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

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&ndash:parasite relationships and the potential public and veterinary health implications of these parasites in arid ecosystems.