Phylogenetic and histopathological characterization of putative Hepatozoon ophisauri-like lineages in rodents from Italy

Acta Parasitologica (2026) 71:129 https://doi.org/10.1007/s11686-026-01304-2 BRIEF REPORT Phylogenetic and histopathological characterization of putative Hepatozoon ophisauri-like lineages in rodents from Italy Davide Sogliani1 · Maria Sampieri1 · Filippo Maria Dini2 · Mattia Calzolari1 Valentina Carta1 · Patrizia Bassi1 · Alicia Rojas3,4 · Giulia Maioli1 · Deborah Torri1 · Received: 5 March 2026 / Accepted: 5 May 2026 © The Author(s) 2026 Abstract Purpose The aim of this study was to describe the occurrence and phylogenetic diversity of Hepatozoon spp. in small mammals from the Emilia-Romagna region, northern Italy, and to assess their genetic relationships with previously described lineages. Methods Tissue samples (spleen and ear) from wild animals belonging to eight small mammal species were examined. Molecular screening was performed using PCR targeting the 18 S rRNA gene of Hepatozoon spp. Positive amplicons were sequenced and analyzed using phylogenetic methods to determine their taxonomic placement, and one positive fresh sample was additionally subjected to histopathological characterization. Results Hepatozoon DNA was detected in two rodent species, the brown rat (Rattus norvegicus) and the European edible dormouse (Glis glis). Sequences showed high identity (98.9–100%) with Hepatozoon sp. previously reported in rodents from Spain and Tanzania, and 98.32–98.9% identity with Hepatozoon ophisauri. Phylogenetic analysis placed the sequences from R. norvegicus within a rodent-associated clade, whereas those from G. glis formed a distinct lineage related to Hepatozoon detected in a great gerbil. Histopathological analysis in a European edible dormouse revealed multifocal pulmonary granulomatous inflammatory lesions associated with intracytoplasmic parasitic zoites and developmental stages consistent with type I and type II meronts. Conclusion This study provides molecular evidence of distinct Hepatozoon lineages in European rodents, potentially representing undescribed taxa, and highlights the need for integrative studies combining molecular, morphological, and ecological data. Keywords Host-parasite interactions · Rodentia · Apicomplexan host range · Tick-borne parasites · Paratenic hosts Introduction Davide Sogliani and Maria Sampieri are shared first authorship. Giulia Maioli 1 Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna, via Antonio Bianchi 9, 25124 Brescia, Italy 2 Department of Veterinary Medical Sciences, University of Bologna, 40064, Ozzano dell’Emilia, Bologna, Italy 3 Laboratory of Helminthology, Faculty of Microbiology, University of Costa Rica, San José, Costa Rica 4 Centro de Investigación en Enfermedades Tropicales, University of Costa Rica, San José, Costa Rica Hepatozoonosis is an arthropod-borne disease caused by apicomplexan protozoa belonging to the family Hepatozoidae and the suborder Adeleorina. Hundreds of Hepatozoon species have been described infecting all classes of terrestrial vertebrates, typically by arthropod vectors, and approximately 120 and 50 Hepatozoon species have been found infecting snakes and mammals, respectively [1]. Hepatozoon species have a heteroxenous life cycle, meaning they require multiple hosts to complete their development. Typically, Hepatozoon species switch between intermediate vertebrate and definitive blood-feeding invertebrate hosts [2]. Diagnosis of Hepatozoon spp. infection in vertebrates is usually through blood smear analyses, but not all infected animals show parasitemia. In addition, molecular 129 Page 2 of 8 techniques such as PCR have also been used to detect the presence of Hepatozoon DNA in mammal tissues [3, 4]. The geographic distribution of Hepatozoon species, particularly those infecting snakes and rodents, is very wide. Hepatozoon species have been identified in various wild animals in Europe [5, 6], the Americas [5, 7, 8], Asia [7], and Africa [8]. In Italy, Hepatozoon spp. are widely distributed, affecting both domestic and wild animals, but investigations have focused mainly on domestic carnivores. Hepatozoon canis is the most extensively studied species having been reported in both domestic dogs and red foxes [9, 10]. In Southern Italy, 14% of hunting dogs have tested positive for H. canis, while a prevalence of 18% was reported among domestic dogs in Sardinia [11, 12]. Higher prevalence has been observed in Central Italy, where 32.5% of dogs tested positive [13]. Other Hepatozoon species, i.e. H. felis and H. silvestris, have been detected in domestic cats in northeastern Italy, with a reported total prevalence of 16.5% [14]. Rodent-associated Hepatozoon species, including H. erhardovae and several novel genotypes, have been reported in small mammals in Europe. Fleas (e.g., Ctenophthalmus spp. and Megabothris turbidus) can act as invertebrate hosts, especially for rodent-associated Hepatozoon species, whereas other Hepatozoon spp. are hypothesized to rely on non-vectorial transmission pathways [15]. On the other hand, H. ophisauri, usually associated with reptiles, has never been reported in European countries but has been detected in rodent blood samples in geographical regions such as Malaysia [16], Pakistan [15] and Taiwan [17]. Like other Hepatozoon species, it is likely transmitted through the ingestion of infected arthropods, mainly ticks [18]. The primary vectors of Hepatozoon species are ticks, particularly Rhipicephalus sanguineus sensu lato, which are highly prevalent in Italy and are known to play a central role in the transmission of H. canis [19, 20]. Molecular detection and characterization of Hepatozoon spp. typically target the 18 S rRNA gene, a robust marker for phylogenetic and epidemiological studies across a wide range of vertebrate and invertebrate hosts [18, 21]. However, due to the highly conserved nature of this locus, the 18 S rRNA gene may lack sufficient taxonomic resolution to discriminate among closely related Hepatozoon species [4, 22]. Despite this, knowledge about the distribution and life cycles of distinct Hepatozoon genotypes in Italy – particularly those infecting wild rodents – remains limited, highlighting the need for further molecular investigations to elucidate their host associations, transmission dynamics, and potential coevolutionary relationships. 13 Acta Parasitologica (2026) 71:129 Materials and Methods Sample Collection and Tissue Analysis The Emilia-Romagna region (Northern Italy) includes the intensively cultivated and urbanized Po Plain, as well as hilly and mountainous areas that create diverse habitats. Nature conservation is supported by a network of protected areas hosting a high diversity of mammalian species [23]. The Emilia-Romagna Regional Council established a wildlife health surveillance plan (Deliberation n. 1763, November 29, 2017) to monitor several infectious and parasitic diseases through the analysis of wild animal carcasses. Small mammal carcasses included in this stu (...truncated)