Nabian et al.: First Detection of Nosema ceranae … Introduction Fcausal agent of Nosemosis in Euro- or several decades, the only known pean honeybees (A. mellifera) was the unicellular microsporidium N. apis (1). During the last 10 years, an increase in in- fections by microsporidian parasites in the honey bee (A. mellifera) has been detected in several European countries (2- 4), while in- creasing numbers of honey bee colony death and low production in the same areas have been reported by beekeepers. In 2006, Higes et al. reported a new microsporidium N. cer- anae, as the main etiological agent of Nose- mosis in Spain (5). Shortly after, the pres- ence of N. ceranae was confirmed in Europe, America, and Asia (6-14). N. ceranae is highly pathogenic in European honeybees and is associated with reduced honey pro- duction and increased winter mortality (5, 15). The spores of the two Nosema species are very similar and can hardly be distinguished by light microscopy, so that in the absence of clear morphological characteristics for species recognition, other techniques using molecular markers may greatly assist in the diagnosis and identification of honeybee mi- crosporidians (5, 16, 17). The PCR tech- nique provides a very sensitive test for de- tecting microsporidian infection because it enables detection of the parasite at very low levels of infection and can reveal all the stages of its life cycle (18, 19). During the last 3 years, an increase in infec- tions by microsporidian parasites in the hon- eybee (A. mellifera) has been detected in Iran. In addition, increasing number of hon- eybee colony death and low production in the same areas has been reported by bee- keepers. In this study the aim was to detect of honeybee microsporidians (N. apis and N. ceranae) using PCR. 90 Former reports of the distribution of Nose- mosis in Iran (20, 21), assumed that only one Nosema species, N. apis, infected honey bees. The prevalence of Nosemosis in hon- eybee colonies in Arasbaran region (North- western Iran) was studied by Lotfi et al. (2009). They showed the highest-level infec- tion in the spring (20). The presence of the reports based on the incidence of colony de- population and high prevalence of nosemosis, with different clinical and epidemiological pattern in Iran, noticed us that its cause may potentially be this addition species. Materials and Methods Samples Based on the retrospective study of the prevalence of hive depopulation in Mazan- daran Province, Northern Iran, six adult bee samples from Savadkouh of Mazandaran were collected and tested for diagnosis of Nosema spp. Spore detection The abdomens of 10 adult honeybees from each sample were macerated in 10 ml of dis- tilled water and the suspension was filtered and centrifuged for 6 min at 800 g. Pellets were analyzed by phase-contrast microscopy (magnification, _400) to verify the presence of spores(22). This methodology was em- ployed for determination of the presence of Nosema spores in all the samples used in this study. DNA extraction For DNA extraction, spore germination was induced with 200 µl of freshly prepared germination buffer (23), and preparations were incubated at 37°C for 15min (24). DNA extraction was conducted using a High Pure PCR template preparation kit (catalog no. 1796828; Roche Diagnostic) as previ-