Quantitative PCR (qPCR) vs culture-dependent detection to assess honey contamination by Paenibacillus larvae

Abstract

The detection/quantification in honey of spores of the bacterial pathogen Paenibacillus larvae, etiological agent of the American Foulbrood (AFB) infectious disease of honey bees, represents a useful diagnostic tool to identify apiaries at risk of outbreaks and apply prevention measures. Plate count is the method in use for analyzing presence and number of spores but its validity needs to be re-assessed since P. larvae field strains showed a low rate of germination in culture media. To this aim, culture-dependent P. larvae detection/quantification was compared in this study with quantitative PCR (qPCR) based analysis for 139 honey samples, each from a different apiary, collected in 2017 and 2018 in the Abruzzo region. According to qPCR based detection, 59.6% samples contained P. larvae spores, while, according to plate count, only 32.3% samples were contaminated. Moreover, the levels of contamination determined with the two methods differed. Many samples (29.5%) were negative with the cultivation assay but positive with the qPCR test, suggesting that not all the field strains were able to develop in plate. This hypothesis was confirmed by re-testing those samples with PLA medium added with germination stimulants and obtaining P. larvae growth. Results strongly suggested the necessity to apply improved culture methods or molecular detection/quantification for a more reliable AFB risk estimation in order to efficiently fight this honey bee plague.