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Westslope Cutthroat Trout Credit: NPS
The Bozeman Fish Health Center (Center) efforts are guided by the Aquatic Animal Health Mission Statement “Working with partners to provide state-of-the-art aquatic animal health services to protect and enhance the health of aquatic animal resources for the continuing benefit of the American Public.” The Center provides diagnostic and inspection services to an eight state region including North Dakota, South Dakota, Kansas, Nebraska, Colorado, Utah, Wyoming, and Montana. The Center is one of nine Fish and Wildlife Service Fish Health Centers nationwide, and is operated as part of the National Fisheries Program. Increasingly, species other than ‘typical’ aquaculture species require the Center’s attention as the Fish and Wildlife Service provides propagation capabilities to species such as Wyoming Toad, Pallid Sturgeon, and endangered mussels. New species may encounter many of the same issues associated with disease outbreaks when introduced to a culture environment: higher densities than encountered in the wild, closed environments, and stress from the presence of, and handling by, people. In order to successfully rear and maintain these important species, the Center also participates in applied research to assist our National Fish Hatcheries and improve recreation and conservation efforts.
The Center‘s laboratories provide service to federal, tribal, state, and commercial fishery programs and partners. Three major areas of responsibility include:
- Inspection testing services for hatchery facilities to facilitate annual health certifications.
- Diagnostic assistance for chronic or acute health problems in cultured and wild stock.
- National Wild Fish Health Survey to detect, monitor and determine the distribution of fish pathogens in free-ranging fish populations.
The Center’s other areas of expertise include:
- Emergency response for aquatic animal Natural Resource Damage Assessment (NRDA) following oil spills, contaminant releases or other aquatic resource incidents.
These services all require the same investigative processes, each of which is conducted in separate laboratories:
- The Bacteriology Laboratory uses traditional and emerging technology to culture and identify bacteria that are present in an organism.
- The Virology Laboratory uses specialized cell lines derived from several species of fish tissue to culture viruses.
- The Parasitology Laboratory uses a variety of procedures to detect and identify parasites present, including the spores found in the cartilage of young salmonids that result in “whirling disease.”.
- The Molecular Laboratory Laboratory uses state-of-the-art technology to analyze genetic material such as DNA or RNA to identify pathogens and potential causes of disease.
- The Histopathology Laboratory uses high-tech equipment and extensive training for microscopic examination of aquatic animal tissues. Individual cells are examined for abnormalities to identify health problems at the microscopic level.
- The National Wild Fish Health Surveyis a US Fish and Wildlife Service-led effort to sample and monitor wild populations of aquatic animals, utilizing the full suite of laboratories and fish health expertise to screen for pathogens and disease in wild populations.
Histology and Histology Preparation »
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Slides, blocks and jars containing fish tissue samples in the histology laboratory.
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Degenerative changes in the kidney tubules of a rainbow trout.
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Bacterial lesion Flavobacterium psychrophilum) in a rainbow trout.
Histology is the microscopic study of tissues, or microanatomy and is a valuable tool in fish health. Aquatic animal organs are carefully collected and placed into a preservative for approximately 48 hours. Once the organs are preserved, the desired tissues are dissected out and then processed using a tissue processor machine. The tissue processor runs the samples through a series of different concentrations of alcohol and water, and then fills the tissue with paraffin wax. The samples are then embedded into wax blocks. The wax blocks are cut into extremely thin slices, and the slices are then transferred to slides and stained. Once the slides are prepared, the highly-trained histopathologist examines them for cellular changes or abnormalities.
Histology allows fish health experts to observe the specific changes or the extent of damage that a pathogen is having on the animal. Histology can show what stage the infection is in, which allows for more targeted recommendations for treatment. Fish often have simultaneous infections with more than one pathogen, and histology helps determine which came first. This can be helpful in determining appropriate treatment options.
Histology is also useful in detecting health issues that are not related to a pathogen. When fish get sick, they often have an underlying problem that has weakened the immune response, making them susceptible to pathogens. Histology can shed light on other potential issues, such as water quality problems or nutritional deficiencies. It can also help detect damage to the organs caused by environmental toxicants that result from oil or chemical spills, heavy metal contamination, or other forms of pollution in the water.
Parasitology Laboratory »
The Center regularly monitors for whirling disease in both hatchery and wild fish populations. Whirling disease is a chronic inflammatory disease caused by a spore-forming parasite Myxobolus cerebralis. This parasite has a complex lifecycle involving salmonid fish and the Tubifex tubifex worm. Clinical signs of whirling disease include whirling or tail-chasing, a black tail, and various skeletal deformities. Fish infected with whirling disease are not able to swim or feed normally and are more susceptible to predators. The mortality rate for juvenile fish affected with whirling disease is high (up to 90% in infected populations), and those that do survive are often deformed and serve as carriers for the disease.
The Center uses a method of spore extraction to detect and diagnose whirling disease. Fish skull bones are excised, mechanically crushed, treated with enzymes, centrifuged and examined for the presence of spores by microscopy. If spores are observed, molecular techniques using genomic DNA are used to confirm that the spores are from M. cerebralis.
Prevention is imperative in controlling this disease. Testing fish prior to stocking or moving, enforcing disease regulations and educating hatchery personnel, field biologists and the public on how the disease is spread will help decrease future infections.
Fish Infected with whirling disease.
*Adapted from M. El Matbouli, T. Fischer Scherl, and R.W. Hoffmann. 1992. Annual Review of Fish Diseases, p. 392.
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Bacteriology Laboratory »
The Bacteriology Laboratory is used for fish health inspections and diagnostic cases that pass through the Bozeman Fish Health Center. The samples are processed and analyzed following approved protocols for the detection and identification of specific bacterial pathogens. Testing for bacterial pathogens ranges from wild fish kills or hatchery mortality events to regular facility fish health inspections. Fish health inspections target specific bacterial pathogens that can cause mortality and disease in a variety of species and are deemed reportable or regulated by federal, state and tribal partners.
Bacterial samples must be carefully collected since contaminating bacteria can grow quickly and mask target species. The selection of tissue samples for bacteriological assays varies depending on the pathogen testing protocols. Organs most commonly tested include the kidney and portions of any organ with visible lesions, however other organs such as the brain, heart or spleen are used for the detection of certain bacterial infections. Samples are inoculated onto appropriate culture media for growth. A variety of methods are used to aid in the isolation and identification of unknown microorganisms. Testing includes gram stain, microscopic examination, biochemical protocols , Enzyme-Linked Immunosorbent Assay (ELISA), bead agglutination, IFAT/DFAT (Indirect and Direct Fluorescent Antibody Test) and antibiotic sensitivity testing.
The bacterium Renibacterium salmoninarum, causes "Bacterial Kidney Disease," or BKD in salmonid fish. Apple-green fluorescence when microscopically examined is considered presumptively positive for R.salmoninarum.
Granulomatous (pustules) lesions in the kidney resulting from Bacterial Kidney Disease - Renibacterium salmoninarum.
Fin rot associated with bacterial cold water disease - Flavobacterium psychrophilum.
Methyl blue stain of Bacterial Gill Disease - Flavobacterium spp.
Molecular Biology Laboratory »
Photo documentation of gel electrophoresis - Missouri River Sturgeon Iridovirus (MRSIV).
The Bozeman Fish Health Center has full molecular capabilities of both conventional Polymerase Chain Reaction (PCR) and Quantitative or real-time PCR (QPCR) for detection of many fish pathogens of concern. The Center also has genetic sequencing capability and expertise for pathogen identification and applied research projects. The field of molecular biology is evolving rapidly, with more sensitive and highly specific protocols for pathogen detection and identification. In addition, development of non-lethal sampling techniques utilizing PCR assays continue to support the conservation of aquatic animal species. For example, a non-lethal fin clip tissue sample can be collected from live fish, and PCR is then used to detect Missouri River Sturgeon Iridovirus (MRSIV) in endangered Pallid sturgeon.
Four steps must occur for a successful PCR assay: 1. Aseptic or sterile sampling of target tissue. 2. Sample extraction of targeted DNA/RNA 3. Amplification of target DNA/RNA. 4. Gel electrophoresis of target DNA/RNA in conventional PCR or, Quantitative PCR (QPCR) assay.
PCR techniques use DNA primers, or short DNA sequences, to amplify segments of genes specific for the target pathogen of interest. DNA or RNA is extracted from fish tissues and laboratory test products, such as bacterial cultures and cell culture supernatant containing viral agents. The product of interest is then amplified using forward and reverse primer sets. Various fish pathogen protocols used are single round PCR, however some protocols utilize the initial amplified product and re-amplify using an additional “nested PCR” technique. The DNA products are then visualized and documented by gel electrophoresis equipment. Methods utilizing Quantitative PCR (QPCR) can be used to quantify or measure the copy number of a target pathogen DNA in a sample, resulting in high accuracy and testing sensitivity.
Thermal cycler used to amplify DNA for conventional PCR.
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Virology Laboratory »
The Center uses numerous fish cell lines in order to detect and identify viral pathogens in federal, state, tribal and commercial hatcheries, as well as in free-ranging populations. The Center also has the capability to monitor for viral pathogens in other aquatic species such as amphibians and mussels. This comprehensive approach will further protect entire aquatic ecosystems.
Viruses are obligate, intracellular parasites that depend on living cells to reproduce. They are very small and cannot be seen by light microscopy. The isolation and identification of viruses is a complex process, and requires sophisticated materials, equipment and training. Standard viral isolation methods use in vitro cell culture (tissue culture) of susceptible cell lines to visualize cytopathic effect (CPE) from virus infection. Once CPE is observed, molecular techniques are used to confirm the specific pathogen. There are no effective treatments for viral infections. Regular monitoring of adult and juvenile fish and other aquatic organisms, as well as proper sanitation and disinfection procedures are the only practical methods of control.
Inoculating samples onto plates containing fish cells.
Infectious Hematopoietic Necrosis Virus (IHNV) Cytopathic effect (CPE) on fish cells.
Preparing plates for virology assay.
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National Wild Fish Health Survey »
When Myxobolus cerebralis (parasite that causes whirling disease) was identified in western rivers in 1996, and wild trout populations crashed, the US Fish and Wildlife Service recognized that no concerted efforts were underway to catalog fish pathogens in the wild. Beginning in 1997, the Service implemented the National Wild Fish Health Survey in conjunction with partnering agencies, and began systematically sampling wild fish populations and developing a database to map the presence of fish pathogens in the wild. Sampling methods for the National Wild Fish Health Survey are similar to a hatchery health inspection where specific pathogens of national or regional concern are targeted. Sampling data is then uploaded into the database, which is accessible to the public. Wild fish kills and mortality events are also investigated and included in the National Wild Fish Health Survey data.
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Newsletters »
Contact Us »
Project Leader: Lacey Hopper
(406) 582-8656 x201
lacey_hopper@fws.gov
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