Investigation of West Nile virus infection in brown bears (ursus arctos) in Turkey

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Eurasian J Vet Sci, 2017, 33, 3, 190-194
190
Eurasian Journal
of Veterinary Sciences
Amaç:
       
 -

Gereç ve Yöntem:
  -
petitive enzyme-linked immunosorbent assay (C-ELISA)


Reverse Transkripsiyon-Polimeraz Zincir Reaksiyonu (RT-

Bulgular:   

Öneri:      -
 -
     

   

Anahtar kelimeler        
PCR
Abstract
Aim: This study is a serological and virological examination
of the West Nile Virus (WNV) in free-ranging brown bears
(Ursus arctos) in Sarikamis district of Kars province of Tur-
ke y.
Materials and Methods: For this purpose, blood samples
from 11 brown bears were tested for antibodies against
WNV using a commercial competitive enzyme-linked immu-
nosorbent assay (C-ELISA). Reverse transcription polymera-
se chain reaction (RT-PCR) technique was used to determine
the presence of West Nile Virus nucleic acid.
Results: 
West Nile virus nucleic acid was not found in samples.
Conclusion:      
bears and other free-ranging mammals in the Turkey is unk-
nown at this time and will require further investigation. This

Keywords: Brown bears, ELISA, RT-PCR, West Nile virus
RESEARCH ARTICLE
Investigation of West Nile virus infection in brown bears (ursus arctos) in Turkey




6
ReceivedAccepted
* yayildirim@hotmail.com
Türkiye’deki boz ayılarda Batı Nil Virüsü’nün araştırılması
Eurasian J Vet Sci, 2017, 33, 3, 190-194

www.eurasianjvetsci.org
Öz

Eurasian J Vet Sci, 2017, 33, 3, 190-194
191
Introduction
West Nile Virus (WNV) can cause a variety of neurological
symptoms such as mild febrile illness, meningitis, encephali-
tis or death in humans, horses, dogs, birds and wild animals
in America, Asia, Africa, Europe and particularly in countries
bordering the Meditterranean Sea (Fauquet et al 2005). Ta-
xonomically, WNV is a member of the Japanese encephalitis
virus complex of the family Flaviviridae, genus Flavivirus,
which also includes Japanese encephalitis virus, St. Louis
encephalitis virus (SLEV), Muray Valey encephalitis virus
(MVEV), and others.
These viruses are mosquito borne, primarily transmitted by
Culex sp, and have wide, overlapping distributions throug-
hout the world (Mackenzie et al 2002). The virus is an en-
veloped virus with a single-stranded, positive sense, 11-kb
RNA genome that transcribes a single polyprotein, cleaved
by host and viral proteases into structural and nonstructural
viral proteins (Chambers et al 1990). Three genetic variants
of WNV have been demonstrated in Russia and these isolates
showed genetically high similarity to those reported from
the United States and Israel (Lvov et al 2004).
Laboratory diagnosis includes the determination of anti-
bodies using enzyme-linked immunosorbent assay (ELISA)
(Dauphin and Zientara 2007), plaque reduction neutralizati-

virus isolation (Lanciotti et al 2000).

diagnosis, ELISA is now routinely used. Padilla et al (2009)


   
WNV in human serum, CSF, brain tissue, mosquito pools, and
avian tissues (Porter et al 1993).
The objective of this study was to investigate the WNV infec-
tion as serologically and virologically in brown bears (Ursus
arctos) in Sarikamis district of Kars province in the nort-

brown bears in Turkey.
Material and Methods
Animals and blood samples
This study evaluated the materials obtained from the study
which aims to place GPS/GPRS collar to the bears captured
  -
gion by Forestry and Water Affairs Ministry, General Directo-
rate of Nature Protection and National Parks (25 December

wildlife in the East Anatolia) and Kuzey Doga Society.
Blood samples were collected from randomly selected 11
       
in Sarikamis district of Kars province of Turkey. Approp-
riately captured bears were taken under anesthesia with
    
blood samples were taken from vena cephalica. For serolo-
gical purposes, serum obtained from the centrifugation of

The blood samples, collected for virological purposes, were
collected in tubes with anticoagulant (EDTA) and centrifuged
at 1500g for 10 min, the buffy coat fraction was removed by
capillary pipette and resuspended in 2 ml of phosphate buf-
fered saline (PBS, 0.01 M, pH 7.2) containing penicillin (100
units/ml), streptomycin (0.1 mg/ml) and amphotericin B
-
se transcription polymerase chain reaction (RT-PCR).
Competitive Enzyme Linked Immunosorbent Assay
(C-ELISA)

Product code: West Nile Competition Multi-species (WNC),
IDvet, Grabels, France) were used to detect anti-pr-E antibo-
dies against the WNV.
-

dilution buffer were added to each well. Following 90 min
  
anti-pr-E antibody peroxidase (HRP) conjugate was added


solution tetramethylbenzidine (TMB) was added to each well


-
velength of 450 nm. According to the kit procedure: the test
is validated if the mean value of the negative control optical
 

󰂫

by the following formula:


  

are considered positive.
iii

Eurasian J Vet Sci, 2017, 33, 3, 190-194
192
Reverse transcription polymerase chain reaction (RT-PCR)
RNA was extracted from leukocyte samples by using the QI-
Aamp Viral RNA Mini Kit (Qiagen GmbH, Hilden, Germany),
     
-

-
mendation.
PCR was performed using the primer pairs (to amplify a
     -
   
of WNV GenBank accession no. NC 001563) and 5'-AG-
    
  -
issenböck et al 2002).
Reverse transcription was performed for 30 min at 50°C. Fol-
lowing an initial denaturation for 15 min at 95°C, the reacti-
on mixture was subjected to 45 cycles of heat denaturation
at 94°C for 30 s, primer annealing at 60°C for 30 s, and DNA

of 10 min at 72°C. Twenty microliters of each PCR product
-
taining ethidium bromide (Sigma, USA). The WNV obtained
from the Bornova Veterinary Control Institute was used as
positive control.
Results
Competitive Enzyme Linked Immunosorbent Assay
(C-ELISA)
         
antibodies using C-ELISA. All animals were negative for an-
tibodies against to WNV.
Reverse transcription polymerase chain reaction (RT-PCR)
West Nile Virus nucleic acid was not found in samples by RT-
PCR (Figure 1).
Discussion

avian neuropathogen. The virus is indigenous to Africa, Asia,
Europe, and Australia, and has recently caused large epide-
mics in Romania, Russia, and Israel. During the summer of

the northeastern U.S. (Petersen and Roehrig 2001).
        
was reported in the 1970s based on the detection of hae-
magglutination-inhibiting (HI) antibodies (Ari 1972, Radda


and sheep respectively (Ari 1972). Five years later, seropre-
   
human sera collected in southeast Turkey, using the HI assay

serosurvey in mammalian species by PRNA.
Positivity rates for the animals varied and were as follows:


blood samples from 70 each cattle, horse, sheep, goat and
water buffalo in northern Turkey for the existence of WNV
antibodies using competitive ELISA (c-ELISA). The c-ELISA

Ergunay et al (2014) reported that seroprevalance for WNV
   

in blood serum samples collected from 423 duck, 389 horse,
102 sheep and 266 human in 15 provinces in Turkey.
-
ology for the all mosquito-borne viruses. Sarikamis district
of Kars province of Northeastern Turkey where the study
was carried out has large forest lands therefore it is suitable

  
annual values of heat, humidity and rainfall of Sarikamis are

heat changes are more dramatic in this region.
Climate conditions of western, central and southern parts
of Turkey were more suitable for mosquitoes than northern
    
brown bears in Turkey. None of the bear samples were po-
sitive for antibodies to WNV by ELISA in this study. WNV-
Figure 1. Samples by RT-PCR
iii

Eurasian J Vet Sci, 2017, 33, 3, 190-194
193
   
51 black bears sampled by PRNT by Farajollahi et al (2003).
In Croatia, Madic et al (1993) detected WNV- neutralizing an-
tibodies in 4 brown bears of the 15 using hemagglutination-
inhibition (HI) test.
The nested and real-time Reverse Transcription polymerase
chain reaction (rRT-PCR) methods have been successful mo-

by identifying the viral genome (Dutton et al 2009, Albayrak

detection of WNV RNA in a variety of clinical samples such as
blood, tissues and mosquito specimens.
-
sitivity for the detection of WNV RNA. Ergunay et al (2014)
tested plasma samples from 256 horses and 266 people in

RNA using nested and rRT-PCR and WNV RNA was detec-
 
(2010) did not detect WNV RNA in wild bird samples. Simi-
larly, WNV RNA was not detected in brown bear samples in
this study.
Conclusion
In conclusion, no antibody response was detected against
WNV in brown bears in Sarikamis district of Turkey. In addi-
tion, WNV nucleic acid was not detected in samples of brown
bears. Although mosquito species known to transmit mos-
quito-borne infections have been observed (Dik et al 2006),
there is no report of acutely infected humans and animals in
Turkey.
This may suggest that WNV infection is not present in Nort-
heastern Turkey. The mosquitoes in this region may not
carry WNV. This study showed that WNV might not become a
risk potential for animals in Northeastern Turkey.
References
-
enza virus but not West Nile virus in wild birds in northern
Turkey. Zoonoses Public Health, 57, 71-75.

nile virus and rift valley fever virus in some of mammalian
species (herbivores) in northern Turkey. J Arthropod-Bor-
ne Dis, 7, 90-93.
Ari A, 1972. Studies on activity and ecology of arboviruses in
Turkey. Turk Hij Tecr Biyol Derg, 32, 134-143.
Chambers TJ, Hahn CS, Galler R, Rice CM, 1990. Flavivirus ge-
nome organization, expression, and replication. Annu Rev
Microbiol, 44, 649-688.
Dauphin G, Zientera S, 2007. West Nile virus: recent trends
in diagnosis and vaccine development. Vaccine, 25, 5563-
5576.

and distribution of Culicoides Latreille, 1809 (Diptera: Ce-
ratopogonidae) in Turkey. J Nat Hist, 40, 1947-1967.
Dutton CJ, Quinnell M, Lindsay R, Delay J, Barker K, 2009.
Paraparesis in a polar bear (Ursus Maritimus) associated
with west nile virus infection. J Zoo Wildl Med, 40, 568-
571.
-
  
 -
cal Surveillance Demonstrates Widespread Circulation of
West Nile Virus in Turkey. Neglected Trop Dis, 8(7), e3028.
Farajollahi A, Panella NA, Carr P, Crans W, Burguess K, Kok-
mar N, 2003. Serologic Evidence of West Nile Virus Infec-
tion in Black Bears (Ursus americanus) from New Jersey. J
Wildl Dis, 39, 894-896.
Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA,
2005. Family Flaviviridae. In: Fauquet CM, Mayo MA, Mani-
loff J, Desselberger U, Ball LA (Eds) Virus Taxonomy-Clas-
       
the International Committee on the Taxonomy of Viruses.
Elsevier Academic Press, San Diego,USA, pp: 739-780.
Lanciotti RS, Kerst AJ, Nasci RS, Godsey MS, Mitchell CJ, Sa-
vage HM, 2000. Rapid detection of West Nile virus from

avian samples by a TaqMan reverse transcriptase-PCR as-
say. J Clin Microbiol, 38, 4066-71.
Lvov DK, Butenko AM, Gromashevsky VL, 2004. West Nile
virus and other zoonotic viruses in Russia: examples of
emerging-reemerging situations. Arch Virol, 18, 85-96.
Mackenzie JS, Barrett ADT, Deubel V, 2002. The Japanese
   -
roduction to the group. Curr Top Microbiol Immunol, 267,
1-10.
Madic J, Huber D, Logovic B, 1993. Serologic survey for se-
lected viral and rickettsial agents of brown bears (Ursus
Arctos) in Croatia. J Wildl Dis, 29, 572-576.
     -
magglutination-inhibiting antibodies in southeastern Ana-
tolian people. Mikrobiyol Bül, 11, 3-17.

Serological evidence of West Nile virus (WNV) in mamma-
lian species in Turkey. Epidemiol Infect, 134, 826-829.
Padilla JA, Rubio EL, Romero EE, Cordobaba L, Cuevas S, Me-
jia F, Calderon R, Milian F, Rosa ATD, Weaver SC, Franco
JGE, Saiz JC, 2009. The continous spread of west nile virus
(WNV): seroprevalance in asymptomatic horses. Epidemi-
ol Infect, 137, 1163-1168.
Petersen LR, Roehring JT, 2001. West Nile virus: a reemer-
ging global pathogen. Emerg Infect Dis, 7, 611-614.
Porter KR, Summers PL, Dubois D, Puri B, Nelson W, Henchal

by the polymerase chain reaction and analysis of nucleo-
tide sequence variation. Am J Trop Med Hyg, 48, 440-446.
Radda A, 1973. Studies on the activity and ecology of arbo-
viruses in Turkey. Zentralbl Bakteriol Mikrobiol Hyg, 225,
iii

Eurasian J Vet Sci, 2017, 33, 3, 190-194
194
19-26.
Weissenböck H, Kolodziejek J, Url A, Lussy H, Rebel-Bauder
B, Nowotny N, 2002. Emergence of Usutu virus, an Mos-
iii  
quito-Borne Flavivirus of the Japanese Encephalitis Virus
Group, Central Europe. Emerg Infect Dis, 8, 652-656.
