Enterocytozoon bieneusi in Italian water buffalo calves (Bubalus bubalis): An exploratory haplotype analysis within Bovidae family in the European context

Microsporidia are an important group of emerging opportunistic parasites in human and non-human animals, with possible zoonotic potential; however, no data are currently available on their presence in commonly farmed species in Italy, particularly regarding Enterocytozoon bieneusi in water buffalo (Bubalus bubalis). This preliminary study investigated the prevalence and genetic variability of E. bieneusi in water buffalo calves in southern Italy. Additionally, given its spread among other members of the Bovidae family, a haplotype-level network analysis was performed using E. bieneusi sequences available in GenBank from two of the most commonly farmed Bovidae species in Europe: cattle (Bos taurus) and water buffalo (B. bubalis). The survey was conducted on four farms between September and December 2023 for a total of 37 buffaloes sampled. DNA extracted from collected faeces was subjected to molecular analysis amplifying ITS region. For the molecular characterization, a phylogenetic analysis was performed by Maximum Likelihood method. For the comparison between our sequences and those available from water buffaloes and cattle from Europe, a haplotype analysis was conducted to obtain a network calculation. Five samples from Italian B. bubalis tested positive for E. bieneusi with an overall prevalence value of 13.5 %. Phylogenetic analysis assigned isolates to three genotypes (YNDCEB-90; A; I) which were shared among different hosts, including humans and clustering in the phylogenetic Group 1 and Group 2. The network analysis identified Hp9 as the most frequently detected haplotype, distributed across multiple countries, including Italy. The second most common haplotype, Hp12, was exclusively found in Italy and Turkey from B. bubalis. The differences in haplotype patterns observed between B. taurus and B. bubalis could shed light on the species-specific interactions of E. bieneusi. The prevalence observed, along with the detection of zoonotic genotypes in water buffaloes, could pose a potential public health concern. The associated risk extends beyond the direct contact with infected animals or their faeces, encompassing possible contamination of the food chain and the environment, including ground and surface water sources.