First evidence of Crimean‐Congo haemorrhagic fever virus circulation in Bosnia and Herzegovina

Abstract Background Crimean–Congo haemorrhagic fever (CCHF) is a widespread tick‐borne zoonosis with reported detection of virus and/or virus‐specific antibodies from over 57 countries across Africa, Asia, Europe and the Middle East and is endemic in the Balkans. Detection of Crimean–Congo Haemorrhagic Fever Virus (CCHFV) antibodies in domestic ruminants has been important in providing initial evidence of virus circulation and in localising CCHFV high‐risk spots for human infection. Objectives The present study investigated the possible exposure of sheep to CCHFV in Bosnia and Herzegovina (B&H). Methods To investigate the presence of anti‐CCHFV antibodies in sheep, all sera (n = 176) were tested using multi‐species double antigen enzyme‐linked immunosorbent assay (ELISA). Reactive sera were further complementary tested by adapted commercial indirect immunofluorescence assay (IFA) using FITC‐conjugated protein G instead of anti‐human immunoglobulins. Results CCHFV specific antibodies were detected in 17 (9.66%) animals using ELISA test. All negative sera were determined as negative by both tests, while 13 out of 17 ELISA‐positive reactors were also determined as unambiguously positive by IFA test. The age group with the highest proportion of seropositive rectors were the oldest animals. Conclusions This is the first report of anti‐CCHFV antibodies in sheep from B&H providing the evidence of CCHFV circulation in the country's sheep population. So far, these findings indicate the circulation of the virus in the westernmost region of the Balkans and point to the potential CCHFV spread further out of this endemic area.


INTRODUCTION
Crimean-Congo haemorrhagic fever virus (CCHFV; family Nairoviridae, genus Orthonairovirus), the causative agent of Crimean-Congo haemorrhagic fever (CCHF) has been recognised as an emerging pathogen of great importance with significant potential as a weapon of biological warfare (Kuhn et al., 2020;WHO, 2021). CCHF is a widespread tickborne zoonosis with reported detection of virus and/or virus-specific antibodies from over 57 countries across Africa, Asia, Europe and the Middle East (Bente et al., 2013). The majority of human infections have an asymptomatic clinical course; however the development of severe clinical forms of the disease in infected people is not such a rare event and should not be neglected (Bente et al., 2013;Portillo et al., 2021).
The transmission occurs through the bite of infected ticks; however, blood and other body fluids of infected animals and human patients represent an additional source of infection (Gunes et al., 2009). The virus is maintained in an enzootic cycle between ixodid ticks, predominantly Hyalomma genus that serves as vectors and reservoirs with transovarial, transstadial, venereal and non-viremic (co-feeding) transmission among them, and wild and domestic vertebrates (Turell, 2007). Domestic ruminants, despite experiencing transient viremia, remain clinically asymptomatic and pose latent sources of infection for ticks and humans (Bente et al., 2013). Despite the well-known CCHF endemicity in the western Balkan region (Messina et al., 2015), there has been no evidence of CCHFV circulation in Bosnia and Herzegovina (B&H). Furthermore, there are no control measures or surveillance programs for CCHF put in place. Detection of CCHFV antibodies in domestic animals has been important in providing initial evidence of virus circulation and in localising CCHFV high-risk spots for human infection (Spengler et al., 2016). Moreover, it is well known that the first reports of the disease in some countries coincided with the introduction of effective control measures (Spengler et al., 2019). Therefore, our study aimed to investigate the possible exposure of sheep to CCHFV in B&H.

MATERIAL AND METHODS
Considering the aim of the study, the most suitable study area was the southeastern part of B&H due to its specific geographic position, that is, the proximity to CCHFV endemic regions in Serbia and Kosovo ( Figure 1). This area is characterised by the moderate continental climate from the north and the Mediterranean climate from the south (Statistical Yearbook of Republika Srpska, 2018), known as a natural habitat for ixodid ticks (Omeragic, 2011), that is, vectors of the disease.
Two municipalities Gacko and Nevesinje with associated local communities were selected for the study. Geographical and bioclimatic features of the selected study area are given in Table 1.
Given that selection of the study area was convenient, the sampling strategy was purposive, organised as risk-based two-stage sampling.
In the first stage, the target population was all sheep flocks located in the previously selected municipalities. Afterwards, the source popula- Optionally, the samples were centrifuged at 3000 × g for 5 min. Serum was pipetted into cryovials and stored at −20 • C until being tested.

RESULTS
Out of 176 serum samples examined by ELISA, 17 (9.66%) were found to be positive for CCHFV. The ELISA test results from both laboratories were congruent, without any significant difference and therefore presented as mean values of results obtained from both testing laboratories (Table 3)

TA B L E 1 Characteristics of the selected study localities and sheep flocks in southwestern Bosnia and Herzegovina
Location/municipality The remaining four samples gave inconclusive results (Table 3). All IFA results in which it was not possible to unambiguously assess the fluorescence of both target proteins (CCHFV-GPC and CCHFV-N) or on one of them while the other was negative were declared as inconclusive results. However, these samples were not further retested.

DISCUSSION
Thus far, there is no scientific data of CCHFV circulation in animals or vectors, nor have the cases of the disease been reported in humans in B&H. Hence, to the best of our knowledge, this study provides early evidence of CCHFV circulation in animals in B&H. The exposure to CCHFV, that is, the evidence of specific antibodies in the sheep population, was confirmed by serological tests. The discrepancy between ELISA and IFA test may be due to known differences in the detectability of sheep immunoglobulins by both kits: while ELISA is reactive to both IgG and IgM, the latter would not be expected to react in the adapted IFA. Indeed, this outcome may reflect different infection stages among sampled animals (Dowall et al., 2012). In this study, older animals were more likely to be seropositive for CCHFV in comparison with younger animals (Table 2). This observation coincides with a recent study of age dependent CCHFV seroprevalences in Mauritanian livestock (Schulz et al., 2021). The simple explanation of this phenomenon is faced in the possibility that older animals are exposed to CCHFV-infected competent vectors longer than younger animals, and thus becoming infected  (Hartlaub et al., 2021). However, considering that there is no scientific evidence of the introduction and circulation of other Orthonairoviruses in the Balkan region, the possibility of serological cross-reactions is substantially low.
Possible factor that could have contributed to the emergence of seropositive individual animals in the southeastern parts of the country is a favourable climatic condition for the life cycle of ticks, especially from the genus Hyalomma. In addition to vectors, the specific geographic position of the investigated area, that is, relative proximity to endemic regions in eastern neighbouring countries (Serbia, Kosovo and Montenegro), could partially contribute to the CCHFV emergence in B&H. This can be explained by the nomadic and semi-nomadic pastoralism and the possibility of illegal cross-border transport and trade of animals from the CCHFV endemic regions to the naïve sheep population in B&H. In such a situation and given the fact that there is no CCHFV surveillance program in B&H, the import of potentially infected animals and vectors cannot be completely ruled out. Based on our results, favourable climatic and ecological conditions with the proven presence of competent vectors in B&H (Omeragic, 2011) as well as reports of CCHF/CCHFV detection in the rest of the Balkan region (Dreshaj et al., 2016), it is reasonable to assume that there is a possibility for CCHFV circulation in B&H as well. However, taking into account a relatively small study area and the number of investigated animals, the results presented here warrant further widespread investigation to get a real picture of the circulation of CCHFV in the whole country. In line with this, our further research will focus on gathering additional serologic as well as molecular evidence of CCHFV circulation in this but also in other regions of B&H, both in ticks and in susceptible vertebrates.