Clinical pathology, parasitological evaluation, and anthelmintic resistance assessment in dogs naturally infected with Uncinaria stenocephala

Uncinaria stenocephala is an increasingly reported canine hookworm in Europe, yet little is known about the clinical relevance of this hookworm under natural conditions. We therefore investigated U. stenocephala infection in domestic dogs from an endemic rural area of Basilicata (southern Italy), focusing on egg-shedding patterns, clinical impact, and anthelmintic susceptibility. Between July and October 2025, fecal and blood samples were collected from 20 naturally Uncinaria -infected dogs and two puppies, as well as three uninfected dogs. Egg shedding was quantified on days − 14, − 7, and 0 (baseline) using the Mini-FLOTAC technique. Dogs were treated with fenbendazole, moxidectin, or milbemycin oxime at day 0 and drug efficacy was evaluated via fecal egg count reduction test (FECRT) by comparison of fecal Uncinaria- egg counts to day 14 post-treatment. Molecular screening was performed to confirm species identity and detect single nucleotide polymorphisms (SNPs) associated with benzimidazole resistance, coupled with an in vitro egg hatch assay (EHA). Marked fluctuations in egg shedding occurred before treatment (i.e., 0–3,105 EPG), with sharp increases and decreases within sampling days (e.g., mean EPG increasing from 122 at day − 14 to 647.2 at day − 7, before decreasing to 162.2 at day 0). Fenbendazole and moxidectin showed high efficacy at the FECRT (i.e., 100% and 99.3% FECR, respectively), whereas milbemycin oxime achieved only 76.8% efficacy. Additionally, the in vitro EHA showed phenotypic benzimidazole susceptibility of IC 50 0.03 (95% CI 0.03 to 0.04) and IC 50 0.09 (95% CI 0.08 to 0.10). Hematological and biochemical values were mostly within reference limits consistent with low-intensity parasitism, but not with clinically significant disease. In particular, mild eosinophilia, slight hypoalbuminemia, and increased β-globulin fractions were the most frequent findings. Amplicon deep sequencing of the ITS-2 rRNA gene and β-tubulin isotype-1 from hookworm egg DNA consistently identified U. stenocephala as the sole hookworm species present, with no canonical benzimidazole resistance-associated SNPs detected at codons 134, 167, 198, or 200 of the β-tubulin isotype-1 gene in either pre- or post-treatment samples. Overall, this study provides an integrated evaluation of U. stenocephala in naturally infected dogs, demonstrating substantial variation in egg-shedding dynamics and high efficacy of fenbendazole and moxidectin, as well as the absence of benzimidazole-resistance-associated mutations. These findings contribute to baseline data for monitoring this underestimated hookworm species and reinforce the need for multi-sample fecal diagnostics and continued surveillance of anthelmintic efficacy.