Detection of Crimean–Congo haemorrhagic fever virus in Hyalomma marginatum ticks, southern France, May 2022 and April 2023

Risk-based sampling

As ticks are the only known natural reservoirs of CCHFV [4], we focused on field collection of ticks. In May 2022, during the peak activity of ticks, we collected ticks mainly from horses in four Mediterranean departments (NUTS-3) on the French mainland bordering Spain (Figure 1). Horses are the likely hosts of adult stages of H. marginatum [5,6].

Cattle are considered good amplifiers of CCHFV and thus enhance local virus circulation [7]. In 2023, we optimised the sampling by collecting ticks from cattle in the Pyrénées-Orientales department (Figure 1) where antibodies against CCHFV were identified in 2021-2022 from cattle (maps showed in https://www.anses.fr/fr/system/files/SABA2020SA0039Ra.pdf). Cattle farms with the highest within-herd seroprevalences were selected, as well as a few seronegative farms in the same zones. In addition, farms with horses (not previously tested with serology), located in the neighbourhood of the seropositive cattle farms were also visited, especially when the cattle farmers did not give their consent to sampling. As Hyalomma ticks are not located within cattle barns but likely in the natural farm environments, specific pastures (e.g. shrublands) were selected. In spring, cattle from different farms are gathered and grazed in such shrublands, and these spring pastures were thus considered as suitable sites for collection of H. marginatum [8]. Ticks were collected in April when adult H. marginatum search for hosts and potentially infect naïve cattle.

Laboratory investigations

Ticks were species identified morphologically by experimented acarologists using relevant identification keys [9,10], and only H. marginatum ticks were included in the further analysis and stored at -80°C.

After being washed in bleach for 30 s and rinsed three times in water, the ticks were crushed individually in 400 µL of Dulbecco’s Modified Eagle Medium (DMEM) (Eurobio Scientific, Les Ulis, France) supplemented with 10% fetal calf serum. Total RNA was extracted using a kit, NucleoMag VET extraction kit (Macherey-Nagel, Düren, Germany), according to the instructions of the manufacturer, using IDEALTM 96 extraction robot (Innovative Diagnostics, Montpellier, France). Then, CCHFV simplex reverse transcription (RT) quantitative PCRs were run, adapted from previously published studies [11,12]. Optimisations, including modifications to the PCR thermoprofile and mix, were carried out by Innovative Diagnostics, and allowed the quickest detection and the best amplification curve for a synthetic RNA used as positive control. The PCR detection limit was estimated to five viral genomic copies.

Positive tick homogenates as well as a set of negative ones were sent for confirmation to the Biosafety Level 4 laboratory of the National Reference Center for Viral Hemorrhagic Fevers (NRC), Lyon, France. After removing tick tissues from suspensions, RNA was extracted using the QIAamp viral RNA mini kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Thereafter, detection of the virus was performed using the QIAGEN OneStep RT-PCR kit (Qiagen), according to a protocol adapted from Wölfel et al. [12].

The NRC selected samples with quantification cycle (Cq) values < 25 for whole genome sequencing. The RNA was subjected to a DNase digest (Turbo DNase, Thermo Fisher Scientific, Waltham, the United States (US)). Then, library preparation was performed using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, US). Sequencing was performed using a MiniSeq platform (Illumina Inc, San Diego, US). The paired-end reads generated were trimmed and de-novo assembled using rnaviralSPAdes (version 3.15.4) on Galaxy platform (version 23.2.rc1) (https://usegalaxy.org/). The CCHFV genomes (segment S, M and L) were searched from the contigs using Basic Local Alignment Search Tool (BLAST) (version 2.14.1). We used the BioEdit software (version 7.1.3.0) (https://bioedit.software.informer.com/) to align the nucleotide sequences obtained with published sequences belonging to the different lineages of CCHFV.

Findings of ticks and Crimean–Congo haemorrhagic fever virus

In 2022, an average of 30 H. marginatum ticks per location, and in 2023 all ticks, were analysed for CCHFV.

In 2022, ticks were collected on 33 horse and three cattle farms. In total, 997 ticks of H. marginatum were identified and CCHFV was detected in 13 (1.3%) ticks (all from the same cattle farm in Pyrénées-Orientales) (Figure 1). In 2023, in Pyrénées-Orientales, ticks were collected on 15 cattle farms, three farms with cattle and horses and three farms with horses. A total of 1,001 H. marginatum ticks were collected and analysed, and 142 (14.2%) were positive for CCHFV. Most of these positive ticks were collected from cattle farms (n = 11), except for 12 ticks from two horse farms (Figure 1).

Considering 2022 and 2023 data, the proportion of infected ticks in positive farms varied from 3.1% to 55.8% (median: 7.4%) or 1 to 66 positive ticks per farm, with four farms with infection rates up to 20%. The Cq values ranged from 18.25 to 40.82 (median:  36.39), which suggested a high viral load in some ticks. At the NRC, CCHFV RNA was confirmed in 132 (85.2%) of 155 samples, and six additional ones among the 114 initially negative samples. All Cq values from the 23 non-confirmed positive samples were high (around 40). All obtained sequences clustered with CCHFV strains, such as the Caceres strain previously isolated from ticks in Spain, within the genotype III (Figure 2).

Discussion

For the first time, CCHFV was detected in resident tick populations in the mainland of France, confirming its circulation. Crimean–Congo haemorrhagic fever is one of the most widespread viral tick-borne zoonoses worldwide with severe consequences for healthcare, such as intensive case management, 5–30% fatality rate in haemorrhagic patients and the need to prevent nosocomial infections [13]. Endemic in Africa, the Middle East and other Asian countries and in the Balkans, CCHF was recently detected in Spain, western Europe [14]. Transmission of the virus to humans occurs predominantly via tick bites or via exposure to infected blood or tissues of viraemic animals or patients [1]. Given the recent findings of H. marginatum, one main competent vector for CCHFV, in the south of France [8,15] near the Spanish border, it was crucial to evaluate the epidemiological situation of CCHFV in France.

We considered that CCHFV circulation in France would be at a low level. Indeed, immature stages of H. marginatum are mainly found on birds and lagomorphs. Most bird species are refractory to CCHFV and thus unable to reinfect naïve ticks, whereas lagomorphs are good amplifiers of the virus. In France, bird populations are sizeable in the south of the country. The populations of hares and rabbits have decreased in France since the outbreak of myxomatosis in the 1950s and the current occurrence of rabbit viral haemorrhagic disease. Consequently, the probability for H. marginatum to become infected at the immature tick stage in France is considered low [7]. Furthermore, adult stages of H. marginatum have a marked trophic preference for horses in France [5], and horses are poor amplifiers of CCHFV (unable to reinfect new ticks) although they are very abundant in the south of France. Thus, the probability for H. marginatum to become infected at the adult stage is considered low [7].

Our findings of a high proportion of infected ticks in some farms further reinforced that this cannot be due to sporadic introduction of infected H. marginatum ticks from endemic countries but local virus transmission. However, such introduction events remain a risk to be monitored, especially to detect the emergence of new CCHFV genotypes [16-18]. All CCHFV isolates sequenced in this study were highly identical and belonged to the same genotype. Ticks infected with CCHFV were predominantly from cattle although many horses were examined, with high proportions of infected ticks in four facilities where very few animals were examined (4–6 animals per farm). This strengthens the importance of cattle in the CCHFV transmission and the possibility of having sampled viraemic animals. As horses are not able to replicate sufficiently CCHFV to infect naïve ticks [19], finding a few infected ticks on horses indicated that these H. marginatum were infected at immature stages or through transovarial transmission, and then maintained the virus along their development.

Because of the risk-based sampling method used, the proportion of infected ticks does not reflect the prevalence of H. marginatum ticks infected with CCHFV. The prevalence of infected ticks is presumably lower which may explain that no human cases have, so far, been notified in France. Additionally, genotype III, which was identified from the first Spanish cases [20], is considered of low virulence. At present, in France, humans may become infected via bites of adult H. marginatum ticks. This risk exists during the activity period of the ticks, namely from April to July. Only ticks seeking hosts in the environment should be considered as possible vehicles for virus transmission; those already attached to animals cannot detach and reattach. Risk areas for CCHFV transmission present conditions conducive to the presence of H. marginatum (open dry natural environments, typical of the Mediterranean area), but infected ticks were not found in all locations infested by H. marginatum and further parameters suitable for local circulation of CCHFV between ticks and animals, remain to be determined. Another CCHFV transmission pathway could be contact with infected animal material, as animals can be viraemic, albeit asymptomatic, during the seasonal activity of H. marginatum e.g. from April to July when adult stages parasite wild and domestic ungulates and from August to October when immature stage parasite lagomorphs. Considering such risks, preventive recommendations towards at-risk populations are needed.