Medical Mycolo gy 2000, 38, 249– 253
Accepted 31 Jaunary 2000
Short communication
Granulomatous skin lesions due to histoplasmosis in a
badger (Meles m eles ) in Austria
B. BAUDER,* A. KUÚ BBER-HEISS,* T. STEINECK,† E. S. KUTTIN* & L. KAUFMAN‡
*Institute of Pathology and Fo rensic V eterinary Medicine, University of Veterinary Medicine, Vienna; †Research Institute
of W ildlife Ecology , University of V eterinary Medicine, V ienna; ‡Myco tic Diseases Branch, C enters for Disease and
C ontrol and Prevention , A tlanta, GA , USA
We describe the Žrst case of histoplasmosis due to infection with Histoplasma
capsulatum var. capsulatum in a wild badger (Meles meles) in Austria. Diagnosis was
established by histopathological and immunohistochemical characterization of yeast
forms in skin lesions and lymph nodes. Although Austria has yet to be regarded as
an endemic region for H. capsulatum, infections of animals and humans exposed to
contaminated soil cannot be excluded.
Keywords Austria, badger, Histoplasma capsulatum var. capsulatum, histoplasmosis
Introduction
Histoplasmosis capsulati is a disease of the mononuclear
phagocytic system (MPS) of humans and animals caused
by the fungus Histoplasma capsulatum var. capsulatum.
Infection occurs through inhalation of spores (frequently
originating from avian and chiropteran habitats), which
reach the respiratory tract and are phagocytosed by alveolar macrophages [1]. After development of intracellular
yeast forms and reproduction by budding, haematogenous dissemination can lead to the involvement of other
organs followed by occurrence of foci of granulomatous
inammation [2,3]. Histoplasmosis is endemic in Africa,
America (especially in central and southern parts) and
Asia, but is a rare disease in Europe. The few cases of
animal and human histoplasmosis reported in Europe
mainly cover southern and eastern parts, whereas western, northern, and central regions appear to be nearly
unaffected. Nevertheless, singular human cases were observed in France [4], Britain [5], Norway [6], Sweden [7]
Correspondence: Mag. Dr Barbara Bauder, Institute of Pathology
and Forensic Veterinary Medicine, University of Veterinary
Medicine, Vienna, Veterinärplatz 1, A-1210 Vienna, Austria. Tel.:
» 43 1250772403; fax: »43 1250772490; e-mail: barbara.bauder@
vu-wien.ac.at
© 2000 ISHAM
and Denmark [8]. Together with corresponding data
from wild animals in Switzerland [9], Denmark [10] and
Germany [11], a possible infection of humans even in
nonendemic European areas cannot be ruled out. This
report presents the Žrst and as yet single case of histoplasmosis in a wild badger (Meles meles) in Austria. The
diagnosis was assessed by macroscopical and histopathological examinations of the animal together with immunohistochemical investigations for characterization of
fungi in tissue sections.
Materials and methods
A nimal
In July 1998, a wild badger was shot by a hunter near
Amstetten, Lower Austria, because of abnormal behaviour. Post-mortem examination was performed at the
Research Institute of Wildlife Ecology, University of
Veterinary Medicine, Vienna, Austria.
Macroscopical and pathohistological examinations
Following macroscopical examination, tissue specimens
of skin, lung, liver, kidney, lymph node, spleen, thyroid
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Bauder et al.
and adrenal gland were Žxed in 7% neutral buffered
formalin and embedded in parafŽn. Sections of 3 mm
were cut and routinely stained with hematoxylin and
eosin (HE). For differentiation of fungi, sections were
stained with periodic acid Schiff (PAS), Grocott’s
methenamine silver stain (GMS), Giemsa, Ziehl Neelsen
stain, alcian blue, mucicarmine and Gram stain. Mycological culture was not attempted.
Immunohistochemical examinations
For immunohistochemical detection of Histoplasma
capsulatum, 3 mm parafŽn-embedded sections were
placed on organo-silane-coated (3Æ-aminopropyltriethoxy-silane) slides and dried for 20 min at 60 °C or
12 h for 37 °C to enhance tissue adherence. Antigen demasking was performed on deparafŽnized and rehydrated sections using microwave oven heating in 0·01 M
citrate-target retrieval buffer pH 6·0. Endogenous peroxidase activity was blocked by incubation with 2%
H2 O2 :methanol. To reduce background staining the sections were incubated with 10% normal rabbit serum for
1 h at room temperature in a humidiŽed chamber. For
immunohistochemical detection of the fungi, the sections were incubated with polyclonal goat-antibodies directed against Histoplasma capsulatum antigen (Immuno
Mycologics, Inc., Norman, OK, USA) overnight at
4 °C in a humidiŽed chamber. After extensive washing
with phosphate-buffered saline (PBS), the sections were
incubated with a biotinylated secondary antibody (antigoat immunoglobulin (Ig)G; Vector, Burlingame, CA,
USA) for 30 min at room temperature in a humidiŽed
chamber. Consecutive steps were performed using the
Vectastain® ABC-kit (Peroxidase ELITE; Vector) according to the manufacturer’s instructions. Visualization of positive reactions was carried out using the
Peroxidase Substrate kit DAB (Vector). Subsequently,
the sections were counterstained with hematoxylin, dehydrated and mounted.
Further immunouorescence investigations for the
classiŽcation of fungi were performed using heterologously absorbed uorescein isothiocyanate (FITC)labeled conjugates for detection of H. capsulatum,
Candida glabrat a, Cryptococcus neoformans and
Sporothrix schenckii [12,13].
Results
Macroscopical examination
Macroscopically, the female badger (6·4 kg) appeared
emaciated and showed multiple nodules and ulcers in the
skin of the head, chest, abdominal wall, perianal region
and all limbs (Fig. 1). The doughy-to-Ž rm lesions were
pea- to chestnut-sized, with their surfaces partially
eroded, and displaceable within the skin. The draining
lymph nodes were moderately enlarged. Apart from multiple traumatic lesions in the thoracic region caused by
the penetration of shotgun pellets, the other organs appeared normal upon gross examination.
Patho histological examination
The macroscopically described lesions were granulomas
with macrophages partly forming multinucleated giant
cells, lymphocytes, plasma cells and small clusters of
granulocytes. They were accompanied by a remarkable
proliferation of connective tissue. Some of the lesions
were exulcerated and succeeded by bacterial colonization.
The macrophages were heavily loaded with oval to spherical yeast-like organisms 2·5 –3 mm in size. Numerous
fungal cells, sometimes showing narrow-based buds (consistent with H. capsulatum var. capsulatum), were not
only localized intracellularly, but also diffusely scattered
all over the poorly vascularized granulation tissue, as
consequence of cell rupture. Some of the cells were distorted and resembled quarter moons or crescents. The
fungi stained moderately with HE and Giemsa revealing
a tiny central nucleus surrounded by a clear zone or
‘halo’ which in turn was encircled by a thin poorly
stained cell wall (Fig. 2), but well with GMS and PAS,
where the ‘halo’ was not evident (Fig. 3). The organisms
did not stain with Ziehl Neelsen, alcian blue, mucicarmine and modiŽed Gram stain. Lots of fungal cells
Further laboratory examinations
Fecal concentration was performed for the examination
of intestinal parasites.
Fig. 1 Multiple nodules and ulcers due to histoplasmosis capsulati
on the badger’s hind limbs.
© 2000 ISHAM, Medical Mycology, 38, 249–253
Histoplasmosis in a badger in Austria
251
Further labo ratory examinations
Parasitological examinations revealed high numbers of
eggs of Capillaria sp. after concentration of feces.
Discussion
Immunohistochemically the fungi showed positive reactions with the polyclonal antibodies raised against H.
capsulat um antigen in the skin lesions as well as in
macrophages of the lymph nodes (Fig. 4). However, some
cells only revealed a low staining intensity.
In immunouorescence assays, the fungi stained positively with the H. capsulatum conjugate, but revealed
negative reactions when incubated with the conjugates for
C. glabrata, C. neoformans and S. schenckii, thereby
conŽrming the classiŽcation of the yeasts as H. capsulatum.
Granulomatous skin lesions of an Austrian wild badger
(Meles meles) were related to histoplasmosis capsulati
based on the characteristic histopathological manifestation, the morphology and distribution of yeast cells
within the affected tissues and on the conventional and
immunohistochemical staining reactions of the fungi. Our
observation of intracellular yeast forms as well as extracellularly localized fungi in areas with heavy destruction
of the mononuclear phagocytic cells within the skin and
the lymph nodes correspond with data reported by others
[14,15].
Interestingly, sections of the lung did not show any
signs of histoplasmosis although the respiratory tract is
regarded as the main portal of entry of Histoplasma
spores. The serious involvement of skin and lymph nodes
could either result from hematogenous dissemination,
with the primary pulmonary manifestation probably having been overlooked due to excessive traumatic thoracic
lesions caused by the shotgun pellets, or it could represent one of the very rarely observed cases of primary
cutaneous histoplasmosis with ulceration and regional
lymphadenitis [16]. Confusion with histoplasmosis
duboisii, due to infection with H. capsulatum var.
duboisii, which causes hardly detectable pulmonary lesions [17,18], but shows a marked tropism to the skin and
the bones, was excluded for several reasons. Natural
infections with H. capsulatum var. duboisii are, with the
exception of one reported case in Japan [19], only known
Fig. 3 Masses of yeast cells diffusely scattered all over the skin,
GMS stain. MagniŽcation × 925.
Fig. 4 Immunohistochemical detection of Histoplasma capsulatum
within macrophages of a lymph node (arrow). MagniŽcation × 550.
Fig. 2 Numerous cells packed with Histoplasma capsulatum in the
skin, Giemsa stain. Notice the clearly visible ‘halo’ (arrow). MagniŽcation × 550.
were found within macrophages in the lymph nodes.
Sections of lung, liver, kidney, spleen, thyroid and adrenal
gland did not show signs of infection due to H. capsulatum
or any other pathological features.
Immunohistochemical examinations
© 2000 ISHAM, Medical Mycology, 38, 249–253
252
Bauder et al.
to occur in humans and nonhuman primates in Africa.
Furthermore, histoplasmosis duboisii can be distinguished from histoplasmosis capsulati on the basis of the
size of the fungal cells in the tissue. Yeast-like forms of
H. capsulatum var. duboisii are larger, moderately thickwalled, measuring 8– 15 mm [2].
As lightly encapsulated C. neoformans cells growing
within histiocytes, epitheloid cells or giant cells, can
mimic the intracellular form of H. capsulatum var. capsulatum, and as those poorly encapsulated cells might stain
negatively in the mucicarmine or alcian blue stain, further immunohistochemical examinations of the tissue sections were performed. The tissue forms of H. capsulat um
have also to be differentiated from those of S. schenckii,
which are usually intracellular and have similar staining
and morphological features. Furthermore, in tissues, C.
glabrata cells might be easily confused with those of H.
capsulatum, because they are of comparable size and may
occur intracellularly. However, C. glabrata cells are often
slightly larger than those of H. capsulatum, and budding
occurs with greater frequency and those buds are usually
attached by a broader base [2]. The results of our immunohistochemical examinations of fungi as well as
those of the immunouorescence assays strongly indicated the organisms to be H. capsulatum var. capsulatum.
The lack of acid-fastness, demonstrated by negative
staining of H. capsulatum cells in the Ziehl Neelsen stain,
enabled us to distinguish the fungi from Blastomyces
dermatitidis [20].
Apart from human cases, histoplasmosis can affect a
number of wild and captive animals, including dogs, cats,
cattle, horses and swine as well as various rodents, bats,
foxes, raccoons, skunks and badgers [2,21]. Post-mortem
examinations of 32 badgers have been performed at the
Viennese University of Veterinary Medicine since 1980,
but the presented case is the Žrst diagnosed with histoplasmosis. The massive infection with Capillaria sp. could
have contributed to immunosuppression facilitating the
establishment of an opportunistic mycosis in the current
badger case. Since histoplasmosis is acquired by the inhalation of airborne spores without revealing any evidence of contagion from one animal to another, and as
badgers’ territories usually cover only few square kilometers [22], the present infection must have an Austrian
origin. Since histoplasmosis, as a consequence of human
immunodeŽciency virus (HIV) infection in humans, is
increasingly occurring as an opportunistic mycosis with a
systemic course even outside Histoplasma-endemic areas
[23], mycologists should be aware of the possibility of
immunocompromised patients acquiring histoplasmosis
even in Austria. To date there are a few reports of
autochthonous histoplasmosis in AIDS patients in Eu-
ropean countries such as Italy [24,25], Germany [23] and
France [4], but there is no case reported in Austria.
Acknowledgements
The authors thank Klaus Bittermann for excellent phototechnical assistance.
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