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8/12/2014
Pathology
of Laboratory
Fishes
Jeffrey C. Wolf, DVM, DACVP
Experimental Pathology
Laboratories (EPL®), Inc.
Sterling, VA
What are Laboratory Fishes?
• Nearly any fish can
be a laboratory fish
What are Laboratory Fishes?
• Nearly any fish can
be a laboratory fish
• >32,000 fish species
• There are more fish
diseases than you
want to learn!
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8/12/2014
Excluded (with some notable exceptions):
• Food fishes
• Foreign laboratory fishes
• Marine fishes
• Wild fishes
• Silverfish
• The band Phish
• Popular fish snacks
Common Laboratory Fish in U.S.
• Zebrafish
• Japanese medaka
• Fathead minnow
• Guppy
• Goldfish
• Rainbow trout
• Channel catfish
• Nile tilapia
Common Laboratory Fish in U.S.
• Zebrafish
• Japanese medaka
-- very hardy and prolific
• Fathead minnow
-- U.S. native species
-- externally dimorphic
• Guppy
-- livebearer
• Goldfish
-- represent coldwater fishes
• Rainbow trout
• Channel catfish
-- larger model
-- surrogate for Atlantic salmon
-- representative catfish species
• Nile tilapia
-- also represents ornamental
tropical fish
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Focus on:
• Diseases likely to be encountered in
laboratory setting
• A few classic diseases
(for you board prep types)
• Of the vast majority of fish that I
evaluate in toxicologic studies,
relatively few have any evidence of
background disease
• Far and away, the most common
disease that you will encounter in
laboratory fishes is …
The 800 lb acid-fast gorilla of fish research…
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striped bass: pale, lumpy, enlarged
spleen due to granulomatous splenitis
striped bass kidney
(Ziel-Neelsen stain):
granulomas with
acid-fast bacterial rods
Mycobacterium spp. (Piscine Tuberculosis)
striped bass: pale, lumpy, enlarged
spleen due to granulomatous splenitis
striped bass kidney
(Ziel-Neelsen stain):
granulomas with
acid-fast bacterial rods
• Gram-positive acid-fast rods; M. marinum, M.
chelonei, M. fortuitum, M. abscessus, M. peregrinum
• Virtually all fishes can be affected (although some
species more susceptible than others)
• Long latent period, low level mortalities
• Numerous variably-sized granulomas in various
tissues, often with necrotic centers
• Relatively resistant fish species may have diffuse
granulomatous inflammation
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Japanese medaka spleen:
diffuse granulomatous
inflammation
Japanese medaka kidney:
numerous acid-fast bacilli
• Gram-positive acid-fast rods; M. marinum, M.
chelonei, M. fortuitum, M. abscessus, M. peregrinum
• Virtually all fishes can be affected (although some
species more susceptible than others)
• Long latent period, low level mortalities
• Numerous variably-sized granulomas in various
tissues, often with necrotic centers
• Relatively resistant fish species may have diffuse
granulomatous inflammation
• Ubiquitous, no cure, difficult to eradicate from
infected populations and environment
• Culture tricky; some labs have PCR assays
• Zoonotic disease
channel catfish: caudal dermal necrosis
channel catfish: gill necrosis
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Flavobacterium columnare (Columnaris Disease)
channel catfish: caudal dermal necrosis
• Gram-negative filamentous rods; Formerly known as
Flexibacter columnaris or Cytophaga columnaris
• Ubiquitous on fish surface and in aquaria
• Serious, highly communicable disease of young
salmonids, catfish and many other fishes
• Associated with stress, predisposing factors include
high water temperature, crowding, injury, and poor
water quality (e.g., low O2, high ammonia)
• Caudal and anal fins may become severely eroded
• Caudal trunk skin sometimes involved: “saddleback
disease”
• Gills common site, and may be only affected area
• Responsive to some water bath treatments
wet mount of skin scraping:
bacteria form “haystacks”
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goldfish: deep ulcers
salmon: deep dermal
and muscle ulcers
salmon kidney: necrosis with
intralesional gram-negative bacteria
Aeromonas salmonicida (Goldfish Ulcer Disease)
goldfish: deep ulcers
salmon: deep dermal
and muscle ulcers
salmon kidney: necrosis with
intralesional gram-negative bacteria
Aeromonas salmonicida (Goldfish Ulcer Disease)
• Gram-negative non-motile short rods; obligate
pathogen, but can survive for long periods off host
• Subcutaneous swelling leads to ulcerative dermatitis
• Chronic disease in salmonids called “furunculosis”
• Splenomegaly, renomegaly, and ascites
• Necrosis of affected tissues with abundant bacterial
colonies, but few inflammatory cells due to
leukocytolytic endotoxin
• Transmission by direct contact with diseased fish,
contaminated water, fomites, and infected eggs
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tilapia: ophthalmitis
tilapia: peri- and myocarditis
tilapia: meningitis
tilapia: Gram+ cocci/diplococci
Streptococcus sp. , e.g., S. iniae
tilapia: ophthalmitis
tilapia: peri- and myocarditis
tilapia: meningitis
tilapia: Gram+ cocci/diplococci
• Primarily beta-hemolytic streptococci
• A major problem in the tilapia industry, and
occasionally affects other fishes
• Associated with overcrowding and poor water quality
in recirculating culture systems
• Either acute fulminating septicemia, or chronic form
limited primarily to the CNS
• Granulomatous meningoencephalitis, polyserositis,
epicarditis, myocarditis, and/or cellulitis
• Should routinely culture brain tissue if Strep
suspected
• Ubiquitous organism, and S. iniae is a potential
zoonotic pathogen
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Streptococcus iniae:
Zoonotic agent
actual S. iniae-infected
patient with cellulitis
wound staged to make a point
• Primarily beta-hemolytic streptococci
• A major problem in the tilapia industry, and
occasionally affects other fishes
• Associated with overcrowding and poor water quality
in recirculating culture systems
• Either acute fulminating septicemia, or chronic form
limited primarily to the CNS
• Granulomatous meningoencephalitis, polyserositis,
epicarditis, myocarditis, and/or cellulitis
• Should routinely culture brain tissue if Strep
suspected
• Ubiquitous organism, and S. iniae is a potential
zoonotic pathogen
• Latest tilapia pathogen: Francisella spp.
Classic
Disease Alert
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exophthalmos
abdominal distention
frontal bone necrosis
peritonitis
focal hepatitis
Gram-negative rods
in renal macrophages
focal hepatitis
Edwardsiella ictaluri (Enteric Septicemia of Catfish,
Hole-in-the Head Disease of Catfish)
exophthalmos
abdominal distention
frontal bone necrosis
peritonitis
focal hepatitis
Gram-negative rods
in renal macrophages
focal hepatitis
Edwardsiella ictaluri (Enteric Septicemia of Catfish,
Hole-in-the Head Disease of Catfish)
• Gram-negative motile pleomorphic curved rod
• Most important disease of channel catfish, affects
fingerlings and yearlings
• Outbreaks seasonally dependent, occur when water
temps are between 24-28oC
• Bacteria enter via nasal route, results in chronic
infection of the olfactory lobe of the brain that causes
necrosis of overlying bone
• Also enteritis, hepatitis, myositis, and interstitial
nephritis; acute and chronic forms
• Can survive for long periods in pond mud and may
be latent in the gut of asymptomatic catfish
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zebrafish: aberrant swimming
zebrafish: brainstem
zebrafish: spinal cord
zebrafish: spinal nerve
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Pseudoloma neurophilium (Microsporidiosis)
zebrafish: spinal cord
zebrafish: spinal nerve
•
•
•
•
Gram-positive intracellular obligate organism
Lately classified with fungi rather than protista
Direct transmission
Most zebrafish colonies in U.S. are affected, although
SPF zebrafish are available from Oregon State U
• Causes neurologic impairment in spinal cord and
myositis in epaxial musculature
• No inflammation unless cysts (xenomas) rupture
• Typical “bedroom slipper” appearance in wet mounts
and histologic sections
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•
•
•
•
•
•
•
•
Gram-positive intracellular obligate organism
Lately classified with fungi rather than protista
Direct transmission
Most zebrafish colonies in U.S. are affected, although
SPF zebrafish are available from OSU
Causes neurologic impairment in spinal cord and
myositis in epaxial musculature
No inflammation unless cysts (xenomas) rupture
Typical “bedroom slipper” appearance in wet mounts
and histologic sections
Spore coats are partially birefringent under polarized
light (spores also gram + and acid-fast)
rabbits: encephalitis and ophthalmitis
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Encephalitozoon cuniculi
rabbits: encephalitis and ophthalmitis
fathead minnow:
microsporidia in
kidney
fathead minnow:
microsporidia in
ovary
Classic
Disease Alert
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8/12/2014
fingerling trout: black tails
fingerling trout:
cartilage
necrosis
associated with
parasitic
spores
fingerling trout:
spinal deformity
Myxobolus cerebralis (Whirling Disease, Blacktail)
fingerling trout: black tails
fingerling trout:
cartilage
necrosis
associated with
parasitic
spores
fingerling trout:
spinal deformity
• Myxozoan parasite; 10-micron oval spore with 2
piriform polar capsules
• Salmonids (rainbow trout most susceptible; brown
trout and Coho salmon are resistant)
• Most severe in young trout from non-endemic areas;
older trout have little cartilage for parasites to feed
on, but instead function as carriers
• Pigmentation changes are caused by damage to
sympathetic nerves adjacent to the spine
• Characteristic frantic tail-chasing behavior (whirling)
that can be triggered by auditory stimuli or feeding
• Whirling behaviors disappear in survivors, spinal
deformities persist
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8/12/2014
polar capsules
highlighted
by Giemsa
stain
tubifex worms
important in
life cycle
goldfish: severe
abdominal distension
goldfish: massively
swollen kidneys
Hofferellus carassii (Kidney Bloater Disease)
goldfish: massively
swollen kidneys
goldfish: severe
abdominal distension
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goldfish: diffuse granulomatous peritonitis
goldfish mesentery:
numerous small
protozoons with
eccentric nuclei
Goldfish Amebiasis
goldfish mesentery:
numerous small
protozoons with
eccentric nuclei
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Goldfish Amebiasis
• Exact taxonomic status remains undetermined
• Necrogranulomatous inflammation of abdominal
cavity, kidney, heart, brain
• Parasites very small (only ~2-4 microns) and have
slightly eccentric nuclei
• Typically located at edges of necrotic areas
• Precise mode of transmission undetermined
Cyprinid Herpesviruses
Cyprinid Herpesviruses
• Cyprinid Herpes 1 – Carp Pox
– Self-limiting dermal lesions in carp and koi
– Focal epidermal hyperplasia
– Can see occasional Cowdry type A
intranuclear inclusion bodies
– Lesions regress in warm weather
• Cyprinid Herpes 2 – Hematopoietic
necrosis virus of goldfish
carp skin:
epidermal plaque
– Juvenile goldfish
– Stressed fish during periods of elevated
temperatures
– Lesions in spleen, kidneys, gills
• Cyprinid Herpes 3 – Koi herpesvirus
– Primarily swollen, necrotic gills
– Reportable disease
goldfish spleen:
karyomegalic
inclusions
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Reportable Diseases for Fish
From APHIS, Animal Health Monitoring &
Surveillance, USDA, 2012
http://www.aphis.usda.gov/vs/nahss/disea
se_status.htm#fish
Classic
Disease Alert
Cichlid: white growth
on fin
Walleye: white growths
Skin: markedly swollen
dermal fibroblasts
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Lymphocystis
• Iridovirus that affects most
fresh and saltwater fishes
• Small white nodules to
cauliflower-like growths
• Fibroblasts undergo
massive cytomegaly
• Basophilic cytoplasmic
inclusions surrounded by
hyaline capsule
• Gains entry through
epidermal abrasions
• Occasionally internal
infections
• Often self-limiting
Cichlid: white growth
on fin
Walleye: white growths
Skin: markedly swollen
dermal fibroblasts
channel catfish:
ulcers, secondary
bacterial infection
channel catfish:
focal epithelial
proliferation causes
white spots
Ichthyophthirius multifilis
("Ich" or White Spot Disease)
channel catfish:
focal epithelial
proliferation causes
white spots
gills: large
ciliate protozoon
with
macronucleus
channel catfish:
ulcers, secondary
bacterial infection
gills: large
ciliate protozoon
with
macronucleus
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• Largest protozoan parasite of fish:
trophozoites up to 100 m diameter
• Oval or horseshoe-shaped nucleus
• Aquarium and hatchery-reared
freshwater fish
• Severely infected fish may succumb
to respiratory impairment
• Trophozoites in skin and gill
surrounded by epithelial hyperplasia
epidermal
hyperplasia
horseshoe-shaped
macronucleus
Direct life cycle
1) Encysted trophozoites (trophonts) leave
the fish and settle to the bottom of the
tank
2) The trophozoites (tomonts) on the
bottom of the tank divide into numerous
tomites (theronts)
3) The motile theronts infect the skin of the
fish
The life cycle takes approximately 4
days to complete. However, it can
be shortened by increasing the
water temperature
gill: free-swimming unattached ciliate protozoons
SEM: ciliate protozoon
with denticular ring
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Trichodina spp. (Trichodiniasis)
gill: free-swimming unattached ciliate protozoons
SEM: ciliate protozoon
with denticular ring
• Saucer-shaped, 50 micron diameter, peritrichal
ciliated protozoans, that have a macro- and a
micronucleus
• Characteristic ring of interlocking denticles
• Low numbers not always associated with disease
• Can cause increased skin and gill mucus,
respiratory distress
• Simple life cycle, reproduces by binary fission
Japanese medaka:
trichodinids in urinary bladder
Japanese medaka: trichodinids
in renal collecting ducts
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gill wet mount:
cyst-like structure
gill lamellae:
individual epithelial
cells distended
with minute organisms
Epitheliocystis (Chlamydia)
gill wet mount:
cyst-like structure
gill lamellae:
individual epithelial
cells distended
with minute organisms
Epitheliocystis (Chlamydia)
• Obligate intracellular parasite;
organisms stain red with Macchiavello
stain
• Affects many species of freshwater
and marine fish
• Grossly, multiple white nodules
in gill lamellae and skin
• Intact cysts rarely induce
inflammation
• Infected fish may be asymptomatic or
show respiratory distress
• Transmission mechanisms not well
understood
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Classic
Disease Alert
striped bass:
cloudy ulcer
white mat
out of water
fungal hyphae
platy:
cottony growth
Saprolegnia spp. (Water Molds)
striped bass:
cloudy ulcer
white mat
out of water
fungal hyphae
platy:
cottony growth
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Saprolegnia spp. (Water Molds)
• Various groups of aquatic oomycete fungi: primarily
Saprolegnia, Achlya, and Aphanomyces spp.
• Affects all species and ages of fresh water and
estuarine fish
• Broad non-septate branching hyphae that produce
motile flagellated zoospores
• Most infections are secondary to trauma or primary
pathogens
• Saprolegnia spp. usually superficial, but other
organisms (e.g., Aphanomyces spp.) can be invasive
female
medaka:
massively
swollen
abdomen
Egg-bound Aged Female
female
medaka:
massively
swollen
abdomen
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Egg-bound Aged Female
• Seen in aged broodstock
• Occurs due to lack of behavioral and/or hormonal
triggers for ovulation (or occasionally oophoritis)
• Egg mass often contains numerous atretic
(degenerating) follicles
• Leads to negative energy balance, starvation
– Egg formation is energy intensive
– Meanwhile, little room in abdomen for food intake
• Results in catabolism and atrophy of adipose
tissue, skeletal and cardiac muscle, parenchymal
and visceral organs
• Very gradual progression
skeletal
muscle from
normal
female
medaka
atrophic
skeletal
muscle from
egg-bound
female
medaka
normal
female
medaka liver
severely
atrophic
liver from
egg-bound
female
26
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yellow perch:
skeletal muscle necrosis
yellow perch:
spinal fractures
yellow perch:
muscle atrophy
Electrocution
yellow perch:
skeletal muscle necrosis
yellow perch:
spinal fractures
yellow perch:
muscle atrophy
Electrocution
yellow perch:
spinal fractures
• Can occur naturally
(lightening strike) or via
human intervention
(electroshock collecting,
short circuits in
aquaculture equipment)
• Fish may have severe
spinal curvature and still
continue to feed
• Histopath:
– Acute: spinal fractures
and myonecrosis
– Chronic: denervation
muscle atrophy,
contralateral contraction
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tilapia:
clear bubbles in fins
tilapia:
clear bubbles in viscera
wet mount of fin:
gas bubbles
Gas Bubble Disease
• Due to supersaturation of the water by a
gas (most often nitrogen)
• Can occur naturally (e.g., heating of the
water, photosynthesis) or in confinement
systems (e.g., pump leaks allowing air to
enter filtration system)
• Signs include exophthalmia, with gas
bubbles in the eyes, oral mucous
membranes, gills and fins
• Histologically, can see ocular lesions
(cataracts, anterior synechia, and
panophthalmitis) and ischemic necrosis of
gill lamellae
• Fish die due to asphyxiation caused by
mechanical disruption of blood flow
wet mount of fin:
gas bubbles
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ventrolateral
pharyngeal
region:
multilobular
masses
Thyroid Follicular Hyperplasia (Goiter)
ventrolateral
pharyngeal
region:
multilobular
masses
Thyroid Follicular Hyperplasia (Goiter)
• Freshwater and marine fish (commercial salt
solutions); certain species seem predisposed
• Causes may include inadequate or excessive
iodine, but more commonly due to substances in
feed or water (goitrogens) that interfere with
thyroid hormone homeostasis
• Hyperplastic thyroids must be differentiated from
follicular cell adenomas and rare thyroid
carcinomas
– Thyroid follicle proliferation in extra-pharyngeal locations
does not necessarily indicate metastasis!
29
8/12/2014
Kidney
Liver
Spleen
Eye
Japanese medaka:
thyroid follicular cell hyperplasia everywhere!
Adenoma
Hyperplastic Thyroid
30
8/12/2014
thyroid follicular cell carcinoma:
compared to goiter, neoplasms are more anaplastic
Kidney Tubules
Hyperplastic Thyroid
Carcinoma
Mitotic Figures
platyfish / swordtail hybrid:
pigmented proliferations
Dark brown pigment
in cytoplasm of
pleomorphic spindle
cells
31
8/12/2014
Melanomas in Platyfish x Swordtail Hybrids
platyfish / swordtail hybrid:
pigmented proliferations
Dark brown pigment
in cytoplasm of
pleomorphic spindle
cells
Melanomas in Platyfish x Swordtail Hybrids
• Unique invasive melanomas occur in offspring
when certain F1 hybrid platyfish/swordtails are
crossed with swordtails
• 25% frequency of melanomas when F1 generation
hybrid females are back-crossed with wild-type
swordtail males
• Tumor development attributed to activity of a
dominant-acting sex-linked oncogene: Xmrk
– Ortholog of human EGFR gene
– Full expression of Xmrk occurs when it is no longer
inhibited by the suppressor gene Diff
• Important animal model for humans because there
are no naturally occurring melanoma models in
rodents
Pigment Cell Tumors of Fish
• 3 types of pigment cells:
– Melanophores
• Dark brown pigment
• Neoplasm: Melanoma
(syn: Melanophoroma)
– Xanthophores
• Red or yellow pigment
• Neoplasm: Xanthophoroma
(syn: Erythrophoroma)
– Iridophores
• Silvery reflective platelets
• Neoplasm: Iridophoroma
(syn: Guanophoroma)
From Ostrander, 2000
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8/12/2014
Iridophoroma under polarized light
female freshwater angelfish:
proliferative lip lesion
Lip Fibromas (Fibropapillomas) of Angel Fish
female freshwater angelfish:
proliferative lip lesion
33
8/12/2014
Lip Fibromas (Fibropapillomas) of Angel Fish
• Tumor of the mucocutaneous junction of the lips
• Only adult female fish are affected
• On cut sections, the tumors are solid white, or white
with cavernous centers that contain clear fluid
• Histologically, dense fibrovascular connective
tissue arranged in whorls, streams and bundles,
covered by a thick stratified squamous epithelium
• The presence of deformed teeth in the some tumors
has led to speculation that these may be
odontogenic neoplasms
• Cause is unknown; however a type "A" retrovirus
was identified in affected tissue in one report
• Laboratory transmission of the disease to other fish
has not occurred
zebrafish intestine:
nematodes embedded in mucosa
nematodes associated with mucosal
epithelial hyperplasia and inflammation
34
8/12/2014
Pseudocapillaria tomentosa
nematodes associated with mucosal
epithelial hyperplasia and inflammation
• Third most problematic infection in zebrafish
research facilities, after mycobacteriosis and
microsporidiosis
• Transmission occurs directly or via oligochaete
worms (e.g., tubifex)
• Diagnosis by visualization of oval eggs with bipolar
plugs in fecal smears
• Anthelmintic treatment possible but not well-tested,
affected facilities tend to depopulate vs. treat
• Presence of these worms has been associated with
intestinal and biliary neoplasia
Kent, M.L., J. K. Bishop-Stewart, J. L. Matthews, and J.M.
Spitsbergen. 2002. Pseudocapillaria tomentosa, a nematode
pathogen of zebrafish (Danio rerio) kept in research colonies
and associated neoplasms. Comp. Med. 52; 362-367.
35
8/12/2014
P. tomentosa-infected zebrafish intestine:
mucinous adenocarcinoma
Liver of P. tomentosa-infected zebrafish:
cholangiocarcinoma
Liver of P. tomentosa-infected zebrafish:
cholangiocarcinoma
36
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Zebrafish abdomen: nodular mass between esophagus and heart
Endocrine System: Anatomy & Physiology
Anterior
Kidney
Brain
Pharynx
Heart
Liver
Zebrafish, Midline Sagittal Section
Ultimobranchial Body Tumor
Zebrafish abdomen: nodular mass between esophagus and heart
37
8/12/2014
Ultimobranchial Body Tumor
• Spontaneous neoplasm of older (>1.5 years)
zebrafish, primarily males
• Certain zebrafish lines predisposed
• Can be up to 50x the size of the normal gland, can
compress adjacent structures
• Malignant invasion of sinus venosus of heart has
been observed in some cases
• Unknown if neoplastic progression is associated
with hormonal changes
zebrafish: humongous fleshy mass protrudes from
ventrally opened abdomen
zebrafish abdomen: humongous basophilic mass
38
8/12/2014
a different zebrafish abdomen: humongous
multinodular basophilic masses
well-differentiated
poorly-differentiated
Seminomas in Zebrafish
well-differentiated
poorly-differentiated
39
8/12/2014
Seminomas in Zebrafish
• Most common spontaneous zebrafish neoplasm
• Occurs in older males
• Most are well-differentiated, spermatocytic-type
seminomas
• Even large seminomas rarely invade locally or
metastasize
Japanese medaka liver: metastatic seminoma partially
obstructs hepatic vein
head of a different Japanese medaka:
tissue beneath brain
40
8/12/2014
Ectopic Gonad Tissue
Sex on the Brain
41
8/12/2014
The Three Most Overdiagnosed / Misdiagnosed
Histopathologic Findings in Fish
1.
Gill edema
–
–
“Epithelial lifting” is frequently an artifact of formalin
fixation
For true gill edema, look for blebbing and/or flocculent
material beneath the elevated epithelium
Actual Gill Edema (and lamellar adhesions)
42
8/12/2014
1.
Gill edema
–
–
2.
fixation
Renal tubular necrosis and glomerular changes
–
–
Tubular necrosis can occur secondary to incidental
intratubular mineralization
Fish glomeruli are morphologically variable, often appear
hypercellular with thickened mesangial matrix
Estrogenic Effects: Lesions in
Kidney of Adult Male FHM
E2-exposed Male
Control Male Kidney
E2 Causes
Nephropathy in
Adult Male
Fish:
Mechanism
Estrogen
Liver
Vitellogenin
Kidney
Nephropathy due to protein overload
43
8/12/2014
1.
Gill edema
–
–
2.
fixation
Renal tubular necrosis and glomerular changes
–
–
3.
Tubular necrosis can occur secondary to incidental
tubular mineralization
Fish glomeruli are morphologically variable, often appear
hypercellular with thickened mesangial matrix
Hepatic lipidosis
–
–
In cultivated fish, hepatocytes tend to be heavily
vacuolated with fat and glycogen due to excess energy
intake
Sick fish more commonly have reduced liver vacuolation
Hepatocyte Vacuolation
Liver from wild red drum
Hepatocyte Vacuolation
•
Less vacuolated:
–
–
•
Wild caught fish
Sick, stressed, or
starving fish
More vacuolated:
–
–
–
Captive-reared fish
Overfed or mal-fed
fish
Toxicant-induced
Liver from wild red drum
Liver from captive red drum
44
8/12/2014
If this wasn’t a shark liver,
it would be Lipidosis
Questions?
45

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