Plate counting is highly unreliable for quantification of Paenibacillus larvae in bee-related samples. To overcome this limitation, we designed a novel quantitative PCR (qPCR) assay for the detection and quantification of P. larvae spores in honey samples, which may be predictive of the AFB health status of corresponding honeybee colonies. The assay is based on the TaqMan technology and amplifies a single-copy metalloproteinase gene of P. larvae, enabling its reliable molecular detection and quantification. To enable absolute quantification, the qPCR standard curve was calibrated by digital PCR. For qPCR, the observed limit of quantification (LOQ) was 13 spores of P. larvae per gram of honey, and the limit of detection (LOD) 3 spores per gram. The developed qPCR assay represents an important contribution to the early diagnostics of American foulbrood by molecular quantification of P. larvae spores in honey samples.
B.03 Paper at an international scientific conference
COBISS.SI-ID: 4881530In this study, we assessed the epidemiological applicability of core genome multilocus sequence typing (cgMLST) and genome-wide single nucleotide polymorphism (SNP) typing for Paenibacillus larvae. A total of 42 ERIC II and nine ERIC I isolates from Slovenia underwent whole-genome sequencing with Illumina technology. cgMLST was performed using ChewBBACA, whereas SNP typing was performed using Snippy. Comparison of cgMLST and SNP typing showed 100% concordance between the three observed ERIC II clusters and a single ERIC I cluster; all clusters were also well supported by the bootstrap values and the corresponding epidemiological data. Median genetic diversity within a single cluster for all four identified clusters was 24 cgMLST alleles (range 2–46) and 30 SNPs (range 1–60). Both methods proved suitable for outbreak cluster delineation. This study improves our understanding of the genetic diversity of P. larvae isolates obtained during outbreak investigation and represents an important basis for establishing an allele/SNP threshold to delineate P. larvae outbreak clones.
B.03 Paper at an international scientific conference
COBISS.SI-ID: 44930307Genetic diversity of P. larvae isolates within the same sample, apiary or beekeeping operation remains poorly understood. In this study, we assessed the genetic diversity of P. larvae from a single beekeeping operation with three apiaries. Isolates were obtained in autumn 2019 from samples of diseased brood and the associated honey, as part of the investigation of an outbreak of American foulbrood. Five isolates were typed per each of the three investigated honey samples and one from each brood sample. All isolates (n=40) were typed using ERIC-PCR and genome-wide single nucleotide polymorphisms (SNPs) using Snippy. All isolates belonged to type ERIC II. The average diversity within the operation was 13 SNPs (range 0–27); the maximum diversity (27 SNPs) was already detected within a single apiary. The phylogenetic tree did not show clear clustering of isolates according to the associated data (sample type, apiary or hive). The monoclonal nature of the outbreak was confirmed, which confirms the transmission of the P. larvae strain within the operation. In the WGS-based investigation of outbreaks of American foulbrood, the genetic diversity of P. larvae isolates at the sample, apiary and beekeeping operation levels should be taken into account. The collected results represent an important contribution to our understanding of the genetic diversity of microbiologically and epidemiologically related P. larvae isolates.
B.03 Paper at an international scientific conference
COBISS.SI-ID: 32901379Members of the project group have worked most closely with beekeepers in the areas of education, expert advice and disease prevention, and diagnosis and remediation of American foulbrood outbreaks. In addition to the activities listed, the knowledge gained from this project has been directly applied to the beekeeping practice, improving some previous experiences. The members of the project group published, among others, professional articles in the Slovenski čebelar magazine, and shared their findings at the meetings of the Expert Association of Professional Beekeepers (2nd and 3rd Pislak Day), at annual meetings of beekeepers within the 42nd and 43rd ApiSlovenia and ApiSlovenia ‘in small’, at the 2nd and 3rd International Conference on Urban Beekeeping and in the preparation of the Good Beekeeping Practice Manual. With their knowledge and experience, the members of the project group cooperated with the Beekeepers' Association of Slovenia and in several working groups within MKGP (Ministry of Agriculture, Forestry and Food) in the preparation of programs in the field of beekeeping, cooperated with UVHVVR (Administration for Food Safety, Veterinary Sector and Plant protection) in the preparation of amendments to the regulations on measures for identification, control, information and prevention of American foulbrood, and participated within the City Municipality of Ljubljana in the preparation of the pilot project 'Shelter for bee swarms'.
F.18 Transfer of new know-how to direct users (seminars, fora, conferences)