4.1. Infection of in vitro reared larvae for the analysis of virulence and pathomechanisms of Paenibacillus larvae
Exposure bioassays (Genersch et al., 2005) are a reliable approach to determine virulence of different P. larvae strains in the laboratory. Briefly, this experiment consists in:
- Rear first-instar larvae from various colonies in 24-well plates, with 10 larvae per well.
- Provide larvae with larval food mixed with a determined quantity of infectious spores during the first 24 hours. Afterwards, larvae will receive normal food every day for the rest of the experiment. A control group receives normal larval diet throughout.
- Each day dead larvae are recorded and examined for AFB infection.
Spore preparations for experimental infection can be performed as described under section 3.4. or as described below (Genersch et al., 2005):
- Resuspend around 100 P. larvae colonies in 300 µl of brain heart infusion broth (BHI).
- Inoculate on Columbia sheep blood agar (CSA) slants.
- Incubate slants containing bacterial suspension at 37°C for 10 days or until sporulation has occurred.
- Collect medium containing spores.
- Calculate spore concentration in the medium in colony forming units after plating different dilutions of the collected medium.
- Count colonies after 6 days of incubation.