Genetic and Biochemical Diversity ofPaenibacillus larvaeIsolated from Tunisian Infected Honey Bee Broods

Chadlia Hamdi,Khaoula Abdi,Alberto Alma,Annalisa Balloi,Elena Gonella,Daniele Daffonchio,Jihène Essanaa,Luigi Sansonno,Abdellatif Boudabous,Elena Crotti,Naima Barbouche,Ameur Cherif

doi:10.1155/2013/479893

Abstract

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a virulent disease of honeybee (Apis mellifera) larvae. In Tunisia, AFB has been detected in many beekeeping areas, where it causes important economic losses, but nothing is known about the diversity of the causing agent. Seventy-five isolates of P. larvae, identified by biochemical tests and 16S rRNA gene sequencing, were obtained from fifteen contaminated broods showing typical AFB symptoms, collected in different locations in the northern part of the country. Using BOX-PCR, a distinct profile of P. larvae with respect to related Paenibacillus species was detected which may be useful for its identification. Some P. larvae-specific bands represented novel potential molecular markers for the species. BOX-PCR fingerprints indicated a relatively high intraspecific diversity among the isolates not described previously with several molecular polymorphisms identifying six genotypes on polyacrylamide gel. Polymorphisms were also detected in several biochemical characters (indol production, nitrate reduction, and methyl red and oxidase tests). Contrary to the relatively high intraspecies molecular and phenotypic diversity, the in vivo virulence of three selected P. larvae genotypes did not differ significantly, suggesting that the genotypic/phenotypic differences are neutral or related to ecological aspects other than virulence.

Highlights

American foulbrood (AFB), a severe and highly contagious disease affecting the larval and pupal stages of honeybee (Apis mellifera), is caused by the bacterium Paenibacillus larvae [1, 2]
The aim of the present work was to characterize a collection of P. larvae isolated from Tunisian diseased brood and to study the genetic and biochemical diversity related to these isolates
Nine reference strains of seven Paenibacillus species phylogenetically related to P. larvae were obtained from the Bacillus Genetic Stock Center (BGSC), USA: Paenibacillus alvei 33A3 and 33A4, Paenibacillus polymyxa ATCC842T, Paenibacillus popilliae 2525 and B2519, Paenibacillus vorticalis 30A1, Paenibacillus thiaminolyticus NRRLB-4156T, Paenibacillus dendritiformis T168, and Paenibacillus macerans BKM B-51

Summary

Introduction

American foulbrood (AFB), a severe and highly contagious disease affecting the larval and pupal stages of honeybee (Apis mellifera), is caused by the bacterium Paenibacillus larvae [1, 2]. AFB is one of the few diseases capable of killing the honeybee colony [3]. An eradication strategy exists with isolation and destruction of infected colonies and burning of contaminated equipments [10, 11]. Different genotypes of P. larvae have been identified in different regions. By using BOX-PCR three genotypes (A, B, and C) have been identified within a worldwide isolate collection [12]. In Germany, four different genotypes of P. larvae, named AB, Ab, ab and αB, have been described by combining BOX A1R and MBO REP1 primers [13, 14].

Objectives

Methods

Results

Conclusion