Histology and Histology Preparation | Parasitology Laboratory
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| Molecular Biology Laboratory
| Virology Laboratory
| Wild Fish Health Survey
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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. These species require the Center to embark on research to assist our National Hatcheries to successfully maintain and rear these species.
The Center ‘s laboratories service federal, tribal, state, and commercial fishery programs. 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 determine the distribution of fish pathogens in free-ranging fish populations.
These Services all require the same investigative processes, each of which is conducted in separate laboratories. The Bacteriology Laboratory uses traditional technology to grow and identify bacteria that are present in the organism. The Virology Laboratory uses cell lines derived from several species of fish to culture viruses. The Parasitology Laboratory assess the type and numbers of parasites present, including the spores found in the cartilage of young salmonids that result in “whirling disease.” In addition, the Center has one of the few Histology laboratories in the Service that use cell abnormalities to diagnosis and confirm diseases. Newer technology includes a Molecular Laboratory that uses DNA and RNA to confirm and identify pathogens. The Wild Fish Health Survey samples wild populations of aquatic animals, and each sampling effort uses the full suite of laboratories to screen for pathogens 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 study of tissues, or microanatomy. It is a valuable tool in fish health. Organs are carefully collected from moribund fish and placed into a preservative for approximately 48 hours. Once the fish or the organs are preserved, the desired tissues are dissected out and then processed using a tissue processor. 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 embedded in wax blocks. The technician takes the blocks and uses a microtome to cut the samples into extremely thin slices. The slices are then transferred to slides and stained. Once the slides are prepared, the histopathologist examines them for cellular changes.
Histology allows us to see the specific changes or the extent of damage that a pathogen is having on the fish. It 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, which can also be helpful in treatment.
Histology is 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 or nutrition. It can detect damage to the organs caused by toxicants that result from chemical spills, heavy metal contamination, or other forms of pollution in the water.
Parasitology Laboratory »
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The Bozeman Fish Health Center (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 symptoms 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.
Bacteriology Laboratory »
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The Bozeman Fish Health Center Bacteriology Laboratory is used for fish health inspections and trouble-shooting cases that pass through the Bozeman Fish Health Center. In the bacteriology laboratory the samples are processed following Blue Book procedures for the detection and identification of bacterial pathogens. Testing for bacterial pathogens ranges from fish kills to annual fish health inspections. For a fish health inspection the target bacterial species tested include Aeromonas salmonicida,Yersinia ruckeri, Edwardsiella ictaluri, and Renibacterium salmoninarum. Other bacterial species that we detect but are not limited to the causative agents for; Bacterial Gill Disease, Columnaris Disease, Edwardsiella tarda Septicemia, Enteric Septicemia, Coldwater Disease and Motile Aeromonas Septicemia.
Samples for culture of bacteria must be taken aseptically as contaminating bacteria can grow quickly, particularly in warmer water environments. 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 or spleen are used for the detection of certain bacterial infections. Samples are inoculated immediately 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 (API system), ELISA Enzyme-Linked Immunosorbent Assay, bead agglutination, IFAT/DFAT (Indirect and Direct Fluorescent Antibody Test) and antibiotic sensitivity testing.
The bacteria Renibacterium salmoninarum, cause "Bacterial Kidney Disease," or BKD. Fluorescence when microscopically examined are considered presumptively positive for R.sal.
Granulomatous (pustules) lesions in the kidney resulting from Bacterial Kidney Disesase - Renibacterium salmoninarum.
Fin rot associated with cold water disease - Flavobacterium psychrophilum.
Methyl blue stain of Bacterial Gill Disease - Flavobacterium spp.
Molecular Biology Laboratory »
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Photo documentation of gel electrophoresis - Missouri River Sturgeon Iridovirus (MRSIV).
The Bozeman Fish Health Center is fortunate to have full molecular capabilities of both Polymerase Chain Reaction (PCR) and Quantitative PCR (QPCR) for many protocols for fish pathogens of concern. The field of molecular biology is evolving rapidly, with new more sensitive and highly specific protocols for pathogens. In addition, non lethal sampling techniques have become very important for the conservation of fish species. For example, a nonlethal fin clip can be collected and PCR used to detect Missouri River Sturgeon Iridovirus (MRSIV).
Some of the fish pathogens of concern that the laboratory preforms PCR for include; Whirling Disease, Bacterial Kidney Disease, Missouri River Sturgeon Iridovirus (MRSIV), Largemouth Bass Virus (LMBV), Koi Herpes Virus (KHV), Cutthroat Trout Virus (CTV), Infectious Pancreatic Necrosis Virus (IPNV), and Infectious Hematopoietic Necrosis Virus (IHNV).
For a successful PCR product four steps must occur; 1. Aseptic sampling of target tissue. 2. Sample extraction of targeted DNA/RNA 3. Amplification of target DNA. 4. Gel electrophoresis of target DNA, Quantitative PCR (QPCR) or reversetranscriptase quantitative PCR (RT-QPCR).
Polymerase Chain Reaction (PCR) techniques use DNA primers to amplify segments of genes specific for the target pathogen of interest. DNA or RNA is extracted from fish tissues and laboratory assay 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 by gel electrophoresis equipment. Methods utilizing Quantitative PCR (QPCR) and reversetranscriptase quantitative PCR (RT-QPCR) can be used to quantify copy number of a target pathogen in a sample.
Thermal cycler used to amplify DNA for PCR.
Quantitative PCR machine used to preform QPCR.
Virology Laboratory »
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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 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.
Wild Fish Health Survey »
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When whirling disease was discovered in western rivers, and wild trout populations crashed, the Service recognized that no concerted efforts were underway to catalog fish pathogens in the wild. Beginning in 1997, the Wild Fish Health Survey, in conjunction with our partners, began systematically sampling wild fish and developing a database to map the presence of fish pathogens in the wild. The Wild Fish Health Survey surveys lakes and streams, and using protocols similar to a hatchery inspection screens for bacterial and viral pathogens, and parasites. Data is then uploaded into the database, which is accessible to the public.
