Manual for the Microscopic
Diagnosis of Proliferative
Liver and Skin Lesions in the
Brown Bullhead (Ameiurus
nebulosus)
Vicki S. Blazer1, John W. Fournie2, Jeffrey C. Wolf5, and
Marilyn J. Wolfe3
National Fish Health Research Laboratory, U.S. Geological Survey, 11649
Leetown Road, Kearneysville, WV25430
U.S. Environmental Protection Agency, National Health and Environmental
Effects Research Laboratory, Gulf Ecology Division, 1 Sabine Island Drive,
Gulf Breeze, FL 32561
3The Registry of Tumors in Lower Animals, Experimental Pathology
Laboratories, Inc., 22866 Shaw Road, Sterling, VA 20166
-------
USGS
science for a changing world
lililll
Sea Grant
Pennsylvania
Pennsylvania Sea Grant, part of the National Sea Grant Program, is a partnership of Penn State,
the Commonwealth of Pennsylvania, and the National Oceanic and Atmospheric Administra-
tion. This publication is available in alternative media upon request. Penn State is committed to
affirmative action, equal opportunity, and the diversity of its workforce. Published in February
2007.
-------
TABLE OF CONTENTS
1.0 Introduction 1
1.1 Background Information 1
1.2 Purpose of the Manual 1
2.0 Specimen Preperation 2
2.1 Field Collection 2
2.2 Specimen Processing 2
3.0 Liver 2
3.1 Normal Liver Histology 3
3.2 Non-neoplastic Hepatic Lesions 4
3.2.1 Proliferation of Macrophage Aggregates 4
3.2.2 Proliferative Response to Parasites 5
3.3 Putatively Preneoplastic Hepatic Lesions - Foci of Cellular Alteration 6
3.3.1 Basophilic Focus 7
3.3.2 Eosinophilic Focus 7
3.3.3 Vacuolated and Clear Cell Foci 8
3.4 Neoplastic Hepatic Lesions 9
3.4.1 Hepatocellular Adenoma 9
3.4.2 Hepatocellular Carcinoma 10
3.5 Non-Neoplastic Biliary Lesions 12
3.5.1 Bile Duct Proliferation and Fibrosis 12
3.5.2 Bile Duct Parasitism 13
3.6 Neoplastic Biliary Lesions 14
3.6.1 Cholangioma 14
3.6.2 Cholangiocarcinoma 15
3.7 Metastatic Neoplasia 16
4.0 Skin 16
4.1 Normal Skin, Barbel, and Oral Cavity Microscopic Appearance 16
4.2 Black Pigmented Skin Lesions 18
4.2.1 Non-Raised Melanistic Spots 18
4.2.2 Raised Black Skin Lesions 19
4.2.2.1 Parasite-Associated Black, Raised Lesions 19
4.2.2.2 Melanotic Hyperplasia 20
4.2.2.3 Melanoma 21
4.2.2.4 Melanotic Papilloma 24
4.3 Pale Areas of Discoloration 25
4.3.1 Non-Raised Areas of Discoloration 25
4.3.2 Raised Pale Areas 26
4.3.2.1 Papilloma 26
4.3.2.2 Squamous Cell Carcinoma 27
4.3.2.3 Miscellaneous Raised Growths 29
5.0 Acknowledgements 29
6.0 References 30
-------
FIGURES
Figure 1. Illustration of liver pieces to be sampled and placed in fixative in the field 2
Figure 2. Microscopic appearance of normal bullhead liver 3
Figure 3. Macrophage aggregates in the liver sections of brown bullhead 4
Figure 4. Grossly visible lesions in brown bullhead liver 5
Figure 5. Helminth parasites and accompanying inflammatory response in bullhead liver 6
Figure 6. Basophilic focus in the liver of brown bullhead 7
Figure 7. Eosinophilic focus in brown bullhead liver 7
Figure 8. Vacuolated cell focus in brown bullhead liver 8
Figure 9. Clear or mixed cell focus in brown bullhead liver 9
Figure 10. Hepatocellular adenoma in brown bullhead liver 10
Figure 11. Hepatocellular carcinoma in brown bullhead liver 11
Figure 12. Normal bile ductule and duct microscopic appearance in brown bullhead liver 12
Figure 13. Bile duct proliferation and fibrosis in brown bullhead liver 13
Figure 14. Bile duct myxozoan parasite of brown bullhead liver 13
Figure 15. Cholangioma within the liver of brown bullhead liver 14
Figure 16. Cholangiocarcinoma within the liver of brown bullhead liver 15
Figure 17. Microscopic appearance of normal skin of brown bullhead 17
Figure 18. Microscopic appearance of normal brown bullhead barbels 17
Figure 19. Microscopic appearance of normal oral cavity 18
Figure 20. Melanistic spots on the skin of brown bullhead 19
Figure 21. Small raised, black areas on the barbels of brown bullhead 20
Figure 22. Slightly raised melanistic lesion on the body surface of brown bullhead 20
Figure 23. Melanoma on the barbel of a brown bullhead 21
Figure 24. Melanoma on the body surface of a brown bullhead 22
Figure 25. Large melanoma on the body surface of a brown bullhead 23
Figure 26. Sessile papilloma on the head of a brown bullhead 24
Figure 27. Non-raised yellowish area of discoloration of the ventral surface of a bullhead 25
Figure 28. Raised growths on the mouth of brown bullhead 26
Figure 29. Microscopic appearance of the papillomas illustrated in Figure 28 27
Figure 30. Squamous cell carcinoma in brown bullhead 28
Figure 31. Osteoma and osteosarcoma in brown bullhead 29
-------
1.0 INTRODUCTION
1.1 Background Information
The Great Lakes Water Quality Agreement, in which the United States and Canada agreed to restore and preserve
the biological, physical and chemical integrity of the Great Lakes Basin Ecosystem, was first signed in 1972. In
1987, a Protocol was signed by both governments which defined Great Lakes Areas of Concern (AOC) as
"geographic areas that fail to meet the general or specific objectives of the agreement where such failure has caused
or is likely to cause impairment of beneficial use of the area's ability to support aquatic life." The U.S. and Cana-
dian governments identified 43 such areas; 26 in the U.S., 12 in Canada and five shared between the U.S. and Can-
ada on connecting river systems. The Great Lakes Water Quality Agreement, as amended via the 1987 protocol,
directs the two federal governments to cooperate with state and provincial governments to develop and implement
Remedial Action Plans (RAPs) for each AOC. The Protocol also called for reports on restorative progress and for
the International Joint Commission (DC) to review RAPs proposed by the 43 Areas of Concern (DC, 1987). Three
AOCs (Collingwood Harbor, Severn Sound and Oswego River) have been delisted and Presque Isle Bay has been
designated an Area of Recovery.
Fourteen beneficial use impairments were identified and the various AOCs have different combinations of these
impairments. One impairment, listed as "fish tumors or other deformities" is defined as occurring when "the inci-
dence rate of fish tumors or other deformities exceeds rates at unimpacted or control sites or when survey data con-
firm the presence of neoplastic or preneoplastic liver tumors in bullhead or suckers" (DC, 1989; www.epa.gov/
lakeerie/buia/reports). Currently, 16 of the 40 AOCs have the presence of tumors or other deformities listed as one
of their beneficial use impairments. It is important to recognize that although neoplasia has been linked with spe-
cific contaminants very little "cause and effect" evidence, particularly well designed exposure studies, exists for
brown bullhead.
1.2 Purpose of the Manual
Over the years numerous surveys have been undertaken to assess the incidence of skin and liver tumors, as well as
other contaminant-associated microscopic lesions at AOCs. However, a major problem is a lack of consistent crite-
ria for evaluating microscopic (histological) changes in bullhead liver and skin. For instance, in some studies there
is no distinction between preneoplastic (altered foci) and neoplastic lesions, or no good descriptions of individual
lesions (Black 1983; Pyron et al. 2001). Some studies included only carcinomas as neoplasia, while in others adeno-
mas, carcinomas, and cholangiomas were included (Baumann et al. 2000) and some included carcinomas, adenomas
and altered foci (Maccubbin and Ersing 1991). Skin tumors are often diagnosed visually without the supporting his-
tological verification. Hence, standardization of the criteria being used to evaluate the impairment is necessary, so
that data from different years, reference sites and Areas of Concern can be adequately compared.
Pennsylvania Department of Environmental Protection, U.S. Environmental Protection Agency Great Lakes Na-
tional Program Office and Pennsylvania Sea Grant sponsored a number of conferences related to Fish Tumors in
Great Lakes Areas of Concern. At the initial meeting, committees were formed to work on 1) standardized moni-
toring protocols and 2) criteria for standardized assessment of skin and liver lesions. This manual is the product of
the Histopathology Subcommittee and the purpose is to describe neoplastic and non-neoplastic proliferative lesions
of the liver and skin of the brown bullhead and suggest terminology that can be consistently used at AOCs through-
out the Great Lakes and other areas.
Field Manual for the Microscopic Diagnosis of Proliferative Liver and Skin Lesions in the Brown Bullhead
-------
2.0 SPECIMEN PREPARATION
2.1 Field Collection
A companion manual "Field Manual for Assessing Internal and External Anomalies in Brown Bullhead (Ameiurus
nebulosus}" by Rafferty and Grazio (2007) provides guidance for the identification of lesions and deformities ob-
served during a gross examination and recommends standard operating procedures for the collection, necropsy, and
preservation of tissue collected in the field. Briefly, external lesions, primarily raised lesions of any color and con-
sistency should be sampled. It is best to remove the whole lesion plus some normal surrounding tissue, if possible.
If the lesion is large, a subsample should be removed. For the identification of preneoplastic or neoplastic liver le-
sions it is necessary to sample all livers as most of the lesions will not be observed grossly. It is suggested that at
least five pieces of liver be placed in fixative in the field (Figure 1). Pieces of skin and liver are generally fixed in
10% buffered formalin, Davidson's (Luna 1992), or Z-Fix (Anatech LTD, Battle Creek, MI).
2.2 Specimen Processing
There have been no studies to statistically validate the number of sections
that need to be examined from an individual bullhead liver to have a given
confidence of diagnosing a particular lesion if present. To our knowledge
the only morphometric study of liver neoplasms and altered foci currently
available was conducted on mummichogs Fundulus heteroclitus, a much
smaller fish species (Stine et al. 2004). In that study six livers (five of
which had visible lesions) were serially sectioned. It was determined that
from 3-15 (mean of 7) sections were required to diagnose neoplasia or al-
tered foci from each liver with 95% accuracy. It is likely that for fish with
larger livers more sections will be required. Hence, it is suggested that
from the five or more pieces collected in the field, six to ten pieces should
be placed in cassettes and routinely processed for embedding in paraffin,
sectioned at a thickness of 4-6 um and stained with hematoxylin and eosin
(Luna 1992; Profet et al. 1992).
Figure 1. Illustration of liver pieces to be
sampled and placed in fixative in the field.
3.0 LIVER
Part of the definition of the beneficial use impairment is "when survey data confirm the presence of neoplastic or
preneoplastic liver tumors in bullhead or suckers". Liver neoplasms and other microscopic liver lesions have been
associated with PAH and other contaminant exposure in brown bullheads (Baumann 1992; Baumann and Har-
shbarger 1995; Baumann and Harshbarger 1998; Pinkney et al. 2001, 2004) and other fish species, including Eng-
lish sole Parophyrs vetulus (Myers et al. 1990), mummichog Fundulus heteroclitus (Vogelbein et al.1990), and
winter flounder Pseudopleuronectes americanus (Gardner et al. 1989). Although it is easy to define and describe
neoplastic lesions, those to be considered preneoplastic are more difficult as there have been no good exposure
studies to document the progression of liver neoplasia in bullhead. While both bile duct proliferation and foci of
cellular alteration may be associated with chemical exposures and are considered preneoplastic in some fish spe-
cies, bile duct proliferation may also be associated with myxozoan parasites and is often reactive in bullhead.
Hence, although bile duct proliferation should be documented, we do not suggest it be considered a chemically-
induced change at AOCs. In addition, there is insufficient evidence to indicate that it is a preneoplastic lesion. Con-
versely, foci of cellular alteration are almost always associated with toxicopathic effects. In wild fish surveys their
presence has been highly correlated with exposure to PAHs, PCBs, DDTs, chlordanes, dieldrin, mercury, pulp mill
effluent and aromatic hydrocarbon metabolites in English sole, white croaker Genyonemus lineatus, Pacific stag-
horn sculpin Leptocottus armatus, starry flounder Platichthys stellatus, dab Limanda limanda, flounder Platichthys
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
-------
flesus, rock sole Lepidopsetta bilineata and Dover sole Microstomus pacificus (reviewed by Au 2004; Feist et al.
2004; Koehler 2004) and are recommended for assessing chronic toxicity (Hinton 1994). Laboratory exposures of a
number of fish species have indicated altered foci to be preneoplastic, particularly basophilic foci (Hendrick et al.
1984; Grizzle and Thiyagarajah 1988; Hinton et al. 1988; Hawkins et al. 1990; Law et al. 1994). Hence, we recom-
mend that these foci of cellular alteration be considered preneoplastic.
3.1 Normal Liver Histology
Bullhead livers, as in most other teleosts, are composed of hepatic tubules. In liver sections from bullheads col-
lected at reference sites observable bile ducts are often sparse and not prominent in sections examined by light mi-
croscopy (Figure 2A and B). Portal veins are commonly surrounded by pancreatic tissue and bile ducts may be ob-
served within this tissue (Figure 2B). Normal hepatocyte appearance can vary from having little to no observable
vacuolization, lipid or glycogen storage (Figure 2A and C), to having extensive vacuolization (Figure 2B and D).
Macrophage aggregates are also observed within pancreatic and hepatic tissue (Figure 2A and C). More detailed
descriptions on normal piscine hepatic histology and hepatotoxicity can be found in (Boorman et al. 1997; Metcalfe
1998; Hinton et al. 2001; Wolf and Wolfe 2005).
V ' * f\ - ' ta! * 1 ' >ti.- •
.,
M - - •-
Figure 2. Microscopic appearance of normal bullhead liver. A. Section of normal liver illustrating the tubular structure, a bile duct
(a) and macrophage aggregate (arrow) within the exocrine pancreatic tissue. Bar = 150 fim. B. Normal liver with highly vacuolated
hepatocytes. Bloodvessels are often surrounded by pancreatic tissue (b) which occasionally contain bile ducts (arrow). Bar = 150
fim. C. Higher magnification of A illustrating the tubular structure and a macrophage aggregate (arrow). Bar = 50 fim. D. Higher
magnification ofB illustrating highly vacuolated hepatocytes, pancreatic tissue (b) and a bile duct (arrow). Bar = 50 fim. H&E
stain.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
-------
3.2 Non-Neoplastic Hepatic Lesions
3.2.1 Proliferation of Macrophage Aggregates
Macrophage aggregates are structures found in the spleen, kidney and liver offish (reviewed by Aguis and Roberts
2003; Wolke 1992). They have been shown to increase with age (Blazer et al. 1987) and in response to environ-
mental stress (Fournie et al. 2001). Hence, in young fish, and/or fish from reference sites, macrophage aggregates
may not be observable (Figure 2B) or they may be present at a low density within the liver or hepatic pancreatic
tissue (Figure 2A and 3 A), while at impacted sites a higher density may be observed (Figure 3B). Three pigments -
melanin, hemosiderin and the lipopigments, lipofuscin/ceroid, can be observed within these structures using the
Perl's Prussian Blue method which stains hemosiderin blue (Luna 1992). In bullheads the lipopigments and he-
mosiderin are most commonly observed. Some have a higher proportion of hemosiderin, an iron-containing pig-
ment, which stains blue (Figure 3B and D), while others contain more of the lipofuscin/ceroid pigments, formed by
oxidation of lipid and retaining a yellowish-brown appearance (Figure 3 A and C).
B
•**:
•
•
Figure 3. Macrophage aggregates in liver sections of brown bullhead stained by the Perl's Prussian Blue method. A. Liver section
from a bullhead collected at a reference site illustrating an aggregate in the hepatopancreatic tissue (a) and another within hepatic
tissue (b). Bar = 100 jim. B. A liver section from a bullhead collected at an AOC illustrating numerous aggregates within hepato-
pancreatic (a) and hepatic (b) tissue. Bar = 100 jim. C. Macrophage aggregate (a) with a moderate amount of hemosiderin (blue)
and ceroid/lipofuscin (arrows). Bar = 20 jim. D. Macrophage aggregate (a) with high levels of hemosiderin (blue). Bar = 20 jim.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
-------
3.2.2 Proliferative Responses to Parasites
Helminth parasites are commonly observed within liver tissue of bullhead collected in some areas. They can in-
clude trematodes, nematodes, acanthocephalans and cestodes. These may be the most commonly observed, grossly
visible lesions (Figure 4A). They may appear as irregularly-shaped, elongate or rounded, pale, raised areas. How-
ever, raised pale areas may also be tumors (Figure 4B).
Figure 4. Grossly visible lesions in brown bullhead liver. A. Numerous pale irregularly-shaped areas, some on the surface of the
liver and some within the parenchyma (yellow arrows). B. Pale discolored areas of variable size and shape on the surface and within
the parenchyma of the liver. These included parasites and the proliferative inflammatory response to them (yellow arrows), illus-
trated in Figure 5, and a cholangiocarcinoma (black arrow), illustrated in Figure 16. Gallbladder is present (white arrow).
Field Manual for the Microscopic Diagnosis of Proliferative Liver and Skin Lesions in the Brown Bullhead
-------
••.;•>, -"-•-
. .-, .••> .;•'.>*• '- •- ' •• •"
•'. fr ""'VVr'' "•'"•:•'••'
'•" ,v
MBHHI
Figure 5. Helminth parasites and the accompanying inflammatory responses in brown bullhead liver. A and B. Cestode parasite (a)
on the surface and extending into the parenchyma of the liver. Bar = 150 jim. C. Remnants of a helminth parasite (a) and extensive
inflammatory response (b) within the liver. Bar = 150 jim. D. Chronic granulomatous inflammatory response (b) and giant cells
(arrows) within the liver of brown bullhead. Bar = 50 jim. H&E stain.
3.3 Putatively Preneoplastic Lesions - Foci of Cellular Alteration
Four categories of foci of cellular alteration are recognized in fish livers stained with hematoxylin and eosin, based
on the tinctorial characteristics of the hepatocyte cytoplasm. In rats, it has been documented that these differences
in staining are due to accumulations of specific components within the cells. Cells within eosinophilic foci are rich
in smooth endoplasmic reticulum, while cells within basophilic foci are rich in RNA. Clear cell foci contain abun-
dant glycogen. Foci of vacuolated cells contain lipid in some fish species and a mixture of lipid and glycogen in
others (reviewed by Bunton 1996). The margins of these foci are generally distinct but the hepatic tubules are ar-
ranged in a relatively normal pattern, merge imperceptibly with the surrounding parenchyma, and little to no com-
pression is observed.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
-------
3.3.1 Basophilic Foci
Basophilic foci are round to irregular clusters of hepatocytes rich in rough endoplasmic reticulum and increased
basophilic staining compared to adjacent cells (Figure 6A). The cells may be smaller than adjacent hepatocytes, but
cellular atypia and mitotic figures are generally not present (Figure 6B).
Figure 6. Basophilic focus in the liver of brown bullhead. A. Discrete focus of hepatocytes (a) staining more basophilic than sur-
rounding (b) hepatocytes. Bar = 150 fim. B. Higher magnification of a basophilic focus (a), illustrating the margin of the focus
(arrow), which merges into the adjacent hepatocytes (b). Bar = 100 fim. H&E stain.
3.3.2 Eosinophilic Focus
Eosinophilic foci are round to irregular areas of hepatocytes with increased eosinophilia compared to adjacent cells
(Figure 7A). The cells may be slightly enlarged with a granular eosinophilic cytoplasm (Figure 7B).
B
b ,
i
f d
Figure 7. Eosinophilic focus in brown bullhead liver. A. Area of hepatocytes with increased eosinophilia (a) when compared to sur-
rounding hepatocytes (b). Arrows indicate the border of the lesion. Bar = 150 fim. B. Hepatocytes within the eosinophilic focus (a)
have a finely granular cytoplasm and are slightly larger than surrounding hepatocytes (b). Bar = 50 fim. H&E stain.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
-------
3.3.3 Vacuolated and Clear Cell Foci
Vacuolated foci are described as round to irregular, containing hepatocytes with medium to large, clear, lipid-
containing, cytoplasmic vacuoles. The nuclei are often displaced to the periphery of these cells (Figure 8). Clear
cell foci, as described in other fish species, are characterized by enlarged hepatocytes with a "ground glass" appear-
ance to the cytoplasm and the nuclei are generally centrally located within the cells. The clear cytoplasm is the re-
sult of glycogen accumulation which is dissolved out in aqueous fixatives. Clear cell foci are less commonly ob-
served than vacuolated foci (Boorman et al. 1997; Feist et al. 2004). In bullhead, these foci often appear to be
mixed foci with cells characteristic of both clear cell and vacuolated foci (Figure 9). Cells within the focus may
have both centrally-located and eccentric nuclei and the ground glass appearance is not as evident.
A
b
Figure 8. Vacuolated Cell Foci. A. Focus (a) with border blending into surrounding hepatocytes (b). Bar = 150 jim. B. Higher
magnification illustrating the lack of compression or encapsulation between the border (arrow) of the focus (a) and surrounding
hepatocytes (b). Bar = 100 jim. C. Cells within the focus have variably-sized clear vacuoles and eccentric nuclei (arrows). Bar = 50
tun. H&E stain.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
-------
.."••
„ -
.
Figure 9. Clear or Mixed Cell Focus. A. Focus (a) with border blending into surrounding hepatocytes (b). Bar = 150 jim. B. Higher
magnification illustrating the lack of compression or encapsulation between the border (arrow) of the focus (a) and surrounding
hepatocytes (b). Bar = 100 jim. C. Some cells within the focus have centrally-located nuclei (arrows), while others are eccentric.
Some cells have cytoplasm with a ground glass appearance (yellow arrows). Bar = 50 jim. H&E stain.
3.4 Neoplastic Hepatocellular Lesions
3.4.1 Hepatocellular Adenoma
Adenomas are discrete lesions which generally have a distinct border. The cells may exhibit altered staining proper-
ties and hence appear more eosinophilic, basophilic or vacuolated compared to the surrounding tissue. Cells are
usually not arranged in normal growth patterns and usually there is very little pleomorphism. Mitotic figures are
rarely observed. Macrophage aggregates, pancreatic tissue and other structures are often missing or sparse within
the neoplastic lesion. Adenomas may be demarcated by compression of the adjacent parenchymal cells (Figure
10B), however in some cases this is not as obvious (Figure 10A).
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
-------
'
Figure 10. Hepatocellular adenoma in the liver of brown bullhead. A. Neoplastic cells within the adenoma (a) stain eosinophilic com-
pared to surrounding hepatocytes (b). Bar = 150 /um. B. Adenoma (a) showing obvious compression (arrows) of surrounding tissue.
Bar = 150 [im. C and D. Higher magnification of the periphery of two adenomas (a) which have some cellular atypia and moderate
compression (arrows) of surrounding tissue. Bar = 50 /um. H&E stain.
3.4.2 Hepatocellular Carcinoma
Hepatocellular carcinomas, malignant hepatic neoplasms, are often diffusely spread throughout the hepatic paren-
chyma (Figure 11A and B) in brown bullhead, but may be distinct foci with irregular borders (Figure 11C and E).
Neoplastic cells invade the adjacent parenchyma (Figure 11D). Cellular pleomorphism and nuclear atypia are key
features (Figure 11F) and there is often an increase in the number of mitotic figures. There can be some tumor giant
cell formation and these may be small or large lesions.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
10
-------
.
|
d
B
e
D
'
Figure 11. Hepatocellular carcinoma in the liver of brown bullhead. A. Foci ofneoplastic cells (a) are observed throughout the nor-
mal hepatic parenchyma (b). Inflammation (c), probably in response to helminth parasites (d) is also observed. Bar = 150 jim. B.
Higher magnification of A, indicating focus ofneoplastic cells (a), normal liver (b) and a macrophage aggregate (e). Bar = 50 jim.
C. Larger, more invasive carcinoma (a) infiltrating normal liver (b). Bar = 150 jim. D. Higher magnification ofC illustrating the
invasiveness of the neoplastic cells (a). Bar = 50 jim. E. Well-differentiated hepatocellular carcinoma (a). Bar = 100 jim. F. Higher
magnification ofE illustrating neoplastic cells which vary in size and shape and may have multiple nucleoli within the nucleus
(arrow). A macrophage aggregate is evident (e). Bar = 50 jim. H&E stain.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
11
-------
3.5 Non-Neoplastic Biliary Lesions
3.5.1 Bile Duct Proliferation and Fibrosis
Normal small and intermediate bile ductules/ducts have columnar epithelium and a thin band of connective tissue
(Figure 12A). Large bile ducts are surrounded by connective tissue and are prominent near the common bile duct
(Figure 12B). Bile duct hyperplasia consists of an increased number of variably-shaped bile ducts, when compared
to the normal appearance (Figure 2A and B). Bile ducts are often scattered throughout the liver and do not form
discrete masses (Figure 13A), although occasionally small clusters are observed (Figure 13B). The biliary epithe-
lium is always well-differentiated (normal-appearing), however there may be moderate fibrosis associated with pro-
liferating bile ducts (Figure 13C). Cholangiofibrosis consists of bile ducts or ductules with extensive periductal fi-
brosis (Figure 13D), however the biliary epithelium is still normal.
:». .-. v. _ .
Figure 12. Bullhead liver showing normal bile ductule and duct. A. Within the hepatic tissue (a) bile ductules (arrow) are com-
monly located within the pancreatic tissue (b) and are bounded by a thin band of connective tissue. Bar = 50 jim. B. Larger ducts
have a thicker layer of connective tissue (arrow) and columnar epithelium, (c). Bar = 50 jim. H&E stain.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
12
-------
B
v.,,^;w- -y'-:^
-^':"''' •
Figure 13. Bile duct proliferation and fibrosis. A. Proliferation of individual bile ductules and ducts (arrows) throughout the liver.
Bar = 150 fim. B. Focal proliferation of bile ducts (a) with a moderate amount of fibrosis around the ducts. Bar = 150 fim. C.
Higher magnification illustrating increased fibrous tissue (b) and normal epithelium (c). Bar = 100 fim. D. Extensive fibrosis (b)
around a focal area of bile duct proliferation. Bar = 50 fim. H&E stain.
3.5.2 Bile Duct Parasitism
Sporoplasms of a myxozoan parasite have been observed within the bile ducts of brown bullhead from numerous
areas. The affected bile ducts are often thickened and in some areas there is evidence of hyperplasia of the epithe-
lium or necrosis (Figure 14). It is currently not known what association this parasite may have on bile duct prolif-
eration or biliary neoplasia.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
13
-------
Figure 14. Bile duct myxozoan parasites of brown bullhead. A. Sporoplasms (a) of a myxozo an parasite within the bile ducts
(arrows). Bar = 150 jim. B. Higher magnification of the sporoplasm (a) containing mature spores (arrows) as well as immature
stages. Spores are elongate with polar capsules on either end. Bar = 20 jim. C. Affected bile ducts may show signs of epithelial hy-
perplasia (arrow) associated with the presence of the sporoplasm (a). Bar = 50 jim. D. Some affected bile ducts show signs of
epithelial necrosis (arrow) associated with the parasite (a). Bar = 50 jim. H&E stain.
3.6 Neoplastic Biliary Lesions
3.6.1 Cholangioma
Cholangiomas are benign tumors of bile ducts within the liver. These are clusters of bile ducts which are well-
differentiated and often have a discrete border between the nodule and surrounding hepatic parenchyma (Figure
15). Many of the bile ducts may be irregularly-shaped and dilated.
T/S*- V *«*>
if^m&r
, .• k * - •*• -^^^-—•
PJ-^' - -
/. - «/»•' •
Figure 15. Cholangioma within the liver of a brown bullhead. A. Nodule ofneoplastic bile ducts (a) is separated from the normal he-
patic tissue (b) by a well-defined border (arrows). Bar = 50 fan. B. Higher magnification illustrating the well-defined border (arrows)
and neoplastic bile ducts (c) which are irregularly shaped and dilated. B. Bar = 25 fan. H&E stain.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
14
-------
3.6.2 Cholangiocarcinoma
Cholangiocarcinomas are malignant tumors of bile ducts. Some tumors contain moderately to well-differentiated
bile ducts, which vary greatly in size and shape and invade into the surrounding parenchyma (Figure 16A and B).
The proliferating bile ducts may be associated with proliferating stroma and inflammation (Figure 16C and D). In
some tumors, there is very little normal duct formation and the neoplastic epithelial cells are pleomorphic with mi-
totic figures and are surrounded by a proliferating spindle cell stroma (Figure 16E and F).
E
i
Figure 16. Cholangiocarcinoma in brown bullhead liver. A. Proliferating neoplastic bile ducts (a) invade into the surrounding he-
patic parenchyma (b). Bar = 150 fim. B. Higher magnification of invading neoplastic bile ducts (a). Bar = 100 fim. C. A Cholangio-
carcinoma in which the proliferating neoplastic bile ducts surround pancreatic tissue (c). Bar = 150 fim. D. Higher magnification
illustrating inflammation (d) around the proliferating bile ducts (a). Bar = 50 fim. E. Small nodule ofundifferentiated bile ducts (a)
separated from normal liver (b) by inflammation. Bar = 100 fim. F. Higher magnification illustrating pleomorphic cells with mitotic
figure (arrow). Bar = 20 fim. H&E stain.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
15
-------
3.7 Metastatic Neoplasia
Metastasis of malignant neoplasms to the liver is rare in fish. However, metastases of dermal melanomas to the
liver have been observed in brown bullhead (M. Wolfe, personal observation).
4.0 SKIN
The presence of external neoplasms, in bullhead and white sucker Catostomus commersoni in the Great Lakes re-
gion and elsewhere, has been used as an indicator of environmental health (Baumann 1992). Skin tumors reported
in brown bullhead include epidermal papillomas, squamous cell carcinomas and melanomas (Sonstegard 1977; Kim
et al. 1989; Bowser et al. 1991). Henry David Thoreau may have been the first to document a raised growth in bull-
head. A study written by Thoreau and reported by Osburn (1925) of bullheads in a pond on Cape Cod, described
lesions varying from melanistic spots to large raised tumors, attributed to "black coccoid bacteria", which were
probably melanosomes (organelles responsible for melanin production). Elevated prevalences of raised skin lesions
have been reported at contaminated and/or industrialized sites in numerous fish species (Smith et al. 1989; Hayes et
al. 1990; Kinae et al. 1990; Okihiro et al. 1993; Mikaelian et al. 2000), although, the association with chemical ex-
posure in bullheads is less conclusive than with liver neoplasia (Poulet et al. 1994; Pinkney et al. 2001). Papillomas
are the most frequently reported cutaneous neoplasms and affect a wide variety of freshwater and saltwater fishes.
Some laboratory studies have indicated a chemical etiology for bullhead papillomas (Black 1983; Grizzle et al.
1984), while others have suggested a viral etiology based on the observation of viral particles (Edwards and Sam-
sonoff 1977). A number of studies have looked for, but not confirmed, the presence of viral particles (Bowser et al.
1991; Poulet et al. 1996) and attempts to transmit the orocutaneous neoplasms have not been successful, suggesting
a multifactorial etiology (Poulet et al. 1993). Viruses have been implicated in the induction of tumors or tumor-like
growths in a number of other fish species (Yamamoto et al. 1985; Lee and Whitfield 1992; Anders and Yoshimizu
1994; Quackenbush et al. 2001). It has been suggested a higher prevalence of skin tumors in fish collected at con-
taminated areas may be explained by a lower resistance or immunosuppression (Au et al. 2004). These raised
growths are most commonly observed on the lips and barbels, however can be found anywhere on the body surface
and fins.
4.1 Normal Skin, Barbel, and Oral Cavity Microscopic Appearance
The histologic appearance of bullhead skin and other tissues is similar to that described for channel catfish Ictalu-
rus punctatus (Grizzle and Rogers 1976). Bullhead skin, like that of other catfishes, does not contain scales. The
skin is composed of the epidermis and underlying dermis and hypodermis and varies in thickness in different areas
of the body. The epidermis is composed of columnar basal cells which become squamous in the outer layer. Goblet
or mucous cells are present in all areas but vary in abundance. Alarm substance, club or fright cells are also com-
mon, while melanophores and taste buds are found in some areas (Figure 17A and B). The dermis is composed of a
compact layer of fibrous connective tissue below the epidermis. In most areas there is a thin layer of melanophores
immediately beneath the epidermis.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead 16
-------
a
Figure 17. Microscopic appearance of normal brown bullhead skin. A. Skin from the lateral surface with numerous alarm sub-
stance cells (a) in the epidermis (between double-headed arrow). The dermis (b) immediately below the epidermis contains a thin
line ofmelanophores (arrow). Bar = 150 jim. B. An area of skin with fewer alarm substance cells (a) and more mucous or goblet
cells (b). The epithelial cells at the base of the epidermis (yellow arrow) are columnar cells, becoming more squamous (black arrow)
at the surface. Bar = 50 fim. H&E stain.
The barbels are composed of a central core of pseudocartilage and nervous tissue, covered with a dermis and epi-
dermis, both generally containing melanophores. Taste buds are very numerous in the epidermis (Figure ISA and
B) of the barbels.
A
;' , ' I;. ;; '•'•.-••
Figure 18. Microscopic appearance of brown bullhead barbels. A. Barbels consist of an epidermis (a), dermis (b) with a layer of
melanophores (arrow), a central portion of nervous tissue (c) and pseudocartilage (d). Bar = 150 fim. B. The epidermis of the bar-
bel has abundant taste buds (e), some alarm substance cells (f) and melanocytes (yellow arrows). Bar = 50 fim. H&E stain.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
17
-------
The oral cavity has an epithelium similar to the outer skin, however it has a more folded appearance (Figure 19A).
Most areas have fewer alarm substance cells, while taste buds are abundant. Teeth are ankylosed to the premaxilla
and dentary bones of the mouth (Figure 19B). The teeth are covered with a thin enamel-like material and formed of
a dentine-like material which surrounds a pulp cavity that contains nerves, blood vessels and fibrous connective
tissue. Modified epithelial cells surround the teeth.
B
\l
Figure 19. Microscopic appearance of the normal oral cavity. A. Mucosal epithelium (between double headed arrow) contains taste
buds (a) and alarm substance cells (b). Bar = 150 jim. B. Teeth (c) extend from the bone through the propia mucosa andmucosa,
and are surrounded by modified epithelial cells (arrows). The subdermal area (d) consists of highly vascularized loose connective
tissue. Bar = 15Qjim. H&E stain.
4.2 Black Pigmented Skin Lesions
Black pigmented lesions in brown bullhead range from non-raised melanistic areas to large raised black areas. In
bullheads, there have been no studies that address the progression of pigmented lesions. It is unknown whether the
non-raised melanistic areas (melanotic hyperpigmentation) are precancerous lesions that progress to melanomas.
We have observed examples of all in wild-caught bullheads (Figures 20-26). The Xiphophorus model has been used
extensively to study molecular and genetic mechanisms involved in melanoma induction (Setlow et al. 1989;
Schartl 1995; Nairn et al. 1996). In this fish species, a classification scheme for spontaneous tumors and neoplastic
lesions induced by UV radiation and exposure to the carcinogen jV-methyl-jV-nitrosourea has been described. These
lesions include nonneoplastic melanotic hyperpigmentation, preneoplastic melanosis and melanomas (Gimenez-
Conti et al. 2001). A correlation between exposure to environmental chemicals and pigment cell neoplasia has been
suggested in some fish species (Kimura et al. 1984; Kinae et al. 1990; Okihiro et al. 1993).
4.2.1 Non-Raised Melanistic Spots
Melanistic areas are non-raised, black pigmented spots on the head, body surface or fins. They can vary greatly in
size and shape (Figure 20A and B). Flistologically, these melanistic lesions consist of increased numbers of melano-
cytes primarily within the epidermis, but sometimes the dermis as well. The melanocyte may have extensive cyto-
plasmic extensions or be in large groups (Figure 20 C and D).
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
18
-------
Figure 20. Melanistic spots. A. Multiple, nonraised, black spots on the lateral body surface. B. A large, irregular, non-raised black
area on the head and body surface. C. Microscopic appearance of a melanistic spot. Epidermis (between double-headed arrow) con-
tains groups ofmelanocytes (black arrow) as well as individual cells with extensive cytoplasmic extensions (yellow arrows). Bar =
150 jim. D. Higher magnification illustrating groups ofmelanocytes of varying sizes (black arrow) and individual melanocytes with
extensive cytoplasmic extensions (yellow arrows). Bar = 50 jim. H&E stain.
4.2.2 Raised Black Skin Lesions
These areas can vary from small raised areas to larger raised areas on fins, body surface, lips and barbels. It is nec-
essary to examine these lesions histologically to determine whether they are parasite-associated raised lesions
(Figure 21), melanotic hyperplasia (Figure 22), melanomas (Figures 23-25), or papillomas with increased melano-
cytes within the proliferating epidermis (Figure 26). Grossly, these may all look very similar.
4.2.2.1 Parasite-Associated Black, Raised Lesions
Some of the raised black areas observed on barbels and the head region are found to contain trematode metacer-
cariae. These parasites appear primarily around and within the nerves of the barbels, and are associated with a pro-
liferation of dermis, underlying connective tissue, dermal melanocytes and in some cases a proliferation of the
overlying epidermis (Figure 21). Similar raised black lesions are known to occur in blue catfish (Ictalurus furca-
tus), associated with Ichthyophonus infections (Fournie, personal observations).
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
19
-------
Figure 21. A. Small raised, black areas (white arrows) on the barbels of a brown bullhead. B. Microscopic appearance of the bar-
bels illustrating parasitic cysts (a) within the nerves (c) and dense connective tissue. In some areas this response is associated with a
proliferation of the epidermis (b). Bar = 150 fim. H&E stain
4.2.2.2 Melanotic Hyperplasia
Slightly raised and irregular pigmented areas have been noted in some bullhead, which microscopically exhibited
epidermal hyperplasia and abnormal-appearing melanin accumulations, and are possibly preneoplastic (Figure 22).
. "t' fn//.€fcv. -.
»
'i •• '
^ .,
,
Figure 22. A. Slightly raised melanistic area on the body surface of a brown bullhead. B. Microscopic appearance of a section of
the area. The epidermis (between the double-headed arrows) is of varying thickness and contains groups of abnormal, spindle-
shaped cells with melanin (a). Normal melanocytes are present in the epidermis and dermis (arrows). Bar = 150 jim. C. Higher mag-
nification showing normal melanocytes in dermis and epidermis (arrows) and groups of abnormal melanocytes in epidermis (a). Bar
= 100 jim. D. Abnormal melanocytes are spindle-shaped and vacuolated (a). Bar = 50 jim. H&E stain.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
20
-------
4.2.2.3 Melanoma
Lesions diagnosed as melanomas range from very small raised areas on the skin or barbels (Figure 23 and 24) to
very large raised areas (Figure 25). A number of types of melanomas similar to those described by Gimenez-Conti
et al. (2001) have been observed in brown bullhead and are illustrated in the following figures. These include the
melanocytic melanomas (Figure 23) which are characterized by a proliferation of melanocytes that are dendritic in
shape and contain variable amounts of pigment.
L%,\3W"'«
Figure 23. Melanoma on the barbel of a brown bullhead. A. Small raised, black area on the barbel. B - D. Histologic appearance of
barbel melanoma. B. Epidermis (arrow) is intact over most of the melanoma (a). Bar = 300 /um. C. The proliferating neoplastic cells
extend into the dense connective tissue of the dermis (b). There are numerous blood vessels throughout the tumor (arrows). Bar = 50
fan. D. In areas there are foci of neoplastic cells (b) within the epidermis (a). The neoplastic cells surround blood vessels (c), are den-
dritic and contain varying amounts of pigment. Bar = 10 fan. H&E stain.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
21
-------
The melanoma on the body surface (Figure 24) is usually a polymorphic melanoma. These tumors are heterogenous
with both heavily and lightly pigmented areas. Cell types within the tumor include melanocytes, epithelioid cells
and macromelanophores.
Figure 24. Melanoma on the body surface of a brown bullhead. A. Grossly visible, raised, black lesion (arrow) on the lateral surface of
a brown bullhead which also has a large discolored lesion (a), covered by fungal growth. B. Microscopic appearance of the melanoma.
In some areas the epidermis covering the tumor is thickened (a) and contains pegs of the neoplastic cells (arrow). The tumor contains
some areas that are less densely stained (b) than others (c). Bar = 300 [im. C. Higher magnification of the tumor illustrating the varia-
bly sized and shaped cells and bizarre growth pattern. Bar = 50 fan. D. Neoplastic cells include very large macromelanophore cells (a)
and cells of relatively normal size (b). Bar = 20 /um. H&E stain.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
22
-------
The large, black raised lesion on the body surface (Figure 25) resembles a spindle cell melanoma histologically,
and is very similar to a dermal melanoma previously described from brown bullhead as having schwannoma-like
differentiation (Sakamoto and White 2002).
'^im&^*mM
Figure 25. Large melanoma on the body surface of a brown bullhead. A. Irregular raised black area on the skin. B. Microscopic ap-
pearance illustrating a "herringbone "pattern with a connective tissue stroma (a). Bar = 150 jim. C. Occasional clusters of rounded,
densely pigmented melanophores (a) were observed. Bar = 100 jim. D. The majority ofneoplastic cells were spindle-shaped with vary-
ing amounts of melanin. Bar = 20 /um. H&E stain.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
23
-------
4.2.2.4 Melanotic Papilloma
Some raised black areas are papillomas (see 4.3.2.1) that are characterized by a proliferation of melanocytes within
the epidermis and along the connective tissue trabeculae (Figure 26).
Figure 26. A. Sessile papilloma (arrow) on the head of a brown bullhead. B. Microscopically, the lesion was composed of proliferat-
ing epithelial cells with melanocytes within these proliferating areas (black arrows). Bar = 150 fim. C. These proliferating epithelial
cells form the typical papillomatous folds supported by the connective tissue stroma lined by melanocytes (white arrows). Bar = 150
fim. D. Higher magnification illustrating accumulations of melanocytes in the stroma (white arrow) and individual melanin-
containing cells in the proliferating epidermis (black arrow). Bar = 100 fim. H&E stain.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
24
-------
4.3 Pale Areas of Discoloration
Areas of discoloration, generally white to yellow, range from small, non-raised to large, raised, multicolored and
multilobed lesions. Microscopically, these areas show epidermal hyperplasia of one or more cell types, papillomas
or squamous cell carcinomas. A number of studies have suggested a progression from epidermal hyperplasia to
papilloma and occasionally to carcinoma in brown bullhead (Lucke and Schlumberger 1941; Smith et al. 1989;
Poulet et al. 1994; Bunton 2000).
4.3.1 Non-Raised Areas of Discoloration
These areas on bullhead are generally gray or yellow areas such as illustrated in Figure 27A. Microscopically, they
appear to be due to proliferation of certain cell types. The pale gray areas contain increased epithelial and mucous
cells, while the yellow areas contain increased mucous and alarm substance cells.
;
•_
. ' / '}f :
' . • ''.•', .aff'ff
Figure 27. A. Non-raised yellow spot on the ventral surface of a brown bullhead. B. Microscopic appearance at the periphery of the
discolored area showing the difference between the normal epithelium (a) and the discolored area (b). Bar = 150 fim. C. Micro-
scopically, the yellow lesions contain areas with increased mucous cells (arrows) and alarm substance cells (c). Bar = 150 fim. D.
Higher magnification of the affected area in B, illustrating the proliferation of mucous cells (arrows) and alarm substance cells (c).
Bar = 100 fim. H&E stain.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
25
-------
4.3.2 Raised Pale Areas
Raised, pale growths can be observed on the lips, fins and body surface and can vary from small, slightly raised,
plaque-like areas to very large multiple, coalescing nodules. In bullheads they are most common in the oral cavity
and on the lips. Most often these growths are papillomas or squamous cell carcinomas.
4.3.2.1 Papilloma
Papillomas are considered benign growths and can range from slightly raised, homogeneous-appearing areas
(Figure 28A) to small raised, slightly eroded and reddened plaques (Figure 28B) to larger, often multiple nodules
(Figure 28C and D) which may vary in color from pale to black (Figure 28C).
Figure 28. Raised growth on the mouth of brown bullhead. All were determined to be papillomas by microscopic evaluation. A. A
slightly raised, plaque-like, pale growth on the lower lip. B. A small, slightly raised, reddened and eroded growth on the upper lip.
C. Larger, multiple raised growths on the lips, some dark (black arrows) and some very pale (white arrows). D. Multiple, raised
growths on the lower lip.
Histologically, the epithelial proliferation causes papillary folds to form which are supported by connective tissue
cords. These folds extend above the normal skin surface and extend into the underlying dermis. Proliferation is pri-
marily of the epidermal malpighian cells. However, in some areas there may be proliferation of mucous or alarm
substance cells causing the area to appear pale, while in others there may be a proliferation of melanocytes giving
the area a dark appearance.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
26
-------
Figure 29. Microscopic appearance of the papillomas illustrated in Fig. 28. A. Proliferating epidermal (malpighian) cells (a) form
folds or papillae supported by connective tissue (b). Neoplastic cells do not infiltrate below the basement membrane (arrow). Bar =
150 fim. B. Raised growths that appear reddened and/or abraded (Figure 28B) contain areas of hemorrhage or congestion (c) and
the outer surfaces may be necrotic (d). Bar = 150 fim. C. Individual growths may appear very pale and translucent (Figure 28C)
due to a proliferation of mucous cells (e). Bar = 100 fim. D. Higher magnification illustrating the well-defined folds of the epidermis
(a) within the connective tissue support (b). Bar = 100 fim. E. The proliferating cells are squamous (f) in the outer regions and co-
lumnar to cuboidal (g) toward the dermis. Bar = 50 fim. F. Proliferating cells vary in size and shape, nuclei are enlarged and may
contain multiple nucleoli (arrow). Bar = 20 fim. H&E stain.
4.3.2.2 Squamous Cell Carcinoma
Squamous cell carcinomas have been described in a number offish species (Fournie et al. 1987; Harshbarger and
Clark 1990; Hanjavanit and Mulcahy 2004) including brown bullhead (Bunton 2000; Pinkney et al. 2001). In a
study of bullhead from the Back River, MD, an area with elevated PAH levels, a progression from hyperplasia to
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
27
-------
papilloma to carcinoma was suggested. Local invasion through the basal lamina occurs as the lesion progresses to
carcinoma (Bunton 2000). Some areas are masses of neoplastic cells in which small islands of supportive tissue,
mucous or alarm substance cells may become entrapped (Figure 30 B). Other areas may contain cords or nests of
neoplastic cells interspersed with fibrous connective tissue (Figure 30 C and D). Carcinomas are characterized by
pleomorphism of the epithelial cells. Large, often polygonal cells, with enlarged nuclei, are observed with a loss of
normal orientation (Figure 30 E and F). They are also characterized by extension through the basement membrane
(Figure 30 F).
Rtft^AhJX V /•*£*• . * *
E
d
F
Figure 30. Squamous cell carcinoma in brown bullhead. A. Large growth on the lower lip and jaw of a brown bullhead. B. Micro-
scopic appearance of the neoplastic growth illustrating the lack of well-defined folds (a) and islands of supporting tissue (b). Bar =
150 fim. C. In some areas, cords (a) or nests (arrows) of neoplastic cells are found within fibrous tissue (b). Bar = 100 fim. D.
Higher magnification of neoplastic cells (a) that have extended through the basal lamina and small nests of neoplastic cells (arrows)
in the dermis and hypodermis. Bar = 50 fim. E. Area of the carcinoma illustrating the lack of normal orientation of the large, often
polygonal cells. Necrotic areas (c), mucous cells (d) and mitotic figure (arrow) may be observed. Bar = 50 fim. F. Invasion of the
neoplastic cells (e) through the basement membrane (double arrow) and into the underlying tissues (b). Bar = 20 fim. H&E stain.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
28
-------
4.3.2.3 Miscellaneous Raised Growths
Occasionally, other tumor types are found to be responsible for the raised growths observed on bullhead fins, body
surface or mouth. An osteoma and osteosarcoma were both diagnosed from the same bullhead. These lesions were
described grossly as a firm, raised mass on the pectoral fin and a hyperplastic lesion on the upper lip, respectively.
The fin lesion was diagnosed microscopically as an osteoma (Figure 31A andB) while the jaw lesion was an
osteosarcoma (Figure 31C and D). To our knowledge, there are no other reports of bony tumors in bullhead, how-
ever individual cases have been described in a few other fish species (Grizzle et al. 1995; Manera and Biavati 1999;
Pereira and Peleteiro 2002). The osteoma is comprised of trabeculae of mature bone, generally with a well-defined
border (Figure 31 A and B). The osteosarcoma contained less mature bone and the periphery was omposed of ir-
regularly-shaped extensions of osteoid surrounded by pleomorphic, basophilic osteoblasts (Figure 31 C andD).
Figure 31. Raised bony tumors on the fin and jaw of a brown bullhead. A. Microscopic appearance of the osteoma on the fin. Tra-
beculae of mature bone (a) with a well-defined border (arrow) in most areas. Bar = 150 fim. B. In some areas of the tumor (a) there
is osteoblastic activity (arrow) on the periphery. Bar = 50 fim. C. Microscopic appearance of an osteosarcoma on the jaw of a
brown bullhead. Periphery is composed of irregularly-shaped extensions of osteoid (arrows). Bar = 150 fim. D. Higher magnifica-
tion illustrating the pleomorphic, basophilic osteoblasts (arrows) surrounding the osteoid branches. Bar = 50 fim. H&E stain.
5.0 ACKNOWLEDGEMENTS
We thank all those within Pennsylvania Department of Environmental Protection and Pennsylvania Sea Grant in-
volved in the collection of fish and other logistical assistance. We appreciate the technical assistance of Kathy
Spring and Darlene Bowling for preparation of histological slides. Funding was provided by the U.S. Geological
Survey, the U.S. Environmental Protection Agency, Pennsylvania Sea Grant and the project was performed, in part
by using the services provided by the National Cancer Institute's Registry of Tumors in Lower Animals, operated
under contract by Experimental Pathology Laboratories, Inc., N02-CB-27034. We appreciate the critical reviews
provided by Paul Bowser, Mark Myers, William Hawkins, Wolfgang Vogelbein and John Harshbarger.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
29
-------
6.0 REFERENCES
Aguis, C. and RJ. Roberts. 2003. Melano-macrophage centres and their role in fish pathology. I Fish Dis. 26:499-
509.
Anders, K. and M. Yoshimizu. 1994. Role of viruses in the induction of skin tumours and tumour-like prolifera-
tions offish. Dis. Aquat. Org. 19:215-232.
Au, D.W.T. 2004. The application of histo-cytopathological biomarkers in marine pollution monitoring: a review.
Mar. Pollut. Bull. 48:817-834.
Baumann, P.C. 1992. The use of tumors in wild fish to assess ecosystem health. Aquat. Ecosystem Health 1:135-
142.
Baumann P.C., V. Cairns, W. Kurey, L. Lambert, I. Smith and R. Thoma. 2000. Fish tumors or other deformities.
Lake Erie Lakewide Management Plan (LaMP) Technical Report Series, Report No. 6, 59 pp.
Baumann, P.C. and J.C. Harshbarger. 1995. Decline in liver neoplasms in wild brown bullhead catfish after coking
plant closes and environmental PAHs plummet. Environ. Health Perspect. 2:168-170.
Baumann, P.C. and J.C. Harshbarger. 1998. Long term trends in liver neoplasm epizootics of brown bullhead in the
Black River, Ohio. Environ. Monitor. Assess. 53:213-223.
Black, JJ. 1983. Epidermal hyperplasia and neoplasia in brown bullheads (Ictalurus nebulosus} in response to re-
peated applications of a PAH containing extract of polluted river sediment. In: Polynuclear aromatic hydro-
carbons: Seventh International Symposium on Formation, Metabolism and Measurment. M.W. Cooke and
A.I Dennis (editors). Battelle Press, Columbus, OH, pp.99-111.
Blazer, V.S., R.E. Wolke, J. Brown and C.A. Powell. 1987. Piscine macrophage aggregate parameters as health
monitors: effect of age, sex, relative weight, season and site quality in largemouth bass (Micropterus sal-
moides). Aquat. Toxicol. 10:199-215.
Boorman, G.A., S. Botts, T.E. Bunton, J.W. Fournie, J.C. Harshbarger, W.E. Hawkins, D.E. Hinton, M.P. Jokinen,
M.S. Okihiro and M.J. Wolfe. 1997. Diagnostic criteria for degenerative, inflammatory, proliferative non-
neoplastic and neoplastic liver lesions in medaka (Oryzias latipes): consensus of a National Toxicology
Program Pathology Working Group. Toxicol. Pathol. 25:202-210.
Bowser, P.R., M.J. Wolfe, J. Reimer and B.S. Shane. 1991. Epizootic papillomas in brown bullheads, Ictalurus
nebulosus from Silver Stream reservoir, New York. Dis. Aquat. Org. 11:117-127.
Bunton, T.E. 1996. Experimental chemical carcinogenesis in fish. Toxicol. Pathol. 24:603-618.
Edwards, M.R. and W.A. Samsonoff 1977. Electron microscopic observations on virus-like particles of a catfish
papilloma. Proc. Annual Electron Micro. Soc. Amer. 35:394-395.
Feist, S.W., T. Lang, G.D. Stentiford and A. Kohler. 2004. Biological effects of contaminants: Use of liver pathol-
ogy of the European flatfish dab (Limanda limanda L.) and flounder (Platichthys Jlesus L.) for monitoring.
ICES Techniques in Marine Environmental Sciences No.38.
Fournie, J.W., J.K. Summers, L.A. Courtney, V.D. Engle and V.S. Blazer. 2001. Utility of splenic macrophage ag-
gregates as an indicator offish exposure to degraded environments. J. Aquat. Animal Health 13:105-116.
Field Manual for the Microscopic Diagnosis of Proliferative Liver and Skin Lesions in the Brown Bullhead
-------
Fournie, J.W., W.K. Vogelbein and R.M. Overstreet. 1987. Squamous cell carcinoma in the gulf menhaden,
Brevoortiapatronus Goode. I FishDis. 10:133-136.
Gardner G.R., RJ. Pruell and L.C. Folmar. 1989. A comparison of both neoplastic and non-neoplastic disorders of
winter flounder (Pseudopleuronectes americanus} from eight areas in New England. Mar. Environ. Res.
28:393-397.
Gimenez-Conti, I, A.D. Woodhead, 1C. Harshbarger, S. Kazianis, RB. Setlow, RS. Nairn andRB. Walter. 2001.
A proposed classification scheme for Xiphophoms melanomas based on histopathologic analyses. Mar. Bio-
technol. 3:S100-S106.
Grizzle, J.M., L. Bunkley-Williams and 1C. Harshbarger. 1995. Renal adenocarcinoma in Mozambique tilapia,
neurofibroma in goldfish, and osteosarcoma in channel catfish from a Puerto Rican hatchery. J. Aquat. Ani-
mal Health 7:178-183.
Grizzle, J.M., P. Melius and D.R. Strength. 1984. Papillomas on fish exposed to chlorinated wastewater effluent. J.
Natl. CancerInst. 73:1133-1142.
Grizzle, J.M. and W.A. Rogers. 1976. Anatomy and Histology of the Channel Catfish. Auburn University Agricul-
tural Experiment Station, Auburn, AL, 94 pp.
Grizzle J.M. and A. Thiyagarajah. 1988. Diethylnitrosamine-induced hepatic neoplasms in the fish Rivulus ocella-
tiis marmoratus. Dis. Aquat. Org. 5:39-50.
Hanjavanit, C. and M. Mulcahy. 2004. Squamous cell carcinoma in rudd Scardinius erythrophthalmus: histopathol-
ogy, ultrastructure, and transmission. Dis. Aquat. Org. 61:215-226.
Harshbarger, 1C. and IB. Clark. 1990. Epizootiology of neoplasms in bony fish of North America. Sci. Total Envi-
ron. 94:1-32.
Hawkins, W.E., W.W. Walker, J.S. Lytle, T.F. Lytle, and RM.Overstreet. 1990. Carcinogenic effects of some
polycyclic aromatic hydrocarbons on the Japanese medaka and guppy in waterborne exposures. Sci. Total
Environ. 94:155-167.
Hayes, M.A., I.R Smith, T.H Rushmore, T.L. Crane, C. Thorn, T.E. Kocal and H.W. Ferguson. 1990. Pathogene-
sis of skin and liver neoplasms in white suckers from industrially polluted areas in Lake Ontario. Sci. Total
Environ. 94:105-123.
Hendricks J.D., T.R Meyers and D.W. Shelton. 1984. Histological progression of hepatic neoplasia in rainbow
trout (Salmo gairdneri}. Natl. Cancer Inst. Monograph 65:321-336.
Hinton, D.E., J.A. Couch, S.J. Teh and L.A. Courtney. 1988. Cytological changes during the progression of neopla-
sia in selected fish species. Aquat. Toxicol. 11:77-112
Hinton, D.E. 1994. Cells, cellular responses, and their markers on chronic toxicity of fishes. In: Aquatic Toxicol-
ogy: Molecular, Biochemical and Cellular Perspectives. Malins, D.C. and G.K. Ostrander (eds.), Lewis
Publishers, Boca Raton, pp.207-239.
Hinton, D.E., H Segner and T. Braunbeck. 2001. Toxic responses of the liver. In: Target Organ Toxicity in Marine
and Freshwater Teleost. D. Schlenk and W.H Benson (eds.), Taylor & Francis, London, pp. 224-268.
International Joint Commission. 1989. Proposed Listing/Delisting Criteria for the Great Lakes Areas of Concern.
Focus on International Joint Commission Activities. Volume 14, Issue 1, insert.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
-------
Kim, J.C.S., E.S. Chao, M.P. Brown and R. Sloan. 1989. Pathology of brown bullhead, Ictalurus nebulosus, from
highly contaminated and relatively clean sections of the Hudson River. Bull. Environ. Contam. Toxicol.
43:144-150.
Kimura, I, N. Taniguchi, H. Kumai, I. Tomita, N. Kinae, K. Yoshizaki, M. Ito and T Ishikawa. 1984. Correlation
of epizootiological observations with experimental data: Chemical induction of chromatophoromas in the
cmaker,Nibeamitsukurii. Natl. Cancer Inst. Monogr. 65:139-154.
Kinae, N., M. Yamashita, I. Tomita, I. Kimura, H. Ishida, H. Kumai and G. Nakamura. 1990. A possible correlation
between environmental chemicals and pigment cell neoplasia in fish. Sci. Total Environ. 94:143-154.
Koehler, A. 2004. The gender-specific risk to liver toxicity and cancer of flounder (Platichthys flesus (L.)) at the
German Wadden Sea coast. Aquat. Toxicol. 70:257-276.
Law J.M., W.E. Hawkins, R.M. Overstreet, W.W. Walker. 1994. Hepatocarcinogenesis in western mosquitofish
(Gambusia affinis) exposed to methylazoxymethanol acetate. J. Comp. Pathol. 110:117-127.
Lee, S. and PJ. Whitfield. 1992. Virus-associated spawning papillomatosis in smelt, Osmerus eperianus L., in the
River Thames. J. FishBiol. 40:503-510.
Lucke, B. and H. Schlumberger. 1941. Transplantable epithelioma of the lip and mouth of catfish. I. Pathology.
Transplantation to anterior chamber of the eye and into the cornea. J. Exp. Med. 74:397-416.
Luna, L.G 1992. Histopathologic Methods and Color Atlas of Special Stains and Tissue Artifacts. American Histo-
labs, Inc.
MacCubbin, A.E. and N. Ersing. 1991. Tumors in fish from the Detroit river. Hydrobiol. 219:301-306.
Manera, M. and S. Biavati. 1999. Branchial osteogenetic neoplasm in barbel Barbus barbus plebejus. Dis. Aquat.
Org. 37:231-236.
Matsumoto, I, TJ. Lynch, S. Grabowski, C.M. Richards, S.L. Lo, C. Clark, D. Kern, J.D. Taylor and T.T. Tchen.
1983. Fish tumor pigment cells: differentiation and comparison to their normal counterparts. Amer. Zool.
23:569-580.
Metcalfe, C.D. 1998. Toxicopathic responses to organic compounds. In: Fish Diseases and Disorders, Volume 2:
Non-infectious Disorders. J.F. Leatherland and P.T.K Woo (eds.), CABI Publishing, Oxon, UK, pp. 133-
162.
Mikaelian, I, Y. de Lafontaine, P. Gagnon, C. Menard, Y. Richard, P. Dumont, L. Pelletier, Y. Mailhot and D.
Martineau. 2000. Prevalence of lip neoplasms of white sucker (Catostomus commersoni) in the St. Law-
rence River basin. Can. J. Fish. Aquat. Sci. 57 (Suppl. 1):174-181.
Myers M.S., IT. Landahl, M.K. Kahn, L.L. Johnson and B.B. McCain. 1990. Overview of studies on liver car-
cinogenesis in English sole from Puget Sound; Evidence for a xenobiotic chemical etiology I: Pathology
and epizootiology. Sci. Total Environ. 94:33-50.
Nairn, R.S., D.C. Morizot, S. Kazianis, A.D. Woodhead and R.B. Setlow. 1996. Nonmammalian models for
sunlight carcinogenesis: genetic analysis of melanoma formation in Xiphophorus hybrid fish. Photochem.
Photobiol. 64:440-448.
Okihiro, M.S., J.A. Whipple, J.M. Groff andD.E. Hinton. 1993. Chromatophoromas and chromatophore hyperpla-
sia in Pacific rockfish (Sebastes spp.). Can. Res. 53:1761-1769.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead 32
-------
Osburn, R.C. 1925. Black tumor of the catfish. Bull. Bur. Fish. 41:9-13.
Pereira, N.M. and M.C. Peleteiro. 2002. Osteoma in the skin of a tawny nurse shark, Nebrius ferrugineus (Lesson).
J. Fish Dis. 25:565-567.
Pinkney, A.E., 1C. Harshbarger, E.B. May and MJ. Melancon. 2001. Tumor prevalence and biomarkers of expo-
sure in brown bullhead (Ameiurus nebulosus) from the tidal Potomac river, USA, watershed. Environ. Toxi-
col. Chem. 20:1196-1205.
Pinkney, A.E., J.C. Harshbarger, E.B. May, and W.L. Reichert. 2004. Tumor prevalence and biomarkers of expo-
sure and response in brown bullhead (Ameiurus nebulosus} from the Anacostia river, Washington, DC and
Tuckahoe river, Maryland, USA. Environ. Toxicol. Chem. 323:638-647.
Poulet, P.M., J.W. Casey, J.M. Spitsbergen. 1993. Studies on the transmissibility and etiology of orocutaneous tu-
mors of brown bullheads Ictalurus nebulosus. Dis. Aquat. Org. 16:97-104.
Poulet, P.M. and J.M. Spitsbergen. 1996. Ultrastructural study of spontaneous orocutaneous papillomas of brown
bullheads Ictalurus nebulosus. Dis. Aquat. Org. 24:93-98.
Poulet, P.M., MJ. Wolfe and J.M. Spitsbergen. 1994. Naturally occurring orocutaneous papillomas and carcinomas
of brown bullheads (Ictalurus nebulosus} in New York State. Vet. Pathol. 31:8-18.
Profet, E.B., B. Mills, J.B. Arrington and L.H. Sobin. 1992. Laboratory Methods in Histotechnology. Armed Forces
Institute of Pathology, Registry of Pathology, Washington, DC.
Pyron M., E.G. Obert and R. Wellington. 2001. Tumor rates and population estimates of brown bullhead (Ameiurus
nebulosus} in Presque Isle Bay, Lake Erie. J. Great Lakes Res. 27:185-190.
Quackenbush, S.A., J. Rovnak, RN. Casey, T.A. Paul, P.R Bowser, C. Sutton and J.W. Casey. 2001. Genetic rela-
tionship of tumor-associated piscine retroviruses. Mar. Biotechnol. 3: S88-S99.
Rafferty, S. and J. Grazio. 2007. Field Manual for Assessing Internal and External Anomalies in Brown Bullhead
(Ameiurus nebulosus).
Sakamoto, K. and M.R. White. 2002. Dermal melanoma with schwannoma-like differentiation in a brown bullhead
catfish (Ictalurus nebulosus}. J. Vet. Diag. Invest. 14:247-250.
Schartl, M. 1995. Platyfish and swordtails: a genetic system for the analysis of molecular mechanisms in tumor for-
mation. Trends Genet. 11:183-189.
Setlow, R.B., A.D. Woodhead and E. Grist. 1989. Animal model for ultraviolet radiation-induced melanoma: platy-
fish-swordtail hybrid. Proc. Natl. Acad. Sci. USA 86:8922-8926.
Smith, I.R., H. Ferguson and M.A. Hayes. 1989. Histopathology and prevalence of epidermal papillomas epidemic
in brown bullhead, Ictalurus nebulosus (Lesueur), and white sucker, Catostomus commersoni (Lacepede),
populations from Ontario, Canada. J. Fish Dis. 12:373-388.
Sonstegard, R.A. 1977. Environmental carcinogenesis studies in fishes of the Great Lakes of North America. Ann.
NY. Acad. Sci. 298:261-269.
Spitsbergen, J.M. and MJ. Wolfe. 1995. The riddle of hepatic neoplasia in brown bullheads from relatively unpol-
luted waters in New York state. Toxicol. Pathol. 23:716-725.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
-------
Stme, C.B., D.L. Smith, W.K. Vogelbem, 1C. Harshbarger, P.R. Gudla, M.M. Lipsky and A.S. Kane. 2004. Mor-
phometry of hepatic neoplasms and altered foci in the mummichog, Fundulus heteroclitus. Toxicol. Pathol.
32:375-383.
Vogelbein, W.K., J.W. Fournie, P.A.Van Veld and RJ. Huggett. 1990. Hepatic neoplasms in the mummichog Fun-
dulus heteroclitus from a creosote-contaminated site. Cancer Res. 50: 5978-5986.
Wolf, J.C. and MJ. Wolfe. 2005. A brief overview of nonneoplastic hepatic toxicity in fish. Toxicol. Pathol. 33:75-
85.
Wolke, R.E. 1992. Piscine macrophage aggregates: a review. Ann. Rev. Fish Dis. 2:91-108.
Yamamoto, T., R.K. Kelly and O. Nielsen. 1985. Morphological differentiation of virus-associated skin tumors of
walleye (Stizostedion vitreum vitreum). Fish Pathol. 20:361-372.
Field Manual for the Microscopic Diagnosis ofProliferative Liver and Skin Lesions in the Brown Bullhead
------- |