Tick surveillance

The tick collection sites were designated within the administrative borders of the counties of Lublin Province (with the exception of Biała Podlaska and Łuków Districts) following the one district-one site principle and taking into account the division into subregions A-D described above (Fig. 3). In total, 18 sites were selected. Ticks were collected from mid-forest clearings surrounded by deciduous trees. Before starting the collection, the plots were checked for the presence of ticks. A 900-m² (30 × 30 m) plot was established in each site. Ticks were collected for 40 min.

The ticks were collected during the vegetation season at monthly intervals (usually every third week of the month) from March to October 2020. The standard flagging method was employed. A 1-m² flannel cloth was used to sweep the vegetation in the plots. Each time after covering ca. 15 m, the cloth was turned over and inspected to detect the presence of ticks attached to the fabric. The specimens collected in this way were transferred with metal tweezers into a 100-cm³ plastic container containing a moist cotton swab to provide approx. 75% relative humidity. Next, the ticks were transported to the laboratory to identify the species and developmental stage of the specimens using a Zeiss STEMI DV4 stereoscopic microscope (Carl Zeiss Light Microscopy, Göttingen, Germany) and a tick identification key⁷.