Novel TaqMan PCR Assay for the Quantification of Paenibacillus larvae Spores in Bee-Related Samples

SIMPLE SUMMARY: American foulbrood (AFB) is the most severe bacterial disease of honeybees, caused by Paenibacillus larvae. Larvae become infected by ingesting food contaminated with P. larvae spores, which are extremely resistant and can remain infectious for decades. Burning affected colonies is widely used to prevent further spread of the disease. The presence of P. larvae spores in bee-related samples is associated with an increased risk of developing clinical symptoms, and spore counts can be used for early detection of at-risk colonies, which should then undergo thorough clinical examination. Because quantification of P. larvae spores by plate counting is time-consuming and unreliable, due to poor and inconsistent germination, molecular quantification is more suitable. To overcome the limitations of available quantification methods, we developed a quantitative PCR (qPCR) assay for reliable quantification of P. larvae that also performs well at low spore counts. The assay was validated for honey and hive debris samples but can be extended to other sample types. Spore counts in AFB-positive colonies were significantly higher than those in asymptomatic colonies, both for honey and hive debris samples. By comparing plate and qPCR counts, the germination rate of P. larvae spores was found to be low and inconsistent. ABSTRACT: Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybees. P. larvae spore counts in bee-related samples correlate with the presence of AFB symptoms and may, therefore, be used to identify at-risk colonies. Here, we constructed a TaqMan-based real-time PCR (qPCR) assay targeting a single-copy chromosomal metalloproteinase gene for reliable quantification of P. larvae. The assay was calibrated using digital PCR (dPCR) to allow absolute quantification of P. larvae spores in honey and hive debris samples. The limits of detection and quantification were 8 and 58 spores/g for honey and 188 and 707 spores/mL for hive debris, respectively. To assess the association between AFB clinical symptoms and spore counts, we quantified spores in honey and hive debris samples originating from honeybee colonies with known severity of clinical symptoms. Spore counts in AFB-positive colonies were significantly higher than those in asymptomatic colonies but did not differ significantly with regard to the severity of clinical symptoms. For honey, the average spore germination rate was 0.52% (range = 0.04–6.05%), indicating poor and inconsistent in vitro germination. The newly developed qPCR assay allows reliable detection and quantification of P. larvae in honey and hive debris samples but can also be extended to other sample types.