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Program Agenda
First Annual CL Davis Symposium on Diagnostic Pathology of Diseases of
Aerial, Terrestrial and Aquatic Wildlife
April 4-6, 2007
Covance Laboratory Learning Center, Madison, WI
Ocean Health & Disease: Coral Reefs & Sea Turtles
Thierry M. Work
USGS-National Wildlife Health Center-Honolulu Field Station
PO Box 50167, Honolulu, HI 96850
Tel: 808 792-9520; Fax: 808 792-9596; [email protected]
Web: www.nwhc.usgs.gov/hfs/Homepage.htm
Slide Topic
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Title
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Outline
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Photo of turtle
Life cycle
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Photo of boat strike,
fishing line
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FP photo
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Map
Comments
Investigation of disease in tropical marine ecosystems
poses particular challenges. You can only stay
underwater for limited time (limited observation), when
animals die, they get eaten (no diagnostic specimens),
and if specimens are found, the heat will decompose
them rapidly. Adding to the complexity is that we have
limited knowledge about the basic physiologic and
anatomic processes for many marine organisms, and
elucidating etiology of diseases is often stymied by lack
of laboratory reagents and inability to culture suspect
agents in the laboratory.
This session will cover some of what we know about
major diseases of reef organisms including marine
turtles, fish, and invertebrates.
There are 7 species of sea turtles worldwide.
One challenge of working with sea turtles is that a
portion of life cycle remains a mystery. Where do
hatchlings go when they go out to sea?
Obvious causes of mortality in turtles include boat strikes
characterized by linear parallel lesions on carapace and
fishing line entanglement and ingestion, which can cause
imbrication of intestines and ulceration of intestinal
mucosa.
By far the most important disease of sea turtles is
fibropapillomatosis (FP) manifested as sessile to
pedunculated smooth to rugose growths arising from the
skin
FP was first documented in Florida in the 1930s but since
has been found wherever marine turtles are found
(tropical oceans). The disease has been documented
mainly in green, loggerhead, and olive ridley turtles.
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FP photo
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Oral tumors
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Internal tumors: heart,
bone, lung
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Internal tumors:
kidney, heart
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Locations
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Graph
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Histogram
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Pie chart
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Histo Skin
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Lung tumor
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Kidney tumor
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Tumor intestines/bone
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Heart tumor
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Pie chart/graph
Tumors can reach very large sizes and impede movement
of limbs and cause loss of vision. FP has been most
intensively studied in Florida and Hawaii. Prevalence in
Florida continues to remain stable at ~20-50% whereas
in Hawaii, the disease peaked at ~60% but has been
steadily declining for the past 5 years for unknown
reasons
Interestingly, oral tumors are a characteristic of FP in
Hawaii that is not found in Florida. Ca. 50% of turtles
with oral tumors also have secondary pulmonary
infections.
Turtles with FP also have internal tumors in various
organs characterized by firm white to grey wellcircumscribed masses.
In some cases, these masses are filled with clear fluid,
and in other cases, larger tumors can have cores of
friable debris.
In Hawaii, tumors are most often seen on the eyes
followed by the mouth, flippers, and seams and scutes.
There also seems to be a predilection for FP to occur
more in the front than the rear of animals.
In most cases, for internal tumors, only one organ is
affected
For both HI and Florida, the most commonly affected
organs for internal tumors include the lungs, heart, and
kidney. Interestingly, in contrast to FL, liver tumors in
HI are rare.
Skin tumors are characterized by acanthotic
orthokeratotic epidermis with prominent rete pegs
overlying a collagen matrix with numerous pleomorphic
plump hapahazardly arranged fibroblasts. In some cases,
keratin cysts are present within connective tissue matrix,
Internal tumors are variations of connective tissue
tumors. In some cases like the lung, tumors are well
circumscribed.
In other cases, tumors infiltrate adjoining tissues causing
atrophy (e.g. note atrophied renal tubules).
In other cases, tumors can be invasive into bone causing
reabsorption. Depending on staining characteristics,
tumors have been classified as fibromas,
myxofibromas…
…or in the case of heart, fibrosarcomas of low-grade
malignancy. In general, tumors have appearance of slow
growth with few mitotic figures.
Severity of disease in turtles is graded from 0 to 3. Most
stranded turtles are TS 2 and 3, and area of tumors
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Map
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Flukes in artery
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Egg diagram
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Chart
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Chart of SCL
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Possible causes of FP
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Okadaic acid
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Immunology
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Flukes/viruses
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Viruses
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Tumor diagram
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Vectors
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Tree
increases with age
In Main HI islands, FP is found in all islands but rarely
on west coast of Hawaii for unknown reasons.
100% of stranded turtles also have infections with
vascular trematodes. Interestingly, turtles from pelagic
stage have little to no infection suggesting these parasites
are acquired once turtles recruit to near shore habitat.
The life cycle is unknown, but it is suspected that it is
similar to schistosomiasis with mollusk intermediate
host.
In HI, turtles are infected by 4 species of trematodes of
which one is probably endemic (not found in other
oceans). The endemic one can be differentiated from 4
other by egg morphology
Eggs in tissue can be quantified in standardized manner
to get an idea of severity of infection. Distribution of
eggs burden in tissues suggests that endemic parasite
makes a small component of trematode community in
turtles.
It also appears that trematodes have a negative effect on
body condition of sea turtles independent of FP status
Several potential causes of FP have been postulated
including UV radiation, flukes, viruses, marine toxins,
and immunosuppression.
Experimentally, OA has caused tumors in mice. This
toxin is produced by Prorocentrum lima, a dinoflagellate.
Although turtles are exposed to OA, the toxin can be
found in both tumored and non-tumored turtles.
Immunology studies on turtles with FP suggests that
those with severe disease are immunosuppressed but that
immunosuppression is not necessarily a prequel to
disease.
Experimental trials in Florida using captive green turtles
revealed that FP could be reproduced using cell free
filterable agents. This along with histopathology
showing intranuclear inclusions in some tumors
suggested a viral cause
Subsequent work in New York and Florida revealed that
a herpes virus is associated with tumors. Incidentally,
turtles also are infected with an endogenous retrovirus.
Using PCR, herpes viral DNA can be found in tumors
but not non-tumored tissue. There also is a gradient with
more viral DNA found in superficial tumors
Viral DNA has been found in a variety of epibionts
associated with turtles.
Phylogeny places the turtle herpes virus in the
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Diagram
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Questions?
Brick scale soldierfish
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alphaherpes category
Unfortunately, because the virus cannot be cultured,
Koch’s postulates remain to be fulfilled.
While quite a bit of information exists on diseases of
aquarium fish, there is very little data on diseases of wild
coral reef fish. Existing literature reviews (Panek 2005)
focus mainly on the Western Atlantic, and major causes
of mortality in reef fish there include blooms of toxic
algae, bacterial infections, and, more rarely, tumors.
Butterfly fish and map In contrast to the Atlantic, tumors in reef fish appear
relatively more common in the Pacific ocean. In Hawaii,
Okihiro (1998) documented chromatophoromas in two
species of butterfly fish (multiband and milletseed).
These tumors were found in fish from Southwest Maui
and Western Lanai
Depth gradient
During extensive surveys in the early 1980s, Okihiro
found that prevalence of tumors ranged from 5-25% with
decreasing prevalence of tumors as depth increased and
increasing prevalence of tumors over time
Fish in wild with
A recent follow up study done in 2005 revealed that
tumors
tumors continue to exist in multiband butterfly fish.
Dead multiband
In multiband butterflyfish, these tumors are characterized
butterflyfish with
by raised sessile grey to iridescent nodular firm masses
tumors
with a homogenous texture on cut surface sometimes
associated with hemorrhage on margins. Tumors appear
most often distributed laterally.
Live milletseed with
In milletseed butterflyfish, tumors are sessile, raised
tumors
nodular dark masses usually appearing to initially arise
from the first dorsal spine.
Dead milletseed with Larger tumors are often ulcerated.
tumors
Histopath of
On histology, tumors are characterized by mixed
milletseed tumor
populations of chromatophores round and spindle cells
mixed with a connective tissue matrix effacing epidermal
architecture and infiltrating underlying skeletal muscle
with occasional nidi of necrosis. Recent surveys in 2005
at the same sites surveyed by Okihiro revealed a much
smaller prevalence of tumors (<5%).
Kole live
Another skin lesion that has been seen in reef fish in the
Pacific are pigment anomalies in goldring surgeonfish.
These are typically distributed laterally at the base of the
tail but more severe cases can encompass the entire body.
Kole dead
Fish with this pigment anomaly have been found on
Maui, Oahu, and the northwestern Hawaiian islands. To
date, no efforts have been made to quantify prevalence,
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Kole histo
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Oahu Map
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Fish photos
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Pie charts
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Micrograph
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Histogram
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Diagram of island
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Graph
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Goatfish photos
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Multiband liver, gill,
heart
however, it appears to be fairly low.
Early lesions manifest as hyperplasia of epidermis.
Advanced lesions consists of masses of round to spindle
shaped cells mixed with chromatophores effacing skin
architecture and infiltrating underlying skeletal muscle.
Etiology for both types of tumors (butterflyfish,
surgeonfish) is unknown.
Monitoring sewer outfalls for liver tumors in fish is
required by EPA for Hawaii. This has presented an
opportunity do survey wild fish for disease agents (in
addition to looking at tumors).
Each year, three species of fish are collected by City and
County of Honolulu (blue lined snapper, brick scale
soldierfish, big eyed scad). Some of these fish are
necropsied to detect presence of liver tumors.
Surveys of fish revealed that prevalence of histozoic
parasites in blue lined snappers was considerably higher
than for other two species of fish. This can probably be
partly explainable by life history patterns (more parasites
in bottom feeders).
The two most commonly encountered parasites in
snappers were a coccidian and an epitheliocystis-like
organism (ELO) in the spleen and kidney. Molecular
work on coccidium at OSU reveals it to be Goussia sp.
ELO stains positive with Giemsa and Gimenez.
Prevalence of infection with coccidium increases with
age of fish, however, prevalence of ELO decreases with
increasing age of fish. Fish seem to mount little to no
inflammatory response to coccidia, however, a chronic
inflammatory response is seen with ELO infections.
Tropical islands are prone to suffer from invasive species
(Plants and animals) because a lot of the native biota lost
adaptive life history traits to defend against predators.
For microorganisms in HI, this has been exemplified by
severe impact of imported avian malaria on native forest
birds. Blue lined snappers were introduced from
Marquesas to HI in 1950s, and this brought up question
as to whether they were sharing parasites with native fish
with which they school.
Surveys over time indicate that prevalence of Goussia
and ELO in snappers remains fairly constant
Question then arose about possibility that snappers,
which school with native goatfish, may be sharing
parasites with these species.
Turns out that, as in many other wild fish, multiband
goatfish are infected at low levels with coccidia in liver,
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Multiband liver,
spleen, caudal kidney
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Orange goatfish
spleen, gill, heart
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Orange goatfish
spleen, gill
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Yellow tail goatfish
spleen and muscle
Yellow stripe goatfish
spleen, muscle, liver
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Graph summary
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Coral reef scene
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Cnidarian diagram
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Anatomy
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Black Band
monogean trematodes in gills, intermediate stage of
parasites in heart and epitheliocystis-like organisms in
gills.
However, multiband goatfish also are infected, albeit at
low prevalence, with ELO and coccidia in spleen and
kidney.
As in multiband, orange goatfish also have infections
with trematodes (eggs, in various organs) and also have
occasional infections with ciliates in gills (trichodinid
ciliates).
However, >90% of orange goatfish infected with
coccidia in spleen. Also see occasional monogean
trematodes in gills.
Ca. 30% of yellow tail goatfish have coccidia in spleen.
Occasional microsporidia are in skeletal muscle.
Ca. 50% of yellow stripe goatfish have coccidia in spleen
with occasional microsporidia in muscle and coccidia in
liver as well.
So, it appears that at least for coccidia, native goatfish
share parasite that is morphologically similar to that of
snappers. The big question remains as to whether these
parasites have detectable effects on fish health
Corals are and indispensable component of reefs
providing nurseries for a variety of fish and invertebrates
and laying the foundation for the diversity of organisms
seen in tropical marine ecosystems. Lose your corals,
you lose your ecosystem.
Corals belong to the cnidaria (animals with stinging
cells).
Corals are colonial sessile organisms that, along with
algae, make up a majority of the biomass of reefs. They
are unique in that they live with symbiotic algae
(zooxanthellae). Anatomically, they are simple
organisms with polyps consisting of tentacles
surrounding a mouth and a digestive system
(gastrovascular canal) that is connected between polyps.
At the cellular level, corals consist of three cell layers:
the calicoblastic epithelium that secretes the skeleton, the
gastrodermis (cells that digest and also harbor
zooxanthellae), the mesoglea (a connective tissue
matrix), and the epidermis (containing supporting cells
and stinging cells or nematocysts).
Coral disease have had the most severe impact in the
Western Atlantic causing massive loss of corals. Of
these, black band disease and vibrio associated bleaching
are the best characterized. The typical lesion of black
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Map of black band
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Bleaching
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Bleaching diagram
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Vibrio associated
bleaching
Vibrio bleaching
Diagram
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Aspergillosis
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Aspergillus diagram
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Aspergillus map
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Dust storm
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Porites Trematodes
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Putative life cycle
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White syndrome
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White syndrome
diagram
band is circular loss of tissues revealing intact white
skeleton separated from intact tissue by a black band.
This black band consists of a consortium of
cyanobacteria and vibrios. In the field, the presence of
disease appears to progress more rapidly with increasing
water temperatures.
Black band was first documented in Central American
and has been found throughout the Caribbean and the
Pacific.
Bleaching is the loss of pigmentation of corals revealing
the underlying white skeleton
The causes of bleaching are due to exit of zooxanthellae
from the gastrodermis leading to loss of tissue pigments.
Can be associated with temperature
In Israel, researchers have characterized a form of
bleaching that is due to infection with Vibrio
In this scenario, the vibrio infects the coral and enters a
viable but non-culturable state. The bacterium produces
a toxins that kills zooxanthellae and induces bleaching.
In the 1980s, researchers noted sea fans with lesions
associated with fungal hyphae. Subsequent
investigations led to discovery of Aspergillus sydowii as
a cause of these lesions
Field research shows that prevalence of seafan lesions
increase with size of sea fan and with increasing depth
suggesting that water motion may play a role
Seafan aspergillosis was initially found in central
America and Venezuela but has since been documented
in multiple islands throughout Caribbean
Research by USGS suggests that dust blown from Africa
may help spread coral disease (Aspergillus spore have
been found in the dust).
In the Pacific, certain species of coral are infected with
the intermediate stage of a fish trematode. These lesions
are characterized by small pink to tan raised nodules
Research in the Pacific has shown that butterfly fish feed
on these lesions preferentially and serve as the definitive
host for the trematode. How corals get infected remains
a mystery.
This is a catch all term for acute tissue loss in corals
revealing white skeleton. White syndrome are often
lethal to coral colonies and are deemed responsible for
coral reef degradation in W. Atlantic. The etiology of
most of these syndromes is unknown.
In the Caribbean, WS has resulted in major shifts in reef
structure with extermination of the dominant coral,
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White syndrome map
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Diagram of disease
investigation
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Pie charts
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Map
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Photodocument
Pathology diagram
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Discoloration photo
GA photo
TL photo
Discoloration Histo
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Tissue loss histo
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GA Histo
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Pie charts
Known causes
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Mixed causes
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White syndrome
Johnston Atoll
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WS Johnston Atoll
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Time line slide
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Acknowledgements
Acropora palmate and its replacement with encrusting
corals and algae
WS was first documented in Caribbean and has since
been found throughout the Western Atlantic.
One of the problems with coral disease research in corals
has been a lack of standardization of nomenclature of
lesions which has led to a lot of confusion
When you look at coral disease literature, not much is
produced regarding morphology, yet morphology is a
critical part of case definitions of disease
Accordingly, in the pacific, emphasis has been on
developing standardized nomenclature of gross lesions in
corals and follow up with histopath
Lesions in corals are broadly characterized as tissue loss,
skeletal growth anomalies, and discoloration
As expected, histology of each of these lesions reveal
different manifestations. For example, bleached corals
manifest loss of zooxanthellae
Tissue loss can be due to infection with ciliates, fungi,
algae or can be uncomplicated
Growth anomalies manifest as proliferation of polyp
basal body wall. So far, no evidence of true neoplasia in
classical sense.
Summary of gross vs histo findings in corals
In some cases, cause of lesion is evident such as
barnacles, predation, or mucus sheathing
In other cases, gross lesions induced by predators
(starfish) can be similar to those of unknown etiology
(white syndromes).
Also a challenge is that determining prevalence of
colonies with lesions can be challenging, particularly for
certain morphologies of corals.
What we do know is that coral diseases can kill colonies
outright. Example photos of a reef taken in 2001 and
2006 reveal >90% loss of live coral.
Coral disease is way behind other animals in level of
knowledge. Lots to learn about in the future.
References:
Herbst LH (1994) Fibropapillomatosis of marine turtles. Annual Review of Fish Diseases
4: 389-425
Okihiro MS (1988) Chromatophoromas in two species of Hawaiian butterflyfish,
Chaetodon multicinctus and C. miliaris. Veterinary Pathology 25: 422-431
Panek F (2005) Epizootics and disease of coral reef fish in the tropical Western Atlantic
and gulf of Mexico. Reviews in Fisheries Science 13: 1-21
Work T, Aeby G (2006) Systematically describing gross lesions in corals. Diseases of
Aquatic Organisms 70: 155-160