4.2.1. Inoculation with known spore concentration solution

Below is one protocol for a field evaluation of AFB prevalence, derived from Evans and Pettis (2005):

  1. Establish clean colonies of equal strength by placing 1.2 kg of worker bees and a marked queen (from resistant or susceptible stock, if desired) into a standard (e.g. Langstroth) hive body.
  2. Following establishment and the initiation of brood rearing (ca. 1 month), inoculate each colony twice (2-4 weeks apart) with spores from a fresh field isolate of P. larvae.
  3. Inoculate by spraying approximately 2,000 immature bees (eggs, embryos, and first-and second-instar larvae) with a sucrose-water (1:10 weight/volume) suspension containing ca. 200 million P. larvae spores.
    Sufficient inoculant for >50 colonies is prepared by macerating and suspending ca. 100 scales (dried larval remains) collected from symptomatic colonies.
  4. Spray inoculation is effective at initiating AFB infection in colonies, with ca. 50 % of managed colonies exhibiting AFB disease one month following a single inoculation, and nearly all of susceptible colonies showing symptoms by three weeks after the second inoculation.