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. 2025 Mar 31:12:1568298.
doi: 10.3389/fvets.2025.1568298. eCollection 2025.

Molecular detection and phylogenetic identification of Toxoplasma gondii-like strains, Hepatozoon ixoxo and Lankesterella sp. in frogs and toads

Mubashra Salim #  1 , Asmat Ullah Khan #  2 , Alaudin Khan Niazi  1 , Amna Aqdas  3 , Turki M Dawoud  4 , Muhammad Usman  5 , Hira Muqaddas  6 , Shakir Ullah  7 , Hanène Belkahia  8 , Adil Khan  9 , Mourad Ben Said  8   10 , Furhan Iqbal  1
Affiliations

Molecular detection and phylogenetic identification of Toxoplasma gondii-like strains, Hepatozoon ixoxo and Lankesterella sp. in frogs and toads

Mubashra Salim et al. Front Vet Sci. .

Abstract

Introduction: Despite Pakistan's rich amphibian fauna, frog and toad species remain largely unexplored regarding blood-borne parasites.

Methods: This study aims to investigate potential infections by toxoplasma gondii and Hepatozoon spp. in four amphibian species (n = 223) collected from various regions in Punjab and Khyber Pakhtunkhwa provinces.

Results: Molecular analyses revealed that 17 out of 223 amphibians (7.6%) were infected with Hepatozoon spp., with the highest infection rates found in Bufo olivaceous (20.0%), followed by Bufo stomaticus (9.3%) and Hoplobatrachus tigerinus (5.05%). DNA sequencing and BLAST analysis confirmed the presence of Hepatozoon ixoxo and Lankesterella sp. phylogenetic analysis of both pathogens demonstrated genetic diversity among the Pakistani isolates, clustering with isolates from birds, amphibians, and reptiles worldwide. To the best of our knowledge, this is first ever report globally where we are documenting that 4.5% of the screened Pakistani anurans, including frogs (H. tigerinus, 8.1%) and toads (B. stomaticus, 1.9%), were infected with toxoplasma gondii-like strains. Parasite prevalence varied between sampling sites and amphibian species. This study represents the first report from Pakistan documenting the prevalence and genetic characterization of Hepatozoon sp., Lankesterella sp., and T. Gondii-like strains among amphibians. We recommend conducting similar large-scale studies across various geo-climatic regions of Pakistan to further explore the epidemiology, genetic diversity, host-parasite interactions, and effective control of these pathogens among local frog and toad species. Identifying genetically related T. Gondii strains in unexpected host animals, such as amphibians, has been crucial for contributing to the elucidation of the parasite's evolutionary history.

Keywords: Bufo olivaceous; Bufo stomaticus; Hoplobatrachus tigerinus; Pakistan; amphibians; prevalence and phylogeny.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Magnified map of Punjab and KPK showing different sampling sites (Multan, Layyah, Sargodha, Upper Dir, Buner, and Mardan).
Figure 2
Figure 2
Phylogenetic tree of Hepatozoon spp. based on the partial 18S rRNA gene sequences. New sequence of Hepatozoon sp. obtained in this study (PP481405) is highlighted in bold. Scale bar represents 0.2 substitutions per nucleotide positions. Bootstrap values are shown as numbers on the nodes.
Figure 3
Figure 3
Phylogenetic tree of Lankesterella sp. based on the partial 18S rRNA gene sequences. The three new sequences of Lankesterella sp. obtained in this study (PP476386, PP544154 and PP541577) are highlighted in bold and presented in box. Scale bar represents 0.07 substitutions per nucleotide position. Bootstrap values are shown as numbers on the nodes.
Figure 4
Figure 4
Phylogenetic tree of Toxoplasma gondii and related strains based on ITS-1 partial sequences. The three new sequences of T. gondii-like strains obtained in this study (OR896860, OR896861, and OR896862) are highlighted in bold and presented in box. Scale bar represents 0.08 substitutions per nucleotide position. Bootstrap values are shown as numbers on the nodes.
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