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Minnesota Fish Kill Investigation Manual
University of Minnesota, College of Veterinary Medicine
2015 Edition
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Revised: August 2015 Page 1
Minnesota Fish Kill Investigation Manual
University of Minnesota, College of Veterinary Medicine
Prepared by S.J. Knowles, S. Massarani and N. Phelps
2015 Edition
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Revised: August 2015 Page 2
Table of Contents
INTRODUCTION ..................................................................................................................................................... 4
CHAPTER 1. COLLECTING INFORMATION ................................................................................................ 5
CHAPTER 2. INVESTIGATING A FISH KILL ................................................................................................ 6
2.A. Standard Protocol and Report Form ........................................................................... 6
2.B. Mobile Kit ..................................................................................................................... 6
CHAPTER 3. SAMPLE COLLECTION AND SUBMISSION ........................................................................ 7
3.A. Collection ..................................................................................................................... 7
3.B. Sample Submission ...................................................................................................... 7
CHAPTER 4. CAUSES OF FISH KILLS IN MINNESOTA ........................................................................... 8
4.A. Environmental Causes ................................................................................................. 8
4.B. Chemical Causes ....................................................................................................... 11
4.C. Infectious Causes ..................................................................................................... 13
4.C.1. Bacteria ..................................................................................................... 13
4.C.2. Fungi and Oomycetes ............................................................................... 17
4.C.3. Parasites ................................................................................................... 18
4.C.4. Viruses ...................................................................................................... 19
4.D. Biological Causes ...................................................................................................... 22
REFERENCES ....................................................................................................................................................... 24
APPENDIX A. STANDARD PROTOCOL FOR FISH KILL INVESTIGATIONS .................................. 25
APPENDIX B. STANDARD INVESTIGATION AND NECROPSY REPORT FORM .......................... 28
APPENDIX C. MOBILE KIT CONTENTS FORM ........................................................................................ 31
APPENDIX D. CONTACTS LIST ..................................................................................................................... 33
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Contributors
Sarah J. Knowles, University of Minnesota College of Veterinary Medicine, 1333 Gortner Avenue, St.
Paul, MN 55108
Sarah Massarani, University of Minnesota College of Veterinary Medicine, 1333 Gortner Avenue, St.
Paul, MN 55108
Dr. Nicholas Phelps, University of Minnesota College of Veterinary Medicine, Veterinary Population
Medicine, 1333 Gortner Avenue, St. Paul, MN 55108
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Introduction
Fish kills, or mass die-offs of five or more fish with similar symptoms in a localized area, are widespread throughout Minnesota and can occur at any time of year. Fish kill events can be caused by a single factor or many working in conjunction, including but not limited to environmental changes, chemical spills, disease, and naturally occurring biological events. Regardless, these events create great concern as Minnesota’s fish populations support very active recreational and aquaculture industries and play an important role in public and ecosystem health. Timely reporting of fish kills allows for rapid and effective investigation. Thorough, standard examination of these events is imperative to the long-term sustainability and health management of the state’s fish populations and aquatic ecosystems. Any and all fish kills encountered, regardless of species, should be reported in a timely manner to the University of Minnesota Fish Kill Reporting Map (z.umn.edu/fishkill). While the public is urged to report fish kills, for safety purposes, only Fish Kill Investigators should handle fish carcasses and fish kill investigations.
This Field Manual contains guidelines to support a rapid and effective response to reported fish kill events.
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Chapter 1. Collecting Information
Some basic information should be collected at the time the fish kill is reported. All information received should be documented (see Appendix B. Standard Investigation and Necropsy Report Form) and used to determine if an investigation should occur.
The reporter of the fish kill should be interviewed for the following information:
o Their name, address, phone number, email address and affiliation (i.e. general public, etc) o The name of the body of water and an address or specific location of the kill site o The date and time the fish kill started and its duration
In conjunction, the following questions (List 1.A) can be utilized to begin investigating the cause and significance of the kill event. List 1.A. Helpful questions to begin investigating the cause of a fish kill. Common responses and probable causes of the kill follow the question.
1. How many species of fish are dying?
One species or family Infectious (virus, fungi, parasites) or Biological?
Multiple species Environmental, Chemical, or Infectious?
2. What size fish are dying?
Small Infectious (bacteria , virus) or Biological?
Large Environmental (low dissolved oxygen (DO))?
3. When did the kill start and how long did it last?
Fish were dead early in the morning Environmental (low DO)?
Lasted 1-3 days Environmental, Chemical, Infectious, or Biological?
Lasted longer than 3 days Chemical or Infectious?
4. What has the weather been like?
Hot temperatures Environmental (low DO, algae bloom) or Infectious?
Sunny Environmental (algae bloom)?
5. Did the water change color before the event?
Yes: Color is green, brown, gray or black Environmental (low DO, algae bloom) or Chemical?
Yes: Color is different from above Chemical?
6. Has there been anything else abnormal about the water?
No, water quality is normal Infectious or Biological?
7. Do the fish look abnormal?
Red spots, dots or hemorrhaging Infectious (bacteria, virus, fungi)?
Small bubbles in skin, fins, gills, or around eyes Environmental (gas supersaturation)?
Eyes are bulging out (“pop-eye” or exophthalmia) Environmental (gas supersaturation) or
Infectious (bacteria, virus)?
Lesions, ulcers or abscesses on skin or fins Infectious?
8. How were the fish behaving before death?
“Gulping” air at the surface Environmental (low DO, algae bloom) or Infectious (bacteria,
fungi)?
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Chapter 2. Investigating a Fish Kill
Time management is essential following a fish kill event. Rapid response and standard investigation procedures are important for improving diagnostic outcomes.
Do not hesitate to contact a fish health professional with questions (see Appendix D. Contacts List)
2.A. Standard Protocol and Report Form
Standard procedures should be followed when conducting a fish kill investigation to ensure all necessary water quality data and fish samples are collected for each and every investigation. Step-by-step guidelines can be found in Appendix A. Standard Protocol for Fish Kill Investigations. A standard report form should also be utilized when documenting and submitting an investigation (see Appendix B. Standard Investigation and Necropsy Report Form).
2.B. Mobile Kit
A mobile kit containing necessary water testing supplies, fish and sample collection tools, personal protective equipment and other gear is valuable when conducting an on-site fish kill investigation. The kit should be assembled and re-stocked prior to any investigation. A list of suggested equipment to include in the kit can be found in Appendix C. Mobile Kit Contents Form.
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Chapter 3. Sample Collection and Submission
3.A. Collection
Fresh fish carcasses (i.e. gills are red or pink in color, normal body coloration is evident, etc.) or fresh tissue samples should be collected accordingly to support effective diagnostic testing. On-site necropsies can be performed if the appropriate dissection tools and collection equipment (e.g. bacterial swabs, vials, 10% buffered formalin, etc.) are available. Step-by-step guidelines for sample collection can be found in Appendix A. Standard Protocol for Fish Kill Investigations. Necropsies should be documented on a standard report form (see Appendix B. Standard Investigation and Necropsy Report Form). All samples collected should be put into the appropriate vials or bags and be kept cold in a container with frozen gel packs or ice then transported to a diagnostic laboratory as soon as possible. If on-site necropsies are not performed, fresh fish carcasses should be placed individually into plastic bags or according to species and kept on frozen gel packs or ice for transport to a laboratory.
3.B. Sample Submission
Collected fish carcasses and tissues should be sent to the University of Minnesota Veterinary Diagnostic Laboratory for proper testing. The lab should be notified prior to submission and the Investigation and Necropsy Report Forms must be included with the samples (see Appendix B. Standard Investigation and Necropsy Report Form). All samples should be sent on frozen gel packs or ice by same-day personal delivery or overnight mail. Laboratory contact information: Attn: Dr. Nick Phelps
Veterinary Diagnostic Laboratory University of Minnesota 1333 Gortner Ave St. Paul, MN 55108 P: 612-624-7450 E: phelp083@umn.edu
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Chapter 4. Causes of Fish Kills in Minnesota
Fish kill events are typically caused by a variety of factors working in conjunction. Often, stressful environmental conditions or biological life events can reduce the immune function of fish leaving them more susceptible to secondary infectious pathogens and death. However, one stressful environmental event or an infectious disease outbreak may be the primary cause of the fish kill. Detailed information on environmental, chemical, infectious and biological causes of fish kills in Minnesota and confirmatory testing methods are provided in the following sections (4.A.-4.D.), respectively.
4.A. Environmental Causes
GENERAL DIFFERENTIALS: o Environmental changes within a body of water induce stress upon fish. These stressors can
increase the susceptibility of fish to secondary pathogens or they can be great enough to be the primary cause of death.
o Generally it is not specific to a single species of fish.
GENERAL TESTS: o Ideally, visual observation and collection of environmental data and samples should be
conducted during or right after death of the fish. o Weather conditions at the time of the kill should be recorded: temperature, cloud cover,
precipitation and wind. o The location should be surveyed for land use and potential runoff (i.e. agricultural,
industrial, municipal and transportation use). o Water quality must be collected on-site: temperature, pH, dissolved oxygen (DO), ammonia,
turbidity, color, and odor. Measurements should be taken at the site of the kill and another site lacking dead fish. Water samples can be collected for further lab testing.
o Fish should be observed and physically examined on-site. Behavior, lesions and other abnormalities should be recorded.
o If the fish cannot be examined on-site, wrap each fish individually in plastic bags and store them on wet ice. Do not freeze the fish or put them on dry ice. Any collected tissue samples should be placed into sterile, sealed containers with 10% buffered formalin. Transport the fish and any other samples to the lab as soon as possible.
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Table 4.A.: Environmental events that are known to cause or are associated with fish kill events
4.A.1. Algae bloom
Environmental
Signs
NON-TOXIC OR TOXIC:
Single species of alga present in large numbers
Low DO levels and corresponding signs post-event
Season: Summer-fall; periods of warm, sunny weather
Heavy precipitation resulting in runoff from developed areas
TOXIC ONLY:
Diatoms and/or dinoflagellates may also be present in water
Dead insects, zooplankton, birds and terrestrial animals may also be present
Event occurs during the day due to photosynthetic activity and toxin production
Fish Affected Non-specific
Clinical Signs Clogged or irritated gills
Increased respiration rate
Tests NON-TOXIC or TOXIC: Algal cell counts from water samples
TOXIC ONLY: Presence of toxins in collected carcasses
4.A.2. Gas supersaturation, “Gas bubble disease”
Environmental
Signs
Rising water temperatures and/or atmospheric changes in a body of water heavily
concentrated with aquatic plants
Can occur downstream from dams, infrastructure or natural barriers
Fish move away from cold water into warm: may occur naturally (i.e. cross a thermocline) or
may move into warm discharges from power plants, dams and other infrastructures
Fish Affected Non-specific
Clinical Signs Bubbles (“gas bubbles”) visible around eyes, under skin, and in the fins and capillaries of gills
Exophthalmia (“pop-eye”) can occur without visible bubbles
Tests Physical examination and microscopy of affected tissues and gills
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4.A.3. Low dissolved oxygen (DO)
Environmental
Signs
Fish are dead early in the morning
DO concentration is low (< 2 ppm)
Water is "pea-soup" green, brown, gray or black in color
Temp: High water temperature
Season: Summer + calm, cloudy, hot weather; Winter + ice and snow cover present,
mortalities occur at any time of day
Prior period of drought
Low water level
High mass of aquatic plants, algae or decaying organic material
Body of water is highly eutrophic from high amount of organic matter
Heavy rainfall may have increased runoff
Turnover event brought anoxic water and decaying organic material up from the bottom of
the water column
Zooplankton and insects are dead or dying
Fish Affected Larger fish and species with high oxygen requirements are affected first
Fish that normally gulp air at surface or have a superior-oriented mouth are NOT affected
Clinical Signs Increased respiration rate
“Gulping" air at surface
Tests DO probe measurement (in ppm)
*NOTE: A low oxygen event can be easily confused with other events (e.g. chemical spill, algae bloom, etc.)
4.A.4. Temperature change
Environmental
Signs
Temp: Prolonged or rapidly elevated or depressed seasonal water temperatures
Rapid changes in solar heat, cloud cover and atmospheric temperature
Abnormal water movements
Rapid precipitation events
Changes in thermal discharges from power generation and production industries
Alterations in water control structure(s)
Commercial fish harvesting and aquaculture presence
Fish Affected Specific species may be affected according to their range of temperature tolerance and
which specific pathogens are virulent during the event
Clinical Signs
Decreased respiration rate Sporadic periods of hyperactivity
Lethargy Hyper or reduced response to stimuli
Loss of equilibrium Reduced predator evasion
Uncoordinated swimming Secondary infection or infestation
Tests Temperature measurement with probe or thermometer
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4.B. Chemical Causes
GENERAL DIFFERENTIALS: o Chemical agents (e.g. toxins, pollutants) can cause a variety of effects on a body of water
and the organisms it supports. The chemical may or may not be visible to the eye. Effects are typically non-characteristic to the specific chemical. As well, organisms can be acutely or chronically affected making it difficult but important to thoroughly investigate these kill events.
o Small fish are most susceptible but all size classes may be affected. o Multiple species will be affected. o Acute effects are severe and cause rapid, large-scale mortality events due to the short-
term exposure of a highly concentrated or toxic chemical. o Chronic, sublethal chemicals or concentrations are usually seen as bioaccumulation and
bio-magnification in fish. Signs of toxicosis or death are generally seen during periods of cold water temperatures or stressful events due to the toxins being released into the bloodstream from the fish’s fat stores. Mortality rates are slower but may be continuous.
GENERAL TESTS: o Water quality must be collected on-site: temperature, pH, dissolved oxygen (DO), ammonia,
turbidity, color, and odor. Measurements should be taken at the site of the kill and another site lacking dead fish. Water samples can be collected for further lab testing.
o The location should be surveyed for land use and potential runoff (i.e. agricultural, industrial, municipal and transportation use).
o Physical examination and observation of freshly dead or dying fish should be conducted on-site. Behavior, lesions and other abnormalities should be recorded.
o Skin scrapes, fin clips and gill clips of these fish should be collected, examined on wet mounts and compared against those from healthy appearing fish.
o Necropsy can be conducted on-site or in the lab to investigate for internal lesions and pathogens. Tissues of significance to collect and examine include gills, blood and brain.
o If the fish cannot be examined on-site, wrap each fish individually in plastic bags and store them on wet ice. Do not freeze the fish or put them on dry ice. Collected tissue samples should be placed into sterile, sealed containers with 10% buffered formalin. Transport the fish and any other samples to the lab as soon as possible.
o Sediment and animal carcasses can also be collected from the kill site and a reference site to be tested in lab for chemical residues. Samples should be transported in sterile, sealed containers and carcasses should be individually wrapped in plastic bags and stored on ice. Do not freeze.
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Table 4.B.1.: Clinical signs seen in fish affected by chemical toxins and pollutants
Target Clinical Signs
Behavior
Lethargy, weakness
Increased respiration rate
Erratic, uncoordinated swimming
Rapid, jerky movements and tremors
Loss of equilibrium
Increased sensitivity to stimuli
Gills
Clogged
Covered in white film
Sloughing of epithelium
Bright red or dark in color
Hemorrhage
Distended opercula
Mouth Covered in white film
Skin Covered in white film
Internal Viscera
Hemorrhage
Blood clots
Blue stomach
Blood Bright red in color
Brown in color
Table 4.B.2.: Characteristic effects of chemical agents on algae, plant, insect and invertebrate
populations in a body of water
Chemical Agent Organisms
Dead or Dying Alive
Herbicide Algae Insects
Insecticide Insects, Invertebrates Algae, Aquatic plants
Acid, Heavy metals, Other
highly toxic substances
Algae, Aquatic plants,
Insects, Invertebrates None
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4.C. Infectious Causes
GENERAL DIFFERENTIALS: o Fish kills involving bacteria, viruses, fungi, or parasites usually occur when a fish population
is or has been under some other environmental or biological stressor(s). The spread of the disease is typically a secondary effect.
o A single fish species or family is generally affected. o Small fish are most susceptible.
GENERAL TESTS: o Physical examination and observation of freshly dead or dying fish should be conducted.
Behavior, lesions and other abnormalities should be recorded. o Skin scrapes, fin clips and gill clips of these fish should be collected, examined on wet
mounts and compared against those from healthy appearing fish. o Necropsy can be conducted on-site or in the lab to investigate for internal lesions and
pathogens. Tissues suspected of or displaying infection or infestation should be collected for further examination and testing.
o For transport to lab, wrap each fish individually in plastic bags and store them on wet ice. Do not freeze the fish or put them on dry ice. Any collected tissues should be placed into sterile, sealed containers with 10% buffered formalin. Transport the fish and any other samples to the lab as soon as possible.
4.C.1. Bacteria……………………………..12
4.C.2. Fungi and Oomycetes……….16
4.C.3. Parasites……………………………17
4.C.4. Viruses………………………………18
4.C.1. Bacteria
GENERAL DIFFERENTIALS:
Most bacterial diseases are due to secondary opportunistic infections following environmental stressors, parasitic infestations, other pathogens, and trauma induced injuries. Typically, the stressful event occurred 10 to 14 days prior to the start of the fish kill.
Many bacterial infections cause similar clinical signs including skin erosion or ulceration (Figure 4.C.1.a.). A key characteristic of ulcers is hemorrhagic or bloody edges of the lesion (Figure 4.C.1.b.).
Figure 4.C.1.a.: Skin ulceration on the dorsal caudal peduncle.
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Figure 4.C.1.b.: Skin ulceration on the left lateral side of the body. Note the hemorrhagic edges.
ADDITIONAL TESTS:
Skin scrapes should be taken from the edge of external lesions. Examine by wet mount observation and gram staining.
Material from the edge of lesions, infected gills or internal organs should be collected using separate sterile swabs and streaked onto appropriate culture plates. An unaffected internal area, or control location, should be swabbed separately and streaked onto culture plates. Following lab culture and isolation, identification tests should be conducted.
Table 4.C.1.: Specific bacterial agents that are known to cause or are associated with fish kill events
4.C.1.a. Aeromonas hydrophila and complex, “Bacterial hemorrhagic septicemia”
Environmental
Signs
Temp: ≥ 10°C
Season: Winter-Spring
Current or previous stressful conditions, especially low but rapidly increasing water
temperature, reduced winter feeding, nutritional deficiencies
High concentrations of organic material in water
Fish Affected Species: All freshwater species, some more susceptible
Clinical Signs
Hemorrhage on body, fins, gills, vent, and
internal organs
Distended abdomen
Inflammation and erosion of mouth Blood-tinged fluid in body cavity
Exophthalmia (“pop-eye”) Soft and swollen kidney
Ulcers Enlarged spleen
Abscesses Lethargy
Samples to
Collect
Kidney + other affected internal organs
Fin clips and skin scrapes
Tests Kidney: Bacterial culture and isolation + identification via biochemical tests
*CAUTION: Bacterium is pathogenic to other cold-blooded vertebrates (i.e. frogs, turtles, reptiles) and
mammals including immunocompromised or wounded humans.
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4.C.1.b. Aeromonas salmonicida, “Furunculosis”
Environmental
Signs
Temp: Cold water 8-20°C; may see with higher water temperature + low DO
Season: Follows seasonal temperature patterns
Fish Affected Species: Freshwater including trout, salmon, minnows, carp, goldfish, perch, chub, pike,
bullheads and catfish
Clinical Signs
Darkened skin color Soft kidney
Hemorrhage at base of fins Enlarged spleen
Ulcerative skin lesions Pale and mottled liver
Raised lesions on muscle that resemble boils
(“furuncles”) Lethargy
Hemorrhage of internal organs
Samples to
Collect Kidney, intestinal material + other affected internal organs
Tests Kidney: Bacterial culture and isolation + identification via biochemical or serological tests
4.C.1.c. Flavobacterium branchiophila, “Bacterial gill disease”
Environmental
Signs
Temp: Increasing water temperatures
Season: Spring-summer
Previous or current stressful conditions
Fish Affected
Size: Small most susceptible
Age: All
Species: Includes trout, salmon, walleye, silver carp, rohu, and catla
Clinical Signs
Pale, swollen and necrotic gills Lethargy
Flared opercula “Gulping” air at surface
Darkened skin color Swimming high in water column
Congested eyes Slow response to stimuli
Samples to
Collect Gill clips
Tests Wet mounts and staining (i.e. gram-stain, simple-stain, etc.)
Histology
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4.C.1.d. Flavobacterium columnaris, “Columnaris disease”
Environmental
Signs
Temp: Warm water >20°C, may occur at lower temps
Season: Seasonal temperature patterns in spring
High concentrations of organic matter in body of water
Fish Affected
Species: Most freshwater fish
Age: All, young more susceptible
Cold water fish that spawn in springtime
Clinical Signs
Grayish colored lesions on body or fins Shallow ulcers
Brown or yellowish colored gill tissue Lethargy
Necrotic skin, fins and/or gills Swimming near the surface
Skin lesions, especially those that cross over
the back (“saddleback lesions”)
FISH MAY DIE WITHOUT ANY GROSS CLINCIAL SIGNS
Samples to
Collect
Gill clips and skin scrapes from edges of lesions
Kidney and spleen
Tests Wet mounts show bacteria that collect into columns (“haystacks”)
Internal organs: Bacterial culture and isolation + identification via Griffin screen
4.C.1.e. Pseudomonas fluorescens, “Pseudomonas septicemia”
Environmental
Signs
Temp: ≥ 10°C
High concentration of organic matter in body of water
Fish Affected Species: All freshwater species
Clinical Signs
Hemorrhage on body, fins and internal
organs Fluid in body cavity
Inflammation and erosion of mouth Soft and swollen kidney
Occasional external skin lesions Enlarged spleen
Exophthalmia (“pop-eye”) Lethargy
Distended abdomen
Samples to
Collect
Kidney + other affected internal organs
Fin clips and skin scrapes
Tests Kidney: Bacterial culture and isolation + identification via biochemical tests
*CAUTION: Bacterium is pathogenic to other cold-blooded vertebrates (i.e. frogs, turtles, reptiles) and
mammals including immunocompromised or wounded humans.
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4.C.2. Fungi and Oomycetes
GENERAL DIFFERENTIALS:
Most of these diseases in fish are secondary opportunistic infections following trauma, disease or death. Fungi and oomycetes can be seen around lesions caused by injuries, bacteria or parasites. However, some species are primary pathogens.
Many fungi and oomycetes can be seen by the naked eye as tufts, nodules or other characteristic epithelial lesions occurring on the external surface of infected fish.
ADDITIONAL TESTS:
Gills, brain, internal organs, and kidney should be examined microscopically on wet mount, squash-prep and/or histology.
If fungi or oomycetes are observed or suspected a sample can be cultured in lab then identified down to species level. Identification can be very difficult and requires advanced taxonomic knowledge
Table 4.C.2.: Specific fungi and oomycetes that are known to cause or are associated with fish kill
events
4.C.2.a. Branchiomyces sp., “Branchiomycosis,” “Gill rot”
Environmental
Signs
Temp: Highest mortalities at ≥25°C.
Body of water is often highly eutrophic with high levels of organic matter
Strong association with poor water quality
Fish Affected Species: Various freshwater fish
May infect a single species or multiple
Clinical Signs
DISEASE IS SPECIFIC TO THE GILLS: Infected
areas necrotic and pale or brownish-grey in
color
May show signs consistent with oxygen
deprivation
May swim listlessly
Samples to
Collect Gill clips
Tests Wet mounts
Histology and staining for species identification
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4.C.2.b. Saprolegnia sp. and other Water molds, “Saprolegniasis,” “Cotton wool disease”
Environmental
Signs
Temp: Cold water
Injuries, malnutrition, temperature shock, external parasitism, and spawning increase
susceptibility
Also infects warm and cold water insect and amphibian species
Fish Affected Species: All freshwater fish
Clinical Signs
Organism appears as focal white to brownish
“cottony tufts” on skin and/or gills
SYSTEMIC INFECTIONS: Masses found in gut
and surrounding viscera with hemorrhage,
necrosis and adhesions
Pale foci of lifted scales surrounded by areas
of redness, may become ulcerated SMALLER FISH: Distended abdomen
Samples to
Collect
Skin scrapes of infected external surfaces
Gill clips
Tests Wet mounts
Isolation by culture
4.C.3. Parasites
GENERAL DIFFERENTIALS:
Many parasitic infestations are not the primary cause of major fish kills but create wounds or act as stressors that render the fish more susceptible to secondary infections or environmental stress.
ADDITIONAL TESTS:
Parasites are easiest to detect in freshly dead fish, thus any collected fish or tissues should be examined promptly. At this time, they will be alive, moving and still attached to their host.
Gill clips, fin clips and skin scrapes, especially taken from behind the fins, should be observed on wet mounts.
Histological preparations of infested tissues can verify presence of the parasite(s).
Parasites can be generally identified down to the levels of class and order easily, but special techniques and expert knowledge is necessary to classify the genus and species.
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4.C.4. Viruses
GENERAL DIFFERENTIALS:
Small sized and fish in early life stages (i.e. fry and fingerlings) are the most susceptible to viral infections.
Lesions and signs are not typically characteristic to a specific virus or consistent across host species. Laboratory tests must confirm the virus.
ADDITIONAL TESTS:
Samples to collect vary according to fish size and life stage. The entire body of small fish (6 cm). Ovaries or ovarian fluid should be collected from sexually mature fish (at least 1 mL).
Suspected infected tissues can only be confirmed in the lab by virus isolation in cell cultures followed by identification using PCR, virus neutralization, molecular techniques, or immunological techniques.
Table 4.C.4.: Specific viral agents that are known to cause fish kill events
4.C.4.a. Infectious Hematopoietic Necrosis virus
Environmental
Signs Temp: ≤ 25°C
Fish Affected Species: Most of the Salmonidae family (i.e. salmon and trout)
Fry and fish in poor health are most susceptible
Clinical Signs
Darkened skin color Anorexia
Pale gills and internal organs Spinal deformities
Exophthalmia (“pop-eye”) Milky white fluid in stomach
External and internal hemorrhaging,
especially at base of the fins
Lethargy with bouts of erratic activity (e.g.
spiral swimming, flashing)
Distended abdomen Swimming high in water column
Fluid buildup (i.e. edema), especially in body
cavity
Blood shows reduced hematocrit,
leukopenia, degeneration of leukocytes and
thrombocytes, large amounts of cellular
debris
Samples to
Collect
Proper tissue samples (according to fish size), especially spleen, kidney, heart and brain
Sexually mature fish: Ovaries or ovarian fluid (at least 1mL)
Blood
Tests Virus isolation in cell culture + identification via PCR, other molecular techniques or
immunological techniques.
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4.C.4.b. Infectious Pancreatic Necrosis virus
Environmental
Signs
Temp: Highest mortality 10-14°C
Low DO and changes in water temperature
Fish Affected
Species: Salmonidae family (especially brook, rainbow and brown trout) + many other
freshwater fish
Age: Fry and fingerlings most susceptible, adults may be affected in stressful conditions
Clinical Signs
Darkened skin color Hemorrhage on skin, at the base of and in
the fins, and on internal viscera
Exophthalmia (“pop-eye”) Stomach and intestines filled with mucoid
material that may extrude from the vent
Pale gills, liver and spleen Erratic swimming (e.g. spiraling or
“corkscrew” swimming)
Distended abdomen
SIGNS VARY BY VIRUS STRAIN
Samples to
Collect
Proper tissue samples (according to fish size)
Sexually mature fish: Ovaries or ovarian fluid (at least 1mL)
Tests Virus isolation in cell culture + identification via PCR, other molecular techniques, or
serological techniques
4.C.4.c. Koi Herpesvirus (KHV)
Environmental
Signs
Temp: Warm water 16-28°C
Season: Spring and summer with rapid water temperature shifts from cold to very warm
Fish Affected Species: Common carp and varieties (especially koi and ghost carp)
Age: All age groups, young most susceptible
Clinical Signs
Discoloration of skin Swollen kidney and spleen
Skin lesions Lethargy
Swollen, necrotic, pale and patchy gills Loss of equilibrium
Sunken looking eyes Erratic swimming prior to death
Samples to
Collect Proper tissue samples (according to fish size), especially gill, kidney, spleen, brain and gut
Tests Virus isolation in cell culture + identification via PCR
Pictures
**REPORTABLE DISEASE**
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4.C.4.d. Spring Viremia of Carp virus (SVC)
Environmental
Signs
Temp: 10-17°C, fry may be affected at temperatures as high as 22-23°C
Season: Spring; may occur in fall if temperatures are in appropriate range
Fish Affected Species: Cyprinidae family (especially carp) + young fish of various species including pike,
perch and largemouth bass
Clinical Signs
Darkened skin color Inflammation or fluid buildup of the vent
often with mucoid fecal casts
Exophthalmia (“pop-eye”) Lethargy and slow reaction to stimuli
Pale gills Decreased respiratory and swimming rates
Hemorrhage on the eyes, gills and skin,
muscles, fat, and internal organs Loss of equilibrium
Distended abdomen Fish gather at water inlet or sides of body
of water
Samples to
Collect Proper tissue samples (according to fish size), especially kidney and liver
Tests Virus isolation in cell culture + identification via RT-PCR, virus neutralization, or
immunological techniques
Pictures
**REPORTABLE DISEASE**
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4.C.4.e. Viral Hemorrhagic Septicemia virus (VHSV)
Environmental
Signs
Temp: Cold water with highest mortality at 9-12°C, upper limit of 18-20°C
Season: Spring
Fish Affected
Species: Many including muskellunge, walleye, yellow perch, smallmouth bass, rock bass,
white bass, black crappie, bluegill, lake whitefish, round goby, gizzard shad, freshwater
drum, and common carp
Clinical Signs
Hemorrhage on the eyes, skin, fins, muscle,
and internal organs (kidney, intestines, swim
bladder)
Lethargy
Pale gills and liver Abnormal and erratic swimming behavior
(e.g. flashing, swimming in circles)
Exophthalmia (“pop-eye”) Swimming near surface
Distended abdomen due to fluid filled body
cavity
ACUTE PHASE: Blood shows anemia and
appears light red and transparent
Samples to
Collect
Proper tissue samples (according to fish size), especially kidney
Sexually mature fish: Ovaries or ovarian fluid (at least 1mL)
Blood
Tests Virus isolation in cell culture + identification via RT-PCR or real-time RT-PCR, virus
neutralization, or immunological techniques
**REPORTABLE DISEASE**
4.D. Biological Causes
GENERAL DIFFERENTIALS: o Naturally occurring biological events can induce stress upon fish populations. This can cause
weakness, lethargy and poor body condition in the fish exhausting their immune system and increasing their susceptibility to secondary pathogens. These events often lead to normally occurring mass fish kill events.
o A single species or age group of fish will be affected depending on the event. o Water quality will be normal.
GENERAL TESTS: o Water quality must be collected on-site: temperature, pH, dissolved oxygen (DO),
ammonia, turbidity, color, and odor. Measurements should be taken at the site of the kill and another site lacking dead fish. Water samples can be collected for further lab testing.
o Physical examination and observation of freshly dead or dying fish should be conducted. Behavior, lesions and other abnormalities should be recorded. Tissues or carcasses suspected of having pathogen infections or infestations should be collected and tested accordingly.
o If the fish cannot be examined on-site, wrap each fish individually in plastic bags and store them on wet ice. Do not freeze or put them on dry ice. Collected tissue samples should be placed into sterile, sealed containers with 10% buffered formalin. Transport the fish and any other samples to the lab as soon as possible.
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Table 4.D.: Common biological events that are known to cause or are associated with fish kill events
Event Spawning Migration Unusual Population Structure
and Density
Environmental
Signs
Mass die-offs during fall or
spring
Water quality is normal, may be
cloudy due to presence of eggs
and sperm
Mass movement of fish
May occur within or outside of
normal migration periods or
patterns
Water quality is normal
High density of fish creating
competition can lead to lack of
prey or habitat, poor body
condition and decreased
immunity
Water quality is normal
Fish Affected
Sexually mature fish of a specific
species (according to natural
spawning time and pattern)
Multiple species may be
affected depending on
chronology of fish spawning in a
body of water
Specific species Specific size or age group of a
specific species
Clinical Signs
Lethargy, weakness
Poor body condition
Secondary infections or infestations of pathogens
Tests Physical examination of fish
Water quality
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Revised: August 2015 Page 24
References
Alaska Department of Fish and Game. Motile Aeromonas and Pseudomonas Septicemia. http://www.adfg.alaska.gov/static/species/disease/pdfs/fishdiseases/motile_aeromonas_and_pseudomo nas_septicemia.pdf (July 2014)
Alaska Department of Fish and Game. Saprolegniasis – Cotton Wool Disease. http://www.adfg.alaska.gov/static/species/disease/pdfs/fishdiseases/saprolegniasis.pdf (July 2014)
Bueno, I. and N. B. D. Phelps. 2014. Retrospective fish kill investigation in Minnesota (2003-2013). Project Report
Donaldson, M. R., S. J. Cooke, D. A. Patterson, and J. S. MacDonald. 2008. Review Paper: Cold shock and fish. Journal of Fish Biology 73: 1491-1530
FAO (Food and Agricultural Organization of the United Nations) – Fisheries and Aquaculture Department. 1996. Parasites, infections and diseases of fishes in Africa: An update. CIFA Technical Paper No. 31. Rome, Italy
Kane, A. S., A. Baya, R. Reimschuessel, K. M. St Pé, C. A. Poukish, and C. P. Driscoll. 1999. Field Sampling and Necropsy Examination of Fish. Virginia Journal of Science 50(4): 345-363
La, V. T. and S. J. Cooke. 2011. Advancing the Science and Practice of Fish Kill Investigations. Reviews in Fisheries Science 19(1): 21-33
Minnesota Department of Natural Resources. 2014. Fish Diseases: Spring Viremia of Carp (SVC). http://www.dnr.state.mn.us/fish_diseases/svc.html (July 2014)
Minnesota Department of Natural Resources. 2014. Fish Diseases: Viral Hemorrhagic Septicemia. http://www.dnr.state.mn.us/fish_diseases/vhs.html (July 2014)
New Jersey Department of Environmental Protection Division of Fish and Wildlife. NJDEP Division of Fish and Wildlife Questions and Answers Concerning Furunculosis. http://www.njfishandwildlife.com/pdf/2014/furunculosis_q-a.pdf (July 2014)
New Jersey Department of Environmental Protection) Division of Fish and Wildlife. 2014. Furunculosis. http://www.njfishandwildlife.com/fishhealth_furunculosis.htm (July 2014)
OIE (World Organization for Animal Health) Aquatic Animal Health Standards Commission. 2014. Manual of diagnostic tests for aquatic animals. Paris, France
Phelps, N. B. D. 2013. Improved Diagnosis and Management of Viral Hemorrhagic Septicemia Virus in Fish (Doctoral dissertation). University of Minnesota Digital Conservancy. http://hdl.handle.net/11299/147222 (July 2014)
Pokorova, D., T. Vesely, V. Piackova, S. Reschova, and J. Hulova. 2005. Current knowledge on koi herpesvirus (KHV): a review. Vet. Med. – Czech 50(4): 139-147
The Center for Food Security and Public Health – Iowa State University. 2007. Spring Viremia of Carp. Fact Sheet
United States Fish and Wildlife Service. 1990. Field Manual for the Investigation of Fish Kills. Ed. Meyer, F. P. and L. A. Barclay. Resource Publication 177
United States Fish and Wildlife Service and American Fisheries Society – Fish Health Section. Fish Health Section Blue Book: Suggested Procedures for the Detection and Identification of Certain Finfish and Shellfish Pathogens. 2012 edition. American Fisheries Society – Fish Health Section, Bethesda, Maryland
Zajac, A. M. and G. A. Conboy. Veterinary Clinical Parasitology. 8th
edition. West Sussex, United Kingdom: Wiley- Blackwell, 2012
http://www.adfg.alaska.gov/static/species/disease/pdfs/fishdiseases/motile_aeromonas_and_pseudomo%09nas_septicemia.pdfhttp://www.adfg.alaska.gov/static/species/disease/pdfs/fishdiseases/motile_aeromonas_and_pseudomo%09nas_septicemia.pdfhttp://www.adfg.alaska.gov/static/species/disease/pdfs/fishdiseases/saprolegniasis.pdfhttp://www.dnr.state.mn.us/fish_diseases/svc.htmlhttp://www.dnr.state.mn.us/fish_diseases/vhs.htmlhttp://www.njfishandwildlife.com/pdf/2014/furunculosis_q-a.pdfhttp://www.njfishandwildlife.com/fishhealth_furunculosis.htmhttp://hdl.handle.net/11299/147222
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Appendix A. Standard Protocol for Fish Kill Investigations
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Standard Protocol for Fish Kill Investigations
Time management is essential following a fish kill event. Rapid response and standard investigation
procedures are important for improving diagnostic outcomes. These guidelines have been prepared to
support the process. Do not hesitate to contact a fish health professional with questions.
Before you go
1) Make sure all fish kill kit supplies are in stock and water quality devices are calibrated
2) Print out a map of the body of water and estimate fish kill location based on:
a) Information from reporter
b) Wind direction
3) Contact either laboratory to discuss investigation strategies and sample submission
a) UMN Veterinary Diagnostic Laboratory: 612-624-7450
b) MN DNR Pathology Laboratory: 651-259-5096
On-site
1) Survey the scene, record findings on Investigation Report Form, and photograph:
a) Behavior and appearance of sick/dying or dead fish
i. Behavior to be noted in dying fish: listless, flashing, spiraling, equilibrium loss,
gasping
b) Weather conditions
c) Location conditions
2) Collect fish samples
a) Collect at least 5 fish from each species
b) Select fish in order of availability:
i. Sick/dying
ii. Freshly dead
iii. Decomposing, then
iv. Healthy
c) Focus on collecting fresh, abnormal fish with clinical signs of disease
d) Place fish on frozen gel packs for transport to the laboratory if on-site necropsies are not
being performed
3) If field necropsies are being performed, for each set of 5 fish follow these steps:
a) Parasitology: If fish samples are fresh, collect gill, fin, and mucus from skin. Wet mount
on a glass microscope slide for off-site examination.
i. Label the slides with sample, species, and fish ID
ii. Carefully store in a container filled with wet paper towels for transport to
laboratory
b) Bacteriology: Swab the kidneys and any internal lesions from each fish. Swab the gills or
wall of the gut for control samples. Label all swabs.
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Revised: August 2015 Page 27
c) Virology: Collect tissue samples from individual fish and place into labeled whirl-pack
bags.
i. For fish less < 3”, collect entire viscera samples.
ii. For fish 3”, collect kidney, spleen and other affected tissue samples (e.g. gills,
swim bladder, ovaries, etc)
iii. Place samples on frozen gel packs within a cooler for transport to laboratory
d) Record findings on On-site Necropsy Report Form
e) Clean up
i. Place dirty dissection tools into a designated “Dirty” container
ii. All scalpel blades, broken microscope slides or cover slips should be discarded
into a designated “Sharps” container
iii. Place carcass remains and disposable supplies into a garbage bag and dispose at
laboratory
iv. Do not leave anything behind or dispose of anything on site. Do not discard
carcass remains into the body of water.
4) Measure water quality/condition, and fill in the data on the report form
a) If fish kill is at a lake or pond, measure the following at both the site where the fish are
collected, and at a location with no dead fish
i. DO
ii. pH
iii. Temperature
b) If the fish kill is at a stream or river, take 3 measurements: upstream, at the fish kill site,
and downstream
c) If a body of water is adjacent to the fish kill body of water, take 4a. measurements
5) Investigators can pass out the fish kill informational cards to individuals in the area if questions
or interest arise
6) Double check to make sure the Report Forms are completed prior to leaving the site
Where to bring samples
1) Samples should be sent on frozen gel packs to the UMN Veterinary Diagnostic Laboratory by
same-day personal delivery or overnight mail
Attn: Dr. Nick Phelps
Veterinary Diagnostic Laboratory
University of Minnesota
1333 Gortner Ave
St. Paul, MN 55108
P: 612-624-7450
E : phelp083@umn.edu
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Appendix B. Standard Investigation and Necropsy Report Form
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Fish Kill Investigation Report Form
Date
Time
Estimated fish kill start: Estimated fish kill duration:
Reporter
Name:________________________
Address:______________________
City: _________________________
State: ______ Zip: ____________
Phone: _______________________
Email: ________________________
Affiliation:
Investigator
Name: __________________________
Address: ________________________
City: ___________________________
State: ______ Zip: ______________
Phone: _________________________
Email: __________________________
Affiliation:
Location Body of water name:
County:
GPS:
Has a fish kill been on this site before? If yes,
when?
Species affected # Dead Wt Length ______________ ______ _____ _______ ______________ ______ _____ _______ ______________ ______ _____ _______ ______________ ______ _____ _______ ______________ ______ _____ _______ ______________ ______ _____ _______ ______________ ______ _____ _______ ______________ ______ _____ _______
Weather
Temperature: _______
Cloud Cover:
Cloudy
Partly cloudy
None
Rain: Yes / No
Wind: Yes / No
List any severe weather conditions:
Surrounding the Body of Water
Farm/Animal Feedlot
Irrigation Runoff
Industrial Plant
Sewage Treatment Plant
Coal/Strip Mining
Construction
Transportation
Unknown
Other:____________________
Water Condition and Quality
Upstream At fish kill site Downstream
Odor Yes / No Yes / No Yes / No
Algal Bloom Yes / No Yes / No Yes / No
Color
Turbidity
Temperature
pH
DO
Ammonia
Additional Comments
Office Use Only
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Revised: August 2015 Page 30
On-site Necropsy Report Form
Species: _______________________________ Number examined: ________
Gross External Exam
Skin: Normal Abnormal Color: ___________________ Hemorrhagic Ulcer Necrotic
Parasites Lesions: ___________________________________________________________________
Other: __________________________________________________________________________________
Location(s): _____________________________________________________________________________
Gills: Normal Pale Mottled Hemorrhagic Necrotic Parasites
Other: __________________________________________________________________________________
Eyes: Normal Exophthalmia/pop-eye Hemorrhagic Parasites Bilateral Unilateral: L / R
Other: __________________________________________________________________________________
Fins: Normal Hemorrhagic Frayed Eroded Necrotic Other: __________________
Samples collected:
Skin scrape: _____________________________________________________________________________
Gill clip: ________________________________________________________________________________
Fin clip: _________________________________________________________________________________
Other: __________________________________________________________________________________
Additional Comments:
Gross Internal Exam
Adipose tissue: Normal Excessive Reduced Hemorrhagic Other: _________________________
Liver: Normal Pale Mottled Enlarged Reduced Hemorrhagic
Texture: ______________________________ Lesions: ____________________________________
Other: __________________________________________________________________________________
Spleen: Normal Pale Enlarged Reduced Texture: __________________________________
Other: __________________________________________________________________________________
Kidney: Normal Pale Enlarged Hemorrhagic Texture: ______________________________
Other: __________________________________________________________________________________
Intestine: Normal Distended: fluid / mucoid Flaccid Hemorrhagic Parasites
Other: _______________________________________________________________________________
Samples collected:
Bacteriology - Swab location(s): _________________________ Control location(s): ___________________
Virology - Tissue(s): _______________________________________________________________________
Other: __________________________________________________________________________________
Additional Comments:
(modified from U.S. Fish and Wildlife Service 1990)
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Appendix C. Mobile Kit Contents Form
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Revised: August 2015 Page 32
Mobile Fish Kill Kit Contents
Forms
Fish Kill Investigation Protocol + Minnesota
DNR Map
Fish Kill Investigation Report & Necropsy
Form
List of contacts
Mobile Kit Contents Checklist
Equipment
10% NBF solution /1
70% alcohol solution /1
Camera/Cell phone camera /1
Disposable cardboard trays /5
Disposable gloves /10
EM containers /5
Folders /1
Forceps /3
Ice box for samples /1
Ice gel packs /3
Iris scissors /3
Large container to store supplies /1
Lighter /1
Paper towels /50
Pens /2
Permanent markers /2
Plastic bags (small and large) /10
Sample collection bags (whirl-pack) /20
Scalpel blades and handles /2
Scissors /1
Scoop net /1
Scotch tape /2
Small containers /2
Tape measure /1
Water Quality
Bottles for collection /3
Dissolved oxygen meter /1
Distilled water /1
pH meter /1
Secchi disk /1
Thermometer /1
Waste Disposal
Garbage bags /10
“Sharps” container /1
Safety
Band-aids /10
Insect repellant /1
Optional
Microscope /1
Microscope cover slip pack /1
Microscope slides pack /1
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Appendix D. Contacts List
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Revised: August 2015 Page 34
Minnesota Veterinary Diagnostic Lab Minnesota DNR Pathology Lab
Dr. Nicholas Phelps Ling Shen
612-624-7450 651-259-5096
phelp083@umn.edu ling.shen@state.mn.us
1333 Gortner Ave 500 Lafayette Rd, Box 25
St. Paul, MN 55108 St. Paul, MN 55155-4025
Minnesota DNR Area Fisheries Officers
Email format: Areaname.Fisheries@state.mn.us
Aitkin, MN 56431
Rick Bruesewitz
218-927-3751
Altura, MN 55910
Adam Moticak
(Crystal Springs Hatchery)
507-796-8000
Baudette, MN 56623
Phil Talmage
218-634-2522
Bemidji, MN 56601
Gary Barnard
218-308-2339
Brainerd, MN 56401
Marc Bacigalupi
218-828-2550
Detroit Lakes, MN 56501
Nathan Olson
218-846-8340
Duluth, MN 55804
Deserae Hendrickson
218-525-0853 x 201
Duluth, MN 55804
Don Schreiner
218-525-0853 x 206
Duluth, MN 55804
Mark Gottwald
(French River Hatchery)
218-525-0867 x 215
Fergus Falls, MN 56537
Jim Wolters
218-739-7576
Glenwood, MN 56334
Dean Beck
320-634-4573
Grand Marais, MN 55604
Steve Persons
218-387-3056
Grand Rapids, MN 55744
Chris Kavanaugh
218-327-4430
Hinckley, MN 55037
Roger Hugill
320-384-7721
Hutchinson, MN 55350
Lee Sundmark
320-234-2550
International Falls, MN 56649
Kevin Peterson
218-286-5220
Lake City, MN 55041
Kevin Stauffer
651-345-3365
Lanesboro, MN 55949
Pat Schmidt
(Lanesboro Hatchery)
507-467-3771
Lanesboro, MN 55949
Steve Klotz
507-467-2442 x 222
Little Falls, MN 56345
Eric Altena
320-616-2450
Montrose, MN 55363
Joe Stewig
763-675-3301
Ortonville, MN 56278
Norm Haukos
320-839-2656
Park Rapids, MN 56470
Doug Kingsley
218-732-4153
Peterson, MN 55962
Shawn Hasse
(Peterson Hatchery)
507-875-2625
Remer, MN 56672
Gary Mattson
218-792-5164
Shakopee, MN 55379
Daryl Ellison
952-496-4141 x 222
Spicer, MN 56288
Dave Coahran
320-796-2161
St. Paul, MN 55106
TJ DeBates
651-259-5831
Tower, MN 55790
Edie Evarts
218-753-2580 x 222
Walker, MN 56484
Doug Schultz
218-547-1683
Waterville, MN 56096
Craig Soupir
507-362-4223 x 222
Windom, MN 56101
Ryan Doorenbos
507-831-2900 x 232
Finland, MN 55603
Dean Paron
218-353-8857
mailto:phelp083@umn.edumailto:ling.shen@state.mn.usmailto:Areaname.Fisheries@state.mn.us
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