Development Of The Molecular Diagnostic Technique For Chalkbrood Disease

Chalkbrood disease in honey bees (Apis mellifera) is an invasive mycosis produced by the fungus, Ascosphaera apis, affecting stretched larvae and causing serious collapse of the colonies. The trend of the disease extensive outbreak has emerged in China in the past few years. Chalkbrood is currently the most serious honeybee disease. The general diagnostic methods for chalkbrood disease based on typical symptoms and morphological characteristics are time-consuming, arduous and unreliable. Thus, it would be difficult to diagnose disease accurately in early stage of its development.The A. apis species-specific primers used in the paper were derived from conserved and variant regions of the DNA sequences in GenBank for the 5.8s ribosomal DNA and the internal transcribed spacers (ITS 1 and ITS 2) using the program Primer-BLAST. Polymerase chain reaction (PCR) techniques utilize highly specific primers and amplify the DNA sequence used as diagnostic molecular marker. The specificity, sensitivity and applicability of the diagnostic molecular marker were tested.(1) The A. apis-specific DNA sequence of 442 bp was amplified and cloned successfully for diagnostic molecular marker of chalkbrood disease.(2) To test the specificity of diagnostic molecular marker, PCR reactions were conducted for the A. apis species-specific primers using DNA extracted from larvae of A. m. ligustica and Apis cerana cerana, Mucor racemosus, Aspergillus flavus, Saccharomyces ceravisiae, E. coli, and A. apis. The results showed that amplification for a specific product of 400~500 bp occurred only for A. apis, and the negative test for other species verify that the diagnostic molecular marker is highly specific to A. apis, and would not amplify DNA from honeybees, bacterial and unrelated fungal species.(3) To determine the sensitivity of diagnostic molecular marker, the PCR tests were applied to serial dilutions of the DNA extracted from A. apis. The lowest concentration that yielded a detectable level of DNA was an average of 1.74×10-7 ng·μL-1, approximated equal to 4.34×10-6 ng DNA added to 25μL PCR reaction. The test result indicated good sensitivity.(4) The PCR reactions were conducted directly for DNAs extracted from the symptomatic and asymptomatic honeybee larvae to detect A. apis. For the symptomatic cadavers, all showed positive for A. apis. The results showed it is a quick and reliable diagnostic method. A. apis could be detected from a part of asymptomatic honeybee larvae, the results showed the diagnostic molecular marker do not relying on the typical symptoms, and can reveal these latent or inapparent infections.(5) A quick and reliable technique based on PCR was developed for the diagnosis of chalkbrood disease using the molecular marker. The technique eliminates the need for culturing samples and the typical symptoms, and could be used to detect these latent or inapparet infections.(6) Using the molecular diagnostic technique, the pathogenetic fungi isolated from chalk-like larval cadavers of A. c. cerana was identified to be A. apis. The result showed that A. apis could infect A. c. cerana. It is the first case of chalkbrood disease in A. c. cerana.