Animal stool samples
A total of 200 fecal samples were collected (each samples about 100 g) from slaughtered cows in the slaughterhouse of Jahrom city and immediately transferred to the Parasitology Laboratory of the Jahrom University of Medical Sciences under sterile conditions during 2019-2020. When the cows were slaughtered, an incision was made in the cow's colon with a scalpel, and in completely sterile conditions, the samples were collected and stored in a refrigerator at 4◦C until DNA extraction (17).
DNA extraction
DNA was extracted from all samples (microscopically positive and negative samples) According to the instructions of Bioneer Company's DNA extraction kit. Purified DNA stored at -20 °C until use. The targets for the nested PCR procedure were the large subunit (LSU) and small subunit (SSU) of the ribosomal DNA (rDNA). Primary primers MSP-1 (5´–TGAATGGTCCCTGT–3´) and Secondary primers MSP-3 (5´–GGAATTCACACCGCCCGTCTAT–3´) target the 3 region of the SSU and recognized a broad range of microsporidian species, including Encephalitozoon spp. and E. bieneusi (8). Primary Primers MSP-2B (5´–GTTCATTCGCACTACT–3´) and Secondary primers MSP-4B (5´–CCAAGCTTATGCTTAAGTCCAGGGAG–3´) target the 5 region of the LSU of E. bieneusi, while Primary Primers MSP-2A (5´–TCACTCGCCGCTACT–3´) and Secondary primers MSP-4A (5´–CCAAGCTTATGCTTAAGTAAGGGT–3´) specifically recognize Encephalitozoon spp. and some other microsporidia but not E. bieneusi. The amplified fragment length by the primers was 300 bp for Encephalitozoon and 500 bp for Enterocytozoon. At first, the samples were examined by the primary primers. Then, by use the secondary primers differentiating the species of Enterocytozoon and Encephalitozoon. Primary PCR reactions were performed in a final volume 25 µl containing 12.5 µl of PCR ready to use mastermix with 1.5 mM MgCl2 (Bionner), 10 µl of each primer, and 3 µl of template DNA under conditions: 95 °C for 5 min followed by 35 cycles of 94 °C for 40 s, 55 °C for 45 s and 72 °C for 45 s, and a final extension of 72 °C for 4 min (8, 18).
The second PCR reactions were performed in a final volume 25 µl containing 12.5 µl of PCR ready to use mastermix with 1.5 mM MgCl2 (Bionner), 10 µl of each primer, and 1 µl from the primary-step PCR product as a template. PCR conditions were as follows: 95 °C for 5 min followed by 35 cycles of 94 °C for 40 s, 57 °C for E. bieneusi / 52 °C for Encephalitoozoon spp. for E. bieneusi 35 s, 72 °C for Encephalitoozoon 40 s, and 72 °C for 5 min as a final extension. Finally, the RFLP-PCR method is used to reaffirming Encephalitozoon spp (8).
Restriction Fragment Length Polymorphism (RFLP-PCR)
RFLP-PCR is used to accurately identify the species. RFLP-PCR was carried out in final volume 15 µl, containing 6 µl of product PCR, 6.5 µl of water, 1.5 µl of buffer and 1 µl of Restriction enzyme. For this work we used Mnl1 Restriction enzyme in under incubation conditions (16 minutes at 37 Cº). RFLP-PCR are precisely determined Encephalitozoon spp. So Encephalitozoon cuniculi will have a band of about (90 and 210 bp), Encephalitozoon inestalience will have a band of about (60 and 160 bp) and Encephalitozoon hellm will have a band of about (80 and 180 bp) (8, 19). Finally, positive samples were sequenced (Bioneer Company). Than compared with other existing sequences by using BLAST and DNAsp and drawn Phylogenetic tree using the MEGA6 (with Neighbor-Joining methods).