European Foulbrood Research Articles

Wild bees and their nests host Paenibacillus bacteria with functional potential of avail

BackgroundIn previous studies, the gram-positive firmicute genus Paenibacillus was found with significant abundances in nests of wild solitary bees. Paenibacillus larvae is well-known for beekeepers as a severe pathogen causing the fatal honey bee disease American foulbrood, and other members of the genus are either secondary invaders of European foulbrood or considered a threat to honey bees. We thus investigated whether Paenibacillus is a common bacterium associated with various wild bees and hence poses a latent threat to honey bees visiting the same flowers.ResultsWe collected 202 samples from 82 individuals or nests of 13 bee species at the same location and screened each for Paenibacillus using high-throughput sequencing-based 16S metabarcoding. We then isolated the identified strain Paenibacillus MBD-MB06 from a solitary bee nest and sequenced its genome. We did find conserved toxin genes and such encoding for chitin-binding proteins, yet none specifically related to foulbrood virulence or chitinases. Phylogenomic analysis revealed a closer relationship to strains of root-associated Paenibacillus rather than strains causing foulbrood or other accompanying diseases. We found anti-microbial evidence within the genome, confirmed by experimental bioassays with strong growth inhibition of selected fungi as well as gram-positive and gram-negative bacteria.ConclusionsThe isolated wild bee associate Paenibacillus MBD-MB06 is a common, but irregularly occurring part of wild bee microbiomes, present on adult body surfaces and guts and within nests especially in megachilids. It was phylogenetically and functionally distinct from harmful members causing honey bee colony diseases, although it shared few conserved proteins putatively toxic to insects that might indicate ancestral predisposition for the evolution of insect pathogens within the group. By contrast, our strain showed anti-microbial capabilities and the genome further indicates abilities for chitin-binding and biofilm-forming, suggesting it is likely a useful associate to avoid fungal penetration of the bee cuticula and a beneficial inhabitant of nests to repress fungal threats in humid and nutrient-rich environments of wild bee nests.

Apis cerana Beekeeping and Sacbrood Disease Management in Vietnam: Review

Beekeeping status of Apis cerana with emphasis of experiences overcoming sacbrood virus disease are presented. Social bee fauna are rich in Vietnam with 6 honeybee species (Apis laboriosa, Apis dorsata, Apis mellifera, Apis cerana, Apis andrenifomis, Apis florea); 8 stingless bee species (Trigona laeviceps, Trigona ventralis, Trigona pagdeni, Trigona gressitti, Trigona fuscobalteata, Trigona capenteri, Trigona scintillans Trigona iridipenis) and 2 bumble bee species (Bumbus haemorrhoidalis, B. breviceps). All of them are native except A. mellifera which was introduced in1887. These bees are slated for conservation by the Ministry of Agriculture & Rural Development. Honey and other bee products are mainly harvested from 3 species including A. cerana, A. mellifera and A. dorsata. The manageable species (A. cerana and A. mellifera) are increasing in number, reaching about 1,500,000 beehives. Vietnam is the second largest honey exporter in Asia, with a total of about 48,000 tons of honey exported to the international market in 2014. A. cerana plays an important role in poverty alleviation in mountainous and remote areas of Vietnam. Honeybee suffers from various diseases of Sacbrood virus disease (SBV), European foulbrood (EFB), Nosema, and parasitic mites of Tropilaelaps mercedes and Varroa destructor. Most of these diseases can be resolved with biocontrol methods. For the parasitic mites, Vietnamese beekeepers usually apply formic acid.

Quality Control of Bee-Collected Pollen Using Bumblebee Microcolonies and Molecular Approaches Reveals No Correlation Between Pollen Quality and Pathogen Presence

Bee-collected pollen is an essential protein source for honey bee and bumblebee colonies. Its quality directly affects bee health. We estimated the quality of pollen samples using bumblebee microcolonies and high-throughput sequencing for the presence of microorganisms. The tested samples of bee-collected pollen were of different quality, as estimated from their effect on the development of bumblebee microcolonies. Based on the pollen quality, we selected a subset of high-quality and low-quality pollen samples to further analyze them for the presence of microorganisms and pathogens. High-throughput sequencing revealed that the most common microorganisms in the bee-collected pollen were Acinetobacter spp. and bacteria of the genera Lactobacillus and Lactococcus. No pathogenic bacteria infectious for honey bees (e.g., those causing American and European foulbrood) or bumblebees have been identified in the analyzed pollen samples. Among potentially harmful microorganisms, there were bacteria from the Enterobacteriaceae family. The fungal pathogens Nosema apis and Nosema ceranae were detected in four samples; Ascosphaera sp. was found in six samples. Several viruses were found in the pollen samples, such as chronic bee paralysis virus, Israeli acute paralysis virus, deformed wing virus, sacbrood virus, and Kashmir bee virus. No correlation between the presence of these microorganisms or viruses and the impact of low-quality pollen samples on the bumblebee development was found. It is possible that factors affecting pollen quality are the absence of certain biologically active compounds or the presence of pesticides.

Prevalence, typing and phylogenetic analysis of Melissococcus plutonius strains from bee colonies of the State of Chihuahua, Mexico.

European foulbrood (EFB) caused by Melissococcus plutonius is an important bee brood disease but, in Mexico, information about this bacterium is limited. We evaluated the prevalence of typical and atypical strains in beehives of seven apicultural regions of the state of Chihuahua, Mexico. We performed MLST and phylogenetic analysis to characterize the isolates. Prevalence was highest 59%, in the region of Chihuahua, and lowest, 14%, in the regions of Cuauhtémoc and Nuevo Casas Grandes. Typical and atypical strains were identified in hives from all regions; however, in the regions of Parral, Cuauhtémoc and Aldama, the atypical strains were only detected in combination with typical strains. We obtained 81 isolates of M. plutonius and identified seven sequence types, of which three were new types. Additionally, we observed a relation between sequence type and the region where the strain was isolated. Phylogenetic analysis and multilocus sequence typing using goeBURST analysis showed that 97.5% of the isolates correspond to the Clonal Complex (CC) 12 and 2.5% to the CC3. Our work is the first molecular characterization of M. plutonius in Mexico and contributes to global information about the epidemiology of this pathogen.

The terpenes of leaves, pollen, and nectar of thyme (Thymus vulgaris) inhibit growth of bee disease-associated microbes

Honey bees are highly prone to infectious diseases, causing colony losses in the worst case. However, they combat diseases through a combination of their innate immune system and social defence behaviours like foraging for health-enhancing plant products (e.g. nectar, pollen and resin). Plant secondary metabolites are not only highly active against bacteria and fungi, they might even enhance selective foraging and feeding decisions in the colony. Here, we tested six major plant terpenes and their corresponding acetates, characterizing six natural Thymus vulgaris chemotypes, for their antimicrobial activity on bacteria associated with European foulbrood. Comparison of the inhibitory activity revealed the highest activity for carvacrol and thymol whereas the acetates mostly did not inhibit bacterial growth. All terpenes and acetates are present in the nectar and pollen of thyme, with pollen containing concentrations higher by several orders of magnitude. The physiological response was tested on forager and freshly emerged bees by means of antennal electroantennography. Both responded much stronger to geraniol and trans-sabinene hydrate compared to carvacrol and thymol. In conclusion, bee-forageable thyme product terpenes (mainly from pollen) yield effective antibiotic activity by reducing the growth of bee disease-associated bacteria and can be detected with different response levels by the honey bees’ antennae. This is a further step forward in understanding the complex pathogen-pollinator-plant network.

Help! There's a funny odour coming from my hive

While not all bee diseases are accompanied by a foul smell, a strange foul smell coming from a bee hive probably indicates a serious problem. This article describes the investigation of such a hive and the steps to be taken following detection of either European foulbrood or American foulbrood, including informing the local bee inspector or Defra, and practical management. It is important that affected hives are not treated with antibiotics in initial management; use of oxytetracycline may be authorised by the Defra inspector on rare occasions for treatment of European foulbrood.

A frameshift mutation in the rRNA large subunit methyltransferase gene rlmA II determines the susceptibility of a honey bee pathogen Melissococcus plutonius to mirosamicin.

American foulbrood (AFB) and European foulbrood (EFB) caused by Paenibacillus larvae and Melissococcus plutonius, respectively, are major bacterial infections of honey bees. Although macrolides (mirosamicin [MRM] and tylosin) have been used to prevent AFB in Japan, macrolide-resistant P. larvae have yet to be found. In this study, we revealed that both MRM-resistant and -susceptible strains exist in Japanese M. plutonius and that a methyltransferase gene (rlmA II ) was disrupted exclusively in MRM-susceptible strains due to a single-nucleotide insertion. The M. plutonius RlmAII modified G748 of 23S rRNA, and the deletion of rlmA II resulted in increased susceptibility to MRM and the loss of modification at G748, suggesting that methylation at G748 by RlmAII confers MRM resistance in M. plutonius. The single-nucleotide mutation in MRM-susceptible strains was easily repaired by spontaneous deletion of the inserted nucleotide; however, intact rlmA II was only found in Japanese M. plutonius and not in a Paraguayan strain tested or any of the whole-genome-sequenced European strains. MRM has been used in apiculture only in Japan. Although M. plutonius is not the target of this drug, the use of MRM as a prophylactic drug for AFB may have influenced the antibiotic susceptibility of the causative agent of EFB.

Comparative Genomics and Description of Putative Virulence Factors of Melissococcus plutonius, the Causative Agent of European Foulbrood Disease in Honey Bees.

In Europe, approximately 84% of cultivated crop species depend on insect pollinators, mainly bees. Apis mellifera (the Western honey bee) is the most important commercial pollinator worldwide. The Gram-positive bacterium Melissococcus plutonius is the causative agent of European foulbrood (EFB), a global honey bee brood disease. In order to detect putative virulence factors, we sequenced and analyzed the genomes of 14 M. plutonius strains, including two reference isolates. The isolates do not show a high diversity in genome size or number of predicted protein-encoding genes, ranging from 2.021 to 2.101 Mbp and 1589 to 1686, respectively. Comparative genomics detected genes that might play a role in EFB pathogenesis and ultimately in the death of the honey bee larvae. These include bacteriocins, bacteria cell surface- and host cell adhesion-associated proteins, an enterococcal polysaccharide antigen, an epsilon toxin, proteolytic enzymes, and capsule-associated proteins. In vivo expression of three putative virulence factors (endo-alpha-N-acetylgalactosaminidase, enhancin and epsilon toxin) was verified using naturally infected larvae. With our strain collection, we show for the first time that genomic differences exist between non-virulent and virulent typical strains, as well as a highly virulent atypical strain, that may contribute to the virulence of M. plutonius. Finally, we also detected a high number of conserved pseudogenes (75 to 156) per genome, which indicates genomic reduction during evolutionary host adaptation.

Complete Genome Sequence of Melissococcus plutonius DAT561, a Strain That Shows an Unusual Growth Profile, Obtained by PacBio Sequencing.

ABSTRACTMelissococcus plutonius is the causative agent of European foulbrood, and its isolates were believed to be remarkably genetically homogeneous. However, recent epidemiological and pathogenic studies have shown this pathogen to be more heterogeneous than expected. Herein, we present the whole-genome sequence of M. plutonius DAT561, a representative atypical strain.

Virulence of Melissococcus plutonius and secondary invaders associated with European foulbrood disease of the honey bee.

European foulbrood is a globally distributed brood disease affecting honey bees. It may lead to lethal infections of larvae and, in severe cases, even to colony collapse. Lately, a profound genetic and phenotypic diversity was documented for the causative agent Melissococcus plutonius. However, experimental work on the impact of diverse M. plutonius strains on hosts with different genetic background is completely lacking and the role of secondary invaders is poorly understood. Here, we address these issues and elucidate the impact and interaction of both host and pathogen on one another. Moreover, we try to unravel the role of secondary bacterial invasions in foulbrood‐diseased larvae. We employed in vitro infections with honey bee larvae from queens with different genetic background and three different M. plutonius strains. Larvae infection experiments showed host‐dependent survival dynamics although M. plutonius strain 49.3 consistently had the highest virulence. This pattern was also reflected in significantly reduced weights of 49.3 strain‐infected larvae compared to the other treatments. No difference was found in groups additionally inoculated with a secondary invader (Enterococcus faecalis or Paenibacillus alvei) neither in terms of larval survival nor weight. These results suggest that host background contributes markedly to the course of the disease but virulence is mainly dependent on pathogen genotype. Secondary invaders following a M. plutonius infection do not increase disease lethality and therefore may just be a colonization of weakened and immunodeficient, or dead larvae.

Molecular detection of Melissococcus plutonius assessed in Africanized honey bee populations (Apis mellifera) in three regions of Colombia

Beekeepers in Colombia are using almost exclusively Africanized hybrids which are considered to be more resistant to diseases and climatic conditions than European-derived honey bee populations. There are no reports of losses of larval and adult bee populations in the country due to infectious diseases. However, there is no official diagnostic system to detect the presence of pathogenic bacteria for Apis mellifera, such as Melissococcus plutonius and Paenibacillus larvae causing European foulbrood and American foulbrood respectively. To assess their presence and prevalence in a large sample sets, representatives of beekeeping populations in the country, we conducted a study on 491 of 5400 registered hives located in three regions in Colombia, analyzing larvae samples with conventional and molecular real-time PCR and molecular taxonomy techniques. Out of the two bacteria targeted, only M. plutonius was detected in 7 of the 491(1.4%) larval samples in all three regions in this sampling campaign. There was no evidence of symptoms consistent with European or American foulbrood. Therefore we are updating and confirming the low prevalence of M. plutonius and the no detection of P. larvae in an extensive sampling survey of Africanized bees at three different regions of Colombia. Our results are consistent with the view that africanized hybrid bees are more more resistant to diseases.

Triplex real-time PCR method for the qualitative detection of European and American foulbrood in honeybee

The bacteria Melissococcus plutonius and Paenibacillus larvae, causative agents of respectively European and American foulbrood, damage honeybee health worldwide. Here, we present a specific and sensitive qualitative triplex real-time PCR method to detect simultaneously those microbial agents and a honeybee gene, validated through a study involving 7 laboratories through Europe.

Bacterial Diseases in Honeybees

Purpose of Review American foulbrood (AFB) and European foulbrood (EFB) are widely distributed and highly infectious bacterial diseases of honeybee brood causing colony losses and considerable economic strain on apiculture globally. In this review, we synthesize the most recent discoveries and achievements made towards understanding the pathogenesis and epidemiology of these two bacterial diseases and present current efforts in finding ways to combat them. Recent Findings Advancements in molecular methods, such as next-generation sequencing, have provided high-resolution insight into the epidemiological parameters and factors of virulence for the foulbroods of honeybees. Summary The recently gained detailed knowledge of the diversity, biogeography, and relatedness of strains and sub-types of the causative bacteria of AFB and EFB provides a background to study their epidemiology at many scales. Such information will help provide a more global perspective on honeybee disease epidemiology for an increasingly international beekeeping industry.

Cow urine as a chemotherapeutic in combating european foul brood in honey bee, Apis mellifera L.

Cow urine as a chemotherapeutic in combating european foul brood in honey bee, <i>Apis mellifera</i> L.

In vitro antibacterial activity of macelignan and corosolic acid against the bacterial bee pathogens Paenibacillus larvae and Melissococcus plutonius

Foulbrood disease, which is caused byPaenibacillus larvae(American foulbrood) orMelissococcus plutonius(European foulbrood disease), is a major threat to honeybees (Apis mellifera) worldwide. Tetracycline derivatives have been used to control these bacteria, but resistant strains have evolved, and the antibiotic derivatives can adversely affect bee health. When foulbrood disease is discovered, beekeepers usually burn the bee hives and equipment. The aim of this study was to investigate thein vitrosusceptibility ofP. larvaeandM. plutoniusto new antibacterial agents. Antibacterial activities of seven compounds prepared as serial two-fold dilutions were assayed using 96-well microtitre plates. Minimum inhibitory concentration values were obtained after 24 h or 48 h of incubation. Antibacterial synergistic activity of tetracycline and the test compounds was evaluated using broth micro-dilution assays with two-fold serial dilutions of the compounds. Among the seven compounds tested, macelignan and corosolic acid showed the strongest anti-bacterial activity. In addition, tetracycline interacted synergistically with corosolic acid to reduceP. larvaeandM. plutoniusgrowth. Even though macelignan and corosolic acid were worth as solely effective agents to treatP. larvaeandM. plutonius, combinatorial treatment with tetracycline would be more useful to overcome toxicity, resistance occurrence and costliness. Further validation studies of these compounds and identification of their targets, as well as actual field tests and bee toxicity studies are still needed. However, macelignan and corosolic acid as natural secondary metabolites would be effective agents for bee foulbrood disease with valuable antibacterial activities.

Biocidal properties of maltose reduced silver nanoparticles against American foulbrood diseases pathogens.

Bee disease caused by spore-forming Paenibacillus larvae and Paenibacillus alvei is a serious problem for honey production. Thus, there is an ongoing effort to find an effective agent that shows broad biocidal activity with minimal environmental hazard. In this study, the biocidal effect of maltose reduced silver nanoparticles (AgNPs) is evaluated against American foulbrood and European foulbrood pathogens. The results demonstrate that the maltose reduced AgNPs are excellent short and long-term biocides against P. larvae isolates. The long-term effect suggests that the Ag+ ions are released from the AgNPs with increasing time in a controlled manner.

High‐level resistance of Melissococcus plutonius clonal complex 3 strains to antimicrobial activity of royal jelly

Melissococcus plutonius is the causative agent of European foulbrood of honey bee larvae. Among its three genetically distinct groups (CC3, CC12 and CC13), CC3 strains have been suggested to be more virulent at the colony level. Honey bee larvae are fed royal or worker jellies by adult bees, and these jellies exhibit antimicrobial activity. Since M. plutonius orally infects larvae via brood food, we herein investigated the resistance of M. plutonius to the antimicrobial activity of royal jelly (RJ). The results obtained revealed that M. plutonius strains were more resistant to RJ and its component, 10-hydroxy-2-decenoic acid, than the other species tested. Moreover, among the M. plutonius strains examined, CC3 strains exhibited the strongest resistance to antimicrobial activity; they temporarily proliferated and survived for a long period in 50% RJ-containing broth. However, resistance was not observed when freshly cultured bacteria were used, it was only detected after a preculture on agar media for five or more days, suggesting that, under certain conditions, CC3 strains change their physiological state to that which is advantageous for survival in brood food. This high-level RJ resistance of CC3 strains may contribute to their virulence in the field.

Bacterial community associated with worker honeybees (Apis mellifera) affected by European foulbrood.

BackgroundMelissococcus 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.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 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.ConclusionsHigh-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.

The immunological dependence of plant-feeding animals on their host’s medical properties may explain part of honey bee colony losses

The honey bee (Apis mellifera) is an important pollinator of agricultural and horticultural crops, but also of wild flowers. The species has been facing declines in many areas of the world, the causes being identified as multifactorial. Recently, it has been theorised that some plant-dwelling animals may develop a dependence on the medicinal properties of their hots plant’s secondary metabolites. Here, the question of honey bee self-medication using organic materials, namely propolis, nectar, honey, honeydew, pollen, wood, and algae for self-medication is addressed. Self-medication in honey bees is a largely unexplored area and thus a comprehensive overview of the field is provided. Prior studies suggest that recent honey bee colony declines are driven by decreased forage plant availability. The problem is expanded and it is suggested, that if honey bees developed a dependence on medical properties of some disappearing plants or materials, this could explain a part of the colony losses observed around the world. To date, convincing evidence points towards self-medication with honey and propolis. Bees also contact plant secondary metabolites, fatty acids, essential oils, and microorganisms that are active against the causative agents of American foulbrood, European foulbrood, nosemosis, chalkbrood, stonebrood, and varroasis. In the future, selected taxa of plants with medicinal properties may be planted to boost honey bee health without chemotherapy. Future directions of research are discussed.

The Curious Case of Achromobacter eurydice, a Gram-Variable Pleomorphic Bacterium Associated with European Foulbrood Disease in Honeybees.

Honeybees are prone to parasite and pathogen infestations/infections due to their social colony life. Bacterial pathogens in particular lead to destructive infections of the brood. European foulbrood is caused by the bacterium Melissococcus plutonius in combination with several other Gram-positive bacteria (Achromobacter eurydice, Bacillus pumilus, Brevibacillus laterosporus, Enterococcus faecalis, Paenibacillus alvei, Paenibacillus dendritiformis) involved as secondary invaders following the initial infection. More than a century ago, A. eurydice was discovered to be associated with European foulbrood and morphologically and biochemically characterized. However, since the 1950s-1960s, only a few studies are known covering the biological relevance of this bacterium. Here, we review the biology, ecology, morphology, and biochemistry and discuss the still unclear systematic classification of A. eurydice.