Gastrointestinal strongyle infections are common in sheep and require accurate species identification for effective diagnosis and control. Morphological identification is limited, and molecular markers such as the internal transcribed spacer 2 (ITS2) region are widely used for species differentiation. However, variability of ITS2 sequences at different biological levels may influence interpretation of molecular data. The aim of this study was to analyze ITS2 sequence variability at three levels—within individual parasites (intra-individual variability), within species (intraspecific variability), and between species (interspecific variability)—using cloned sequences obtained from morphologically identified adult strongyles in sheep in Slovenia. Adult nematodes were collected from four naturally infected sheep, and five species were identified: Trichostrongylus axei, Trichostrongylus colubriformis, Teladorsagia circumcincta, Cooperia curticei and Chabertia ovina. A total of 45 ITS2 sequences were obtained from 15 parasite specimens (three clones per specimen) and analyzed using sequence alignment and phylogenetic methods. Intra-individual variability was generally low, with identical sequences observed in some species and minor differences in others. Intraspecific variability ranged from zero to 3.8%, depending on the species, while clear interspecific differences enabled reliable species discrimination. Phylogenetic analysis confirmed clustering of Slovenian isolates with corresponding reference sequences. These findings indicate that ITS2 is a suitable marker for distinguishing major gastrointestinal strongyle species, while highlighting the presence of intra-individual and intraspecific variation that should be considered when interpreting molecular data. The results represent preliminary ITS2 sequence data for strongyles in sheep in Slovenia and provide a basis for future molecular studies.

To Cite This Article: Šimenc Kramar L and Posedi J, 2026. ITS2-based molecular identification and phylogenetic analysis of major gastrointestinal strongyles in sheep from Slovenia. Pak Vet J, 46(5): 1140-1151. http://dx.doi.org/10.29261/pakvetj/2026.102