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Acquired Melanosis Caused by Acorn Ingestion in the Nero Siciliano Pig

G. Lanteri, F. Marino, G. Laganà, E. Bellocco, D. Barreca, L. Liotta, A. Sfacteria and B. Macrì
Vet Pathol 2009 46: 329
DOI: 10.1354/vp.46-2-329

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 ENVIRONMENTAL PATHOBIOLOGY
Vet Pathol 46:329–333 (2009)

Acquired Melanosis Caused by Acorn Ingestion in the

Nero Siciliano Pig
G. LANTERI, F. MARINO, G. LAGANÀ, E. BELLOCCO, D. BARRECA, L. LIOTTA, A. SFACTERIA, AND B. MACRÌ
Dipartimento Sanità Pubblica Veterinaria (GL, FM, AS, BM), Chimica Organica e Biologica
(GL, EB, DB), and MOBIFIPA (LL), Università degli Studi di Messina, Polo Universitario
dell’Annunziata, 98168 Messina, Italy

Abstract. In this study, an acquired pigmentation in Nero Siciliano pigs is reported and evaluated by
a multidisciplinary approach to support the hypothesis it is caused by an ingested material. A total of 18
pigs were studied. Fourteen conventionally slaughtered animals showed black discoloration of lymph
nodes. The lymph nodes were normal in size and shape but showed diffuse black discoloration of the
cortex and medulla. Melanosis of fat was observed in 2 animals and was limited to the back.
Histochemical tests performed on tissues enabled identification and differentiation of the pigment.
Immunohistochemical staining for macrophage markers showed macrophages containing a variable
amount of melanin-like granules. Stains for human melanoma, as well as S-100 protein, did not show
any reaction. Histochemical methods for tyrosinase showed colorimetric patterns that confirmed the
presence of the enzyme in acorns. The activity was mostly latent. A high tannin content was
demonstrated, reaching about 76% of the total phenolic compounds. Our data, and the well-known steps
on melanin formation, permit us to hypothesize that swine tyrosinase could act on phenolic substances
found in acorns. Tyrosinase activation could take place in genetically predisposed swine after acorns are
eaten, and this event could increase the biosynthesis and the anomalous storage of melanin.

Key words: Acorn; immunohistochemistry; melanosis; Nero Siciliano pigs; pathogenesis.

Introduction discoloration of tissue by iron sulfide formed by

Melanosis is a disorder in which anomalous
storage of melanin occurs. It has been reported in
domestic animals, but rarely described in swine as a
generalized pigmentation23 or as a localized disor-
der of the mammary gland,5 thymus, and retro
pharyngeal lymph nodes.32 Granules of melanin are
produced by melanocytes, which can release them
to melanophages, and these cells can store the
pigment and carry it to distant sites. Abnormal
storage of melanin can be caused by modification
of the processes of production or transport. The
term ‘‘melanosis’’ is used for a group of conditions
characterized by an abnormal black or brownish-
black pigmentation of an organ or tissue. It does
not confirm that the pigment is melanin. For
example, in melanosis coli the pigment granules
contain lipofuscin, in melanosis ilei the pigment
granules may contain hemosiderin, and in melano-
sis esophagi the pigment is melanin.26 The term
‘‘pseudomelanosis’’ is used for the blue-green
329
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the reaction of hydrogen sulfide generated by
putrefactive bacteria on iron from hemoglobin
released from lysed erythrocytes; it usually takes a
day or more to develop after the death. Pseudo-
melanosis, associated with dietary compounds, has
rarely been reported in swine6 and guinea pigs.16
These pigmentations could have economic impor-
tance affecting carcass quality, decreasing consum-
er appeal and the marketability of the pigs, but they
do not pose a health risk for the consumer. ‘‘True’’
melanosis in swine is usually congenital, and in
some breeds, such as Sinclair, Duroc-Jersey,
Hormel, Vietnamese pot-bellied and their cross-
breeds, it has been be linked to tumors.7,11,21,27
Awareness of the pathogenesis of melanosis will aid
its differentiation from melanoma, lipofuscinosis,
hemosiderosis, and ceroid pigmentation.
The Nero Siciliano pig, native to rural areas in
north Sicily, Italy, lives freely in the woods of the
Nebrodi mountains (maximum altitude 1,800 me-
ters above sea level). This pig is found in fossil
330 Lanteri, Marino, Laganà, Bellocco, Barreca, Liotta, Sfacteria, and Macrı̀
remains and in writings from the Greek period
(VIII–VI BCE). The breed shows good adaptation
to the often unfavorable environmental conditions
and lives on undergrowth products (roots, tubers,
acorns, uncultivated fruits, etc.). Exploitation of
free living native breeds not only provides high-
quality meat products and limits imports. The
addition of these production could allow wider and
more rational utilization and recognition of the
value of marginal land.8
As yet, little is known of the prevalence and
incidence of diseases in such breeds.15,25 In this
study, an abnormal pigmentation occurring occa-
sionally in Nero Siciliano pigs is reported and
evaluated by a multidisciplinary approach to
support the hypothesis that the cause is related to
ingested materials.
Materials and Methods
Over a 3-year period, a total of 18 Nero Siciliano pigs
of heterogeneous genetic background were studied.
Thirteen animals (13), conventionally slaughtered in
winter 2005, after containment for 3 months in movable
shelters while fed on acorns, showed black discoloration
of all the lymph nodes. In 2 of these pigs, pigmentation
also involved the subcutaneous fat in the lumbar region.
Five pigs comprising a second group were slaughtered at
the same age (12 months) as the first group, in late
spring 2007, when acorns are less available or no longer
available at all. These pigs, belonging to the same breed,
coming from the same farm, and fattened in the same
place, were examined postmortem at the slaughterhouse.
Only 1 subject of the second group had black
pigmentation, localized in the retro-pharyngeal lymph
nodes.
Lymph nodes and fat tissue samples were collected
both unfixed and fixed in 10% buffered formalin. Serial
5-mm cryostat sections were stained with hematoxylin
and eosin (HE), and deparaffinized sections were stained
using HE, Nile blue, Millon and Lillie stains, and
decolorized with hydrogen peroxide and melanin bleach.
Retro-pharyngeal lymph node samples collected from 5
subjects, belonging to the first group, with melanosis,
were also fixed in a 10% buffered formalin solution and
routinely processed for paraffin embedding and sec-
tioned at 5 mm. The following immunohistochemical
stains were applied using the Avidin-biotin complex
technique: macrophage marker (Novocastra, Newcastle
Upon Tyne, UK; dilution 1 : 100), mouse monoclonal
antibody with specificity toward macrophages, mono-
cytes and histiocytes, monoclonal mouse anti-human
melanoma antibody (Dako, Glostrup, Denmark; dilu-
tion 1 : 25, 1 : 50, 1 : 100, 1 : 200),9 S-100 (Dako; dilution
1 : 1,000), and a calcium-binding protein detected in
almost all benign nevi and malignant melanocytic
tumors of the skin. Additionally, the Millon reaction28
was carried out on lymph node sections to show
tyrosine. Biochemical and histochemical evaluations
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Vet Pathol 46:2, 2009
Fig. 1. Acorns collected in the Nebrodi area used to
feed Nero Siciliano pigs during fattening. Bar 5 1 cm.
were carried out on acorns (Fig. 1) collected in the
forest area where the pigs were fattened. The presence of
tyrosinase was shown by qualitative and quantitative
assays. For the qualitative methods, histochemistry was
performed on cryostat sections of acorns, and the Okun
method20 was used in an assay on extracted acorn liquid.
Quantitative determination of tyrosinase extracted from
acorns was performed by spectrophotometric analysis
using catechol as a substrate.31 The activity of tyrosinase
in the crude extract was determined in triplicate by
melanin-like pigments formed in the polymerization of
quinone. The reaction mixture, in a final volume of
1.0 ml, contained 10 mM catechol and 20 ml enzyme
preparation in 50 mM phosphate buffer (pH 7.0). One
activity unit (U) is defined as the amount of enzyme that
causes an increase of absorbance at 410 nm of 0.001 per
minute at pH 7.0 at 25uC. A total phenolic compounds
analysis was carried out on a methanolic acorn extract
by the Folin-Ciocalteau reagent30; the data were
expressed as gallic acid equivalents. Tannin content
was monitored using binding properties of polyvinyl
polypyrrolidone.24 All values were expressed as a mean
of three replications 6 SD.
Results
At macroscopic evaluation, the lymph nodes
were normal in size and shape, and showed a
diffuse black color consistently involving the cortex
and sometimes the medulla (Fig. 2). The melanotic
fat tissue observed in 2 animals was limited to the
back region, and the cut surface of the fat tissue
was mottled (Fig. 3).
Lymph node sections stained with HE showed
infiltrating macrophages with brown/black granu-
lar pigment (Fig. 4). Normal melanocytes were
present in the skin. To identify and differentiate the
pigment further, histochemical tests were carried
out. Staining with Nile blue (hydrogen sulphate)
Vet Pathol 46:2, 2009 Acquired Melanosis in the Nero Siciliano Pig
Fig. 2. Nero Siciliano pig. Cut surface of a retro-
pharyngeal lymph node showing melanosis. Bar 5 1 cm.
showed a dark green pigment, typical of melanin,
differentiating it from lipofuscin, which would have
stained blue. Additionally, 2 decolorizing agents,
melanin bleach and hydrogen peroxide, applied for
24 hours made the pigment totally disappear, as
would be expected only for melanin. Millon’s
reaction to tyrosine, which is a precursor of
melanin, was positive too, giving a red-yellowish
staining of the lymph nodes. Finally, Lillie’s
method,3 a highly specific reaction, showed dark
green granules within the macrophages (Fig. 5),
giving further confirmation of the presence of
melanin. The positive immunohistochemical reac-
tion for macrophage marker, as well as the negative
staining for S-100 and human melanoma, com-
bined with the histologic appearance support the
hypothesis that this is melanosis rather than
melanoma.
The histochemical and biochemical methods for
tyrosinase showed colorimetric patterns that con-
firmed the presence of this enzyme in acorns. The
results obtained by the quantitative determination
of the tyrosinase activity in acorn extracts from the
samples analyzed are shown in Table 1; the activity
was present mainly in latent form, as reported for
other vegetable species.10,19 The amount of phenolic
compounds was 0.137 6 0.012 mg gallic acid
equivalents/mg of acorn tissue. Results also showed
a strong tannin content, reaching about 76% of the
total phenolic compounds.
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331
Fig. 3. Nero Siciliano pig. Melanotic fat tissue from
the region of the back. Bar 5 1 cm.
Discussion
Melanosis is considered an unusual congenital
pigmentation in swine, only occasionally observed
in the slaughter house. The aim of the present
paper was to identify an acquired pigmentation
detected in a local swine breed and to relate it to the
ingestion of acorns.
Our results showed a high content of phenols in
acorns. These substances have various physiologic
functions, and some authors have recently reported
their effect on melanogenesis.4,14,17,18,29 For this
reason, we hypothesize an involvement of phenols
in this lesion, probably acting as a substrate instead
of tyrosine. Our data and current understanding of
the process of melanin formation permit us to
hypothesize that swine tyrosinase, being a poly-
phenol oxidase, could accelerate the use of phenol-
rich substrates in acorns. After acorns are eaten,
tyrosinase activation could take place in genetically
predisposed pig breeds, leading to increased bio-
synthesis of melanin-like pigment, followed by
anomalous storage. This hypothesis is supported
by the different prevalence of melanosis in the 2
groups studied: 100% (13/13) in pigs fattened in
acorn season versus 20% (1/5) in those ones
fattened in late spring, when acorns are less
available or no longer available at all. Moreover,
melanosis has never been found in Nero Siciliano
pigs reared in intensive systems. In conclusion, the
data reported defend the hypothesis that ingestion
of acorns can cause acquired melanosis. Kitt13
published a picture showing dark lymph nodes of
a pig fed on acorns, without any explanation with
regard to the nature of the pigment. Carta6
reported a prevalence of 100% in half-wild pigs
fed on acorns in Sardinia, Italy. He called this
332 Lanteri, Marino, Laganà, Bellocco, Barreca, Liotta, Sfacteria, and Macrı̀
Fig. 4. Lymph node. Histologic section showing melanin granules within macrophages. HE 403. Bar 5 30 mm.
Fig. 5. Lymph node. Lillie’s method showed dark green granules within macrophages (303). Bar 5 50 mm.
pigmentation ‘‘pseudomelanosis’’ and recognized
the cause in tannins. It is interesting that acorn
bread is a typical local food for people in the same
Italian region. More recently, there have been
reports of melanosis in slaughtered swine.1,32
Modern husbandry and intensive farming do not
include use of acorns as feed. Basic commercial pig
feed has a very low concentration of phenolic
compounds. This could explain why this pigmen-
tation is rare in farm animals. Using the movable
shelter farming system, this swine breed could be
proposed as a model for the study of unusual
lesions, such as melanosis, which today have
disappeared in other breeds intensively reared.
Acorns in ground cover are frequently damaged
by parasite attack.12,22 Further investigations are
needed to understand the parasite’s role, if any, in
eliciting melanosis in the tissues of this black swine
breed. Acquired melanosis has been described in
man’s intestines, associated with the use of
vegetable-derived laxatives,2 supporting the possi-
bility of the cause being ingested material. In our
study, a melanin-like pigment was found, and other
possible pigmentations have been excluded histo-
chemically. Immunohistochemical and histological
findings permit us to exclude melanoma, which is a
likely cause of abnormal melanin deposition. As a
Table 1. Acorn tyrosinase activity.*
U ml21 min21
Cotyledon acorn 1,660 6 173
Embryo acorn 1,100 6 115
Total acorn 1,530 6 204
* Values perform the mean of 3 different experiments 6 SD.
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Vet Pathol 46:2, 2009
final confirmation, we are now carrying out a trial
to evoke acquired melanosis by feeding Nero
Siciliano pigs on acorns and using white pigs as
controls.
Acknowledgement
The authors are particularly grateful to Prof.
Vincenzo Chiofalo, Coordinating Sect. Animal Produc-
tion University of Messina, Italy, for providing data on
this local pig and for the contact with the farmers.
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