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Detection of Crimean-Congo hemorrhagic fever virus-specific IgG antibodies in ruminants residing in Central and Western Macedonia, Greece
Accepted Manuscript Title: Detection of Crimean-Congo hemorrhagic fever virus-specific IgG antibodies in ruminants residing in Central and Western Macedonia, Greece Author: Isolde Schuster Serafeim C. Chaintoutis Chrysostomos I. Dovas Martin H. Groschup Marc Mertens PII: DOI: Reference:
S1877-959X(17)30106-1 http://dx.doi.org/doi:10.1016/j.ttbdis.2017.02.009 TTBDIS 796
To appear in: Received date: Revised date: Accepted date:
19-9-2016 6-2-2017 23-2-2017
Please cite this article as: Schuster, I., Chaintoutis, S.C., Dovas, C.I., Groschup, M.H., Mertens, M.,Detection of Crimean-Congo hemorrhagic fever virus-specific IgG antibodies in ruminants residing in Central and Western Macedonia, Greece, Ticks and Tick-borne Diseases (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.02.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Detection of Crimean-Congo hemorrhagic fever virus-specific IgG antibodies in
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ruminants residing in Central and Western Macedonia, Greece
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Isolde Schuster1,*, Serafeim C. Chaintoutis1,2,*, Chrysostomos I. Dovas2, Martin H.
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Groschup1, Marc Mertens1
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*both authors contributed equally
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Greifswald-Insel Riems, Germany.
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Institute for Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut,
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Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki,
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Thessaloniki, Greece.
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Diagnostic Laboratory, Department of Clinical Sciences, School of Veterinary
Address correspondence to:
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Marc Mertens
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Institute for Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut,
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Suedufer 10, 17493 Greifswald-Insel Riems, Germany
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[email protected]
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Abstract
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Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus which causes
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lethal hemorrhagic fever in humans. Although, several reports regarding CCHFV
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antibody prevalence in humans exist in Greece, information about the current
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distribution is limited. The aim of the present study is to investigate the prevalence
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of CCHFV-specific IgG antibodies in cattle and sheep in Macedonia-Greece. The
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samplings were performed during spring 2013, in 5 regional units of Central
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Macedonia (Chalkidiki, Imathia, Kilkis, Pella and Thessaloniki) and in the 4 regional
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units of Western Macedonia (Grevena, Florina, Kastoria and Kozani). Specifically,
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sera from 538 cattle and 81 sheep underwent testing against CCHFV-specific IgG
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antibodies. Antiviral immune responses were observed in 31 cattle (6%, 95% CI: 4-
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8%) and in one sheep (1%, 95% CI: 0-8%). The total seroprevalence in the cattle
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sampled in Central Macedonia was 7% (28 out of 396, 95% CI: 5-10%). Within Central
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Macedonia, the highest seroprevalence was detected in Chalkidiki (38%, 95% CI: 23-
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56%), which was significantly higher (p<0.01) compared to the overall
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seroprevalence detected in cattle. In Western Macedonia, the total seroprevalence
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in cattle was 2% (3 out of 142, 95% CI: 1-7%). The 3 seropositive cattle were residing
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in the regional unit of Grevena. The one IgG-positive sheep serum was obtained from
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an animal residing in Thessaloniki. In this regional unit, the prevalence in sheep (2%,
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95% CI: 0-10%) was much lower compared to the prevalence in cattle (12%, 95% CI:
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6-22%), but significance was not achieved (p=0.03). The here presented
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seroepidemiological study demonstrates high transmission risk to human in specific
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geographical areas, which should be communicated to national and local public
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health authorities, so as to intensify preventive measures for public health
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protection.
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Keywords
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Crimean-Congo hemorrhagic fever virus, Greece, Macedonia, Seroprevalence, Cattle,
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Sheep
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1. Introduction
Crimean-Congo hemorrhagic fever virus (CCHFV) is a single-stranded RNA
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virus, and belongs to the family Bunyaviridae (genus Nairovirus). This zoonotic
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arbovirus is primarily transmitted by the bites of infected Ixodidae ticks. The main
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CCHFV vector (Hyalomma marginatum) prefers the Mediterranean climate of North
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Africa and southern Europe, with low to moderate levels of humidity and a long dry
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season during the summer months (Estrada-Peña et al., 2011; Whitehouse, 2004). A
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study which was conducted in the region of Macedonia during 1983-86 indicated
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that at least one CCHFV tick vector (Hyalomma marginatum, the main vector
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species) and one suspected vector (Rhipicephalus bursa, the species which was
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present in the highest percentage) occurring in all bioclimatic zones (Papadopoulos
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et al., 1996). A more recent study indicated presence of Hyalomma ticks in wild
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resident birds in Greece (Diakou et al., 2016). Besides ticks, the virus can be transmitted by direct contact with blood, or
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tissues of viremic animals, or humans (Whitehouse, 2004). CCHFV infection in
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humans can cause a severe hemorrhagic disease (Crimean-Congo hemorrhagic fever;
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CCHF) with high lethality rates. A wide spectrum of wild and domestic animals can be
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infected, which play a crucial role in virus amplification and spread (Shepherd et al.,
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1989; Smirnova ,1979). In contrast to humans, these animals do not show clinical
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signs, but seroconversion can be observed in most infected animals (Spengler et al.,
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2016). It has been shown that the detection of CCHFV-specific immunoglobulins in
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animal species (e.g. ruminants) can successfully be used as indicator for the
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circulation of CCHFV in an area (Whitehouse, 2004; Spengler et al., 2016).
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The first Greek CCHFV strain (named “AP-92”) was isolated from a pool of
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Rhipicephalus bursa ticks, collected from goats residing at Vergina village (regional
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unit of Imathia, Central Macedonia) in May 1975 (Papadopoulos and Koptopoulos,
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1980). After the detection of the AP-92 strain, seroprevalence studies were initiated
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in humans, to investigate the level of CCHFV circulation in the regional unit of
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Imathia (Central Macedonia) in 1980-81. Sera from 4 out of 65 humans (6.2%) were
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found positive for antibodies against CCHFV, both by indirect immunofluorescence
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assay (IFA) and by hemagglutination-inhibition (HAI) test (Antoniadis and Casals,
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1982). During 1981-88, a large number (n = 3,388) of sera obtained from farmers and
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shepherds residing in 25 regional units of Greece were analyzed by IFA, showing the
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presence of anti-CCHFV antibodies in 1.1% of them (Antoniadis et al., 1990). Towards
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the end of June 2008 the Hellenic Center for Disease Control and Prevention
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(HCDCP) was notified about the first human case of CCHF, which occurred in the
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regional unit of Rhodopi (Thrace) and had a fatal outcome (Maltezou et al., 2009).
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Virus identification via sequencing indicated that the causative strain differed from
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the AP-92 strain by 25% (Papa et al., 2010). Another seroprevalence study was
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conducted in 1,178 residents of Eastern Macedonia and Thrace (regional units of
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Drama, Kavala, Xanthi, Rhodopi and Evros) in 2008-09, resulting in the detection of
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CCHFV-specific IgG antibodies by enzyme-linked immunosorbent assay (ELISA) in 37
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sera (3.1%) (Papa et al., 2011). During 2009-10, ELISA-testing of 1,611 sera collected
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from 28 regional units of Greece indicated presence of CCHFV-specific IgG antibodies
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in 68 (4.2%) participants (Sidira et al., 2011). During 2010-11, ELISA-testing of 277
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residents of the regional units of Imathia and Pella, resulted in detection of CCHFV-
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specific IgG antibodies in 6 sera (2.2%) (Sidira et al., 2013). Another novel CCHFV
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strain was detected in a pool of R. bursa ticks, which were collected from sheep
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residing in the regional unit of Kastoria during 2012. Molecular characterization of
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the strain indicated 9.7% differences from the AP-92 strain (Papa et al., 2014). During
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2012, 207 human sera which were collected from the regional unit of Achaia
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(Peloponnese peninsula, Southern Greece) were tested by ELISA and IFA, revealing
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the presence of CCHFV-specific IgG antibodies in 7 sera (3.4%), whereas testing for
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specific IgM antibodies was negative (Sargianou et al., 2013). Finally, 24 (14.4%) out
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of 166 sera collected from residents of the regional unit of Thesprotia during 2010-
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12, were positive for CCHFV-specific IgG antibodies (Papa et al., 2013).
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Data regarding the circulation of the virus in animals are very limited. During
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1969-78, precipitins against CCHFV were detected in 139 (32.9%) out of 422 goat
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sera, and in 34 (11.6%) out of 294 sheep sera, which were obtained from numerous
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locations of Northern Greece and which were analyzed by agar gel immunodiffusion
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test (Papadopoulos and Koptopoulos, 1980). Additionally, in 2012 a more recent
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small-scale survey was conducted in 40 sheep from 4 flocks located in the regional
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unit of Kastoria, which revealed CCHFV-specific IgG antibodies in 10 sera (25%) (Papa
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et al., 2014). Nonetheless, the total information about CCHFV circulation in
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ruminants in Greece is limited, especially in cattle, where no serological
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investigations have been performed. Therefore, the aim of the present study was to
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investigate the prevalence of CCHFV-specific IgG antibodies in cattle and sheep in
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Macedonia-Greece.
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2. Materials and methods
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2.1. Ethics statement Serum sampling was performed in compliance with ethical principles for diagnostic purposes. Cattle sera were obtained from clinically healthy animals,
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within the framework of serological testing for Enzootic Bovine Leukosis (EBL). This
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activity is performed under the national program for control and eradication of EBL,
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which is supported by the Greek Government and national veterinary services.
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Sheep sera were also obtained from clinically healthy animals.
2.2. Study area and samples
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The study was conducted in the regions Western Macedonia (NUTS;
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Nomenclature of Territorial Units for Statistics 13) and Central Macedonia (NUTS 3)
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during March-May 2013. In total 538 Holstein-Friesian cattle and 81 Chios cross-bred
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sheep were included. The age of the cattle ranged between 3-5 years and they were
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residing in 28 farms of the study area. Specifically, 20 farms were located in Central
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Macedonia in the NUTS level 3 equivalent geographical areas (regional units) of
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Chalkidiki, Imathia, Kilkis, Pella and Thessaloniki, and 8 farms were located in
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Western Macedonia in the regional units of Florina, Grevena, Kastoria and Kozani
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(Fig. 1, Table 1). The age of the sampled sheep ranged between 2-4 years. They were
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residing in 6 flocks in Central Macedonia in the regional units of Kilkis and
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Thessaloniki (Fig. 1, Table 1). The obtained sera were stored at -80 °C until they were
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analyzed.
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2.3. Serological testing
The serological screening of the 619 ruminant sera was performed by using
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indirect in-house ELISAs. The antigen used in these methods is a recombinant N-
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protein of the CCHFV Kosovo Hoti strain, which was isolated from a fatal human case
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in the Republic of Kosovo in 2001 (Duh et al., 2006). The sequence identity between
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the S-segments of CCHFV strain Kosovo Hoti and strain AP92 and between strain
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Kosovo Hoti and strain Rhodopi is 84% and 98% on nucleotide and 93% and 99% on
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amino acid level, respectively.
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Testing of bovine and sheep sera via the in-house tests has been described
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previously (Mertens et al., 2015; Schuster et al., 2016a). Based on the performance
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of the tests, sera with a negative result were interpreted as negative. All sera with
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positive or inconclusive results were analyzed with an adapted, commercial indirect
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confirmation ELISA (Vector-Best, Novosibirsk, Russia), according to previously
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described modifications (Mertens et al., 2015; Schuster et al., 2016a). The
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confirmation ELISA based on inactivated virus derived of a CCHFV strain from
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Uzbekistan. Sera which were positive or inconclusive in one of the ELISAs (screening
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or confirmation) and positive in the other one (screening or confirmation) were
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interpreted as positive. The result for sera with inconclusive screening ELISA and
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confirmation ELISA result remain inconclusive. Finally, all sera which were positive in
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the screening ELISA and negative in the confirmatory ELISA were analyzed by a
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confirmatory IFA. Specifically, an indirect IFA (Euroimmun, Lübeck, Germany) for the
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detection of CCHFV-specific antibodies in human sera was used, with adaptations for
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testing of cattle (Mertens et al., 2015) and sheep (Schuster et al., 2016a) sera,
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respectively. The IFA is based on cells transfected with Gc- and N-protein of a CCHFV
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strain from Nigeria.
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In this case, the result of the IFA (positive, negative or inconclusive) was considered
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the final result for the presence of CCHFV-specific immunoglobulins.
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2.4. Data analysis
The comparison of prevalence rates based on Fisher’s Exact Test. The data
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were corrected using the Bonferroni-Method. The tests were done on the
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significance level of p<0.05 (95% confidence interval). Because of the Bonferroni
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correction (p-value / no. of statistical tests), the p-value for the results had to be
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lower than 0.01 (p<0.01) to be statistically significant.
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3. Results
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The total CCHFV seroprevalence in cattle and sheep was 5% (32 positive sera
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out of 619, 95% CI: 4-7%) (Table 1). In addition to the positive sera, 8 cattle sera and
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one sheep serum had an inconclusive reaction (1%, 95% CI: 1-3%).
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Specifically, 31 (6%, 95% CI: 4-8%) out of 538 cattle were positive for CCHFVspecific antibodies. The total seroprevalence in the cattle sampled in Central
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Macedonia was 7% (28 out of 396, 95% CI: 5-10%). The highest seroprevalence in
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Central Macedonia was detected in Chalkidiki, where 13 (38%, 95% CI: 22-56%) out
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of 34 samples were positive. More specifically, these 13 seropositive animals were
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residing in one out of the two farms sampled from Chalkidiki (13 out of 28, 46%; 95%
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CI: 28-66%). Two (2%, 95% CI: 0.2-6%) sera out of 113 were positive in Imathia. The
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two seropositive animals were residing in two different farms, out of the 6 tested.
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Moreover, 5 (4%, 95% CI: 1-9%) out of 121 sera were positive three farms of Pella,
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out of the 7 farms tested. Eight (12%, 95% CI: 5-22%) more seropositive cattle were
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found out of 68 tested in Thessaloniki. In this regional unit, 3 cattle farms located in
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different areas were tested, and seropositive cattle were found in all of them.
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CCHFV-specific IgG antibodies were not detected in any of the 60 cattle sampled
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from Kilkis (95% CI: 0-6%) (Fig. 1, Table 1). In Western Macedonia, the total
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seroprevalence in cattle was 2% (3 out of 142, 95% CI: 0.4-6%). Specifically, the 3
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(8%, 95% CI: 2-21%) seropositive cattle were found in both cattle farms sampled in
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Grevena (n=38). No seropositive cattle were detected in Florina (95% CI: 0-6%),
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Kastoria (95% CI: 0-19%), or Kozani (95% CI: 0-13%).
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Regarding sheep, only one (1%, 95% CI: 0-7%) out of the 81 animals tested
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was seropositive in Central Macedonia. The serum which was tested positive for
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CCHFV-specific antibodies was collected from an animal from Thessaloniki (1 out of
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62, 2%, 95% CI: 0-9%). No CCHFV-specific IgG antibodies were detected in sheep
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from Kilkis (95% CI: 0-18%) (Fig. 1, Table 1).
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4. Discussion
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The endemic presence of CCHFV in the Balkan Peninsula and Turkey has already been documented (Bente et al., 2013, Mertens et al., 2013). As a result of
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this endemic status, sporadic cases or outbreaks occur every year (Mertens et al.,
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2013). In Greece, although the presence of CCHFV-specific IgG antibodies in humans
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has been reported in several regional units, only one clinical case was reported.
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Concerning animal seroprevalence, one study was performed recently, and although
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it is limited, the results demonstrate a wide circulation of CCHFV (Papa et al., 2014). To our knowledge, this recent study comprises the most extensive
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seroepidemiological evaluation of CCHFV circulation in animals in Greece, as well as
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the first report on the presence of CCHFV-specific IgG antibodies in cattle residing in
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Greece. The seroepidemiological study revealed higher, not significantly (p=0.03),
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but still higher prevalence in Central Macedonia, compared to Western Macedonia.
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In fact, in most of the regional units sampled from Western Macedonia (Florina,
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Kozani and Kastoria), no specific antibodies were detected in cattle. On the contrary,
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in Chalkidiki-Central Macedonia, a very high prevalence (38%) was observed, which
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was significantly higher (p<0.01) than the overall prevalence found in cattle within
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this study. Since all seropositive cattle were residing in one farm, it might be an
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isolated local CCHFV circulation in that area. With 12%, the prevalence in
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Thessaloniki was relative high as well. In the regional unit of Imathia, the area where
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the first CCHFV strain (AP-92) was isolated, the prevalence of CCHFV-specific
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antibodies was relative low (2%). The overall prevalence in sheep (1%) from Central
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Macedonia was not significantly lower (p=0.04) compared to the respective
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prevalence in cattle (7%). Like in cattle, no CCHFV-specific antibodies were detected
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in sheep from Kilkis. In Thessaloniki, the prevalence in sheep (2%) was much lower
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compared to the prevalence in cattle (12%), but not significantly (p=0.03). In general,
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several factors can alter the tick parasitism status of ruminants (e.g. the use of
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arthropod repellents, climate conditions) and therewith the infection status as well.
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More research is needed to clarify the susceptibility and the impact of different
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animal species in the amplification and maintenance of CCHFV in a geographical area
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(Spengler et al., 2016).
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Detection of CCHFV-specific antibodies in domestic animals has been important in
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providing evidence of circulating virus and in localizing CCHFV foci and increased risk
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for human infection (Spengler et al., 2016). The large number of animals, which were
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sampled in Western and Central Macedonia provide important information
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regarding the levels of CCHFV transmission in the study area. The fact that the
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sampled animals were young indicates that CCHFV circulation in the study area
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occurred recently. Nevertheless, in order to investigate CCHFV dynamics in its true
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extent, the serological surveillance plan should include samplings from sentinel
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animals, i.e. juvenile, seronegative cattle and sheep without maternal immunity.
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Further investigations of the prevalence of CCHFV-specific antibodies in other areas
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and other animal species will contribute to the understanding of the transmission
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cycle of CCHFV (Whitehouse, 2004; Spengler et al., 2016)).
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5. Conclusion
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The results of the seroepidemiological studies indicate a persisting circulation of
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CCHFV in specific geographical areas in Greece and therewith a high risk for human
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CCHFV infections. Those results should be communicated to national and local public
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health authorities, in order to intensify preventive measures for public health
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protection. In addition, further epidemiological studies are needed to determine the
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distribution of CCHFV in the whole country.
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Author disclosure statement
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No competing interests that could influence the results of this study exist.
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Acknowledgements
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This study was funded in parts by the EU grant FP7-261504 EDENext
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(http://www.edenext.eu). The content of this publication is the sole responsibility of
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the authors and does not necessarily reflect the views of the European Commission.
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Funding was also received by resources of the Operational Programme “Education
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and Lifelong Learning”, of the European Social Fund and the NSRF 2007-2013
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(project “IKY scholarships”). The funders had no role in study design, data collection
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and analysis, decision to publish, or preparation of the manuscript. We gratefully
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acknowledge the Thessaloniki Center of Veterinary Institutes, and especially G.
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Anastasiadis, for providing samples.
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Schuster, I. Mertens, M., Mrenoshki, S., Staubach, C., Mertens, C., Brüning, F.,
325
Wernike, K., Hechinger, S., Berxholi, K., Mitrov, D., Groschup, M.H., 2016a. Sheep
326
and goats as indicator animals for the circulation of CCHFV in the environment. Exp.
327
Appl. Acarol. 68, 337–346.
328
Shepherd, A.J., Leman, P.A., Swanepoel, R., 1989. Viremia and antibody response of
329
small African and laboratory animals to Crimean-Congo hemorrhagic fever virus
330
infection. Am J Trop Med Hyg. 40(5), 541–547.
331
Sidira, P., Maltezou, H. C., Haidich, A.-B., Papa, A., 2011. Seroepidemiological study
332
of Crimean- Congo haemorrhagic fever in Greece , 2009-2010. Clin. Microbiol. Infect.
333
18, E16–E19.
334
Sidira, P., Nikza, P., Danis, K., Panagiotopoulos, T., Samara, D., Maltezou, H.C., Papa,
335
A., 2013. Prevalence of Crimean-Congo hemorrhagic fever virus antibodies in Greek
336
residents in the area where the AP92 strain was isolated. Hippokratia 17, 322–325.
337
Smirnova, S.E., 1979. A Comparative Study of the Crimean Hemorrhagic Fever-Congo
338
Group of Viruses. Arch. Virol. 62(2), 137–143.
339
Spengler, J.R., Bergeron, É., Rollin, P.E., 2016. Seroepidemiological Studies of
340
Crimean-Congo Hemorrhagic Fever Virus in Domestic and Wild Animals. PLoS Negl.
341
Trop. Dis. 10, 1–28.
342
Whitehouse, C.A., 2004. Crimean-Congo hemorrhagic fever. Antiviral Res. 64, 145–
343
160.
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344 345 346
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Figure legends
348
Fig. 1. Prevalence of CCHFV-specific IgG antibodies in cattle and sheep in Northern
349
Greece, i.e. in Western Macedonia (NUTS 13, blue) and in Central Macedonia (NUTS
350
3, yellow). The total number of animals which were tested per regional unit is also
351
depicted. C: cattle, S: sheep, N/A: not applicable. (Figure modified from Wikimedia
352
Commons, https://commons.wikimedia.org/w/index.php?curid=24489143)
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Table 1. Seroepidemiological study in cattle and sheep in Central and Western
356
Macedonia, Greece.
Total No. of sera
No. of IgG
% IgG positive sera
Region / Regional unit
included in the study
tested
positive sera
(95% CI)
Cattle
28
538
31
6 (4-8)
Central Macedonia
20
396
28
7 (5-10)
regional unit of Chalkidiki
2
34
13
38 (22-56)
regional unit of Imathia
6
113
2
2 (0.2-6)
regional unit of Kilkis
2
60
0
0 (0-6)
regional unit of Pella
7
121
5
4 (1-9)
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No. of farms/flocks
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Animal Species
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regional unit of Thessaloniki
3
68
8
12 (5-22)
Western Macedonia
6
142
3
2 (0.4-6)
regional unit of Florina
3
60
0
0 (0-6)
regional unit of Grevena
2
38
3
8 (2-21)
regional unit of Kastoria
1
18
0
0 (0-19)
regional unit of Kozani
2
26
0
0 (0-13)
Central Macedonia
6
81
1
1 (0-7)
regional unit of Kilkis
1
19
0
0 (0-18)
regional unit of Thessaloniki
5
62
1
2 (0-9)
Total (study area, cattle and sheep)
34
619
32
5 (4-7)
Sheep
357 358 359
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S1877-959X(17)30106-1 http://dx.doi.org/doi:10.1016/j.ttbdis.2017.02.009 TTBDIS 796
To appear in: Received date: Revised date: Accepted date:
19-9-2016 6-2-2017 23-2-2017
Please cite this article as: Schuster, I., Chaintoutis, S.C., Dovas, C.I., Groschup, M.H., Mertens, M.,Detection of Crimean-Congo hemorrhagic fever virus-specific IgG antibodies in ruminants residing in Central and Western Macedonia, Greece, Ticks and Tick-borne Diseases (2017), http://dx.doi.org/10.1016/j.ttbdis.2017.02.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Detection of Crimean-Congo hemorrhagic fever virus-specific IgG antibodies in
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ruminants residing in Central and Western Macedonia, Greece
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Isolde Schuster1,*, Serafeim C. Chaintoutis1,2,*, Chrysostomos I. Dovas2, Martin H.
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Groschup1, Marc Mertens1
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*both authors contributed equally
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Greifswald-Insel Riems, Germany.
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Institute for Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut,
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Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki,
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Thessaloniki, Greece.
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Diagnostic Laboratory, Department of Clinical Sciences, School of Veterinary
Address correspondence to:
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Marc Mertens
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Institute for Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut,
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Suedufer 10, 17493 Greifswald-Insel Riems, Germany
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[email protected]
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Abstract
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Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus which causes
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lethal hemorrhagic fever in humans. Although, several reports regarding CCHFV
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antibody prevalence in humans exist in Greece, information about the current
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distribution is limited. The aim of the present study is to investigate the prevalence
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of CCHFV-specific IgG antibodies in cattle and sheep in Macedonia-Greece. The
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samplings were performed during spring 2013, in 5 regional units of Central
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Macedonia (Chalkidiki, Imathia, Kilkis, Pella and Thessaloniki) and in the 4 regional
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units of Western Macedonia (Grevena, Florina, Kastoria and Kozani). Specifically,
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sera from 538 cattle and 81 sheep underwent testing against CCHFV-specific IgG
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antibodies. Antiviral immune responses were observed in 31 cattle (6%, 95% CI: 4-
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8%) and in one sheep (1%, 95% CI: 0-8%). The total seroprevalence in the cattle
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sampled in Central Macedonia was 7% (28 out of 396, 95% CI: 5-10%). Within Central
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Macedonia, the highest seroprevalence was detected in Chalkidiki (38%, 95% CI: 23-
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56%), which was significantly higher (p<0.01) compared to the overall
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seroprevalence detected in cattle. In Western Macedonia, the total seroprevalence
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in cattle was 2% (3 out of 142, 95% CI: 1-7%). The 3 seropositive cattle were residing
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in the regional unit of Grevena. The one IgG-positive sheep serum was obtained from
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an animal residing in Thessaloniki. In this regional unit, the prevalence in sheep (2%,
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95% CI: 0-10%) was much lower compared to the prevalence in cattle (12%, 95% CI:
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6-22%), but significance was not achieved (p=0.03). The here presented
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seroepidemiological study demonstrates high transmission risk to human in specific
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geographical areas, which should be communicated to national and local public
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health authorities, so as to intensify preventive measures for public health
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protection.
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Keywords
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Crimean-Congo hemorrhagic fever virus, Greece, Macedonia, Seroprevalence, Cattle,
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Sheep
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1. Introduction
Crimean-Congo hemorrhagic fever virus (CCHFV) is a single-stranded RNA
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virus, and belongs to the family Bunyaviridae (genus Nairovirus). This zoonotic
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arbovirus is primarily transmitted by the bites of infected Ixodidae ticks. The main
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CCHFV vector (Hyalomma marginatum) prefers the Mediterranean climate of North
55
Africa and southern Europe, with low to moderate levels of humidity and a long dry
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season during the summer months (Estrada-Peña et al., 2011; Whitehouse, 2004). A
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study which was conducted in the region of Macedonia during 1983-86 indicated
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that at least one CCHFV tick vector (Hyalomma marginatum, the main vector
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species) and one suspected vector (Rhipicephalus bursa, the species which was
60
present in the highest percentage) occurring in all bioclimatic zones (Papadopoulos
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et al., 1996). A more recent study indicated presence of Hyalomma ticks in wild
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resident birds in Greece (Diakou et al., 2016). Besides ticks, the virus can be transmitted by direct contact with blood, or
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tissues of viremic animals, or humans (Whitehouse, 2004). CCHFV infection in
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humans can cause a severe hemorrhagic disease (Crimean-Congo hemorrhagic fever;
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CCHF) with high lethality rates. A wide spectrum of wild and domestic animals can be
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infected, which play a crucial role in virus amplification and spread (Shepherd et al.,
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1989; Smirnova ,1979). In contrast to humans, these animals do not show clinical
69
signs, but seroconversion can be observed in most infected animals (Spengler et al.,
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2016). It has been shown that the detection of CCHFV-specific immunoglobulins in
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animal species (e.g. ruminants) can successfully be used as indicator for the
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circulation of CCHFV in an area (Whitehouse, 2004; Spengler et al., 2016).
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The first Greek CCHFV strain (named “AP-92”) was isolated from a pool of
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Rhipicephalus bursa ticks, collected from goats residing at Vergina village (regional
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unit of Imathia, Central Macedonia) in May 1975 (Papadopoulos and Koptopoulos,
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1980). After the detection of the AP-92 strain, seroprevalence studies were initiated
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in humans, to investigate the level of CCHFV circulation in the regional unit of
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Imathia (Central Macedonia) in 1980-81. Sera from 4 out of 65 humans (6.2%) were
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found positive for antibodies against CCHFV, both by indirect immunofluorescence
80
assay (IFA) and by hemagglutination-inhibition (HAI) test (Antoniadis and Casals,
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1982). During 1981-88, a large number (n = 3,388) of sera obtained from farmers and
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shepherds residing in 25 regional units of Greece were analyzed by IFA, showing the
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presence of anti-CCHFV antibodies in 1.1% of them (Antoniadis et al., 1990). Towards
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the end of June 2008 the Hellenic Center for Disease Control and Prevention
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(HCDCP) was notified about the first human case of CCHF, which occurred in the
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regional unit of Rhodopi (Thrace) and had a fatal outcome (Maltezou et al., 2009).
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Virus identification via sequencing indicated that the causative strain differed from
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the AP-92 strain by 25% (Papa et al., 2010). Another seroprevalence study was
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conducted in 1,178 residents of Eastern Macedonia and Thrace (regional units of
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Drama, Kavala, Xanthi, Rhodopi and Evros) in 2008-09, resulting in the detection of
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CCHFV-specific IgG antibodies by enzyme-linked immunosorbent assay (ELISA) in 37
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sera (3.1%) (Papa et al., 2011). During 2009-10, ELISA-testing of 1,611 sera collected
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from 28 regional units of Greece indicated presence of CCHFV-specific IgG antibodies
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in 68 (4.2%) participants (Sidira et al., 2011). During 2010-11, ELISA-testing of 277
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residents of the regional units of Imathia and Pella, resulted in detection of CCHFV-
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specific IgG antibodies in 6 sera (2.2%) (Sidira et al., 2013). Another novel CCHFV
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strain was detected in a pool of R. bursa ticks, which were collected from sheep
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residing in the regional unit of Kastoria during 2012. Molecular characterization of
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the strain indicated 9.7% differences from the AP-92 strain (Papa et al., 2014). During
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2012, 207 human sera which were collected from the regional unit of Achaia
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(Peloponnese peninsula, Southern Greece) were tested by ELISA and IFA, revealing
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the presence of CCHFV-specific IgG antibodies in 7 sera (3.4%), whereas testing for
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specific IgM antibodies was negative (Sargianou et al., 2013). Finally, 24 (14.4%) out
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of 166 sera collected from residents of the regional unit of Thesprotia during 2010-
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12, were positive for CCHFV-specific IgG antibodies (Papa et al., 2013).
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Data regarding the circulation of the virus in animals are very limited. During
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1969-78, precipitins against CCHFV were detected in 139 (32.9%) out of 422 goat
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sera, and in 34 (11.6%) out of 294 sheep sera, which were obtained from numerous
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locations of Northern Greece and which were analyzed by agar gel immunodiffusion
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test (Papadopoulos and Koptopoulos, 1980). Additionally, in 2012 a more recent
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small-scale survey was conducted in 40 sheep from 4 flocks located in the regional
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unit of Kastoria, which revealed CCHFV-specific IgG antibodies in 10 sera (25%) (Papa
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et al., 2014). Nonetheless, the total information about CCHFV circulation in
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ruminants in Greece is limited, especially in cattle, where no serological
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investigations have been performed. Therefore, the aim of the present study was to
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investigate the prevalence of CCHFV-specific IgG antibodies in cattle and sheep in
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Macedonia-Greece.
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2. Materials and methods
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2.1. Ethics statement Serum sampling was performed in compliance with ethical principles for diagnostic purposes. Cattle sera were obtained from clinically healthy animals,
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within the framework of serological testing for Enzootic Bovine Leukosis (EBL). This
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activity is performed under the national program for control and eradication of EBL,
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which is supported by the Greek Government and national veterinary services.
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Sheep sera were also obtained from clinically healthy animals.
2.2. Study area and samples
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The study was conducted in the regions Western Macedonia (NUTS;
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Nomenclature of Territorial Units for Statistics 13) and Central Macedonia (NUTS 3)
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during March-May 2013. In total 538 Holstein-Friesian cattle and 81 Chios cross-bred
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sheep were included. The age of the cattle ranged between 3-5 years and they were
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residing in 28 farms of the study area. Specifically, 20 farms were located in Central
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Macedonia in the NUTS level 3 equivalent geographical areas (regional units) of
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Chalkidiki, Imathia, Kilkis, Pella and Thessaloniki, and 8 farms were located in
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Western Macedonia in the regional units of Florina, Grevena, Kastoria and Kozani
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(Fig. 1, Table 1). The age of the sampled sheep ranged between 2-4 years. They were
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residing in 6 flocks in Central Macedonia in the regional units of Kilkis and
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Thessaloniki (Fig. 1, Table 1). The obtained sera were stored at -80 °C until they were
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analyzed.
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2.3. Serological testing
The serological screening of the 619 ruminant sera was performed by using
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indirect in-house ELISAs. The antigen used in these methods is a recombinant N-
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protein of the CCHFV Kosovo Hoti strain, which was isolated from a fatal human case
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in the Republic of Kosovo in 2001 (Duh et al., 2006). The sequence identity between
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the S-segments of CCHFV strain Kosovo Hoti and strain AP92 and between strain
Page 5 of 14
Kosovo Hoti and strain Rhodopi is 84% and 98% on nucleotide and 93% and 99% on
149
amino acid level, respectively.
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Testing of bovine and sheep sera via the in-house tests has been described
151
previously (Mertens et al., 2015; Schuster et al., 2016a). Based on the performance
152
of the tests, sera with a negative result were interpreted as negative. All sera with
153
positive or inconclusive results were analyzed with an adapted, commercial indirect
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confirmation ELISA (Vector-Best, Novosibirsk, Russia), according to previously
155
described modifications (Mertens et al., 2015; Schuster et al., 2016a). The
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confirmation ELISA based on inactivated virus derived of a CCHFV strain from
157
Uzbekistan. Sera which were positive or inconclusive in one of the ELISAs (screening
158
or confirmation) and positive in the other one (screening or confirmation) were
159
interpreted as positive. The result for sera with inconclusive screening ELISA and
160
confirmation ELISA result remain inconclusive. Finally, all sera which were positive in
161
the screening ELISA and negative in the confirmatory ELISA were analyzed by a
162
confirmatory IFA. Specifically, an indirect IFA (Euroimmun, Lübeck, Germany) for the
163
detection of CCHFV-specific antibodies in human sera was used, with adaptations for
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testing of cattle (Mertens et al., 2015) and sheep (Schuster et al., 2016a) sera,
165
respectively. The IFA is based on cells transfected with Gc- and N-protein of a CCHFV
166
strain from Nigeria.
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In this case, the result of the IFA (positive, negative or inconclusive) was considered
168
the final result for the presence of CCHFV-specific immunoglobulins.
170 171
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2.4. Data analysis
The comparison of prevalence rates based on Fisher’s Exact Test. The data
172
were corrected using the Bonferroni-Method. The tests were done on the
173
significance level of p<0.05 (95% confidence interval). Because of the Bonferroni
174
correction (p-value / no. of statistical tests), the p-value for the results had to be
175
lower than 0.01 (p<0.01) to be statistically significant.
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3. Results
178
The total CCHFV seroprevalence in cattle and sheep was 5% (32 positive sera
179
out of 619, 95% CI: 4-7%) (Table 1). In addition to the positive sera, 8 cattle sera and
180
one sheep serum had an inconclusive reaction (1%, 95% CI: 1-3%).
181
Specifically, 31 (6%, 95% CI: 4-8%) out of 538 cattle were positive for CCHFVspecific antibodies. The total seroprevalence in the cattle sampled in Central
183
Macedonia was 7% (28 out of 396, 95% CI: 5-10%). The highest seroprevalence in
184
Central Macedonia was detected in Chalkidiki, where 13 (38%, 95% CI: 22-56%) out
185
of 34 samples were positive. More specifically, these 13 seropositive animals were
186
residing in one out of the two farms sampled from Chalkidiki (13 out of 28, 46%; 95%
187
CI: 28-66%). Two (2%, 95% CI: 0.2-6%) sera out of 113 were positive in Imathia. The
188
two seropositive animals were residing in two different farms, out of the 6 tested.
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Moreover, 5 (4%, 95% CI: 1-9%) out of 121 sera were positive three farms of Pella,
190
out of the 7 farms tested. Eight (12%, 95% CI: 5-22%) more seropositive cattle were
191
found out of 68 tested in Thessaloniki. In this regional unit, 3 cattle farms located in
192
different areas were tested, and seropositive cattle were found in all of them.
193
CCHFV-specific IgG antibodies were not detected in any of the 60 cattle sampled
194
from Kilkis (95% CI: 0-6%) (Fig. 1, Table 1). In Western Macedonia, the total
195
seroprevalence in cattle was 2% (3 out of 142, 95% CI: 0.4-6%). Specifically, the 3
196
(8%, 95% CI: 2-21%) seropositive cattle were found in both cattle farms sampled in
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Grevena (n=38). No seropositive cattle were detected in Florina (95% CI: 0-6%),
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Kastoria (95% CI: 0-19%), or Kozani (95% CI: 0-13%).
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Regarding sheep, only one (1%, 95% CI: 0-7%) out of the 81 animals tested
200
was seropositive in Central Macedonia. The serum which was tested positive for
201
CCHFV-specific antibodies was collected from an animal from Thessaloniki (1 out of
202
62, 2%, 95% CI: 0-9%). No CCHFV-specific IgG antibodies were detected in sheep
203
from Kilkis (95% CI: 0-18%) (Fig. 1, Table 1).
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4. Discussion
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The endemic presence of CCHFV in the Balkan Peninsula and Turkey has already been documented (Bente et al., 2013, Mertens et al., 2013). As a result of
208
this endemic status, sporadic cases or outbreaks occur every year (Mertens et al.,
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2013). In Greece, although the presence of CCHFV-specific IgG antibodies in humans
210
has been reported in several regional units, only one clinical case was reported.
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Concerning animal seroprevalence, one study was performed recently, and although
212
it is limited, the results demonstrate a wide circulation of CCHFV (Papa et al., 2014). To our knowledge, this recent study comprises the most extensive
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seroepidemiological evaluation of CCHFV circulation in animals in Greece, as well as
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the first report on the presence of CCHFV-specific IgG antibodies in cattle residing in
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Greece. The seroepidemiological study revealed higher, not significantly (p=0.03),
217
but still higher prevalence in Central Macedonia, compared to Western Macedonia.
218
In fact, in most of the regional units sampled from Western Macedonia (Florina,
219
Kozani and Kastoria), no specific antibodies were detected in cattle. On the contrary,
220
in Chalkidiki-Central Macedonia, a very high prevalence (38%) was observed, which
221
was significantly higher (p<0.01) than the overall prevalence found in cattle within
222
this study. Since all seropositive cattle were residing in one farm, it might be an
223
isolated local CCHFV circulation in that area. With 12%, the prevalence in
224
Thessaloniki was relative high as well. In the regional unit of Imathia, the area where
225
the first CCHFV strain (AP-92) was isolated, the prevalence of CCHFV-specific
226
antibodies was relative low (2%). The overall prevalence in sheep (1%) from Central
227
Macedonia was not significantly lower (p=0.04) compared to the respective
228
prevalence in cattle (7%). Like in cattle, no CCHFV-specific antibodies were detected
229
in sheep from Kilkis. In Thessaloniki, the prevalence in sheep (2%) was much lower
230
compared to the prevalence in cattle (12%), but not significantly (p=0.03). In general,
231
several factors can alter the tick parasitism status of ruminants (e.g. the use of
232
arthropod repellents, climate conditions) and therewith the infection status as well.
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More research is needed to clarify the susceptibility and the impact of different
234
animal species in the amplification and maintenance of CCHFV in a geographical area
235
(Spengler et al., 2016).
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Detection of CCHFV-specific antibodies in domestic animals has been important in
237
providing evidence of circulating virus and in localizing CCHFV foci and increased risk
238
for human infection (Spengler et al., 2016). The large number of animals, which were
239
sampled in Western and Central Macedonia provide important information
240
regarding the levels of CCHFV transmission in the study area. The fact that the
241
sampled animals were young indicates that CCHFV circulation in the study area
242
occurred recently. Nevertheless, in order to investigate CCHFV dynamics in its true
243
extent, the serological surveillance plan should include samplings from sentinel
244
animals, i.e. juvenile, seronegative cattle and sheep without maternal immunity.
245
Further investigations of the prevalence of CCHFV-specific antibodies in other areas
246
and other animal species will contribute to the understanding of the transmission
247
cycle of CCHFV (Whitehouse, 2004; Spengler et al., 2016)).
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5. Conclusion
250
The results of the seroepidemiological studies indicate a persisting circulation of
251
CCHFV in specific geographical areas in Greece and therewith a high risk for human
252
CCHFV infections. Those results should be communicated to national and local public
253
health authorities, in order to intensify preventive measures for public health
254
protection. In addition, further epidemiological studies are needed to determine the
255
distribution of CCHFV in the whole country.
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Author disclosure statement
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No competing interests that could influence the results of this study exist.
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Acknowledgements
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This study was funded in parts by the EU grant FP7-261504 EDENext
263
(http://www.edenext.eu). The content of this publication is the sole responsibility of
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the authors and does not necessarily reflect the views of the European Commission.
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Funding was also received by resources of the Operational Programme “Education
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and Lifelong Learning”, of the European Social Fund and the NSRF 2007-2013
267
(project “IKY scholarships”). The funders had no role in study design, data collection
268
and analysis, decision to publish, or preparation of the manuscript. We gratefully
269
acknowledge the Thessaloniki Center of Veterinary Institutes, and especially G.
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Anastasiadis, for providing samples.
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Figure legends
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Fig. 1. Prevalence of CCHFV-specific IgG antibodies in cattle and sheep in Northern
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Greece, i.e. in Western Macedonia (NUTS 13, blue) and in Central Macedonia (NUTS
350
3, yellow). The total number of animals which were tested per regional unit is also
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depicted. C: cattle, S: sheep, N/A: not applicable. (Figure modified from Wikimedia
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Commons, https://commons.wikimedia.org/w/index.php?curid=24489143)
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Table 1. Seroepidemiological study in cattle and sheep in Central and Western
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Macedonia, Greece.
Total No. of sera
No. of IgG
% IgG positive sera
Region / Regional unit
included in the study
tested
positive sera
(95% CI)
Cattle
28
538
31
6 (4-8)
Central Macedonia
20
396
28
7 (5-10)
regional unit of Chalkidiki
2
34
13
38 (22-56)
regional unit of Imathia
6
113
2
2 (0.2-6)
regional unit of Kilkis
2
60
0
0 (0-6)
regional unit of Pella
7
121
5
4 (1-9)
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No. of farms/flocks
d
Animal Species
M
us
355
regional unit of Thessaloniki
3
68
8
12 (5-22)
Western Macedonia
6
142
3
2 (0.4-6)
regional unit of Florina
3
60
0
0 (0-6)
regional unit of Grevena
2
38
3
8 (2-21)
regional unit of Kastoria
1
18
0
0 (0-19)
regional unit of Kozani
2
26
0
0 (0-13)
Central Macedonia
6
81
1
1 (0-7)
regional unit of Kilkis
1
19
0
0 (0-18)
regional unit of Thessaloniki
5
62
1
2 (0-9)
Total (study area, cattle and sheep)
34
619
32
5 (4-7)
Sheep
357 358 359
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