A PCR-based method that permits specific detection of Paenibacillus larvae subsp. larvae, the cause of American Foulbrood of honey bees, at the subspecies level

Abstract

Aims: A reliable procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American Foulbrood disease of honey bees (Apis mellifera L.) based on the polymerase chain reaction (PCR) and subspeciesspecific primers is described. Methods and Results: By using ERIC-PCR, an amplicon of ca 970 bp was found among P. l. larvae strains but not in other closely related species. Based on the nucleotide sequence data of this amplicon, we designed the pair of oligonucleotides KAT 1 and KAT 2, which were assayed as primers in a PCR reaction. A PCR amplicon of the expected size ca 550 bp was only found in P. l. larvae strains. Conclusions: This PCR assay provides a specific detection for P. l. larvae. Significance and Impact of the Study: The developed PCR assay is highly specific because can differentiate Paenibacillus larvae subsp. larvae from the closely related Paenibacillus larvae subsp. pulvifaciens. The technique can be directly used to detect presence or absence of P. l. larvae spores in honey bee brood samples and contaminated honeys.

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.…”

“…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).…”

Microorganisms in Honey

“…Several molecular techniques have been developed for the detection of pathogens like Paenibacillus larvae, Melissococcus plutonius, Nosema ceranae and Nosema apis [129,146,147], Ascophera apis and Ascophera ceranae, and A. flavus [129,148]. Among them can highlight the simple PCR [149][150][151], NESTED-PCR [152], RT-PCR [153,154], immunology-based tests (ELISA), and probe-based hybridization analysis [155]. The main advantages of these techniques would be less needed for sample treatment which often can be applied directly to the honeybee products, fast technique, specificity, and sensitivity of detection.…”

Prevalence of honeybee viruses in the Czech Republic and coinfections with other honeybee disease

“…P. larvae can be identified using several methods (de Graaf et al 2006). The most efficient is to use PCR to detect it in honey, larvae, pupae or beehive-debris (Govan et al 1999;Dobbelaere et al 2001;Bakonyi et al 2003;Alippi et al 2004;Ryba et al 2009). This method is mainly based on the amplification of the 16S rRNA of P. larvae, which is used as the target gene for PCR.…”