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Applied and Environmental Microbiology, July 2002, p. 3655-3660, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3655-3660.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Adriana M. Alippi,^1* Ana Claudia López,² and O. Mario Aguilar²

Centro de Investigaciones de Fitopatología, Facultad de Ciencias Agrarias y Forestales,¹ Instituto de Bioquímica y Biología Molecular, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 1900 La Plata, Argentina²

Received 13 December 2001/ Accepted 17 April 2002

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.

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* Corresponding author. Mailing address: Centro de Investigaciones de Fitopatología, Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, calles 60 y 118, c.c. 31, 1900 La Plata, Argentina. Phone: 54 221 4236758, ext. 423. Fax: 54 221 425-2346. E-mail: alippi{at}biol.unlp.edu.ar.

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Applied and Environmental Microbiology, July 2002, p. 3655-3660, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3655-3660.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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