Genetic characterisation of Cryptosporidium spp., Encephalitozoon spp., and Enterocytozoon bieneusi in European brown hare (Lepus europaeus) in Central Europe.

The European brown hare (Lepus europaeus) is an ecologically important small game mammal whose populations have declined in many European countries. A total of 370 faecal samples collected between 2022 and 2024 from hares in the Czechia and Slovakia were molecularly screened for the presence of Cryptosporidium spp. (18S rRNA, gp60), Giardia intestinalis (TPI), Encephalitozoon spp., and Enterocytozoon bieneusi (ITS). qPCR analysis confirmed the presence of Cryptosporidium spp. (n = 15; 4.1%; CI: 2.5-6.6%), Encephalitozoon spp. (n = 34; 9.2%; CI: 6.7-12.6%), and E. bieneusi (n = 16; 4.3%; CI: 2.6-6.9%). Sequence analyses revealed the presence of C. parvum (n = 2), C. sciurinum (n = 3), C. ubiquitum (n = 5), and Cryptosporidium sp. deer mouse genotype IV (n = 5). At the gp60 locus, C. parvum belonged to subtypes IIaA15G1R1 and IIaA23G1R1, C. sciurinum to subtypes VIIIcA10G2R1 (n = 2) and VIIIbA11G1R1 (n = 1), and C. ubiquitum to subtypes XIIa (n = 3) and XIId (n = 2). Five E. bieneusi genotypes were identified: D (n = 8), CHALT1 (n = 1), WildBoar3 (n = 1), CHN-F1 (n = 2), and C (n = 4). Encephalitozoon sequencing revealed E. cuniculi genotypes I (n = 6), II (n = 26), and III (n = 1), and E. hellem (n = 1). Giardia intestinalis was not detected. Cryptosporidium sp. deer mouse genotype IV is reported here for the first time in Europe.