In August 2023, a 42-year-old male patient was referred to the Experimental Zooprophylactic Institute of Southern Italy with a tick attached to his neck. The patient had noticed the tick 48 hours after a hike in a rural area of Salerno Province, Campania region, southern Italy. After obtaining the patient's signed informed consent, we removed the tick with fine-tipped tweezers, checked the patient's skin for other ticks, and collected a 7-mL blood specimen from his cephalic vein. We used a 2-mL aliquot placed in a Vacutainer (https://www.bd.com) K3-EDTA tube for complete blood count on a CELL-DYN 3700 Hematology Analyzer (Abbott, https://www.abbott.com). We added the remaining 5 mL to a Vacutainer clot activator serum tube for biochemical analysis on a SAT 450 Random Access Analyzer (KPM Analytics, https://www.kpmanalytics.com) after centrifugation for 15 min at 1,500 g at room temperature. We classified the tick with regard to developmental stage (larva, nymph, or adult), sex (male or female), and feeding status (engorged or not engorged) by using a Leica MS5 stereomicroscope (https://www.leica-microsystems.com). Then we taxonomically identified the tick by using morphological keys. In addition, we looked for pathogenic microorganisms by performing Romanowsky staining using Diff Stain Quick Kit (ProEko, https://www.proekosrl.com) on smears of the gut, hemolymph, and salivary glands of the tick and peripheral blood of the patient.

We extracted DNA from the tick and the patient's blood by using QIAamp DNA Blood and Tissue kit (QIAGEN, https://www.qiagen.com) and molecularly tested it for tickborne pathogens (8,9) (Table 1). The tick was also molecularly identified at species level by the amplification of a 248-bp partial fragment of the 16S rRNA gene, with forward (5'-CTGCTCAATGATTTTTTAAATTGCTGT-3') and reverse (5'-TTACGCTGTTATCCCTAGAG-3') primers, by using the following thermocycling conditions: 95°C for 10 minutes of initial denaturation followed by 35 cycles of 94°C for 45 seconds, 58°C for 60 seconds, 72°C for 60 seconds, and 72°C for 7 minutes of final extension. We ran all PCRs in a final volume of 50 μL including 5 μL of 10× PCR buffer II, 6 μL of 25 mmol MgCl, 5 μL of 1.25 mmol of dNTPs, 0.5 μL of 100 pmol/μL of each primer, and 1.25 U of AmpliTaq Gold (Applied Biosystems, https://www.thermofisher.com). We sequenced the purified amplicons in both directions by using a BigDye Terminator v3.1 Cycle Sequencing Kit in a 3130 Genetic Analyzer (Applied Biosystems), then used Geneious version 9.0 (https://www.geneious.com) for editing and analysis. We compared the resulting sequences with those available in the GenBank database by using Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and performed the phylogenetic analysis by using the maximum-likelihood method based on the general time reversible model with gamma distribution to assess evolutionary differences among sites (+G) selected by best-fit model (10) with MEGA X software (11). Our study was approved by the Experimental Zooprophylactic Institute of Southern Italy within the framework of a memorandum of agreement (authorization no. IZSM-DIMBA/23) with the Interdisciplinary Department of Medicine, University of Bari Aldo Moro, according to national regulations.

We identified the tick removed from the patient as an engorged female Haemaphysalis punctata (GenBank accession no. PP419005). According to PCR testing, the tick and the patient's blood scored positive for a fragment of the E. canis 16S rRNA gene. The sequences we obtained (GenBank accession nos. OR518413 and OR506261) were identical to each other (100% query cover) and to those of E. canis obtained from a red fox (Vulpes vulpes) in the same study area, humans from United States and Venezuela, and dogs from Mediterranean Basin countries (Italy, Greece, Israel, Egypt, and Turkey) (Figure). We confirmed the molecular identification of E. canis from the tick and the patient's blood by amplifying the groEL gene. The sequences obtained were identical to each other (GenBank accession no. PP839296). Other pathogens were not detected by PCR or cytologic examination of stained smears. No major clinicopathologic abnormalities were detected in the patient, except severely increased alanine aminotransferase and mildly decreased total leukocyte count and aspartate aminotransferase (Table 2). Three days after the first visit, the patient complained of mild symptoms (i.e., fever of 38°C, headache, muscle pain, and malaise) which spontaneously healed within a week, without antimicrobial drug treatment.