*2.4. Ectoparasite Screening*

We screened all carcasses for arthropod ectoparasites. Ectoparasites were collected by combing the fur, checking the skin, and carefully inspecting each carcass's storage bag. We also collected and stored any ectoparasites that left the carcass during the dissection process. Ectoparasites were stored in 70% ethanol in microcentrifuge tubes (one tube per parasite species per host individual) at −20 ◦C.

Ectoparasites were counted per species and life stage. Tick and flea species were identified using established identification keys [20,21]. Lice and mite species were identified by Herman J.W.M. Cremers (Utrecht University, Utrecht, The Netherlands), who also confirmed flea species identity.

#### *2.5. Gastrointestinal Parasite Screening*

Gastrointestinal parasites were isolated following previously described protocols [22]. Briefly, mouse intestines were separated individually into small (duodenum up to ileum) and large intestine (including caecum and colon), and their contents were homogenized in phosphate bu ffered saline. Parasites were collected by sieving the suspension over a 63 μm mesh size sieve. The stomach was screened for parasites macroscopically and microscopically. Isolated parasites were counted, sexed, identified morphologically, and stored in 70% ethanol.

#### *2.6. Screening for Microparasites*

DNA and RNA were extracted from tissue samples in a diagnostic laboratory using a robot (MagNA Pure Compact Extraction Robot; Roche, Basel, Switzerland) and Nucleic Acid Isolation Kit I (Roche, Roche, Basel, Switzerland) following the manufacturer's instructions. To detect potential cross-contamination, we included negative controls in each extraction batch. To minimize contamination and false positives, all main steps (extraction, PCR mix preparation, sample addition, and (q)PCR analyses) were performed in separate air-locked dedicated labs.

We analyzed all nucleic acid extractions with di fferent (multiplex) qPCRs carried out on a LightCycler 480 (Roche Diagnostics Nederland B.V, Almere, The Netherlands). These qPCRs were performed as described previously and based on gene fragments specific for the (vector-borne) microparasites of interest: *Borrelia burgdorferi* s. l. (two targets, [23]), *Borrelia miyamotoi* [24], *Anaplasma phagocytophilum* [25,26], *Candidatus* Neoehrlichia mikurensis [27], *Rickettsia* spp. [28], *Leptospira* spp. [29], and *Bartonella* spp. [30]. We also analyzed the nucleic acid extractions for the presence of four viruses: tick-borne encephalitis virus, Tula hantavirus, Puumala hantavirus, and Eyach virus. For the first three, we performed reverse transcription real-time PCRs as previously described [31–34]. Positive (plasmid) controls and negative (water) controls were used on every plate.

We used methodology that has not been previously described for two microparasites: *Spiroplasma ixodetes* and Eyach virus. We screened for *S. ixodetes* using exactly the same conditions as for *B. burgdorferi* s. l. but with (rpoBF) 5-TGTTGGACCAAACGAAGTTG-3 and (rpoBR) 5-CCAACAATTGGTGTTTGGGG-3 as primers and (rpoBP) 5-(FAM)GCTAACCGTGCTTT AATGGG(BHQ1)-3 as the probe. For the Eyach virus, we performed a reverse transcription real-time PCR targeting the VP2 of the Eyach virus genome with (EyachF) 5-TGGCTGACAAC ATGACGGATA-3 and (EyachR) 5-GGCCTCACGATACTTTCGATT-3 as primers and (EyachP) 5-ACGGGCTCGGTACTTCGGTTGAGAT-3 as the probe. We used 20 μL with TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), 5 μL of sample, and 0.2 μM for the primers and probes for the qPCR, which was performed with a 20 min reverse transcription step at 50 ◦C, denaturation at 95 ◦C for 30 s, and 50 cycles of 95 ◦C for 10 s and 60 ◦C for 30 s.

Samples positive for *Bartonella* spp. were subjected to conventional PCR and sequencing to determine species identity [35,36]. *Bartonella* sequences were compared with reference sequences from Genbank using the unweighted pair group method with arithmetic mean-based (UPGMA) hierarchical clustering.
