Nejvíce citovaný článek - PubMed ID 28634639
European foulbrood (EFB) is an infectious disease of honey bees caused by the bacterium Melissococcus plutonius. A method for DNA isolation and conventional PCR diagnosis was developed using hive debris, which was non-invasively collected on paper sheets placed on the bottom boards of hives. Field trials utilized 23 honey bee colonies with clinically positive symptoms and 21 colonies without symptoms. Bayes statistics were applied to calculate the comparable parameters for EFB diagnostics when using honey, hive debris, or samples of adult bees. The reliability of the conventional PCR was 100% at 6.7 × 103 Colony Forming Unit of M. plutonius in 1 g of debris. The sensitivity of the method for the sampled honey, hive debris, and adult bees was 0.867, 0.714, and 1.000, respectively. The specificity for the tested matrices was 0.842, 0.800, and 0.833. The predictive values for the positive tests from selected populations with 52% prevalence were 0.813, 0.833, and 0.842, and the real accuracies were 0.853, 0.750, and 0.912, for the honey, hive debris, and adult bees, respectively. It was concluded that hive debris can effectively be utilized to non-invasively monitor EFB in honey bee colonies.
- Klíčová slova
- European foulbrood, Melissococcus plutonius, PCR, hive debris, honey bee,
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Melissococcus plutonius is an entomopathogenic bacterium that causes European foulbrood (EFB), a honeybee (Apis mellifera L.) disease that necessitates quarantine in some countries. In Czechia, positive evidence of EFB was absent for almost 40 years, until an outbreak in the Krkonose Mountains National Park in 2015. This occurrence of EFB gave us the opportunity to study the epizootiology of EFB by focusing on the microbiome of honeybee workers, which act as vectors of honeybee diseases within and between colonies. METHODS: The study included worker bees collected from brood combs of colonies (i) with no signs of EFB (EFB0), (ii) without clinical symptoms but located at an apiary showing clinical signs of EFB (EFB1), and (iii) with clinical symptoms of EFB (EFB2). In total, 49 samples from 27 honeybee colonies were included in the dataset evaluated in this study. Each biological sample consisted of 10 surface-sterilized worker bees processed for DNA extraction. All subjects were analyzed using conventional PCR and by metabarcoding analysis based on the 16S rRNA gene V1-V3 region, as performed through Illumina MiSeq amplicon sequencing. RESULTS: The bees from EFB2 colonies with clinical symptoms exhibited a 75-fold-higher incidence of M. plutonius than those from EFB1 asymptomatic colonies. Melissococcus plutonius was identified in all EFB1 colonies as well as in some of the control colonies. The proportions of Fructobacillus fructosus, Lactobacillus kunkeei, Gilliamella apicola, Frischella perrara, and Bifidobacterium coryneforme were higher in EFB2 than in EFB1, whereas Lactobacillus mellis was significantly higher in EFB2 than in EFB0. Snodgrassella alvi and L. melliventris, L. helsingborgensis and, L. kullabergensis exhibited higher proportion in EFB1 than in EFB2 and EFB0. The occurrence of Bartonella apis and Commensalibacter intestini were higher in EFB0 than in EFB2 and EFB1. Enterococcus faecalis incidence was highest in EFB2. CONCLUSIONS: High-throughput Illumina sequencing permitted a semi-quantitative analysis of the presence of M. plutonius within the honeybee worker microbiome. The results of this study indicate that worker bees from EFB-diseased colonies are capable of transmitting M. plutonius due to the greatly increased incidence of the pathogen. The presence of M. plutonius sequences in control colonies supports the hypothesis that this pathogen exists in an enzootic state. The bacterial groups synergic to both the colonies with clinical signs of EFB and the EFB-asymptomatic colonies could be candidates for probiotics. This study confirms that E. faecalis is a secondary invader to M. plutonius; however, other putative secondary invaders were not identified in this study.
