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Israel Journal of Veterinary Medicine  Vol. 70 (3)  September 2015 Yildirim, Y. 52
Molecular and Serological Investigation of Akabane Virus Infection
in Cattle in Kars-Turkey
Yildirim, Y.,
1
Gökçe, G.,
2
Kirmizigül, A.H.,
2
Erkiliç, E.E.,
2
Yilmaz, V.,
1
Tan, M.T.
3
and Özgünlük, İ.
4
1
Kafkas University, Faculty of Veterinary Medicine, Department of Virology, Kars-Turkey.
2
Kafkas University, Faculty of Veterinary Medicine, Department of Internal Medicine, Kars-Turkey.
3
Adnan Menderes University, Faculty of Veterinary Medicine, Department of Virology, Aydın-Turkey.
4
Harran University, Faculty of Veterinary Medicine, Department of Virology, Şanlıurfa-Turkey.
*
Corresponding Author: Dr. Yildirim, Y., Department of Virology, Faculty of Veterinary Medicine, University of Kafkas, Kars 36300, Turkey.
Tel: +90 474 2426807/5136, Fax: +90 474 2426847. Email: yayildirim@hotmail.com
ABSTRACT
Akabane virus (AKAV) is an insect-transmitted disease in ruminants that causes abortions, mummifed
fetuses, premature birth, stillbirth and congenital arthrogryposis hydranencephaly. Tis study was a serological
and virological examination of the AKA virus in herds with and without abortion problems. Blood serum
samples were collected for this purpose from 326 cows chosen at random; four of these cattle herds had
abortion problems (n=138) and 5 cattle herds did not have abortion problems (n=188). In the same way,
blood serum samples were obtained from 25 bulls in herds with abortion problems and 25 bulls in herds
without abortion problems. Te blood serum samples were tested with the Competitive Enzyme Linked
Immunosorbent Assay (C-ELISA) for the presence of the AKAV antibody. AKAV seroprevalence rates
were found to be 2.2% in cows and 2% in bulls. Serological data in cows was found to be 2.9% in herds with
abortion problems and 1.6% in herds without abortion problems. In the bulls, seropositive results were only
identifed in one bull’s blood sample from the herds with abortion problems. In the virological studies, the
presence of AKAV nucleic acid was examined in 10 randomly selected seronegative and 8 seropositive blood
samples using reverse transcription polymerase chain reaction (RT-PCR). AKAV nucleic acid was not found
in any of the seropositive or seronegative samples.
Keywords: Akabane Virus; Cattle; C-ELISA; RT-PCR; Turkey.
INTRODUCTION
Akabane is a pathological infection spread by biting midges
(Culicoides spp.) in cattle, sheep and goats. In ruminants, the
disease is observed particularly in the rainy part of the sum-
mer when the vectors are active. Te agent has been isolated
from midges and infected cattle in many countries (1-5).
Te akabane virus (AKAV) generally causes short-lived
subclinical viremia without signifcant clinical symptoms
in sensitive ruminants. Te infection results in stillbirths,
abortions, and congenital arthrogryposis-hydranencephaly
(AH) syndrome in the 2
nd
and 3
rd
trimesters of pregnancy
with lower incidence rates in the 1
st
trimester (5-7). However,
certain strains of AKAV are known to cause encephalomy-
elitis in adult cattle and calves (8-10). Signifcant economic
losses may occur, depending on how the pathogenesis of the
AKAV infection develops in pregnant animals. Akabane virus
is a members of the Simbu serogroup, genus Orthobunyavirus,
family Bunyaviridae (11, 12).
AKAV infection was confrmed by histopathology, im-
munohistochemistry, serology, and genetic analysis (13). In
order to determine the specifc antibodies that have devel-
oped against AKAV, methods such as serum neutralization
(SN), hemagglutination inhibition (HAI) and enzyme linked
immunosorbent assay (ELISA) can be used (14, 15). In addi-
Israel Journal of Veterinary Medicine  Vol. 70 (3)  September 2015 53 Akabane Virus in Cattle in Kars-Turkey
tion, RT-PCR and real-time RT-PCR techniques are used
to identify the viral genome of AKAV (9, 13).
Te aim of this study was to defne, in cattle, the mo-
lecular and serology of AKAV, which has been shown to be
active in western Turkey, in three provinces in North-eastern
Anatolia. In this way, we expected to obtain virological and
seroepidemiological datas authorizing some hypotheses on
the possible AKAV circulation pathways among the neigh-
boring regions. Another aim is to determine possible the role
played by the akabane virus infection in cases of abortion in
cows.
MATERIAL AND METHODS
Animals and Serum Samples
Blood serum samples were collected by jugular vein puncture
into vacuum tubes with clot activator from randomly selected
326 cattle and 50 bull reared in private small scale produc-
tion units from three provinces, i.e. Igdır, Kars and Ardahan
(Figure 1). Of the blood serum samples collected from the
cattle, 138 came from four diferent herds in the areas of
Ardahan and Iğdır, and 188 came from fve diferent herds
without an abortion problem in the Kars region (Table 1).
Te samples of bull blood sera came from animals in the 9
diferent herds of cattle used for sampling (Table 1). After
clotting at room temperature for 15-30 minutes and centrifu-
gation at 3000 g, at 4°C for 10 minutes, sera were carefully
harvested, and stored at –20°C until analysis.
Competitive Enzyme Linked Immunosorbent Assay
(C-ELISA)
For detection of AKA virus antibodies a competitive ELISA
system (ID VET, Productc code: AKAC, France) was used.
Tests were performed according to the manufacturer’s in-
structions. Briefy, 25 µl of test sera, and controls diluted at
1:2 in dilution bufer were added to each well. Following 90
min incubation at 37°C all wells were washed three times and
anti-Akabane-HRP conjugate was added in all well as a 100
µl. Washings were performed again after 30 min incubation
at 37°C. In the fnal step, 100 µl substrate solution tetrameth-
ylbenzidine (TMB) was added to each well and incubated
for 15 min at 21°C and the reaction was stopped by adding
100 µl 0.5M H2SO4. Te OD of each well was read using
an ELISA reader at a wavelength of 450 nm. According to
the kit procedure: the test is validated if the mean value of
the negative control optical density (ODnc) is greater than
0.6 and the mean value of the positive control optical density
(ODpc) is less than 50% of the ODnc.
Te competition percentage (S/N) for each sample was
calculated by the following formula:
S/N = (ODsample ÷ ODpc) × 100
Samples presenting a competition percentage: greater
than or equal to 40% are considered negative, between 30 and
40% are considered doubtful, less than 30% are considered
positive.
Extraction and Reverse Transcription Polymerase
Chain Reaction (RT-PCR)
Ten samples randomly selected from seronegative samples
and eight seropositive samples were extracted using a Figure 1: Geographical positioning of the Turkish provinces in which
the study was performed.
Table 1: Te distribution of samples based on province and herds
Herd
No.
Province
Blood serum
samples from
herds of cattle
without an
abortion problem
Blood serum
samples from
herds of cattle
with an abortion
problem
Bull blood
serum
samples
Animal number Animal number
Animal
number
I Ardahan – 13 6
II Ardahan – 22 6
III Iğdır – 11 6
IV Iğdır – 92 7
V Kars 40 – 6
VI Kars 40 – 5
VII Kars 40 – 5
VIII Kars 40 – 5
IX Kars 28 – 4
Total 188 138 50
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (3)  September 2015 Yildirim, Y. 54
High Pure Viral Nucleic Acid Kit (Roche, Mannheim,
Germany), according to the manufacturer’s recommenda-
tion. Amplifcation of cDNAs by OIAGEN OneStep
RT-PCR kit (Qiagen, Hilden, Germany) was per-
formed using the primer pairs AKAI172F/AKAI560R
( 5' - CAGAAGAAGGCCAAGATGGT- 3' / 5' -
AAGTTGACATCCATTCCATC-3') for detecting the viral
S RNA segment (16). Reverse transcription was conducted
at 50°C for 30 min. Tis mixture was then heated for 95°C
for 15 min to stop the reaction and to activate HotStart
Taq DNA polymerase. Te resulting cDNA was amplifed
by 35 cycles of denaturation at 94°C for 30 s, annealing at
55°C for 30 s and extension at 72°C for 45 s followed by one
step of fnal extension at 72°C for 10 min. Five microliters
of each PCR product was analyzed in 1.5% agarose (Prona
agarosa-BIOMAX, Ardoz-Madrid, Spain) gels containing
ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA).
Akabane virus obtained from the Ankara University Faculty
of Veterinary Medicine Virology Department was used as
positive control.
Statistical Analysis
Statistical analysis was carried out with Statistical Package
for Social Sciences software (Chicago, IL., USA. ) (17).
Signifcant diferences between male and female were evalu-
ated using the chi-square (χ
2
).
Furthermore, in view of the climatic diferences and the
herds with and without abortion problems, statistical analyses
were performed using the Chi-Square (χ
2
) test to determine
whether or not there were signifcant diferences between
seropositive values in the Iğdır region and other two areas.
A P value < 0.05 was regarded as a signifcant diference
between groups compared.
RESULTS
C-ELISA
Te seropositive rate for AKAV infection in the 326 cows
collected as part of the study was 2.15% and for bulls 2.00%.
When examined on the basis of herds, seropositivity in all of
the blood sera obtained from herds with abortion problems
was 2.90%, and seropositivity among these herds ranged from
0% to 4.55%. AKAV seropositivity in the blood sera obtained
from herds without abortion problems was 1.6%, and seroposi-
tivity among these herds ranged from 0% to 5.0% (Table 2).
In terms of AKAV serology in bulls, on the other hand,
seropositivity was not found in blood samples from herds
without abortion problems, and specifc antibodies formed
against the AKA virus were found in the blood serum sample
of only one bull from herd III, which had abortion problems.
When the regions where the sampling for the study was
conducted are evaluated in terms of AKAV seropositivity
(cow and bull serum combined), it was 2.13% in Ardahan,
3.45% in Iğdır and 1.41% in Kars (Table 3).
RT-PCR
Specifc PCR products of 389 base pairs were not detected
in the total 18 sera samples (Figure 2).
Statistical Analysis comparing diferent regions
Taking into consideration male and female cattle, herds with
and without abortion problems and the climatic diferences,
no signifcant diferences (P > 0.05) were found as a result
Table 2: Te seroprevalence of AKAV infection according to the
sampled herds
Herd
No
Cattle samples Bull samples
Te number
of materials
C-ELISA
Te number
of materials
C-ELISA
+ % + %
I* 13 0 0 6 0 0
II* 22 1 4.55 6 0 0
III* 11 0 0 6 1 16.67
IV* 92 3 3.26 7 0 0
V** 40 0 0 6 0 0
VI** 40 0 0 5 0 0
VII** 40 0 0 5 0 0
VIII** 40 2 5.00 5 0 0
IX** 28 1 3.57 4 0 0
Total 326 7 2.15 50 1 2.00
* Cattle herds with abortion problems.
** Cattle herds without abortion problems.
Table 3: Te seropositivity rates of AKAV infection according to
provinces from which the were materials obtained
Province
Animal Number
(cattle+bull)
Number of
positive sera
prevalence (%)
Ardahan 47 1 2.13
Iğdır 116 4 3.45
Kars 213 3 1.41
Total 376 8 2.13
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (3)  September 2015 55 Akabane Virus in Cattle in Kars-Turkey
of the statistical analyses of the seropositive numbers in the
Iğdır region compared with the other two regions.
DISCUSSION
To date, AKA virus has been found in Australia, China,
Japan, Israel, Korea, Taiwan, and Saudi Arabia (2, 4, 5,
18-21). Its antibodies have been found in cattle from many
countries in Southeast Asia, Middle East, and Africa (22-24).
Te seropositive rate for AKAV infection in the 326 cows
collected as part of the study was 2.2% and for bulls it was
2%. When examined on a herd basis, total seropositivity in
the blood serum obtained from herds with abortion prob-
lems was 2.9% while seropositivity among the herds varied
between 0% and 4.6%. AKAV seropositivity in the blood
serums obtained from herds without abortion problems was
1.6%, and seropositivity among these herds ranged from 0%
to 5%. In terms of AKAV serology in bulls, on the other
hand, seropositivity was not found in bull blood samples from
herds without abortion problems, and specifc antibodies
formed against the AKA virus were found in the blood serum
sample of only one bull from herd III, which had abortion
problems.
When the diferences in seroprevalence rates between the
diferent sample regions are examined, the reason for the high
seroprevalence in the Iğdır region is thought to be due to the
hotter climate and a more widespread vector population.
However, the diference in seropositivity was not statistically
signifcant (P > 0.05). Similarly, statistical analyses found that
the seroprevalence diferences between males and females
and between herds with and without abortion problems were
insignifcant (P > 0.05).
In studies conducted in Turkey and around the world (21,
25-27), the seroprevalence of the akabane infection has been
found to vary between 0.14% and 22%. Te reasons for the
diferent AKAV seroprevalence rates found in cattle both in
our study and other studies maybe dependent on factors such
as the number of animals sampled, local climatic conditions,
breed sensitivity, age, vector distribution and sampling time.
Te RT-PCR and real-time RT-PCR methods have been
successful molecular tools for the confrmatory diagnosis of
Akabane disease by identifying the viral genome (3, 4). Tey
have allowed the detection of AKAV RNA in a variety of
clinical samples such as blood, tissues, fetuses and swabs. In
this study serum samples were used. Several PCR assays have
been developed for the detection of AKAV (5, 16, 28, 29). Te
RT-PCR has the advantages of speed, specifcity, and sensi-
tivity for the detection of AKAV RNA. Specifc probes have
been developed to detect AKAV using PCR technique. Te
use of PCR allows sensitive confrmation of the presence of
AKAV in infected animals and may also be useful for phyloge-
netic and epizootiological studies in both natural and AKAV
susceptible hosts. We used the primer pairs AKAI172F/
AKAI560R (5'-CAGAAGAAGGCCAAGATGGT-3'/5'-
AAGTTGACATCCATTCCATC-3') for viral S RNA
segment (16) and AKAV RNA was not detected in 18 sera
samples. Even though this study found no specifc DNA
product (389 bp in length), this situation could be due to a
low rate of infection or a lack of infectivity in serum samples
tested.
In recent years, with the importation of cattle and sheep
from abroad, the incidence of many diseases, especially abor-
tion diseases, has increased in Turkey, posing serious threats
to cattle and sheep industry. Surveys on abortion diseases in
domestic cattle and sheep have been carried out, but most
were restricted to Infectious Bovine Rhinotracheitis (IBR),
Bovine Viral Diarrhea (BVD), Blue tongue (BT), brucel-
losis, and other diseases. In spite of akabane being one of
the important pathogenic microorganisms causing abortion,
akabane virus has not received adquate attention in ruminants
in Turkey. In order to develop an efective prevention and
control program to fght against abortion diseases in rumi-
nants, it is important to obtain epidemiological information
on the prevalence of akabane disease in cattle and sheep.
To this end, we found specifc antibody against akabane
virus and studied the prevalence rate of the abortion disease
caused by akabane virus in cattle from various North-eastern
Figure 2: Te result of AKAV PCR in serum samples. Line M: 100 bp
DNA Ladder; Lines 1,2: positive control (389 bp); Lines 3,4: Samples
used for the study; Line 5: negative control.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (3)  September 2015 Yildirim, Y. 56
provinces of Turkey. Even though we could not identify the
viral genome in the molecular study that was conducted,
demonstrating the presence of the disease serologically is an
important piece of data.
Considering the Turkey’s geographical location, this
country forms a bridge between Asia-Europe for transition
of many contagious diseases, such as the AKAV infection.
Further transversal epidemiological serosurvey would be
necessary for exploring the AKAV infection occurrence in
this world area regrouping all borderline provinces of Turkey,
Georgia, Armenia, Azerbaijan and Iran and for evidence and
the preferential routes of virus circulation.
In conclusion, the results obtained in the present study
showed that AKAV is not common in North-eastern Anatolia
region, which is the most important cattle production area in
Turkey. Consequently, sanitary programs for preventing and
controlling the akabane disease may be based not only on
vaccination plans and vector eradication but also on control
of cattle movement between neighboring countries.
ACKNOWLEDGEMENT
Tis project was supported by the Commission for the
Scientifc Research Projects of Kafkas University (2011-
VF-04). Te authors thank Ankara University Faculty of
Veterinary Medicine Virology Department for providing the
PCR positive control virus.
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