https://doi.org/10.52973/rcfcv-e33271
Received: 26/05/2023 Accepted: 17/07/2023 Published: 01/08/2023
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Revista Científica, FCV-LUZ / Vol. XXXIII, rcfcv-e33271, 1 – 5
ABSTRACT
Brucellosis is a zoonotic disease that affects a large number of people
and animals, causing physical disability, workforce loss and signicant
economic losses in the livestock industry. In the current study, it was
aimed to determine and compare the levels of tumor necrosis factor
alpha (TNF–α), interferon gamma (IFN–γ), Procalcitonin (PCT) and
Neopterin in the blood serums of cattle with brucellosis and vaccinated
against brucellosis. The materials of this study consisted of a total 48
blood serums belonging to three basic groups, each consisting of 16
animals. Disease group (1st group) were divided into two subgrups each
consisting of 8 animals that 21st day after abortion and seropositive
7 months pregnant, the vaccinated (2nd group) and the control (3rd
group) groups were divided into two subgroups, each consisting of
8 animals that gave birth 21 days ago and 7 months pregnant. IFN–γ
and PCT levels were determined by sandwich enzyme immunoassay,
TNF–α and Neopterin levels were determined using competitive
inhibition enzyme immunoassay method by using ELISA device. In this
study, TNF–α, PCT and Neopterin levels measured in the blood serums
of the Brucella seropositive (1st), conjunctival Brucella abortus S19
vaccine administered (2nd) and unvaccinated Brucella seronegative
control groups were compared and no signicant difference could be
determined between the subgroups of the groups (P>0.05). There were
a signicant differences between 1st, 2nd, and 3rd groups (P<0.05).
IFN–γ levels determined in the blood serums of 1st, 2nd and 3rd groups
were compared and nosignicant differences were found between the
subgroups of 2nd and 3rd groups (P>0.05), but there were a signicant
differences between the subgroups of the 1st group (P<0.05). Similarly, a
signicant differences were determined between 1st, 2nd and 3rd groups
in terms of IFN–γ levels (P<0.05). As a result, it was thought that detecting
very high serum TNF–α, IFN–γ, neopterin levels in cattle with brucellosis
would be helpful in the diagnosis and follow–up of brucellosis. However,
it was concluded that there is a need for controlled studies comparing
more herds with brucellosis to determine whether the relevant cytokines
can be used in the diagnosis of brucellosis.
Key words: Brucellosis; tumor necrosis factor alpha; interferon
gamma; procalcitonin; neopterin
RESUMEN
La brucelosis es una enfermedad zoonótica que afecta a un gran número
de personas y animales, provocando discapacidad física, pérdida de
mano de obra e importantes pérdidas económicas en la industria
ganadera. En este estudio se tuvo como objetivo determinar y comparar
los niveles de factor de necrosis tumoral alfa (TNF–α), interferón gamma
(IFN–γ), procalcitonina (PCT) y neopterina en el suero sanguíneo de
bovinos, tanto con brucelosis como vacunados contra la brucelosis.
Los materiales de este estudio consistieron en un total de 48 sueros
sanguíneos pertenecientes a tres grupos básicos, cada uno compuesto
por 16 animales. El grupo de enfermedad (1
er
grupo) se dividió en dos
subgrupos, cada uno compuesto por 8 animales, uno 21 días después del
aborto y el otro, seropositivos a los 7 meses de gestación. Los grupos
vacunado (2
do
grupo) y control (3
er
grupo) se dividieron en dos subgrupos,
cada uno compuesto por 8 animales, uno en el que parieron 21 días antes
y en el otro con 7 meses de gestación. Mediante el uso de un dispositivo
ELISA, los niveles de IFN–γ y PCT se determinaron con un inmunoensayo
enzimático tipo sándwich, mientras que los niveles de TNF–α y neopterina
se determinaron a través del método de inmunoensayo enzimático de
inhibición competitiva. En este estudio, se compararon los niveles de
TNF–α, PCT y neopterina medidos en el suero sanguíneo de los grupos
de control seropositivos a Brucella (1
ro
), a los que se les administró la
vacuna conjuctival Brucella abortus S19 de la Brucella (2
do
) y seronegativos
en Brucella no vacunados, no pudiéndose determinar una diferencia
signicativa entre los subgrupos (P>0,05). Hubo diferencias signicativas
entre los grupos 1, 2 y 3 (P<0,05). Se compararon los niveles de IFN–γ en
el suero sanguíneo de los grupos 1, 2 y 3, y no se encontraron diferencias
signicativas entre los subgrupos del 2
do
y 3
er
grupo (P>0,05) pero sí hubo
diferencias signicativas entre los subgrupos del 1er grupo (P<0,05).
Del mismo modo, en referencia a los niveles de IFN–γ, se determinaron
diferencias significativas entre los grupos 1, 2 y 3 (P<0,05). Como
resultado, se pensó que la detección de niveles séricos muy altos de
TNF–α, IFN–γ y neopterina en ganado bovino con brucelosis sería útil en
el diagnóstico y seguimiento de la brucelosis. Sin embargo, se concluyó
que existe la necesidad de estudios controlados que comparen más
rebaños con brucelosis para determinar si las citoquinas relevantes
pueden usarse en el diagnóstico de brucelosis.
Palabras clave: Brucelosis; factor de necrosis tumoral alfa;
interferón gamma; procalcitonina; neopterina
Evaluation of tumor necrosis Factor Alpha, Interferon Gamma,
Procalcitonin and Neopterin levels in Brucella seropositive cattle
Evaluación de los niveles de factor de necrosis tumoral alfa, interferón gamma, procalcitonina y
neopterina en bovinos seropositivos para Brucella
Nevin Tuzcu
1
, Mehmet Tuzcu
2
, Gokhan Akcakavak
3
*
1
Selcuk University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology. Selcuklu, Konya, Turkey.
2
Selcuk University, Faculty of Veterinary, Medicine Department of Pathology. Selcuklu, Konya, Turkey.
3
Yozgat Bozok University, Faculty of Veterinary Medicine, Department of Pathology. Sorgun, Yozgat, Turkey.
*Corresponding Author: gokhan.akcakavak@bozok.edu.tr
Assessment of serum cytokine levels in Brucella seropositive cattle / Tuzcu et al. __________________________________________________
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INTRODUCTION
Although brucellosis is basically an animal disease, it is also
known as one of the most important zoonotic diseases, since
more than 500,000 human cases are reported Worldwide every
year [1]. Brucellosis affects a large number of people, causing
physical disability and workforce loss as well as causing signicant
economic losses in the livestock industry and negatively affecting
the sustainable livestock production [2, 3].
Brucella species are Gram–negative, facultative intracellular bacteria,
in the form of cocci, coccobacillus or short rods, 0.5–0.7 µm in width
and 0.6–1.5 µm in length. The edges are slightly convex and the ends
are rounded. They are appear singly or in pairs, short chains or small
clusters on stained preparations. Sometimes the agents can be seen
in the form of 3–5 chains in preparations made from liquid media [2, 3].
Brucella abortus is the primary agent of infection in the cattle (Bos
taurus and Bos indicus),and it can also infect buffalo (Bubalis bubalus),
bison (Bison bison), sheep (Ovis aries), pig (Sus scrofa domesticus),
camel (Camelus), deer (Dama), horse (Equus caballus), dog (Canis
lupus familiaris) and humans [3, 4]. B. melitensis may cause bovine
brucellosis, and B. suis may rarely cause chronic infection of the
mammary glands in cattle in some Countries, especially in Southern
Europe and Western Asia, where cattle are kept together with sheep
and goats [3, 5].
The most signicant feature of Brucella infections is that the agent
can proliferate both in the phagocytic cells of the reticuloendothelial
system and non–phagocytic cells such as trophoblasts [6]. Cellular
immune response is more important than humoral immune response
in brucellosis [7]. T cell subgroups, macrophages, cytokines released
from these cells and interaction between cytokines play asignicant
role in protective immunity against intracellular pathogens [7, 8].
Neopterin is a cytokine synthesized from monocytes and
macrophages as a result of stimulation of interferon–gamma (IFN–γ)
released from active T lymphocytes. Neopterin is a sensitive indicator
of cellular immunity [9]. IFN–γ is the most signicant macrophage
stimulating cytokine and are synthesized by natural killer (NK), T helper
(Th) and cytotoxic T (Ts) cells. It enhances phagocytosis of macrophages,
stimulates Th1 differentiation and prevents Th2 proliferation [10, 11].
Procalcitonin (PCT) is the precursor of calcitonin hormone produced
in thyroid C cells and responsible in calcium homeostasis [12]. It has
been reported by various investigators that the normal serum levels of
PCT increase one hundredfold in human septicemias [12, 13].
T helper (Th1/Th2) stability plays a signicant role in the formation
of resistance or susceptibility to brucellosis, and cytokines play
asignicant role in the pathogenesis of brucellosis [11, 14]. Studies
in mice (Mus musculus) have shown that type 1 (Th1) cellular immune
response is stimulated in brucellosis. Type 1 cellular response
progresses under the control of tumor necrosis factor alpha (TNF–α),
interferon gamma (IFN–γ), interleukin–12 (IL–12) produced at the
beginning of the disease [10, 15].
There are various studies in the eld of Human Medicine on the role
of cytokines at post–treatment response and follow–up complications
in the pathogenesis of brucellosis [7, 10, 16, 17, 18, 19]. However, studies
on farm animals considered as the source of brucellosis are limited
[20, 21]. In this study, it was aimed to determine and compare the
levels of TNF–α, IFN–γ, PCT and Neopterin in the blood serums of
cattle with brucellosis and vaccinated against brucellosis.
MATERIAL AND METHODS
The samples of the study consisted of 48 blood serums belonging
to three basic groups each consisting of 16 animals. These groups
were the disease group (Brucellosis was diagnosed by the Ministry of
Agriculture and Forestry), the vaccinated group (Brucella abortus S19
conjunctival vaccination was applied to 4–10 months old animals at a
dose of 50 µL ), and the control group (blood was taken three times
with 2 months intervals and Brucella antibody was found negative).
The groups divided into two subgroups consisting of 8 animals were
as follows; disease group (DG) being 21st day after abortion (DG1) and
seropositive 7 months pregnant (DG2), the vaccinated group (VG) and
the control group (CG) to gave birth 21 days ago (VG1/CG1) and being 7
months pregnant (VG2/CG2). The blood samples of the study groups
were taken from 5 different dairy farms that were diagnosed with
brucellosis by the Ministry of Agriculture and Forestry after abortion
complaints and from 2 different dairy farms that were certied free
from Brucella disease between 2014 and 2016. The blood serum
samples were stored at -20°C in a deep freezer (Nuve, DF 590, Ankara,
Turkey) to be used in serological tests.The presence of Brucella
antibodies in the CG, DG and VG were determined by Rose Bengal
plate agglutination test (RBT) and antibody titers were determined
by serum microagglutination test (MAT) according to kit procedures
with commercial kits (Rose Bendoll, SAT–A–DOLL) [3]. TNF–α and
Neopterin levels were determined by competitive inhibition enzyme
immunoassay method, IFN–γ and PCT levels were determined by
sandwich enzyme immunoassay according to kit procedures with
commercial kits (Cusabio, PRC, USA) using ELISA device (Thermo
Fisher, Multiskan FC, USA).
Statistical analysis
Statistical program SPSS (Inc., Chicago, USA 14.0) was used in
the analysis of the obtained data. Comparisons between groups
were evaluated with ANOVA and post hoc Duncan test. The limit of
signicance was accepted as P<0.05.
RESULTS AND DISCUSSION
In the study, slide agglutination tests were applied to the control
group animals 3 times in 2–month periods and all of them were
found to be seronegative. The presence of antibodies in the blood
serums taken from the DG1 and DG2 were scored with the slide
agglutination test, and the antibody titers were determined by the
serum microagglutination test. According to the determined values,
it was determined that DG1 and DG2 groups were statistically similar
among themselves, and VG1 and VG2 groups were statistically similar
among themselves (P>0.05). However, when the antibody titers were
compared between the DG and VG, a statistically important difference
was determined (P<0.05), values were given in TABLE I.
TNF–α, IFN–γ, PCT and Neopterin levels determined in the study
groups were given in TABLE II. In the study, TNF–α, PCT and Neopterin
levels determined in the blood serums of cattle belonging to DG, VG and
CG were compared. While no statistically important difference could
be determined between the subgroups of the groups (P>0.05), there
was a statistically important difference between the groups (P<0.05).
In the study, when the IFN–γ values determined in the blood
serums of cattle belonging to DG, VG and CG were compared; While
no signicant difference could be determined amongthe subgroups
of the VG and CG (P>0.05), there were a statistically important
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TABLE I
Scores determined by slide agglutination test and antibody titers determined by serum microagglutination test of study groups
DG VG CG
DG1 DG2 VG1 VG2 CG1 CG2
Score Antibody Titer Score Antibody Titer Score Antibody Titer Score Antibody Titer Score Antibody Titer Score Antibody Titer
++++ 1/320 +++ 1/160 + 1/10 + 1/10 – – – –
+++ 1/320 +++ 1/320 + 1/20 + 1/20 – – – –
+++ 1/160 +++ 1/160 + 1/20 + 1/10 – – – –
++++ 1/320 +++ 1/320 ++ 1/20 + 1/20 – – – –
++++ 1/320 +++ 1/160 + 1/10 + 1/10 – – – –
+++ 1/320 ++ 1/160 + 1/20 + 1/20 – – – –
+++ 1/320 +++ 1/320 + 1/20 + 1/10 – – – –
++++ 1/160 +++ 1/160 + 1/10 + 1/10 – – – –
++++ 1/320 +++ 1/160 + 1/10 + 1/10 – – – –
(DG; Disease group, DG1; 21st day after abortion, DG2; seropositive 7 months pregnant, VG; vaccinated group, CG; control group, VG1/ CG1; gave birth 21 days ago,
VG2/ CG2; 7 months pregnant, N; Negative)
TABLE II
Averages and standard deviations of TNF–α, IFN–γ, PCT and neopterin levels measured in study groups
Biomarkers
DG VG CG
DG1 DG2 VG1 VG2 CG1 CG2
TNF–α
(ng·mL
-1
)
4.51 ± 1.58
a
4.56 ± 1.64
a
1.94 ± 0.38
b
1.83 ± 0.26
b
0.92 ± 0.24
c
0.88 ± 0.12
c
IFN–γ
(ng·mL
-1
)
633.04 ± 246.53
a
494.03 ± 197.84
b
140.43 ± 60.42
c
141.54 ± 54.28
c
48.14 ± 12.14
d
48.32 ± 12.28
d
PCT
(ng·mL
-1
)
139.24 ± 45.89
a
31.86 ± 4.44
b
30.9 ± 4.24
b
31.51 ± 4.38
b
30.71 ± 3.62
b
28.88 ± 3.02
b
Neopterin
(ng·mL
-1
)
8.80 ± 2.99
a
7.31 ± 2.49
a
4.02 ± 1.18
b
3.89 ± 1.22
b
1.92 ± 0.46
c
1.67 ± 0.22
c
a,b,c
The difference among groups having different letters on the same line were statistically signicant (P<0,05).(DG; Disease group,
DG1; 21st day after abortion, DG2; seropositive 7 months pregnant, VG; vaccinated group, CG; control group, VG1/CG1; gave birth
21 days ago, VG2/CG2; 7 months pregnant)
differences between the subgroups of the DG (P<0.05). Similarly,
when the DG, VG and CG were compared, it was determined that there
were a statistically important differences (P<0.05).
Serum agglutination test (SAT) is an agglutination test that can
detect IgM antibodies very well, but has a lower specicity in detecting
IgG antibodies, since the pH of the antigen prepared with a suspension
of the agent in phenol saline is close to neutral, such as 7.2, Although
it is a sensitive test, it is recommended to be used in combination
with other tests [22]. In this study, as suggested in the literature and
stated in the regulation prepared for free farms by the Ministry of
Agriculture and Forestry, seronegativity was conrmed by applying
Brucella microagglutination and slide agglutination tests to control
animals 3 times in 2–month periods to determine seronegativity.
The presence of Brucella antibodies in the serums of the DG and
VG were determined by the slide agglutination test and scored, and
the antibody titers were determined by the serum microagglutination
test. When the antibody titers were compared between DG and VG, it
was noted that there was a statistically important difference (P<0.05).
The denitive diagnosis of brucellosis is made by bacterial growth
from clinical samples. However, since it is not always possible to
bacterial growth, serological tests gain importance in diagnosis.
The TNF–α receptor complex induces many biological activities in the
target cell. TNF–α is released from activated T cells and macrophages
as a proinammatory cytokine [23]. Palmer et al. [24] vaccinated 10
pregnant cattle with intravenous B. abortus RB51, 5 pregnant cattle
subcutaneously with B. abortus RB51, 5 pregnant cattle subcutaneously
with B. abortus S19, 2 cattle in the control group injected subcutaneous
non–pyrogen solution. They found that placentatitis occurred after 8–12
weeks in intravenously vaccinated cattle, and TNF–α levels increased,
and there was no difference among the subcutaneously vaccinated
group and the control group. Akbulut et al. [19] determined TNF–α
levels in 28 brucellosis cases and 20 healthy individuals in their study
on humans, and reported that the TNF–α levels were statistically
signicantly higher in the patient group compared to the control group.
Similarly, in the expression study of Sahiwal cattle vaccinated with
Brucella abortus S19 by Kumar et al. [25] was reported that IFN–γ,
TNF–α, IL6, and IL10 genes show initial downregulation and then
upregulation. In this study, the mean of TNF–α levels measured in
blood serums taken from DG1, DG2, VG1, VG2, CG1, CG2 were determined
as 4.51, 4.56, 1.94, 1.83, 0.92 and 0.88 ng·mL
-1
,respectively. The levels
of TNF–α determined in the CG subgroups are similiar to the levels
determined by Ercan et al. [26] in healthy cattle. TNF–α averages
determined in DG were found to be higher than other groups (VG and
CG) and were statistically important (P<0.05).
Assessment of serum cytokine levels in Brucella seropositive cattle / Tuzcu et al. __________________________________________________
4 of 5
In this study, the average of IFN–γ levels measured in blood serums
taken from seropositive 21 days ago, seropositive 7 months 21 days
pregnant vaccinated, 7 months pregnant vaccinated, seronegative
who gave birth 21 days ago, and 7 months pregnant seronegative
groups were determined as averaged 633.04, 494.03, 140.43, 141.54,
48.14 and 48.32 ng·mL
-1
, respectively. IFN–γ averages, which were
determined as 48.14 ng·mL
-1
and 48.32 ng·mL
-1
in the control group,
were similiar with the levels determined by Ercan et al. [26] in healthy
cattle. IFN–γ averages determined in the disease groups were found
to be higher compared to the vaccinated groups and control groups,
and this difference was statistically important (P<0.05). Similar results
obtained in this study with the study of Ahmed et al. [7], in which they
determined IFN–γ levels in 27 patients with acute brucellosis and 15
healthy adult individuals, IFN–γ levels were found to be statistically
signicantly higher in the brucellosis group compared to the control
group (P<0.05). Diez–Ruiz et al. [16] reported that serum IFN–γ and
Neopterin levels were found to be signicantly higher in patients
with brucellosis than in the healthy control group. Akbulut et al. [19]
compared serum cytokine levels in 35 patients with brucellosis and a
control group of 20 people, and reported that the averages of serum
IFN–γ and TNF–α levels were higher in patients with brucellosis than
in the control group. El–Boshy et al. [20] compared B. abortus and
B. melitensis infected camels with healthy camels and reported that
they found lower TNF–α and IFN–γ levels in camels with brucellosis.
In the study of Odbileg et al. [27] in camels, cytokine levels produced
by peripheral blood mononuclear cells in response to B. abortus
S19 vaccine were determined and it was revealed that IFN–γ level
increased during the rst week after vaccination. They detected low
level of TNF–α expression compared to the control group. In this study,
TNF–α and IFN–γ levels measured in the serums of the VG were found
to be higher than CG. In studies, low TNF–α in brucellosis patients was
attributed to the short half–life of TNF–α Ahmed et al. [7], while high
TNF–α could be explained by its being a proinammatory mediator
and a high IFN–γ level.
It has been shown in different studies that the serum levels of
PCT, which is measured below 0.1 ng·mL
-1
in the blood serums of
healthy individuals, increases at least ve times in bacterial infections,
exceeds 10 ng·mL
-1
and even exceeds 1,000 ng·mL
-1
[12, 13, 28]. In this
study, the averages of PCT levels in blood serums taken from DG1, DG2,
VG1, VG2, CG1, CG2 were determined as 139.24, 31.86, 30.9, 31.51, 30.71
and 28.88 ng·mL
-1
, respectively. Serum PCT levels determined in the
CG, VG and DG2 were similar with the results determined in healthy
cattle by Ercan et al. [26]. The fact that the PCT level determined in the
abortion group was higher than the other groups is consistent with the
studies showing that the PCT level increased in bacterial infections
[12, 13, 28, 29]. Undetermining difference between the other groups
and the control group may be related to the short half–life of PCT.
Neopterin is a cytokine synthesized from monocytes and macrophages
as a result of stimulation of IFN–γ released from active T lymphocytes.
Neopterin is a sensitive indicator of cellular immunity Ercan et al. [26].
Irmak et al. [30] investigated the diagnostic value of Neopterin levels
in the follow–up of treatment in 20 patients with brucellos is and
reported that Neopterin levels could be used in the follow–up of
patients with Brucellosis and evaluating the success of treatment.
Diez–Ruiz et al. [16] reported that serum IFN–γ and Neopterin levels
in patients with brucellosis were signicantly higher than the healthy
control group. Akbulut et al. [19] investigated serum neopterin levels
in 30 brucellosis and 30 healthy control groups. They reported that
serum Neopterin levels in patients with brucellosis were signicantly
higher than the healthy CG group. In this study, the averages of
neopterin levels in blood serums taken from DG1, DG2, VG1, VG2, CG1,
CG2 were determined as 8.80, 7.31, 4.02, 3.89, 1.92 and 1.67 ng·mL
-1
,
respectively. The averages of Neopterin, which were determined
as 1.92 ng·mL
-1
and 1.67 ng·mL
-1
in CG were similar with the results
determined in the healthy cattle by Ercan et al. [26]. The averages
of Neopterin levels in DG were found to be higher than the VG, CG.
This difference was statistically important (P<0.05). The determined
results are compatible with the literature [16, 19, 30].
There are few studies investigating cytokine levels in farm animals
to elucidate the pathogenesis of brucellosis [20, 27]. In this study,
serum levels of biological markers such as TNF–α, IFN–γ, Neopterin
and PCT, which are used in the diagnosis and prognosis of infectious
diseases in human medicine, were tried to be revealed in Brucellosis
and Brucella–vaccinated cows.
CONCLUSION
In conclusion, although the fact that serum TNF–α, IFN–γ, Neopterin
levels were determined to be quite high in cattle with brucellosis is
thought to be helpful in the diagnosis and monitoring of brucellosis, it
was concluded that there is a need for controlled studies comparing
TNF–α, IFN–γ, Neopterin levels in more herds with brucellosis in order
to determine whether TNF–α, IFN–γ, Neopterin levels can be used in
the diagnosis of brucellosis in the cattle.
Conict of interest
There is no conict of interest between the authors.
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