2007 CL Davis Pathology of Genetically Engineered Rodents and Aquatic Species
J. Spitsbergen
Zebrafish II Disease - Model - Husbandry July 27, 2007
Lack of Database of Pathologic
Lesions in Zebrafish
• Compared to most laboratory animals,
particularly rodents, the database on
zebrafish diseases and pathologic lesions is
very sparse.
• Despite the widespread use of zebrafish as an
animal model for understanding the genetics
of human development and disease for the
past 40 years, relatively little information is
available regarding normal aging lesions,
unique disease susceptibility, and patterns of
specific tumors in the wide variety of wildwildtype and mutant lines used in research
colonies around the world.
Diseases of
Zebrafish
Jan Spitsbergen,
Spitsbergen, DVM, Ph.D.,
Diplomate American College of
Veterinary Pathologists
Oregon State University
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Infectious Diseases of
Zebrafish
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Bacterial Diseases of Zebrafish
• The pneumatic duct of zebrafish is the
Achilles heel of this species, serving as
a portal for systemic fungal and
bacterial infections.
• Early stages of most bacterial and
fungal infections target the gas bladder
first, entering by the pneumatic duct.
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Skin Hemorrhages in
Septicemia
• Most bacterial diseases of zebrafish are
opportunistic infections.
• Gram negative septicemia causes
ascites, skin hemorrhages and skin
ulcers. Aeromonas, Pseudomonas, and
other ubiquitous organisms can be
involved.
• Most cases of gram negative septicemia
affect just 1 or 2 fish from a tank.
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Ascites and “Dropsy”
• Ascites is one of the most common
lesions seen in bacterial septicemia.
• Sometimes both ascites and edema
of the scale pockets (“
(“dropsy”
dropsy”) are
evident in septicemias.
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2007 CL Davis Pathology of Genetically Engineered Rodents and Aquatic Species
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Ascites and “Dropsy”
Mycobacteriosis in Zebrafish
• Even in well managed zebrafish colonies,
piscine mycobacteriosis is a common problem
in fish older than 1 year of age.
• Mycobacteria marinum, M. chelonae
(=abscessus
), M. fortuitum and other species
(=abscessus),
are causative agents. With M. chelonae,
often just the gas bladder is inflamed.
• Typically 10% or fewer fish per tank are
affected, but occasionally a “hot”
hot” strain
(especially M. haemophilum) will devastate a
lab, causing high mortality, and requiring
depopulation.
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Mycobacteriosis in Zebrafish
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Skin Ulcer Due to Piscine
Mycobaceriosis
• Common clinical signs include
emaciation, skin ulcers, hyperemia of
head.
• Internal lesions may or may not be
seen grossly. Spleen and kidney may
be greatly enlarged, pale and mottled.
• Infection is transmitted by water and by
cannabilism.
cannabilism. We do not yet know if the
bacteria are vertically transmitted.
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Mycobacterium haemophilum
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Mycobacterium haemophilum
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2007 CL Davis Pathology of Genetically Engineered Rodents and Aquatic Species
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M. Haemophilum is often
AcidAcid-fast Mycobacteria in Smears
and Tissue Sections
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Neurotropic
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Growing Use of Zebrafish Model
to Study Bacterial Pathogenesis
Gliding Bacteria
• Several species of filamentous gliding
bacteria (Flavobacterium columnare,
columnare, F.
branchiophilum)
branchiophilum) cause skin and gill
disease in zebrafish.
zebrafish.
• Bacterial gill disease and fin rot are
typically associated with crowding,
stress and poor water quality, for
example after long shipping times.
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Zebrafish model for Understanding
Streptococcal Pathogenesis
• Investigators at Wayne State University
in MI are using zebrafish to understand
the pathogenesis of necrotizing fasciitis
caused by Streptococcus.
• Several streptococcal species cause
disease in warmwater aquaculture and
pose a zoonotic risk to humans handling
the fish.
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•
•
•
•
•
•
Streptococcus
Mycobacteria
Salmonella
Edwardsiella
Bacillus anthracis
Clostridium difficle cardiotoxicity
(caspase(caspase-3 inhibitors prevent
cardiotoxicity)
cardiotoxicity)
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Fungal Diseases of Zebrafish
• Even in old zebrafish (2.5(2.5-3 yr old) and
in fish stressed by exposure to
mutagens and carcinogens, external
fungal infections are exceedingly rare.
• We have observed occasional systemic
fungal infection caused by
chromomycosis (Exophiala).
Exophiala).
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Systemic Chromomycosis
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Fungal Diseases of Zebrafish
• High levels of the fungus Lecythophora
mutabilis in the aquatic environment
caused epizootic mortality of young
zebrafish fry.
• Lecythophora mutabilis did not invade
tissues of fish, but large amounts of
environmental fungus debris clogged
gills and guts of young fish impairing
digestion and respiration.
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Piscinoodinium pillulare on
Gills
Systemic Chromomycosis
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Parasites of Zebrafish
• Appropriate quarantine of new adults and
bleaching of all eggs brought into a colony
should eliminate ectoparasitic protozoans.
protozoans.
• Zebrafish broodstock obtained from pet
stores or pond culture operations in Florida
are commonly colonized by the dinoflagellate
Piscinoodinium pillulare.
pillulare.
• Because of the direct life cycle, this agent can
rapidly cause high mortality or severe skin
and gill lesions.
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Microsporidiosis of Zebrafish
• The microsporidian parasite Pseudoloma
neurophila is the most important parasite
occurring in well managed zebrafish colonies.
• Even with strict quarantine and egg bleaching
procedures, this parasite can be introduced
into wellwell-managed facilities, suggesting
vertical transmission within eggs.
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Microsporidiosis of Zebrafish
• Microsporidiosis can be transmitted by
cohabitation and cannibalism.
• We strongly suspect vertical transmission of
the disease because the parasite has been
observed inside eggs of zebrafish.
zebrafish.
• Dr. Mike Kent of Oregon State University is
suggesting PCR testing of each potential
broodstock female in quarantine in order to
eliminate microsporidiosis from colonies.
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Microsporidiosis of Zebrafish
• Lesions seen in infected fish include
emaciation and spinal curvature,
however, most infections are
inapparent.
inapparent.
• Up to 50% of the brain volume of adult
fish can be occupied by parasite
xenomas,
xenomas, so this disease can interfere
with behavioral and disease studies that
relate to brain function in adult fish.
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Microscopic Lesions of
Microsporidiosis
Gross Lesions of Microsporidiosis
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Microsporidiosis in Brain
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Myzxozoa of Zebrafish
• Occasionally Thelohanellus is seen in gill
epithelium associated with gill
hyperplasia.
• Surprisingly 10 diagnostic cases from
closed colonies living indoors in fish
tanks showed Myxidium trophozoites in
mesonephric duct or kidney tubules.
Occasional sporocyts of this parasite
seen near kidney.
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Myxozoan in
Mesonephric Duct
Myzxozoa of Zebrafish
• Since myxosporidians typically require an
invertebrate as intermediate host, we are
speculating how closed indoor colonies of
zebrafish support these parasites.
• We think the most likely explanation is
that oligochaete invertebrates have
colonized biofilms in aquaculture systems
and act as intermediate hosts to the
myxozoa.
myxozoa.
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Capillariasis in Zebrafish
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Capillaria Wet Mount
• The only helminth commonly seen in zebrafish
diagnostic cases is the gut nematode
Pseudocapillaria tomentosa.
tomentosa.
• Routine quarantine of incoming adults and
bleaching of eggs before introduction into the
colony will eliminate this parasite.
• The parasite is observed commonly in Florida
wildwild-type line zebrafish raised in natural ponds.
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Capillariasis in Zebrafish
Viral Diseases of Zebrafish
• This parasite is a particular problem in tumor
studies as it is associated with an elevated
incidence of gut tumors, which occur in close
association with the parasite.
• Intense inflammation and gut mucosal
epithelial hyperplasia occur near the parasite.
• Parasite infection can also enhance
carcinogencarcinogen-induced gut tumorigenesis.
• Despite the widespread intense use of
zebrafish worldwide in biomedical
research for the past decade, a
pathogenic virus has not yet been
isolated or visualized in zebrafish
tissues.
• Dr. Michael Kent and I have
aggressively looked for viral agents in a
variety of tumors and in moribund fish.
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Viral Diseases of Zebrafish
Use of Zebrafish Model for Study of
Viral Pathogenesis and Immunity
• All wellwell-studied vertebrate species have
pathogenic viruses, so we need to be
vigilant to find them in zebrafish.
zebrafish.
• Salmonid infectious hematopoietic
necrosis and infectious pancreatic
necrosis viruses infect zebrafish and
their cell lines, but do not cause
disease.
• In the past 5 years a number of viruses that
do not naturally infect zebrafish have been
studied in zebrafish in order to better
understand the genetics (fish and virus) and
other mechanisms influencing pathogenesis.
• Snakehead rhabdovirus and viral hemorrhagic
septicemia virus (VHSV) can experimentally
cause severe lesions and high mortality in
zebrafish.
zebrafish.
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NonNon-infectious Diseases of
Zebrafish
Nephrocalcinosis
• Nephrocalcinosis: deposition of calcium salts
in kidney tubules or collecting ducts.
• This lesion is associated with high dissolved
carbon dioxide in waters, or with excessive
calcium or magnesium in diets.
• Sodium bicarbonate is preferred to calcium
carbonate as a buffer in aquaculture systems
to minimize this problem.
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Heart Disease: Pericardial Edema
and Dilated Cardiomyopathy
• Pericardial edema is seen much more
commonly in recirculating aquaculture
systems and is suspected to be associated
with a toxicant in the water. Some mutant
lines are more prone to pericardial edema
(smu).
smu).
• Dilated cardiomyopathy is rare, but hearts
can be enlarged 5X normal size. Cause
uncertain. Fish seem remarkably normal
clinically.
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• This lesion is usually mild in the
colonies we work closely with.
• Clinical signs are not typically seen
except in very severe cases.
• So far, no zebrafish have had lesions
severe enough to observe grossly.
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Dilated Cardiomyopathy
• Usually no significant pathologic lesion
is seen in the cardiac myocytes, so the
cause of the dysfunction is unclear.
• In some cases in recirculating sytems a
higher incidence of severe verrucous
endocardiosis of heart valves is
associated with dilated cardiomyopathy
of ventricle or atrium.
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J. Spitsbergen
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Dilated Cardiomyopathy
Pericardial Edema
• Interestingly the typical lesions seen in
mammal tissues in congestive
cardiomyopathy are not seen in
zebrafish.
zebrafish.
• For example, no chronic passive
congestion of liver is observed.
• When chronic passive congestion of
spleen is seen, it is usually associated
with ultimobranchial neoplasia, not
heart disease.
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Congestive Cardiomyopathy
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Verrucous Endocardiosis
• A degenerative, proliferative lesion of
endocardium similar to the aging lesions seen
in heart valves of dogs occurred in zebrafish
over 1.5 years of age.
• Verrucous endocardiosis occurred most
commonly on any of the three heart valves,
but also occurred occasionally on the
endocardial surface of the atrium, ventricle or
bulbus arteriosus.
arteriosus.
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Verrucous Endocardiosis
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Normal Heart
• Verrucous endocardiosis (VE) was most
commonly an incidental finding as is the
case in old dogs.
• However, occasionally florid VE resulted
in flow restrictions and caused dilated
cardiomyopathy (VE of AV valve) of
atrium or ventricle (VE of VB valve).
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J. Spitsbergen
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Verrucous Endocardiosis AV Valve
with Congestive Cardiomyopathy
Toxicity of Plastics to Fry
• Zebrafish fry are much more fragile
than fry of trout and salmon, especially
the very fastidious fry of the relatively
inbred TU and AB lines.
• Any new system components to be
used with fry must be tested carefully
for toxicity.
• Avoid black plastic (such as OO-rings).
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EggEgg-associated
Inflammation and Fibroplasia
EggEgg-associated
Inflammation and Fibroplasia
• Granulomatous and fibrotic changes are
commonly seen in diagnostic cases and
in colonies where microsporidiosis is
common, and fish are squeezed for
spawning.
• Females of the AB line in such colonies
frequently become “eggbound”
eggbound”.
• Interestingly in our flowflow-through
systems in which we do not have a high
incidence of microsporidiosis and in
which we use only natural mating, we
do not see eggegg-associated fibroplasia.
• We also see very few eggbound
females in these flowflow-through systems
with only natural mating.
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My Current Research
Zfish II - 52
Acknowledgements
• Developing lines of zebrafish showing high incidences
of specific types of neoplasia to allow studies of
molecular pathogenesis and drug discovery.
• Studying mechanisms of synergy between gut
nematode parasites and gut carcinogens.
• Determining whether cyprinid herpesvirus 1, the
cause of skin papillomas in carp and koi can infect
zebrafish and cause skin tumors.
• Collaborating with scientists at University of Oregon
to develop zebrafish models for study of Fanconi
anemia.
• Collaborating with scientists at Oregon Health
Sciences University to develop zebrafish models for
skin cancer.
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• Marine and Freshwater Biomedical Sciences and
Environmental Health Sciences Centers at Oregon
State University funded by the National Institutes
of Environmental Health Sciences
• Zebrafish International Resource Center at the
University of Oregon funded by the National
Center for Research Resources
• The John Fryer Salmon Disease Laboratory at
Oregon State University
• Research Grants from the National Institutes of
Environmental Health Sciences
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Why Zebrafish?
The Promise of the Zebrafish
Model for Cancer Research and
Drug Discovery
Jan Spitsbergen,
Spitsbergen, DVM, Ph.D.,
Diplomate American College of Veterinary
Pathologists;Center for Fish Disease
Research, Oregon State University
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• A vertebrate model whose husbandry
costs and space requirements are small
compared to mammals
• Unrivaled database on genetics,
genomics and molecular development
among submammalian vertebrate
models
• Can be raised for the first week or 2 of
life in 96 or 384 well microtiter plates
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Cancer Research in Zebrafish
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Zebrafish Histology
• Zebrafish is a pathologist’
pathologist’s dream—
dream—all
major tissues can be evaluated on a
small number of tissue sections on
about 3 microscope slides
• Most carcinogens active in humans are
active in zebrafish
• Zebrafish share with humans many of
the cytochrome P450 enzymes for
carcinogen activation (and
detoxification)
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Recent Advances in Zebrafish
Cancer Research
Zebrafish Tumors
• Studies of spontaneous and induced tumors
in zebrafish show that they develop a wide
variety of epithelial, mesenchymal,
neuroepithelial,
neuroepithelial, and neural crest neoplasms.
• Zebrafish develop a number of histologic
types of neoplasia similar to those in humans
but that rarely occur even in carcinogencarcinogentreated vertebrates of other species
(chordoma,
chordoma, hepatoblastoma, ocular
medulloepithelioma,
medulloepithelioma, olfactory
esthesioneuroblastoma)
esthesioneuroblastoma)
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• Recent studies have used inducible, tissuetissuespecific transgene expression systems with
fluorescent markers (GFP, RFP, YFP) to
control tumor development and to visualize
tumor initiation and progression
• Targeted expression of human MYCN to
zebrafish pancreatic islets results in
neuroendocrine carcinoma.
• Targeted expression of mouse c-Myc to
zebrafish T lymphocytes induces T cell acute
lymphoblastic leukemia.
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J. Spitsbergen
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Zebrafish Models for
Melanoma
Neat Tools for Study of Cancer in
Zebrafish
• Several laboratories around the world are
developing zebrafish models for study of
molecular pathogenesis of melanoma and for
drug discovery to treat melanoma.
• Homozygous p53 deficient zebrafish rapidly
develop melanomas due to expression of
human BRAF with an activating mutation
under control of the zebrafish transcription
factor (Mitfa
(Mitfa)) targeting expression to
melanocytes.
• Pigment mutants that are transparent as adults will
greatly aid visualization of tumor formation and
progression, especially with fluorescent markers in
tumor cells.
• Cloned fish with transplantable tumors. Transparent
mutants would be best for these studies.
• “Nude mouse alternatives”
alternatives”
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– Phylonix,
Phylonix, a biotech company in Boston is injecting human
tumors into zebrafish embryos and fry prior to
immunocompetency as a drug discovery tool.
– Gamma irradiated adult fish will tolerate allografts and
xenografts.
xenografts.
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Zebrafish Tumor Studies
Carcinogenesis Studies in
Florida wt Zebrafish
• I have focused my research program over the
past decade on spontaneous and induced
neoplasia of zebrafish.
zebrafish.
• Initially I studied responses of Florida wt
zebrafish to a panel of structurally diverse
carcinogens.
• More recently I have focused on identifying
and developing lines of zebrafish showing
high incidences of specific histologic types of
neoplasia to facilitate understanding of
molecular pathogenesis of neoplasia.
• The Florida wt line of zebrafish was quite
responsive to most carcinogens when
treated during early life stages, showing a
wide variety of epithelial, mesenchymal,
neural and neural crest neoplasia.
• However, incidences of particular
histologic types of neoplasia were
relatively low in the Florida wt line treated
with carcinogens.
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Crowding and
Tumor Incidence
Colony Surveillance
• The optimal density of zebrafish for
tumors studies has not been
established.
• Several genetics labs crowd their fish
much more than we do at OSU.
• From studies with rainbow trout we
know that any factor which reduces
growth rate or stunts fish reduces
tumor incidence in carcinogen studies.
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• I recommend that any lab wishing to study
tumors of zebrafish conduct surveillance to
establish their baseline tumor incidences in
retired broodstock.
broodstock.
• Feeding a diet known to be low in natural
carcinogens such as Aquatox (Ziegler) can
help to minimize tumor background.
• Extreme caution is needed when adding new
components to a recirculating system—
system—many
plastics are quite toxic to young zebrafish.
zebrafish.
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How to Get There from Here
Strategy for Developing
TumorTumor-Sensitive Lines of Zebrafish
• I believe that the key to success in optimizing
tumor models in zebrafish will be to utilize
compound mutant zebrafish to maximize the
incidence and shorten the latency for tumor
formation.
• Ideally one would want a line that develops
high incidences of neoplasia as embryos or
young fry.
• Early development of definitive neoplasia in
the lines will make the most efficient models
for anticancer drug discovery.
• Based on the data available today, lines of
zebrafish with combinations of inactivating
mutations in tumor suppressor genes and
activating mutations in oncogenes will likely
develop specific histologic types of neoplasia
rapidly.
• Use of carcinogens and tumor promoters may
be appropriate to understand the
pathogenesis of specific types of human
neoplasia.
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Conducting Tumor Studies with
Mutant Lines
Methods for Tumor Studies
• To assure statistical significance, one
usually requires groups of 5050-100 fish
at sampling times at which histology is
evaluated.
• It is best to use replicate groups for
each treatment.
• To end up with 5050-100 fish at sampling
time, it is prudent to start with twice
that number of fish.
• Most neoplasms of zebrafish are not
visible grossly, even in our highly
sensitive lines.
• Extensive complete histology is required
to confirm tumor incidences in specific
lines of zebrafish.
zebrafish.
• For embryos and fry, I prefer glycol
methacrylate resin as less tissue
distortion occurs.
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Zebrafish Models for
Drug Discovery
Zebrafish Model for
Drug Discovery
• At least 6 review papers on use of
zebrafish in drug discovery have been
published since 2002.
• Much enthusiasm is present among
biotechnology companies for use of the
zebrafish model, but major pharma are
still relatively reluctant to use the
model.
• Since embryos and young fry can be raised in
96 or 384 well plates, the system is efficient
for highhigh-throughput robotic assays.
• Most hydrophilic as well as hydrophobic
agents are absorbed from the culture
medium.
• Very small amounts of agents are needed for
assay.
• Good for structurestructure-activity studies in lead
compound optimization—
optimization—can determine if
structure of molecule alters uptake or toxicity
as well as pharmacologic effect.
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Advantages of Whole Animal
Model for Drug Discovery
PhenotypePhenotype-Based Drug Discovery
• Can assess more complex biological
processes than a single cell culture
• Can tell if a disease process is altered, not
just a specific target molecule or cell
• Integrates absorption, distribution,
metabolism, and toxicity
• Can examine synergism and antagonism
between agents requiring metabolism or
acting at surface receptors
• Can either approach certain known
target molecules or signaling pathways,
or can assess phenotypic effects in
embryos/fry.
• Many drugs in current use in mammals
were discovered by “serendipity”
serendipity”, based
on phenotypic evaluation of treated
animals.
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Future Needs for Mainstreaming
Zebrafish in Drug Discovery
Validation of Zebrafish Model for
Drug Discovery
• Need validation of zebrafish responses to
a wide variety of pharmaceuticals.
• As yet no standard chemotherapy
regimens for specific mammal tumors
have been applied in a similar zebrafish
model.
• We need to try various chemo regimens
for their activity against specific
zebrafish tumors.
– So far a variety of classes of drugs for a
diverse array of disease categories have
acted similarly in zebrafish and mammals.
– For example drugs causing Parkinsonism
(MPTP) and those ameliorating it (deprenyl
(deprenyl))
work similarly in zebrafish.
zebrafish. Antibiotics,
antimitotics,
antimitotics, anticoagulants work similarly.
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Summary
Summary
• The zebrafish is a powerful model for
clarifying the role of specific genes and
the interactions of multiple genes in the
oncogenesis process in many tissues
relevant to human disease.
• Much remains to be optimized in order
to have the best models for study of
specific histologic types of cancer in
nearly all organs.
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• The zebrafish is a highly efficient and
relatively low cost system for highhighthroughput antianti-cancer drug discovery.
• A whole animal vertebrate system is a
better predictor of complex biological
phenomena than a tissue culture of a
single cell type.
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Acknowledgements
• Marine and Freshwater Biomedical Sciences and
Environmental Health Sciences Centers at Oregon
State University funded by the National Institutes
of Environmental Health Sciences;
Sciences; NIEHS
Research Grants
• Zebrafish International Resource Center at the
University of Oregon funded by the National
Center for Research Resources
• The John Fryer Salmon Disease Laboratory at
Oregon State University
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Diet and Husbandry System
Strongly Influence Tumor Incidences
in Zebrafish of the AB Line at
Two Years of Age
Jan Spitsbergen and
Michael Kent
Center for Fish Disease
Research,
Oregon State University
Monte Westerfield
Zebrafish International
Resource Center,
University of Oregon
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Background
Background
• Zebrafish were the first fish species in
which laboratory experiments confirmed
that carcinogens active in mammals
caused cancer in fish.
• These experiments were conducted by
Dr. Clyde Dawe at the National Cancer
Institute in the 1960s.
• Little additional carcinogenesis work was
done with zebrafish until the past decade.
• Dr. Jerry Hendricks of Oregon State
University obtained funding from the U.S.
Army to study effects of a panel of
structurally diverse carcinogens on Florida
wildwild-type zebrafish exposed at the egg, fry
or 2 month old juvenile stages.
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Background
Background
• The Florida wt line of zebrafish was quite
responsive to most carcinogens when
treated during early life stages, showing a
wide variety of epithelial, mesenchymal,
neural and neural crest neoplasia.
• However, incidences of particular
histologic types of neoplasia were
relatively low in the Florida wt line treated
with carcinogens.
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• In the last decade, growing interest
among several research groups has
focused on use of the zebrafish model
to study neoplasia.
• I have focused my research over the
past 10 years on spontaneous and
carcinogencarcinogen-induced neoplasia in a
variety of wildwild-type and mutant lines.
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2007 CL Davis Pathology of Genetically Engineered Rodents and Aquatic Species
J. Spitsbergen
Zebrafish II Disease - Model - Husbandry July 27, 2007
Background
Experimental Design
• I soon realized that zebrafish fed commercial
diets and maintained in recirculating
aquaculture systems appeared to have
different patterns of tumors than zebrafish
fed a semisemi-purified diet (OTD) in a flowflowthrough system.
• From studies with laboratory mammals and
humans we know that diet can profoundly
influence tumor incidences.
• Replicate tanks of 80 zebrafish fed
either a mixture of commercial diets or
semisemi-purified diet (OTD) twice daily plus
brine shrimp once daily.
• Fish were raised at 3 sites, two with
flowflow-through systems receiving well
water, one with a recently established
recirculating system with a fluidized
sand biofilter.
biofilter.
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Experimental Design
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Diet/Husbandry and Neoplasia
• Fish sampled at 22 months postpost-fertilization
for necropsy to document gross and
histologic lesions
• A few fish were sampled prior to the
scheduled termination date due to cachexia,
distended abdomens or other gross lesions.
• Sagittal step sections were prepared at 9
levels between the middle of the L eye and
the middle of the R eye.
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Neoplasia in Zebrafish in FlowFlowthrough Systems
• Fraction of fish with
any neoplasm when
fed OTD: 9/222 (4%).
• Fraction of fish with
any neoplasm when
fed commercial diet
mix: 4/218 (2%).
• Dietary influence not
significant using chichisquare test.
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• Most common histologic
type of tumor was
seminoma: 8/224 males
(4%).
• Most seminomas were
1-2 mm in diameter, a
few were up to 4 mm.
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60
50
40
30
Flow-through
Fluidized sand
20
10
0
OTD
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Neoplasia in Zebrafish in FlowFlowthrough Systems
• Other histologic types of tumors occurred
rarely (1/440; 0.2% incidence).
• These tumors types: 1 hepatocellular
adenoma, 1 adenoma of pneumatic duct, 1
acinar cell carcinoma of pancreas (ACCA), 1
ultimobranchial adenoma, 1 malignant
peripheral nerve sheath neoplasm (MPNS)
involving skeletal muscle, spine and
abdominal viscera
• Most of these neoplasms microscopic except
ACCA (4 mm) and MPNS (7 mm)
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Malignant Nerve Sheath
Neoplasm Near Spine
Neoplasia in Zebrafish in
Recirculating Systems
• Diet showed a strong interaction with the
recirculating husbandry system—
system—fraction of fish
with neoplasia was 20/94 (21%) when fed OTD
and 59/108 (55%) when fed commercial diet mix.
• Neoplasms in fish in the recirculating system were
often grossly visible and up to 10 mm in diameter.
• A wide variety of tumor types occurred in fish
from the recirculating system, including liver, gut,
pancreas and some very unusual tumors such as
rhabdomyoma of heart.
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Multiple Histologic Types of
Neoplasia Per Fish
Neoplasia in Zebrafish in
Recirculating Systems
• No fish from a flowflow-through system showed
more than 1 histologic type of neoplasm.
• 2% of fish fed OTD in a recirculating system
showed 2 or more histologic types of
neoplasia per fish.
• 17% of fish fed commercial diet in a
recirculating system showed 2 or more
histologic types of neoplasia per fish.
• Only fish from recirculating systems had 3 or
more histologic types of neoplasia per fish.
• Liver was the most common organ affected
by neoplasia, with tumors in 41/202 (20%)
fish.
• Gut was the second most commonly affected
organ with neoplasia occurring in esophagus
or intestine in 18/202 (9%) fish.
• Seminomas in testis occurred in 16/202 fish
(16/124 males).
• Neoplasia occurred in exocrine pancreas in
8/202 fish (4%).
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Liver Neoplasia in Zebrafish in
Recirculating Systems
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Hepatocellular Adenoma
• Incidence of liver neoplasia in fish fed OTD
6/94 (6%) was significantly lower than in fish
fed commercial diet 35/108 (32%). P <0.00
using chichi-square test.
• Histologic types of hepatic neoplasia were
similar in fish fed either diet in the
recirculating system, with hepatocellulr
adenoma most common, hepatocellular
carcinoma less common, and hepatoblastoma
or cholangiocellular carcinoma occurring
rarely.
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Gut Neoplasia in Zebrafish in
Recirculating Systems
Intestinal Carcinoma
• Incidences of gut neoplasia in fish fed OTD
4/94 (4%) were significantly lower than those
in fish fed commmercial diet 14/108 (13%).
P=0.05 using chichi-square test.
• Small cell carcinomas of the transition zone
from esophagus to intestine (intestinal bulb)
or ampulla of Vater occurred most commonly
followed by adenocarcinoma of intestine or
distal esophagus, then squamous cell
carcinoma of distal esophagus or transition
zone.
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Small Cell Carcinoma of
Intestine at Ampulla of Vater
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Seminomas in Zebrafish in
Recirculating Systems
• Seminomas occurred more frequently in
males from the recirculating system
(16/124=13%) than in males from the flowflowthrough systems (8/224=4%; p=0.002 with
chichi-square test), although in neither system
type was diet a significant influence on
seminoma incidence.
• Most seminomas in males in recirculating
systems were over 2 mm in diameter, with
several 88-10 mm.
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Seminoma, Gross Lesion
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Seminoma, Histology
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Neoplasms of Exocrine Pancreas in
Zebrafish in Recirculating Systems
• Incidence of neoplasia was far greater in
zebrafish in recirculating systems compared
to fish in flowflow-through systems 8/202 vs
1/440 (p=0.007 with chichi-square test).
• Neoplasia of exocrine pancreas also occurred
more commonly in fish fed commercial diet in
recirculating systems 1/94 vs 7/108 (p=0.05(p=0.050.10 using chichi-square test without or with
Yates correction).
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Pancreatic Acinar Cell Carcinoma
Neoplasms of Exocrine Pancreas in
Zebrafish in Recirculating Systems
• Most common
histologic type of
neoplasia in exocrine
pancreas was acinar
cell carcinoma. Several
8-10 mm.
• One ductal carcinoma
and 1 ductal adenoma
occurred.
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Other Neoplasia in Zebrafish in
Recirculating Systems
Epithelial Neoplasms
• 5 ultimobranchial
adenomas
• 1 papilloma, vent
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Normal Ultimobranchial
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Lymphohemopoietic
Neoplasms
• 1 lymphosarcoma,
lymphosarcoma,
disseminated
• 1 erythroleukemia,
erythroleukemia,
disseminated
• 1 granulocytic
sarcoma of
gut/liver
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Ultimobranchial Adenoma
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Rhabdomyoma of
Ventricle of Heart
Mesenchymal Neoplasms in
Zebrafish in Recirculating Systems
•
•
•
•
•
•
•
5 rhabdomyomas,
rhabdomyomas, ventricle of heart
1 hemangioma, bulbus of heart
1 rhabdomyosarcoma, skeletal muscle
1 fibroma, upper jaw
1 chondroma,
chondroma, lower jaw
1 chordoma,
chordoma, spine
1 myxoma, ovary
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Rhabdomyoma of
Ventricle of Heart
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Rhabdomyosarcoma of
Skeletal Muscle
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The Mystery of Hepatic
Megalocytosis in Zebrafish
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The Mystery of Hepatic
Megalocytosis in Zebrafish
• In diagnostic cases from labs around the
world and in certain lots of fish from
recirculating systems with fluidized sand
filters we see frequent mild to severe
hepatocyte megalocytosis,
megalocytosis, with cytoplasm
and nuclei up to 5050-100 X normal size.
• We do not see hepatocyte megalocytosis in
untreated control fish of any line born and
raised in flow through systems and fed semisemipurified diet.
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2007 CL Davis Pathology of Genetically Engineered Rodents and Aquatic Species
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Zebrafish II Disease - Model - Husbandry July 27, 2007
Hepatic Megalocytosis
Causes of Hepatic Megalocytosis
• Data from many vertebrates including
zebrafish indicate that hepatocyte
megalocytosis is caused by toxicant
damage to DNA or the mitotic
apparatus.
• In carcinogen studies we often see high
incidences of hepatic megalocytosis in
zebrafish.
zebrafish.
• Dietary factors probably play a role—
role—
certain commercial diets are known to
contain naturally occurring nitrosamine
carcinogens from imperfectly handled
fish meal. Paramecium cultures may
contribute.
• Microbial activity in fluidized sand
filters, biofilms,
biofilms, algae, and other system
components may contribute.
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Hepatic Megalocytosis
Studies of Retired Broodstock
• Tanks of fish with birth dates within the same
month, but not on the same day are often
not concordant for megalocytosis.
megalocytosis.
• The oldest fish sampled for colony
surveillance (2(2-3.5 yr) are not more likely
than younger fish to have hepatic
megalocytosis.
megalocytosis.
• These findings suggest that brief “spikes”
spikes” of
toxicant occur during critical sensitive life
stages.
• Within a given recirculating system, the
mix of toxicant(s) in episodic spikes
varies from time to time.
• Some cohorts of retired AB broodstock
from a particular recirculating system
show elevated tumors, but no
megalocytosis.
megalocytosis.
• Some lots show tumors and
megalocytosis,
megalocytosis, and some lots show only
megalocytosis.
megalocytosis.
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Both Hepatocellular Carcinoma
and Hepatocyte Megalocytosis
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Implications of Naturally Occurring
Toxicants in Recirculating Systems
• In addition to increasing the spontaneous
incidences of neoplasia, the toxicant(s)
causing megalocytosis and neoplasia may
also cause other health problems, reducing
early life stage survival, longevity,
reproductive potential, immune competence
and disease resistance.
• Since these intensive recirculating systems
are highly desirable in urban and 3rd world
aquaculture systems, we must optimize these
systems to minimize toxicant exposure.
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2007 CL Davis Pathology of Genetically Engineered Rodents and Aquatic Species
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Zebrafish II Disease - Model - Husbandry July 27, 2007
Remedies to Toxicant Problems
in Recirculating Systems
• Increase water turnover.
• Minimize nitrosamines in fish diet-Ziegler Aquatox
diet--Ziegler
diet is pretested to minimize nitrosamines in fishmeal
component.
• Possible use of adsorbants to reduce NN-nitroso
carcinogens or other toxicants (zeolite
(zeolite,, activated
carbon etc.)
• We have studied fish health and disease in a variety
of more traditional recirculating aquaculture systems
for over 20 years and have not before observed
health problems such as hepatocyte megalocytosis or
elevated tumor incidences associated with
recirculating systems.
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Conclusions
• Both commercial diets containing fishmeal
and recirculating aquaculture systems with
fluidized sand biofilters may contribute
toxicants that impair the health of fish,
damage liver cells, and increase the
incidences of neoplasia in fish populations.
• Optimization of fish health will require careful
study of the design of aquaculture systems to
minimize adverse effects on fish health.
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Acknowledgements
• Marine and Freshwater Biomedical Sciences
and Environmental Health Sciences Centers at
Oregon State University funded by the
National Institutes of Environmental Health
Sciences
• Zebrafish International Resource Center at
the University of Oregon funded by the
National Center for Research Resources
• The John Fryer Salmon Disease Laboratory at
Oregon State University
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