MATERIAL AND METHODS
Ticks were collected during July 2019 from different locations in south-eastern Romania. The sampling consisted in environmental and body-surface of domestic ruminants, from five sites across Tulcea county. Tulcea county (latitude: 44.64°S–45.42°N; longitude: 28.00°W–29.65°E) is situated in southeast Romania and forms the northern part of Dobrogea province. Vegetation is represented by submediterranean broadleaf forests and xerophytic pastures with shrubs. The county is bordered by the river Danube (W and N), and the Black Sea (E), and is neighboring Ukraine (N). Ground-feeding birds and small and larger mammals, including livestock, are abundant in this area.
Questing ticks collection was performed by dragging. The surface covered at each collection sites was ∼1 hectare and the flag was examined after every 10 m of dragging, at the end of each segment. Between every 10 m of dragging, every transect was separated by 20 m. Tick sampling in Tulcea county was performed only once at each site (Table 1). Engorged ticks were collected on domestic ruminants. The whole body of each sampled animal was inspected for the presence of tick infestations by palpation, mainly on the ears and along the nape of the neck, perineum, scrotum or udder, and tail base. Ticks collected from each animal were kept alive in separate vials and labeled. Live ticks were transported in vials (within a closed secondary container) to the laboratory. Prior handling, ticks were cooled by placing the vials in ice. Developmental stages were identified under a stereomicroscope using standard morphological identification keys. After the identification, each tick was cut lengthwise, using sterile scalpels on an ice-cold surface Petri dish, in a class II laminar flow hood. Half was used for additional processing and the remainder was stored at −80°C. Ticks were transferred to Institut Pasteur (Paris, France) and analysed by real time RT-qPCR. For maintaining the integrity of RNA, ticks were introduced in RNA later solution (Invitrogen, France), according to best security practices of storing and transport.