Standard methods for nosema research.

Authors: Ingemar Fries, Marie-Pierre Chauzat, Yan-Ping Chen, Vincent Doublet, Elke Genersch, Sebastian Gisder, Mariano Higes, Dino P McMahon, Raquel Martín-Hernández, Myrsini Natsopoulou, Robert J Paxton, Gina Tanner, Thomas C Webster, Geoffrey R Williams.

Table of contents

 

Authors
Summary
1. Introduction
2. Method type
   2.1. Field methods

      2.1.1. Colony level infection
         2.1.1.1. Sampling
         2.1.1.2. Sample size
         2.1.1.3. Clinical signs and symptoms of infection
      2.1.2. Colony level infection dynamics
         2.1.2.1. Sampling period and sampling frequency
      2.1.3. Standardising field trials
         2.1.3.1. Selection of colonies
         2.1.3.2. Behaviour of infected bees
         2.1.3.3. Parameters to record
 
  2.2. Laboratory methods
      2.2.1. Colony and individual level infection
         2.2.1.1. Spore counts
         2.2.1.2. Individual bees or pooled samples
         2.2.1.3. Parts to examine
      2.2.2. Nosema species identification
         2.2.2.1. Light microscopy (LM)
            2.2.2.1.1. Giemsa staining
            2.2.2.1.2. Toluidine staining
        2.2.2.2. Transmission electron microscopy (TEM)
        2.2.2.3. Molecular detection of Nosema spp. (N. apis , N. ceranae and N. bombi)
           2.2.2.3.1. Nosema DNA extraction
           2.2.2.3.2. Multiplex PCR for detection of Nosema and differentiation between N. apis, N. bombi and N. ceranae
              2.2.2.3.2.1. PCR reaction mix
              2.2.2.3.2.2. Visualization
              2.2.2.3.2.3. Controls
           2.2.2.3.3. Realtime PCR for quantification of N. apis and N. ceranae
              2.2.2.3.3.1. PCR reaction mix
              2.2.2.3.3.2. Quantification
              2.2.2.3.3.3. Controls
      2.2.3. Standardising cage trials
         2.2.3.1. Source of bees
            2.2.3.1.1. Source colonies
            2.2.3.1.2. Age of bees
         2.2.3.2. Type of cages
         2.2.3.3. Type of food
         2.2.3.4. Incubation conditions
      2.2.4. Inoculation methods
         2.2.4.1. Spore source
          2.2.4.2. Spore suspension
             2.2.4.2.1. Purification
                2.2.4.2.1.1. Centrifugation
                2.2.4.2.1.2. Triangulation
                2.2.4.2.1.3. Density gradient
             2.2.4.2.2. Spore suspension concentration
             2.2.4.2.3. Storage of stock suspension
         2.2.4.3. Handling of bees
         2.2.4.4. Individual feeding
         2.2.4.5. Group feeding
      2.2.5. Viability control of Nosema spores
         2.2.5.1. Colouration test for spore viability
         2.2.5.2. In vivo test for spore viability
         2.2.5.3. In vitro test for spore viability
      2.2.6. In vitro rearing of Nosema spp.: cell culture systems
         2.2.6.1 Infection of primary honey bee ventricular cells
         2.2.6.2. Infection of heterologous lepidopteran cell lines
      2.2.7. Experiment type
         2.2.7.1. Determination of infectious dose
            2.2.7.1.1. Study of dose effects
            2.2.7.1.2. Effects of different infection doses
         2.2.7.2. Course of infection in individual bees
         2.2.7.3. Longevity of infected bees
3. Future perspectives
4. References