Molecular screening for microsporidians

All 1904 individuals were screened for the presence of microsporidians using a short (c.350 bp long) diagnostic fragment of the small ribosomal subunit (SSU rDNA) marker. The microsporidia-specific primer V1f (forward) (5′-CAC CAG GTT GAT TCT GCC TGA C-3′) [52] paired with newly designed UNIr (reverse) (5′-TCA GGC TCC CTC TCC GGA AT-3′) was used. The use of this short fragment maximised the ability to detect the presence of microsporidians even in case of low infection intensity. As negative and positive controls in PCR reactions, we used, respectively, water and microsporidian DNA (Dictyocoela roeselum). The PCR program consisted of an initial denaturing phase at 95 °C for 2 min, followed by 35 cycles of 95 °C for 20 s, 57 °C for 20 s and 72 °C for 20 s, and a final extension at 72 °C for 5 min. The PCR products were visualised by electrophoresis after 20 min migration under 100V in 2% agarose gel.