Abstract Book of EAVLD2012 - eavld congress 2012

S3 - O - 07
DETECTION OF WNV ENZOOTIC CIRCULATION IN HORSES WITH NEUROLOGICAL SIGNS AND IN
CAPTIVE SENTINEL CHICKENS IN THE PREFECTURE OF THESSALONIKI, GREECE
Chrysostomos I. Dovas 1 , Serafeim C. Chaintoutis 1 , Alexandra Chaskopoulou 2 , Nikolaos Diakakis 3 ,
Ilias Bouzalas 1 , Maria Papanastassopoulou 1 , Kostas Danis 4 , Anna Papa 5 , Orestis Papadopoulos 1
1
Aristotle University of Thessaloniki, Faculty of Veterinary Medicine, Laboratory of Microbiology and Infectious Diseases, Thessaloniki, Greece
2
United States Department of Agriculture-Agricultural Research Service, European Biological Control Laboratory, Thessaloniki, Greece
3
Aristotle University of Thessaloniki, Faculty of Veterinary Medicine, Clinic of Companion Animals, Thessaloniki, Greece
4
Hellenic Centre for Disease Control and Prevention, Athens, Greece
5
Aristotle University of Thessaloniki, Faculty of Medicine, A’ Laboratory of Microbiology, Thessaloniki, Greece
WNV, detection, characterization, horses, sentinel chickens
Introduction
During 2010, a large outbreak of West Nile virus (WNV)
infections occurred in Greece, with 262 reported human cases.
Among them, 197 were diagnosed with West Nile neuroinvasive
disease (WNND) and 35 patients died. The epidemic reoccurred
in 2011. Laboratory methods, including RT-PCR, ELISA,
microtiter plaque reduction neutralization test (micro-PRNT),
indirect immunofluorescence antibody test (IFAT) and virus
isolation, were used to detect WNV enzootic circulation in horses
with neurological signs (passive surveillance), as well as in
captive sentinel chickens (active surveillance), prior to the onset
of human WNND cases in the prefecture of Thessaloniki, Greece.
Materials & methods
Blood samples were obtained from 17 horses with neurological
signs, during the epidemic of 2010 (with the first case being
reported on August 1). Furthermore, blood was collected from
one horse which showed ataxia during the epidemic of 2011, on
August 13. Also, in May 2011, 7 coops of 6 chickens each were
placed at the edges of Thessaloniki City and blood samples were
collected weekly until October. In conjunction to blood sampling
from chicken, mosquitoes were sampled using 28 CO 2 -baited
CDC light traps.
The sera obtained from the 18 horses were assayed with two IgM
capture ELISA (MAC-ELISA) kits (IDEXX IgM WNV Ab Test and
IDVet ID Screen West Nile IgM Capture) for the detection of
WNV specific IgM antibodies, sign of recent infection, as well for
specific IgG antibodies, using IFAT. Plasma samples from these
horses were tested with real-time RT-PCR for WNV (LSI TaqVet
West Nile Virus – Dual IPC). Chicken sera were assayed by
competitive ELISA (cELISA) (IDVet ID Screen West Nile
Competition) for the detection of specific antibodies, and positive
results were confirmed by micro-PRNT. Plasma samples from the
seropositive chickens were screened using a WNV specific RT-
PCR and confirmed using nested RT-PCR, employing specific
primers for the WNV “Nea Santa-Greece-2010” strain, followed
by sequencing of the NS3 gene for further molecular
characterization. Vero cell cultures were inoculated with plasma
samples from the RT-PCR positive chickens, for virus isolation.
The collected mosquitoes were being counted and
morphologically identified. 146 pooled samples, each one
consisting of 50 mosquitoes of the same species (123 Culex
pipiens and 23 Culex modestus pools) were examined by three
different WNV specific real-time RT-PCR protocols, as well as
with the nested RT-PCR protocol,
Results
WNV specific antibodies were detected in all 18 horse cases of
2010 and 2011, while the IgG IFAT titer was high (>250) in all of
them. Three horses were found positive with real-time RT-PCR
(Ct ~39). Regarding chickens, seroconversion was detected in 11
birds (10 in Western Thessaloniki). The first seroconversion
occurred on June 29, a month before the onset of human cases
in the area. The seroconversion rate peaked on August 17 with 4
seropositive birds and the last seroconversion occurred on
September 28. WNV was detected in 2/11 seropositive chickens
with RT-PCR, from samples taken one week prior to
seroconversion. According to BLAST algorithm, the partial NS3
sequence presented highest nucleotide sequence identity
(99,73%) to the strain “Nea Santa-Greece-2010”, responsible for
the 2010 Greek epidemic. The inferred partial NS3 amino acid
sequence was 100% identical to that of the “Nea Santa-Greece-
2010”. The strains maintained the amino acid substitution H 249 P,
which may be associated with increased virulence. WNV was
successfully isolated in cell cultures. Chicken serum neutralizing
antibody titers to WNV ranged between 20 and 40. Culex
mosquito populations peaked in mid-June, ~10 days prior to the
first chicken seroconversion, remained high throughout the
summer and showed a significant drop in September. The most
prevalent mosquito species in both residential and agricultural
areas were Culex pipiens, followed by Culex modestus Ficalbi.
Only one out of the 123 Culex pipiens pools tested was found
positive for the WNV “Nea Santa-Greece-2010” strain using the
nested RT-PCR, while the application of all different real-time RT-
PCR protocols resulted in detection of additional mosquito
flaviviruses, indicating problems of detection specificity.
Figure 1. Location of mosquito traps (n=28) and sentinel chicken
coops for WNV surveillance in Thessaloniki, 2011. Blue bullets
represent the mosquito traps, while yellow dots represent the
location of the 7 chicken coops. The 10 seroconverted chickens
belonged to the coops No. 1, 2 and 3 (Western Thessaloniki),
while one more chicken belonged to the coop No. 5, in Eastern
Thessaloniki.
Discussion & conclusion
With passive surveillance, 18 clinical cases of horses were
detected mainly in suburban areas of Thessaloniki. Unfortunately
there could not be successfully used as an early warning system,
given that the first human case in the prefecture of Thessaloniki
was recorded 15 days prior to the onset of clinical cases in
horses, both in 2010 and 2011. On the contrary, with the second
surveillance system, it was shown that the “Nea Santa-Greece-
2010” strain became endemic in Greece, as it was detected,
molecularly identified and isolated early in 2011. Most
importantly, enzootic circulation of WNV was successfully
detected one month prior to human cases. As a result, health
authorities were informed in a timely manner and were facilitated
the successful implementation of preparedness plans to protect
public health and minimize the impact of the epidemic of 2011. In
regards to the use of real-time RT-PCR for vector surveillance,
the methods need further optimization and validation, concerning
the specificity of WNV detection and results should be further
confirmed by sequencing.
References
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G, Kapsimali, V, Tsakris, A, Kansouzidou, A, Tsiodras, S, Vakalis, N,
Bonovas, S, Kremastinou, J (2011). Ongoing outbreak of West Nile virus
infection in humans, Greece, July to August 2011. Eurosurveillance,
16(34), pii=19951.