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.

Bee Research Institute at Dol Libcice nad Vltavou Czechia

Crop Research Institute Prague Czechia

Czech Hydrometeorological Institute Prague Czechia

Department of Zoology and Fisheries Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Czechia

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Alippi AM. A comparison of laboratory techniques for the detection of significant bacteria of the honey bee, Apis mellifera, in Argentina. Journal of Apicultural Research. 1991;30(2):75–80. doi: 10.1080/00218839.1991.11101237. DOI

Alippi AM, Reynaldi FJ. Inhibition of the growth of Paenibacillus larvae, the causal agent of American foulbrood of honeybees, by selected strains of aerobic spore-forming bacteria isolated from apiarian sources. Journal of Invertebrate Pathology. 2006;91(3):141–146. doi: 10.1016/j.jip.2005.12.002. PubMed DOI

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research. 1997;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. PubMed DOI PMC

Anderson MJ, Ellingsen KE, McArdle BH. Multivariate dispersion as a measure of beta diversity. Ecology Letters. 2006;9(6):683–693. doi: 10.1111/j.1461-0248.2006.00926.x. PubMed DOI

Arai R, Tominaga K, Wu M, Okura M, Ito K, Okamura N, Onishi H, Osaki M, Sugimura Y, Yoshiyama M, Takamatsu D. Diversity of Melissococcus plutonius from honeybee larvae in Japan and experimental reproduction of European foulbrood with cultured atypical isolates. PLOS ONE. 2012;7(3):e33708. doi: 10.1371/journal.pone.0033708. PubMed DOI PMC

Audisio MC, Torres MJ, Sabate DC, Ibarguren C, Apella MC. Properties of different lactic acid bacteria isolated from Apis mellifera L. bee-gut. Microbiological Research. 2011;166(1):1–13. doi: 10.1016/j.micres.2010.01.003. PubMed DOI

Bailey L. Aetiology of European foulbrood; a disease of the larval honeybee. Nature. 1956;178(4542):1130–1130. doi: 10.1038/1781130a0. DOI

Bailey L. European foul brood: a disease of the larval honeybee (Apis mellifera L.) caused by a combination of Streptococcus pluton (Bacillus pluton White) and Bacterium eurydice White. Nature. 1957a;180(4596):1214–1215. doi: 10.1038/1801214a0. DOI

Bailey L. The isolation and cultural characteristics of Streptococcus pluton and further observations on Bacterium eurydice. Journal of General Microbiology. 1957b;17(1):39–48. doi: 10.1099/00221287-17-1-39. PubMed DOI

Bailey L. An improved method for the isolation of Streptococcus pluton, and observations on its distribution and ecology. Journal of Insect Pathology. 1959;1(1):80–85.

Bailey L. The epizootiology of European foulbrood of the larval honey bee, Apis mellifera Linnaeus. Journal of Insect Pathology. 1960;2(2):67–83.

Bailey L. The pathogenicity for honey-bee larvae of microorganisms associated with European foulbrood. Journal of Insect Pathology. 1963;5(2):198–205.

Bailey L. Melissococcus pluton, the cause of European foulbrood of honey bees (Apis spp.) Journal of Applied Bacteriology. 1983;55(1):65–69. doi: 10.1111/j.1365-2672.1983.tb02648.x. DOI

Bailey L. Melissococcus pluton and European foul brood. Bee World. 1985;66(4):134–136. doi: 10.1080/0005772x.1985.11098843. DOI

Bailey L, Ball BV. Honey bee pathology. 2nd Edition Academic Press; London: 1991.

Bailey L, Collins MD. Reclassification of ‘Streptococcus pluton’ (White) in a new genus Melissococcus, as Melissococcus pluton nom. rev.; comb. nov. Journal of Applied Bacteriology. 1982;53(2):215–217. doi: 10.1111/j.1365-2672.1982.tb04679.x. DOI

Belloy L, Imdorf A, Fries I, Forsgren E, Berthoud H, Kuhn R, Charriere J-D. Spatial distribution of Melissococcus plutonius in adult honey bees collected from apiaries and colonies with and without symptoms of European foulbrood. Apidologie. 2007;38(2):136–140. doi: 10.1051/apido:2006069. DOI

Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I, Lipman DJ, Ostell J, Sayers EW. GenBank. Nucleic Acids Research. 2013;41(Database issue):D36–D42. doi: 10.1093/nar/gks1195. PubMed DOI PMC

Borcard D, Gillet F, Legendre P. Numerical ecology with R. Springer; New York: 2011.

Bottacini F, Milani C, Turroni F, Sanchez B, Foroni E, Duranti S, Serafini F, Viappiani A, Strati F, Ferrarini A, Delledonne M, Henrissat B, Coutinho P, Fitzgerald GF, Margolles A, Van Sinderen D, Ventura M. Bifidobacterium asteroides PRL2011 genome analysis reveals clues for colonization of the insect gut. PLOS ONE. 2012;7(9):e44229. doi: 10.1371/journal.pone.0044229. PubMed DOI PMC

Budge GE, Barrett B, Jones B, Pietravalle S, Marris G, Chantawannakul P, Thwaites R, Hall J, Cuthbertson AGS, Brown MA. The occurrence of Melissococcus plutonius in healthy colonies of Apis mellifera and the efficacy of European foulbrood control measures. Journal of Invertebrate Pathology. 2010;105(2):164–170. doi: 10.1016/j.jip.2010.06.004. PubMed DOI

Budge GE, Jones B, Powell M, Anderson L, Laurenson L, Pietravalle S, Marris G, Haynes E, Thwaites R, Bew J, Wilkins S, Brown M. Recent advances in our understanding of European foulbrood in England and Wales. In: Jensen AB, Forsgren E, Genersch E, editors. Proceedings of the COLOSS workshop: the future of brood disease research—guidelines, methods and development, Copenhagen, Denmark, April 10–12, 2011; Bern. 2011. p. 7.

Budge GE, Shirley MD, Jones B, Quill E, Tomkies V, Feil EJ, Brown MA, Haynes EG. Molecular epidemiology and population structure of the honey bee brood pathogen Melissococcus plutonius. ISME Journal. 2014;8(8):1588–1597. doi: 10.1038/ismej.2014.20. PubMed DOI PMC

Cariveau DP, Powell JE, Koch H, Winfree R, Moran NA. Variation in gut microbial communities and its association with pathogen infection in wild bumble bees (Bombus) ISME Journal. 2014a;8(12):2369–2379. doi: 10.1038/ismej.2014.68. PubMed DOI PMC

Cariveau DP, Powell JE, Koch H, Winfree R, Moran NA. Erratum to “Variation in gut microbial communities and its association with pathogen infection in wild bumble bees (Bombus) [ISME J. 8(12):2369–2379 (2014)] ISME Journal. 2014b;8(12):2550–2551. doi: 10.1038/ismej.2014.180. PubMed DOI PMC

Chao A, Chazdon RL, Colwell RK, Shen T-J. A new statistical approach for assessing similarity of species composition with incidence and abundance data. Ecology Letters. 2005;8(2):148–159. doi: 10.1111/j.1461-0248.2004.00707.x. DOI

Chiodini RJ, Dowd SE, Chamberlin WM, Galandiuk S, Davis B, Glassing A. Microbial population differentials between mucosal and submucosal intestinal tissues in advanced Crohn’s disease of the ileum. PLOS ONE. 2015;10(7):e0134382. doi: 10.1371/journal.pone.0134382. PubMed DOI PMC

Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, Brown CT, Porras-Alfaro A, Kuske CR, Tiedje JM. Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Research. 2014;42(Database issue):D633–D642. doi: 10.1093/nar/gkt1244. PubMed DOI PMC

Dahle B, Sorum H, Weideman JE. European foulbrood in Norway: how to address a major outbreak after 30 years absence. In: Jensen AB, Forsgren E, Genersch E, editors. Proceedings of the COLOSS workshop: the future of brood disease research—guidelines, methods and development, Copenhagen, Denmark, April 10–12, 2011; Bern. 2011. p. 9.

Dietemann V, Ellis JD, Neumann P. The COLOSS BeeBook. Vol. 2—Standard methods for Apis mellifera pest and pathogen research. Bern: COLOSS (Prevention of honey bee COlony LOSSes); 2013.

Djordjevic SP, Noone K, Smith L, Hornitzky MAZ. Development of a hemi-nested PCR assay for the specific detection of Melissococcus pluton. Journal of Apicultural Research. 1998;37(3):165–174. doi: 10.1080/00218839.1998.11100968. DOI

Dray S, Legendre P, Blanchet G. packfor: forward selection with permutation (Canoco p. 46). R-Forge, The R Project for Statistical Computing. http://R-Forge.R-project.org/projects/sedar/ [6 August 2016];2013

Edgar RC. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nature Methods. 2013;10(10):996–998. doi: 10.1038/nmeth.2604. PubMed DOI

Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics. 2011;27(16):2194–2200. doi: 10.1093/bioinformatics/btr381. PubMed DOI PMC

Emery O, Schmidt K, Engel P. Immune system stimulation by the gut symbiont Frischella perrara in the honey bee (Apis mellifera) Molecular Ecology. 2017;26(9):2576–2590. doi: 10.1111/mec.14058. PubMed DOI

Endo A, Salminen S. Honeybees and beehives are rich sources for fructophilic lactic acid bacteria. Systematic and Applied Microbiology. 2013;36(6):444–448. doi: 10.1016/j.syapm.2013.06.002. PubMed DOI

Engel P, James RR, Koga R, Kwong WK, McFrederick QS, Moran NA. Standard methods for research on Apis mellifera gut symbionts. Journal of Apicultural Research. 2013;52:UNSP 52.4.07

Engel P, Kwong WK, McFrederick Q, Anderson KE, Barribeau SM, Chandler JA, Cornman RS, Dainat J, De Miranda JR, Doublet V, Emery O, Evans JD, Farinelli L, Flenniken ML, Granberg F, Grasis JA, Gauthier L, Hayer J, Koch H, Kocher S, Martinson VG, Moran N, Munoz-Torres M, Newton I, Paxton RJ, Powell E, Sadd BM, Schmid-Hempel P, Schmid-Hempel R, Song SJ, Schwarz RS, VanEngelsdorp D, Dainat B. The bee microbiome: impact on bee health and model for evolution and ecology of host-microbe interactions. mBio. 2016;7(2):e02164-15. doi: 10.1128/mBio.02164-15. PubMed DOI PMC

Erban T, Ledvinka O, Kamler M, Nesvorna M, Hortova B, Tyl J, Titera D, Markovic M, Hubert J. Honeybee (Apis mellifera)—associated bacterial community affected by American foulbrood: detection of Paenibacillus larvae via microbiome analysis. Scientific Reports. 2017a;7(1):5084. doi: 10.1038/s41598-017-05076-8. PubMed DOI PMC

Erban T, Ledvinka O, Nesvorna M, Hubert J. Experimental manipulation shows a greater influence of population than dietary perturbation on the microbiome of Tyrophagus putrescentiae. Applied and Environmental Microbiology. 2017b;83(9):e00128-17. doi: 10.1128/AEM.00128-17. PubMed DOI PMC

Erler S, Lewkowski O, Poehlein A, Forsgren E. The curious case of Achromobacter eurydice, a Gram-variable pleomorphic bacterium associated with European foulbrood disease in honeybees. Microbial Ecology. 2017 doi: 10.1007/s00248-017-1007-x. In Press. PubMed DOI

Evans JD, Armstrong T-N. Antagonistic interactions between honey bee bacterial symbionts and implications for disease. BMC Ecology. 2006;6:4. doi: 10.1186/1472-6785-6-4. PubMed DOI PMC

Forfert N, Natsopoulou ME, Frey E, Rosenkranz P, Paxton RJ, Moritz RFA. Parasites and pathogens of the honeybee (Apis mellifera) and their influence on inter-colonial transmission. PLOS ONE. 2015;10(10):e0140337. doi: 10.1371/journal.pone.0140337. PubMed DOI PMC

Forsgren E. European foulbrood in honey bees. Journal of Invertebrate Pathology. 2010;103(Supplement 1):S5–S9. doi: 10.1016/j.jip.2009.06.016. PubMed DOI

Forsgren E, Budge GE, Charriere J-D, Hornitzky MAZ. Standard methods for European foulbrood research. Journal of Apicultural Research. 2013;52(1):52.1.12. doi: 10.3896/IBRA.1.52.1.12. DOI

Forsgren E, Lundhagen AC, Imdorf A, Fries I. Distribution of Melissococcus plutonius in honeybee colonies with and without symptoms of European foulbrood. Microbial Ecology. 2005;50(3):369–374. doi: 10.1007/s00248-004-0188-2. PubMed DOI

Fries I, Camazine S. Implications of horizontal and vertical pathogen transmission for honey bee epidemiology. Apidologie. 2001;32(3):199–214. doi: 10.1051/apido:2001122. DOI

Gaggia F, Baffoni L, Stenico V, Alberoni D, Buglione E, Lilli A, Di Gioia D, Porrini C. Microbial investigation on honey bee larvae showing atypical symptoms of European foulbrood. Bulletin of Insectology. 2015;68(2):321–327.

Garrido-Bailon E, Higes M, Martinez-Salvador A, Antunez K, Botias C, Meana A, Prieto L, Martin-Hernandez R. The prevalence of the honeybee brood pathogens Ascosphaera apis, Paenibacillus larvae and Melissococcus plutonius in Spanish apiaries determined with a new multiplex PCR assay. Microbial Biotechnology. 2013;6(6):731–739. doi: 10.1111/1751-7915.12070. PubMed DOI PMC

Govan VA, Brozel V, Allsopp MH, Davison S. A PCR detection method for rapid identification of Melissococcus pluton in honeybee larvae. Applied and Environmental Microbiology. 1998;64(5):1983–1985. PubMed PMC

Hammer O, Harper DAT, Ryan PD. PAST: paleontological statistics software package for education and data analysis. Palaeontologia Electronica. 2001;4(1):4.

Haynes E, Helgason T, Young JPW, Thwaites R, Budge GE. A typing scheme for the honeybee pathogen Melissococcus plutonius allows detection of disease transmission events and a study of the distribution of variants. Environmental Microbiology Reports. 2013;5(4):525–529. doi: 10.1111/1758-2229.12057. PubMed DOI

Hornitzky MAZ, Smith LA. Sensitivity of Australian Melissococcus pluton isolates to oxytetracycline hydrochloride. Australian Journal of Experimental Agriculture. 1999;39(7):881–883. doi: 10.1071/EA99064. DOI

Hornitzky MAZ, Wilson SC. A system for the diagnosis of the major bacterial brood diseases of honeybees. Journal of Apicultural Research. 1989;28(4):191–195. doi: 10.1080/00218839.1989.11101183. DOI

Hubert J, Bicianova M, Ledvinka O, Kamler M, Lester PJ, Nesvorna M, Kopecky J, Erban T. Changes in the bacteriome of honey bees associated with the parasite Varroa destructor, and pathogens Nosema and Lotmaria passim. Microbial Ecology. 2017;73(3):685–698. doi: 10.1007/s00248-016-0869-7. PubMed DOI

Hubert J, Kamler M, Nesvorna M, Ledvinka O, Kopecky J, Erban T. Comparison of Varroa destructor and worker honeybee microbiota within hives indicates shared bacteria. Microbial Ecology. 2016;72(2):448–459. doi: 10.1007/s00248-016-0776-y. PubMed DOI

Kakumanu ML, Reeves AM, Anderson TD, Rodrigues RR, Williams MA. Honey bee gut microbiome is altered by in-hive pesticide exposures. Frontiers in Microbiology. 2016;7 doi: 10.3389/fmicb.2016.01255. Article 1255. PubMed DOI PMC

Kamler M, Tyl J, Nesvorna M, Hubert J, Merta J, Karesova B, Titera D. The European foulbrood—rediscovered infection of honeybee in the Czech Republic. [Hniloba vceliho plodu - znovuobjevena infekce vcelstev v Ceske republice] Veterinarstvi. 2016;66(6):435–438. [in Czech with English summary]

Killer J, Dubna S, Sedlacek I, Svec P. Lactobacillus apis sp. nov., from the stomach of honeybees (Apis mellifera), having an in vitro inhibitory effect on the causative agents of American and European foulbrood. International Journal of Systematic and Evolutionary Microbiology. 2014;64(1):152–157. doi: 10.1099/ijs.0.053033-0. PubMed DOI

Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Applied and Environmental Microbiology. 2013;79(17):5112–5120. doi: 10.1128/AEM.01043-13. PubMed DOI PMC

Kutner MH, Nachtsheim CJ, Neter J, Li W. Applied linear statistical models. 5th Edition McGraw-Hill Irwin; Boston: 2005.

Kuzysinova K, Mudronova D, Toporcak J, Molnar L, Javorsky P. The use of probiotics, essential oils and fatty acids in the control of American foulbrood and other bee diseases. Journal of Apicultural Research. 2016;55(5):386–395. doi: 10.1080/00218839.2016.1252067. DOI

KVSH Specific veterinary precautions in the course of incidence of the dangerous European foulbrood disease in breeding of honeybees on the territory of the Hradec Kralove Region, to prevent spread and overcome the outbreak. 2015. [6 August 2016]. Regulation of the State Veterinary Administration of the Czech Republic, ref. no. SVS/2015/084740-H, 19th August 2015. [Mimoradna veterinarni opatreni pri vyskytu nebezpecne nakazy hniloba vceliho plodu v chovech vcel v regionu Kralovehradeckeho kraje, k zamezeni jejiho sireni a k jejimu zdolani. Narizeni Statni veterinarni spravy, c. j. SVS/2015/084740-H, 19. 8. 2015.] Hradec Kralove, Czechia: Krajska veterinarni sprava Statni veterinarni spravy pro Kralovehradecky kraj (KVSH) [in Czech]. http://eagri.cz/public/web/file/417238/Narizeni_MVO_hniloba_vceliho_plodu_c.j._SVS_2015_084740_H.pdf .

Kwong WK, Moran NA. Gut microbial communities of social bees. Nature Reviews Microbiology. 2016;14(6):374–384. doi: 10.1038/nrmicro.2016.43. PubMed DOI PMC

Lane DJ. 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M, editors. Nucleic acid techniques in bacterial systematics. John Wiley and Sons; Chichester: 1991. pp. 115–175.

Legendre P, Legendre L. Numerical ecology. 3rd Edition Elsevier; Amsterdam: 2012.

Lee FJ, Rusch DB, Stewart FJ, Mattila HR, Newton ILG. Saccharide breakdown and fermentation by the honey bee gut microbiome. Environmental Microbiology. 2015;17(3):796–815. doi: 10.1111/1462-2920.12526. PubMed DOI

MA CR Decree No 299/2003 Coll., on measures for prevention and eradication of epizooties and zoonoses. Collection of Laws of the Czech Republic, part 102/2003, 17th September 2003. [Vyhlaska c. 299/2003 Sb, o opatrenich pro predchazeni a zdolavani nakaz a nemoci prenosnych ze zvirat na cloveka] 2003. [6 August 2016]. Prague: Ministry of Agriculture of the Czech Republic [in Czech]. http://eagri.cz/public/web/mze/legislativa/pravni-predpisy-mze/tematicky-prehled/Legislativa-MZe_uplna-zneni_Vyhlaska-2003-299-veterinarnipece.html .

MA CR Decree No 72/2013 Coll., which modifies the decree No 299/2003 Coll. on measures for prevention and eradication of epizooties and zoonoses, as amended. Collection of Laws of the Czech Republic, part 34/2013, 25th March 2013. [Vyhlaska c. 72/2013 Sb, kterou se meni vyhlaska c. 299/2003 Sb, o opatrenich pro predchazeni a zdolavani nakaz a nemoci prenosnych ze zvirat na cloveka, ve zneni pozdejsich predpisu] 2013. [6 August 2016]. Prague: Ministry of Agriculture of the Czech Republic [in Czech]. http://eagri.cz/public/web/mze/legislativa/pravni-predpisy-mze/tematicky-prehled/Legislativa-MZe_uplna-zneni_vyhlaska-2013-72-novela-299-2003.html .

McKee BA, Djordjevic SP, Goodman RD, Hornitzky MA. The detection of Melissococcus pluton in honey bees (Apis mellifera) and their products using a hemi-nested PCR. Apidologie. 2003;34(1):19–27. doi: 10.1051/apido:2002047. DOI

McKee BA, Goodman RD, Hornitzky MA. The transmission of European foulbrood (Melissococcus plutonius) to artificially reared honey bee larvae (Apis mellifera) Journal of Apicultural Research. 2004;43(3):93–100. doi: 10.1080/00218839.2004.11101117. DOI

Moran NA. Genomics of the honey bee microbiome. Current Opinion in Insect Science. 2015;10:22–28. doi: 10.1016/j.cois.2015.04.003. PubMed DOI PMC

Nakamura K, Yamazaki Y, Shiraishi A, Kobayashi S, Harada M, Yoshiyama M, Osaki M, Okura M, Takamatsu D. Virulence differences among Melissococcus plutonius strains with different genetic backgrounds in Apis mellifera larvae under an improved experimental condition. Scientific Reports. 2016;6:33329. doi: 10.1038/srep33329. PubMed DOI PMC

OIE . Manual of diagnostic tests and vaccines for terrestrial animals: mammals, birds, and bees. Paris: OIE—World Organisation for Animal Health; 2008. Chapter 2.2.3: European foulbrood of honey bees.

OIE . Manual of diagnostic tests and vaccines for terrestrial animals 2016. Paris: OIE—World Organisation for Animal Health; 2016a. Chapter 2.2.3: European foulbrood of honey bees (infection of honey bees with Melissococcus plutonius)

OIE OIE-Listed diseases, infections and infestations in force in 2016. 2016b. [6 August 2016]. Paris: OIE—World Organisation for Animal Health. http://www.oie.int/en/animal-health-in-the-world/oie-listed-diseases-2016/

Oksanen J, Blanchet FG, Kindt R, Legendre P, Minchin PR, O’Hara RB, Simpson GL, Solymos P, Stevens MHH, Wagner H. vegan: Community Ecology Package. CRAN—The Comprehensive R Archive Network. http://CRAN.R-project.org/package=vegan. [6 August 2016];2016

Olofsson TC, Vasquez A. Detection and identification of a novel lactic acid bacterial flora within the honey stomach of the honeybee Apis mellifera. Current Microbiology. 2008;57(4):356–363. doi: 10.1007/s00284-008-9202-0. PubMed DOI

Ondov BD, Bergman NH, Phillippy AM. Interactive metagenomic visualization in a web browser. BMC Bioinformatics. 2011;12:385. doi: 10.1186/1471-2105-12-385. PubMed DOI PMC

Pinnock DE, Featherstone NE. Detection and quantification of Melissococcus pluton infection in honeybee colonies by means of enzyme-linked immunosorbent assay. Journal of Apicultural Research. 1984;23(3):168–170. doi: 10.1080/00218839.1984.11100627. DOI

Poltev VI. Diseases of bees. [Bolezni pchel.] 3rd Edition. Sel’khozgiz; Moscow & Leningrad: 1950. [in Russian]

QGIS Development Team . Beaverton: Open Source Geospatial Foundation (OSGeo); 2009. [19 June 2017].

Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glockner FO. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Research. 2013;41(Database issue):D590–D596. doi: 10.1093/nar/gks1219. PubMed DOI PMC

Rada V, Machova M, Huk J, Marounek M, Duskova D. Microflora in the honeybee digestive tract: counts, characteristics and sensitivity to veterinary drugs. Apidologie. 1997;28(6):357–365. doi: 10.1051/apido:19970603. DOI

Raymann K, Shaffer Z, Moran NA. Antibiotic exposure perturbs the gut microbiota and elevates mortality in honeybees. PLOS Biology. 2017;15(3):e2001861. doi: 10.1371/journal.pbio.2001861. PubMed DOI PMC

Roetschi A, Berthoud H, Kuhn R, Imdorf A. Infection rate based on quantitative real-time PCR of Melissococcus plutonius, the causal agent of European foulbrood, in honeybee colonies before and after apiary sanitation. Apidologie. 2008;39(3):362–371. doi: 10.1051/apido:200819. DOI

Schloss PD. Evaluating different approaches that test whether microbial communities have the same structure. ISME Journal. 2008;2(3):265–275. doi: 10.1038/ismej.2008.5. PubMed DOI

Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Applied and Environmental Microbiology. 2009;75(23):7537–7541. doi: 10.1128/AEM.01541-09. PubMed DOI PMC

Spivak M, Reuter GS. Resistance to American foulbrood disease by honey bee colonies Apis mellifera bred for hygienic behavior. Apidologie. 2001;32(6):555–565. doi: 10.1051/apido:2001103. DOI

Stewart Jr CN, Excoffier L. Assessing population genetic structure and variability with RAPD data: application to Vaccinium macrocarpon (American cranberry) Journal of Evolutionary Biology. 1996;9(2):153–171. doi: 10.1046/j.1420-9101.1996.9020153.x. DOI

Takamatsu D, Morinishi K, Arai R, Sakamoto A, Okura M, Osaki M. Typing of Melissococcus plutonius isolated from European and Japanese honeybees suggests spread of sequence types across borders and between different Apis species. Veterinary Microbiology. 2014;171(1–2):221–226. doi: 10.1016/j.vetmic.2014.03.036. PubMed DOI

Takamatsu D, Sato M, Yoshiyama M. Infection of Melissococcus plutonius clonal complex 12 strain in European honeybee larvae is essentially confined to the digestive tract. Journal of Veterinary Medical Science. 2016;78(1):29–34. doi: 10.1292/jvms.15-0405. PubMed DOI PMC

Tarr HLA. Studies on European foul brood of bees IV. On the attempted cultivation of Bacillus Pluton, the susceptibility of individual larvae to inoculation with this organism and its localization within its host. Annals of Applied Biology. 1938;25(4):815–821. doi: 10.1111/j.1744-7348.1938.tb02356.x. DOI

Tomkies V, Flint J, Johnson G, Waite R, Wilkins S, Danks C, Watkins M, Cuthbertson AGS, Carpana E, Marris G, Budge G, Brown MA. Development and validation of a novel field test kit for European foulbrood. Apidologie. 2009;40(1):63–72. doi: 10.1051/apido:2008060. DOI

Tozkar CO, Kence M, Kence A, Huang Q, Evans JD. Metatranscriptomic analyses of honey bee colonies. Frontiers in Genetics. 2015;6 doi: 10.3389/fgene.2015.00100. Article 100. PubMed DOI PMC

Truper HG, De’ Clari L. Taxonomic note: erratum and correction of further specific epithets formed as substantives (nouns) ‘in apposition’. International Journal of Systematic Bacteriology. 1998;48(2):615–615. doi: 10.1099/00207713-48-2-615. DOI

Vasquez A, Forsgren E, Fries I, Paxton RJ, Flaberg E, Szekely L, Olofsson TC. Symbionts as major modulators of insect health: lactic acid bacteria and honeybees. PLOS ONE. 2012;7(3):e33188. doi: 10.1371/journal.pone.0033188. PubMed DOI PMC

Waite R, Brown M, Thompson H. Hygienic behavior in honey bees in the UK: a preliminary study. Bee World. 2003;84(1):19–26. doi: 10.1080/0005772X.2003.11099567. DOI

Warnes GR, Bolker B, Bonebakker L, Gentleman R, Huber W, Liaw A, Lumley T, Maechler M, Magnusson A, Moeller S, Schwartz M, Venables B. gplots: Various R programming tools for plotting data. CRAN—The Comprehensive R Archive Network. https://CRAN.R-project.org/package=gplots. [6 August 2016];2016

White GF. The cause of European foul brood. Circular No. 157. Washington D.C.: U. S. Department of Agriculture, Bureau of Entomology; 1912.

Wilkins S, Brown MA, Cuthbertson AGS. The incidence of honey bee pests and diseases in England and Wales. Pest Management Science. 2007;63(11):1062–1068. doi: 10.1002/ps.1461. PubMed DOI

Wu M, Sugimura Y, Iwata K, Takaya N, Takamatsu D, Kobayashi M, Taylor D, Kimura K, Yoshiyama M. Inhibitory effect of gut bacteria from the Japanese honey bee, Apis cerana japonica, against Melissococcus plutonius, the causal agent of European foulbrood disease. Journal of Insect Science. 2014;14 doi: 10.1093/jis/14.1.129. Article 129. PubMed DOI PMC

Zheng H, Nishida A, Kwong WK, Koch H, Engel P, Steele MI, Moran NA. Metabolism of toxic sugars by strains of the bee gut symbiont Gilliamella apicola. mBio. 2016;7(6):e01326-16. doi: 10.1128/mBio.01326-16. PubMed DOI PMC

Screening of Honey Bee Pathogens in the Czech Republic and Their Prevalence in Various Habitats

Melissococcus plutonius Can Be Effectively and Economically Detected Using Hive Debris and Conventional PCR

Honeybee (Apis mellifera)-associated bacterial community affected by American foulbrood: detection of Paenibacillus larvae via microbiome analysis