Standard methods for American foulbrood research.

Authors: Dirk C de Graaf, Adriana M Alippi, Karina Antúnez, Katherine A Aronstein, Giles Budge, Dieter De Koker, Lina De Smet, Douglas W Dingman, Jay D Evans, Leonard J Foster, Anne Fünfhaus, Eva Garcia-Gonzalez, Aleš Gregorc, Hannelie Human, K Daniel Murray, Bach Kim Nguyen, Lena Poppinga, Marla Spivak, Dennis VanEngelsdorp, Selwyn Wilkins and Elke Genersch.

Table of contents



1. Introduction

2. Working with Paenibacillus larvae

   2.1. Biosafety measures
   2.2. Strains
   2.3. Sampling for AFB monitoring or diagnosis
3. Basic microbiological techniques
   3.1. Cultivation
   3.2. Identification

      3.2.1. Bacterial DNA extraction
      3.2.2. Polymerase chain reaction

   3.3. Genotyping
      3.3.1. PCR amplification of repetitive elements
      3.3.2. Pulsed-field gel electrophoresis  Preparation of genomic DNA agarose plugs
 Restriction enzyme digestion Gel loading and electrophoresis
   3.4. In vitro sporulation of Paenibacillus larvae
      3.4.1. Sporulation on solid growth medium
      3.4.2. Sporulation in liquid growth medium
   3.5. Long term conservation of vegetative cells
      3.5.1. Short-term preservation
      3.5.2. Preservation via ultra-low freezing
      3.5.3. Preservation via lyophilization
   3.6. Measuring susceptibility/resistance to antibiotics of Paenibacillus larvae
      3.6.1. Determination of minimal inhibitory concentrations (MICs)
      3.6.2. Determination of antibiotic susceptibility testing by the disc diffusion method Preparation of discs Preparation of plates Determination of resistance/susceptibility
4. Experimental infection
   4.1. Infection of in vitro reared larvae for the analysis of virulence and pathomechanisms of Paenibacillus larvae
      4.1.1. Protocol for exposure bioassays
      4.1.2. Analysis of generated data
   4.2. Experimental infection of a bee colony
      4.2.1. Inoculation with known spore concentration solution
      4.2.2. Inoculation with diseased brood
   4.3. Measuring colony resistance to AFB
      4.3.1. Surveying inoculated colonies
      4.3.2. Colony resistance
5. ‘omics and other sophisticated techniques
   5.1. Paenibacillus larvae gene expression
      5.1.1. Reference gene selection Sample collection and storage RNA and cDNA preparation Primer design and secondary structures qRT-PCR reactions qRT-PCR program qRT-PCR analysis
      5.1.2. Differential gene expression
   5.2. Comparative genome analysis within the species Paenibacillus larvae using suppression subtractive hybridization
   5.3. Conventional proteomics using two-dimensional gel electrophoresis
   5.4. Differential proteomics of Paenibacillus larvae

      5.4.1. Sample preparation Extract proteins Reduce, alkylate and digest proteins to peptides Clean up peptides Label peptides with stable isotopes Clean up peptides Mass spectrometry analysis
   5.5. Expression of heterologous proteins in Paenibacillus larvae
      5.5.1. Transformation of Paenibacillus larvae
      5.5.2. Detection of GFP-expression
   5.6. Fluorescent in situ hybridization for the detection of Paenibacillus larvae
      5.6.1. Preparation and embedding of larval tissues
      5.6.2. Performing fluorescence in situ hybridization
6. Final remarks
7. Acknowledgements
8. References