J Arthropod-Borne Dis, March 2019, 13(1): 9–16 H Gevorgyan et al.: Evidence of Crimean-Congo … 9 http://jad.tums.ac.ir Published Online: April 27, 2019 Original Article Evidence of Crimean-Congo Haemorrhagic Fever Virus Occurrence in Ixodidae Ticks of Armenia *Hasmik Gevorgyan1,2, Gohar G. Grigoryan1, Hripsime A. Atoyan1, Martin Rukhkyan1, Astghik Hakobyan2, Hovakim Zakaryan3, Sargis A. Aghayan4 1Scientific Center of Zoology and Hydroecology, Yerevan, Armenia 2National Institute of Health, MOH RA, Yerevan, Armenia 3Institute of Molecular Biology NAS RA, Yerevan, Armenia 4Laboratory of Zoology, Research Institute of Biology, Yerevan State University, Yerevan, Armenia (Received 3 Mar 2018; accepted 8 Oct 2018) Abstract Background: Crimean-Congo hemorrhagic fever (CCHF) causes serious health problems in humans. Though ticks of the genera Hyalomma play a significant role in the CCHF virus transmission it was also found in 31 other tick species. Methods: Totally, 1412 ticks from 8 remote sites in Armenia during 2016 were sampled, pooled (3-5 ticks per pool) and tested for the presence of CCHFV antigen using ELISA test. Results: From 359 tick pools, 132 were CCHF virus antigen-positive. From 6 tick species, four species (Rhipicephalus sanguineus, R. annulatus, R. bursa, Hyalomma marginatum) were positive for the virus antigen and R. sanguineus was the most prevalent (37.9%). Dermacentor marginatus and Ixodes ricinus revealed no positive pools, but both revealed delectable but very low virus antigen titers. The highest infection rate (50%) was observed in R. sanguineus, whereas H. marginatus rate of infection was 1 out of 17 pools. Conclusion: For the first time in the last four decades CCHF virus antigen was detected in Ixodid ticks of Armenia. This finding substantiates the role of R. sanguineus in the disease epidemiology; however, the role of H. marginatum in the CCHF virus circulation in the country could not be excluded. Keywords: CCHF; Ixodid ticks; Armenia; ELISA; Infection rate Introduction Ixodid ticks present significant epidemio- logic and health concerns in the transmission of dangerous arboviruses to humans (1, 2). In addition, the potential for person to person transmission makes the control of the spread of infection more difficult and unpredictable (3, 4). The Crimean-Congo hemorrhagic fever (CCHF) is one of the zoonotic viral infections that manifests with influenza-like symptoms and can progress to hemorrhagic disease with mortality rate ranging from 5% to 50% (5, 6). There are several routes and sources of human infection with CCHF virus, e.g., a direct tick bite, contact with virus-containing killed ticks, direct contact with blood and other tissues of infected animals or humans (3, 7-9). The caus- ative agent is CCHF virus (CCHFV), a mem- ber of the Bunyaviridae, genus Nairovirus. The virus has been isolated from at least 31 species of ticks belonging to Ixodidae (hard ticks) and Argasidae (soft ticks) families (10). A number of ticks in genera like Rhipiceph- alus and Dermacentor are able to care CCHF virus, but ticks of the genus Hyalomma are the essential vector for the pathogen transmission (11, 12). Except direct, through bites, disease transmission there are additional factors that can potentially increase the virus survival in nature. The livestock maintains the stable cir- culation of the virus in nature. The virus rep- licates to high titers in some organs of the car- rier livestock and usually causes only subclini- *Corresponding author: Dr Hasmik Gevorgyan, E-mail: hasgevorgian@yandex.ru J Arthropod-Borne Dis, March 2019, 13(1): 9–16 H Gevorgyan et al.: Evidence of Crimean-Congo … 10 http://jad.tums.ac.ir Published Online: April 27, 2019 cal disease in domestic animals, that goes un- noticed and untreated (13). The member of the crow family (Corvidae) are the reservoir of the ticks immature stage, whereas, infected an- imals are the main reservoirs for fully devel- oped adult stage of ticks (14). Epidemics and sporadic CCHF cases have been reported from Eastern Europe, Russia, Af- rica, the Middle East, and Central Asia (15-17). Tick-born arbovirus infections present epi- demiologic and health challenge in Armenia, as well. Being represented by a distinct vari- ety of landscape zones with several climate zones in relatively small territory, the country- side supports potential distribution of the vec- tor tick species (18). Although historical re- view related to arbovirus surveillance in Ar- menia has revealed the circulation of differ- ent arthropod-transmitted viruses (19), the first detection of CCHFV in ticks and the only laboratory-confirmed severe case of human CCHFV disease in Armenia is dated back to 1970s (20). To the best of our knowledge there are no published reports about CCHFV ac- tivity in the country in the last 5 decades. In spite of recent worsening of CCHFV situation in neighboring Turkey and Iran (9, 21–24), there is no consistent monitoring of CCHF key vectors distribution and abundance in Armenia. Moreover, there are no efforts to determine the prevalence and the level of CCHFV in ixodid ticks. This study was designed to study the pos- sible presence of CCHF virus in ixodid ticks in target provinces of Armenia. The initial stud- ies are highly relevant in the context of trans- formed socio-economic conditions, climatic and environmental changes, and particularly in the context of a significant interest towards tick- born arboviral infections worldwide (25). Materials and Methods The ticks’ collection was conducted in 2016 and covered 8 locations in five provinces of Armenia that included southern, central and northern parts of the country (Fig. 1). The se- lection of these territories was based on pre- liminary but limited knowledge of high level of Hyalomma and Rhipicephalus ticks abundance in these areas (26). Ticks were collected using two methodologies - by flagging in pasture lands of each locality and direct collection of the ticks from livestock. The sampling was linked to the ticks’ sea- sonal activities and covered period from Apr to Oct 2016 (27). The field sampling of free- living questing ticks was performed using flan- nel strips (1000x100mm). At each pastureland, 5 areas were randomly selected. Each select- ed area was walked in a slow manner drag- ging the flannel through the grass. At the end of each area caught ticks were collected. For collection of ticks from host animals (cattle and sheep), 10 animals were randomly select- ed for examination in each herd. Ticks were collected with forceps. Each individually col- lected tick was placed in separate vials, labeled and kept alive until species identification. Spe- cies identification was implemented under the light microscope with magnification of 100x, using the published taxonomic keys and spe- cies accounts (28). Overall, 1412 ticks were sampled of which only 548 were identified to species level. In 864 cases the identification was difficult due to in- adequate life stage or the damage of the sample. However, these “unidentified species” were pooled in groups according to sampling area and tested separately to determine whether or not these ticks may contain any titer of CCHFV antigen. Prior to serological detection of CCHFV antigen, each collected tick was rinsed with 70% ethanol and then placed in physiological solution with antibiotics (mixture of 100U/mL penicillin, 100μg/mL streptomycin). Sampled ticks were clustered and labeled according to genera, collection area, season, and placed in separate vials and stored at -18 °C for further processing. Stored ticks were pooled into groups of J Arthropod-Borne Dis, March 2019, 13(1): 9–16 H Gevorgyan et al.: Evidence of Crimean-Congo … 11 http://jad.tums.ac.ir Published Online: April 27, 2019 three to five (depending on size) by species and collection area. All pooled ticks were put in the liquid nitrogen for 20min then homogenized using glass pestle. Homogenized samples were resuspended in 200μl of dilution buffer in case of flat ticks and 400μl in case of engorged ticks. After the short vortexing tubes were centri- fuged at 2000rpm for 5min and supernatant was collected to test for CCHF viral antigen using antigen-capture ELISA (VectoCrimea- CHF ELISA, Vector-Best, Russia) (29). The sensitivity and specificity of the standard pan- els of positive and negative samples were in- dicated as 100%. Based on obtained optical density (OD) values we calculated critical OD (ODcr) according to the following formula: ODcr= ODm (K-)+ 0.2 where ODm (K-) is the medium value of negative controls’ OD. The sample was considered as positive if OD sam- ple was higher than ODcr. According to the manufacturer protocol if the OD sample was higher than 0.8*ODcr but less than ODcr the reading was considered as inconclusive, i.e., doubtful. The data were analyzed using pre- viously published technique developed for pooled sample studies (30). Results Of 1412 collected ticks only 548 were iden- tified to species level which belonged to four genera of the family Ixodidae: Rhipicephalus, Hyalomma, Ixodes, Dermacentor. The domi- nant tick species were from the genera Rhip- icephalus (364 ticks, 66.4%). Further, 6 tick species were identified, and R. sanguineus was identified as the most common tick species (n= 208, 37.9%) followed by R. annulatus (n= 68, 12.4%), R. bursa (n= 88, 16%), D. margina- tus (n= 64, 11.7%), I. ricinus (n= 53, 9.5%) and H. marginatum (n= 67, 12.4%). The analysis of tick species according to geographic location showed that Rhipicepha- lus spp. was the most abundant tick species in Kotayk region. I. ricinus and R. annulatus are the most recorded species in Gegharkunik Province, while sheep in Ararat and Tavush provinces suffered from D. marginatus ticks. Collected ticks were separated into 137 pools of identified tick species and 222 pools of not identified and assayed using ELISA test. From 137 pools comprising six tick spe- cies, 38 tested positive for the CCHFV anti- gen. These pools included four tick species (R. sanguineus, R. annulatus, R. bursa, H. mar- ginatum) and were registered in all surveyed areas. Noteworthy, R. sanguineus showed the highest CCHFV infection rate meanwhile only 1 of 17 H. marginatum pools showed positive response. The other six species contained doubt- ful (non-interpretable) pools (Table 1). Identified and non-identified tick pools were also clustered to analyze occurrence of CCHF virus antigen in carriers in targeted ge- ographic areas. Totally, 359 pools were test- ed. The observation of ticks’ infection rate in studied areas is shown in Table 2. The highest tick infection level was found in Kotayk and Tavush Provinces. Ticks from other areas demonstrated uneven distribution, with lower prevalence in southern and central part of the country. Table 1. Serological results of Crimean-Congo hemorrhagic fever antigen detection in pools according to tick species* Tick species No ticks Infection rate No Pools Positive pools Doubtful pools Rhipicephalus annulatus 65 107.73 17 6 1 Dermacentor marginatus 68 0.00 16 0 2 Hyalomma marginatum 70 17.85 17 1 3 Ixodes ricinus 51 0.00 13 0 1 Rhipicephalus bursa 86 71.87 22 5 3 J Arthropod-Borne Dis, March 2019, 13(1): 9–16 H Gevorgyan et al.: Evidence of Crimean-Congo … 12 http://jad.tums.ac.ir Published Online: April 27, 2019 Rhipicephalus sanguineus 208 175.30 52 26 4 Total 548 137 38 14 *Infection rate per 1000 ticks and 95% confidence interval Table 2. Results of CCHFV antigen detection in tick pools according to geographic location* Town/village (Province) Infection rate No Pools Positive Pools Doubtful Pools Zorak (Ararat) 0.00 16 0 2 Chambarak (Gegharqunik) 42.80 39 6 2 Tsaghkadzor (Kotayk) 195.79 37 20 3 Khndzoresk (Syunik) 10.87 25 1 2 Dilijan (Tavush) 156.65 220 100 18 Ijevan (Tavush) 65.76 8 2 0 Artsvaberd (Tavush) 73.58 14 3 3 Total 359 132 30 *Infection rate per 1000 ticks and 95% Confidence Interval Fig. 1. Study area Discussion Changes in climatic, environmental, social and anthropogenic factors have contributed to the spread of CCHF infection in new areas and increased incidence in endemic regions. Armenia is one of the countries where no studies on circulation of CCHF virus among ticks and livestock were conducted during last 5 decades. The first alarming data on the CCHF risk in Armenia occurred after the detection of AGDP antibodies to the virus in cattle sera from 5 areas of the country (31). The next evidence of the virus was the isola- tion of CCHF strains from H. marginatum (5), H. anatolicum (1), R. bursa (1), Boophilus (Rhipicephalus) annulatus (1), R. rossicus (1) (32). We have embarked on this study based on these 2 references (31, 32). The specific study areas were selected based on prelim- inary known distribution of Hyalomma and Rhipicephalus ticks in certain territories, as well as contrasting bio-climatic conditions be- tween targeted provinces. Our samples were represented predomi- nately by Rhipicephalus, Hyalomma, Ixodes, Dermacentor genera distinguished by their high magnitude of populations and variety of domestic and wild animal hosts (15, 18). Ticks were identified, pooled and analyzed using ELISA according to species and geographic location. From 1412 collected tick samples by Table 1. Continued … J Arthropod-Borne Dis, March 2019, 13(1): 9–16 H Gevorgyan et al.: Evidence of Crimean-Congo … 13 http://jad.tums.ac.ir Published Online: April 27, 2019 the time ticks were delivered to the laborato- ry the condition of 60% of ticks were not ap- propriate for the species identification. Since the aim of this study was to determine the pres- ence of CCHF virus in ticks in a specific area identified for the study, we pooled together the unidentified ticks and analyzed these pools only according to geographic area. The Rhipicephalus is the most prevalent genus in Kotayk region. I. ricinus and R. an- nulatus are the most prevalent species in Gegharkunik Province. The most abundant tick species in the current study is R. san- guineus which is, in general, in agreement with published data (34, 35). Comparison of results obtained from dif- ferent regions of the county showed that ticks collected from central regions were more in- fected than those from southern and northern regions. First, this can be explained by the com- position of sampled ticks from this region, where R. sanguineus the most abundant spe- cies that were also showing the highest infec- tion rate among all other tick species. Second, it could be due to the concentration of live- stock and the quality of breeding management including poor hygienic conditions of livestock breeding sites. Among all areas studied, we identified the highest ticks’ infection rate in Kotayk Prov- ince with prevalence of 54.05%. The second highest prevalence was identified in Dilijan town (Tavush Province) where along with R. sanguineus two more species (I. ricinus and R. bursa) were obtained. Prevalence of infected ticks was decreased in the north – Ijevan (25%) and Artsvaberd (21.5%) as well as in the east -Chambarak (15%). According to our data in Artsvaberd and Ijevan the most abundant tick species were R. bursa and in Chambarak - R. annulatus, demonstrating that the infection prevalence in study sites coincides with tick species abundance and infection rates. While in previously published study no virus was ob- served in H. detritum, H. scupense, I. ricinus, or Argas persicus (31), in our study one pool of I. ricinus and 2 pools of D. marginatum demonstrated low-level titers of CCHFV an- tigen. Some of ticks could be the virus carriers and these finding may suggest that further stud- ies may be necessary to determine the role of these other tick species in the epidemiology of the CCHFV transmission. In our opinion, a significant attention should be devoted to the recognized vector of CCHF virus - H. mar- ginatum. In our study from 17 H. marginatum ticks only one pool showed positive response and two were rated as doubtful. If compared with Rhipicephalus genera figures, there are indications to consider the latter as an accepta- ble vector for the virus in Armenia. At the same time, however, relatively high number of uni- dentified tick species with significant number of positive pools (132) may avert from an objec- tive assessment of Hyalomma impact on CCHF cases in Armenia. Therefore, a geographically wider area and more directed studies should be conducted to be able to answer this question. According to scientific consensus, although, many tick genera are capable of becoming in- fected with CCHF virus, several species of ge- nus Hyalomma are the principal vector for CCHF virus. To some extent our results and the only serologically proven human CCHF case recorded in 1974 (20) support this statement. Hyalomma and Rhipicephalus ticks are widely distributed and abundant in Armenia, but Rhip- icephalus spp. is likely to have a greater role in the circulation of CCHFV in Armenia and by serving as a virus reservoir. Hence, the oc- currence of the pathogen in ticks can represent a significant risk for human population and this risk should not be overlooked. Further studies should be focused on ani- mals’ seroprevalence and virus genetic diver- sity for identification of high-risk areas for hu- man infection. Conclusion For the first time in the last five decades, CCHF virus antigen was detected in tick sam- J Arthropod-Borne Dis, March 2019, 13(1): 9–16 H Gevorgyan et al.: Evidence of Crimean-Congo … 14 http://jad.tums.ac.ir Published Online: April 27, 2019 ples in Armenia. Serological test (ELISA) en- abled to prove the existence of CCHFV anti- gen mostly in Rhipicephalus ticks. Our results showed the important role of R. sanguineus tick species in supporting of CCHFV circu- lation in the natural foci, although without of- ficially registered cases of human CCHF. More- over, the highest level of infection based on collection area is registered in Kotayk region which coincided with abundance of R. san- guineus. Our results demonstrated the neces- sity to conduct PCR-based studies to deter- mine genetic diversity of CCHFV in the coun- try. Further, country-wide investigations in- cluding surveys of domestic animal sera and the risk assessment of human exposure to in- fected tick bite may be necessary. This data can be used as foundation for development of a country-wide epidemiologic study to iden- tify unrecognized CCHF foci in Armenia. Acknowledgements This work was made possible by a research grant from the Armenian National Science and Education Fund (ANSEF) based in New York, USA (grant number: zoo-4143). 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