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):
- 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.
- 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.
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.
- 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.