Bull Vet Inst Pulawy 59, 295-301, 2015

DOI: 10.1515/bvip-2015-0043

Effects of flunixin and florfenicol

combined with vitamins E and/or C
on selected immune mechanisms in cattle
under conditions of adaptive stress

Renata Urban-Chmiel1, Rafał Stachura1,2, Piotr Hola2,

Andrzej Puchalski1, Marta Dec1, Andrzej Wernicki1
1Sub-department of Veterinary Prevention and Avian Diseases,
Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine,
University of Life Sciences, 20-033 Lublin, Poland
2Agromarina Farm, Kulczyn-Kolonia 48, 22-235 Hańsk, Poland.

renata.urban@up.lublin.pl

Received: November 11, 2014 Accepted: June 08, 2015

Abstract

The aim of the study was to evaluate the effect of flunixin and florfenicol administered in combination with vitamin E or C
on selected leukocyte immune mechanisms and on the inflammatory process during the first few weeks in the feedlot. Fifty
calves divided into 5 groups (n = 10) received florfenicol and flunixin with vitamin E or C. Blood was collected on the 1st, 3rd,
7th, 14th, 21st, and 28th d of the experiment. Intracellular metabolism (NBT), apoptosis, chemotaxis, susceptibility to
M. haemolytica leukotoxin, and expression of β2-integrins were determined in leukocytes. The symptoms of respiratory tract
infection were observed in 40% of calves in control group, while in the other groups the morbidity rate ranged from 10% to 20%.
Leukocytes showed decreased NBT, and the mean values for apoptosis ranged from 14% to 24%. The lowest percentage of
apoptotic cells was observed in the calves that received florfenicol with flunixin and vitamins E and C. The chemotactic activity
confirmed the significant inhibitory effect of the preparations on migration of the cells. A significant decrease (P ≤ 0.05) in the
susceptibility of leukocytes to leukotoxin was noted in the group that received florfenicol and flunixin with vitamin E. Expression
of β2-integrin receptors was the lowest in calves receiving florfenicol with flunixin and vitamin E or C. The application of an
antibiotic and a non-steroidal anti-inflammatory drug with antioxidants protected the leukocytes involved in defence against
M. haemolytica virulence factors and effectively limited oxidative stress in the calves.

Keywords: calves, respiratory diseases, apoptosis, florfenicol, flunixin, vitamin E, vitamin C, cellular immunity.

Introduction respiratory syndrome in cattle are about £60 million

Bovine respiratory disease complex (BRDC) is
a serious health and economic problem in feedlot cattle
herds due to the involvement of numerous etiological
agents, species-specific predispositions, and production
technology.
Herd losses (16%-30%) resulting from increased
morbidity rate, growth inhibition, reduced weight gain,
decreased feed consumption and conversion (14%-
20%), as well as high costs of prevention and treatment
necessitate comprehensive measures to reduce
economic losses (22, 23). In Great Britain, the total
costs incurred for prevention and treatment of
a year, while in the European Union annual
expenditures equal €576 million (5).
Numerous infectious agents are involved in the
aetiology of BRDC, including bovine respiratory
syncytial virus (BRSV), parainfluenza-3 virus (PI-3),
bovine viral diarrhoea virus (BVDV), herpesviruses
BHV-1 and BHV-3, coronaviruses, and bacteria such
as Mannheimia haemolytica, Pasteurella multocida,
Histophilus somni, Klebsiella pneumoniae,
Actinobacillus spp., and mycoplasmas (3, 10, 11, 23,
30). A fundamental role in the aetiopathogenesis of the
syndrome is played by viruses, which damage cells of
the ciliary epithelium and impair phagocytosis

© 2015 R. Urban-Chmiel et al. This is an open access article distributed under the Creative Commons Attribution-
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296 R. Urban-Chmiel et al./Bull Vet Inst Pulawy/59 (2015) 295-301
mechanisms, preceding the development of pathological
changes most often induced by bacteria. The
immunosuppressive effect of viruses involves inhibition
of neutrophil migration and activity, proliferation of
B cells, impairment of NK cell activity, induction of
apoptosis in CD4+ T cells, and induction of the
production and release of proinflammatory cytokines
(IL-1, 8, TNFα). Impairment of defence mechanisms of
the lungs and the efficiency of bronchus-associated
lymphoid tissue creates favourable conditions for the
colonisation and development of bacteria, as well as for
the expression of their pathogenic factors (12, 15).
Environmental and infectious agents usually play
a synergistic role in the disease process. The most
important predisposing factors are: stress associated
with transport (excessive speed, noise, vibrations),
microclimatic parameters, limited access to water,
social stress, loading and unloading, and excessive
physical exertion associated with maintaining
a standing position (9, 19, 28).
An increased morbidity rate in calves with
symptoms of BRDC occurs during the first 45 d in the
feedlot. One of the factors determining morbidity is
body weight upon entering the feedlot. Calves with low
body weight (<180 kg) are more susceptible to illness,
partly due to a decreased resistance to the negative
effect of environmental stress factors associated with
transport and adaptation (4, 19).
The aim of the study was to evaluate the effect of
flunixin with florfenicol in combination with vitamin E
and/or C on selected leukocyte defence mechanisms
and on the course of the inflammatory process in cattle
during the first few weeks in the feedlot.
Material and Methods
Animals. The study was performed on a group of
50 young Simmental beef cattle (males), aged
6 months, with a body weight of about 160 kg. Prior to
entering the feedlot, all animals underwent an
evaluation of their clinical condition. In addition,
ELISA (Cypress Diagnostics, Belgium) was conducted
to evaluate antibodies against bovine respiratory
syncytial virus (BRSV), and the immunodiffusion test
TRU RSV (Meridian Sciences, Belgium) was used for
direct detection of the F and G genes of the virus in
upper respiratory tract secretions. Both tests were
performed and interpreted according to the
manufacturer’s recommendations.
During the feedlot period, the animals received
total mixed rations – TMR (ground grain, rapeseed,
rolled cereals, hay, straw, spent grain, and maize silage)
in amounts ranging from 3.5 to 4 kg, together with 2 kg
CJ feed mix per calf, as well as hay ad libitum.
The animals were divided into 5 equal groups.
Group 1 was the control. The animals in the
experimental groups received once, on the first day in
their feedlot, florfenicol and flunixin in combination
with vitamin E or C (groups 2 and 3), florfenicol and
flunixin in combination with vitamins E and C (group
4), or florfenicol and flunixin without vitamin
supplements (group 5). The animals were clinically
examined, including measurements of rectal
temperature, on days 3, 7, 14, 21, and 28 in the feedlot.
Blood was collected for sera and into EDTA tubes
(to obtain the cell fraction) on the day the preparations
were administered and on days 3, 7, 14, 21, and 28 of
the experiment.
Isolation of leukocytes. Leukocytes were isolated
by density gradient separation using Histopaque-1083,
according to an earlier study (26). The cell suspension
obtained, with a density of 5 × 106cells/mL, was
suspended in RPMI 1640 medium with 7% FBS,
penicillin G (100 U/mL), and streptomycin (100
μg/mL). The viability of the cells was evaluated by
flow cytometry of cells stained with propidium iodide.
Fluorescence was measured in a flow cytometer (Epics
XL Beckman-Coulter, Comesa CH-Werfen Company,
USA) at 488 nm (20).
Nitrotetrazolium blue reduction assay. The
metabolic activity of the leukocytes was determined by
nitrotetrazolium blue reduction (NBT), according to an
earlier study by Urban-Chmiel et al. (25). A cell
suspension with a density of 5.5 × 106 cells/mL was
incubated for 1.5 h at 37ºC in a 10 mM solution of
iodoacetamide (Sigma, Germany) containing 1 mg/mL
of NBT dye (Sigma-Aldrich, Germany). Absorbance
was read using a microplate reader (BioRad, model
680, USA) at a wavelength of 550 nm.
Leukocyte apoptosis. Apoptotic cells were
identified using a FITC Annexin V Apoptosis
Detection Kit I (BD Pharmingen™), according to the
procedure recommended by the manufacturer.
Cytometric analysis was performed at 488 nm after
adding 400 μL of 10 × Annexin V Binding Buffer to
each sample.
Evaluation of the chemotactic activity. The
chemotactic activity of the neutrophils was determined
using a 48-well Boyden chamber (R&D Systems, USA)
with a nitrocellulose membrane, 3 μm in diameter. The
number of migrating cells was determined under an
optical microscope (Olympus, Japan) at 40 x objective
magnification. Chemotactic activity (%) was
determined according to Alves et al. (2).
Leukocyte susceptibility to M. haemolytica
leukotoxin. Leukocyte sensitivity to leukotoxin (Lkt)
was determined by the MTT assay, according to Vega
et al. (27). The Lkt unit was calculated as the reciprocal
of the culture supernatant dilution causing a 10% death
of bovine leukocytes. Each well of the microplate
(Nunc) was filled with 100 µL of leukocyte suspension
with a density of 2.5 × 106 cells/mL. The plate was
incubated at 37ºC for 1 h, and then 100 µL of Lkt in
RPMI 1640 medium was added to each well. Following
incubation (45 min at 37ºC), 20 µL of MTT
dye (5 mg/mL) (3-(4,5-dimethylthiazol–2-yl)–2,5
diphenyltetrazolium bromide, Sigma, Germany) was
 R. Urban-Chmiel et al./Bull Vet Inst Pulawy/59 (2015) 295-301
added to each well and the plate was incubated at 37ºC
for 4 h. Then 100 µL of 0.04M HCl solution in
isopropyl alcohol was added to each well. To test for
non-specific reactions, samples were prepared using
RPMI 1640 medium with MTT dye and cell suspension
alone with MTT dye. The results were analysed using
a Model 680 microplate reader (BioRad, USA) at 550
and 630 nm. Lkt was obtained from the supernatant of
a reference strain of M. haemolytica serotype 1,
according to Urban-Chmiel et al. (24).
Expression of β2-integrin receptors. Expression
of β2-integrin receptors (CD11a/CD18) was evaluated
using flow cytometry, according to Leite et al. (13).
Cells suspended in PBS (pH 7.4) at a density of
2 × 106/mL were incubated with bovine anti-CD18
antibodies (BAT 75A, VMRD, USA) diluted 1:100 in
PBS with 5% BSA. The cells were washed in PBS with
1% bovine albumin (BSA, Sigma, Germany) and
incubated in FITC-conjugated anti-IgG mouse
monoclonal antibodies (BioKom, UK), diluted 1:100,
for 30 min at room temperature, and then analysed in
a cytometer (FAX, Beckman, USA). The results were
expressed as the percentage of the cells with active
receptors present in the cell membranes.
Statistical analysis. Statistical analysis was
performed using Statistica 10.0 software (Statsoft,
USA). The results were analysed by one-way ANOVA
to compare differences between treatments, and the
post-hoc differences were measured using Tukey’s test.
Correlation of the results was analysed using Pearson’s
coefficient. The correlations between parameters were
determined using Pearson's linear correlation
coefficient. Differences were considered significant at
P < 0.05.
Fig. 1. Mean absorbance values obtained in the NBT test for leukocytes isolated from calves on different days in the feedlot (S ± SD)
* P ≤ 0.05 in comparison to control; Y axis – absorbance 550 nm; X axis – days in feedlot
** P ≤ 0.05 among experimental groups
297
Results
Other than sporadic coughing, no significant
clinical symptoms of respiratory syndrome were
observed in the animals before they entered the feedlot.
Average values for core body temperature were within
physiological ranges for this species. Tests for the
presence of BRSV in the herd showed positive results
in ELISA and in the TRU RSV immunodiffusion test.
The percentage of positive titres was 28% and 32%
respectively, which confirms infection with this virus in
the group of young beef cattle.
Among 50 animals studied, clinical respiratory
symptoms (serous exudate from the nose, intense
coughing, dyspnoea) and a body core temperature over
39.5ºC were observed in 40% of calves from the
control group between days 3 and 14 in the feedlot
(group 1). The total morbidity rate in all animals was
16%, while mortality was 4% and affected only the
control group.
NBT assay used to determine the intracellular
metabolism of leukocytes collected on different days of
the feedlot period showed a statistically significant
(P ≤ 0.05) reduction in absorbance in groups 2 and 4 in
comparison with the controls on days 3, 7, 14, and 21.
In the case of group 4, the differences in mean
absorbance values were statistically significant
(P ≤ 0.05) in comparison with the other experimental
groups (Fig. 1). A significant reduction in NBT in
comparison with the control group was also observed in
group 5 on days 7 and 21 and in group 3 on days 14
and 21 (Fig. 1). The highest absorbance values, ranging
from 0.4 to 0.56, were observed on day 3.
298 R. Urban-Chmiel et al./Bull Vet Inst Pulawy/59 (2015) 295-301

The average values for cellular apoptosis in the
experimental groups on different days of the feedlot
period ranged from 14% to 24% (Table 1). The lowest
percentage of apoptotic cells was observed in the
animals from group 4. The highest percentage of
apoptotic cells was observed in group 5 which received
florfenicol and flunixin without vitamins. The results
obtained were not statistically significant in comparison
with the controls (Table 1). However, statistically
significant differences (P ≤ 0.05) were observed
between group 5, which received florfenicol with
flunixin without of vitamins, and group 4, which
received flunixin and florfenicol with vitamins E and C
(Table 1.)
The chemotactic activity of neutrophils in
different groups of calves confirmed that the
preparations administered to the calves had
a significant effect on cell migration induced by the
chemotactic factor. A statistically significant (P  0.05)
reduction in chemotaxis, in comparison with the control
group, was observed on days 3 and 7 of the feedlot period
in the calves which received florfenicol and flunixin, and
on days 21 and 28 in those which received florfenicol
and flunixin with vitamin C (Fig. 2). Statistically
significant differences in the chemotaxis of cells, in
comparison with the remaining experimental groups,
were observed in group 5 on day 3 and in group 3 on
days 21 and 28 (Fig. 2).
A significant reduction (P ≤ 0.05) in leukocyte
susceptibility to Lkt, in comparison with the controls,
was observed in group 2 on all days of the experiment
(Fig. 3). For group 3, a significant reduction (P ≤ 0.05)
in leukocyte susceptibility to Lkt was observed on days
7, 14, and 21 in the feedlot. In the case of group 5,
significant differences were observed on days 14 and
21 (Fig. 3). In the remaining groups, despite differences
in absolute values, there were no statistically significant
differences in comparison with control group. In the
case of group 2, statistically significant differences
(P ≤ 0.05) in the susceptibility of leukocytes to Lkt
were also noted on day 3 in comparison with the other
experimental groups (Fig. 3).

Table 1. Average apoptosis of leukocytes obtained from calves on particular days in the feedlot (mean ± SE)
Days in feedlot
3 7 14 21 28
Group 1 19.3 ± 4.5 17.2 ± 3.1 18.5 ± 4.5 16.4 ± 5 16.2 ± 5.2
Group 2 16.6 ± 4.8 19.7 ± 5.4 18.4 ± 4.4 17.4 ± 4.8 15.8 ± 5.2
Group 3 17.1 ± 3.8 16.8 ± 4.1 16.5 ± 3.8 15.8 ± 4.2 15.9 ± 4.5
Group 4 15.4 ± 4.2 15.5 ± 5.5 14.1 ± 4.8 14.4 ± 6.2 14.3 ± 1.2
Group 5 23.9 ± 5.6** 21.2 ± 2.9** 19.8 ± 4.1** 19 ± 4.8** 17.8 ± 4.2
** P ≤ 0.05 among the experimental groups

Fig. 2. Chemotaxis of the cells isolated from calves on different days of the feedlot period (S±SD)
* P ≤ 0.05 in comparison with the control group
** P ≤ 0.05 in comparison with other experimental groups
 R. Urban-Chmiel et al./Bull Vet Inst Pulawy/59 (2015) 295-301 299

Fig. 3. Susceptibility of leukocytes to M. haemolytica Lkt on different days in the feedlot

* P ≤ 0.05 in comparison with the control group

Fig. 4. Expression of β2-integrin receptor fragments (CD18/CD11a) in the membranes of leukocytes isolated from calves on different days in the
feedlot
* P ≤ 0.05 in comparison with control group
**
P ≤ 0.05 in comparison with group 4

Table 2. Correlation coefficients between expression of β2-integrin receptors and leukocyte susceptibility to M. haemolytica Lkt (r < 0.05)
F-florfenicol, Fl- flunixin

Correlation coefficients
Group
Control (Gr.1) F+Fl+vit.E (Gr.2) F+Fl+vit.C (Gr.3) F+Fl+vit.E&C (Gr.4) F+Fl (Gr.5)
Group 2 0.72* - - - -
Group 3 0.74* 0.8* - - -
Group 4 0.72* 0.6* 0.49 - -
Group 5 0.74* 0.6* 0.4 0.7* -

* Significant correlation

Furthermore, immunofluorescent staining and percentage of expression of β2-integrin receptors was

flow cytometric analysis revealed the differences in the the lowest in the animals which received florfenicol
expression of β2-integrin receptors in leukocytes of and flunixin in combination with vitamin E or C
calves from different experimental groups. The (groups 2 and 3); the values obtained were 50%
300 R. Urban-Chmiel et al./Bull Vet Inst Pulawy/59 (2015) 295-301
(Fig. 4). Statistically significant differences in
comparison with the controls were observed in group 2
on days 3, 7, and 14, and in group 3 on day 14 in the
feedlot. A significant (P ≤ 0.05) decrease in the
expression of β2-integrin receptors in leukocyte
membranes was also observed in the group receiving
florfenicol and flunixin without antioxidants (group 5)
on days 7 and 14. Moreover, in comparison with group
4, statistically significant differences were noted in the
expression of β2-integrin receptors in group 2 on days
3, 14, 21, and 28 in the feedlot, and in group 3 on days
3 and 28.
The analysis of the correlation coefficients
between the expression of β2-integrin receptors and
leukocyte susceptibility to Lkt showed that these
parameters were correlated in all experimental groups.
The coefficients had values of r > 0.7. The correlations
in different experimental groups ranged from r = 0.4 to
r = 0.8 (Table 2).
Discussion
The study showed a varied inhibitory effect of
florfenicol and flunixin, alone or in combination with
vitamins E and/or C, on cellular mechanisms of
systemic defence, which resulted in reduced
chemotaxis of neutrophils on different days in the
feedlot. The strongest protective effect of leukocytes
against the cytotoxic effect of M. haemolytica Lkt was
observed following the application of florfenicol and
flunixin in combination with vitamin E. This resulted in
a significant decrease in the susceptibility of cells to
Lkt, correlated with a decrease in the expression of
M. haemolytica receptors of β2-integrin fragments
CD18/CD11a, which are specific for leukotoxin (27). It
should be noted that increased susceptibility of
leukocytes to leukotoxin is a key element in the
development of respiratory syndrome induced by
M. haemolytica in cattle.
The changes observed may be due to the inhibition
of the inflammatory reaction, which contributed to the
reduction in the morbidity rate during the first few
weeks in the feedlot. Infiltration of neutrophils induced
by proinflammatory factors in the early stage of
inflammation has been shown to be a significant
element of the defence process. A prolonged process of
leukocyte activity increases the amount of proteolytic
enzymes and free radicals released, and the latter in
turn increase damage to cells involved in protection
against respiratory infections (1, 18). In a study
conducted on cows in the perinatal period, Pinotti et al.
(14) observed a slight increase in chemotactic activity
following administration of α-tocopherol, which,
according to the authors, may be the effect of systemic
immunomobilisation in the cows during this period.
Another study (16) confirmed that antioxidants may
inhibit the inflammatory response within pulmonary
tissue, resulting in reduced chemotaxis and
phagocytosis of neutrophils isolated from mice.
The positive effect of the preparations used is
evidenced by the results obtained in the evaluation of
the intracellular metabolism of leukocytes with the
NBT test. The statistically significant (P ≤ 0.05)
reduction in absorbance on different days of the feedlot
period indicates a beneficial effect on the stability of
intracellular structures, which indirectly indicates
a reduction in oxidative stress in the cells, expressed as
decreased free radical production. According to
Esfandiari et al. (8) and Sabeur and Ball (17), the
reduction reaction of nitrotetrazolium blue can be used
to evaluate the level of oxidative stress on the basis of
the amount of oxygen radicals produced and released
by the cells, which has been confirmed in studies on
humans and rats.
The substantial increase in the resistance of
leukocytes to the cytotoxic effect of M. haemolytica
Lkt, accompanied by reduced expression of β2-integrin
membrane receptors, confirms the significant effect of
the preparations on the cells’ defence mechanisms
directed against virulence factors of the bacteria. The
high correlation (r > 0.5) between the susceptibility of
leukocytes to Lkt and expression of β2-integrin
receptors may be a measurable indicator in determining
the susceptibility of calves to infections induced by
M. haemolytica. The protective effect on cells involved
in the immune response in the lungs may significantly
reduce morbidity in cattle with symptoms of respiratory
syndrome.
The positive effect of the preparations is also
evidenced by the approximately threefold lower
morbidity rate in the young beef cattle in comparison
with the controls. The results are similar to those
obtained by Chirase et al. (6), who administered
vitamins E and C to calves and achieved a substantial
decrease in their morbidity and mortality rates in
comparison to calves that did not receive antioxidants.
Ekstrand-Hammarström et al. (7) demonstrated that
administration of vitamin E can inhibit the
inflammatory process by inhibiting the production of
proinflammatory cytokines, reducing production of
acute phase proteins and weakening the inflammatory
response of epithelial cells in the lungs. Moreover, in
a study by Weingarten et al. (29), in calves with
symptoms of respiratory syndrome which were
administered florfenicol and flunixin, a reduction in
the disease process was observed in the bronchi and
lungs, expressed as abatement of clinical symptoms.
To sum up, it should be emphasised that
simultaneous administration of an antibiotic and a non-
steroid anti-inflammatory drug together with
antioxidants protected leukocytes involved in defence
against the virulence factors of M. haemolytica. The
preparations also effectively limited the development of
oxidative stress and inflammatory process in the feedlot
calves, resulting in increased resistance to disease. The
 R. Urban-Chmiel et al./Bull Vet Inst Pulawy/59 (2015) 295-301
presented findings may be useful in development of
effective adjuvant therapy.
Conflict of Interests Statement: The authors declare
that there is no conflict of interest regarding the
publication of this article.
Animal Rights Statement: The experiment was
conducted in accordance with the Local Ethics
Commission.
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