Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)-American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I-IV (strain DSM 7030-ERIC I, DSM 25430-ERIC II, LMG 16252-ERIC III, DSM 3615-ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius.
- MeSH
- Enterococcaceae * MeSH
- larva mikrobiologie MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- Paenibacillus larvae * genetika MeSH
- Paenibacillus * genetika MeSH
- plazmidy genetika MeSH
- transpozibilní elementy DNA MeSH
- včely genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Social honey bees, Apis mellifera, host a set of distinct microbiota, which is similar across the continents and various honey bee species. Some of these bacteria, such as lactobacilli, have been linked to immunity and defence against pathogens. Pathogen defence is crucial, particularly in larval stages, as many pathogens affect the brood. However, information on larval microbiota is conflicting. Seven developmental stages and drones were sampled from 3 colonies at each of the 4 geographic locations of A. mellifera carnica, and the samples were maintained separately for analysis. We analysed the variation and abundance of important bacterial groups and taxa in the collected bees. Major bacterial groups were evaluated over the entire life of honey bee individuals, where digestive tracts of same aged bees were sampled in the course of time. The results showed that the microbial tract of 6-day-old 5th instar larvae were nearly equally rich in total microbial counts per total digestive tract weight as foraging bees, showing a high percentage of various lactobacilli (Firmicutes) and Gilliamella apicola (Gammaproteobacteria 1). However, during pupation, microbial counts were significantly reduced but recovered quickly by 6 days post-emergence. Between emergence and day 6, imago reached the highest counts of Firmicutes and Gammaproteobacteria, which then gradually declined with bee age. Redundancy analysis conducted using denaturing gradient gel electrophoresis identified bacterial species that were characteristic of each developmental stage. The results suggest that 3-day 4th instar larvae contain low microbial counts that increase 2-fold by day 6 and then decrease during pupation. Microbial succession of the imago begins soon after emergence. We found that bacterial counts do not show only yearly cycles within a colony, but vary on the individual level. Sampling and pooling adult bees or 6th day larvae may lead to high errors and variability, as both of these stages may be undergoing dynamic succession.
- MeSH
- Bacteria klasifikace genetika izolace a purifikace MeSH
- denaturační gradientová gelová elektroforéza MeSH
- DNA bakterií genetika MeSH
- ekosystém MeSH
- gastrointestinální trakt mikrobiologie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- Lactobacillaceae genetika MeSH
- RNA ribozomální 16S genetika MeSH
- střevní mikroflóra * MeSH
- včely embryologie růst a vývoj mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
