J. Vet. Med. A 52, 202–208 (2005)
2005 Blackwell Verlag, Berlin
ISSN 0931–184X
University of Turin, Department of Animal Pathology, Grugliasco (TO), Italy
Effects of Low-dose Dexamethasone on Thymus Morphology and Immunological
Parameters in Veal Calves
B. Biolatti1,5, E. Bollo1, F. T. Cannizzo1, G. Zancanaro1, M. Tarantola2, M. Dacasto3, M. Cantiello3,
M. Carletti1, P. G. Biolatti1 and G. Barbarino4
Addresses of authors: 1Department of Animal Pathology, University of Turin, Via L. Da Vinci 44, 10095 Grugliasco (TO), Italy;
2
Department of Animal Production, University of Turin, Via L. Da Vinci 44, 10095 Grugliasco (TO), Italy; 3Department of
Public Health, Comparative Pathology and Veterinary Hygiene, University of Padua, Agripolis, Via Romea 16, 35020 Legnaro
(PD), Italy; 4Regione Piemonte, Direzione di Sanità Pubblica, Corso Stati Uniti 1, 10128 Torino, Italy; 5Corresponding author:
Tel.: +39 011 6709033; fax: +39 011 6709031; E-mail: bartolomeo.biolatti@unito.it
With 9 figures
Received for publication December 8, 2004
Summary
Glucocorticoids are often illegally used in association with
anabolic steroids as growth promoters in veal calves and beef
production. An experimental administration of dexamethasone was carried out in veal calves in order to assess the role of
low doses of exogenous glucocorticoids on induction of thymus atrophy and on the immune response. Three groups of
five veal calves each were included in this study: group D was
administered 0.4 mg/day of dexamethasone-21-phosphate per os
for 25 days; group V was administered 2 mg of dexamethasone-21-isonicotinate i.m. at days 14 and 21, and group K
served as control. At slaughter, the weight of the thymus was
severely reduced in group D and in group V, compared with
control animals. Lesions included severe lymphoid depletion
and hyperplasia of adipose tissue. In situ evaluation of apoptosis in thymus, showed a reduction of the percentage of
positive nuclear areas of animals belonging to group V in
comparison with control animals. An overall decrease of
lymphocyte proliferative response was detected after treatment
with short acting dexamethasone, while antibody response was
not affected by treatments.
and Kaeberle, 1981; Jayappa and Loken, 1983; Oldham and
Howard, 1992; Burton et al., 1995; Doherty et al., 1995;
Burton and Kehrli, 1996; Nonnecke et al., 1997; Bureau et al.,
1998; Lan et al., 1998; Anderson et al., 1999; Moiré et al.,
2002), depression of the ability of T cells to produce interleukin (IL)-2 and interference with their responses to mitogens
(Eckblad et al., 1984; Pruett et al., 1987; Oldham and Howard,
1992; Roth et al., 1994) have been described.
In the last 15 years, farmers have progressively reduced the
dosages of illegal administration of these drugs to avoid the
penalties of the public veterinary services. As a consequence,
the thymus becomes only partially atrophic in treated animals,
making the detection of alterations by veterinary inspectors a
difficult task.
The aim of this study was to describe the alterations of the
morphology of the thymus following the administration of
low-dose glucocorticoids as growth promoters in veal calves,
often resorted to in farm practice, and to evaluate their effect
on peripheral blood mononuclear cell proliferation and
immunoglobulin concentration.
Materials and Methods
Introduction
Animals and experimental design
Dexamethasone is a potent synthetic analogue of hydrocortisone that has a long history of use in veterinary medicine for
the treatment of a range of metabolic diseases and inflammatory disorders in farm animals. Animal diseases in which
dexamethasone is an effective treatment include inflammation,
shock and stress. Although its use in animals is primarily
therapeutic, in some European countries glucorticoids are
often illegally used in association with anabolic steroids as
growth promoters, in veal calves and beef production, in order
to improve quality and quantity of meat (Van de Wal et al.,
1975; Istasse et al., 1989; Sabbe and Vander Beken, 2001;
Courtheyn et al., 2002). Their use has also been associated
with generalized immunosuppression and consequent exacerbation of infectious diseases (Callow and Parker, 1969; Sheffey
and Davies, 1972; Roth and Kaeberle, 1982; Ilott et al., 1997).
Early reduction of the thymus in young animals treated with
therapeutic dosages of corticosteroid (Guarda et al., 1983;
Canese et al., 1985), as well as changes in the composition of
the population of circulating mononuclear leucocytes (Roth
Three groups of five male veal calves each, aged 130 days, were
included in the study. Group D was administered 0.4 mg/day
of dexamethasone-21-phosphate (D21P) per os for 23 days
starting from day 0, for a total of 9.2 mg/animal. Group V was
administered 2 mg of dexamethasone-21-isonicotinate (D21I)
i.m. at days 14 and 21 for a total of 4 mg/animal. Group K
served as control. The animals were fed liquid milk replacer,
twice a day, increasing gradually from 13 to 16 l/day. After a
month, a quantity of 0.5 kg/day of straw was added to the diet
in according to the indications of the European Commission
(97/182/EC). Calves were weighed at weekly intervals and
slaughtered at 165 days of age.
U.S. Copyright Clearance Center Code Statement:
Processing of thymic tissue
The thymus of each animal was collected and weighed soon
after slaughtering. The weight ratio of the thymus was
calculated according to the following formula: weight of organ
(kg)/body weight of veal calves (kg) · 100. Tissue samples
0931–184X/2005/5204–0202 $15.00/0
www.blackwell-synergy.com
Low Doses of Dexamethasone in Veal Calves
were fixed in 10% neutral buffered formalin overnight at room
temperature, and paraffin embedded according to routine
histological procedures. Representative sections of each sample were haematoxylin–eosin stained for histological examination.
In situ detection of apoptosis
In situ detection of apoptosis was performed using terminal
deoxynucleotidyl transferase biotin-dUTP nick end labelling
(TUNEL). This method is based on the addition of labelled
deoxynucleotide triphosphate to the 3¢-OH ends of DNA
fragments catalysed by deoxynucleotidyl transferase (TdT).
This enzyme more selectively detects apoptotic rather than
necrotic cells (Gold, 1994). The reaction was revealed immunohistochemically by means of anti-digoxigenin antibody peroxidase conjugated.
Representative 4-lm-thick sections of each sample were
stained by means of ApopTag In Situ Apoptosis Detection
Kit (Intergen Company, Purchase, NY, USA) for identification of apoptotic nuclei. Briefly, sections were deparaffinized,
rehydrated and treated with proteinase K (Sigma, St Louis,
MO, USA) 20 lg/ml for 15 min at room temperature. The
slides were then washed in distilled water and endogenous
peroxidases are blocked by means of 3% hydrogen peroxide in
phosphate-buffered saline (PBS) for 5 min at room temperature. Subsequently, the sections were allowed to react with
TdT-enzyme for 1 h at 37C and then incubated with the antidigoxigenin-peroxidate antibody for 30 min in a humidified
chamber at room temperature. The reaction was developed in
a solution of diaminobenzidine and H2O2 for 5 min, counterstained with 0.5% (w/v) methyl green for 10 min and destained
in n-butanol. A negative control was obtained by omitting the
TdT in the reaction mixture during the labelling steps.
Quantitation of apoptosis in thymus
The apoptotic cells were identified by light microscopic
examination at ·200 magnification. For each slide, 16
randomly selected fields were examined by means of ImagePro Plus software (Media Cybernetics, Silver Spring, MD,
USA). In each field, the total area of apoptotic and negative
nuclei was evaluated. The incidence of thymocyte apoptosis
was expressed as percentage of positive nuclear area
(mean ± SEM). Mean and SEM were calculated for numerical values. Statgraphics Plus software (Statistical Graphic
Corp., Rockville, MD, USA) was used for one-way anova
with Student–Newman–Keuls test for comparison of multiple
samples. In all comparisons, probability values of P < 0.05
were considered statistically significant.
Isolation of peripheral blood mononuclear cells and lymphocyte
proliferation assay
Blood samples were aseptically collected from the external
jugular vein with evacuating tubes containing acid-citratedextrose (ACD) as anticoagulant, at days 0, 4, 10, 14, 21 and
28 from calves of groups K and D, and at days 0, 4, 10 and 18
from calves of group V. The peripheral blood mononuclear
cells were isolated on Ficoll-gradient as previously described
(Moiré et al., 2002). For proliferation experiments, 2 · 105
cells were cultured in quadruplicate in 200 ll RPMI-1640
203
10% foetal calf serum (FCS) (Sigma) in 96-well flat-bottomed
plates (Nunc A/S, Roskilde, Denmark) for 90 h with 5 lg/ml
concanavalin A (ConA), 10 lg/ml phytohaemagglutinin
(PHA) and 5 lg/ml pokeweed mitogen (PWM) (Sigma). The
cells were pulsed for 6 h with 1 lCi per well [3H]-thymidine
(ICN Biomedicals, Irvine, CA, USA), harvested on glass fibre
discs and the label incorporation assessed by liquid scintillation counting. Results were expressed as stimulation index (SI:
counts per minute (cpm) with mitogen/cpm without mitogen).
SI values were normalized, applying the formula: SI ¼
(T/K ) 1) (where T indicates treated animals, and K control
animals). Statistical analysis of SI was performed by repeatedmeasure anova for each group and, between groups, at each
sampling times.
IgG, IgM and IgA concentrations
Immunoglobulin G, IgM and IgA concentrations were determined in serum samples, collected at days 0, 4, 10, 14, 21 and
28 from calves of groups K and D, and at days 0, 4, 10 and 18
from calves of group V, by radial immunodiffusion assay
(Bethyl Lab., Montgomery, AL, USA) in comparison with a
standard curve, according to the manufacturer’s instructions.
Statistical analysis of Ig concentrations was performed by
repeated-measure anova for each group and, between groups,
at each sampling times.
Results
Body weight and thymus morphology
The increase in body weight of experimental groups during the
treatment time is shown in Fig. 1. The average weight of
animal of groups K increased from 176 to 215 kg from the
beginning to the end of the treatment; for group D the average
of the body weight increased from 166.3 to 193.8 kg; for group
V, the average of the body weight increased from 170 to
210.2 kg. No statistically relevant difference was detected
between groups D, V and K.
At slaughter, the thymus of all treated and control animals
was always detectable. Figure 2 shows the changes in the
thymus weight of normal and treated animals. The average
weight of total thymus was 551.2, 221.2 and 373.6 g in animal
of groups K, D and V respectively. The administration of
dexamethasone to young calves resulted in a remarkable
thymic weight loss. In particular, a reduction of 60% and 32%
in thymus weight of the group treated with D21P and D21I
respectively, was observed. Statistically relevant differences
(P < 0.05) were detected between groups D and V, and group
K for the total weight of the thymi, and between group D and
group K for the cervical portion of the thymus. Figure 3 shows
the thymus/body weight ratio in treated and control animals,
ranging from 0.124% of group D, to 0.179% of group V, to
0.268% of group K. Statistically significant (P < 0.05)
differences were detected only between the thymus/body
weight ratio of animals of group D compared with control
animals.
Already at gross examination, a fatty infiltration either in
the thoracic and cervical part of the thymus both in group D
and V was evident (Fig. 4). Histologically, the thymus of
control animals showed the typical structure of the gland in
young animals, characterized by accumulation of small
B. Biolatti et al.
204
230
220
Body weight (kg)
210
200
K
D
V
190
180
170
160
Fig. 1. Body weights in control
veal calves (K) and in calves
treated with dexamethasone-21phosphate (D) and dexamethasone-21-isonicotinate (V). Data are
expressed as mean ± SEM.
150
1
2
3
4
5
Sampling weeks
700
600
Thymus weight (g)
500
*
K
400
300
D
V
*
200
*
*
100
0
Total thymus
Cervical thymus
Toracic thymus
Apoptosis in thymus
0.3
Thymus/body weight ratio (%)
Fig. 2. Thymus weight in control
veal calves (K) and in calves
treated with dexamethasone-21phosphate (D) and dexamethasone-21-isonicotinate (V). Data are
expressed as mean ± SEM.
*Significant differences at
P < 0.05 compared with control
animals (Student–Neuman–Keuls
test).
0.2
K
D
V
*
0.1
0.0
K
D
V
Fig. 3. Thymus/body weight ratio in control veal calves (K) and in
calves treated with dexamethasone-21-phosphate (D) and dexamethasone-21-isonicotinate (V). Data are expressed as mean ± SEM.
*Significant difference at P < 0.05 compared with control animals
(Student–Newman–Keuls test).
lymphocytes more densely packed in the cortex than medulla,
with almost absence of adipose tissue both in the cortex and
medulla (Fig. 5a). The thymus of group D and V showed
severe atrophy of the cortex associated with hyperplasia of
adipose tissue which replaces most of the outer part of the
cortex (Fig 5b and c), while the medulla showed a depletion of
lymphocytes.
Apoptotic cells were detected in thymus of treated and
control animals, scattered in the thymic medulla and more
densely distributed throughout the cortex (Fig. 6). The
apoptotic signal was localized in the nucleus of lymphocytes.
The staining of the apoptotic nuclei was not uniform and
was related to different stages of apoptosis. In some cells, the
nuclear staining was homogenous, while in other cells the
nuclear staining was more intense in the periphery. In few
cells cluster of apoptotic fragments were detected within the
cytoplasm of thymic macrophages. Quantitative analysis of
apoptosis in thymic tissues of treated and control animals
(Fig. 7) showed percentages of positive nuclear area ranging
from 0.60 for group K, to 0.57 for group D, to 0.43 for
group V. A statistically significant (P < 0.05) reduction of
the percentage of positive nuclear areas for animals belonging to group V in comparison with control animals was
detected.
Lymphocyte proliferation assay
Figure 8 shows the results of proliferation responses to ConA,
PHA and PWM. An overall significant reduction in lymphocyte proliferation for ConA, PHA and PWM of animals of
group D was observed, although statistically significant only
Low Doses of Dexamethasone in Veal Calves
205
Fig. 4. Gross appearance of the thymus in control veal calves (a) and in calves treated with dexamethasone-21-phosphate (b) and
dexamethasone-21-isonicotinate (c). A remarkable reduction of the thymus weight accompanied by fatty infiltration in calves of groups D and V
may be seen.
Fig. 5. Histological appearance of the thymus in control veal calves (a) and in calves treated with dexamethasone-21-phosphate (b) and
dexamethasone-21-isonicotinate (c). Severe atrophy of the cortex associated with hyperplasia of adipose tissue, replacing most part of the cortex
in groups D and V (haematoxylin and eosin, 50·).
Fig. 6. Apoptosis in thymus of calves of control veal calves (a) and in calves treated with dexamethasone-21-phosphate (b) and dexamethasone21-isonicotinate (c). Clusters of apoptotic cells and isolated positive cells are scattered throughout the thymic medulla (Apoptag In Situ
Apoptosis staining; 400·).
for ConA after 4 and 21 days of treatment, and for PHA after
14 days of treatment, while an increase of the stimulation
index was detected at day 28 compared with control animals.
Results for animals of group V were contradictory: a general
trend of increase was revealed for ConA, while a decrease was
observed for PHA (statistically significant at day 18 compared
with controls) and for PWM.
treated animals, differences in IgG concentrations were statistically relevant only for animals belonging to group V at days 10
and 18 compared with controls. As regards the other Ig
subclasses, IgM showed an overall decrease in their concentration in dexamethasone-21-phosphate treated animals and an
increase in dexamethasone-21-isonicotinate treated calves. For
IgA, an overall decrease in their concentration was observed in
all treated animals at all sampling times, although not statistically relevant.
IgG, IgM and IgA concentrations
Figure 9 shows the results of IgG, IgM and IgA concentrations
in blood of control and treated animals. Although an overall
increase in IgG concentrations was detected in both dexamethasone-21-phosphate and dexamethasone-21-isonicotinate
Discussion
Although it has long been recognised that synthetic glucocorticoids have immunosuppressive activities, dexamethasone and
B. Biolatti et al.
% positive nuclear area
206
0.8
K
D
V
*
0.4
0.0
Fig. 7. Expression of apoptosis in control veal calves (K) and in calves
treated with dexamethasone-21-phosphate (D) and dexamethasone21-isonicotinate (V). Data are expressed as mean ± SEM. *Significant
difference at P < 0.05 compared with control animals (Student–
Neuman–Keuls test).
other corticosteroids are frequently used as illegal growth
promoters in livestock production, because of improved feed
intake, increased live weight gain, reduced feed conversion
ratio, reduced nitrogen retention and increased water retention
and fat content (Istasse et al., 1989). A few studies have shown
(b)
20
10
0
–10
–20
–30
(T–K)/K*100
(T–K)/K*100
(a)
a significant reduction of the thymic tissue, namely cortical
atrophy and fatty infiltration, following the use of therapeutic
doses of synthetic corticosteroid (Guarda et al., 1983, 1990;
Groot et al., 1998; Schilt et al., 1998). These findings have
been correlated with apoptosis of thymocytes (Sun et al.,
1992), a finding also related to elevated endogenous corticosterone levels in mice (Gruber et al., 1994; Tarcic et al., 1998).
Our findings demonstrate that low dosages of dexamethasone administered as growth promoter in veal calves, according to a protocol often illegally adopted in farm practice, can
induce thymic atrophy, resulting in a significant reduction of
the thymus/body weight ratio. Evaluation of apoptotic
phenomena in the thymus revealed a significant reduction
of specific processes of cell death in dexamethasone-21isonicotinate treated animals at the time of slaughter, although
this experimental group showed an overall reduction of the
thymus weight. An increase of apoptotic processes induced by
dexamethasone in the early phase of treatment could account
for the observed changes in treated animals; further studies are
needed in order to understand the corticosteroid action on the
thymic lymphocytes apoptosis, possibly evaluating the progression of changes in thymic tissue at early phase and during
the treatment.
*
*
*
0
4
10
14
21
28
25
20
15
10
5
0
–5
–10
0
Sampling times
4
10
18
Sampling times
0
–20
–40
–60
–80
(T–K)/K*100
(d)
(T–K)/K*100
(c)
*
0
4
10
14
21
28
20
10
0
–10
–20
–30
–40
–50
–60
Sampling times
*
0
4
10
18
Sampling times
(f)
0
–10
–20
–30
–40
–50
–60
–70
(T–K)/K*100
(T–K)/K*100
(e)
40
30
20
10
0
–10
–20
0
4
10
14
Sampling times
21
28
0
4
10
18
Sampling times
Fig. 8. Effect of dexamethasone-21-phosphate and dexamethasone-21-isonicotinate treatment on lymphocyte proliferative response of calves to
mitogens. (a) group D, ConA; (b) group V, ConA; (c) group D, PHA; (d) group V, PHA; (e) group D, PWM; (f) group V, PWM. Each data point
represents the mean of calves of each group. SI values were normalized, applying the formula: SI ¼ (T/K ) 1) (where T indicates treated animals,
and K control animals). *Significant difference at P < 0.05 compared with control animals (Student–Newman–Keuls test).
Low Doses of Dexamethasone in Veal Calves
207
1200
mg/dl
1000
800
K
D
600
400
200
0
0
4
10
14
21
(b) 1400
1200
1000
800
600
400
200
0
*
*
K
mg/dl
(a) 1400
V
0
28
(c) 120
(d) 250
100
200
K
D
*
mg/dl
mg/dl
80
40
18
150
K
V
100
50
20
0
0
0
4
10
14
21
28
0
4
Sampling times
10
18
Sampling times
(e) 14
12
(f) 14
12
10
10
8
K
D
6
4
mg/dl
mg/dl
10
Sampling times
Sampling times
60
4
8
6
K
V
*
4
2
2
0
0
0
4
10
14
21
28
Sampling times
0
4
10
18
Sampling times
Fig. 9. Immunoglobulin concentrations in blood of control calves and of dexamethasone-21-phosphate and dexamethasone-21-isonicotinate
treated calves). (a), (b) IgG; (c), (d) IgM; (e), (f) IgA. *Significant differences at P < 0.05 compared with control animals (Student–Newman–
Keuls test).
Results on the ability of dexamethasone in inducing
suppression of lymphocyte proliferation to mitogens both in
in vivo and in vitro experiments, as well as depression of
antibody synthesis, are controversial, mainly because of the
use of high doses of dexamethasone (Anderson et al., 1999;
Moiré et al., 2002). Our data demonstrate an overall decrease
of lymphocyte proliferative response to ConA, PHA and
PWM after treatment with dexamethasone-21 isonicotinate,
while for dexamethasone-21 phosphate, results are conflicting.
These findings confirm previous investigations in both conventional and gnotobiotic calves, in which dexamethasone,
although at therapeutic dosages, was highly inhibitory for
ConA, PHA and PWM responses (Muscoplat et al., 1975;
Pruett et al., 1987; Oldham and Howard, 1992).
The effects of corticosteroids on B cell function in cattle
have not yet been fully elucidated: pharmacological dosages
of corticosteroids appear to reduce antibody production in
previously sensitized cattle (Roth and Flaming, 1990), but
other studies have revealed that B cells are resistant to the
effects of corticosteroids (Doherty et al., 1995). Finally,
other authors have demonstrated a reduction in in vitro
secretion of IgM by peripheral blood mononuclear leukocytes
following dexamethasone treatment in Holstein bulls
(Nonnecke et al., 1997). In our study, the administration
of low dosages of dexamethasone did not significantly affect
the secretion of Igs, although a more accurate investigation
on changes in the composition and functional activity of
B cells in treated animals could provide more detailed
information.
In conclusion, the potential of illegally administered anabolic doses of corticosteroids to reduce the immunological
responsiveness in beef cattle should be considered. Important
implications are represented by susceptibility of animals to
infections, response to vaccination, animal welfare and
improvement of animal productivity.
Moreover, the results from the present investigation suggest
that thymus weight and histology may be good indicators of
illegal corticosteroid administration in veals, although a
discrimination between a possible anabolic treatment and a
therapeutic treatment must be achieved. Solutions to improve
controls in cattle for the illegal use of growth promoters should
therefore include the application of screening methods based
on the measurement of body parameters and on histological
investigations.
208
Acknowledgements
This work was supported by a Grant from the Direzione di
Sanità Pubblica, Assessorato alla Sanità, Regione Piemonte,
Italy.
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