Iranian J Parasitol: Vol. 6, No.3, 2011, pp.89 -95 ously described (15). Negative controls in- cluding uninfected honeybees were proc- essed in parallel to detect possible contami- nation. PCR design methodology The 16 SrRNA locus was selected to per- form N. apis and N. ceranae PCR. Published sequences in the GenBank database from N. apis( accession number U26534, DQ235446) and N.ceranae (accession num- ber U26533, DQ078785) were compiled. Specific primers for both N. apis and N. cer- anae were visually selected taking into ac- count that primer sequences were specific to each of the two species. The selected prim- ers were 321 Apis for N. apis (FOR5’-GGG GGC ATG TCT TTG ACG TAC TAT GTA-3’, 321APIS REV 5’-GGG GGG CGT TTA AAA TGT GAA ACA ACT ATG-3’) and 218 MITOC for N. ceranae (FOR 5’-CGG CGA CGA TGT GAT ATG AAA ATA TTA A-3’) 218MITOC: (REV 5’-CCC GGT CAT TCT CAA ACA AAA AAC CG-3’). In the single- gradient PCR test of both primers, the annealing temperature was 61.8. PCR was performed using a Mastercycler gradient (Eppendorf) in 50µl volumes con- taining 25 µl of High Fidelity PCR Master Mixture( catalog no.12140314001;Roche Diagnostic), 0.4 µM of each primer, 0.4 mMof each deoxynucleoside triphosphate, 3mMcl2mg, 0.2mg/ml bovine serum, 0.1%TritonX-100, and 5 µl of Nosema tem- plate DNA. The thermocycler program consisted of 94◦C for 15 second followed by 25 cycles of 15s at 94◦C, 30s at 61.8◦C and 45s at 72◦C and a final extension step at 72◦C for 7 min. Nega- tive controls (from DNA extraction) were included in all PCR experiments. Amplified PCR products were electrophoresed through 1.5% agarose TAE gel in standard TAE buffer, stained with ethidium bromide, and 91 visualized using UV illumination. All six samples and controls were checked some times. The PCR products were purified with a QIA quick PCR purification Kit ( catalog no. 28104;QIAGEN) as previously described ( 5) and fully sequenced in both directions. These sequences were aligned and compared to the N. ceranae and N. apis consensus us- ing Sequencher;GeneCodes, AnnAr- bor,MI).Sequence similarity analyses were performed using BLAST database search. Results Adult honey bee samples from 3 apiaries in Savadkouh (Mazandaran Province) were collected. All of the analyzed samples were positive for presence of Nosema spore (Fig.1). DNA was extracted from spores and amplified with common primer pair derived from 16SrRNA of Nosema spores. The am- plified DNA fragment revealed that the iso- lation of DNA from spores was successfully performed (Fig.2, Lane1-6). The 16SrRNA gene has seen successfully applied to iden- tify Nosema. Also the application of the mentioned primer pairs (MITOC and APIS) could easily differentiate Two different spe- cies ( N. apis and N. ceranae). PCR and sequencing analyses of both strands using the designed primers (321 APIS and 218MITOC primers) yielded the expected amplicon sizes (218 bp for N. cer- anae) and sequences. Neither primer interac- tion nor undesirable amplicons were de- tected in agarose gels. None of the samples was identical to N. apis (U26534, DQ235446). To verify the species diagnosis three PCR products were sequenced and the partial 16SrRNA sequences were submitted to GenBank (accession numbers U26533 and DQ078785). The generated consensus se- quences created from both forward and re-