Welcome to Standardized Procedures (QA/QC)

Where We’ve Been

Historically, fish disease diagnosis and hatchery inspections performed by the U.S. Fish and Wildlife Service Aquatic Animal Health Program (FWS-AAHP) relied upon the standard protocols presented in the American Fisheries Society- Fish Health Section “Bluebook”, titled Suggested Procedures for Detection and Identification of Certain Finfish and Shellfish Pathogens (Thoesen 1994). Today, there has been much effort and cooperation in the aquatic animal health field to promote national and international standardization for aquatic animal health. The Office International des Epizooties has long been recognized as the European Union and international authority on aquatic animal health (OIE, 2004), and the recently updated Bluebook (Fifth Edition 2003) is a comparable reference in the United States.

In 1997, the U.S. Fish and Wildlife Service initiated the National Wild Fish Health Survey (NWFHS), a congressional initiative to study fish diseases in wild fish. The purpose of the survey is to determine the prevalence and distribution of fish pathogens to gain a better understanding of aquatic animal health in natural populations. Fish Health specialists immediately understood the importance of utilizing the most sensitive detection methods, and incorporated advances in molecular diagnostics, such as the Polymerase Chain Reaction (PCR) assay, into their testing methods. The FWS Aquatic Animal Health Program also recognized the extreme importance of developing standardized procedures and laboratory methods for the 9 Fish Health Centers conducting the surveys across the country.

This necessity to impose the most sensitive testing methods and strict standardization was accomplished with the publication of the USFWS – National Wild Fish Health Survey Laboratory Procedures Manual (True, 2004). This detailed set of SOP’s addressed all aspects of the Survey from field collection, through laboratory assays, to final reporting of the data in a national database. The deviation from standard “Bluebook” protocol presented the FWS-AAH laboratories with the opportunity to field test and benchmark advanced molecular techniques developed for detection of pathogen genome segments from fish tissues. This “bench testing” effort has contributed to the validation and acceptance process of these methods for detection of fish pathogens. Annual revisions of the NWFHS Manual are accomplished by FWS fish health specialists to reflect advancing molecular techniques and continually improve the sensitivity of these detection methods.

The FWS-AAHP strong commitment to QA/QC, demonstrated in the quality of the NWFHS Manual, led to the evolution of a complete set of protocols and procedures for all aquatic animal health functions. This Handbook of the U.S. Fish and Wildlife Service – Fish Health Procedures and Protocols, is a compilation of procedure manuals that consist of the following volumes:

Volume 1: National Wild Fish Health Survey Laboratory Procedures Manual
Volume 2: Standard Procedures for Aquatic Animal Health Inspections Manual
Volume 3: Aquatic animal Husbandry Practices
Volume 4: Risk Assessment
Volume 5: Quality Assurance/Quality Control Procedures
Volume 6: Non-lethal Sampling Procedures
Volume 7: Miscellaneous Techniques and Procedures
Volume 8: Aquatic Animal Health Policies
Volume 9: Triploid Grass Carp Inspection Program
Volume 10: Sample Agreements

Concurrently, the FWS-AAHP standardization effort led to a successful collaboration with AFS-FHS to revise the Bluebook in 2003, and include a new section, Standard Procedures for Aquatic Animal Health Inspections, which is assembled by many individuals with academic and field expertise. This is a compilation of minimal standards and methodologies determined to be most appropriate for detecting the presence of specific pathogens when carrying out health inspections on aquatic animals. These inspections may be used for intrastate, interstate, or international movement of animals. However, the final decision to require the use of these protocols remains in the hands of those regulating bodies requesting/requiring health inspections. Nonetheless, the combined efforts and expertise of the United States Fish and Wildlife Service Fish Health Centers and the American Fisheries Society Fish Health Section has resulted in an impressive manual which is a major accomplishment towards national standards for aquatic animal health inspections.

The partnership of AFS and FWS-AAHP is also an important endeavor towards standardizing international trade and commerce for aquaculture in the United States and Europe. The Inspection Manual Committees are dedicated to aligning aquatic animal health inspections with the international health codes and procedures specified by the Office International des Epizooties (OIE), whenever possible. In addition, several FWS-AAH personnel are active participants on the Joint Subcommittee on Aquaculture (JSA) Task Force and Working Groups. The mission of the Task Force is to develop a National Aquatic Animal Health Plan (NAAHP) which provides for national and international commerce of aquatic animals and protection of cultured and wild aquatic animals from foreign pests and diseases, to meet U.S government trade obligations.

Current Work

The FWS-AAHP provides excellence in science and continues to be a leader in aquatic animal health in the United States. In addition to accomplishments towards national standardization, the Fish Health Centers maintain a comprehensive Quality Improvement (QI) program, which includes standardized reagents, applied studies, and continuing education and training for our fish health specialists.
The following examples of standardized procedures illustrate some of the current work, and extra measures, we pursue to ensure high quality data and excellence in the laboratory services we provide for our hatchery program and our partners.

Standardized Biologics and Reagents for all Viral Testing
Tissue culture cell lines undergo rigorous testing to comply with high standards for virology testing for our hatchery inspection program and the National Wild Fish Health Survey:

• Only use high quality, characterized cell lines from American Type Culture Collection (ATCC).
• Conduct bi-annual mycoplasma testing to ensure cell cultures perform optimally.
• Conduct annual viral sensitivity testing to ensure cell culture are sensitive to target pathogens.
• Conduct periodic Viral Workshops with USGS – Western Fish Disease Laboratory to assess sensitivity of cell lines to newly emerging viral pathogens and strains. In 2004, all cell lines used by FWS-AAHP were tested for susceptibility to Spring Viremia Carp viral strains (North American and Asian strains) to ensure viral testing would detect this newly emerging fish pathogen.
• Assisted in accession of EPC cell line (Fijan 1983) to ATCC to secure this important cell line for future use.

Standardized Reagents and Reference (Negative Control) Materials for Immunological Assays
Renibacterium salmoninarum, the causative agent of Bacterial Kidney Disease (BKD) is a significant pathogen for salmonid fish (salmon, trout and grayling). The FWS-AAH program developed assay standards for the Enzyme Immunosorbent Assay (ELISA) for this fish pathogen and conducted repeated testing among state and federal laboratories to ensure standardization and optimal performance of this important detection method.

• Developed a Negative-Control Tissue (NCT) for use in ELISA.
• Performed Quality Assurance testing and analysis of commercial antibodies sources for ELISA:
  • Tested performance of various antibody lots
  • Conducted ring testing among multiple laboratories
    • Idaho Department of Fish and Game (IDFG)
    • USGS-Western Fish Disease Center (USGS-WFDC)
    • Oregon State University (OSU)
    • USFWS – California/Nevada Fish Health Center

Bacteriology: Utilize high quality, standardized culture controls for identification of bacterial fish pathogens, obtained directly from ATCC, or similar commercial sources such as KWIK-STIK PLUS Microorganisms (minimizes passage number of ATCC reference strains).

Conduct Quality Assurance Studies and Applied Research
Prioritize and select 1-2 assay validation studies per year to assess performance, or modify procedures to improve sensitivity or efficiency.

• Conducted comparative studies of Quantitative PCR (QPCR) and ELISA to determine sensitivity and detection limits for a QPCR assay for R.salmoninarum.
• Assisted in field testing a newly developed R.salmoninarum PCR positive control reference material.
• Conducting assay optimization studies to determine effects of pooling fish tissue samples on detection of R.salmoninarum by Fluorescent Antibody Technique (FAT) - in progress.
• Conducting comparative analysis of PCR confirmation method using two tissue types used in detection of Myxobolus cerebralis (Whirling Disease) -in progress.

Through the efforts of our Fish Health Centers, the public, states, tribes, and non-government organizations can be confident that our Aquatic Animal Health Program will provide high quality technical information, and continue to protect and conserve the health of our aquatic animal species for the benefit of the American people.