Development and Evaluation of PCR Assays for the Detection of Paenibacillus larvae in Honey Samples: Comparison with Isolation and Biochemical Characterization

Abstract

PCR assays were developed for the direct detection of Paenibacillus larvae in honey samples and compared with isolation and biochemical characterization procedures. Different primer pairs, designed from the 16S rRNA and the metalloproteinase precursor gene regions, and different DNA extraction methods were tested and compared. The sensitivity of the reactions was evaluated by serial dilutions of DNA extracts obtained from P. larvae cultures. The specificity of the primers was assessed by analyzing related Paenibacillus and Bacillus strains isolated from honey. The PCR assays also amplified these related bacteria, but at lower sensitivity. In the next step, the PCR assays were applied to contaminated honey and other bee products originating from 15 countries. Lysozyme treatment followed by proteinase K digestion was determined to be the best DNA extraction method for P. larvae spores. The most sensitive primer pair detected P. larvae in 18 of 23 contaminated honey samples, as well as in pollen, wax, and brood. Honey specimens containing saprophyte bacilli and paenibacilli, but not P. larvae, were PCR negative. Although the isolation and biochemical identification method (BioLog) showed higher sensitivity and specificity, PCR proved to be a valuable technique for largescale screening of honey samples for American foulbrood, especially considering its rapidity and moderate costs.

Characterization of selected Gram-negative non-fermenting bacteria isolated from honey bees (Apis mellifera carnica)

“…All samples were taken from different combs. Five milliliters of collected nectar was mixed with 5 mL of sterile water and vortexed for 30 s. Two hundred microliters of diluted nectar was directly inoculated on blood agar plates and on PYE agar and incubated at 28°C for 48 h. For the isolation of bacteria from honey, the method described by Bakonyi et al (2003) to detect the causative agent of American foulbrood (P. larvae) was slightly modified to keep non-spore-forming bacteria alive and germinable. Two hundred microliters of diluted honey was directly inoculated on two blood agar plates.…”

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

“…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).…”

Güney Marmara Bölgesindeki Bal Arılarının Yavru Çürüklüğü Hastalığı etkenlerinin PZR ve Kültürel Metodlar ile Belirlenmesi

“…Larval remains from brood comb were collected with a sterile swab for the bacterial isolation and suspended in 5 ml of sterile distilled water [8,10,11,14] . The suspension was incubated at room temperature for 10 min and separated into two samples.…”

“…Molecular techniques have also been developed for the identification of P. larvae and M. plutonius. PCRbased methods for detecting P. larvae have been described by several authors [1,[8][9][10][11][12][13] . The sequences of detection primers were based on 16S rRNA gene of P. larvae and M. plutonius [8,10,[12][13][14] .…”

“…There are many sensitive and selective culture media exist to identify the bacterial agents in honeybees. Biochemical characteristics and microscopy of suspected materials are used for routine identification of bacterial agents [8][9][10][11] . Molecular techniques have also been developed for the identification of P. larvae and M. plutonius.…”