Molecular surveillance reveals high prevalence of Hepatozoon sp. in wild canids of Central Italy

Introduction: Tick-borne pathogens (TBPs) are of growing concern for animal health due to their increasing distribution and impact on both domestic and wild hosts. However, the epidemiological role of wild canids, particularly foxes and wolves, remains poorly understood, and data on the circulation of these pathogens in wildlife are still limited. Evidence from Europe has highlighted the widespread occurrence of several TBPs in wild canid populations, suggesting that they may contribute to the maintenance and dissemination of these agents in natural ecosystems. Among them, Hepatozoon spp., mainly transmitted by the ingestion of the tick Rhipicephalus sanguineus, is an emerging pathogen with a broad host range, increasing veterinary importance, and growing attention in domestic animals, particularly dogs and cats, due to its expanding distribution and clinical relevance. Importantly, the genus Hepatozoon comprises a large number of species and shows considerable genetic diversity, with multiple lineages reported across different host species and geographic regions. Nevertheless, knowledge regarding its circulation and epidemiological significance in wildlife remains scarce. Therefore, this study was conducted to investigate the occurrence of Hepatozoon spp. in wild canids and to provide further insights into their potential role as reservoirs of infection. Materials and Methods: A total of 310 spleen samples were collected from 168 wolves and 142 red foxes living in Abruzzo region and border areas during routine necropsies carried out as part of wildlife health monitoring programs at IZSAM. Spleen samples were stored at – 80 °C until molecular examination. DNA was extracted using the Maxwell® RSC Genomic DNA Kit (Promega) following the manufacturer's protocol. Samples were tested for the presence of Hepatozoon spp. DNA by real-time PCR using the commercial kit HepSpp qPCR (genetic PCR solutionsTM). Results and Discussion Of the 168 wolves examined, 155 tested positive for Hepatozoon spp. (92%), while 118 of 142 red foxes were positive (83%). To the best of our knowledge, these are the first data available on the occurrence of Hepatozoon spp. in wildlife from the Abruzzo region. The prevalence values observed in the present study are among the highest reported in Italy and in Europe. In red foxes, previous studies have reported highly variable prevalence rates, ranging from 5% in northern Italy to 50% in southern regions, whereas in wolves prevalence values between 76% and 92% have been documented. The results obtained here therefore confirm the intense circulation of Hepatozoon spp. among wild canids in central Italy. At the European level, Hepatozoon spp. is recognized as one of the most widespread tick-borne protozoans infecting wild carnivores. However, prevalence rates appear highly variable among host species and geographic areas. In wolves, infection rates ranging from approximately 35% to 90% have been reported across Europe, with occasional reports of 100% positivity. Similarly, prevalence in red foxes has been reported to vary markedly, ranging from approximately 20% to 90% depending on the country and study area. The remarkably high prevalence detected in both wolves and foxes further supports the hypothesis that Hepatozoon spp. has relatively low pathogenicity in its hosts. Similarly, in domestic dogs, H. canis infection is frequently subclinical, although severe clinical disease may occur in case of high parasitemia and/or co-infections. The high prevalence observed in both wolves and foxes may be largely explained by the feeding ecology of these species. Wolves, as apex predators, regularly consume large prey that may carry attached ticks, whereas red foxes exhibit highly opportunistic feeding behavior and commonly scavenge carcasses. Consequently, both species are likely to ingest infected ticks repeatedly throughout their lifetime, resulting in a cumulative exposure that may contribute to the remarkably high prevalence detected in the present study. A limitation of the present study is that molecular identification was restricted to the genus level, preventing species-level characterization of the detected parasites. Given the considerable genetic diversity reported within the genus Hepatozoon, future studies should incorporate sequencing and phylogenetic analyses to identify the circulating species and genotypes, thereby improving our understanding of transmission dynamics, host sharing, and pathogen circulation at the wildlife–domestic animal interface.