The molecular prevalence of Ancylostoma caninum was determined in stray and pet dogs in Lahore, Pakistan from July 2014 to August 2015. A total of 500 dog faecal samples were first evaluated using a sedimentation technique and further through a PCR assay targeting the ITS-2 region of the A. caninum genome. Overall, 130 (26%) samples were positive for hookworm infestation by microscopic examination. Only microscopically positive samples were processed for PCR and 89 (17.6%) were positive for A. caninum. Sequence analysis of amplicons showed 100% homology with A. caninum and the genotypes clustered in one clade with Brazilian A. caninum hookworms. There was a significantly (P < 0.05) increased prevalence in male dogs younger than six months old. Labrador retrievers had higher A. caninum incidence compared to German shepherds and other breeds. To the best of our knowledge, this is the first report of the molecular prevalence of A. caninum in dogs in Pakistan. The novel results of the present study allow us to conclude that A. caninum is more prevalent in pet dogs, especially puppies, and this can be a potential threat for humans that come into contact with such animals. Therefore, the routine monitoring of pets, especially the more susceptible breeds, is essential for disease control.
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