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

The detection/quantification in honey of spores of the bacterial pathogen Paenibacillus larvae, the etiological agent of the American Foulbrood (AFB) infectious disease of honey bee, represents a useful diagnostic tool to identify apiaries at risk of infection and carry out focused prevention measures. Plate count is currently the recommended method used to analyze presence and number of spores for this pathogen. However, its validity needs to be re-assessed since P. larvae field strains have a low rate of germination in culture media. Therefore, in this study, culture-dependent P. larvae detection/quantification was compared with quantitative PCR (qPCR) based analysis for 139 honey samples collected in 2017 and 2018 from as many apiaries in the Abruzzo region. According to qPCR based detection, 58.27% samples were positive for P. larvae, while only 33.8 % samples were positive by plate count. Moreover, the levels of contamination with the two methods differed for most samples. Potential false positives were obtained by plate count for 12.9% samples that were negative with the qPCR test. On the other hand, 26.6% samples were culture negative but qPCR positive, suggesting that not all the field strains were able to develop in plate. This was confirmed by obtaining P. larvae growth from those samples after supplementing the medium with germination stimulants. Results strongly suggested the necessity to improve culture methods or replace them with molecular detection/quantification for a more reliable AFB risk estimation.