# First Molecular Evidence and Phylogeny of Hepatozoon sp. and Theileria sp. in Saudi Rodents

## TL;DR

This study reports the first molecular evidence of Hepatozoon and Theileria parasites in Saudi rodents, revealing new genetic lineages and their ecological roles.

## Contribution

The study provides the first molecular detection and phylogenetic analysis of Hepatozoon and Theileria in Saudi rodents, revealing novel haplotypes and host associations.

## Key findings

- Hepatozoon was detected in four rodent species in Saudi Arabia, with a star-like haplogroup indicating host specificity.
- Saudi Hepatozoon sequences clustered with reptile and rodent parasites, distinct from those in carnivores.
- Theileria showed low genetic diversity in Saudi rodents, clustering with a single widespread haplotype.

## Abstract

Apicomplexan species of the genera Hepatozoon and Theileria are known to infect both domestic and wild animals. This study, performed using molecular tools, provides the first report of Hepatozoon sp. in rodent species (Arvicanthis niloticus, Gerbillus cheesmani, G. nanus, and Rattus rattus) from Saudi Arabia. Additionally, Theileria sp. was detected in G. nanus and R. rattus. Genetic analyses revealed that Saudi Hepatozoon haplotypes form a star-like haplogroup, indicating host specificity to local rodent species. Phylogenetic analyses generated trees with similar topologies, revealing two sister clades: one comprising Hepatozoon spp. sequences from rodents and reptiles forming a distinct clade, separate from those found in felids and canids. These findings confirm that rodents play a key role in the epidemiology of reptile-associated Hepatozoon spp. rather than species linked to Carnivora. The Hepatozoon sequences from Saudi Arabia formed a well-supported cluster, revealing novel haplotypes primarily associated with rodents. This study also highlights the lack of genetic diversity in Theileria sp. in Saudi Arabia, with a single haplotype clustering with sequences from ruminants and rodents across multiple regions.

Rodents are among the most abundant and ecologically diverse mammals, playing key roles in terrestrial ecosystems and often serving as reservoirs for various zoonotic and wildlife pathogens. Among these are protozoan parasites of the genera Hepatozoon and Theileria, which are known to infect a wide range of domestic and wild animals worldwide. However, little is known about the diversity and phylogenetic relationships of these hemoprotozoans in rodent hosts, particularly in the Arabian Peninsula. The aim of this study was to investigate the presence and genetic diversity of Hepatozoon sp. and Theileria sp. in rodents from different regions of Saudi Arabia and to determine potential reservoir species. A total of 111 rodents were captured and identified by molecular analysis of the mitochondrial 16S rRNA gene. Screening for parasites was performed using PCR amplification of the 18S rRNA gene, followed by sequencing, haplotype analysis, and phylogenetic reconstruction using both maximum likelihood and Bayesian inference methods. Our results represent the first molecular detection of Hepatozoon sp. in Arvicanthis niloticus (31.3%), Gerbillus cheesmani (26.5%), G. nanus (28.5%), and Rattus rattus (32.0%) and of Theileria sp. in G. nanus (21.5%) and R. rattus (24.0%) in Saudi Arabia. Haplotype network analysis revealed seven distinct Hepatozoon haplotypes forming a star-like cluster, suggesting host specificity. One divergent haplotype (Hap_2), 19 mutation steps apart, may represent a novel lineage. Phylogenetic analyses grouped Saudi Hepatozoon sequences with those from reptiles and rodents, forming a clade distinct from sequences isolated from felids and canids. In contrast, Theileria sequences showed low diversity, clustering with a single widespread haplotype found in rodents and ruminants in several regions. These findings significantly expand the current knowledge on rodent-associated apicomplexan parasites in Saudi Arabia, revealing novel Hepatozoon haplotypes and highlighting the role of rodents in the transmission of reptile-associated Hepatozoon spp. This study provides basic molecular data crucial to understanding host–parasite relationships and the potential public and veterinary health implications of these parasites in arid ecosystems.