Differentiation of Paenibacillus larvae subsp. larvae , the Cause of American Foulbrood of Honeybees, by Using PCR and Restriction Fragment Analysis of Genes Encoding 16S rRNA

Abstract

A rapid procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American foulbrood (AFB) disease of honeybees (Apis mellifera L.), based on PCR and restriction fragment analysis of the 16S rRNA genes (rDNA) is described. Eighty-six bacterial strains belonging to 39 species of the genera Paenibacillus, Bacillus, Brevibacillus, and Virgibacillus were characterized. Amplified rDNA was digested with seven restriction endonucleases. The combined data from restriction analysis enabled us to distinguish 35 profiles. Cluster analysis revealed that P. larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens formed a group with about 90% similarity; however, the P. larvae subsp. larvae restriction fragment length polymorphism pattern produced by endonuclease HaeIII was found to be unique and distinguishable among other closely related bacteria. This pattern was associated with DNA extracted directly from honeybee brood samples showing positive AFB clinical signs that yielded the restriction profile characteristic of P. larvae subsp. larvae, while no amplification product was obtained from healthy larvae. The method described here is particularly useful because of the short time required to carry it out and because it allows the differentiation of P. larvae subsp. larvae-infected larvae from all other species found in apiarian sources.

Güney Marmara Bölgesindeki Bal Arılarının Yavru Çürüklüğü Hastalığı etkenlerinin PZR ve Kültürel Metodlar ile Belirlenmesi

“…Larval remains from brood comb were collected with a sterile swab for the bacterial isolation and suspended in 5 ml of sterile distilled water [8,10,11,14] . The suspension was incubated at room temperature for 10 min and separated into two samples.…”

“…DNA was eluted with 200 μl of elution buffer and stored at -20°C. Bacterial DNA was isolated using the OIAamp DNA minikit (Qiagen) as instructed by the manufacturer [8,[10][11][12]14,17] . [10][11][12] .…”

“…PCR amplified products were examined on agarose (0.8%) gel electrophoresis. DNA amplified by PCR was stained with ethidium bromide and visualised with UV transilluminator [10][11][12]14,17,18] .…”

“…PCRbased methods for detecting P. larvae have been described by several authors [1,[8][9][10][11][12][13] . The sequences of detection primers were based on 16S rRNA gene of P. larvae and M. plutonius [8,10,[12][13][14] .…”

Detection of novel probiotic bacterium Lactobacillus spp. in the workers of Indian honeybee, Apis cerana indica

“…So far, most studies on honeybee microflora have focused on diseasecausing microorganisms (Alippi et al, 2002), while much less emphasis has been given to non-pathogenic microorganisms and their potential benefit for individual bees or whole colonies. However, there is growing awareness of the importance of the composition of the intestinal micro-flora for health and growth of honeybees (Gilliam, 1979;Gilliam et al, 1988a;Gilliam et al, 1997;Dillon & Dillon, 2004).…”