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Subpopulation of lymphocytes CD4 + and CD8 + in peripheral blood of sheep

with zearalenone mycotoxicosis

Article  in  Bulletin- Veterinary Institute in Pulawy · January 2011

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Bull Vet Inst Pulawy 55, 241-246, 2011

SUBPOPULATION OF LYMPHOCYTES CD4+ AND CD8+

IN PERIPHERAL BLOOD OF SHEEP
WITH ZEARALENONE MYCOTOXICOSIS
KRZYSZTOF KOSTRO, MAGDALENA GAJĘCKA1, URSZULA LISIECKA, BARBARA MAJER-
DZIEDZIC2, KAZIMIERZ OBREMSKI1, ŁUKASZ ZIELONKA1, AND MACIEJ GAJĘCKI1
Department of Epizootiology and Clinic of Infectious Diseases, Faculty of Veterinary Medicine,
University of Life Sciences in Lublin, 20-612 Lublin, Poland
1
Chair of Veterinary Prevention and Feed Hygiene, Faculty of Veterinary Medicine,
University of Warmia and Mazury in Olsztyn, 10-718 Olsztyn, Poland
2
Subdepartment of Veterinary Microbiology, Institute of Biological Bases of Animal Diseases,
Faculty of Veterinary Medicine, University of Life Sciences in Lublin, 20-033 Lublin, Poland
kkostro@wp.pl

Received: January 14, 2011 Accepted: May 6, 2011

Abstract
In sheep raised in chamber system and fed concentrate fodder containing high dose of zearalenone and its metabolite (α-
zearalenol), the clinical course of zearalenonosis was diagnosed. Control animals were kept on grazing-land from the spring to the
late autumn, and only in winter time they were fed shredded wheat with the lowest concentration of zearalenone. These sheep
showed no clinical symptoms of the mycotoxicosis. Zearalenone and α-zearalenol cause immunosuppression, which means that
lymphocyte T CD4+ and lymphocyte T CD8+ percentages decline and CD4/CD8 ratio increases when compared with control
animals. Zearalenone mycotoxin suppression effect modulates cellular immunological response, which is essential in suppression of
post-vaccination immunity and infectious diseases development despite applying normal specific immunoprophylaxis.

Key words: sheep, zearalenone, mycotoxicosis, lymphocytes, immunosuppression, flow cytometry.

Zearalenone mycotoxin belongs to a Material and Methods

remarkable group of mycotoxins, formed as metabolites
of filamentous fungi from the genus Fusarium sp. The
phenol ring present in the molecule of zearalenone
enables bounding to oestrogen receptors of the uterus,
vagina, ovary, and oviduct, enhancing cell proliferation
and in the end increased synthesis of RNA and proteins
in cells of the reproductive tract (3, 12, 15, 22).
Zearalenone acts in animals as feminising agent even at
low concentrations, but higher concentrations (50-100
ppm) disturb reproduction hindering fertilisation,
ovulation, embryos implantation and their normal
development, and impairment of newborn animals (14,
23, 24). Previous achievements on the action of
zearalenone on the activity of immunological system in
polygastric animals are fragmentary and often
incoherent (8). Therefore, our studies should clarify the
relationship between the presence of zearalenone in
blood of sick sheep and formation of subpopulation of
lymphocytes CD4+ and CD8+ playing a fundamental role
in the development of specific immune response and in
immunoregulation.
The aim of the study was the evaluation of the
expression of CD4+ and CD8+ on T lymphocytes of
peripheral blood of sheep with natural zearalenone
mycotoxicosis.
Experimental animals. The study was carried
out in a sheep farm with 150 females and 50 males of
synthetic meet-prolific line BPC (37.5% Polish lowland
sheep, 12.5% Finish or Romanowska sheep, 25%
Berrichon du Cher, 25% Charolaise) and SPC (37.5%
Polish lowland sheep, 12.5% Finish or Romanowska
sheep, 25% Suffolk, 25% Charolaise) being in a
continuous productive cycle. Local Ethics Commission
has approved the studies.
The animals were kept in two breeding
systems. The first group of sheep (50 females and 15
males) was kept in the stabling system under a roof
throughout the year without seasonal pasture grazing.
The animals were fed the concentrate feed containing
soy, wheat, and corn pellets in 1:1:2 proportion, as well
as wheat bran with added vitamins and minerals. Bulky
feed consisted of straw and hay in proportion 2:1. The
animals received water ad libitum. Lambs were with
their mothers from the birth until they reached slaughter
weight. In this group, zearalenone mycotoxicosis
developed regardless of the season and manifested itself
in the prolapse of the anus and rectal mucosa. The
prolapsed mucosa was in light to dark red colour and
was covered with grey fibrinous exudate. The affected
sheep showed increased intestinal peristalsis manifesting
242
itself in continuous tensing without defecation. The
animals were stooping or lying with their necks
stretched. In the final stage of the disease, intense
peristaltic contractions caused the rectal and intestinal
prolapse, which was the direct cause of 35 deaths.
Despite veterinary interventions, i.e. administration of
tranquilisers and muscle relaxants, as well as purse-
string sutures put in around the reponated rectum and
anus, necrosis of the mucosa occurred, which led to its
perforation and peritonitis.
In the second system, the sheep (100 females
and 35 males), were kept on a pasture from spring until
late autumn and additionally fed wheat pellets and hay.
The animals had unrestricted access to water. Lambs
stayed with their mothers for 70 d; however, from day
14, they additionally received crushed oat, meadow hay,
and dry sugar beet ad libitum. In this group, the clinical
symptoms of the disease were not observed; however,
isolated deaths of some lambs caused by mechanical
injuries were noted.
The examinations were performed on 10 sheep
from the first breeding system (group I) and on 10 sheep
from the second breeding system (control group II). To
provide uniformity of the material, only sheep in the
first stage of the disease (i.e. prolapse of the anus and
rectum) were included (Fig. 1).
In both groups, the concentration of
zearalenone and lymphocyte subpopulations were
determined in blood samples collected from the jugular
vein. Furthermore, the presence of zearalenone was
determined in the samples of individual ingredients of
concentrates (soy, wheat bran and corn pellets, wheat
bran and pressed oat) constituting the dietary intake of
sheep from group II.
Determination of zearalenone and α-
zearalenol in concentrates. The detection and
quantitative determinations were performed using the
HPLC method. Analyses involved injection of 100 μl of
the sample on the chromatographic column maintaining
the conditions similar to the ones for preparation of
standard curve.
Determination of zearalenone and α-
zearalenol in blood plasma. The blood samples were
immediately placed in the pre-chilled tubes containing
heparin and centrifuged at 1,500 x g for 20 min at 4°C.
Qualitative and quantitative determinations of
zearalenone and its metabolites were performed by
HPLC analysis.
Determination of lymphocyte surface
antigens CD4+ and CD8+. Blood from sheep of both
groups was collected from the jugular vein into 2 ml
EDTA tubes. Monoclonal antibodies conjugated with
fluorochromes (CD8:FITC and CD4 RPE, Serotec
Immunological Excellence, England) were used. The
antibodies were specific for ovine CD4+ and CD8+
lymphocyte antigens. All tests were done according to
the manufacturer’s procedure. For all probes, the flow
cytometry method was applied (flow cytometer- Epics
XL, Beckman-Coulter, Comesa CH-Werfen Company,
USA), and a single step labelling was conducted.
Statistical analysis. The obtained results were
statistically analysed with Statistica 5.0 programme. The
significance of differences between control group and
investigated group was verified by t-Student test.
Results
Results of mycotoxicological analysis of
concentrates used for feeding sheep are presented in
Table 1. All fodder samples contained zearalenone
mycotoxin, in both forms: the parent compound, i.e.
zearalenone and its metabolite - α-zearalenol. The
highest levels of zearalenone were found in ground corn
meal (14.49 μg/kg) and in wheat bran (13.62 μg/kg),
whereas α-zearalenol was detected in ground wheat
meal (4.05 μg/kg).
Table 2 illustrates the plasma levels of
zearalenone in the affected sheep. Zearalenone, as the
parent compound, was present in all plasma samples and
its concentration ranged from 4.44 to 15.68 ng/kg. α-
zearalenol was detected in six out of ten examined
plasma samples; in samples 1 and 2 its levels were
higher than those of the parent substance, 13.76 and 7.64
ng/kg, respectively.
Cytometric analysis of the subpopulations of
CD4+ and CD8+ lymphocytes as well as the ratio of
CD4+:CD8+ lymphocytes are presented in Figs 2, 3, and
4. Fig. 2 demonstrates that the percentage of CD4 +
lymphocytes was significantly (P≤0.01) lower in the
experimental group (group I) than in controls (group II),
both in individual sheep and in relation to its mean
values in groups. In group I, the percentage of CD4 +
lymphocytes ranged from 18.40 to 28.10% with the
mean value of 24.07+/-0.9. In group II, the extreme
percentages of CD4 + lymphocytes were found to be
26.3% and 40.2% with the mean value of 31.7+/-1.5.
Similar results were obtained for the
percentages of CD8+ lymphocytes in the affected sheep
compared to controls, both in individual sheep and in
relation to mean values in groups (Fig. 3).
The ratio of CD4/CD8 lymphocytes in group I
was higher than that in controls and ranged from 1.51 to
2.76. In group II, the lowest value of the CD4/CD8
lymphocyte ratio was 1.1 while the highest one - 1.81,
and these two values were within normal limits for
sheep (Fig. 4).
Discussion
The clinical or subclinical course of
zearalenonosis in ruminants is highly dependent on the
species, breed, and age of animals, as well as feed and
season, as the incidence of intoxications in the autumn-
winter period is higher. Moreover, our findings show
that the system of maintenance and feeding of animals is
extremely important, which is indicated by the presence
of overt zearalenonosis in the experimental group of
sheep (group I). The animals of this group were kept in
sheepfolds throughout the year and were fed
concentrates with high levels of zearalenone and its
metabolite (α-zearalenol) (Table 1). This facilitated the
accumulation of zearalenone, reaching the dose, which
induced the clinical symptoms of the disease.
 243

Table 1
Concentrations of zearalenone and α-zearalenol in concentrates
Zearalenone α-zearalenol
Concentrate type
μg/kg
Corn pellets 13.62 3.44
Soy pellets 3.87 2.87
Wheat pellets 3.07 4.05
Wheat barn 14.49 3.46

Table 2
Concentrations of zearalenone and α-zearalenol (μg/kg) in blood plasma of the affected sheep (group I)
Zearalenone α-zearalenol
No. of sheep.

1 6.04 13.76
2 6.52 7.64
3 4.64 0.00
4 8.68 5.84
5 4.44 0.00
6 10.36 5.72
7 9.76 0.00
8 7.24 4.16
9 12.08 10.08
10 15.68 0.00

Fig. 1. Entire prolapse of the anal and rectal mucous membrane in sheep with zearalenosis.
 244

Fig. 2. Percentage of CD4+ lymphocytes in peripheral blood in sheep with zearalenone mycotoxicosis and in controls
(α+/-SD).

Fig. 3. Percentage of CD8+ lymphocytes in peripheral blood in sheep with zearalenone mycotoxicosis and in controls
(α+/-SD).

Fig. 4. The CD4+:CD8+ ratio in peripheral blood in sheep with zearalenone mycotoxicosis and in controls (α+/-SD).
No clinical symptoms were observed in the
control sheep (group II), which spent the spring-late
autumn period on the pasture and were fed concentrates
containing ground wheat only during winter, when the
lowest levels of zearalenone were found.
The typical symptoms accompanying
zearalenonosis in sheep include the 10%-20% decrease
in fertility, metritis, and abortions. Our study revealed
that zearalenonosis in sheep may also be accompanied
by prolapses of the anus and rectal mucosa, increased
intestinal peristalsis or complete prolapse of the rectum
with intestines in the final stage. Such symptoms were
the direct cause of numerous deaths observed in the
affected sheep (group I). The pathomechanism of
clinical alimentary symptoms in sheep with zearalenone
mycotoxicosis is difficult to explain as the literature
lacks any reports on the subject. Thanks to the presence
of a phenol ring in its structure, zearalenone is capable
of binding to the oestrogen receptor competitively to
oestradiol (the duodenum and large intestine) (2, 17).
Detoxication of Fusarium mycotoxins,
including zearalenone (ZEA), occurs mainly in the liver
and, to lesser degree, in the gastrointestinal tract; in
ruminants most likely in the terminal part of the
masseter with the bacterial flora (20). These changes
precede absorption. Almost 90% of ZEA is reduced to
α-ZOL and β-ZOL. According to Dänicke et al. (4),
ZEA and its metabolites are detected in ruminant bile in
the amounts reaching 68% β - ZOL, 24% α - ZOL, and
8% - ZEA. Recently, a number of reports concerning
biotransformation of mycotoxins in various animal
species by intestinal microorganisms were published.
The majority of them describe changes in
deoxynivalenol and Fusarium mycotoxin levels in pigs
(5, 7, 10). As far as ZEA is concerned, it was suggested
that intestinal microorganisms hydrolyse the parent
compound only to α-ZOL (11). Our results confirm that
biotransformation processes occur earlier in the plant
material (Table 1), which is demonstrated by the
presence of α-zearalenol, whose hormonal activity is
several times higher than that of the parent substance,
245
i.e. zearalenone. On the other hand, results of
mycotoxicological analysis, especially samples 1 and 2,
which contained higher amounts of α-zearalenol than the
parent compound, demonstrate that intoxication was
long lasting or biotransformation, or rather
biostimulation of zearalenone (i.e. enzymatic conversion
of zearalenone to α-zearalenol), was quicker than the
process of detoxication (21).
Beside their toxic action, Fusarium mycotoxins
show adverse immunosuppressive effects impairing
cellular and humoral mechanisms of non-specific and
specific immunity (19). A few studies concerning the
effects of Fusarium mycotoxins on the immune system
indicate imbalance in the production of cytokines by
helper lymphocytes Th1 and Th2 (1). In pigs receiving
the feed with fumonisin B1, the level of IL-4 decreased
whereas the production and release of IFN-γ by T
lymphocytes increased (6). In piglets, fumonisin B 1
inhibited the specific humoral immunity, which was
manifested in decreased serum levels of specific
antibodies after vaccination, and reduced expression of
mRNA for IL-10 (19). Moreover, in vitro studies
demonstrated that fumonisin B1 had proinflammatory
properties visible in increased production of nitrous
oxide by macrophages in rats and increased TNF-α
synthesis by LPS – stimulated macrophages in mice (1).
Our results demonstrate explicitly that in sheep,
zearalenone and its metabolite (α-zearalenol) induced a
significant decrease in the percentage of lymphocytes
TCD4+ and TCD8+ as well as an increase in their ratio
compared to the control group. There is lack of reports
in the literature concerning immune phenomena during
natural zearalenone mycotoxicosis in sheep and
therefore it is difficult to confront our findings. Similar
results concerning mice receiving zearalenone were
published by Ben Salah-Abbes et al. (1), who
demonstrated that the percentage of CD3+, CD4+, and
CD8+ T lymphocytes as well as percentage of B and NK
cells in the peripheral blood decreased significantly.
Moreover, levels of proinflammatory cytokines and G
and serum M immunoglobulins decreased. Most likely,
 246
immunosuppression accompanying the zearalenone
mycotoxin-induced intoxication cannot be explained by
a single mechanism.
In the immune response to infection or vaccine
antigen, the helping/inducing TCD4+ and
+
suppressive/cytotoxic TCD8 lymphocytes act as
regulatory cells; their proportion determines the profile
of the induced immune response (9, 16). In healthy
animals, their suitable number and appropriate
TCD4+/TCD8+ ratio are maintained. This fact was
confirmed by our results in control sheep (group II)
where the CD4+:CD8+ proportions were within normal
limits (13). However, in the affected sheep (group I), the
percentage of CD4+ and CD8+ lymphocytes was found
to be significantly reduced and their ratio markedly
increased when compared to group II. Mycotoxins could
both stimulate and suppress immunological system,
leading often to hypersensitivity (8). Thus, it may be
assumed that the suppressive action of zearalenone
observed in our study modulates the cellular specific
immune response. Immunosuppression of cellular
mechanisms of anti-infectious immunity plays a
significant role in overcoming the post-vaccination
immunity in sheep, which may result in increased
susceptibility of animals to infections and decreased
effectiveness of vaccines used for specific
immunoprophylaxis (18, 19).
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