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2005 European Society of Veterinary Dermatology 373
Veterinary Dermatology 2005, 16, 373384
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Alterations of epidermal proliferation and cytokeratin expression inskin biopsies from heavy draught horses with chronic pastern
dermatitis
FLORIAN GEBUREK*, BERNHARD OHNESORGE*, ECKEHARD DEEGEN*, RENATEDOELEKE and MARION HEWICKER-TRAUTWEIN
*Klinik fr Pferde, Tierrztliche Hochschule Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germany,
Institut fr Pathologie, Tierrztliche Hochschule Hannover, Bnteweg 17, D-30559 Hannover, Germany
(
Received
16 August
2004; accepted
17 August
2005)
Abstract
We report the historical, clinical and histopathological characteristics of skin lesions in biopsies from
37 heavy draught horses with chronic pastern dermatitis. The skin lesions were divided into four macroscopic
groups: scaling (group I, n
= 5), hyperkeratotic and hyperplastic plaque-like lesions (group II, n
= 14), nodular
skin masses (group III, n
= 16) and verrucous skin lesions (group IV, n
= 2). The principal histological findings
were hyperkeratosis and epidermal hyperplasia. There was a gradual increase in epidermal hyperplasia fromgroups I to IV, suggesting that the lesions represent different stages of disease. In all cases, there was perivascular
dermatitis dominated by T lymphocytes with an increase in MHC class II-positive dendritic-like cells. Immuno-
histochemical labelling for cytokeratins CK5/6(4), CK10 and CK14 indicated a change in their expression
pattern. This correlated with the degree of epidermal hyperplasia, indicating abnormal differentiation of
keratinocytes. There was a statistically significant correlation between the severity of skin lesions and several other
factors including increasing age, increasing cannon circumference, prominence of anatomical structures such as
fetlock tufts of hairs, ergots and chestnuts, and bulges in the fetlock region.
INTRODUCTION
Pastern dermatitis, also known as greasy heel, scratches
or mud fever in the Anglo-American literature, or as
mauke in the German literature, is a progressive
inflammatory skin lesion involving the posterior
pasterns of horses.
13
Pastern dermatitis occurs in all
breeds, but is most common in heavy draught horses
with feathering.
3
Affected horses initially show oedema
and scaling that progresses to exudation and crusting.
Pastern dermatitis is thought to be multifactorial with
many potential causes, e.g. infection with bacteria
(
Staphylococcus
spp., Dermatophilus congolensis
),
dermatophytosis, chorioptic mange, trombiculosis,photosensitization, vasculitis or contact with chemical
irritants.
3
Recently, the occurrence of papillomatous
pastern dermatitis associated with spirochetes and
nematodes identified as Pelodera strongyloides
in a
Tennessee Walking Horse was reported.
4
When the
aetiology is indeterminable, the term idiopathic
pastern dermatitis is used.
13
In these chronic cases,
the skin of the posterior pasterns becomes hyperkera-
totic and lichenified. Finally, hyperplasia is visible as
papillomatous or multifocal circumscribed verrucous
masses referred to as grapes.
13
These chronic, verru-
cous pastern lesions, which almost exclusively occur in
heavy draught horses, are known as warzenmauke
(
warze
= wart in English; muche
= German medievalword for a lameness-inducing disease of horses) in
the German veterinary literature. Although chronic
hyperplastic skin lesions of the pasterns of heavy
draught horses have been described for centuries, detailed
descriptions of the histopathology are not available,
except from older reports or dissertations mainly by
German or French authors.
59
The purpose of this study was to describe the clinical
and histopathological features of chronic pastern
dermatitis in biopsies from 37 heavy draught horses of
different breeds. The proliferative activity of epidermal
basal cells and expression of cytokeratins were charac-terized immunohistochemically with antibodies to the
proliferation marker Ki-67 and cytokeratins CK5/6(4),
CK10 and CK14. The dermal cellular immune response
was assessed with antibodies for leucocyte and MHC
class II antigens.
MATERIALS AND METHODS
Historical and clinical details
Data from 37 heavy draught horses of different breeds
with chronic pastern dermatitis were collected (Table 1).
For each horse, this included details of housing, feedingregimes, amount of work, use for breeding, condition
of the hair/hooves, duration of the skin lesions, presence
or absence of white markings or pruritus in the foot
Correspondence: M. Hewicker-Trautwein, Institut fr Pathologie,
Tierrztliche Hochschule Hannover, Bnteweg 17, D-30559 Hannover,
Germany. E-mail: [email protected]
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region and presence or absence of other skin lesions.
The cleanliness of stable facilities and quality of the
air and light were graded subjectively as good = 1,moderate = 2 or bad = 3. The ground where horses
were kept outdoors was recorded as pasture/soil, sand,
or rubber meadows. Coat condition was graded subjec-
tively as good = 1 (clean, smooth, shiny), moderate = 2
(partly dirty, partly rough, partly dull) or bad = 3 (dirty,
rough, dull). The condition of the hooves was graded
as good = 1 (clean, no trimming necessary, little or no
thrush) or bad = 2 (dirty, trimming necessary, noticeable
thrush).
A scoring system was developed for evaluating the
pastern skin lesions according to their type, extent and
severity. The clinical extent and macroscopic appear-
ance of skin lesions were recorded and documented bypreparing schematic drawings and by colour photography.
Parameters recorded were erythema, exudation, crust
formation, scaling, hyperkeratotic and hyperplastic
plaque-like lesions, erosions/excoriations, greasiness and
malodorous skin surface, nodular masses, or verrucous
masses with rugged surfaces. Each of these nine features(if observed) was multiplied by 13, depending on the
severity of the lesion (1 = mild, 2 = moderate, 3 = severe).
These were added together to give a total severity score
for each horse. The cannon circumference of all four
feet was measured in centimetres with a tape measure
at the smallest circumference of the metacarpal and
metatarsal bone immediately beneath the carpal or
tarsal joint. The average cannon circumference was
then calculated. Normal anatomical structures, i.e.
fetlock tufts of hairs (feathering), ergots, chestnuts
and bulges in the fetlock region were recorded. Skin
scrapings were examined for Chorioptes
sp.
Biopsy specimens
From each horse, at least one punch biopsy of 6 mm in
diameter was taken from the diseased skin. The location
Table 1. Clinical data, sites and numbers of biopsy specimens and macroscopic lesions on the feet of 37 heavy draught horses with chronic
pastern dermatitis
Case no. Cold blood breed Age (years) Sex
Cannon circumference
(in cm)
Biopsied
foot/feet
Total number
of biopsies
Macroscopic
lesion
1 Mecklenburgisch* 8 MC 27.0 LH, RH 3 Sc
2 Rhenisch German 6 MC 32.0 LH 2 Sc
3 Rhenisch German 9 F 27.5 RH 2 Sc4 Westphalian 5 F 28.25 RH 2 Sc
5 Lower Saxon 2 F 26.0 LH 2 Sc
6 Unknown 5 MC 26.75 LF, RH 4 Hhp
7 Westphalian 3 M 28.0 RH 2 Hhp
8 Altmrkisch* 17 F 31.0 RH 2 Hhp
9 Rhenisch German 4 M 29.0 LH 1 Hhp
10 Westphalian 3 M 27.5 RH 1 Hhp
11 Rhenisch German 5 M 31.0 LH 2 Hhp
12 Rhenisch German 6 M 33.0 LH 1 Hhp
13 Westphalian 4 M 28.0 LH 2 Hhp
14 Westphalian 4 M 27.5 LH 2 Hhp
15 Belgian Draught 10 M 31.0 LH 2 Hhp
16 Mecklenburgisch 7 F 29.0 RH 2 Hhp
17 Rhenisch German 5 M 32.0 RH 2 Hhp
18 Mecklenburgisch 7 MC 28.75 RH 2 Hhp19 Lower Saxon 3 F 26.0 LH 2 Hhp
20 Belgian Draught 6 M 32.0 RF 3 Nm
21 Rhenisch German 9 M 32.5 RH 2 Nm
22 Percheron 15 M 34.5 LH 2 Nm
23 Percheron 13 M 33.5 LH 2 Nm
24 Percheron 9 MC 34.0 RH 2 Nm
25 Rhenisch German 19 MC 31.25 RH 2 Nm
26 Westphalian 7 M 33.0 LH 2 Nm
27 Westphalian 10 M 29.5 RH 1 Nm
28 Saxon 13 M 37.5 RH 2 Nm
29 Westphalian 8 M 31.5 LH 2 Nm
30 Rhenisch German 8 M 29.5 LH 2 Nm
31 Rhenisch German 7 M 31.5 LH 2 Nm
32 Polish 14 F 24.5 LH 2 Nm
33 Saxon 7 M 33.5 LH 1 Nm34 Altmrkisch 8 M 31.25 RH 2 Nm
35 Lower Saxon 9 F 27.0 RH 2 Nm
36 Westphalian 10 M 32.0 LH 3 Vm
37 Rhenisch German 12 F 37.0 RH 1 Vm
C1 Altmrkisch 3 M 27,5 LH 1 None
C2 Rhenisch German 3 M 28.5 RH 1 None
M, male; MC, male castrated; F, female; LH, left hind; RH, right hind; LF, left front; RF, right front; C, control horse; *East German
Coldblood; Sc, scaling; Hhp, hyperkeratotic/hyperplastic plaques; Nm, nodular masses; Vm, verruccous masses.
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Pastern dermatitis in heavy draught horses 375
and total number of biopsies taken from the 37 cases
are given in Table 1. Tissue specimens were fixed in 4%
neutral buffered formaldehyde, embedded in paraffin
wax, cut at 4
m, mounted on coated slides (SuperFrost
Plus, Menzel-Glser, Wiesbaden, Germany) and stained
with haematoxylin and eosin or cresyl echt violet for
demonstration of mast cells. Selected sections were
stained with by the Warthin Starry silver staining
method. For comparison, from all 37 horses one 6-mm
punch biopsy was taken from the macroscopically
normal skin of the lateral neck. Control 6-mm skin
biopsies were included from two heavy draught horses
without clinical dermatological abnormality. These
were taken from the pasterns (C 1 and C 2 in Table 1)
and from the neck of horse C2.
Antibodies and immunohistochemical labellingprocedures
Antibodies used for immunohistochemical labellingare shown in Table 2.
Paraffin sections were dewaxed and rehydrated. Endo-
genous peroxidase activity was blocked by incubation
for 30 min in a solution of 0.5% H
2
O
2
in 85% ethanol.
For antigen retrieval, sections stained with antibodies
to Ki-67 or cytokeratins were pretreated by heating in
a microwave oven for 2
5 min in citric acid buffer
(0.01 mol L
1
, pH 6.0). For detection of CD3 and CD79a
antigens, sections were pretreated with unmasking
fluid G (Biologo, Kronshagen, Germany) for 10 min at
95
C. Sections stained with antibodies to MHC class
II antigens were incubated for 15 min at room temper-ature (rt) in a 0.25% solution of Triton X-100, rinsed in
phosphate-buffered saline (PBS), and pretreated with
a 0.05% pronase E (Merck, Darmstadt, Germany)
solution for 20 min at 37
C. Following incubation with
inactivated normal goat serum for 20 min at rt and
incubation with the primary antibodies (1820 h at
4
C) biotin-conjugated goat-antimouse IgG (H + L)
or goat-antirabbit IgG (H + L) (both from Vector
Laboratories, Burlingame, USA), diluted 1 : 200 in
PBS containing 10% inactivated normal horse serum
were used as second antibodies for 30 min at rt. After
incubation with the ABC solution (Vectastain Elite
ABC Kit, Vector Laboratories, Burlin-game, CA,USA) for 30 min at rt, peroxidase activity was detected
by incubation in 3-amino-9-ethylcarbazol solution
(AEC-Plus, Dako, Hamburg, Germany). Sections were
counterstained with Mayers haematoxylin. Negative
control sections, in which the primary antibody was
replaced by appropriately diluted normal mouse or
rabbit serum, were included in all staining runs.
Positive control tissues were equine lymph node for
CD3, CD79a, MHC II and Ki-67 antigens and normal
human and equine skin for CK5/6(4), CK10 and
CK14.
Statistical analysis
SAS software (version 6.04, Statistical Analysis Insti-
tute, Cary, NC, USA) was used for statistical analysis.
The clinical and morphological data were either direct
and quantitative (age, cannon circumference) or qual-
itative, grouped or graded (hygienic condition of the
stable, character of the ground where the horses were
kept on outdoors, feeding, amount of work, use of
stallions for breeding, grooming condition of the hair/
hooves, white markings in the foot region, fetlock tuftsof hairs, ergots, chestnuts, anatomically normal bulges
in the fetlock region, pruritus). The previously mentioned
parameters were correlated using Spearmans rank
correlation.
Differences in clinical severity for paired data were
compared with Wilcoxons signed-rank test, i.e. use of
stallions for breeding, grooming condition of hooves,
anatomically normal bulges in the fetlock region, white
markings in the foot region, pruritus and frequency of
cleaning stables. KruskalWallis test was used when
more than two groups had to be compared, i.e. sex,
amount of work, grooming condition, type of groundhorses were kept on. Means and standard deviations
were calculated. Differences were considered signifi-
cant at P
< 0.05.
Immunolabelled cells were counted in pastern skin
sections and compared to the number in skin sections
from the neck. Immunolabelling for Ki-67 was quan-
tified by counting basal epidermal cells with Ki-67-
positive nuclei in interfollicular epidermal areas in five
high-power fields (HPF) at 400
magnification. The
field of view was 600
m. Mast cells, CD3-, CD79a- and
MHC class II-positive cells were counted in five HPF
(400
) in the upper dermis. The fields were selected to
contain perivascular inflammatory foci. The meannumbers of positively labelled nuclei or cells, respec-
tively, were documented, standard deviations were
calculated and correlation was tested by Spearmans
Table 2. Antibodies used for immunohistochemical staining
Antibody Clone or identification Specificity Dilution Source
Anti-Ki-67 MIB-1 Human Ki-67 1 : 75 Dako
Anti-CK5/6(4) D5/16B4 Human CK 5/6(4) 1 : 3000 Dako
Anti-CK10 DE-K10 Human CK10 1 : 80 Dako
Anti-CK14 LL002 Human CK14 1 : 1000/1 : 50* Novocastra, Newcastle upon Tyne, UK
Anti-CD3 A0452 Human CD3 1 : 300 DakoAnti-CD79a HM57 Human CD79a 1 : 60 Dako
Anti-MHC II H42A Equine MHC II 1 : 120 VMRD, Pullman, Washington, USA
*In biopsies with moderate and severe epidermal hyperplasia the 1 : 1000 dilution resulted in a very weak staining intensity. Therefore, the
sections were also stained with a lower antibody dilution of 1 : 50.
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rank correlation. To compare differences between
numbers of cells, Wilcoxons signed-rank test was used.
RESULTS
Clinical and macroscopic findings
The data obtained from the owners or animal attend-
ants indicated that in the majority of horses (
n
= 17),
the skin lesions had been present for at least 2 years. In
10 cases, the lesions had not been noted or recognized
by the owners or animal attendants as pastern dermatitis.Some horses had additional dermatological diseases
such as facial alopecia (case no. 21), sweet itch (case
nos 3, 23, 25, 35) or inflammation of elbows and knees
(case no. 15). Hoof disease (case no. 6) and ulcerating
subsolar abscesses (case no. 1) were each found in one
horse. All other horses were clinically healthy apart
from the pastern dermatitis.
Based on macroscopic findings, the pastern skin
lesions were divided into four different groups (Table 1).
In horses of group I (
n
= 5), scaling was the predominant
sign (Fig. 1). Lesions in horses of group II (
n
= 14)
were characterized by hyperkeratotic and hyperplastic
plaque-like lesions (Fig. 2) as well as areas with scaling.In horses of group III (
n
= 16), nodular skin masses
were present (Figs 3 and 4), and horses of group IV
(
n
= 2) were affected by verrucous skin lesions with
rugged surfaces (Figs 5 and 6). Horses of groups III
and IV also had scaling and plaque-like lesions and
greasy, malodorous skin with focal excoriations/erosions
(Fig. 6).
Statistical analysis revealed a significant correlation
between the severity of skin lesions and age (
P =
0.0005),
increasing mean cannon circumference (
P