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Infectious Salmon Anemia Program Standards

USDA APHIS Veterinary Services

Maine Department of Marine Resources

Maine Aquaculture Association

Version January 2010

The U.S. Department of Agriculture (USDA)

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Persons with disabilities who require alternative means for communication of program information

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USDA’s TARGET Center at (202) 720-2600 (voice

And TDD).

To file a complaint of discrimination, write USDA, Director, Office of Civil Rights, Room 326-W, Whitten Building, 1400 Independence Avenue, SW, Washington, DC 20250-9410 or call (202)

720-5964 (voice and TDD). USDA is an Equal

Opportunity provider and employer.

The United States Department of Agriculture, (USDA), Animal and Plant Health

Inspection Services, (APHIS), Veterinary Services, (VS) Infectious Salmon Anemia

Program Standards, (ISA), hereafter referred to as the “Standards”, establishes recommended procedures for the prevention and containment of Infectious Salmon Anemia (ISA) from farm raised Atlantic salmon. These standards provide industry, accredited veterinarians, approved laboratory personnel and regulators a template to follow to meet the goals of this Federal program. Indemnity payments, an integral part of this program, may be made to producers at a set schedule provided established procedures and standards described herein are followed, and provided that adequate funding is made available. It is further understood that these standards must be open to amendment as science and circumstances evolve. These standards were originally written in 2001 by the USDA, APHIS, VS after numerous meetings, consultations, and discussions with members the Standards Committee of the Maine/USDA, APHIS, VS, ISA

Joint Working Group on ISA. This current version of the Standards resulted from recommendations put forward by the ISA Technical Board through the end of 2009.

Table of Contents (See endnotes)1

INTRODUCTION

MAJOR ISA PROGRAM COMPONENTS:

PART I. DEFINITIONS

PART II. DISEASE SURVEILLANCE AND INVESTIGATION

A. BASIC REQUIREMENTS

B. SURVEILLANCE REQUIREMENTS

C. SAMPLE SPECIFICATIONS

D. DIAGNOSTIC TESTS AND DIAGNOSTIC TESTING STANDARDS

E. APPROVED LABORATORIES

F. BIOSECURITY AUDITS

G. REPORTING

PART III. STANDARDS FOR DISEASE CONTROL ACTIONS AND MANAGEMENT

A. ISA PROGRAM CATEGORIES

B. QUARANTINES

C. INDEMNITY

D. DEPOPULATION

E. PERMITTING

F. CLEANING AND DISINFECTION

G. FALLOWING

PART IV. ADMINISTRATIVE PROCEDURES

A. SUPERVISION OF THE ISA PROGRAM

B. ACCESS TO SITES

C. ISA TECHNICAL BOARD

D. CONFIDENTIALITY

REFERENCES

APPENDIX A: FISH HEALTH AND BIOSECURITY GUIDELINES

APPENDIX B: DISINFECTION GUIDELINES

APPENDIX C: INTEGRATED PEST MANAGEMENT GUIDELINES

APPENDIX D: ISA PROGRAM CONTACTS

APPENDIX E: USDA APHIS ISA PROGRAM FLOW CHARTS

APPENDIX F: USDA SAMPLE SUBMISSION FORM

INDEX

LIST OF TABLES

TABLE 1. SAMPLE SPECIFICATIONS FOR ISA PROGRAM APPROVED TESTS

TABLE 2. ISA PROGRAM CATEGORIES AND DESCRIPTION

TABLE 3: CLEANING AND DISINFECTION LEVELS AND SPECIFICATIONS:

TABLE 4: FALLOWING BETWEEN YEAR CLASSES

Introduction

The agent responsible for ISA is a single stranded, enveloped RNA virus composed of 8 genomic segments. It is related to the influenza viruses but belongs to a distinct genus, Isavirus

(ICTVdB Index of Viruses), within the family Orthomyxoviridae. Viral particles are spherical with a diameter ranging from 45 to 140 nm (Dannevig 1995). Intracytoplasmic inclusions of

2.5 µm have been observed in infected endothelial cells. The presence of a hemagglutinin capable of binding erythrocytes of Atlantic salmon, rainbow trout, cod, and carp is believed to facilitate binding and infection of host cells (Falk et al. 1997). The virus attacks endothelial cells, producing hemorrhage in affected fish (Nylund et al. 1995). Variable gross lesions may include ascites, petechiae along the GI tract, in mesenteric fat, on the swim bladder and kidney capsule, as well as congestion and necrosis in the liver, spleen and kidneys. As its name implies, ISA causes anemia. Hematocrits in fish showing clinical disease may range from near normal to as low as 2 or 3%. Outwardly, affected fish may also show a variety of associated signs including pale gills, exophthalmia or bulging eyes (with or without hemorrhage), lethargy, and darkened skin surfaces (Evensen et al. 1991).

Distribution of the ISA virus (ISAV) is widespread. The virus has affected fish in Norway, Scotland, the Faroe Islands, New Brunswick, Canada, the Cobscook Bay area of Maine, and

Chile. Byrne et al. (1998) describes a hemorrhagic kidney syndrome in Atlantic salmon from the east coast of Canada which appears in retrospect to be the first account of ISA in North

America.

ISAV may have evolved from among wild populations in North America as is believed to be the case in European populations (Plarre et al. 2005). Sequence comparisons of ISAV isolates have identified two general subtypes, one termed North American and the other European

(Kibenge et al. 2001; Krossøy et al. 2001); molecular studies on isolates from Norway, Scotland, and Canada, appear to indicate that these two subtypes diverged more than 100 years ago (Krossøy et al. 2001). Genotype variation within the subtypes is evident, and apparent pathogenicity varies from non-virulent to virulent (Ritchie et al. 2001; Mjaaland et al. 2005;

Kibenge et al. 2006). In Maine, the first genotype of ISAV that was isolated was determined to be identical to the pathogenic New Brunswick genotype of virus described by Mullins et al.

(1998). In subsequent years, additional pathogenic genotypes were detected at sites in both

Maine and New Brunswick (Micro Technologies Inc. & New Brunswick Department of

Agriculture & Aquaculture, pers. comm.). A second genotype of ISAV (i.e. HPR0 genotype) that does not appear to cause pathology to date, clinical disease or increased mortality has been repeatedly detected by RT-PCR since 2003 at many sites in Maine and New Brunswick (Micro

Technologies Inc., pers. comm.; Cook-Versloot et al. 2004). Since the apparently non-pathogenic genotype is not currently detectable by IFAT, has not been successfully cultured to date and currently does not produce pathology or mortality, its detection solely by RT-PCR, with confirmation by genomic sequencing, does not automatically trigger disease control action parameters. Nevertheless, the increased complexity of multiple genotypes, differing impacts and regulatory responses have mandated the addition of regular sequencing protocols to the diagnostic process. As new discoveries regarding different genotypes and their pathogenicity are made, so will our view of which genotypes are pathogenic and which are apparently non-pathogenic.

The ISA virus is transmitted mainly through viral particles shed in fish mucus, feces or tissue wastes, or through direct contact with other infected fish (Rolland & Nylund 1998).

Epidemiological investigations indicate that the virus is also spread through untreated waste products and water coming from harvest operations, natural circulation of water between infected and uninfected sites, fish processing plants, and shared equipment and gear that has not been properly disinfected at marine sites (Vagsholm et al. 1994, Jarp & Karlsen 1997, Jarp

1999, McClure et al. 2005, Hammell et al. 2005, Gustafson et al. 2005, Ellis et al. 2006).

While there is no validated evidence to indicate that ISA virus is vertically transmitted (i.e.

from parent to offspring; Melville & Griffiths 1999), an investigation of ISA in first-feed

Atlantic salmon fry raises the possibility of infection at very early life stages (Nylund et al.

1999). ISA can also be transmitted indirectly. Sea lice (Lepeophtheirus salmonis and Caligus sp.) are potential pathogen vectors and may increase disease susceptibility through added stress on fish, which may result in substantial infection and mortality (Rolland & Nylund 1998).

Significant morbidity and mortality due to ISAV are specific to Atlantic salmon held in marine net pens, although experimentally the virus can infect and replicate in a variety of species, including herring (Nylund et al. 2002), rainbow trout (Nylund et al. 1997), and brown trout

(Nylund et al. 1995),. While the segment of the ISA viral genome corresponding to a primer used in RT-PCR assays has been detected in wild Atlantic salmon, neither the presence of replicating virus nor mortality have been documented in wild free-ranging Atlantic salmon. (G.

Olivier, DFO Moncton NB, Canada; P. Barbash, USFWS, Lamar Fish Health Center-- pers.

comm.). A variety of marine species may act as ISA virus carriers or reservoirs capable of infecting cultured fish. Although no disease transmission has been documented, experimental laboratory challenges have demonstrated the potential of viral infection from brown trout

(Salmo trutta L.) to Atlantic salmon through cohabitation (Nylund & Jakobsen 1995; Rolland

& Nylund 1998).

ISA virus does not infect humans or other mammals, since the virus is inactivated at temperatures below the typical mammalian internal temperatures of 37C- 40C (Falk et al.

1997). The virus is also inactivated by external heat, extreme pH changes and disinfectants containing chlorine or iodine compounds (Torgersen 1997; Torgersen 1998; Smail et al. 2004;

NB-DAFA/Washburn & Gillis report 1998).

Experience with the disease at marine sites in Europe and North America indicates that, if uncontrolled, the virus spreads variably from site to site. Anecdotal observations indicate that onset of disease may be extended by several months in marine netpens, depending on the length of time the fish have been in saltwater, water temperature, vaccination status, sea lice numbers, nutrition, site management procedures, stress placed on the fish by environmental or management conditions, and the status of the fishes’ immune system. Cumulative mortality due to the disease varies greatly from a very low percentage (near zero) to more than 50% of the fish population. Strategic management practices, including aggressive depopulation of affected cages, supported and facilitated by an effective indemnification program, have reduced actual and potential losses due to ISA (Sandi McGeachy/DAFA, Nell Halse/NBSGA; pers.

comm.). Ongoing efforts regarding ISA management at US and New Brunswick farms associate swift detection/removal of infected cages with a consequent reduction in disease spread at an affected site (Gustafson et al. 2006), compared to sites where other (or slower) management approaches are taken (McClure et al. 2004 ). In the United States, clinical disease from ISA has been confined to Cobscook and Passamaquoddy Bays, the latter being an international body of water shared by Maine and New Brunswick, Canada. It has also been documented off nearby Campobello Island (New Brusnwick) sites as well as in sites located along the International Passage abutting Deer Island in New Brunswick.

Major ISA Program Components

The guiding principles for ISA management, as for most infectious diseases, include aggressive surveillance, risk identification, reduction and avoidance, strict biosecurity, selective depopulation, and site fallowing. While elimination of the ISA virus from the marine environment may not be realistic because of the complexity of the marine ecosystem, elimination of the disease from aquaculture operations appears to be an achievable goal.

Information from Norway, Scotland, and North America has shown that the prevalence and spread of disease among cages and sites, and the number of fish lost to disease, can be variably controlled through the development and deployment of effective management techniques.

These include strict biosecurity and disinfection practices, selective age-grouping by location, the use of site-specific personnel and equipment, and ongoing surveillance programs coupled with aggressive depopulation of affected fish cages. Fish health regulatory agencies in other countries, and many companies through their Best Management Practices, have developed similar approaches to disease management for ISA.

The farmed salmon industry in Maine and maritime Canada uses highly qualified personnel experienced in all aspects of fish culture, husbandry, and health management on both sides of the international border. Most industry members have established in-house procedures for increased disease surveillance and a working relationship with aquaculture veterinarians and diagnostic laboratories to provide further technical expertise. The industry complies with mandated regulatory fish health inspections to meet import and export regulatory requirements for fish. Current requirements in Maine include health inspections for smolts before transfer from freshwater hatcheries to marine sites and lethal testing of broodstock at spawning.

Additional elective diagnostic sampling is also conducted to meet individual company management protocols and production requirements.

The industry has worked with academia and regulatory agencies to establish biosecurity protocols for aquaculture operations. Exposure to ISA risks has been assessed through a series of biosecurity audits conducted over the last 8 years by both independent auditors as well as through internal company audits. Audits were initiated in 2001 to assess biosecurity and the risk of disease transmission through shared equipment such as boats traveling between sites in

Canada and the United States or among United States’ sites. Fish Health Zones were previously established in Maine Department of Marine Resources (DMR) regulation. With the detection of ISA virus and disease in Maine an ISA Increased Risk Zone designation was established in February 2001 by DMR for Cobscook and Passamaquoddy Bays. In the event that additional areas in Maine experience clinical infection by a pathogenic genotype of ISAV, and/or develop disease, the Increased Risk Zones will be modified accordingly. To insure that objectives for ISA control are consistent across the industry, DMR established regulations to define the ISA clinically diseased areas, require mandatory ISA virus surveillance, and restrict finfish aquaculture vessel and equipment movement. In addition, these ISA Standards were developed to specify the testing procedures to confirm ISA virus, stipulate chain of custody requirements, standardize sampling requirement protocols, identify responsible inspectors, and stipulate consequent action upon detection of ISA virus. The agency has incorporated these emergency measures into the DMR Chapter 24 Regulations (see section 24.21).

In January of 2002, the Maine farmed salmon industry developed a Bay Management Program encompassing single year-class stocking, site allocation, and strategies for the transfer of fertilized eggs (and subsequent fingerlings) and smolts. In view of the uncertainty surrounding potential vertical transmission of ISA virus, the aquaculture industry has voluntarily adopted precautionary measures that apply to many areas of fish husbandry to limit this risk, including broodstock screening for ISA virus and thorough egg disinfection protocols at fertilization and water hardening. Due to the shared water resources, as well as shared personnel and equipment by companies raising fish in both New Brunswick and Maine, coordination of ISA management programs between New Brunswick, Canada, and Maine is considered essential and is an ongoing focus of attention by the regulatory authorities of both countries.

In summary, this federally sponsored ISA program consists of seven components, which include surveillance, testing, disease reporting, disease control and biosecurity, quarantine, depopulation, and indemnity (subject to availability of funding). Surveillance ensures that resources and producers’ attention will be directed at routine and regularly scheduled inspections and health assessments of fish so that ISAV infection will be quickly diagnosed when present. Testing with the best and most scientifically sound assays at an USDA APHIS-approved laboratory facilitates a prompt and accurate diagnosis. Reporting procedures ensure that once infected or diseased fish are identified, control measures can proceed rapidly.

Disease control practices such as biosecurity measures and integrated pest management, also elements required for this program, will also mitigate pathogen and disease spread. Prompt depopulation of cages of infected fish eliminates their continuing transmission risk. Finally, indemnity, when available, provides financial relief to producers while encouraging prompt reporting and compliance with all of the ISA Program Standards.

Part I. Definitions

Accredited veterinarian: a veterinarian holding a current state veterinary license who has also fulfilled the accreditation requirements of United States Department of Agriculture Animal and

Plant Health Inspection Service (USDA, APHIS).

Action Plan: a document that defines response contingencies for a particular threat such as a pathogen or disease.

Active site: a finfish lease site as designated by the Maine DMR, and at which fish are present.

Approved fish processing plant: a fish processing plant that is approved by the USDA

APHIS ISA Program Veterinarian, undergoes audits for biosecurity and treats waste-water and blood water prior to discharge into surrounding waters to sufficiently inactivate any ISA virus that may be present.

Approved laboratory: a laboratory that is approved, by USDA APHIS, to conduct official diagnostic tests for ISA virus (Reference VS memorandum 567.2).

Assays: specific diagnostic tests for the detection of pathogens.

Atlantic salmon: for this document, all strains of Salmo salar being raised or maintained under cultivation conditions in hatcheries or other freshwater facilities, or in marine net-pens.

AVIC: the USDA APHIS, Veterinary Services, Area Veterinarian in Charge responsible for the administration and management of federal animal disease programs in states.

Biosecurity: procedures designed to eliminate or lessen physical or economic losses involving farmed stocks. Risks of acquiring or transmitting pathogens are assessed and factored into a comprehensive program involving aspects of site design, stock selection, and husbandry practices, therapeutants, veterinary supervision, and many other management variables (See

Appendix A2 ).

Biosecurity audit: onsite visit to a hatchery, marine site, processing plant, vessel or facility servicing or involved with aquaculture operations to assess biosecurity and/or audit management practices for compliance with recommended or generally accepted biosecurity protocols (See Appendix A).

Bloodwater: water mixed with blood from harvested fish. This may be found in stun or bleed tanks, boat holds, container boxes, or processing plants.

Broodstock: reproductively mature fish that have been selected or used as a part of a defined breeding program and designated as broodstock no earlier than month 28 post-introduction into sea water. These fish will be separately valued as ‘broodstock’ for any indemnification purposes.

Broodstock candidates: a group of animals from which it is anticipated that the final broodstock will be selected. These fish will be separately valued as ‘production fish’ for any indemnification purposes. .

Carryover: fish from a previous year class held on a site after the subsequent year class has been placed on that site.

Cell culture: See Virology.

Clinical signs: any visual signs of disease by gross external or internal examination.

CPE: cytopathic effect, or the observable disruption of a cell culture monolayer by a virus.

Many viruses have specific types of CPE, which may be confirmed through RT-PCR, using pathogen-specific primer sequences.

Depopulation: removal of all fish of a defined fish population within a cage, lease site or other venue with the intent of eliminating a disease outbreak.

Dipnet: apparatus for removing moribund or dead fish from the surface water of cages.

Disease: a syndrome including clinical signs, impairment and/or mortality resulting from infection with a pathogen or from other causes such as water quality, environmental factors, nutrition, genetics, etc.

Diseases of Regulatory Concern: infectious diseases that have been demonstrated to cause a significant increase in the risk of mortality among salmonid populations in the State of Maine.

Diseases of Regulatory concern are defined in Maine DMR Chapter 24 Regulations (see section 24.21).

DMR: Maine Department of Marine Resources.

Fallow: complete depopulation of all ISAV-susceptible cultured aquatic organism populations within a marine site or defined management zone. Fallowing is coupled with cleaning and disinfection protocol of the depopulated area. The levels of cleaning and disinfection are established in Part III: Disease Control Actions and Management.

Fallowing time: the period of time following the complete emptying, cleaning and disinfection of an active cage or site, and prior to restocking. The start, end and duration of the fallow will be determined by the ISA Program Veterinarian. See also Part III: Disease Control Actions and

Management.

Fish Health Zones: defined marine geographic areas as designated in Maine DMR Chapter 24 regulations (see section 24.01).

Genotype: A group of evolutionarily related viruses possessing a defined degree of nucleotide sequence relatedness (The defined degree of relatedness will vary by virus and gene or region of genome being compared).

Genotyping: Determination of the genetic similarity of a particular ISAV isolate to described

ISAV isolate groupings that are based on variations in genomic sequences targeted, such as partial sequences from the segment 6 HPR region or segment 8.

Gross Pathology: any visual signs of disease in fish organs or tissues by gross external or internal examination.

Harvesting: the removal of fish from netpens, generally for transportation to a processing plant. Removal usually occurs by means of either containment in a boat hold (live or dead), or by containment in refrigerated boxes after slaughter on the site.

Horizontal transmission: infection spread from one fish to another through means including close physical proximity that facilitates contact with infected tissues or products, contaminated equipment, sea lice, untreated processing plant effluent, infected wild fish and other mechanisms.

Incubation: period of time between infection with ISAV and the appearance of clinical signs;

generally thought to be around 3 weeks but which may be variable depending on other factors.

Indemnity: compensation paid to farmers in exchange for depopulation of a fish population.

IFAT: indirect fluorescent antibody test; an assay that makes use of the binding capacity of specific antibodies to selected antigens. The IFAT makes use of a fluorescently tagged secondary antibody that binds to a primary antibody specific to the target antigen or pathogen.

Detection of the pathogen is based on evaluation of the resulting fluorescent patterns.

Import: to land on, bring into, or deposit, in any place subject to the jurisdiction of the State of

Maine from outside the State of Maine.

Industry: All entities involved in the production of marketable farm-raised Atlantic salmon, including freshwater hatchery owners or lessors, marine lease-site holders,, and processors operating in Maine.

Inspection: an on-site visit and/or a statistically-based sampling of fish on the facility and the resulting laboratory tests and inspection reports conducted by an inspector in accordance with the testing requirements and procedures set forth in this plan.

Inspector: means an APHIS-accredited, licensed veterinarian, an AFS-certified (American

Fisheries Society-certified) fish health inspector, or other persons recognized by federal or state agencies with responsibility for fish health or transfer in the state from which the fish or gametes originate.

Introduce: to import, or to land on, bring into or deposit in any place subject to the jurisdiction of the State of Maine from any restricted areas within the State of Maine.

ISA: infectious salmon anemia; the clinical disease resulting on a variable basis from infection by a pathogenic genotype of ISA virus. Signs include hemorrhage and anemia, which may result in very pale gills or reduced hematocrit. Other external signs may include exophthalmia, lethargy, epithelial darkening, petechiae (pinpoint hemorrhages) on the skin, or nothing at all.

Sero-sanguinous fluid accumulations (i.e. ascites) are often observed in peritoneal and pericardial cavities. Internally, surfaces of kidneys, livers, intestines, or swim bladders may show signs of petechiae or hemorrhage. Mortality due to ISA varies.

ISAV: Infectious salmon anemia virus; an infectious pathogen with numerous genotypes, some of which are capable of infecting several species of finfish. Morbidity and mortality have only been documented in Salmo salar.

ISA Program Site Categories: staged categories of ISA status at marine pen sites following a review of official diagnostic tests by the ISA Program Veterinarian in conjunction with input from the ISA Technical Board if necessary. The ISA Program Site Categories are defined in

Table 2.

ISA Program Veterinarian: USDA APHIS VS veterinarian assigned to manage the ISA

Program for USDA APHIS VS in Maine, and who reports to the Area Veterinarian in Charge for Maine.

ISA Risk Zone: a Fish Health Zone or subsection classified with respect to ISA risk, based upon epidemiological data and analysis.

ISA Technical Board: a group of five people consisting of the USDA APHIS ISA Program

Veterinarian, one Maine DMR representative and 2 industry representatives, plus a nominal chairperson selected by the committee representatives. The duties of the board are to make recommendations to the ISA Program Veterinarian, the USDA APHIS Area Veterinarian in

Charge (AVIC) and/or Maine DMR Commissioner after being called together to consider positive lab results, epidemiological data, audit reports or other information regarding reported disease risks or conditions requiring action under the terms of this program. A quorum will be all four voting representatives. The chairperson only votes in the case of a tie and acts as a facilitator.

Negative site: an active site testing negative for ISAV while involved with active participation in an official surveillance program, including inspection by a veterinarian (See ISA Case

Definition Category 1).

Netpens: also called “cages”; plastic or steel structures of differing sizes and shapes designed to contain variable numbers of fish.

Non-pathogenic: not capable of inducing disease (i.e. no clinical signs/gross pathology observed and no increased mortality in a population).

Official Diagnostic Test: procedures for the detection of ISAV performed by a USDA APHIS

VS approved laboratory: including 1) Indirect Fluorescent Antibody Test (IFAT), 2) Gross pathology, 3) Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and 4) Virus isolation.

Owner: any person, partnership, company or corporation with a proprietary interest in the fish culture facility being inspected.

Pathogen: an agent (bacteria, viruses, fungi, parasites, etc.) capable of causing pathological changes in tissues. Not all pathogens cause clinical disease, and not all diseases are caused by pathogens.

Pathogenic: capable of causing disease (i.e. clinical signs/gross pathology observed and increased mortality in a population).

Permit: a USDA APHIS Veterinary Services document entitled Permit for Movement of

Restricted Animals (VS form 1-27) which allows movement of animals from ISA infected or suspect premises to insure biosecurity.

Processing plant: any facility where Atlantic salmon (including products such as fillets used for value-added purposes) are taken for processing or rendering into a marketable product.

Production fish: fish destined for market or harvest.

Qualified source/hatchery: an established source/hatchery that has had 3 consecutive annual inspections in which pathogens as described in Maine State Regulations Chapter 24.21

(subsection D) have not been detected; or a new hatchery that has had 3 successive negative annual inspections over a continuous 2 year period.

Quarantine: no movement of live fish to or from the site except for processing or slaughter as approved and permitted by USDA or the Maine DMR. In addition 1) no visitors to site except necessary fish health personnel; 2) institution of a biosecurity program approved by the

Commissioner of Maine DMR; and 3) disposition of deceased and quarantined fish in a manner approved by the DMR Commissioner.

Restriction: no movement of live fish off or to the site and institution of a biosecurity program approved by the Commissioner of Maine DMR.

RT-PCR: reverse-transcriptase polymerase chain reaction, a process where RNA or DNA is multiplied in a cyclic fashion using a special, sequence-specific primer that binds to the genome of a particular organism if it is present in even extremely small numbers. The resulting amplification of the RNA or DNA of that organism is made visible by means of photographing a stained gel in which the amplified genomic fragments have been separated based on their size.

Sampler: a person designated by an accredited site veterinarian for the collection of surveillance and diagnostic samples at the direction of the veterinarian, and using an established chain-of-custody document for the respective sample. Samplers will be trained by an accredited veterinarian and approved by the USDA APHIS ISA Program Veterinarian.

Identification of the sampler must be referenced in all documents related to surveillance activities at a site. The sampler’s role is limited to completing the complement of required surveillance submissions and/or processing samples for submission.

Sea lice: copepod arthropods belonging to either the Lepeophtheirus or Caligus genera. Both species infest Atlantic salmon and live in the mucus layer, where they attach and suck blood or cause sores. Studies indicate that Lepeophtheirus may be a vector capable of transmitting ISAV to Atlantic salmon (Nylund et al. 1993; Nylund et al. 1994). Both species can cause stress on fish, which adversely affects the immune response.

Sequence: noun The order of nucleotide bases in a DNA or RNA molecule; the order of amino acids in a protein molecule; the order of amino acids translated from a DNA or RNA molecule.

verb To determine the genetic code (nucleotides or amino acid translation) of a specific portion of the ISAV genome.

Single Year-Class Site: a marine site containing only one year-class of salmon at a time. That year-class of fish is entirely removed and fallow implemented prior to introducing salmon of another year class to that site.

Site: (or ‘marine site’ or ‘finfish leased site’) a specific leased area in saltwater where fish are raised. Sites in Maine waters must be identified with the specific site identification code issued by the Maine DMR.

Site identification number: a finfish aquaculture lease site identification number assigned by the DMR and unique for each site.

Site veterinarian: a licensed and accredited veterinarian with fish health responsibility for one or more marine site.

Smolt: the stage at which Atlantic salmon are capable of physically and metabolically transforming to accommodate large osmotic differences as they move from a freshwater to a saltwater habitat.

Surveillance: a program designed to detect or monitor a pathogen or disease through periodic sampling and testing fish within cages or sites. Programs are generally formulated to establish the presence or absence of a particular pathogen or disease using a statistically defined sampling plan. The sampling plan allows an estimate of prevalence or the extent of either infection or disease in a population within a specified level of probability.

Transfer permit: a permit issued by the Commissioner of Marine Resources (DMR) that authorizes the recipient to transfer live fish, fertilized eggs or gametes to or from designated geographical area(s) in the coastal waters of Maine during a specified time period. A transfer permit may not be issued until the DMR has reviewed fish health inspection reports.

Vertical transmission: infection passing from one generation to another. True vertical infection involves a transmissible virus within the egg or sperm that cannot be eliminated by external disinfection protocols.

Virology: (aka ‘cell culture’ or ‘virus isolation’) growth of a virus on a particular cell line that is susceptible to being infected by that virus. In theory, only one virion, or virus particle, is needed for infection, growth, and replication.

Year class: grouping of a population of fish of the same age/hatch year and/or grouping by year of saltwater transfer.

Part II. Disease Surveillance and Investigation

A. Basic requirements

An ongoing disease surveillance program to facilitate early detection of both pathogen and disease is essential for effective prevention, management, and control of a wide variety of aquatic diseases including ISA. The following basic requirements for the ISA Program are necessary and mandatory for industry for effective ISA control in Maine:

a. Each industry member or company with Atlantic salmon grow-out sites in Maine will establish and maintain a veterinary-client-patient relationship with a licensed and APHIS-accredited (hereafter ‘accredited’) veterinarian. The accredited veterinarian will have responsibility for implementing all provisions of the ISA

Program Standards at participating sites under his/her supervision.

b. Accredited veterinarians will conduct surveillance on behalf of their industry clients/leaseholders, and adhere to testing and reporting procedures for ISAV/ISA as described in these Standards.

c. Individual salmon-producing companies will develop and implement biosecurity protocols for use at marine sites, processing plants (if applicable) and vessels engaged in aquaculture operations throughout Maine (See Appendix A: Fish Health and Biosecurity Guidelines). In addition to the inputs of these personnel, biosecurity protocols will be periodically reviewed by the USDA APHIS ISA

Program Veterinarian.

d. Each company shall develop a site-specific (and year-class specific, if applicable)

ISA action plan for the prevention, control and management of ISA. Involvement of the company or service veterinarian and fish site health manager is essential.

Site-specific ISA action plans will be reviewed as necessary by the USDA APHIS

ISA Program Veterinarian.

e. Industry participation in the Integrated Pest Management Program for the Control of Sea Lice on Salmonids in Maine is mandatory (see Appendix C: Integrated Pest

Management) as studies have indicated that Lepeophtheirus sea lice may be a vector capable of transmitting ISAV to Atlantic salmon (Nylund et al. 1993;

Nylund et al. 1994).

f. Industry will submit to the USDA APHIS ISA Program Veterinarian fish inventory information for every leased finfish site with site and cage identifiers. Fish inventory information must include the numbers, age, hatchery of origin, date of saltwater transfer, vaccination status, and previous therapeutant history for all fish in the unit. This will provide APHIS VS with data necessary to establish disease control actions, complete epidemiological assessments, and increase the ability to effectively monitor fish populations.

g. Industry will maintain and make available to USDA APHIS VS mortality data for each site and pen in production. This can be accomplished utilizing existing industry records systems and log sheets.

h. Sites will cooperate and assist USDA APHIS VS with the completion of biosecurity audits at marine sites, processing plants, and vessels involved in salmonid aquaculture (See Part II F).

i. Documentation required in items c, d, e, and f, above must be submitted to the ISA

B. Surveillance Requirements

Participation in the ISAV surveillance protocol as detailed in this section is mandatory for all active salmonid lease sites to become and remain eligible for indemnification under the

ISA program in the event that funds become available. Minimum surveillance activities will involve onsite inspection by an accredited veterinarian or technician under his/her supervision, as well as collection of appropriate samples for ISAV assays. In addition, USDA APHIS personnel under the direction of the ISA Program Veterinarian may collect samples as needed as part of this surveillance. The schedule of veterinary surveillance inspections depends upon each site’s specified ISA Program Site Category (See Part III. A.

Table 2 for Category Definitions). This schedule will be as follows:

Monthly: Sites meeting the criteria for ISA Program Site Category 1 in either the low or high risk zone.

Biweekly: Sites meeting the criteria for ISA Program Site Category 2 as well as all other marine sites within the same bay which are considered potentially exposed. Also, if a pathogenic detection of ISAV has been made in the lower end of New Brunswick’s

ABMA1 (around Deer Island or Campobello Island), all Maine sites in Cobscook &

Passamaquoddy Bays will increase sampling to biweekly.

Weekly: Sites meeting the criteria for ISA Program Site Categories 3 thru 5.

As Needed: The inspection schedule will be increased to resolve issues of ISA site category. Sampling frequency may also be adjusted as needed at the discretion of the

ISA Program Veterinarian and/or the ISA Technical Board.

Note: New sites, ISA Program Site Category 6 sites, or inactive sites returning to active sites must initiate surveillance activities within 30 days of introduction or transfer of salmonids to the site.

1. Veterinary surveillance inspections shall consist of visual inspections of all cages on a site, review of weekly and/or most recent mortality figures, and collection of diagnostic samples for testing as described in these Standards. Inspection reports and other documents showing surveillance activities must be maintained and made available to the ISA Program

Veterinarian.

2. The accredited site veterinarian will be responsible for personally conducting a minimum of one monthly veterinary surveillance inspection of each respective site for which she/he has fish health responsibility. This should coincide with a scheduled dive to collect mortalities.

Fish samples for ISA Program testing as described in Part II D may be collected by the accredited veterinarian or a USDA APHIS VS authorized sampler at the direction of the site veterinarian. More frequent surveillance requirements as outlined above or at the recommendation of the ISA technical board or ISA Program Veterinarian should include veterinary surveillance inspections as defined by the ISA program standard definitions.

3. USDA APHIS VS-authorized personnel may also conduct sampling as necessary to comply with these program Standards. This sampling option will be coordinated by the ISA

Program Veterinarian after consultation with the producer and their accredited veterinarian.

4. When completion of harvest of all fish at a site is anticipated within the next month, a waiver of veterinary surveillance inspections may be obtained on a case by case basis from the Program Veterinarian.

C. Sample specifications

Sampling and diagnostic evaluation of fish tissues will be accomplished in conjunction with veterinary surveillance inspections conducted as above. In cooperation with producers’ schedules, divers, and timeliness for sample submission, sites will be sampled as follows:

1. Appropriate numbers of moribund or recently dead fish will be collected per scheduled veterinary surveillance inspection. A minimum of 10 targeted fish per site should be collected whenever possible. Moribund may be interpreted to include slow-swimming, ‘pinhead’ (stunted or dwarf fish), otherwise deformed, dark or exophthalmic fish.

2. Samples are to be collected by an accredited veterinarian or technician under his/her supervision, state inspector, federal inspector, or laboratory contract personnel as appropriate. Chain-of-custody documentation on fish samples submitted for ISA Program testing under the surveillance program must be submitted to the USDA APHIS ISA

3. Samples will be tested in accordance with diagnostic testing protocols in Part II D and by

USDA APHIS-approved laboratories (See Part II E).

4. Sample specifications: Table 1 below lists sample specifications for the ISA assays to be performed: the number of fish per pool, the required tissues, and the collection technique used for each test.

Table 1: Sample Specifications for ISA Program Approved Assays: Fish Pool, Tissues and Collection Vessel

TEST

Fish Pool Tissue Collecting vessel/ Preservative/media

RT-PCR Single fish 0.25 cm3 mid kidney 2 ml tube/RNA

Later IFAT Single fish 2-3 Mid-kidney impressions Frosted end slide

Virology Single fish/cage (up to 5 fish per pool)

Kidney, spleen, heart Specimen cup/ PBS

D. Diagnostic Tests and Diagnostic Testing Standards

To insure that surveillance and diagnostic procedures are standardized throughout the industry the following diagnostic tests will be considered official. The official diagnostic tests for the

ISA Program include the following tests:

a) IFAT

b) Gross pathology

c) RT-PCR

d) Virus Isolation

All official diagnostic tests will be performed by a USDA APHIS-approved laboratory. ISA diagnostic test procedures will be performed according to the protocols established and filed with USDA APHIS VS.

Surveillance will utilize RT-PCR as the primary screening diagnostic test. IFAT impression smears will be acetone-fixed and archived. IFAT slides corresponding to any tissue sample testing positive by RT-PCR will be subsequently tested. Tissue samples for virus isolation should be collected during inspections of a Category 2 or higher numbered site at the discretion of the accredited veterinarian, the Program Veterinarian and/or the ISA Technical Board. All tissue samples and positive supernatants will be archived (for at least a year) after laboratory testing has been completed. At least the initial positive RT-PCR from each cage will be genetically sequenced. Additional or subsequent diagnostic tests on archived samples will be determined following consultation among the USDA APHIS ISA Program Veterinarian, the approved laboratory, NVSL, and the site accredited veterinarian.

On a first-time basis, samples testing positive for ISAV from previously negative sites (i.e. ISA

Program Category 1 sites) by any USDA APHIS-approved laboratory other than the National

Veterinary Services Laboratories (NVSL) in Ames, Iowa will be sent to NVSL for confirmatory testing. Archived original tissues should be submitted to NVSL for this confirmation.

Resolution of inconclusive or conflicting diagnostic tests will be determined by the USDA

APHIS ISA Program Veterinarian following consultation with the site accredited veterinarian, the approved laboratories, and NVSL. If a sample or site status remains unresolved after consultation with these bodies then referral to the ISA Technical Board is required. Issues surrounding conflicting or otherwise questionable results may be resolved through the following (independently or through combinations):

1. Additional tests performed on archived samples by the approved laboratory.

2. Additional tests on archived samples performed by the NVSL.

3. Additional samples from the site or cage with testing by the USDA APHIS-approved laboratory and/or NVSL.

4. Increased sampling frequency from the cage in question, additional cages or net pen site.

5. Consultation with the USDA APHIS ISA Technical Board.

The ISA Program Veterinarian shall consider recommendations made by the ISA Technical

Board and make final determination on all ISA Program issues.

E. Approved laboratories

All ISA program tests must be conducted at USDA APHIS-approved laboratories. Approved laboratories must report all positive test results within 24 hours of completion to the ISA

Program Veterinarian, Maine DMR and the accredited veterinarian. All reports must reference all information required in Part II G (Reporting).

F. Biosecurity audits

Biosecurity protocols as specified in the Maine Aquaculture Association’s Bay Area

Management Agreement are to be followed at all marine sites. Periodic assessments of existing biosecurity measures will be coordinated through the ISA Program Veterinarian. Audits of marine sites, fish processing plants, marine vessels, or other epidemiological components may be completed by trained and experienced personnel as assigned by the ISA Program

Veterinarian.

G. Reporting

Accurate and timely reporting of all activities related to this program is essential. Reports and documents required include but are not limited to laboratory test reports; cage/site depopulation reports; cleaning and disinfection certificates; fish inventory documents; and permits. All surveillance reports and actions regarding ISA control measures must reference the site identification code. All surveillance documents, laboratory reports, and other documents as required will be forwarded to the ISA Program Veterinarian. A flow chart reflecting the reporting mechanism is provided in Appendix E. Reporting responsibilities and requirements are as follows:

Accredited Veterinarians: Accredited veterinarians shall, in accordance with Title 9 Code of

Federal Regulations Part 161.3, report to the ISA Program Veterinarian and Maine’s DMR any suspected or confirmed cases of ISA within 24 hours of learning of the positive test results or diagnosis. Accredited veterinarians shall submit all diagnostic samples for ISA to an approved laboratory in accordance with instructions provided by the ISA Program Veterinarian or Area Veterinarian in Charge (AVIC) and shall complete any and all procedures as instructed by this program by the ISA Program Veterinarian or AVIC. Accredited veterinarians shall use either USDA APHIS Specimen Submission Form (VS form 10-4) or other approved form developed by the ISA Program Veterinarian.

The specimen submission form under this program is shown in Appendix F. It must be filled in completely and must be signed and dated by the submitting accredited veterinarian.

Approved Laboratories: The approved laboratory shall report results of surveillance laboratory tests to the ISA Program Veterinarian and the accredited veterinarian within 2 weeks of receiving the samples submitted for ISA surveillance. The approved laboratory shall within

24 hours of test completion report to the ISA Program Veterinarian, the Commissioner of the

Maine DMR, and to the accredited veterinarian all test results for any official test for ISA. If viral culture is required to resolve the results of ISA assays, the finalized report of viral culture results must submitted to the ISA Program Veterinarian within 30 days from the day of sample receipt by a USDA APHIS-approved laboratory.

All surveillance documents and laboratory reports will be forwarded to the ISA Program

Veterinarian. A flow chart reflecting the reporting mechanism is provided in Appendix E.

ISA Program Veterinarian: The ISA Program Veterinarian is responsible for overall field management of the ISA program in Maine and reports directly to the Area Veterinarian in

Charge (AVIC) for New England. The ISA Program Veterinarian may also consult with the

USDA APHIS VS Regional Director, and headquarters staff as necessary to carry out this program. Major duties and responsibilities for this veterinarian include but are not limited to:

assigning personnel as needed to carry out program goals; collating surveillance reports from industry, site veterinarians, approved laboratories; resolving the accuracy of the respective surveillance reports; determining ISA Program Site Categories; reviewing laboratory procedures; processing indemnity payment; and meeting with industry on program related issues. The ISA Program Veterinarian will, under conditions of confidentiality, have access to records of transfer permits and production information. USDA APHIS may utilize this information in epidemiological and economic investigations conducted by USDA staff or designees approved by the AVIC and for the generation of reports and information for utilization of the USDA APHIS ISA Technical Board in resolution of ISA incidents. The ISA

Program Veterinarian will be responsible for producing summaries of surveillance activities (as needed for significant changes in program or site status) for submission to: 1) the USDA

APHIS VS AVIC, 2) the Commissioner of DMR, and 3) members of the USDA APHIS ISA

Technical Board.

Part III. Standards for Disease Control Actions and Management

A. ISA Program Categories

ISA Program Categories and their descriptions are found in Table 2. The purpose of the classification system is to provide the ISA Program Veterinarian more information for sites so that further evaluation, epidemiological investigation, and testing can be designed, ordered, and initiated as appropriate.

Table 2: ISA Program Categories and Description

ISA Program Category

Category Description

Category 1 (presumed negative)

ISAV has not been detected at a cage or site participating in active ISAV monthly surveillance testing; considered negative for ISA.

Category 2 (suspect)

ISAV has been detected by at least one diagnostic test in at least one fish; considered suspect needing further evaluation and testing within 7 days.

Category 3 (infected)

A pathogenic genotype of ISAV has been detected by at least two diagnostic tests in at least two fish from the same cage. For subsequent cages on an infected site, a cage may be deemed Category 3 based on two fish found positive by one test (PCR, IFAT, gross pathology) accompanied by a positive virus isolation from individual samples…