Zebrafish Biology, Health, and Diseases

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Zebrafish Biology, Health, and Diseases

• Claudia Harper, DVM, Dipl. ACLAM

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Presentation Outline

• Zebrafish as a Biomedical Research Model
• Non Protocol Sources of Variation in Aquatic
  Biomedical Research
• Disease Prevention
• Clinical Signs of Disease
• Zebrafih Pathogens
• Bacteria
• Mycosis
• Virus
• Protozoa

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Zebrafish and Research

• Biological pathways
• Carcinogenicity
• Drug development
• Genomics
• Genetics
• Behavioral
• Molecular
• Mutagenesis screening
• Toxicology
• Vertebrate development
• Other

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Non Protocol Sources of Variation on Aquatic
Biomedical Research

• Maintaining healthy animals minimizes non
  protocol sources of variation associated with
  disease and in apparent infection
• The widespread use of genetically engineered fish
  is likely to modify current concepts of aquatic
  diseases because of novel or unpredictable
  interaction among genetic alterations which can
  alter disease expression in genetically
  engineered fish

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Non Protocol Sources of Variation

• Sources of variation can include any factor that
  may impact histology, organogenesis, behavior,
  gene regulation, physiology and immunology etc.
• These variables have been well described in
  rodent models used as biomedical research. These
  variables include genetics, bacteria, protozoan,
  viruses, fungal agents and environmental
  parameters.
• There are few publications regarding non protocol
  variables in zebrafish.

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Examples of Possible Non Protocol Sources of
Variation in Aquatic Biomedical Research

• Disease
• Environmental parameters
• Fish genetics
• Health status
• Husbandry practices
• Nutrition
• Etcetera

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Poor Water Quality - Gill Hyperplasia
10x
40x
Gills are involved in respiration, excretion of
nitrogenous waste, ion balance
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Disease Prevention
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Diseases Prevention

• Source of Fish, Water and Feed
• Ekkwill water life resource pond reared in
  Gibsonton Florida, Aquatic Ecosystems in Florida,
  Scientific hatcheries in California, ZIRC in
  Oregon Storage of feed
• Quarantine Program / Bleach eggs
• Established Traffic Flow - Clean to Dirty
• Maintenance of life support systems. Ex.
  Ultraviolet light can minimize the number of
  infectious agents in the water.
• Husbandry practices which minimizes the presence
  of biological substrate.
• Cleanliness of facility.
• Disinfection of systems between groups of fish to
  prevent disease transmission by equipment,
  personnel, or water.

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Diseases Prevention

• Wash hands with soap upon entering the facility
• Disinfect footwear and wheels of carts
• Reduce the number of visitors to a minimum
• Monitor water quality parameters
• Sentinel programs
• Reduce stressful conditions - susceptibility to
  disease
• Crowding, low water flow, poor nutrition, poor
  water quality, rapid water fluctuations, fish
  handling

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Stress in Fish
INCREASING DEGREE OF STRESS
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Stress Disease Prevention

• Physiology
• Elevated cortisol and catecholamine (head kidney)
• Elevated blood glucose
• Hematology and CBC chemistry profile
• Gene regulations
• Increased expression of Myostatin zfMSTN-1 and-2
  gene expression in spleens from stressed fish -
  Myostatin is a member of the TGF-beta superfamily
  and a potent negative regulator of muscle growth
  and development
• Immune modulation
• Stress impairs the production and release of
  antibodies. Temperature stress severely limits
  the fishs ability to release antibodies leading
  to increased disease susceptibility
• Cell Signaling
• Heat shock proteins

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Why Do Laboratory Zebrafish Get Sick

• Handling
• Dermal abrasion
• Net induced injury
• Egg stripping
• Pathogens
• Parasites
• Bacteria
• Poor water quality
• Stress
• Overcrowding

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Signs of Disease

• Eyes
• Exophthalmia
• Gill
• coloration
• Fins / Skin
• Frayed fins
• Ulceration
• Excess mucus
• Discoloration
• Skeletal deformities
• Body Condition
• Abdominal distention
• Emaciation
• Urogenital pore
• Fecal casts

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BACTERIA
Electron micrograph of Aeromonas hydrophila

• Bacteria
• Mycobacterium spp.
• Flexibacter columnaris
• Aeromonas hydrophila

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Flexibacter columnaris

• ETIOLOGY
• Gram-negative slender rods (3-8 microns)
• Gliding bacteria
•  
• TRANSMISSION
• Opportunistic ubiquitous pathogen
• Fry are the most susceptible. All ages may be
  affected.
• Skin damage maybe needed to initiate infection
• CLINICAL PRESENTATION
• Flared opercula and fish face the water current
• Mucus strands from gills
• Lethargy and anorexia
• Skin lesions and frayed fins

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Flexibacter columnaris

• DIAGNOSIS
• Culture bacteria from lesions (skin or gills)
• Wet mount preparation of gills
• Flavobacterium branchiophilum - non motile
• Flexibacter columnaris gliding bacteria
• Histopathology of skin or gills
• proliferation of the epithelium resulting in
  clubbing and fusion of the lamella. Necrosis of
  the gill lamella occurs in serious cases.
•  

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Flexibacter columnaris
Abnormal Gill Histology
Normal Gill Histology
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Motile Aeromonad Septicemia

• ETIOLOGY
• Aeromonas hydrophila is an opportunistic gram
  negative motile bacteria
• HISTORY CLINICAL PRESENTATION
• Effects many different freshwater species
• Associated with stress and overcrowding
• The clinical signs and lesions are variable
• Most common finding is hemorrhage in skin, fins,
  oral cavity and muscles with superficial
  ulceration of the epidermis.
• Exophthalmus and ascites are common findings.
• Splenomegaly and swollen kidneys are common
  findings.
• Occasionally cavitating ulcers maybe seen.
• Necrosis of multiple organs such as spleen,
  liver, kidney and heart maybe observed.
• Acute to chronic mortality
• Myositis

Pullium et al. 2000
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Motile Aeromonad Septicemia contd

• DIAGNOSIS
• Histology dermatitis / myositis
• Culture Rimmler-Shotts 22 to 25C
• PREVENTION
• Avoid stress stocking density, water quality
• Avoid elevated water temperature
• UV approximately 5 000 microW sec/cm2
• TREATMENT
• Stress born disease - Remove the environmental
  stress that caused the outbreak
• Antibiotics may not be necessary
• Oxytetracycline, sulfamethoxine-ormetoprim,
  chloramphenicol, nifurpirinol

Urogenital pore
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Mycobacteriosis
Astrofsky et al. 2000

• ETIOLOGY
• M. marinum, M. abscessus, M. chelonae and M.
  fortuitum
• Ubiquitous acid-fast bacteria found in the soil
  and water
• HISTORY MODE OF TRANSMISSION
• Poor water quality
• Colonize biofilms on tanks and pipes
• Stressed fish (overcrowding or immunocompromised
  animals)
• CLINICAL PRESENTATION
• Subclinical, lethargy, anorexia
• Chronic, systemic bacterial infections with low
  level mortality
• Skin ulcers, raised scales and frayed fins
• Decreased reproductive performance
• Presence of multiple, white nodules in various
  visceral organs
• DIAGNOSIS
• Histology acid-fast-positive, rod-shaped
  bacteria
• Culture on selective media

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Mycobacterium marinumGranulomatous nephritis and
hepatitis
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Mycobacteriosis

• PREVENTION
• Disinfection
• Quarantine
• Maintain good water quality
• Minimize stress
• TREATMENT
• No known treatment
• ZOONOSIS
• Fish tank granuloma, swimming pool granuloma

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Mainous ME, Smith SA. 2005. Efficacy of common
disinfectants against Mycobacterium marinum.
Journal of Aquatic Animal Health 17284288
The most effective disinfectants against M.
marinum are 70 and 50 ethyl alcohol, benzyl-
4-chlorophenol-2-phenylphenol (Lysol), and sodium
chlorite (Clidox-S). They can reduce the number
of detectable M. marinum to zero within 1 min of
contact time. Sodium hypochlorite (Ultra-
Clorox) was moderately effective but required at
least 10 min of contact time to reduce bacterial
counts and 20 min of contact time to eliminate
the organism
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Nematodes

• Nematodes / Platyhelminths
• Pseudocapillaria tomentosa
• Tubellarians
• Stylaria

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Intestinal Capillariasis

• ETIOLOGY
• Metazoan parasite Pseudocapillaria tomentosa
• Broad host specificity infecting approx. 25
  fishes in the Cyprinid family
• Capillarids infect all classes of vertebrates and
  are pathogenic as they invade tissues
• TRANSMISSION
• Indirect life cycle intermediate hosts such as
  oligochaete worms
• Tubifex tubifex can serve as a host
• Direct transmission can also occur
• CLINICAL PRESENTATION
• Reduced growth potential
• Decrease reproductive rate
• Wasting Disease
• Intestinal carcinomas

Fig 1. Double operculated Capillaria egg
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Intestinal Capillariasis

• DIAGNOSIS
• Microscopic examination
• fresh feces allows visualization of the egg
• gut content allows visualization of the worm
• PREVENTION
• Quarantine
• Do not feed worms
• TREATMENT
• No treatment tested in zebrafish
• Ivermectin low therapeutic index and thus
  dangerous
• Fenbendazole

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Intestinal Capillariasis(Kent et al. 2002)
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Tubellarian

• ETIOLOGY
• Free living tubellarian, trematode
• Opportunistic organism in fresh water aquaria
• TRANSMISSION
• Direct transmission
• CLINICAL PRESENTATION
• Infection is load dependant
• Affects skin and gills
• DIAGNOSIS
• Wet mount of gills or skin
• PREVENTION
• Minimize organic load accumulation
• TREATMENT
• Improve water quality and husbandry

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Stylaria

• ETIOLOGY
• Free living oligochaete
• TRANSMISSION
• Direct transmission
• CLINICAL PRESENTATION
• None
• DIAGNOSIS
• Wet mount of organic waste
• RISK
• Intermediate host for many pathogens
• PREVENTION
• Minimize organic load accumulation

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WATER MOLDS MICROSPORIDIAN

• Microsporidian
• Pseudoloma neurophilia
• Mold
• Saprolegnia sp.
• Acremonium sp.
• Lecythophora mutabilis

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Microsporodiosis

• ETIOLOGY
• Neural microsporidiosis
• Pseudoloma neurophila obligate intracellular
  parasites
• CLINICAL DISEASE
• Emaciation
• Spinal curvature (lordosis, kyphosis, scoliosis)
• TRANSMISSION
• Horizontal and vertical transmission
• PATHOLOGY DIAGNOSIS
• PCR whole body
• Wet mounts central nervous system (laborious)
• Histology Acid fast stain and HE
• Xenoma (spores) in central nervous system
• Spinal cord and hindbrain
• CONTROL AND TREATMENT

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Microsporidiosis, Pseudoloma neurophiliain
Zebrafish Brain Muscle
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Typical Water Mold Infection

• ETIOLOGY
• Saprolegnia spp. is an opportunistic fresh water
  fungus
• Most common fungal infection of fresh water and
  estuarine fish
• Class Oomycetes
• TRANSMISSION
• Direct transmission and affects all species and
  ages
• The fungi are normal water inhabitants that
  invade the traumatized epidermis.
• Infection acquired from inanimate sources such as
  fungi sporulating on inanimate sources releasing
  motile zoospores (dead fish or eggs)
• Improper handling, bacterial or viral skin
  diseases, and trauma are the major causes of the
  disease.
• CLINICAL PRESENTATION
• White to brown cotton-like growths on skin, fins,
  gills, traumatized epidermis
• Superficial lesions start on small surface and
  spread rapidly
• Fish die due to osmotic or respiratory problems
  if the affected area of skin or gills is large.

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Typical Water Mold Infection

• DIAGNOSIS
• Wet mount broad nonseptate branching hyphae
  motile flagellated zoospores in the terminal
  sporangia skin or gills
• Culture
• Potato dextrose agar pH 5.6 (yeast fungi)
• Cottonseed Agar
• Cornmeal Agar
• PREVENTION TREATMENT
• Avoid skin damage and predisposing stress
• Water molds cannot be eliminated from any culture
  system
• Prolonged salt immersion 1-5 ppt (1-5 gm / L)

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Lecythophora mutabilis (Dykstra et al. 2001)

• ETIOLOGY
• Ubiquitous, opportunistic fungal agent
• TRANSMISSION
• Direct transmission
• CLINICAL PRESENTATION
• Lethargy, reduced appetite, mortality
• Fry ( 5-24 days post hatch) most commonly
• affected
• Biofilm around the head occluding of oral cavity,
  
• starvation, asphyxiation
• DIAGNOSIS
• Culture
• PREVENTION TREATMENT
• Maintain total hardness and calcium within normal
  levels

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Lecythophora mutabilis (Dykstra et al. 2001)
eyes
Oral cavity
Fungal hyphae
gills
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Protozoan

• Tetrahymena sp.
• Oodinium sp.
• Ichthyophthirius multifilis
• Trichodina sp.
• Ichthyobodo sp.
• Epistylis

RM Durborow. 2003. SRAC Publication No. 4701
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Chilodonella

• ETIOLOGY
• Oval flat protozoan
• Parallel rows of cilia and a notched anterior end
• TRANSMISSION
• Direct transmission
• Parasite glides over the fish gill and skin
  surface
• CLINICAL PRESENTATION
• Whitish or bluish sheen on body
• Heavy infection can cause mortality
• DIAGNOSIS
• Wet mount Free swimming parasite with erratic
  swimming behavior
• Histopathology skin or gill
• TREATMENT
• Salt bath
• Copper Sulfate

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Tetrahymenosis

• ETIOLOGY
• Tetrahymena spp.
• Ciliated protozoa that have worldwide
  distribution in FW SW
• Related to Ichthyophthirius multifiliis
• TRANSMISSION
• Direct contact
• Mode of entry into the host has not been well
  documented
• Commonly found
• Resident of biofilter
• Food source
• CLINICAL PRESENTATION
• Sudden, high mortality in 30-day-old fry - 100
  mortality
• Epithelial necrosis and hemorrhage, myositis,
  exophthalmos due to peri-ocular tissue invasion,
  subdermal muscle tissue necrosis with minimal
  inflammatory
• Colonize the surface epithelium and cause excess
  mucus production
• DIAGNOSIS
• Wet mount, histopathology gills, skin or
  internal organs

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Trichodiniasis

• ETIOLOGY
• Trichodina sp.
• Ciliated protozoans
• Saucer-shaped, 50 microns diameter, cilia, macro
  micro nucleus
• HISTORY AND CLINICAL PRESENTATION
• Not always associated with disease.
• Flashing and become lethargic
• Skin -increase in mucus production (white to
  bluish haze)
• - ulcers and the fins may fray
• Respiratory distress.
• TRANSMISSION
• Direct contact with infected fish and or
  contaminated water.
• DIAGNOSIS
• Wet mount of skin and gills
• Histologically, masses of organisms are attached
  by adhesive discs to the skin.
• The underlying epithelial cells undergo necrosis.
  
• There is secondary hyperplasia and hypertrophy of
  the gill epithelium.

RM Durborow. 2003. SRAC Publication No. 4701
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Costiosis

• ETIOLOGY
• Ichthyobodo spp.
• Protozoa
• 6-12 microns long with flagella smallest
  parasite
• Piriform shaped Stalked protozoa
• HISTORY AND CLINICAL PRESENTATION
• Commonly affects hatchery raised fish and
  attaches to the skin or gills (feeds on fish)
• Flashing
• Skin -increase in mucus production (white to
  bluish haze)
• Respiratory distress (flaring of gills).
• Mortalities
• TRANSMISSION
• Direct contact
• DIAGNOSIS
• Wet mount (40X)
• Histology of skin and gills

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VIRUS
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Lymphocystis

• ETIOLOGY
• Iridovirus DNA virus affecting freshwater and
  saltwater fishes
• Most common viral infection - reported in over
  125 species of FW and SW fish
• TRANSMISSION CLINICAL PRESENTATION
• causes diseases that range in severity from
  subclinical to lethal
• White yellow raised cauliflower-like lesions on
  fin and skin
• Epidermal abrasions acts as port of entry.
  Infected cells are enlarged and rupture or
  slough off and which release the viral particles
  into the water.
• Disease runs its course in 4 or more weeks
  (depending on species and water temperature)
  Complications
• Lesions on the mouth may lead to difficulty in
  feeding and difficulty breathing if gills are
  affected.
• Prone to secondary bacterial or fungal infections
  
• Lethargy and low mortality rate
• DIAGNOSIS
• Wet mount skin or gills showing massively
  enlarged dermal fibroblasts
• Histology Virus infects dermal fibroblast and
  causes cytomegaly
• TREATMENT
• Isolate affected animals as viral particles in
  the water can infect other fish.

Picture from http//www.aquariumpharm.com/disease/
lymphocystis.html
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Drugs and Ammonia Detoxification BacteriaFrom
Noga EJ. 1996. Fish Diseases Diagnosis and
Treatment. P.65
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Conclusions on Zebrafish Diseasesin Biomedical
Research

• Most diseases in zebrafish research settings can
  be prevented by good husbandry, good water
  quality and preventive programs.
• More research and publications are needed which
  specifically pertain to zebrafish.