The occurrence of Melissococcus plutonius in healthy colonies of Apis mellifera and the efficacy of European foulbrood control measures

BackgroundMelissococcus plutoniusis an entomopathogenic bacterium that causes European foulbrood (EFB), a honeybee (Apis melliferaL.) 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.MethodsThe 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.ResultsThe bees from EFB2 colonies with clinical symptoms exhibited a 75-fold-higher incidence ofM. plutoniusthan those from EFB1 asymptomatic colonies.Melissococcus plutoniuswas identified in all EFB1 colonies as well as in some of the control colonies. The proportions ofFructobacillus fructosus,Lactobacillus kunkeei,Gilliamella apicola,Frischella perrara, andBifidobacterium coryneformewere higher in EFB2 than in EFB1, whereasLactobacillus melliswas significantly higher in EFB2 than in EFB0.Snodgrassella alviandL. melliventris,L. helsingborgensisand,L. kullabergensisexhibited higher proportion in EFB1 than in EFB2 and EFB0. The occurrence ofBartonella apisandCommensalibacter intestiniwere higher in EFB0 than in EFB2 and EFB1.Enterococcus faecalisincidence was highest in EFB2.ConclusionsHigh-throughput Illumina sequencing permitted a semi-quantitative analysis of the presence ofM. plutoniuswithin the honeybee worker microbiome. The results of this study indicate that worker bees from EFB-diseased colonies are capable of transmittingM. plutoniusdue to the greatly increased incidence of the pathogen. The presence ofM. plutoniussequences 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 thatE. faecalisis a secondary invader toM. plutonius; however, other putative secondary invaders were not identified in this study.

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The transmission of European foulbrood (Melissococcus plutonius) to artificially reared honey bee larvae (Apis mellifera)

Journal of Apicultural Research ◽

10.1080/00218839.2004.11101117 ◽

2004 ◽

Vol 43 (3) ◽

pp. 93-100 ◽

Cited By ~ 22

Author(s):

Ben Alexander McKee ◽

Russell David Goodman ◽

Michael Alan Hornitzky

Keyword(s):

Apis Mellifera ◽

Honey Bee ◽

Melissococcus Plutonius ◽

European Foulbrood ◽

Honey Bee Larvae

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European foulbrood in Czechia after 40 years: application of next-generation sequencing to analyze Melissococcus plutonius transmission and influence on the bacteriome of Apis mellifera

10.7287/peerj.preprints.2618v1 ◽

2016 ◽

Cited By ~ 1

Author(s):

Tomas Erban ◽

Ondrej Ledvinka ◽

Martin Kamler ◽

Bronislava Hortova ◽

Marta Nesvorna ◽

...

Keyword(s):

Apis Mellifera ◽

Next Generation Sequencing ◽

Pathogenic Bacteria ◽

Clinical Symptoms ◽

Rrna Gene ◽

Next Generation ◽

Colony Losses ◽

Melissococcus Plutonius ◽

European Foulbrood ◽

Generation Sequencing

Worker honeybees (Apis mellifera) transmit Melissococcus plutonius between colonies. However, the transmission of M. plutonius, which causes European foulbrood (EFB), is poorly understood. To analyze the first EFB outbreak in 40 years in Czechia, we collected 49 hive worker samples from 18 beehives in two diseased apiaries for bacteriome analysis of the V1-V3 portion of the 16S rRNA gene. When we compared control samples obtained outside of the EFB zone, bees from an EFB apiaries containing colonies without clinical symptoms and bees from colonies with EFB clinical symptoms, there was a 100-fold higher occurrence of M. plutonius in colonies with EFB symptoms. The presence of M. plutonius in controls indicated that this pathogen exists in an enzootic state. EFB influenced the core bacteria in the worker bacteriome because the number of Snodgrassella alvi, Lactobacillus mellis, Lactobacillus melliventris, and Fructobacillus fructosus sequences increased, while Bartonella apis, Frischella perrara, and Commensalibacter intestine sequences decreased. Together, the results of this study suggest worker bees from EFB-diseased apiaries serve as vectors of M. plutonius, and eliminating such colonies is an appropriate method to overcome disease outbreaks. Because M. plutonius exists in honeybee colonies in an enzootic state, there may be similar abundances in control colonies outside the EFB zone to those in asymptomatic colonies from EFB apiaries. High-throughput Illumina next-generation sequencing permitted the quantitative interpretation of M. plutonius within the honeybee worker bacteriome. Future studies focusing on honeybee diseases, colony losses, detection of bacterial pathogens and interactions of bacteriome with pathogenic bacteria will benefit of this study.

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European foulbrood in Czechia after 40 years: application of next-generation sequencing to analyze Melissococcus plutonius transmission and influence on the bacteriome of Apis mellifera

10.7287/peerj.preprints.2618 ◽

2016 ◽

Author(s):

Tomas Erban ◽

Ondrej Ledvinka ◽

Martin Kamler ◽

Bronislava Hortova ◽

Marta Nesvorna ◽

...

Keyword(s):

Apis Mellifera ◽

Next Generation Sequencing ◽

Pathogenic Bacteria ◽

Clinical Symptoms ◽

Rrna Gene ◽

Next Generation ◽

Colony Losses ◽

Melissococcus Plutonius ◽

European Foulbrood ◽

Generation Sequencing

Worker honeybees (Apis mellifera) transmit Melissococcus plutonius between colonies. However, the transmission of M. plutonius, which causes European foulbrood (EFB), is poorly understood. To analyze the first EFB outbreak in 40 years in Czechia, we collected 49 hive worker samples from 18 beehives in two diseased apiaries for bacteriome analysis of the V1-V3 portion of the 16S rRNA gene. When we compared control samples obtained outside of the EFB zone, bees from an EFB apiaries containing colonies without clinical symptoms and bees from colonies with EFB clinical symptoms, there was a 100-fold higher occurrence of M. plutonius in colonies with EFB symptoms. The presence of M. plutonius in controls indicated that this pathogen exists in an enzootic state. EFB influenced the core bacteria in the worker bacteriome because the number of Snodgrassella alvi, Lactobacillus mellis, Lactobacillus melliventris, and Fructobacillus fructosus sequences increased, while Bartonella apis, Frischella perrara, and Commensalibacter intestine sequences decreased. Together, the results of this study suggest worker bees from EFB-diseased apiaries serve as vectors of M. plutonius, and eliminating such colonies is an appropriate method to overcome disease outbreaks. Because M. plutonius exists in honeybee colonies in an enzootic state, there may be similar abundances in control colonies outside the EFB zone to those in asymptomatic colonies from EFB apiaries. High-throughput Illumina next-generation sequencing permitted the quantitative interpretation of M. plutonius within the honeybee worker bacteriome. Future studies focusing on honeybee diseases, colony losses, detection of bacterial pathogens and interactions of bacteriome with pathogenic bacteria will benefit of this study.

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Pathogenesis, Epidemiology and Variants of Melissococcus plutonius (Ex White), the Causal Agent of European Foulbrood

Journal of Apicultural Science ◽

10.2478/jas-2020-0030 ◽

2020 ◽

Vol 64 (2) ◽

pp. 173-188

Author(s):

Adrián Ponce de León-Door ◽

Gerardo Pérez-Ordóñez ◽

Alejandro Romo-Chacón ◽

Claudio Rios-Velasco ◽

José D. J. Órnelas-Paz ◽

...

Keyword(s):

Morphological Characteristics ◽

Etiologic Agent ◽

Infection Process ◽

Control Measures ◽

Molecular Tools ◽

Bacterial Diseases ◽

Bacterial Development ◽

Melissococcus Plutonius ◽

European Foulbrood

AbstractThe bacterium Melissococcus plutonius is the etiologic agent of the European foulbrood (EFB), one of the most harmful bacterial diseases that causes the larvae of bees to have an intestinal infection. Although EFB has been known for more than a century and is practically present in all countries where beekeeping is practiced, the disease has been little studied compared to American foulbrood. Recently, great advances have been made to understand the disease and the interaction between the pathogen and its host. This review summarizes the research and advances to understand the disease. First, the morphological characteristics of M. plutonius, the infection process and bacterial development in the gut of the larva are described. Also, the epidemiological distribution of EFB and factors that favor the development of the disease as well as the classification of M. plutonius according its genomic and phenotype characteristics are reported. Finally, the new molecular tools for the study of M. plutonius, possible virulence factors in its genome, the issue of current EFB control measures and possible alternatives to the use of antibiotics are addressed.

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Susceptibility of Apis mellifera Larvae of Different Ages to Infection from Melissococcus plutonius, an European Foulbrood Disease-causing Pathogen

Journal of Apiculture ◽

10.17519/apiculture.2021.06.36.2.47 ◽

2021 ◽

Vol 36 (2) ◽

pp. 47-54

Author(s):

A-Tai Truong ◽

Seonmi Kim ◽

Mi-Sun Yoo ◽

Yun Sang Cho ◽

ByoungSu Yoon

Keyword(s):

Apis Mellifera ◽

Melissococcus Plutonius ◽

European Foulbrood ◽

Different Ages

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A scientific note on improved isolation methods for Melissococcus plutonius from diseased Apis mellifera larvae

Apidologie ◽

10.1007/s13592-018-0572-4 ◽

2018 ◽

Vol 49 (4) ◽

pp. 459-461 ◽

Cited By ~ 2

Author(s):

Yuka Nakai ◽

Michika Ishihara ◽

Rie Arai ◽

Daisuke Takamatsu

Keyword(s):

Apis Mellifera ◽

Melissococcus Plutonius ◽

Scientific Note ◽

Isolation Methods

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Click-conjugated photon-upconversion nanoparticles in an immunoassay for honeybee pathogen Melissococcus plutonius

Nanoscale ◽

10.1039/c9nr01246j ◽

2019 ◽

Vol 11 (17) ◽

pp. 8343-8351 ◽

Cited By ~ 6

Author(s):

Veronika Poláchová ◽

Matěj Pastucha ◽

Zuzana Mikušová ◽

Matthias J. Mickert ◽

Antonín Hlaváček ◽

...

Keyword(s):

Click Chemistry ◽

Upconversion Nanoparticles ◽

Melissococcus Plutonius ◽

European Foulbrood

A method for the conjugation of photon-upconversion nanoparticles with streptavidin via copper-free click-chemistry was introduced, and the prepared label was applied in an immunoassay for European foulbrood diagnosis.

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