Detection of Paenibacillus larvae spores in the debris and wax of honey bee by the Tween 80 method

The aim of the present study was to validate a new method of detection of Paenibacillus larvae spores in the debris and wax of honey bee and compare it with the method commonly used in the Czech Republic, i.e. the method based on dissolving wax components of samples in toluene or benzene and releasing them in a liquid medium. The new method builds on homogenization of the material at the temperature of 70 +/- 2 degrees C, using distilled water and homogenization agent Tween 80. The spores are transferred from the suspension thus created to a liquid medium diluted and treated by heat (90 +/- 2 degrees C) in order to destroy vegetative saprophytic microflora. This medium is then inoculated on solid culture media (blood agar and MYPGP with nalidixic acid) at individual doses of 0.2 ml. After five to eight-day incubation at 37 +/- 1 degrees C, the suspected P. larvae colonies are counted and subjected to confirmation. The count of colonies multiplied by 100 represents the number of spores in I g of the material. Eleven debris control samples from circular test and 10 demonstrably positive debris samples containing P. larvae spores were used to compare both methods. The new method was independently tested on other 1,509 field samples of winter debris collected in protection areas around American foulbrood (AFB) outbreaks and examined from 1 January 2006 to 10 May 2006. Of these 1,509 field samples, 46 were P. larvae positive. The comparison of the number of spores found concurrently by both methods in 21 control samples and their benchmarking by the paired t-test method showed that the Tween method was significantly more effective in detection of P. larvae spores in the examined material than the commonly used Toluene method (the t value was -3.524). The test results indicate that the Tween method, as soon as it is tested and validated at other research sites, can be effective in examining the debris and wax of honey bee. Unlike Toluene method based on organic solvents, the Tween method is more acceptable from the point of view of fire protection, environment compatibility, waste disposal and occupational health and safety of laboratory staff. From the scientific perspective, the Tween method could play an important role in early diagnostics and subsequent control of the incidence of American foulbrood not only in the Czech Republic, but also in other countries worldwide.