Melissococcus plutonius
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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.
- Klíčová slova
- Apis mellifera, Melissococcus plutonius, Paenibacillus larvae, American foulbrood, European foulbrood, Multiplex PCR,
- 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
- Názvy látek
- transpozibilní elementy DNA MeSH
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
Host-specific Lactobacillus and Bifidobacterium species constitute the core microbiota of the honey bee digestive tract and are recognized for their probiotic properties. One of the properties of these bacteria is the inhibition of bacterial pathogens such as Paenibacillus larvae and Melissococcus plutonius, the causative agents of American and European foulbrood, respectively. Additionally, Serratia marcescens has emerged as a relevant opportunistic pathogen. Although several previously published studies have examined the inhibition of selected bacterial pathogens of bees by members of the bee physiological microbiota, none have simultaneously investigated the inhibition of multiple clinical isolates of P. larvae, M. plutonius, and S. marcescens using a wide range of bifidobacterial and lactobacilli strains isolated from various locations within a single country. Thus, this study evaluated the antimicrobial potential of Lactobacillus and Bifidobacterium strains against these pathogens, with a focus on strain-dependent inhibition. A total of 111 bacterial strains (62 Lactobacillus and 49 Bifidobacterium) were isolated from the digestive tracts of honey bees collected from eight sites across the Czech Republic. Using 16S rRNA gene sequencing, the isolates were classified and tested in vitro against four P. larvae isolates, one M. plutonius isolate, and the S. marcescens strain sicaria in modified BHI medium. Twenty-eight strains (~26%) exhibited strong inhibition (≥21 mm) against at least two P. larvae isolates, while 12 strains showed moderate inhibition (16-20 mm) against all four isolates. Inhibition of M. plutonius and S. marcescens was observed in three and twenty strains, respectively. The most effective strains belonged to Bifidobacterium asteroides, B. choladohabitans, B. polysaccharolyticum, Lactobacillus apis, L. helsingborgensis, L. kullabergensis, and L. melliventris. These results underscore the strain-dependent nature of antimicrobial activity and highlight the importance of selecting probiotic strains with broad-spectrum pathogen inhibition to support honey bee health.
- Klíčová slova
- Melissococcus plutonius, Paenibacillus larvae, Serratia marcescens strain sicaria, bifidobacteria, honey bee probiotics, in vitro inhibition, lactobacilli,
- Publikační typ
- časopisecké články MeSH
European foulbrood (EFB) is an infectious disease affecting honeybee larvae caused by the bacterium Melissococcus plutonius. The enzyme-linked immunosorbent assay (ELISA) is the gold standard for antibody-based bacteria detection, however, its sensitivity is not high enough to reveal early-stage EFB infection. Photon-upconversion nanoparticles (UCNPs) are lanthanide-doped nanomaterials that emit light of shorter wavelength under near-infrared (NIR) excitation and thus avoid optical background interference. After conjugation with specific biorecognition molecules, UCNPs can be used as ultrasensitive labels in immunoassays. Here, we introduce a method for conjugation of UCNPs with streptavidin based on copper-free click chemistry, which involves surface modification of UCNPs with alkyne-modified bovine serum albumin (BSA) that prevents the non-specific binding and provides reactive groups for conjugation with streptavidin-azide. To develop a sandwich upconversion-linked immunosorbent assay (ULISA) for M. plutonius detection, we have prepared a rabbit polyclonal anti-Melissococcus antibody. The specific capture of the bacteria was followed by binding of biotinylated antibody and UCNP-BSA-streptavidin conjugate for a highly sensitive upconversion readout. The assay yielded an LOD of 340 CFU mL-1 with a wide working range up to 109 CFU mL-1, which is 400 times better than the LOD of the conventional ELISA. The practical applicability of the ULISA was successfully demonstrated by detecting M. plutonius in spiked real samples of bees, larvae and bottom hive debris. These results show a great potential of the assay for early diagnosis of EFB, which can prevent uncontrolled spreading of the infection and losses of honeybee colonies.
- MeSH
- Enterococcaceae imunologie izolace a purifikace MeSH
- fotony MeSH
- imunoanalýza metody MeSH
- larva imunologie metabolismus MeSH
- limita detekce MeSH
- nanočástice chemie MeSH
- oxid křemičitý chemie MeSH
- protilátky bakteriální imunologie MeSH
- včely růst a vývoj mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- oxid křemičitý MeSH
- protilátky bakteriální 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.
A taxonomic study was performed on Gram-stain-positive, catalase-negative and regular rod-shaped bacterial strains R4B(T) and R4C, isolated from the stomachs of honeybees. 16S rRNA gene sequence analysis revealed that the phylogenetic position of the novel strains was within the genus Lactobacillus; the highest sequence similarity to R4B(T) was shown by Lactobacillus acidophilus BCRC 10695(T) (93.6 %). Lower sequence similarities were found to other obligately homofermentative lactobacilli. A PCR-DGGE method could detect the sequence of the 16S rRNA gene of strain R4B(T) at different developmental stages of honeybees occurring in two different locations in the Czech Republic. The distinctiveness of the strains from other lactobacilli was also confirmed by analysis of sequences of other phylogenetic markers applicable to the taxonomy of the genus Lactobacillus, ribotyping and rep-PCR analysis. The DNA G+C content of strain R4B(T) was 41.3 mol%. The predominant cellular fatty acids of strain R4B(T) were C18 : 1ω9c, summed C19 : 1ω6c/C19 : 0 cyclo ω10c, C16 : 0, summed C18 : 1ω7c/C18 : 1ω6c and summed C16 : 1ω7c/C16 : 1ω6c. The major polar lipids of strain R4B(T) were glycolipids, lipids and phospholipids. Phenotypic and phylogenetic characteristics also confirmed the independent status of the strains at the species level. Interestingly, strain R4B(T) was able to inhibit growth in vitro of Paenibacillus larvae subsp. larvae (causal agent of American foulbrood in honeybees) and Melissococcus plutonius (causal agent of European foulbrood). The name Lactobacillus apis sp. nov. is proposed for this novel taxon; the type strain is R4B(T) ( = CCM 8403(T) = LMG 26964(T)).
- MeSH
- antibióza * MeSH
- DNA bakterií genetika MeSH
- Enterococcaceae patogenita MeSH
- fosfolipidy chemie MeSH
- fylogeneze * MeSH
- Lactobacillus klasifikace genetika izolace a purifikace fyziologie MeSH
- mastné kyseliny chemie MeSH
- molekulární sekvence - údaje MeSH
- Paenibacillus patogenita MeSH
- ribotypizace MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- techniky typizace bakterií MeSH
- včely mikrobiologie MeSH
- žaludek mikrobiologie MeSH
- zastoupení bazí MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
- Spojené státy americké MeSH
- Názvy látek
- DNA bakterií MeSH
- fosfolipidy MeSH
- mastné kyseliny MeSH
- RNA ribozomální 16S MeSH
