Neglected but widespread: molecular evidence of the distribution and genetic diversity of Klossiella equi in Europe

Klossiella equi is the only known Apicomplexan adeleorinid protist infecting the renal parenchyma of equids. Its monoxenous life cycle begins with ingestion of sporocysts via contaminated feed or water; sporozoites migrate to the renal epithelium, where merogony, gametogony, and sporogony occur, with sporocysts excreted in urine (Vetterling and Thompson, 1972, J. Parasitol., 58: 589–594). Infections are usually asymptomatic, but heavy burdens may cause renal tubular rupture and lymphoplasmacytic interstitial nephritis. Despite its worldwide distribution, data from Europe are limited. This study aimed to assess the molecular prevalence of K. equi in European equids. Between 2023 and 2025, 284 kidney samples were opportunistically collected during postmortem examination from horses (277), mules (6), and one donkey across Italy (46), Netherlands (100), Spain (30), Romania (96), and Czech Republic (12). Additionally, 82 urine samples were obtained from a subset of horses from Spain (28), Romania (51), Czech Republic (3). Kidney tissue from the corticomedullary junction was sampled. Approximately 0.25 g of tissue was stored at −20 °C until DNA extraction. Urine samples (4–15 mL) were centrifuged at 1,500 rpm for 5 minutes, and up to 0.25 g of pellet was used for DNA isolation. Detection of K. equi DNA was performed by conventional PCR targeting the mitochondrial multicopy rRNA gene array (12S/16S), located between cytochrome c oxidase subunits 1 (COI) and 3 (COIII), using primers Api_LSUG_F and Haem_RNA_14_R (Léveillé et al., 2019, J. Parasitol., 105: 29). A subset of 38 positive samples was further analysed by PCR targeting partial COI and cytochrome b oxidase (cytB). A COI-based phylogenetic tree was constructed using unique Adeleorina sequences from GenBank. Cohen’s kappa (k) was estimated to evaluate agreement between kidneys and urine samples. Risk factors (country, age and sex) were analysed using Fisher’s Exact Test. Klossiella equi DNA was detected in 33.1% of the equines. Significant differences (p<0.05) were reported among countries with highest prevalence in Spain (73.3%), followed by Italy (43.5%), Netherlands (33.0%), Romania (17.7%), and Czech Republic (16.7%). Cohen’s kappa was 0.5 suggesting a moderate agreement (31.0% in kidneys; 25.6% in urine). No difference was observed between sex (p=1.00), whereas age was significant (p<0.001), with highest prevalence in young adults (56.3%) and lowest in foals (12.5%). A dominant “European haplotype” was reported across all countries; the remaining minor variants were restricted to Italy. The greatest divergence occurred in one donkey (Campania region) and one mule (Latium region). These findings confirm the widespread presence of K. equi in European horses. Given its tropism for renal tubular epithelium and association with inflammatory lesions, detection of K. equi should be considered in cases of equine nephropathies.