VECTOR-BORNE AND ZOONOTIC DISEASES Volume 12, Number 9, 2012 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2011.0768
ORIGINAL ARTICLES
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
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 fatal, hemorrhagic fever. CCHFV is the most geographically widespread tick-borne virus of medical importance (Ergonul 2006). CCHFV is endemic in many areas in Africa, Asia, and in southeastern Europe. It produced outbreaks in Bulgaria, Albania, and Kosovo, southwestern Russia, Ukraine, Turkey (Maltezou et al. 2010), and recently emerged as a human pathogen in Greece (Papa et al. 2008). CCHFV activity was documented in Hungary by serological data (Horva´th 1976), and in Moldova Republic by direct detection in ticks (Chumakov et al. 1974). Circulation of CCHFV in Romania has not been documented to date. CCHFV circulates in nature in a tick-vertebrate-tick cycle, transmission being linked to ticks from the genus Hyalomma. H. marginatum, the main vector of CCHFV in Europe, is a two-host tick (Ergonul 2006): larvae and nymphs feed on small mammals and ground-feeding birds, while adults feed on large mammals like roe deer, wild boar, and livestock. CCHFV can be transmitted vertically in H. marginatum, which serves both as reservoir and vector for this pathogen. According to the old records reporting the presence of this tick species in Romania, its distribution is limited to the south, southeast, and southwest of the country (Feider 1965). Recent unpublished data by Ceianu and collaborators confirmed the presence of H. marginatum on sheep, cows, and horses in
southeast Romania, across the Dobrogea historical province. Among livestock raised in this area, sheep are the most 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. Materials and Methods Samples Serum samples to test for CCHFV antibodies were obtained from the serum bank of the Institute for Diagnostic and Animal Health (IDAH). Sera had been collected from sheep in different localities in the county of Tulcea. Most of the sera (433) were collected in August and September 2008, and only 43 samples were collected at the beginning of the summer in June, 2008 (Table 1). Six to 162 samples were analyzed per locality. Data on the animals’ breed, sex, and age were not available. Negative control sheep sera were obtained from the serum bank of the Vector-Borne Infections Laboratory in Cantacuzino Institute, collected in 1999 from a tick-borne encephalitis focus in central Transylvania, an area where the H. marginatum was not recorded to date. Study area Tulcea county (latitude: 44.64S–45.42N; longitude: 28.00W–29.65E) is situated in southeast Romania and forms the northern part of Dobrogea province. The county is 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, Unite´ de Ge´ne´tique Mole´culaire des Bunyavirus, Paris, France.
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CCHFV IN ROMANIA
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Table 1. Presence of IgG Antibodies Against Crimean-Congo Hemorrhagic Fever Virus in Sheep from Tulcea District, 2008
Locality 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Garvan Isaccea Parches Tulcea Mahmudia Ilganii de Jos Pardina Chilia Veche Salcioara Visina Baia Ceamurlia de Sus Total (%)
Time of collection (month) August September August, September September August August August August June, August August August June
Number of IgG-positive sera/number of serum samples tested 0/7 0/6 0/19 0/9 0/30 2/6 21/162 16/20 6/29 13/21 59/123 14/39
Sea (E), and is neighboring Ukraine (N). It is a complex geo-morphological territory comprising Danube Delta and Danube flood plain, Macin hercinic massive, and plateau areas that descend in terraces towards the Razim-Sinoe lagoons of the Black Sea. The average annual temperature is between 10 and 11C, precipitation is less than 400 mm/ year, and drought is frequent. Winter may be severe in the continental area, and milder close to the lagoon border, due to the influence of the Black Sea. Vegetation is represented by submediterranean broadleaf forests and xerophytic pastures with shrubs (Bıˆndiu 1971). Ground-feeding birds and small and larger mammals, including livestock, are abundant. Agriculture and animal breeding are the major activities in the area. Collection sites were distributed in rural and peri-urban pastures from the flood plain of the river Danube, in the Casimcea Plateau, the saline plains and brackish water marshes bordering the Razim lagoon, and the core of the Danube Delta. Serum samples were taken from sheep in 12 localities in Tulcea county (Fig. 1).
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 Serological assays Heat-inactivated sheep sera were tested for the presence of IgG antibodies specific to CCHFV using a recombinant nucleoprotein antigen sandwich ELISA following the protocol described by Garcia and associates (2006), with minor modifications. Recombinant CCHFV antigen and normal control antigen used for background subtraction were incubated overnight at 4C, and HRP-labeled anti-sheep IgG antibody (Bethyl Laboratories, Montgomery, TX) was used at 1:6000. Twenty sera found to be positive in this test were also tested in direct ELISA using the native CCHFV antigen. All IgG-positive sera were tested for the presence of IgM antibodies specific to CCHFV in an IgM capture ELISA (Garcia et al. 2006). For IgM detection assay, plates were coated overnight at 4C with anti-sheep IgM (Bethyl Laboratories) diluted 1:200 in 0.05% PBS. Sera diluted 1:100 were incubated 1 h at 37C. Direct IgM ELISA using native antigen was also used for testing a limited number (20) of IgG-positive sera. Results In 5 out of 476 sheep serum samples tested, the results were non-interpretable because of the high background OD, and these were not included in the analysis. Out of the remaining 471 tested samples, 131 (27.8%) had IgG antibodies against CCHFV. In the recombinant antigen IgG ELISA test the average OD for positive sera was 1.140 (OD range 0.357–1.998), and in the direct ELISA with cellular antigen, it was 2.046 (OD range 0.986–2.929). The IgG-positive sera in the recombinant CCHFV antigen EIA test were also found to be positive when tested against the native CCHFV antigen. In negative sera the OD ranged from 0.045–0.236 in the recombinant antigen sandwich ELISA test, and from 0.213– 0.364 in the cellular antigen direct ELISA. The sera that tested positive for IgG antibodies did not show IgM-specific antibodies to CCHFV. Localities where the animals resided and the results obtained for each site of origin are presented in Table 1 and Fig. 1. No serum was positive among the samples collected in sites situated in the flood plain of the Danube (sites 1, 2, 3, and 4), and the Sfantu-Gheorghe river branch (site 5). In the case of samples collected in three localities (sites 6, 7, and 8) situated in the Danube Delta, 39 out of 188 tested sera (20.7%) presented IgG antibodies to CCHFV. In samples from four localities (sites 9, 10, 11, and 12), situated in an area between the Casimcea Plateau and the Razim border, 92 sera were IgG-positive out of 212 tested (43.3%). The highest number of positive samples was collected from the locality of Baia (site 11), where 59 (47.9%) out of 123 tested samples presented IgG antibodies specific to CCHFV. Discussion This is the first report documenting, via indirect serological evidence, the circulation of CCHFV in Romania. IgG antibodies to CCHFV were detected in the sera of sheep from the district of Tulcea. A sandwich immunoenzymatic assay with a recombinant nucleoprotein antigen of CCHFV was used for testing sheep sera. Antibodies to related nairoviruses (Hazara virus and Dugbe virus), or to other viruses from the Bunya-
CEIANU ET AL. viridae family like Puumala virus, have been shown not to cross-react with the recombinant nucleoprotein of CCHFV (Marriot et al. 1994; Garcia et al. 2006). Sera presenting nonspecific reactivity after background OD subtraction were excluded from the analysis. Therefore the IgG antibodies detected in the animal sera had a specific reactivity to the nucleoprotein antigen of CCHFV. The serological investigation took place within the area of distribution of H. marginatum in Romania. In a previous study of ticks, H. marginatum, the main vector of CCHFV in Europe, was recorded in Dobrogea, and represented 37% of ticks infecting livestock from the central plateau of this province (Ceianu et al. unpublished data). It seems that both abiotic and biotic factors in the region are favorable to the development of the H. marginatum tick. The proximity of the Black Sea explains the maintenance of warm weather later into the fall, a condition that is likely to favor the development of nymphs, and their molting into adults, which can better survive over the winter (Estrada-Pen˜a et al. 2011). There is molecular evidence for the hypothesis that birds may be involved in the dissemination of CCHFV via migration (Hewson et al. 2004). The area we investigated comprises one of the most active bird migration pathways in southeastern Europe, for both spring and fall migrations, linking the region to the Middle East and Africa to the South, and to Asia towards the East. CCHFV infection in livestock does not cause clinical disease; however, domestic animals are important for CCHFV epidemiology. Not only are they hosts for adult vectors, but they also may amplify the virus and infect other ticks during their short-lived viremia, and thus may introduce the virus into new areas via movement and importation of tick-infested and virus-infected livestock, which may transmit the virus to humans through contact with blood and tissues of viremic animals (Gale et al, 2010). Levels of seroprevalence in livestock similar to the ones we found in sheep were associated with the occurrence of overt human disease in other regions where CCHFV is endemic (Papa et al. 2009; Telmadarraiy et al. 2010). In Romania, however, CCHFV human disease has not been reported to date. Results of the present study suggest that CCHFV circulates in Romania; thus increased awareness among public health professionals is necessary for the eventual detection of human cases. Genetic detection of the virus in ticks or patients will provide information about the strains circulating in the country. Acknowledgments Sheep sera were provided by the late Dr. Aurelia Ionescu (Institute for Diagnostic and Animal Health, Bucharest) in the framework of the EVIRNET partnership project. Her expertise is sadly missed, as is her friendly, straightforward, and courageous personality. C. Ceianu and R. Panculescu-Gatej were funded for this work by the Romanian Agency for Research (EVIRNET/ CEEX 86/2006/Viasan Program and PN 09-22-01-02, Nucleus Program); the international collaboration was supported by the international network for capacity building for the control of emerging viral vector-borne zoonotic diseases, or ArboZoonet.
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Address correspondence to: Cornelia Svetlana Ceianu Laboratory for Vector-Borne Infections and Medical Entomology Cantacuzino NIRDMI ITV EM Splaiul Independentei 103 Bucharest 050096 Romania E-mail:
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