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First Serologic Evidence for the Circulation of Crimean-Congo Hemorrhagic

Fever Virus in Romania

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DOI: 10.1089/vbz.2011.0768 · Source: PubMed

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VECTOR-BORNE AND ZOONOTIC DISEASES
Volume 12, Number 9, 2012
ORIGINAL ARTICLES
ª Mary Ann Liebert, Inc.
DOI: 10.1089/vbz.2011.0768

First Serologic Evidence for the Circulation of Crimean-Congo

Hemorrhagic Fever Virus in Romania

Cornelia Svetlana Ceianu,1 Raluca Ioana Panculescu-Gatej,1 Daniel Coudrier,2 and Michele Bouloy 2

Abstract

Serum samples from sheep in localities situated in the county of Tulcea, Northern Dobrogea, were tested with an
IgG sandwich ELISA using a recombinant Crimean-Congo hemorrhagic fever virus (CCHFV) antigen. In all, 131
sera out of 471 tested (27.8%) had IgG antibodies specific to CCHFV. This is the first evidence for the circulation
of CCHFV virus in Romania.

Key Words: Antibodies—Crimean-Congo hemorrhagic fever virus—Romania—Sheep.

Introduction southeast Romania, across the Dobrogea historical province.

Among livestock raised in this area, sheep are the most

C rimean-Congo hemorrhagic fever virus (CCHFV)

belongs to the genus Nairovirus in the Bunyaviridae
family, and is a human pathogen that can cause a severe, often
common. The objective of the present study was to investigate
whether sheep in the county of Tulcea, situated in northern
Dobrogea, have antibodies to CCHFV.
fatal, hemorrhagic fever. CCHFV is the most geographically
widespread tick-borne virus of medical importance (Ergonul
Materials and Methods
2006). CCHFV is endemic in many areas in Africa, Asia, and in
southeastern Europe. It produced outbreaks in Bulgaria, Al- Samples
bania, and Kosovo, southwestern Russia, Ukraine, Turkey
Serum samples to test for CCHFV antibodies were obtained
(Maltezou et al. 2010), and recently emerged as a human
from the serum bank of the Institute for Diagnostic and Ani-
pathogen in Greece (Papa et al. 2008). CCHFV activity was
mal Health (IDAH). Sera had been collected from sheep in
documented in Hungary by serological data (Horváth 1976),
different localities in the county of Tulcea. Most of the sera
and in Moldova Republic by direct detection in ticks (Chu-
(433) were collected in August and September 2008, and only
makov et al. 1974). Circulation of CCHFV in Romania has not
43 samples were collected at the beginning of the summer in
been documented to date.
June, 2008 (Table 1). Six to 162 samples were analyzed per
CCHFV circulates in nature in a tick-vertebrate-tick cy-
locality. Data on the animals’ breed, sex, and age were not
cle, transmission being linked to ticks from the genus
available.
Hyalomma. H. marginatum, the main vector of CCHFV in
Negative control sheep sera were obtained from the serum
Europe, is a two-host tick (Ergonul 2006): larvae and
bank of the Vector-Borne Infections Laboratory in Cantacu-
nymphs feed on small mammals and ground-feeding birds,
zino Institute, collected in 1999 from a tick-borne encephalitis
while adults feed on large mammals like roe deer, wild
focus in central Transylvania, an area where the H. margin-
boar, and livestock. CCHFV can be transmitted vertically in
atum was not recorded to date.
H. marginatum, which serves both as reservoir and vector
for this pathogen.
Study area
According to the old records reporting the presence of this
tick species in Romania, its distribution is limited to the south, Tulcea county (latitude: 44.64S–45.42N; longitude:
southeast, and southwest of the country (Feider 1965). Recent 28.00W–29.65E) is situated in southeast Romania and
unpublished data by Ceianu and collaborators confirmed the forms the northern part of Dobrogea province. The county is
presence of H. marginatum on sheep, cows, and horses in bordered by the river Danube (W and N), and the Black

1
Cantacuzino National Institute for Research and Development for Microbiology and Immunology, Laboratory for Vector-Borne
Infections, Bucharest, Romania.
2
Pasteur Institute, Unité de Génétique Moléculaire des Bunyavirus, Paris, France.

718
CCHFV IN ROMANIA 719

Table 1. Presence of IgG Antibodies Against Sea (E), and is neighboring Ukraine (N). It is a complex
Crimean-Congo Hemorrhagic Fever Virus geo-morphological territory comprising Danube Delta and
in Sheep from Tulcea District, 2008 Danube flood plain, Macin hercinic massive, and plateau
areas that descend in terraces towards the Razim-Sinoe la-
Number of
Time of IgG-positive goons of the Black Sea. The average annual temperature is
collection sera/number of between 10 and 11C, precipitation is less than 400 mm/
Locality (month) serum samples tested year, and drought is frequent. Winter may be severe in the
continental area, and milder close to the lagoon border, due
1. Garvan August 0/7 to the influence of the Black Sea. Vegetation is represented by
2. Isaccea September 0/6 submediterranean broadleaf forests and xerophytic pastures
3. Parches August, September 0/19 with shrubs (Bı̂ndiu 1971). Ground-feeding birds and small
4. Tulcea September 0/9 and larger mammals, including livestock, are abundant.
5. Mahmudia August 0/30
Agriculture and animal breeding are the major activities in
6. Ilganii de Jos August 2/6
7. Pardina August 21/162 the area.
8. Chilia Veche August 16/20 Collection sites were distributed in rural and peri-urban
9. Salcioara June, August 6/29 pastures from the flood plain of the river Danube, in the Ca-
10. Visina August 13/21 simcea Plateau, the saline plains and brackish water marshes
11. Baia August 59/123 bordering the Razim lagoon, and the core of the Danube
12. Ceamurlia June 14/39 Delta. Serum samples were taken from sheep in 12 localities in
de Sus Tulcea county (Fig. 1).
Total (%) 131/471 (27.8)

FIG. 1. Map showing the distribution of sheep serum collection sites in the county of Tulcea, and the position of Tulcea
County on a map of Romania. Empty squares indicate sites where samples negative for Crimean-Congo hemorrhagic fever
virus (CCHFV)-specific IgG antibodies were collected. Solid squares indicate sites where CCHFV IgG-positive sera were
found (Mount., mountains; pl., plateau).
720 CEIANU ET AL.

Serological assays viridae family like Puumala virus, have been shown not to
cross-react with the recombinant nucleoprotein of CCHFV
Heat-inactivated sheep sera were tested for the presence
(Marriot et al. 1994; Garcia et al. 2006). Sera presenting non-
of IgG antibodies specific to CCHFV using a recombinant
specific reactivity after background OD subtraction were
nucleoprotein antigen sandwich ELISA following the pro-
excluded from the analysis. Therefore the IgG antibodies
tocol described by Garcia and associates (2006), with minor
detected in the animal sera had a specific reactivity to the
modifications. Recombinant CCHFV antigen and normal
nucleoprotein antigen of CCHFV.
control antigen used for background subtraction were in-
The serological investigation took place within the area of
cubated overnight at 4C, and HRP-labeled anti-sheep IgG
distribution of H. marginatum in Romania. In a previous study
antibody (Bethyl Laboratories, Montgomery, TX) was used
of ticks, H. marginatum, the main vector of CCHFV in Europe,
at 1:6000. Twenty sera found to be positive in this test were
was recorded in Dobrogea, and represented 37% of ticks in-
also tested in direct ELISA using the native CCHFV antigen.
fecting livestock from the central plateau of this province
All IgG-positive sera were tested for the presence of IgM
(Ceianu et al. unpublished data). It seems that both abiotic
antibodies specific to CCHFV in an IgM capture ELISA
and biotic factors in the region are favorable to the develop-
(Garcia et al. 2006). For IgM detection assay, plates were
ment of the H. marginatum tick. The proximity of the Black Sea
coated overnight at 4C with anti-sheep IgM (Bethyl La-
explains the maintenance of warm weather later into the fall, a
boratories) diluted 1:200 in 0.05% PBS. Sera diluted 1:100
condition that is likely to favor the development of nymphs,
were incubated 1 h at 37C. Direct IgM ELISA using native
and their molting into adults, which can better survive over
antigen was also used for testing a limited number (20) of
the winter (Estrada-Peña et al. 2011).
IgG-positive sera.
There is molecular evidence for the hypothesis that birds
may be involved in the dissemination of CCHFV via migra-
Results tion (Hewson et al. 2004). The area we investigated comprises
one of the most active bird migration pathways in south-
In 5 out of 476 sheep serum samples tested, the results were
eastern Europe, for both spring and fall migrations, linking
non-interpretable because of the high background OD, and
the region to the Middle East and Africa to the South, and to
these were not included in the analysis. Out of the remaining
Asia towards the East.
471 tested samples, 131 (27.8%) had IgG antibodies against
CCHFV infection in livestock does not cause clinical dis-
CCHFV. In the recombinant antigen IgG ELISA test the av-
ease; however, domestic animals are important for CCHFV
erage OD for positive sera was 1.140 (OD range 0.357–1.998),
epidemiology. Not only are they hosts for adult vectors, but
and in the direct ELISA with cellular antigen, it was 2.046 (OD
they also may amplify the virus and infect other ticks during
range 0.986–2.929). The IgG-positive sera in the recombinant
their short-lived viremia, and thus may introduce the virus
CCHFV antigen EIA test were also found to be positive when
into new areas via movement and importation of tick-infested
tested against the native CCHFV antigen.
and virus-infected livestock, which may transmit the virus to
In negative sera the OD ranged from 0.045–0.236 in the
humans through contact with blood and tissues of viremic
recombinant antigen sandwich ELISA test, and from 0.213–
animals (Gale et al, 2010).
0.364 in the cellular antigen direct ELISA.
Levels of seroprevalence in livestock similar to the ones we
The sera that tested positive for IgG antibodies did not
found in sheep were associated with the occurrence of overt
show IgM-specific antibodies to CCHFV.
human disease in other regions where CCHFV is endemic
Localities where the animals resided and the results ob-
(Papa et al. 2009; Telmadarraiy et al. 2010). In Romania,
tained for each site of origin are presented in Table 1 and Fig. 1.
however, CCHFV human disease has not been reported to
No serum was positive among the samples collected in sites
date.
situated in the flood plain of the Danube (sites 1, 2, 3, and 4),
Results of the present study suggest that CCHFV circulates
and the Sfantu-Gheorghe river branch (site 5).
in Romania; thus increased awareness among public health
In the case of samples collected in three localities (sites 6, 7,
professionals is necessary for the eventual detection of human
and 8) situated in the Danube Delta, 39 out of 188 tested sera
cases. Genetic detection of the virus in ticks or patients will
(20.7%) presented IgG antibodies to CCHFV.
provide information about the strains circulating in the
In samples from four localities (sites 9, 10, 11, and 12), si-
country.
tuated in an area between the Casimcea Plateau and the Ra-
zim border, 92 sera were IgG-positive out of 212 tested
(43.3%). The highest number of positive samples was col- Acknowledgments
lected from the locality of Baia (site 11), where 59 (47.9%) out
Sheep sera were provided by the late Dr. Aurelia Ionescu
of 123 tested samples presented IgG antibodies specific to
(Institute for Diagnostic and Animal Health, Bucharest) in the
CCHFV.
framework of the EVIRNET partnership project. Her expertise
is sadly missed, as is her friendly, straightforward, and cou-
Discussion
rageous personality.
This is the first report documenting, via indirect serological C. Ceianu and R. Panculescu-Gatej were funded for this
evidence, the circulation of CCHFV in Romania. IgG anti- work by the Romanian Agency for Research (EVIRNET/
bodies to CCHFV were detected in the sera of sheep from the CEEX 86/2006/Viasan Program and PN 09-22-01-02, Nucleus
district of Tulcea. A sandwich immunoenzymatic assay with a Program); the international collaboration was supported by
recombinant nucleoprotein antigen of CCHFV was used for the international network for capacity building for the control
testing sheep sera. Antibodies to related nairoviruses (Hazara of emerging viral vector-borne zoonotic diseases, or Arbo-
virus and Dugbe virus), or to other viruses from the Bunya- Zoonet.
 CCHFV IN ROMANIA 721

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