Journal of Fish Diseases 2005, 28, 13–22
Vaccine-associated granulomatous inflammation and
melanin accumulation in Atlantic salmon, Salmo salar L.,
white muscle
E O Koppang1, E Haugarvoll1, I Hordvik2, L Aune1 and T T Poppe1
1 Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, Oslo, Norway
2 Department of Fisheries and Marine Biology, HiB, University of Bergen, Norway
Abstract
The purpose of this study was to investigate the
nature of variably sized pigmented foci encountered
in fillets of farmed Atlantic salmon, Salmo salar L.
The material was sampled on the fillet production
line and on salmon farms from fish with an average
size of 3 kg from various producers. The fish had
been routinely vaccinated by injection. Gross
pathology, histology, immunohistochemistry using
antisera against major histocompatibility complex
(MHC) class II b chain and transmission electron
microscopy (TEM) were used to characterize the
changes. Macroscopically, melanized foci were seen
penetrating from the peritoneum deep into the
abdominal wall, sometimes right through to the
skin, and also embedded in the caudal musculature.
Histological investigation revealed muscle degener-
ation and necrosis, fibrosis and granulomatous
inflammation containing varying numbers of
melano-macrophages. Vacuoles, either empty or
containing heterogeneous material, were frequently
seen. The presence of abundant MHC class II+
cells indicated an active inflammatory condition.
TEM showed large extracellular vacuoles and
leucocytes containing homogeneous material of
lipid-like appearance. The results showed that the
melanized foci in Atlantic salmon fillet resulted
from an inflammatory condition probably induced
by vaccination. The described condition is not
known in wild salmon and in farmed salmon where
injection vaccination is not applied.
Correspondence Trygve T Poppe, Department of Basic Sci-
ences and Aquatic Medicine, Norwegian School of Veterinary
Science, Ullevålsveien 72, Box 8146 Dep., 0033 Oslo, Norway
(e-mail: trygve.poppe@veths.no)
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Keywords: Atlantic salmon, inflammation, melano-
macrophage, major histocompatibility complex
class II, mineral oil, vaccine.
Introduction
Various pathological conditions may be associated
with abnormal pigmentation in tissues and organs.
Such pigments may either be of exogenous or
endogenous origin. Endogenous pigments include
derivates of lipids, haemoglobin, porphyrins and
melanin. The term melanosis is used to describe
the presence of melanin in abnormal locations
(Thomson 1984). In vertebrates, melanin is syn-
thesized by melanocytes and organized in melano-
somes, which are lysosome-related intracellular
organelles (Orlow 1995; Raposo, Fevrier, Stoorvo-
gel & Marks 2002). Mammalian melanocytes
originate from the embryonic neural tube (Sulai-
mon & Kitchell 2003) and it has been observed
that such cells can migrate into inflamed tissue
(Thomson 1984).
Inflammatory reactions and tissue regeneration
in salmonids seem similar to those of mammals
(Finn & Nielson 1971), but have in addition been
associated with the involvement of so-called
melano-macrophages (Roberts 1975; Agius &
Roberts 2003). The origin of melanosomes in
melanin-containing viscerally located cells in fish is
not clear (Agius & Roberts 2003), but Sichel,
Scalia, Mondio & Corsaro (1997) suggested that
melanogenesis in poikilothermic vertebrates may
occur in mesenchyme-derived cells of the haema-
topoietic lineage. Although teleost melano-macro-
phages have been ascribed macrophage-like
properties, their functions and significance are
 Journal of Fish Diseases 2005, 28, 13–22
not well understood (Agius & Roberts 2003). It
has been speculated that the synthesis and actions
of melanin in various cell populations are
associated with non-specific immune mechanisms,
which is an appealing hypothesis that could explain
its presence in visceral organs and in the
peritoneum of poikilothermic vertebrates (Mack-
intosh 2001).
Most injectable vaccines used in fish farming are
based on or contain non-metabolizable mineral oils
which induce stronger inflammatory reactions than
metabolizable oils (Spickler & Roth 2003). Mineral
oils are long chain hydrocarbons that are extremely
resistant to chemical and biochemical reactions.
Normally, they are residues from petroleum
distillation (Murray, Cohen & Hardegree 1972).
Adverse effects of mineral oil-adjuvanted vaccines
have been well documented in various animals.
Injections with water-in-oil emulsions, or Freund’s
incomplete adjuvant have caused distribution of
mineral oils to different locations and induced
granulomatous inflammatory responses. The sever-
ity of these responses has made oil-adjuvanted
vaccines generally unacceptable for applications in
man (Gupta, Relyveld, Lindblad, Bizzini, Ben-
Efraim & Gupta 1993; Spickler & Roth 2003).
Recent investigations have also shown that oil
adjuvants may induce autoimmunity and immune
complex-mediated glomerulonephritis (Shaheen,
Satoh, Richards, Yoshida, Shaw, Jennette & Reeves
1999; Satoh, Kuroda, Yoshida, Behney, Mizutani,
Akaogi, Nacionales, Lorenson, Rosenbauer &
Reeves 2003; Kuroda, Akaogi, Nacionales, Wasdo,
Szabo, Reeves & Satoh 2004). Studies addressing
such effects of mineral oil-adjuvanted vaccines have
not been reported in fish, but localized inflamma-
tory reactions within the abdominal cavity have
been described (Lillehaug, Lunder & Poppe 1992;
Midtlyng 1996; Poppe & Breck 1997; Mutoloki,
Alexandersen & Evensen 2004). The most common
side-effects have been granulomatous peritonitis
with melanization and adhesions between visceral
organs and the peritoneum, retarded growth, and
consequent downgrading during processing. These
side-effects have caused considerable concern, partly
because of the reduced quality of the carcass and
partly because of animal welfare considerations
(Poppe, Barnes & Midtlyng 2002). Recent inves-
tigations indicate that pathological vaccine-induced
reactions and changes in fish may not only be
confined to the structures of the abdominal cavity,
but also, as has been reported in mammals, affect
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E O Koppang et al. Vaccine-associated pathology in salmon muscle
other organs such as the eyes (Koppang, Bjerkås,
Bjerkås, Sveier & Hordvik 2003; Koppang, Hau-
garvoll, Hordvik, Poppe & Bjerkås 2004).
According to industry representatives, pigmented
foci in Atlantic salmon, Salmo salar L., musculature
have occurred sporadically over many years, but the
problem appears to be rapidly escalating and has
become a serious quality problem for the entire
salmon industry. Particular concern is related to
cassations, downgrading, extra work and loss when
trimming the fillets. The purpose of this study was
to clarify the nature of the pigmented areas in the
musculature of farmed Atlantic salmon.
Materials and methods
Fish
The material was collected from farmed Atlantic
salmon from several producers on the fillet
production line over a 2-day period. The salmon
were eviscerated before arriving at the processing
factory (Fig. 1), precluding any investigations of
internal organs. The fish were about 3 kg, trans-
ported on ice and received for further processing
within 24 h post-mortem. In addition, fish from
various farms were examined and autopsy and
sampling performed on site.
Sampling
Pigmented areas in the fillet (Fig. 2) were inspected,
dissected and fixed in 10% neutral buffered forma-
lin for 24 h for light microscopy and immunohist-
ochemistry. Blocks of pigmented muscle, 1 mm3,
were fixed in 2.5% glutaraldehyde in 0.5 m caco-
dylate buffer for transmission electron microscopy
(TEM).
Light microscopy
Material for light microscopy was processed rou-
tinely, paraffin embedded, sectioned (4 lm),
mounted on glass slides and stained with haema-
toxylin and eosin (H&E), elastin–van Gieson
(EVG) for elastin and fibrin identification and oil
red O for lipids. To identify the presence of possible
microorganisms, slides were Gram-stained for bac-
teria, Ziel–Neelson-stained for mycobacteria and
periodic acid-Schiff (PAS)-stained for fungi. All
staining techniques followed standard procedures
(Bancroft & Gamble 2002).
 Journal of Fish Diseases 2005, 28, 13–22 E O Koppang et al. Vaccine-associated pathology in salmon muscle

Figure 1 Gross pathological changes in the carcass of an Atlantic salmon. The pericardial cavity (a) is normal, but severe melanization is
apparent in the abdominal cavity (b). Melanized musculature subjacent to the peritoneum is seen on the cut surface (arrow).
Figure 2 A melanized area in the musculature of an Atlantic salmon. The peritoneum is removed and darker foci are seen in a dark to
grey area involving five myosepta. The lesion is situated laterally in the fish, covering the area of the lateral organ. Note the contraction in
the musculature, disrupting the curves of the intramuscular septa.

Hordvik, Bjerkås, Torvund, Aune, Thevarajan &

Immunohistochemistry
Endresen 2003). Negative controls used rabbit
Sections were subjected to immunohistochemistry pre-immune serum. Tissue sections were de-waxed
using polyclonal antisera against major histocom- in xylene and rehydrated in graded ethanol baths.
patibility complex (MHC) class II (Koppang, To unmask MHC class II epitopes, the sections
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 Journal of Fish Diseases 2005, 28, 13–22
were subsequently autoclaved in a solution of
0.01 m citrate buffer, pH 6.0, and kept at 120 °C
for 15 min, cooled to room temperature and then
transferred to PBS.
To prevent non-specific binding, the sections
were incubated for 20 min in normal caprine serum
diluted 1:50 in 5% bovine serum albumin (BSA) in
Tris-buffer solution (TBS), 0.05 m Tris-HCl, pH
7.6, diluted 1:10 in 0.9% NaCl (TBS) before
incubation overnight at 4 °C with pre- or post-
immune isolated sera, diluted 1:1000 in 1% BSA in
TBS. The following morning, the sections were
washed in PBS and incubated at room temperature
for 30 min with biotinylated goat anti-rabbit
immunoglobulin G (IgG) (Vector Laboratories,
Burlingame, CA, USA) diluted 1:200 in 1% BSA in
TBS. The sections were subsequently washed in
PBS.
To inhibit endogenous peroxidase activity, the
sections were treated with 3% H2O2 in methanol
for 10 min and then washed in PBS 0.01 m, pH
7.3. The inhibition step was followed by incuba-
tion for 30–35 min in avidin–biotin complex/
horseradish peroxidase (ABC/HRP complex; PK-
4000; Vector Laboratories) according to the manu-
facturer’s instructions. After washing in PBS,
bound antibody was detected using 3-amino-9-
ethyl-carbazole (AEC) (Sigma Chemical Company,
St Louis, MO, USA) for 17 min at room tem-
perature.
The sections were washed in distilled water and
either left unstained or counterstained with haem-
atoxylin for 45 s, washed in distilled water and
mounted with polyvinyl alcohol mounting med-
ium, pH 8.2, (Ullevål Apotek, Oslo, Norway) for
light microscopic analysis. Sections treated with
pre-immune rabbit antiserum served as negative
controls in the experiments.
Semi-thin sections/transmission electron
microscopy
The glutaraldehyde-fixed material was post-fixed in
2% OsO4 in 0.1 m cacodylate buffer, pH 7.2 for
2 h, dehydrated in ascending concentrations of
ethanol and embedded in LX 112 Resin (Ladd
Research Industries, Williston, VT, USA). Semi-
thin (1 lm) sections were cut, mounted on glass
slides and stained with toluidine blue. Ultra-thin
sections were mounted on uncoated copper grids,
stained with uranyl acetate and lead citrate using
standard methodology and examined in a TEM.
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E O Koppang et al. Vaccine-associated pathology in salmon muscle
Results
Gross pathology
Remnants of adhesions between visceral organs and
the body wall were evident in many fillets, partic-
ularly near the dorsal part of the septum transver-
sum. Many of these adhesions were also heavily
pigmented (Fig. 1). At inspection, approximately
20% of the fillets on the production line showed grey
or black foci in the musculature (Figs 1 & 2). The
changes were typically seen in the peritoneum in the
hepatic region, in the area of the lateral line organ,
appearing to be bilaterally distributed in the fish.
Some foci were also recognized in the musculature
caudal to the abdominal cavity. Generally, pigmen-
ted areas varied in diameter from pinhead size up to
4–5 cm and could involve 2–5 myosepta (Fig. 2).
Black and grey foci were in most cases surrounded by
light grey or whitish patches in the myosepta
(Fig. 2). Both myosepta and intermyotomal septa
were involved in varying degrees of scar retraction
(Fig. 2). Upon incision, some foci were superficial
and involved only the peritoneum and subjacent
connective tissue, while others extended deep into
the musculature including the intermyotomal septa,
and even affected the skin on the other side of the
fillet. On palpation, these changes were frequently
indurated with an increased texture. The smaller foci
were typically dark grey or black, while larger
changes were diffusely light or bluish grey (Fig. 2).
In fish inspected at various fish farms, the muscle
changes were invariably associated with peritonitis.
Light microscopy
Microscopy of discoloured areas revealed extensive
infiltration of mononuclear leucocyte-like cells
between the muscle cells, which were extensively
and severely degenerated or necrotic (Fig. 3).
Granulomas in the intermyotomal septa were
frequent and contained melano-macrophages, epi-
thelioid-like cells, multinucleated giant cells
(MGCs) of the Langhan’s type and encapsulated
vesicles with crystal-like content (Fig. 4). Two
kinds of large vesicles were seen embedded in the
muscle. One form was filled with macrophage-like
cells, debris and small droplets containing a homo-
geneous material. Occasional haemorrhage was seen
within these structures (Fig. 5). The other form of
vesicle was round to oval and empty (Fig. 6). Both
forms were surrounded by epithelioid-like cells and
fibrogranulomatous tissue, which was revealed using
 Journal of Fish Diseases 2005, 28, 13–22
Figure 3 Histological image of affected musculature in a
melanized area as seen in Fig. 2. Myocytes showing various
degrees of degeneration are seen. One myocyte is totally replaced
by mononuclear inflammatory-like cells (arrows) (H&E,
bar ¼ 100 lm).
EVG staining (Fig. 7). The filled forms contained
cells that were oil red O positive, as was the
homogeneous material within them, although it
stained more weakly (Fig. 8). Both forms were
frequently coated with flattened melanosome-con-
taining cells. Occasionally, Langhan’s giant cells
were seen adjacent to the deposits (Fig. 9).
Apart from these vesicles, melano-macrophages
were present in varying degrees throughout the
lesions. There was no positive oil red O reaction
outside the filled vesicles, but EVG staining was
positive and showed that extensive fibrinogenesis
had taken place in the affected tissue. The lesions
were dominated by fibroblasts, macrophage-like
cells, epithelioid cells and melano-macrophages,
changes consistent with granulomatous inflamma-
tion. Gram, Ziel–Neelson and PAS staining failed
to reveal bacteria or fungi.
Immunohistochemistry
Large proportions of macrophage-like and epithe-
lioid-like cells surrounding the muscle cells were
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E O Koppang et al. Vaccine-associated pathology in salmon muscle
Figure 4 Granuloma in an intermyotomal septa. The granuloma
consists of foci containing a crystallogenous matrix surrounded
by epithelioid-like cells and melano-macrophages. A multinucle-
ated giant cell is seen (arrow) (H&E, bar ¼ 50 lm).
MHC class II+ (Fig. 10). In most cases, the empty
vacuoles were lined with MHC class II+ cells,
frequently thin, elongated melano-macrophages.
Aggregates of MHC class II+ cells were seen in
the intermyotomal septa (not illustrated).
Semi-thin sections/transmission electron
microscopy
Semi-thin toluidine blue-stained sections revealed
large masses of a yellow-greenish-stained homo-
geneous substance within large and smaller vesi-
cles embedded in the musculature. This
colouration concurs with that seen when fat-
containing material is similarly treated. However,
morphologically the substance was not confined
to lipid cells, but was intimately surrounded by
fibrogranulomatous tissue (Fig. 11). TEM
revealed the presence of large droplets of a
homogeneous material surrounded by flattened
cells frequently containing melanosomes (Fig. 12)
and small droplets within phagocytic compart-
ments of macrophage-like cells (Fig. 13). Other-
wise, typical epithelioid cells were detected
 Journal of Fish Diseases 2005, 28, 13–22 E O Koppang et al. Vaccine-associated pathology in salmon muscle

Figure 5 A large vesicle embedded in an intermyotomal septum containing macrophage-like cells, debris and a fresh haemorrhage
(arrow) (H&E, bar ¼ 500 lm).
Figure 6 Empty vesicles surrounded by granulomatous tissue embedded in the white musculature. Note adjacent, seemingly unaffected
muscle cells (H&E, bar ¼ 200 lm).
Figure 7 Vesicles embedded in the white musculature surrounded by fibrogranulomatous tissue (red staining) (EVG, bar ¼ 500 lm).
Figure 8 Reaction against oil (red staining) in a vesicle as shown in Fig. 5. Homogeneous masses (asterisk) and macrophage-like cells
show positive reactions. Note the melano-macrophages in the vesicle wall (arrows) (oil red O, bar ¼ 50 lm).
Figure 9 High magnification of the wall of a vesicle as seen in Fig. 6. The wall contains a multinucleated giant cell (MGC)(arrow),
epithelioid-like cells, small vacuoles and is lined towards the lumen of the greater vesicle with melanosome-containing cells, probably
swollen melano-macrophages (H&E, bar ¼ 40 lm).
Figure 10 Muscle cells infiltrated with MHC class II+ cells. One muscle cell is unaffected (asterisk). One fibre shows severe
degeneration (arrowhead), whereas one is invaded by MHC class II+ cells (red reaction) (MHC class II immunostain, haematoxylin
counterstain, bar ¼ 100 lm).

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 Journal of Fish Diseases 2005, 28, 13–22
Figure 11 Semi-thin section of red musculature. Masses of unorganized lipid droplets (green-yellow colour) are seen surrounded by
granulomatous tissue with bundles of fibrin (dark-red colour). Muscle cells are visible at the upper left corner of the image (toluidine
blue, bar ¼ 20 lm).
between muscle cells. Microorganisms were not
detected in any samples investigated by TEM.
Discussion
In the present study, we have shown that the
frequently reported pigmented changes in the white
muscle of vaccinated Atlantic salmon can be
classified as a granulomatous inflammatory condi-
tion similar to that of the foreign-body type
containing macrophage-like cells, epithelioid cells,
MGCs and melano-macrophages. The absence of
known pathogens or other explanations leaves
intraperitoneal vaccination the most probable caus-
ative aetiological factor for these changes. The
majority of the granulomatous foci were located
adjacent to the peritoneal cavity, but not in the
ventral midline, where the vaccine had been
injected. Our findings showed that accumulations
of oil droplets were present in the musculature
associated with the lesions. These oil droplets were
associated with extensive granulomatous processes
and were coated with MHC class II+ cells. Based
on previous studies on the distribution of, and
inflammatory reactions against, mineral oil-adjuv-
anted vaccines in mammals (Gupta et al. 1993), we
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E O Koppang et al. Vaccine-associated pathology in salmon muscle
believe the pigmented changes encountered in
salmon fillets are a consequence of vaccine local-
ization followed by a foreign body reaction. The
greyish to black appearance of the changes was a
reflection of the accumulation of melano-macro-
phages. Processing for TEM leaves lipids in the
tissue, and because of the lack of organization in
lipid cells, their large size and the intimate presence
of granulomatous tissue, we interpreted the yellow-
greenish masses in semi-thin toluidine blue stained
sections as oil adjuvant. Further, we interpreted the
debris-containing vesicles as structures in the pro-
cess of organization, containing residues of mineral
oil.
Toxic side-effects of adjuvants are problems that
are continuously debated. In production animals,
water-in-oil vaccines are used only when a strong
adjuvant is needed and inflammation is not a
drawback (Spickler & Roth 2003). The major
concerns are the ability of mineral oil-adjuvanted
vaccines to form granulomas both at the injection
site and elsewhere, and the induction of auto-
immune reactions.
To the best of our knowledge, there are no
published reports on the distribution and pharma-
cokinetics of injected mineral oils in fish. In
 Journal of Fish Diseases 2005, 28, 13–22
Figure 12 Ultra-thin section of a vesicle filled with homogeneous
material that appears as lipid surrounded by dendrites from
melano-macrophages (uranyl acetate and lead citrate,
bar ¼ 1 lm).
poultry, Franchini, Piretti, Tubertini, Govoni &
Sapigini (1984) discovered rapid diffusion to all
tissues following hydrocarbon injection, and espe-
cially to those with rich vascularization. The
hydrocarbons were eliminated from the hens
through their eggs. In non-laying hens, elimination
from the body was extremely slow. However,
hydrocarbons injected in one leg could not be
detected in the other leg of the same animal (Piretti,
Franchini & Zanetello 1982). In rats, experiments
have shown that injected radio-labelled hydrocar-
bons are finally distributed to the liver, and most
prominently to the fat tissue (Bollinger 1970). In
this context, it is worth noting that salmonids store
lipids in their red and white musculature. Although
discussed (Kuroda et al. 2004), the exposure to
hydrocarbons from food seems not to represent any
health risk to humans (Nash, Gettings, Diembeck,
Chudowski & Kraus 1996).
To determine the chemical composition of the
oil droplets in the melanized foci would require the
use of gas chromatography/mass spectrometry
(Kuroda et al. 2004). Together with pharmacolo-
gical studies using radio-labelled hydrocarbons for
distribution studies, this is an obvious task for the
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E O Koppang et al. Vaccine-associated pathology in salmon muscle
Figure 13 Ultra-thin section of a cell containing multiple small
vesicles filled with lipid-like material (uranyl acetate and lead
citrate, bar ¼ 300 nm).
vaccine producers, and it is both peculiar and
disturbing that such information is not at hand
from the industry.
Compared with mammals, inflammatory reac-
tions in teleosts have been little studied and are
poorly understood. A fundamental problem in fish
pathology is the lack of specific cell markers. Only
the use of such markers can justifiably define
specific leucocyte populations.
In this report, we have used the term macro-
phage-like cells instead of macrophages. It is not
uncommon for fish pathologists to classify cells as
ÔmacrophagesÕ based purely on morphological
characteristics. However, when T and B cells
undergo blast formation they are impossible to
distinguish morphologically from macrophages
unless specific markers are used (Manjo & Joris
1996). In homeothermic animal anatomy, a
number of different criteria have to be fulfilled
to justify any classification. The principal charac-
ters used to identify macrophages are their
morphology, the presence of non-specific esterase
and endogenous peroxidase activities, pinocytic
and phagocytic abilities, the presence of mem-
brane receptors for the Fc region of IgG and for
 Journal of Fish Diseases 2005, 28, 13–22
complement, and their stainability with specific
monoclonal antibodies. It is, however, sufficient to
recognize a minimum of three of these features to
define a cell as a macrophage (Leenen & Camp-
bell 1993). These requirements can presently not
be met using standard histological and histochem-
ical methods. However, the use of MHC class II
antisera enabled us to demonstrate an immune
induction (Glimcher & Kara 1992) in cells
surrounding vacuoles, muscle fibres and in
degenerated myocytes, and based on the assump-
tion that inflammatory reactions follow the same
cellular sequences as in mammals (Finn &
Nielson 1971), it is likely that most of the
MHC class II+ cells were macrophages and
stimulated epithelioid cells. We have previously
published similar reactions in the uvea of Atlantic
salmon suffering from a severe post-vaccine con-
dition (Koppang et al. 2004).
In chronic inflammations, recruited macrophages
may differentiate into epithelioid cells and MGCs,
which are the hallmarks of granulomas. These cells
may be MHC class II+, depending on their age and
stimulation (Izaki, Tanji, Okuma, Shimoda, Hsu-
Oyama, Hibino & Kitamura 1991). As such cells
were present in the described lesions, they should be
classified as granulomatous.
White muscle pigmentation in farmed salmon
has been reported in vaccinated fish from British
Columbia, Canada (S. Raverty, personal commu-
nication), Scotland (H. W. Ferguson, personal
communication) and Chile (T.T. Poppe, unpub-
lished observations), but not in Tasmania,
Australia, where salmon are not subjected to
intraperitoneal vaccination (B. Nowak, personal
communication), and not in wild fish. Together
with this information, our results strongly suggest
that macrocomponents of the vaccine are dis-
seminated from the injection site throughout the
body to different organs and tissues, where foreign
body inflammatory reactions are initiated. With a
lesion prevalence of 10–30% as reported by
several producers, this is not a question of
inaccurate placement of the vaccine injection,
but a generalized, unpredictable and highly unat-
tractive side-effect of vaccination probably because
of the use of mineral oil adjuvants.
Acknowledgement
The authors thank The Professor of Pathology at
the Dental Faculty, University of Oslo, Hanna S.
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E O Koppang et al. Vaccine-associated pathology in salmon muscle
Koppang, for discussion of the results and critical
reading of the manuscript.
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Received: 14 June 2004
Revision received: 18 August 2004
Accepted: 19 August 2004