## 2 Materials and Methods  
One-day-old _Apis mellifera_ L. bees were obtained by placing frames with a breaking brood deriving from one queen bee in an incubator at the temperature of 30 \({}^{\circ}\)C and an air humidity of 70% [12]. The experiment was carried out on 160 worker bees that were kept in wooden cages settled down by 40 specimens. Worker honeybees were fed with 50% (\(w/v\)) sucrose solution. On the 3 rd day after emergence, the honeybees were divided into two groups: a control group (cages 1 and 2), in which honeybees were left uninfected, and an experimental group (cages 3 and 4), in which honeybees were infected by administering sugar syrup with purified _N. ceranae_ spores. The spores were purified to an 85% purity level via the centrifugation of the spore suspension at 5000 G for 5 minutes to produce a pellet of spores. After that, the supernatant was discarded and the pelleted spores were resuspend in distilled water via vortexing for 5 seconds. The whole procedure was repeated 3 times to create an _N. ceranae_ spore suspension with an 85% purity level [12]. Honeybees were individually fed with freshly purified 50,000 _N. ceranae_ spores/\(\upmu\)L in 2 \(\upmu\)L of 50% (\(w/v\)) sucrose solution. Uninfected controls were fed with 2 \(\upmu\)L of sucrose solution without spores. Honeybees that had not consumed the entire inoculum were excluded from the experiment [12; 13]. of the number of _N. ceranae_ spores was estimated using a hemocytometer and an Olympus BX61 light microscope (Olympus Corporation, (Tokyo, Japan)) [12]. The total DNA from the uninfected and _N. ceranae_-infected _A. mellifera_ was isolated from the pooled whole abotomers of 3 bees using the DNEasy Blood and Tissue Kit (Qiagen) according to the manufacturer's instructions. The 100 \(\upmu\)L of honeybee homogenate was added to 180 \(\upmu\)L of lysis buffer and 20 \(\upmu\)L of proteinase K, and the total DNA was isolated using the DNEasy Blood and Tissue Kit (Qiagen) according to the manufacturer's instructions. Every isolate was used as a template for the detection of _N. apis_ and _N. ceranae_-specific 165 rDNA by performing a PCR test with specific primers: 321-APIS for _N. apis_ (5\({}^{\prime}\)-GGGGGCATGTCTTTGACGTACTATGTAG-3\({}^{\prime}\), 5\({}^{\prime}\)-GGGGGCGTTTAAAATGTGAACAACTATG-3\({}^{\prime}\)) and 218-MITOC for _N. ceranae_ (5\({}^{\prime}\)-CGGCAGATGATGAAATATATAA-3\({}^{\prime}\), 5\({}^{\prime}\)-CCCGGTCATTCTCAAACAAAAACCG-3\({}^{\prime}\)) [12; 14]. To establish the relative quantification of _N. ceranae_ DNA, a real-time PCR was applied according to earlier studies [15; 16; 17]. The analysis was completed using the specific 165 SU rDNA region for the _N. ceranae_ and _Apis mellifera_\(\beta\)-actin primer pairs, and utilizing the Biometra TOptical Thermal Cycler (Biometra GmbH 846-070-000, TProfessional Basis, Gottingen, Germany). The levels of nucleic acid in _N. ceranae_ were quantified in all samples and each well in the PCR plate was loaded with 50 ng of DNA. Standard positive curves were used, and no template controls were run with each plate. Standard curve quantification was used to convert the resulting cycle threshold (CT) values to the number of copies of _N. ceranae_ present in each sample. Serial dilutions using purified PCR products with known concentrations from 1 \(\times\) 10\({}^{9}\) to 1 \(\times\) 10\({}^{2}\) copies/\(\upmu\)L were used to prepare the standard curve. The products of the amplifications were separated on 2% agarose gel to exclude the presence of primer-dimer structures and non-specific products. The correlation coefficient values were calculated for each sample to ensure the repeatability of the amplicons. The analyses were performed in tree repetitions and in triplicate technical runs. The copy number was expressed as the average _N. ceranae_ copy number per bee. After 21 days, the intestines isolated from 40 healthy and 40 infected bees were prepared.