Development and Preliminary Evaluation of a qPCR Workflow for Assessing Nosema ceranae Infection Levels in Honey Bees (Apis mellifera)

Nosemosis, caused by the microsporidian Nosema ceranae, is a major debilitating disease of the honey bee (Apis mellifera), and an accurate assessment of infection level is a prerequisite for diagnosis and control strategy development. This study aimed to develop and preliminarily evaluate a qPCR workflow for assessing N. ceranae infection levels in honey bees, with particular attention to sample pretreatment and calibration approach. A candidate DNA extraction workflow incorporating additional homogenization and an automated nucleic acid extraction system was selected based on preliminary comparisons of sample pretreatment conditions. In quantitative PCR (qPCR) targeting the Hsp70 gene, a 10-fold dilution of the extracted DNA showed the most favorable calibration characteristics among the tested dilution conditions and was selected for subsequent analyses. When infected samples were quantified using the two calibration approaches, spore-calibrated qPCR estimates exceeded microscopic spore counts by a mean of 0.803 log10 units. Plasmid-calibrated DNA copy outputs were numerically approximately two orders of magnitude lower than microscopic spore counts, although DNA copy number and spore number represent different quantities. These results show that calibration approach affects numerical outputs under the tested conditions, but do not establish quantitative accuracy or isolate the contribution of matrix mismatch.