Molecular Typing of Paenibacillus larvae Strains Isolated from Bulgarian Apiaries Based on Repetitive Element Polymerase Chain Reaction (Rep-PCR)

Abstract

The aim of the present study was to perform molecular typing of Paenibacillus larvae (P. larvae) isolates from Bulgarian apiaries with repetitive element polymerase chain reaction (rep-PCR) using BOX A1R, MBO REP1, and ERIC primers. A total of 96 isolates collected from brood combs with clinical symptoms of American foulbrood originating from apiaries located in different geographical regions of Bulgaria, a reference strain P. larvae NBIMCC 8478 and 30 commercial honey samples with Bulgarian origin were included in the study. Rep-PCR fingerprinting analysis revealed two genotypes ab and AB of P. larvae isolates from brood combs and honey samples. A combination of genotypes ab/AB was detected in one apiary and honey sample. The prevailing genotype ab was found in 78.1 % of brood combs isolates as well as in the reference strain whereas genotype AB was determined in 21.9 % of isolates. The examination of honey samples confirmed the preponderance of ab genotype which was demonstrated in 20 of 30 samples analyzed. In conclusion, the genetic epidemiology of P. larvae revealed two genotypes--ab and AB for Bulgarian strains. Developed protocols for molecular typing of P. larvae are reliable and may be used to trace the source of infection.

Analysis of the Global Population Structure of Paenibacillus larvae and Outbreak Investigation of American Foulbrood Using a Stable wgMLST Scheme

“…Genersch et al (5) classified the strains LMG 16252 and DSM 3615 into two separate ERIC types, whereas Ebeling et al (24) categorized these strains as ERIC III/IV type according to MLST analysis. ERIC-PCR is known to exhibit limited reproducibility and inter-laboratory comparability (26). Based on the high genetic distance between this wgMLST clade and the remaining clades as well as the uncertainty regarding the ERIC types of both strains, the present data suggest this wgMLST clade may represent a novel ERIC type (or more than one ERIC type, as observed in ERIC III/IV clade) and should thus be further investigated and characterized.…”

Proteinase pattern of honeybee prepupae from healthy and American Foulbrood infected bees investigated by zymography

“…Those affected by American Foulbrood were collected by tweezers, only if still alive; dead prepupae were discarded. AFB was confirmed by AFB Diagnostic Test Kit (Vita Europe Ltd) followed by P. larvae isolation and microscopic identification . Five bacterial colonies (A, B, C, D, and E) with typical P. larvae morphology obtained on TCBS agar were subjected to DNA extraction.…”

Biogeography of Paenibacillus larvae, the causative agent of American foulbrood, using a new multilocus sequence typing scheme

“…The above typing schemes have been used to categorize crude patterns of P. larvae distribution in Europe (Genersch and Otten, 2003 ; Pentikäinen et al ., 2008 ; Loncaric et al ., 2009 ; Di Pinto et al ., 2011 ), the Americas (Alippi et al ., 2004 ; Antúnez et al ., 2007 ) and Australasia (Alippi et al ., 2004 ). However, rep-PCR methods have the disadvantage that they are difficult to repeat or to standardize between laboratories and therefore comparisons between different studies is difficult (Rusenova et al ., 2013 ). Recently, it was shown that the four ERIC genotypes can also be discriminated via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Schäfer et al ., 2014 ), providing a cost-effective, reliable and highly reproducible alternative tool for P. larvae ERIC typing.…”