Despite Pakistan's rich reptilian diversity, wild lizards have largely been unexplored for vector-borne parasites. This study reports the prevalence and phylogenetic assessment of Hepatozoon ophisauri and Toxoplasma gondii DNA in the blood samples of 101 wild lizards from Khyber Pakhtunkhwa, Pakistan, captured at altitudes of 1200 to 2250 m above sea level between March 2022 and June 2023. PCR-based molecular analysis identified H. ophisauri DNA in three (3%) lizards of all Laudakia tuberculata. Additionally, 45 (44.5%) lizards tested positive for T. gondii DNA, including L. agrorensis, L. pakistanica, L. tuberculata, and Abblepharus pannonicus. Sequence analysis and BLAST confirmed the presence of H. ophisauri and T. gondii. Phylogenetic analysis showed genetic diversity among the H. ophisauri and T. gondii nucleotide sequences, clustering with reference sequences from reptiles, birds, and ticks. Toxoplasma gondii prevalence varied among species, with the highest rates in L. agrorensis and A. pannonicus (67%), followed by L. pakistanica (45%) and L. tuberculata (43%). This study is the first from Pakistan to report high T. gondii prevalence and low H. ophisauri prevalence in wild lizards. Similar and larger-scale studies in unexplored regions of Pakistan are needed to enhance understanding of these pathogens' genetic diversity and host-parasite interactions.

Department of Basic Sciences Higher Institute of Biotechnology of Sidi Thabet University of Manouba Manouba 2010 Tunisia

Department of Botany and Zoology Bacha Khan University Charsadda 24420 Khyber Pakhtunkhwa Pakistan

Department of Dental Industry Techniques College of Health and Medical Techniques Al Mustaqbal University Babylon 51001 Iraq

Department of Zoology Emerson University Multan Multan 60800 Pakistan

Department of Zoology Ghazi University Dera Ghazi Khan 32200 Pakistan

Department of Zoology Shaheed Benazir Bhutto University Dir Upper Sheringal 18050 Khyber Pakhtunkhwa Pakistan

Department of Zoology The Women University Multan Multan 60800 Pakistan

Institute of Zoology Bahauddin Zakariya University Multan 60800 Pakistan

Laboratory of Microbiology National School of Veterinary Medicine of Sidi Thabet University of Manouba Manouba 2010 Tunisia

Medical Laboratory Technology Mazaya University College Annasiriyah Dhi Qar Iraq

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