Seroprevalence of Crimean-Congo Hemorrhagic Fever Virus, Bulgaria

VECTOR-BORNE AND ZOONOTIC DISEASES Volume 22, Number 7, 2022 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2021.0091 Open camera or QR reader and scan code to access this article and other resources online. Seroprevalence of Crimean-Congo Hemorrhagic Fever Among Small Ruminants from Southern Romania Downloaded by 54.146.243.225 from www.liebertpub.com at 03/05/24. For personal use only. Bianca Bratuleanu,1,2 Adriana Anita,2 Sarah Temmam,1,3 Anca Dascalu,2 Luciana Crivei,2 Andreea Cozma,2 Philippe Pourquier,4 Gheorghe Savuta,2 Marc Eloit,1,3,5 and Dragos Anita2 Abstract Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease that can be contracted by direct contact with viremic animals or humans. Domestic animals are accidental hosts and contribute to the spread and amplification of the virus. The main objective of this study was to provide updated information related to CCHF virus (CCHFV) infection in Southern Romania by assessing the seroprevalence of CCHF in small ruminants (sheep and goats) using a double-antigen sandwich enzyme-linked immunosorbent assay and by detection of CCHFV in engorged ticks and serum samples using real-time RT-PCR. The overall seroprevalence of CCHF in small ruminants was 37.7% (95% CI 31.7 to 43.7). No statistical seroprevalence difference was observed between the two species of ruminants ( p = 0.76), but a significant difference was established between the locations ( p < 0.01). No CCHFV RNA was detected in tick pools and small ruminant’s sera tested by real-time RT-PCR, although the high seroprevalence to CCHFV among ruminants indicates that CCHV or a closely related virus circulates in Southern Romania. Keywords: tick-borne disease, Crimean-Congo hemorrhagic fever, double antigen sandwich ELISA, real-time RT-PCR, small ruminant Introduction C rimean-Congo hemorrhagic fever (CCHF) is a tickborne disease caused by CCHF virus (CCHFV), an arbovirus of the Nairoviridae family (Bente et al. 2013). CCHF is considered the most important tick-borne viral disease of humans, existing over a large geographic area, including Africa, South Europe, and parts of Asia (Hoogstraal 1979, Whitehouse 2004, Negredo et al. 2017). Within SouthEastern Europe, human cases have been reported in Bulgaria, the Republic of Kosovo, Albania, Greece, and Ukraine (Whitehouse 2004, Mertens et al. 2013). The natural cycle of CCHFV includes transovarial and transstadial transmission among ixodid ticks and a cycle involving different vertebrates (goats, sheep, horses, pigs, camels, birds, rodents, and hares). Although several tick genera can be infected with CCHFV, ticks of the genus Hyalomma are considered the main vector of the virus (Bente et al. 2013). In contrast to humans, domestic animals do not show clinical signs of illness, and act as accidental hosts, which amplify and disseminate the virus. Seroepidemiological studies in endemic regions indicate that various domestic animals (sheep, goats, and cattle) could be asymptomatically infected with CCHFV. Therefore, they can act as sentinels, providing initial evidence of the silent circulation of the virus, and allow identification and localization of CCHFV foci, which could be a risk for human health. In Romania, there is a lack of data concerning the circulation of CCHFV: the main vector is present in the studied 1 Pathogen Discovery Laboratory, Institut Pasteur, Paris, France. Regional Center of Advanced Research for Emerging Diseases, Zoonoses and Food Safety (ROVETEMERG), ‘‘Ion Ionescu de la Brad’’ University of Life Sciences, Iasi, Romania. 3 OIE Collaborating Centre for Detection and Identification in Humans of Emerging Animal Pathogens, Institute Pasteur, Paris, France. 4 IDVet, Grabels, France. 5 Alfort National Veterinary School, Maisons-Alfort, France. 2 397 398 BRATULEANU ET AL. area (Southern Romania) and several studies have shown a high seroprevalence in animals (Ceianu et al. 2012, Raileanu et al. 2015), but the virus has never been detected in ticks and no human case has been reported so far. The aim of this study was to assess the seroprevalence of CCHFV in small ruminants using a double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) to determine the prevalence of CCHFV infection in small ruminants from this region. The study was completed by the search of CCHFV RNA in engorged ticks and serum samples collected from small ruminants in South region of the country. Materials and Methods Downloaded by 54.146.243.225 from www.liebertpub.com at 03/05/24. For personal use only. Animal blood and tick sample collection Serum samples from 250 sheep and goats, collected during 2019–2020, were investigated. Animal samples were collected in five sites across Tulcea County (Cataloi, Baia, Slava Rusa, Slava Cercheza, and Somova) (Fig. 1) and stored at -80°C before use. In addition, 96 serum samples from sheep from mainland France were used as reference population, as there is no evidence for CCHFV circulation in continental France. Finally, 169 Rhipicephalus sanguineus ticks were FIG. 1. collected from the investigated ruminants from Cataloi location and treated as previously described (Bratuleanu et al. 2021). The detection of antibodies against CCHFV nucleoprotein in animal sera was performed using ID Screen CCHF Double Antigen Multispecies (IDvet, Grabels, France), following the manufacturer’s instructions (Sas et al. 2018a). RNA extraction Ticks and serum samples were transferred to Institute Pasteur (Paris, France) to be analyzed by real-time RT-qPCR targeting CCHFV. To ensure the maintenance of the integrity of RNA and virus inactivation, ticks were introduced in RNA later solution (Invitrogen, France), according to security practices of storing and transport. Nucleic acid extraction steps were conducted in a BSL3 laboratory. Ticks were pooled before extraction, resulting in a final number of 14 pools. Total RNA was extracted from crushed materials using TRIzol reagent (Invitrogen, USA) and RNeasy mini kit (Qiagen, Germany) according to the manufacturers’ recommendations. Sixty-one serum samples from Cataloi and Slava Rusa were pooled before extraction, resulting in a final number of Sampling locations of ruminant sera and ticks in Southern Romania. CCHF PREVALENCE IN RUMINANTS FROM ROMANIA seven pools. Total nucleic acid extraction from serum samples was performed using QIAamp Cador kit (Qiagen) with nuclease pretreatment. Directive 2010/63/EU and approved by the Research Ethics Committee of the Faculty of Veterinary Medicine, IULS Romania. Determination of tick species Results To determine the species (and not only the genus) of analyzed ticks, we took advantage of concomitant sequencing of tick transcriptome and the Barcode of Life Data Systems (BOLD) as previously described (Bratuleanu et al. 2021). Briefly, all trimmed reads were mapped onto the Ixodidae BOLD database, de novo assembled after extraction of mapped reads, and submitted to the BOLD Identification System. The identification was confirmed by BlastN, all ticks analyzed in this study being identified as R. sanguineus. The global CCHFV antibody seroprevalence rate for tested animals was 37.7% (95% CI 31.7 to 43.7). The estimated seroprevalence in sheep was 29.8% (95% CI 23.2 to 36.5), (54/181) and 57.7% (95% CI 46.3 to 69.2) in goats (41/71). Overall results and prevalence of CCHFV infection in different species and collection sites are shown in Table 1. A significant difference was observed between Romanian sheep and the reference population from France ( p < 0.01). No statistical seroprevalence difference was observed between these two species of ruminants from Romania ( p = 0.76). However, a significant difference was established between the five locations ( p < 0.01), with 93.1% (95% CI 83.8 to 102.3) positivity in animals at Somova and 0% at Baia. No CCHFV RNA was detected from the tick pools and small ruminant’s sera tested by real-time RT-PCR. Moreover, using the pan-Nairovirus PCR system, no positive sample was identified (Bratuleanu et al. 2021). Detection of CCHFV and pan-Nairovirus in R. sanguineus and serum samples by real-time RT-PCR Downloaded by 54.146.243.225 from www.liebertpub.com at 03/05/24. For personal use only. 399 Viral RNA was first reverse transcribed to cDNA using SuperScript IV Reverse Transcriptase kit (Invitrogen) and random hexamers. Primer sets specific for each CCHFV genotype (N = 6) and one degenerated for detection of all genotypes were used to screen tick and serum samples as previously described (Sas et al. 2018b). The PCR protocol was adapted for a real-time SYBR Green format. For regulatory reasons regarding biosafety level, it was not possible to use a positive control. As negative control, nuclease-free water was used to rule out cross-contamination of reagents and surfaces. A pan-Nairovirus PCR system, capable of detecting 14 viruses representative of the genus, was also used to identify other nairoviruses (Honig et al. 2004). Statistical analysis Statistical analysis was performed on commercially available software (SPSS 17.0; IBM), using chi-square test and Fisher’s exact test. Statistical significance was defined as p < 0.05. The study was conducted in accordance with the Discussions This study provides an updated overview of CCHFV seroprevalence in small ruminants from Tulcea County. This area shows a high degree of biodiversity, comprising one of the most active bird migration pathways in South-Eastern Europe, and it serves as breeding sites for small ruminants. The results of this study revealed a high seroprevalence, indicating the importance of sheep and goats in CCHFV ecology in Romania, in the same region where the presence of CCHFV antibodies in animals has been detected previously (Ceianu et al. 2012). CCHFV is endemic in Balkan Peninsula, where Turkey and Bulgaria are mainly affected. Table 1. Anti-Crimean-Congo Hemorrhagic Fever Virus Antibodies Prevalence in Small Ruminants from Southern Romania Species Collection site Sheep Goat Cataloi 77 35 Baia 20 0 Slava Rusa 9 Slava Cercheza Somova Total Reference population (France) Sampling period Overall Positive/ Rhipicephalus Seroprevalence Seroprevalence prevalence, % goats, % sheep, % sanguineus tested 95% CI 95% CI 95% CI ticks animals August 2019 October 2020 August 2019 54/112 169 31.2 20.8 to 41.5 0 85.7 74.1 to 97.3 0 48.2 38.9 to 57.4 0 0/20 NA 23 August 2019 October 2020 1/32 NA 11.1 -9.42 to 31.64 0 3.1 -2.9 to 9.15 59 0 August 2019 13/59 NA 0 27/29 NA 22 11.4 to 32.6 100 100 84.6 65 to 104.2 22 11.4 to 32.6 93.1 83.8 to 102.3 16 13 August 2019 181 71 95/252 169 — 6/96 — 57.7 46.3 to 69.2 — 37.7 31.7 to 43.7 — 96 95% CI, 95% confidence interval. 29.8 23.2 to 36.5 6.3 1.4 to 11.1 Downloaded by 54.146.243.225 from www.liebertpub.com at 03/05/24. For personal use only. 400 In Bulgaria and Hungary, countries that are on the border with Romania, CCHFV is endemic, which suggests that the virus could also circulate in Romania. Small ruminants have been recognized as CCHFV hosts in certain endemic regions and have been epidemiologically linked to human cases (Gergova and Kamarinchev 2013, Földes et al. 2019, Magyar et al. 2021). Domestic animals (sheep and goats) that serve as amplifying hosts are frequently tested seropositive (Spengler et al. 2016). Our results revealed seropositive results in small ruminants from four out of five localities, suggesting a uniform circulation of the virus in Southern Romania. However, similar seroprevalence rates in domestic animals were also reported in other countries, although using different reagents in most cases: Pakistan (36.2%) (Zohaib et al. 2020), Pakistan (32.6% in sheep and 18.9% in goats) (Zohaib et al. 2020), Albania (46% in sheep and 28% in goats) (Lugaj et al. 2014), and Republic of Macedonia (75% in sheep and 59% in goats) (Schuster et al. 2016, Mertens et al. 2013); lower rates were detected in Greece (25%) (Papa et al. 2014), Kosovo (18.4%) (Fajs et al. 2014), Bulgaria (22.7% in goats and 7.7% in cattle) (Christova et al. 2018), and Corsica (3.1% in goats and 2.5% in sheep) (Grech-Angelini et al. 2020), whereas an even higher seroprevalence was found in Turkey (57% and 74%) (Mertens et al. 2016). In Romania, only few serological studies with limited data regarding the CCHFV circulation are available. Ceianu et al. (2012) tested 471 sheep serum samples from different localities from Tulcea county, obtaining a prevalence of 27.8% for IgG antibodies against CCHFV. However, a more recent study conducted by Raileanu in 2015 reported an overall prevalence of 74% IgG antibodies among 90 domestic ruminants from Tulcea and Constant, a counties (Raileanu et al. 2015). In the study conducted by Raileanu, 74 Rhipicephalus sp. ticks, collected from the positive animals, were tested negative using the same RT-PCR method, as in our study. It is puzzling that such high seroprevalence was observed in Romania without any recorded human case. This is not unique. Some countries record human cases that parallel animal seroprevalence (Yen et al. 1985, Mostafavi et al. 2013, Zohaib et al. 2020), while in other areas, high level of seroprevalence can be detected without any evidence for human cases. For example, Bulgaria reported a high seroprevalence in ruminants in the absence of reported human cases, in many regions (Christova et al. 2018). While there appears to be an association between the presence of infected ticks and detection of seropositive animals at the population level, viral RNA detection in attached ticks is not linked to seropositivity of the infested host, and vice versa (Zeller et al. 1997). Another possible hypothesis is that different CCHFV biotypes with different pathogenicity for humans exist in the field, while not detected up to now, or, alternatively, that another prevalent Nairovirus with antigenic cross-reactivity with CCHFV may circulate. To complete our serological results and try to identify the CCHFV strain responsible for infection of these animals, Rhipicephalus sp. ticks were collected from small ruminants, in one location from where we had collected the largest number of serum samples. First, our results did not show any significant correlation between ruminant tick infestation (mainly Rhipicephalus) and animal’s seropositivity. Indeed, the main CCHFV vector is Hyalomma sp., but ticks of the genera Rhipicephalus are also capable of transmitting the BRATULEANU ET AL. virus (Zeller et al. 1997). Second, we did not find any CCHFV-positive tick; our results could be related to tick species collected, Hyalomma sp. ticks not being tested, which could be considered a limitation of our study. Conclusions In summary, our results indicate the circulation of CCHFV or another close Nairovirus among small ruminants in Southern Romania. Further studies should focus on increasing the number and species diversity of collected ticks and in expanding the geographical area of research, and should add NGS sequencing, to broaden the spectrum of detection of nairoviruses throughout Romania. Authors’ Contributions B.B.: conceptualization, formal analysis, resources, investigation, writing-original draft, and visualization; A.A.: investigation and resources; S.T.: conceptualization and reviewing; A.D.: investigation; L.C.: investigation; A.C.: investigation; P.P.: funding acquisition; G.S.: conceptualization, supervision, reviewing, and editing; M.E.: conceptualization, supervision, reviewing, and editing; D.A.: investigation, resources, and editing. All authors have read and approved the final article. Author Disclosure Statement One of the authors (P.P.) works at the IDvet company (310, rue Louis Pasteur, Grabels, France), which commercializes this ELISA kit. However, this does not alter the authors’ adherence to the principles of good scientific practice and to relevant policies on sharing data and materials. The authors declare no other competing interest. Funding Information Funded by Agence Nationale de la Recherche France, grant no. ANR-10-LABX-62-IBEID. References Bente DA, Forrester NL, Watts DM, McAuley AJ, et al. Crimean-Congo hemorrhagic fever: History, epidemiology, pathogenesis, clinical syndrome and genetic diversity. Antiviral Res 2013; 100:159–189. Bratuleanu BE, Temmam S, Chrétien D, Regnault B, et al. The virome of Rhipicephalus, Dermacentor and Haemaphysalis ticks from Eastern Romania includes novel viruses with potential relevance for public health. Transbound Emerg Dis 2022; 69:1387–1403. Ceianu CS, Panculescu-Gatej RI, Coudrier D, Bouloy M. First serologic evidence for the circulation of Crimean-Congo hemorrhagic fever virus in Romania. Vector Borne Zoonotic Dis 2012; 12:718–721. Christova I, Panayotova E, Groschup MH, Trifonova I, et al. High seroprevalence for Crimean-Congo haemorrhagic fever virus in ruminants in the absence of reported human cases in many regions of Bulgaria. Exp Appl Acarol 2018; 75:227–234. Fajs L, Humolli I, Saksida A, Knap N, et al. Prevalence of Crimean-Congo hemorrhagic fever virus in healthy population, livestock and ticks in Kosovo. PLoS One 2014; 9: e110982. Downloaded by 54.146.243.225 from www.liebertpub.com at 03/05/24. For personal use only. CCHF PREVALENCE IN RUMINANTS FROM ROMANIA Földes F, Madai M, Németh V, Zana B, et al. Serologic survey of the Crimean-Congo haemorrhagic fever virus infection among wild rodents in Hungary. Ticks Tick Borne Dis 2019; 10:101258. Gergova I, Kamarinchev B. Comparison of the prevalence of Crimean-Congo hemorrhagic fever virus in endemic and nonendemic Bulgarian locations. J Vector Borne Dis 2013; 50: 265–270. Grech-Angelini S, Lancelot R, Ferraris O, Peyrefitte CN, et al. Crimean-Congo hemorrhagic fever virus antibodies among livestock on Corsica, France, 2014–2016. Emerg Infect Dis 2020; 26:1041–1044. Honig JE, Osborne JC, Nichol ST. The high genetic variation of viruses of the genus Nairovirus reflects the diversity of their predominant tick hosts. Virology 2004; 318:10–16. Hoogstraal H. The epidemiology of tick-borne Crimean-Congo hemorrhagic fever in Asia, Europe, and Africa. J Med Entomol 1979; 15:307–417. Lugaj A, Mertens M, Groschup M, Bërxholi K. Serological survey of CCHFV in cattle in 10 regions of Albania. Int J Res Appl Nat Soc Sci (IMPACT IJRANSS) 2014; 2:55–60. Magyar N, Kis Z, Barabás É, Nagy A, et al. New geographical area on the map of Crimean-Congo hemorrhagic fever virus: First serological evidence in the Hungarian population. Ticks Tick Borne Dis 2021; 12:101555. Mertens M, Schmidt K, Ozkul A, Groschup MH. The impact of Crimean-Congo hemorrhagic fever virus on public health. Antiviral Res 2013; 98:248–260. Mertens M, Schuster I, Sas MA, Vatansever Z, et al. CrimeanCongo Hemorrhagic Fever Virus in Bulgaria and Turkey. Vector Borne Zoonotic Dis 2016; 16:619–623. Mertens M, Vatansever Z, Mrenoshki S, Krstevski K, et al. Circulation of Crimean-Congo Hemorrhagic Fever Virus in the former Yugoslav Republic of Macedonia revealed by screening of cattle sera using a novel enzyme-linked immunosorbent assay. PLoS Negl Trop Dis 2015; 9:e0003519. Mostafavi E, Haghdoost A, Khakifirouz S, Chinikar S. Spatial analysis of Crimean Congo hemorrhagic fever in Iran. Am J Trop Med Hyg 2013; 89:1135–1141. Negredo A, de la Calle-Prieto F, Palencia-Herrejón E, MoraRillo M, et al. Autochthonous Crimean-Congo hemorrhagic fever in Spain. N Engl J Med 2017; 377:154–161. Papa A, Chaligiannis I, Kontana N, Sourba T, et al. A novel AP92-like Crimean-Congo hemorrhagic fever virus strain, Greece. Ticks Tick Borne Dis 2014; 5:590–593. 401 Raileanu C, Anita A, Porea D, Savuta G. Serologic evidence of Crimean-Congo haemorrhagic fever infection in small ruminants in Southeastern Romania. Epidemiol Sante Anim 2015; 67:145–149. Sas MA, Comtet L, Donnet F, Mertens M, et al. A novel double-antigen sandwich ELISA for the species-independent detection of Crimean-Congo hemorrhagic fever virus-specific antibodies. Antiviral Res 2018a; 151:24–26. Sas MA, Vina-Rodriguez A, Mertens M, Eiden M, et al. A onestep multiplex real-time RT-PCR for the universal detection of all currently known CCHFV genotypes. J Virol Methods 2018b; 255:38–43. Schuster I, Mertens M, Mrenoshki S, Staubach C, et al. Sheep and goats as indicator animals for the circulation of CCHFV in the environment. Exp Appl Acarol 2016; 68:337–346. Spengler JR, Bergeron É, Rollin PE. Seroepidemiological Studies of Crimean-Congo Hemorrhagic Fever Virus in Domestic and Wild Animals. PLoS Negl Trop Dis 2016; 10: e0004210. Whitehouse CA. Crimean-Congo hemorrhagic fever. Antiviral Res 2004; 64:145–160. Yen YC, Kong LX, Lee L, Zhang YQ, et al. Characteristics of Crimean-Congo hemorrhagic fever virus (Xinjiang strain) in China. Am J Trop Med Hyg 1985; 34:1179–1182. Zeller HG, Cornet JP, Diop A, Camicas JL. Crimean-Congo hemorrhagic fever in ticks (Acari: Ixodidae) and ruminants: Field observations of an epizootic in Bandia, Senegal (1989– 1992). J Med Entomol 1997; 34:511–516. Zohaib A, Saqib M, Athar MA, Hussain MH, et al. CrimeanCongo hemorrhagic fever virus in humans and Livestock, Pakistan, 2015–2017. Emerg Infect Dis 2020; 26:773–777. Address correspondence to: Dragos Anita Regional Center of Advanced Research for Emerging Diseases, Zoonoses and Food Safety (ROVETEMERG) ‘‘Ion Ionescu de la Brad’’ University of Life Sciences 3, Mihail Sadoveanu Alley Iasi 700490 Romania E-mail: [email protected]