ABSTRACT
American foulbrood, because of its virulence and worldwide spread, is currently one of the most dangerous diseases of honey bees. Quick diagnosis of this disease is therefore vitally important. For its successful eradication, however, all the hives in the region must be tested. This is time consuming and costly. Therefore, a fast and sensitive method of detecting American foulbrood is needed. Here we present a method that significantly reduces the number of tests needed by combining batches of samples from different hives. The results of this method were verified by testing each sample. A simulation study was used to compare the efficiency of the new method with testing all the samples and to develop a decision tool for determining when best to use the new method. The method is suitable for testing large numbers of samples (over 100) when the incidence of the disease is low (10% or less).
Subject(s)
Bees/microbiology , Nucleic Acids/analysis , Paenibacillus/isolation & purification , Polymerase Chain Reaction/methods , Spores, Bacterial/isolation & purification , Animals , Computer Simulation , Decision Support Techniques , Paenibacillus/physiology , Polymerase Chain Reaction/economics , Sensitivity and Specificity , Spores, Bacterial/physiologyABSTRACT
The objective of this work was to create a fast and sensitive method of detecting Paenibacillus larvae from beehive debris based on PCR that does not require long-lasting cultivation steps. Various methods of extracting spores from beehive debris were compared: the original method of extraction of spores into toluene, and alternative spore extraction methods into Tween 80, into water, into isopropanol and into 95% ethanol. Isolation of DNA from various spore extractions was evaluated too. Best results were provided by isolation of DNA using the QIAamp DNA Mini Kit, without heat treatment. DNA of spores was detected by PCR from 0.25 g of beeswax debris, with the detected titer of 10(5) in 1g according to the cultivation tests.
