fluid from the test well(s) showing CPE, dilute the filtrate with BSS to 10-1 and 10-3, and inoculate 0.1 mL of each of the dilutions into fresh duplicate cultures of the same cell line. If CPE is again observed, proceed with the serum neutralization test or molecular testing. If the FHO has reason to believe a particular virus is present, the FHO can request molecular testing of the initial cell culture. However, caution must be used, and this should only be done if the molecular test has been bench validated and is shown to be sensitive enough to detect the targeted virus from an initial cell culture. Note that molecular testing does not replace subculture. If a molecular test of the initial culture yields a negative result, but the subculture is positive, serum neutralization and/or molecular testing of the subculture must be performed.

F. SERUM NEUTRALIZATION TEST

Presumptive identification of the agent producing CPE may be made on the basis of clinical evidence at sampling and on the type of CPE produced. Identification is accomplished by neutralization of the agent with specific antiserum or any other approved confirmatory methods outlined in section G or detection using molecular biology. When using specific antiserum, failure to obtain any degree of neutralization using antisera prepared against known viruses will usually indicate the presence of a previously unrecognized virus or an atypical serotype of a known salmonid virus.

Procedure

  1. Use a dilution of antiserum sufficient to neutralize an equal volume of a suspension containing 102-103 TCID50 per mL of the homologous virus.
  2. Filter the fluid from a culture showing CPE through a 0.45 μm pore diameter membrane filter. Dilute the filtrate 10-2 and 10-6 with sterile BSS .
    1. Mix 0.3 mL of normal serum with 0.3 mL of each of the dilutions of sample.
    2. Mix 0.3 mL of normal serum with 0.3 mL of each of the dilutions of test sample.
    3. In the same manner, perform the serum neutralization test on the positive controls using homologous antiserum and normal serum.
  3. Incubate the reaction mixtures at 15o C for 30-60 min and then inoculate 0.2 mL of each mixture into duplicate cultures of the cell line in which the virus was isolated.
  4. Incubate the cultures at 15o C and observe for the production and inhibition of CPE. Inhibition of CPE by a particular antiserum, but not by normal serum, identifies the virus.

G. MOLECULAR DETECTION -PCR

Identification of a viral agent can be made using molecular detection with polymerase chain reaction PCR. The molecular techniques should be bench validated or follow procedures set out for specific viruses in the OIE Manual of Diagnostic Tests for Aquatic Animals. Following PCR, DNA sequencing can also be used to determine the strain of the virus in the case of known viruses. It can also be used to help elucidate taxonomy of novel virus types.

H. OTHER CONFIRMATORY METHODS

The approved methodology for detection of viruses is based upon isolation, followed by serological identification or molecular detection. Methods of confirming the identification of the cell culture isolates are not limited to the suggested serum neutralization test (X F). Other acceptable immunoserological tests may be used, including fluorescent-antibody microscopy, immunoperoxidase techniques, enzyme-linked immunosorbent assays, microtitration and microneutralization, plaque neutralization, complement fixation and immunoelectron microscopy.

XI. PROCEDURES FOR THE DETECTION OF CERTAIN PARASITES

A. SCOPE

The absence of two myxosporean disease agents must be verified. These agents are Myxobolus cerebralis and Ceratomyxa shasta. Any other parasites detected which the Fish Health Official considers important should be reported.

It is neither cost effective nor biologically justifiable to continue looking for evidence of Ceratomyxa shasta from fish that do not show any clinical signs of disease (the parasite has, thus far, never been detected in Canada east of the Rocky Mountains). Routine examination of stained smears of intestinal material taken from each fish collected for Fish Health Protection Regulations (FHPR) inspection is not required, except where the fish shows clinical signs of infection. Visual screening that reveals no clinical evidence of infection by C. shasta should be recorded by checking "not detected" in the Fish Health Certificate box. The "not tested" box is not checked off if visual screening is performed.

If a client requires C. shasta screening for reasons over and above FHPR certification, this can still be undertaken under an agreement between the FHO and the client. A Fish Health Officer can choose whether or not to provide the diagnostic screening or prepare the slides for examination under an alternate arrangement. Clients should be informed prior to each inspection as to whether or not they require complete sample screening (i.e. examination of intestinal smears) for C. shasta, in order to avoid the need for them to kill more fish for such screening at a later date.

B. PROCEDURES FOR MYXOBOLUS CEREBRALIS

  1. Fish for this procedure must be at least 120 days old, and preferably fresh. Frozen, but not formalin-preserved specimens may also be used. All glassware and equipment used in processing samples must be carefully cleaned to avoid carry-over of spores.
  2. Decapitate fish and deflesh heads after heating in water at 45-50o C until the brain has coagulated. Remove the brain (and any attached spinal cord) intact. Discard to avoid possible contamination of skull material with spores of the related parasite Myxobulus neurobius that might be mistaken for M. cerebralis.
  3. Either of the following procedures may be now used:
    1. Digestion Method4
      1. Pool up to approximately 100 g of the resulting skull material and macerate finely. Small initial fragment size will facilitate complete and rapid digestion.
      2. Place in a beaker and add 25 mL of freshly prepared pepsin digest solution (1.0g of pepsin dissolved in 100 mL of 0.5% HC1) to each gram of macerated material.
      3. Mix well, let settle for 2 min and examine a sample from the surface of the supernatant at 400-450X magnification for typical spores using phase contrast microscopy.
      4. If spores are not detected, incubate the mixture at 35-40o C for 1-1.5 h with gentle agitation. A cloudy, grayish suspension, free of large particles, should be present at the end of the digest period.
      5. Place 50mL of the suspension into conical, screw cap centrifuge tubes designed to hold 50mL. Centrifuge at 1200 x g for 15 min at room temperature. Discard the supernatant and resuspend the pellet in 1.0mL of distilled water. The contents of up to five tubes can be combined into one sample. Examine microscopically for spores as before.
      6. If no spores are detected, bring the volume of each sample to approximately 6mL with distilled water. Layer the contents of each tube onto 3mL of 55% aqueous glucose solution contained in a 12mL conical centrifuge tube. Centrifuge at 1200 x g for 30 min at room temperature.
      7. Withdraw pelleted material with a Pasteur pipette and examine at least 25 microscope fields per sample as given in XI B 3a. (iii). Observation of spores at any stage in the procedure constitutes a positive result.
    2. Plankton centrifuge method (O'Grodnick 1975 and Prasher et al. 1971)
      1. Pool up to 100 g of skull material and macerate in a blender for five min with distilled water. Up to 200 mL of water per 100 g of skull material can be used.
      2. Remove the macerated material and vacuum filter through a fine (0.5-1.0 mm) wire mesh. If the filter clogs, rinse with distilled water to clear it allowing the rinse water to mix with the filtrate. Coarse material such as large bone chips are removed by this procedure and can be discarded.
      3. Place the filtrate in a separatory funnel located to discharge into a plankton centrifuge. Run the centrifuge at high speed while adding the filtrate in slowly.
      4. Centrifuge until all water has been removed. Scrape the residue from the wall of the centrifuge and place in a small bottle. Add five volumes of distilled water. Do not dilute beyond a total of 30 mL. Cap and shake the bottle until the material is uniformly suspended.
      5. Place a drop of the suspension on a slide or hemocytometer if quantification is required.
      6. Examine at least 25 fields of the slide at 450X magnification for the presence of M. cerebralis spores using phase contrast microscopy.
  4. Confirmatory identification of M. cerebralis spores must be based on the morphological characters given by Lom and Hoffman (1971)

C. PROCEDURE FOR CERATOMYXA SHASTA

  1. Fish for the following procedures must be at least 120 days old; either fresh (preferably) or frozen specimens may be used.
  2. The preferred organs for examination for C. shasta spores are the intestine and gall bladder. Peritoneal fluid may also contain spores. Microscope slides of tissue, fluid and purulent material can be prepared and examined as required by either of two methods:
    1. Wet mounts: Prepare wet mounts by gently mixing sufficient material in one or two drops of saline (0.9% NaCl) on a standard microscope slide to give a reasonably dilute suspension, cover with a cover slip and examine at 400-450x magnification, using phase contrast microscopy.
    2. Dried smears: Smear material to be examined on a standard microscope slide, allow to air dry, stain 30-60 sec with Loeffler's methylene blue (dissolve 0.3 g of methylene blue chloride in 30 mL of 95% ethanol and add 100 mL of 0.01% aqueous KOH), rinse with water and air dry. Add a drop of immersion oil or water to each smear, cover with a cover slip and examine at 400-450x magnification for the presence of spores. The polar capsules and extended polar filaments stain an intense blue and the sporoplasm stains a pale blue.
  3. For fish less than 7.5 cm in fork length, material for examination can be expressed from the intestines. In addition, if nodular lesions are present in any tissue, or if ascetic fluid is evident, prepare smears or wet mounts of this tissue or fluid.
  4. For fish 7.5 cm or more in fork length, open the body cavity and collect material lightly scraped from the interior wall of the upper intestine or gall bladder. If nodular lesions are observed on or in any tissue, especially the pyloric caeca, or if any abnormal accumulations of fluid are found, they must be examined for spores of C. shasta.
  5. For each smear or wet mount at least 25 microscope fields must be examined for spores of C. shasta.
  6. The identification of C. shasta spores must be based on the diagnostic characters given by Johnson et al. (1979). Pre-spore stages of C. shasta may be found without accompanying spores; by themselves they are not diagnostic for the organism and their presence indicates that further samples and search for the characteristic spores should be made (Noble 1950; Yamamoto and Sanders 1979).

Additional information on identification of fish disease agents can be found in McDaniel (1979).

XII. EGG DISINFECTION PROCEDURES

Transmission of disease agents (bacteria, virus, fungus, parasites) can occur through the transfer of contaminated fish eggs disease agents present on the surface of the egg or in the water that the eggs are contained. Egg disinfection using a chemical that is applied to the water will reduce the load of disease agents on the surface of the egg and in the water. Chemical disinfection of eggs will normally not affect disease agents present inside the egg tissue. Egg disinfection is only effective when chemical disinfection is combined with a comprehensive biosecurity plan that includes the use of a clean water source to rinse and hold eggs, the use of clean (disinfected equipment) and through the establishment and maintenance of healthy brood stock.

The acquisition of clean water that is virtually free of pathogens can be achieved through a number of strategies and technologies; describing these processes is beyond the scope of this document.

The goal of the egg disinfection procedure herein is to reduce the risk of transferring pathogens present on the surface of eggs or in the water containing eggs; other FHPR and facility biosecurity strategies contribute towards reducing the likelihood of pathogens getting established inside (and outside) eggs.

Salmonid eggs are disinfected as green eggs following fertilization and water hardening, or as early eyed eggs. The following is a suggested procedure for egg disinfection, utilizing iodophors. Iodophor disinfectants are some of the most effective egg disinfectants; iodophors combine an effective pathogen killing while minimizing toxicity to the eggs and personnel handling the material. Iodophors for disinfection are usually povidone or polyalcoholic complexes of iodine in which the solubilized iodine confers its broad spectrum germicidal activity, but is not as corrosive or irritating as in its elemental form. A number of topical disinfectants of this type are available commercially in North America however, only OvadineTM and Parasite-S are registered for use to disinfect fish eggs in Canada. These products have Drug Identification Numbers (DIN) indicative of their registration status. Product label instructions outline their proper use. Some iodophor compounds (besides OvadineTM) have DIN's but are not labelled for use with fish or fish eggs (e.g., Wescodyne®). Other compounds for use in chemically disinfecting eggs may be similarly effective to that of OvadineTM but may require a veterinary prescription to use.

The following is a summary of part of the label instructions for OvadineTM with additional information also included:

A. PREPARATION OF THE DISINFECTANT

  1. Please read the complete label directions for OvadineTM; this includes the 'Directions for Use', 'Warnings' and 'Cautions'.
  2. Dilute the stock OvadineTM solution (10% povidone-iodine or 1% available iodine) to create a solution containing 100 parts per million (ppm) of available iodine. This involves mixing 10 ml of stock OvadineTM per litre of clean water. The disinfectant must be prepared in water with a low organic content to minimize loss of the free iodine. Use a plastic, glass, stainless steel or fibreglass tank for preparing the holding of the solution.
  3. Check the pH of the diluted disinfectant and, if necessary, adjust to 6.5-7.5 using 8% aqueous sodium bicarbonate (baking soda)

B. DISINFECTION PROCEDURE

  1. Use a fresh solution of diluted disinfectant.
  2. To avoid temperature shock, adjust the disinfectant solution to the same temperature as the subsequent egg incubation temperature.
  3. In the case of freshly fertilized eggs, allow eggs to water harden one hour before disinfection.
  4. Immerse water hardened green eggs or early eyed eggs in the disinfectant for 10 min.
  5. Treat approximately 2000 eggs per litre before discarding the disinfectant.
  6. Rinse eggs thoroughly in uncontaminated water after the disinfection treatment.
  7. Arrange the egg handling process to ensure that disinfected eggs do not have subsequent contact with contaminated equipment, water or personnel.

Diluted iodophors can also be used to disinfect work surfaces, utensils, nets and other equipment used during the egg taking process, but it is important to rinse thoroughly in clean, uncontaminated water following the disinfection to avoid contamination of eggs or inadvertent exposure of personnel to iodophors.

References

Amandi, A., S.F. Hiu, J.S. Rohovec and J.L. Fryer. 1982. Isolation and characterization of Edwardsiella tarda from fall chinook salmon (Oncorhynchus tshawytscha). Appl. Envir. Microbiol. 43:1380-1384.

Anacker, R.L. and E.J. Ordal. 1959. Studies on the myxobacterium Chondrococcus columnaris. 1. Seriological typing. J. Bacteriol. 78:25-32.

Bullock, G. L., B.R. Griffin and H.M. Stuckey. 1980. Detection of Corynebacterium salmoninus by direct fluorescent antibody test. Can. J. Fish. Aquat. Sci. 37:719-721.

Bullock, G.L. and H.M. Stuckey. 1975. Fluorescent antibody identification and detection of the Corynebacterium causing kidney disease of salmonids. J. Fish. Res. Board Can. 32:2224-2227.

Chen, P.K., G.L. Bullock, H.M. Stuckey and A.C. Bullock. 1974. Serological diagnosis of corynebacterial kidney disease of salmonids. J. Fish. Res. Board Can. 31:1939-1940.

Edwards, P.R. and W.H. Ewing. 1972. Identification of Enterobacteriaceae, 3rd ed. Burgess Publishing Co., Minneapolis, MN. 362 p.

Evelyn, T.P.T. 1971. An aberrant strain of the bacterial fish pathogen Aeromonas salmonicida isolated from a marine host, the sablefish (Anoplopoma fimbria) and from two species of cultured Pacific salmon. J. Fish. Res. Board Can. 28:1629-1634.

Fijan, N.N. 1969. Antibiotic additives for the isolation of Chondrococcus columnaris from fish. Appl. Micro. 17:333-334.

Griffin, P.J., S.F. Snieszko and S.B. Friddle. 1952. A more comprehensive description of Bacterium salmonicida. Trans. Am. Fish. Soc. 82:129-138.

Johnson, K.A., J.E. Sanders and J.L. Fryer. 1979. Ceratomyxa shasta in salmonids. U.S. Fish and Wild. Serv., Fish Dis. Leafl. No. 58. Washington, DC. 11 p.

Lom, J. and G.L. Hoffman. 1971. Morphology of the spores of Myxosoma cerebralis and M. cartilaginis (Hoffman, Putz, and Dunbar 1965). J. Parasitol. 57(6):1302-1308.

MacFaddin, J.F. 1980. Biochemical tests for the identification of medical bacteria, 2nd ed. Williams and Wilkins. Baltimore, MD. 527 p.

Markiw, M.E. and K.Wolf. 1974a. Myxosoma cerebralis: Isolation and concentration from fish skeletal elements – sequential enzymatic digestions and purification by differential centrifugation. J. Fish. Res. Board Can. 31:15-20.

Markiw, M.E. and K.Wolf. 1974b. Myxosoma cerebralis: Comparative sensitivity of spore detection methods. J. Fish. Res. Board Can. 31:1597-1600.

McDaniel, D. 1979. Fish Health Bluebook: procedures for the detection and identification of certain fish pathogens. Fish. Health. Am. Fish. Soc., Bethesda, MD. 118 p.

Noble, E.R. 1950. On a myxosporidian (protozoan) parasite of California trout. J. Parasitol 36:457-460.

O'Grodnick, J. J. 1975. Whirling disease Myxosoma cerebralis: Spore concentration using the continuous plankton centrifuge. J. Wild. Dis. 11:54-57.

OIE Aquatic Animal Health Standards Commission. 2011. OIE Manual of Diagnostic Tests for Aquatic Animals.

Ossiander, F.J. and G. Wedemeyer. 1973. Computer program for sample sizes required to determine disease incidence in fish populations. J. Fish. Res. Board Can. 30:1383-1384.

Paterson, W.D. 1974. Biochemical and serological differentiation of several pigment producing aeromonads. J. Fish. Res. Board Can. 31:1259-1261.

Paterson, W.D., D. Douey and D. Desautels. 1980. Isolation and identification of an atypical Aeromonas salmonicida strain causing epizootic losses among Atlantic salmon (Salmo salar) reared in a Nova Scotian hatchery. Can. J. Fish. Aquat. Sci. 37:2236-2241.

Prasher, J. B., W. M. Tidd and R. A. Tubb. 1971. Techniques for extracting and quantitatively studying the spore stage of the protozoan parasite Myxosoma cerebralis. Prog. Fish-Cult. 33:193-196.

Rabb, L., J.W. Cornick and L.A. McDermott. 1964. A microscopic slide agglutination test for the presumptive diagnosis of furunculosis in fish. Prog. Fish-Cult. 26:118-119.

Sanders, J.E. and J.L. Fryer. 1980. Renibacterium salmoninarum gen. nov. sp. nov., the causative agent of bacterial kidney disease. Int. J. Syst. Bacteriol. 30(2):496-502.

Shieh, H. S. 1980. Studies on the nutrition of a fish pathogen, Flexibacter columnaris. Microbios Letters 13:129-133.

Simon R.C and B. S. Schill 1984. Table of sample size requirements for detection of fish infected by pathogens: three confidence levels for different prevalence and various populations sizes. J. FishDis.7 (6 ): 515-520

Stevenson, R.M.W. and J.G. Daly. 1982. Biochemical and serological characteristics of Ontario isolates of Yersinia ruckeri. Can. J. Fish. Aquat. Sci. 39:870-876.

Taylor, E.L., S.J. Coli and D.R. Junell. 1973. Attempts to control whirling disease by continuous drug feeding. J. Wild. Dis. 9:320-325.

Wolf, K. 1970. Guidelines for virological examination of fishes. In: A symposium on diseases of fish and shellfish. Am. Fish. Soc., Spec. Publ. No. 5. Washington DC. p327-340.

Wolf, K. 1970. Guidelines for virological examination of fishes. In: A symposium on diseases of fish and shellfish. Am. Fish. Soc., Spec. Publ. No. 5. Washington DC. p327-340.

Yamamoto, T. and J.E. Sanders. 1979. Light and electron microscopic observations of sporogenesis in the myxosporidium, Ceratomyxa shasta (Noble 1950). J. Fish Dis. 2:411-428.

APPENDIX 1

NATIONAL REGISTRY OF AQUATIC ANIMAL HEALTH

The National Registry of Aquatic Animal health is a data centre for the documentation and Dissemination of information pertaining to fish diseases outlined in the Fish Health Protection Regulations within Canada. Fish Health Laboratory Reports of all disease examinations in Canada, not only those specifically for certification purposes but also those from routine periodic sampling programs should be submitted routinely to the Registry by Fish Health Officials.

The National Registry of Aquatic Animal Health will:

  1. Serve in conjunction with CFIA as the lead in the event of national fish health emergencies;
  2. Provide health histories of sources of live fish and eggs
  3. Maintain and provide lists of certified Canadian fish production facilities under the FHPR;
  4. Maintain and provide lists of Local Fish Health Officers and Fish Health Officials approved by the Minister;

The address for the Registry is:

The National Registry of Aquatic Animal Health
Fisheries and Oceans Canada
Ottawa ON Canada
K1A 0E6

Tel. 613-949-7522 Fax: 613-993-7665 E-mail: nrfd@dfo-mpo.gc.ca

APPENDIX 2

REGIONAL ADMINISTRATIVE AUTHORITIES

The Fish Health Protection Regulations are administered and applied by various provincial and regional authorities. In some areas provincial authorities are involved while other areas are a federal responsibility. The addresses follow:

PROVINCIAL FISHERIES AUTHORITIES
ALBERTA Alberta Fish and Wildlife
Fisheries Management Branch
Sustainable Resource Development
6909 - 116 Street
Edmonton, AB T6H 4P2
Tel.: 780-427-8288
MANITOBA Manitoba Water Stewardship
Box 20
200 Saulteaux Crescent
Winnipeg MB R3J 3W3
Tel.: 204-945-7789
ONTARIO Ontario Ministry of Natural Resources
Fish and Wildlife Branch
300 Water Street
Peterborough, ON K9J 8M5
Tel: 705-755-1928
QUEBEC Direction du développement de la faune
Société de la faune et des parcs du Québec
Édifice Marie-Guyart, 11 étage, boîte
92
675, boul. René-Levesque Est
Québec
QC G1R 5V7
Tel : 418-521-3875 Ext. 4496
SASKATCHEWAN Saskatchewan Ministry of Environment
Fish and Wildlife Branch
3211 Albert Street, 2nd Floor
Regina, SK S4S 5W6
Tel.: 306-787-2467

REGIONAL FEDERAL FISHERIES AUTHORITIES
ONTARIO, MANITOBA,
SASKACHEWAN, ALBERTA,
NUNAVUT TERRITORY
Fisheries and Oceans Canada
Freshwater Institute
501 University Avenue
Winnipeg MB R3T 2N6
Tel: 204-983-0607
NEWFOUNDLAND Fisheries and Oceans Canada
Newfoundland Regional Office
P.O. Box 5667
St. John's NL A1C 5X1
Tel: 709-772-2891
NORTHWEST TERRITORIES Fisheries and Oceans Canada
42043 Mackenzie Hwy
Hay River, NWT X0E 0R9
Tel: 867-874-5575
NEW BRUNSWICK
NOVA SCOTIA
PRINCE EDWARD ISLAND
Fisheries and Oceans Canada
Gulf Region
P.O. Box 5030
Moncton, NB E1C 9B6
Tel: 506-851-3107
YUKON & MARINE AREAS OF
BRITISH COLUMBIA
Fisheries and Oceans Canada
Pacific Biological Station
3190 Hammond Bay Road
Nanaimo BC V9T 6N7
Tel: 250-756-7072

APPENDIX 3

QUALIFICATIONS OF FISH HEALTH OFFICIALS

A Fish Health Official carries the burden of a great deal of responsibility and therefore must be knowledgeable in the field of fish health and thoroughly familiar with the methodology for diagnosing fish diseases and their causative infectious agents. The following criteria represent the minimum requirements for acceptance by the Canadian Government as a Fish Health Official.

A. EDUCATION AND EXPERIENCE

  1. Bachelor's degree or equivalent in any field of fisheries or biological science and two years' experience in the diagnosis of fish diseases, including experience in bacteriological, virological, and parasitological techniques.
  2. Veterinary, master's, or doctoral degree or equivalent with evidence of training in bacteriology, virology, and parasitology and one year's experience in the diagnosis of fish diseases.

B. LABORATORY FACILITIES

Laboratory facilities adequate for microbiological and parasitological evaluation of samples must be available to the proposed Fish Health Official.

C. APPLICATION PROCEDURE

To be considered for approval as a Fish Health Official, a candidate must submit curriculum vitae, three specimen signatures, and a description of available laboratory facilities and equipment to the National Registry of Aquatic Animal Health, and complete a knowledge based questionnaire.

APPENDIX 4

FISH HEALTH CERTIFICATE

The fish health certificate has been revised.

Fish Health Certificate (PDF)

APPENDIX 5

FISH HEALTH LABORATORY REPORT

This report form has been revised. An example is attached to assist you with completing the new form. The form allows detailed review of the diagnostic methods and results. The additional information recorded can be used by the Local Fish Health Officer in reviewing an application for an interprovincial carrying permit. Even though a range of tests is listed, the methods of the Manual of Compliance and its amendments are the standard upon which Canadian interprovincial carrying permits are based. The change allows flexibility to use this Laboratory Report form for fish health certification purposes other than FHPR, e.g. for OIE-based trade requirements.

Fish Health Protection Regulations Laboratory Report (PDF)

APPENDIX 6

Scenario #1.

Equal intensity sampling. For any given lot, equal intensity sampling is only recommended when it is assumed that all units within a lot have the same probability of being infected. In the absence of such information, the greatest probability of detecting infected fish in the facility is to assume that all groups either have the same likelihood of being infected. In this case, the optimal sampling strategy is to sample each group/unit with equal intensity (i.e., equal sample sizes).

For any given lot split among two or more holding units, approximately equal numbers of fish should be selected from each individual holding unit.

For example, if a facility is comprised of the following holding units:
Lot ID Species # of fish age class
1 Coho salmon 250 brood (terminal spawners)
2 Coho salmon 35,000 17 months
2 Coho salmon 40,000 3 months
2 Coho salmon 47,000 7 months

For a 95% confidence level and apparent prevalence of approximately 5% (and >40,000-population size), 60 need to be sampled on every production and broodstock lots. For lot#1, 52 Coho brood fish should be sampled (tissue samples must be collected from all fish). For lot#2, 60 Coho salmon should be sampled: twenty (20) seventeen month old, 20 three month old and 20 seven month should be collected. For facilities that hold more than one species, the same process is repeated for each species.

Scenario #2.

Sampling relative to age class. It is assumed, based on available data, that certain age class (given a lot has more than one unit, and units are representative of age class), have a greater probability of infection for the pathogen(s) of concern (e.g. for viral agents, brood and sac fry may be the most likely to show the pathogen. For other pathogens such as Myxobolus cerebralis fish in the mid-age range may be the most useful.). These units should be sampled accordingly to their probability of infection.

For example, if a facility is comprised of the following holding units:
Lot ID Species # of fish age class
1 Rainbow trout 280 brood (non-terminal spawners)
2 Rainbow trout 35,000 10 months
2 Rainbow trout 40,000 3 months
2 Rainbow trout 47,000 17 months

For a 95% confidence level and apparent prevalence of approximately 5% (and >40,000-population size), 60 need to be sampled per production lot, assuming that for the pathogens of concern, scientific knowledge demonstrates that the younger production fish are at greater risk of infection (all other sources of risk are considered equal). For lot#1, 28 (10% of population size up to 30) brood fish should be sampled plus 27 reproductive fluids (balance of sample required to make up a total sample of 55 based on a lot size larger than 250 and smaller than 500 fish, as per Table 2). For lot#2, 60 three months old and 0 seventeen and ten months old should be collected (sampling of production fish should be preferably targeted to those younger than nine months of age). For facilities that hold more than one species, the same process is repeated for each species.

Scenario #3.

Sampling proportional to group size. It is assumed, based on available data, that the larger the group, the greater the probability of infection for the disease(s) of concern (fish are to be sampled as in scenario#2, except that stratification should be done according to group size as opposed to age class).

For example, if a facility is comprised of the following holding units:
Lot ID Species # of fish age class
1 Atlantic salmon 280 brood (non-terminal spawners)
2 Atlantic salmon 25,000 10 months
2 Atlantic salmon 60,000 3 months
2 Atlantic salmon 135,000 17 months

For a 95% confidence level and apparent prevalence of approximately 5% (and >40,000-population size), 60 need to be sampled per production lot. Assuming that for the pathogens of concern, scientific knowledge demonstrates that the younger production fish are at greater risk of infection (all other sources of risk are considered equal). For lot#1, 28 (10% of population size up to 30) brood fish should be sampled plus 27 reproductive fluids (balance of sample required to make up a total sample of 55 based on a lot size larger than 250 and smaller than 500 fish, as per Table 2). For lot#2, 60 seventeen months old and 0 three and ten months old should be collected. For facilities that hold more than one species, the same process is repeated for each species.

FACILITY-BASED SAMPLING

In the proposed sampling regime, a sample size of "n" fish is collected at each facility inspection (note that not all of the fish required to achieve the recommended sample size need to be collected at the same time). Unless knowledge/data indicates otherwise, the greatest probability of detecting infected fish in the facility is to assume that all groups have the same likelihood of being infected. In this case, the optimal sampling strategy is to sample each group with equal intensity (i.e., equal sample sizes; in other words, only the first requirement above is met).

The sampling regime is based on the objective of detecting at least one fish infected with a pathogen of concern, assuming a more conservative apparent prevalence of infection of 2% (to adjust for diagnostic error) and confidence level of 95% (refer to Table 2 of sample sizes). Based on purely statistical grounds, the new unit of concern (hatchery rather than lot) allows making more conservative assumptions (same level of confidence and lower level of detectable prevalence), without increasing the number of fish required to be sampled.

The sample should consist of broodstock and production fish (the production fish should be preferably younger than 9 months). The ratio of production fish to broodstock does not have to be fixed, but should range somewhere between 50%: 50% to 70%: 30%.

The total number of broodfish to be sampled should be divided equally (unless detection data for the pathogens of concern show otherwise) among all species present on the facility. The number of production fish should also be divided equally among all species present on the facility. If a lot is split among two or more holding units, approximately equal numbers of fish should be selected from each individual holding unit (again, unless detection data for the pathogens of concern show otherwise).

For example, if a facility is comprised of the following holding units:
Lot ID Species # of fish age class
1 Atlantic salmon 250 brood (repeat spawners)
2 Brook trout 130 brood (repeat spawners)
3 Brook trout 35,000 4 months
4 Lake trout 40,000 18 months
4 Lake trout 20,000 2 months

For a 95% confidence level and apparent prevalence of approximately 2% (and >40,000-population size), 150 fish need to be sampled (refer to Table 2). If one chose the 70% production: 30% broodfish ratio, 38 brood fish (10% of all spawners) and 7 reproductive fluids (balance to make up to required 45 broodfish samples as per the 30% ratio of 150 fish) and 105 production fish need to be sampled from the facility. Of the 45 brood fish, 23 fish should be sampled from lot#1 and 13 fish and 9 reproductive fluids (for a total of 22) from lot#2. Of the 105 production fish, 53 would be collected from lot#3, and 52 from lot #4 (26 from each of the two fish from each of the two holding units respectively).

On the other hand, if the same facility is sampled only for viruses (for egg testing only), given the same 70% production: 30% broodfish ratio, sampling of broodstock remain unchanged (as above), whereas, of the 105 production fish, 52 fish could (the decision to stratify remains to the discretion of the FHO) be collected from lot#3, and 53 from the two months old unit (eighteen months old) from lot #4.

It is recommended that a 95% confidence level and an apparent prevalence of 2% (in large populations) be used to establish the sample size needed to detect at least one test-positive fish.


1 can be rounded to 60 as in the OIE Code and most sampling reference manual for matters of practicality and international consistency. Therefore, consistently rounded to 60 throughout these guidelines.

2 The lethal sampling of only 10% of all the spawners does not conform to the sampling rates shown in Table 2; it is intended to conserve small but valuable populations of spawners that may live to spawn again.

3 The products specified have proven satisfactory for the purposes indicated; this, however, does not imply that other products may not be equally satisfactory.

4 The procedure outlined above is a modification of the pepsin-trypsin digest method of Markiw and Wolf (1974a and 1974b).

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