A real-time PCR-based strategy for the detection of Paenibacillus larvae vegetative cells and spores to improve the diagnosis and the screening of American foulbrood

Abstract

Aim: To develop a real‐time PCR‐based strategy for the detection of Paenibacillus larvae vegetative cells and spores to improve the diagnosis and the screening of American foulbrood (AFB), the most harmful pathology of honeybee brood. Methods and Results: A real‐time PCR that allowed selective identification and quantification of P. larvae 16S rRNA sequence was developed. Using standard samples quantified by flow cytometry, detection limits of 37·5 vegetative cells ml−1 and 10 spores ml−1 were determined. Compared to spread plate method, this real‐time PCR‐based strategy allowed, in only 2 h, the detection of P. larvae in contaminated honeys. No false‐positive results were obtained. Moreover, its detection limit was 100 times lower than that of the culture method (2 vs 200 spores g−1 of honey). Conclusion: A rapid, selective, with low detection limit, sensitive and specific method to detect and quantify vegetative cells and spores of P. larvae is now available. Significance and Impact of Study: In addition to honey samples, this real‐time PCR‐based strategy may be also applied to confirm AFB diagnosis in honeybee brood and to screen other apiary supplies and products (bees, pollen, wax), thus broadening the control of AFB spreading.

Prognostic value of using bee and hive debris samples for the detection of American foulbrood disease in honey bee colonies

“…Incubation of the samples in a rich nutrient medium for 1 h at 37°C considerably improved the protocol, presumably because the spore envelope is altered and the bacterial DNA has become more accessible for extraction. Martinez et al (2010) reported an assay detection limit of 2 P. larvae spores g −1 honey contrasting to earlier reports of a detection limit of 10 5 spores g −1 of honey (Ryba et al 2009) and 283 spores g −1 of honey (Alippi et al 2004). These differences may very well be due to differences in extraction methods, highlighting the possibility that comparative evaluation of DNA extraction and purification may be more important than comparisons of different amplification methods.…”

“…A pilot experiment was performed to evaluate two previously published extraction protocols for isolating genomic bacterial DNA from P. larvae spores (Martinez et al 2010;Ryba et al 2009). A stock spore suspension of P. larvae (CCUG 48979 Forsgren et al 2008) spores/mL) of the suspension were prepared.…”

“…Bees and hive debris from healthy colonies were spiked with different concentrations of P. larvae spores (Table I). Briefly, one whole bee or 1 g debris was crushed in 1 mL of spore suspension, before being subjected to DNA extraction using two commercial extraction kits, QIAamp® DNA Mini Kit (Qiagen) (Ryba et al 2009) and UltraClean Soil DNA isolation kit (Mo Bio Laboratories Inc., Martinez et al 2010), following the manufacturer's instructions.…”

“…The quantitative real-time PCR assay used here was modified from Martinez et al (2010). Briefly, the reaction contained 10 μL of SsoFast™ EvaGreen® Supermix (Bio-Rad), 0.4 μM each of primers PL2-Fw and PL2-Rev, 2 μL of template, and the final reaction volume was adjusted to 20 μL with nucleasefree water.…”

“…Conventional PCR has been used to detect P. larvae in brood, foulbrood scales and in honey (Alippi et al 2004;Bakonyi et al 2003;Dobbelaere et al 2001;Lauro et al 2003) as well as in beehive debris (Ryba et al 2009). More recently quantitative, real-time PCR-based methods have been developed for the quantification of P. larvae in honey and larvae (Han et al 2008;Martinez et al 2010).…”