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Research Article *Corresponding author Martin H. Groschup, Institute of Novel and Emerging Infectious Diseases at the Friedrich-Loeffler-Institut, Crimean-Congo Hemorrhagic Südufer 10, 17493 Greifswald-Island of Riems, Germany, Tel: 49-38351-7-1163; Email: Submitted: 19 December 2017 Fever Virus Infections in Cattle Accepted: 25 January 2018 Published: 29 January 2018 in ISSN: 2378-931X Copyright Arber Taraku1, Miriam Andrada Sas2, Arta Lugaj3, Bejo Bizhga4, © 2018 Groschup et al. Kristaq Berxholi4, and Martin H. Groschup2* OPEN ACCESS 1Faculty of Veterinary Medicine, Agricultural University of Tirana, Albania 2Institute of Novel and Emerging Infectious Diseases, Federal Research Institute for Animal Health, Germany Keywords 3Departments of Biology, University “Ismail Qemali”, Albania • Orthonairovirus; Nairoviridae; IgG antibodies; Cattle; 4Department of Preclinical Subjects, Agricultural University of Tirana, Albania ELISA

Abstract Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic vector-born viral disease named for the causative agent, Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is a member of the family Nairoviridae, genus Orthonairovirus. The virus is mainly transmitted via tick bites. Other transmission pathways are contact to blood, other bodily fluids and tissues of viremic animals or human patients. The case fatality rate in humans lies between 2 and 80%. Unlike humans, animals do not show clinical signs but they develop a stable antibody titer after a short viremia (< 2 weeks). Hence, seroepidemiological studies in livestock are very useful as risk indicator for CCHF in humans. The aim of this study was to examine the distribution of CCHFV infections among cattle in different . This survey was carried out in 2013-2014. Blood samples were taken from the jugular vein of 932 cattle in Kosovo. These sera were collected from nine different districts of Kosovo (Suhareke, Rahovec, Malisheve, Kline, Decan, , , Peje and Gjakove) and were tested with different serological assays (ELISA and IFA) at Friedrich-Loeffler-Institut (FLI), Greifswald, Germany. Through these techniques it was possible to identify CCHFV-specific IgG antibodies in serum samples of infected animals. Specific IgG antibodies were detected in cattle from all sampled areas and detected prevalence were substantial in some districts (43, 64% in Malisheve, followed by 25.25% in Rahovec). The overall seroprevalence was 19.21% (179 positives), with major CCHFV risk areas in Malisheve and Rahovec.

ABBREVIATIONS CCHFV: Crimean-Congo Hemorrhagic Fever Virus; in Albania, Bulgaria and Kosovo, while infection rates and case numbers in most other countries are fairly unknown [10,12]. Clinical symptoms can be mild and flu-like but also severe FLI: Friedrich-Loeffler-Institut; ELISA: Enzyme-Linked hemorrhagic fevers do occur. Case fatality rates vary between ImmunosorbentINTRODUCTION Assays; IFA: Immunofluorescence Assays 5% (Turkey) [13] and 80% (China) [14-16], depending on the The Crimean–Congo hemorrhagic fever virus (CCHFV) is a circulating virus strain, the healthcare system and the general awareness. In contrary to humans, other mammals do not show Nairoviridae of any signs of illness but elicit IgG antibodies after a short viremia tick-borne virus belonging to the genus Orthonairovirus, family and therefore can serve as risk indicators for CCHF circulating RNA viruses. The new genus Orthonairovirus includes 12 species, which are grouped in seven serogroups. in the environment. CCHFV is endemic in Kosovo, which2 is a landlocked country with a surface area of 10,877 km in the CCHF serogroup contains CCHF virus and Hazara virus. CCHF virus circulates in nature in an enzootic tick-vertebrate-tick cycle. center of the Balkan Peninsula and has borders with Macedonia, Ticks of the genus Hyalomma are the main vectors and reservoirs Albania, and Montenegro [17]. The first cases of CCHF in Kosovo were registered in 1954 in Nishor village (three fatal of the virus [1, 2-5] and define the worldwide. distribution of CCHFV [2,3,6, 7]. In Europe, Hyalomma marginatum ticks play cases) [17,18] and in 1957 a family outbreak leading to eight the most important role in CCHFV transmission The geographic fatalities was described [19]. Cases were again detected in 1989 distribution of the diseases is closely linked to the distribution (7 cases) and 1991 (9 cases). Since 1992 the disease has been of Hyalomma ticks which are found up to 46°N (latitude North) considered to be present in sporadic or epidemic form, notably [2,8,9]. Hence, humans become infected primarily when working with an outbreak in 1995 (46 cases) [20]. Based on the records outdoors or as health care workers with infected patients. Human of the Institute of Public Health of Kosovo, from 1995 to August cases have been reported from more than 30 countries of Asia, 2013, 228 cases of CCHF have been reported in Kosovo, with the South-Eastern Europe and Africa [2,10]. In Turkey, more than fatality rate of 25.5% [21]. Whereas human clinical cases have 1,000 human CCHF cases are reported annually in some years of been studied extensively, there is only limited knowledge about the last decade [10,11]. In Europe, human cases occur regularly the overall seroprevalence of the disease in Kosovo [21-24]. Cite this article: Taraku A, Sas MA, Lugaj A, Bizhga B, Berxholi K, et al. (2018) Crimean-Congo Hemorrhagic Fever Virus Infections in Cattle in Kosovo. J Vet Med Res 5(1): 1119. Groschup et al. (2018) Email:

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Figure 1 Kosova map and the study areas.

Table 1: Region/Location Serum sample tested (Final result) The results obtained from indirect ELISA for detection of CCHFV-specific IgG antibodies in cattle. (village) Animal species Total samples Positive samples Negative Antibody prevalence % Suharekë Cattle 220 10.2 Rahovec Cattle 198 148 25.25 245 25 Malishevë Cattle 72 93 43.65 50 Klinë Cattle 4 8.0 165 Decan Cattle 77 10 12.98 50 46 Drenas Cattle 49 44 10.2 67 Prizren Cattle 48 43 10.41 5 Pejë Cattle 3 47 6.0 5 Gjakovë Cattle 10 50 Total Cattle 932 179 753 19.2% CI=0.95 50 5 45 Abbreviations

: CCHFV: Crimean-Congo Hemorrhagic Fever Virus; ELISA: Enzyme-Linked Immunosorbent Assays; CI: Continuous Integration. MATERIALS AND METHODS in cattle adapted commercial CCHFV-IgG-ELISA (Vector-Best, Serum samples re-tested again in a cattle adapted commercial CCHFV-IgG-IFA This survey was carried out in 2013-2014 in 9 municipalities Novosibirsk, Russia). In case of divergent results, samples were of Kosovo. Blood samples were taken from the jugular vein of 932 collected in endemic regions of southwest region of Kosovo and cattle in (Suhareke, Rahovec, Malisheve, Kline, Decan, Drenas, also(Euroimmun, in a non-endemic Lübeck, Germany) region (Drenas) for final of confirmation. Kosovo, also Sera to cover were Prizren, Peje and Gjakove). Serum samples were collected in different ecologic conditions. regions where human cases already occurred as refer to the RESULTS AND DISCUSSION figureSerological below. analysis

A total of 932 bovine serum samples were tested for CCHFV- ith a specific IgG antibodies. The data presented in Table 1 indicate the positiveAll collected or inconclusive sera were result tested in theusing in-house a cattle ELISA specific were in-house rerun CCHFV-IgG-ELISA [as described before (10, 25)]. Samples w presence of CCHFV-specific antibodies in 9 districts of Kosovo: 10.2% in Suharekë, 25.25% in Rahovec, 43.65 % in Malishevë, J Vet Med Res 5(1): 1119 (2018) 2/4 Groschup et al. (2018) Email:

Central Bringing Excellence in Open Access 8.0 % in Klinë, 12.98 % in Decan, 10.2% in Drenas, 10.41% in . replication. Fields virology, 4th edn. In: HowleyDMKaPM, editor. 2. WhitehousePhiladelphia, CA.PA: Lippincott-Raven Crimean-Congo hemorrhagic 200. fever. Antiviral Res. Prizren,These 6.0% data in strongly Pejë and indicate 10.0% inthe Gjakovë presence and circulation of

3. 2004; 64: 145-160. Crimean-Congo hemorrhagic fever [15]virus among cattle in all different districts of Kosovo. The CCHFV-specific IgG antibody Charrel RN, Attoui H, Butenko AM, Clegg JC, Deubel V, Frolova TV, et al. Malisheve is already known as a CCHFV hotspot for human Tick-borne virus diseases of human interest in Europe. Clin Microbiol prevalence among cattle in Malisheve was very high (43.64%). infections and many hemorrhagic cases occurred in this district 4. Infect.Ergönü 2004;l O. Crimean-Congo 10: 1040-1055. haemorrhagic fever. Lancet Infect Dis. in the past. This correlates well with the high seroprevalence detected in cattle now. One explanation for this accumulation is 2006;Fisher-Hoch 6: 203-214. SP. Lessons from nosocomial viral haemorrhagic fever

5. the high number of Hyalomma ticks in this part of the country [26]. outbreaks.Swanepoel BrR, MedShepherd Bull. 2005;AJ, Leman 73-74: PA, 123-137. Shepherd SP, McGillivray GM, The other municipality with a high prevalence of CCHFV-specific Erasmus MJ , et al. Epidemiologic and clinical features of Crimean- areas in Kosovo have lower seroprevalences than Malisheve and 6. Congo hemorrhagic fever in southern Africa. Am J Trop Med Hyg. antibodies is Rahovec (25.25 %). The results show that all other

7. emphasizeRahovec respectively: that Prizren Decani is located 12.99%, near Priezreni the has 10.42%,region in Drenasi Kukes 1987; 36: 120-132. 10.20%, 10.0%, 8% and 6%. It is important to Hoogstraal H. The epidemiology of tick-borne Crimean-Congo hemorrhagic fever in Asia, Europe, and Africa. J Med Entomol. 1979; (Albania). A total of 48 cattle from Prizren were examined and 5 8. 15: 307-417.á antibody positive sera were found, which gives a seroprevalence Hub lek Z, Rudolf I. Tick-borne viruses in Europe. Parasitol Res. 2012; of 10.42%. This fits very well to the known seroprevalence in 9. 111: 9-36. Has-Kukes (16.67 %) [27] and shows that the cross-border circulation of CCHFV virus in the neighboring countries Kosovo Charrel RN, Attoui H, Butenko AM, Clegg JC, Deubel V, Frolova TV, et al. Tick-borne virus diseases of human interest in Europe. Clin Microbiol and Albania. The results of our study clearly show an overlap population and in animals in Kosovo. From an ecological point of 10. Infect.Marc M, 2004; Zati 10:V, Slavcho 1040-1055. M, Kiril K, Jovana S, Igor Di, et.al. Circulation view,between the theresults incidence are consistent rates of with CCHF the invegetation the healthy and climate human of Crimean-Congo Hemorrhagic Fever Virus in the Former Yugoslav present in Kosovo. Seroprevalence is highest in regions with less dense vegetation, where the main occupation is farming Republic of Macedonia Revealed by Screening of Cattle Sera Using a Novel Enzyme-linked Immunosorbent Assay. PLOS Negl Trop Dis. 11. 2015;Maltezou 9. HC, Andonova L, Andraghetti R, Bouloy M, Ergonul O, et al. and animal breeding. Agriculture in these areas is limited to individual small farms and people commonly get infected by seroprevalence are mostly forest or mountainous areas at higher Crimean-Congo hemorrhagic fever in Europe: current situation calls tick bites while working outside. On the other hand, areas of low 12. forMertens preparedness. M, Schmidt Euro K, Ozkul Surveill. A, Groschup 2010; 15:19504. MH. The impact of Crimean- and low virulent CCHF strains. Low, if at all, virulent strains (so- altitude. Antibodies can be elicited in humans and animals by high Congo hemorrhagic fever virus on public health. Antiviral Res. 2013; 13. 98: 248-260. called AP92-like) have been described to circulate in some parts epidemiology of Crimean-Congo hemorrhagic fever in Turkey, 2002- of Greece and Turkey. Hence the detection of CCHFV antibodies Yilmaz GR, Buzgan T, Irmak H, Safran A, Uzun R, Cevik MA, et al. The in human or animal cohorts should always be interpreted taking 14. Yen YC, Kong LX, Lee L, Zhang YQ, Li F, Cai BJ, et.al. Characteristics into account the presence or absence of human cases. In Kosovo 2007. Int J Infect Dis. 2009; 13: 380-386. the corresponding national prevalence was much lower (4.74%) of Crimean-Congo hemorrhagic fever virus (Xinjiang strain) in China. 19.21% of 932 bovines were antibody positive, while in Albania

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Cite this article Taraku A, Sas MA, Lugaj A, Bizhga B, Berxholi K, et al. (2018) Crimean-Congo Hemorrhagic Fever Virus Infections in Cattle in Kosovo. J Vet Med Res 5(1): 1119.

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