Ancylostoma caninum and Uncinaria stenocephala hookworms: Morphological and molecular differentiation and epidemiological data in Southern Italy.

Ancylostoma caninum and Uncinaria stenocephala are the most prevalent canine hookworms worldwide, both having veterinary and public health importance. Considering the emergence of anthelmintic resistance in A. caninum and the inefficacy of milbemycin oxime against U. stenocephala, the accurate diagnosis is essential, especially in areas where they occur in sympatry. Here, we differentiated hookworm species in naturally infected dogs from southern Italy, using coproculture and morphometric analysis of third-stage larvae (L3) and eggs, along with molecular characterization. Dog fecal samples (n = 278) from Basilicata and Apulia regions were screened, with 39.2 % testing positive for hookworm eggs. Two coproculture protocols (incubation at 15-18 °C and 21°C) were applied. Morphologically, U. stenocephala (n = 682) and A. caninum (n = 159) larvae exhibited similar dimensions (mean body length of 582.7 µm ± 39.5; width of 28.8 µm ± 2.7; and mean body length: 584.2 µm ± 39.5; width: 29.0 µm ± 2.9, respectively), with 32 well-defined intestinal cells in the first species and denser lipid granules that hindered cell visualization, in the latter. Egg morphometry supported species identification for U. stenocephala (83.5 µm ± 6.3 µm and 45.9 µm ± 5.3 µm) and A. caninum (61.5 µm ± 19-44 µm ± 8.4 µm). Molecular analysis based on the 18S rRNA gene and ITS-1 fragment confirmed the species, with sequences showing 98-99 % nucleotide identity. Moreover, U. stenocephala was more prevalent (85.1 %; 74/87) than A. caninum (26.4 %; 23/87), including coinfections (11.5 %). This study shows the value of morphological identification for the diagnosis of canine hookworms in co-endemic areas, with direct implications for treatment decisions and parasitic surveillance.