Phospholipase A2 activity is required for immune defense of European (Apis mellifera) and Asian (Apis cerana) honeybees against American foulbrood pathogen, Paenibacillus larvae.

Honeybees require an efficient immune system to defend against microbial pathogens. The American foulbrood pathogen, Paenibacillus larvae, is lethal to honeybees and one of the main causes of colony collapse. This study investigated the immune responses of Apis mellifera and Apis cerana honeybees against the bacterial pathogen P. larvae. Both species of honeybee larvae exhibited significant mortality even at 102 103 cfu/mL of P. larvae by diet-feeding, although A. mellifera appeared to be more tolerant to the bacterial pathogen than A. cerana. Upon bacterial infection, the two honeybee species expressed both cellular and humoral immune responses. Hemocytes of both species exhibited characteristic spreading behaviors, accompanied by cytoskeletal extension along with F-actin growth, and formed nodules. Larvae of both species also expressed an antimicrobial peptide called apolipophorin III (ApoLpIII) in response to bacterial infection. However, these immune responses were significantly suppressed by a specific inhibitor to phospholipase A2 (PLA2). Each honeybee genome encodes four PLA2 genes (PLA2A ~ PLA2D), representing four orthologous combinations between the two species. In response to P. larvae infection, both species significantly up-regulated PLA2 enzyme activities and the expression of all four PLA2 genes. To determine the roles of the four PLA2s in the immune responses, RNA interference (RNAi) was performed by injecting gene-specific double stranded RNAs (dsRNAs). All four RNAi treatments significantly suppressed the immune responses, and specific inhibition of the two secretory PLA2s (PLA2A and PLA2B) potently suppressed nodule formation and ApoLpIII expression. These results demonstrate the cellular and humoral immune responses of A. mellifera and A. cerana against P. larvae. This study suggests that eicosanoids play a crucial role in mediating common immune responses in two closely related honeybees.

Use of Dicranum polysetum extract against Paenibacillus larvae causing American Foulbrood under in vivo and in vitro conditions / Uso del extracto de Dicranum polysetum contra larvas de Paenibacillus causantes de la loque americana en condiciones in vivo e in vitro

Recent research shows that Dicranum species can be used to ameliorate the negative effects of honeybee bacterial diseases and that novel compounds isolated from these species may have the potential to treat bacterial diseases. This study aimed to investigate the efficacy of Dicranum polysetum Sw. against American Foulbrood using toxicity and larval model. The effectiveness of D. polysetum Sw. ethanol extract in combating AFB was investigated in vitro and in vivo. This study is important in finding an alternative treatment or prophylactic method to prevent American Foulbrood disease in honey bee colonies. Spore and vegetative forms of Paenibacillus larvae PB31B with ethanol extract of D. polysetum were tested on 2040 honey bee larvae under controlled conditions. Total phenolic and flavonoid contents of D. polysetum ethanol extracts were determined as 80.72 mg/GAE(Gallic acid equivalent) and 303.20 µg/mL, respectively. DPPH(2,2-diphenyl-1-picrylhydrazyl) radical scavenging percent inhibition value was calculated as 4.32%. In Spodoptera frugiperda (Sf9) and Lymantria dispar (LD652) cell lines, the cytotoxic activities of D. polysetum extract were below 20% at 50 µg/mL. The extract was shown to considerably decrease infection in the larvae, and the infection was clinically halted when the extract was administered during the first 24 h after spore contamination. The fact that the extract contains potent antimicrobial/antioxidant activity does not reduce larval viability and live weight, and does not interact with royal jelly is a promising development, particularly regarding its use to treat early-stage AFB infection.(AU)

Use of Dicranum polysetum extract against Paenibacillus larvae causing American Foulbrood under in vivo and in vitro conditions.

Recent research shows that Dicranum species can be used to ameliorate the negative effects of honeybee bacterial diseases and that novel compounds isolated from these species may have the potential to treat bacterial diseases. This study aimed to investigate the efficacy of Dicranum polysetum Sw. against American Foulbrood using toxicity and larval model. The effectiveness of D. polysetum Sw. ethanol extract in combating AFB was investigated in vitro and in vivo. This study is important in finding an alternative treatment or prophylactic method to prevent American Foulbrood disease in honey bee colonies. Spore and vegetative forms of Paenibacillus larvae PB31B with ethanol extract of D. polysetum were tested on 2040 honey bee larvae under controlled conditions. Total phenolic and flavonoid contents of D. polysetum ethanol extracts were determined as 80.72 mg/GAE(Gallic acid equivalent) and 303.20 µg/mL, respectively. DPPH(2,2-diphenyl-1-picrylhydrazyl) radical scavenging percent inhibition value was calculated as 4.32%. In Spodoptera frugiperda (Sf9) and Lymantria dispar (LD652) cell lines, the cytotoxic activities of D. polysetum extract were below 20% at 50 µg/mL. The extract was shown to considerably decrease infection in the larvae, and the infection was clinically halted when the extract was administered during the first 24 h after spore contamination. The fact that the extract contains potent antimicrobial/antioxidant activity does not reduce larval viability and live weight, and does not interact with royal jelly is a promising development, particularly regarding its use to treat early-stage AFB infection.

Comparison of individual hive and apiary-level sample types for spores of Paenibacillus larvae in Saskatchewan honey bee operations.

Three commercial honey bee operations in Saskatchewan, Canada, with outbreaks of American foulbrood (AFB) and recent or ongoing metaphylactic antibiotic use were intensively sampled to detect spores of Paenibacillus larvae during the summer of 2019. Here, we compared spore concentrations in different sample types within individual hives, assessed the surrogacy potential of honey collected from honey supers in place of brood chamber honey or adult bees within hives, and evaluated the ability of pooled, extracted honey to predict the degree of spore contamination identified through individual hive testing. Samples of honey and bees from hives within apiaries with a recent, confirmed case of AFB in a single hive (index apiaries) and apiaries without clinical evidence of AFB (unaffected apiaries), as well as pooled, apiary-level honey samples from end-of-season extraction, were collected and cultured to detect and enumerate spores. Only a few hives were heavily contaminated by spores in any given apiary. All operations were different from one another with regard to both the overall degree of spore contamination across apiaries and the distribution of spores between index apiaries and unaffected apiaries. Within operations, individual hive spore concentrations in unaffected apiaries were significantly different from index apiaries in the brood chamber (BC) honey, honey super (HS) honey, and BC bees of one of three operations. Across all operations, BC honey was best for discriminating index apiaries from unaffected apiaries (p = 0.001), followed by HS honey (p = 0.06), and BC bees (p = 0.398). HS honey positively correlated with both BC honey (rs = 0.76, p < 0.0001) and bees (rs = 0.50, p < 0.0001) and may be useful as a surrogate for either. Spore concentrations in pooled, extracted honey seem to have predictive potential for overall spore contamination within each operation and may have prognostic value in assessing the risk of future AFB outbreaks at the apiary (or operation) level.

Paenibacillus larvae bacteriophages: obscure past, promising future.

Paenibacillus larvae is a Gram-positive, spore-forming bacterium that is the causative agent of American foulbrood (AFB), the most devastating bacterial disease of the honeybee. P. larvae is antibiotic resistant, complicating treatment efforts. Bacteriophages that target P. larvae are rapidly emerging as a promising treatment. The first P. larvae phages were isolated in the 1950s, but as P. larvae was not antibiotic resistant at the time, interest in them remained scant. Interest in P. larvae phages has grown rapidly since the first P. larvae phage genome was sequenced in 2013. Since then, the number of sequenced P. larvae phage genomes has reached 48 and is set to grow further. All sequenced P. larvae phages encode a conserved N-acetylmuramoyl-l-alanine amidase that is responsible for cleaving the peptidoglycan cell wall of P. larvae. All P. larvae phages also encode either an integrase, excisionase or Cro/CI, indicating that they are temperate. In the last few years, several studies have been published on using P. larvae phages and the P. larvae phage amidase as treatments for AFB. Studies were conducted on infected larvae in vitro and also on hives in the field. The phages have a prophylactic effect, preventing infection, and also a curative effect, helping resolve infection. P. larvae phages have a narrow range, lysing only P. larvae, and are unable to lyse even related Paenibacillus species. P. larvae phages thus appear to be safe to use and effective as treatment for AFB, and interest in them in the coming years will continue to grow.

Feeding Honeybee Colonies with Honeybee-Specific Lactic Acid Bacteria (Hbs-LAB) Does Not Affect Colony-Level Hbs-LAB Composition or Paenibacillus larvae Spore Levels, Although American Foulbrood Affected Colonies Harbor a More Diverse Hbs-LAB Community.

The main current methods for controlling American Foulbrood (AFB) in honeybees, caused by the bacterial pathogen Paenibacillus larvae, are enforced incineration or prophylactic antibiotic treatment, neither of which is fully satisfactory. This has led to an increased interest in the natural relationships between the pathogenic and mutualistic microorganisms of the honeybee microbiome, in particular, the antagonistic effects of Honeybee-Specific Lactic Acid Bacteria (hbs-LAB) against P. larvae. We investigated whether supplemental administration of these bacteria affected P. larvae infection at colony level over an entire flowering season. Over the season, the supplements affected neither colony-level hbs-LAB composition nor naturally subclinical or clinical P. larvae spore levels. The composition of hbs-LAB in colonies was, however, more diverse in apiaries with a history of clinical AFB, although this was also unrelated to P. larvae spore levels. During the experiments, we also showed that qPCR could detect a wider range of hbs-LAB, with higher specificity and sensitivity than mass spectrometry. Honeybee colonies are complex super-organisms where social immune defenses, natural homeostatic mechanisms, and microbiome diversity and function play a major role in disease resistance. This means that observations made at the individual bee level cannot be simply extrapolated to infer similar effects at colony level. Although individual laboratory larval assays have clearly demonstrated the antagonistic effects of hbs-LAB on P. larvae infection, the results from the experiments presented here indicate that direct conversion of such practice to colony-level administration of live hbs-LAB is not effective.

Honeybee-Specific Lactic Acid Bacterium Supplements Have No Effect on American Foulbrood-Infected Honeybee Colonies.

Paenibacillus larvae, the causative agent of American foulbrood (AFB), is the primary bacterial pathogen affecting honeybees and beekeeping. The main methods for controlling AFB are incineration of diseased colonies or prophylactic antibiotic treatment (e.g., with tylosin), neither of which is fully satisfactory. The search for superior means for controlling AFB has led to an increased interest in the natural relationships between the honeybee-pathogenic and mutualistic microorganisms and, in particular, the antagonistic effects of honeybee-specific lactic acid bacteria (hbs-LAB) against P. larvae These effects have been demonstrated only on individual larvae in controlled laboratory bioassays. Here we investigated whether supplemental administration of hbs-LAB had a similar beneficial effect on P. larvae infection at colony level. We compared experimentally AFB-infected colonies treated with hbs-LAB supplements to untreated and tylosin-treated colonies and recorded AFB symptoms, bacterial spore levels, and two measures of colony health. To account for the complexity of a bee colony, we focused on (Bayesian) probabilities and magnitudes of effect sizes. Tylosin reduced AFB disease symptoms but also had a negative effect on colony strength. The tylosin treatment did not, however, affect P. larvae spore levels and might therefore "mask" the potential for disease. hbs-LAB tended to reduce brood size in the short term but was unlikely to affect AFB symptoms or spores. These results do not contradict demonstrated antagonistic effects of hbs-LAB against P. larvae at the individual bee level but rather suggest that supplementary administration of hbs-LAB may not be the most effective way to harness these beneficial effects at the colony level.IMPORTANCE The previously demonstrated antagonistic effects of honeybee-derived bacterial microbiota on the infectivity and pathogenicity of P. larvae in laboratory bioassays have identified a possible new approach to AFB control. However, honeybee colonies are complex superorganisms where social immune defenses play a major role in resistance against disease at the colony level. Few studies have investigated the effect of beneficial microorganisms on bee diseases at the colony level. Effects observed at the individual bee level do not necessarily translate into similar effects at the colony level. This study partially fills this gap by showing that, unlike at the individual level, hbs-LAB supplements did not affect AFB symptoms at the colony level. The inference is that the mechanisms regulating the honeybee microbial dynamics within a colony are too strong to manipulate positively through supplemental feeding of live hbs-LAB and that new potential remedies identified through laboratory research have to be tested thoroughly in situ, in colonies.

An integrated management strategy to prevent outbreaks and eliminate infection pressure of American foulbrood disease in a commercial beekeeping operation.

The bacterial disease American Foulbrood (AFB), caused by the Gram-positive bacterium Paenibacillus larvae, is considered the most contagious and destructive infectious disease affecting honeybees world-wide. The resilient nature of P. larvae bacterial spores presents a difficult problem for the control of AFB. Burning clinically symptomatic colonies is widely considered the only workable strategy to prevent further spread of the disease. Antibiotic use is banned in EU countries, and although used commonly in the U.S. and Canada, it only masks symptoms and does not prevent the further spread of the disease. Not surprisingly, there is an increased demand for chemical-free strategies to prevent and control of AFB. The aim of this study was to implement a management program with a long-term perspective to reduce infection pressure and eliminate AFB outbreaks. The study was conducted within a commercial beekeeping operation in central Sweden that has previously experienced reoccurring AFB outbreaks. For 5 years, P. larvae were cultured from adult bee samples taken in the fall. The following spring, any identified sub-clinically infected colonies were shaken onto new material and quarantined from the rest of the beekeeping operation. After the first year clinical symptoms were not again observed, and during the 5 years of the study the proportion of apiaries harbouring P. larvae spores decreased from 74% to 4%. A multinomial regression analysis also clearly demonstrated that the proportion of infected colonies with the highest levels of spore counts disproportionately declined so that by the end of the study the only remaining infected apiaries were in the lowest spore count category (the three higher spore count categories having been eradicated). These results demonstrate the importance of management practices on AFB disease epidemiology. Early detection of subclinical spore prevelance and quarantine management as presented here can provide an effective sustainable chemical-free preventive solution to reduce both the incidence of AFB outbreaks and continued transmission risk at a large-scale.

Swarming motility and biofilm formation of Paenibacillus larvae, the etiological agent of American Foulbrood of honey bees (Apis mellifera).

American Foulbrood is a worldwide distributed, fatal disease of the brood of the Western honey bee (Apis mellifera). The causative agent of this fatal brood disease is the Gram-positive, spore-forming bacterium Paenibacillus larvae, which can be classified into four different genotypes (ERIC I-IV), with ERIC I and II being the ones isolated from contemporary AFB outbreaks. P. larvae is a peritrichously flagellated bacterium and, hence, we hypothesized that P. larvae is capable of coordinated and cooperative multicellular behaviors like swarming motility and biofilm formation. In order to analyze these behaviors of P. larvae, we firstly established appropriate functional assays. Using these assays we demonstrated that P. larvae ERIC II, but not P. larvae ERIC I, was capable of swarming. Swarming motility was hampered in a P. larvae ERIC II-mutant lacking production of paenilarvin, an iturin-like lipopeptide exclusively expressed by this genotype. Both genotypes were able to form free floating biofilm aggregates loosely attached to the walls of the culture wells. Visualizing the biofilms by Congo red and thioflavin S staining suggested structural differences between the biofilms formed. Biofilm formation was shown to be independent from paenilarvin production because the paenilarvin deficient mutant was comparably able to form a biofilm.

Lactobacillus kunkeei strains decreased the infection by honey bee pathogens Paenibacillus larvae and Nosema ceranae.

Due to their social behaviour, honey bees can be infected by a wide range of pathogens including the microsporidia Nosema ceranae and the bacteria Paenibacillus larvae. The use of probiotics as food additives for the control or prevention of infectious diseases is a widely used approach to improve human and animal health. In this work, we generated a mixture of four Lactobacillus kunkeei strains isolated from the gut microbial community of bees, and evaluated its potential beneficial effect on larvae and adult bees. Its administration in controlled laboratory models was safe for larvae and bees; it did not affect the expression of immune-related genes and it was able to decrease the mortality associated to P. larvae infection in larvae and the counts of N. ceranae spores from adult honey bees. These promising results suggest that this beneficial microorganism's mixture may be an attractive strategy to improve bee health. Field studies are being carried out to evaluate its effect in naturally infected colonies.

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

Honeybee (Apis mellifera L.) workers act as passive vectors of Paenibacillus larvae spores, which cause the quarantine disease American foulbrood (AFB). We assessed the relative proportions of P. larvae within the honeybee microbiome using metabarcoding analysis of the 16 S rRNA gene. The microbiome was analyzed in workers outside of the AFB zone (control - AFB0), in workers from asymptomatic colonies in an AFB apiary (AFB1), and in workers from colonies exhibiting clinical AFB symptoms (AFB2). The microbiome was processed for the entire community and for a cut-off microbiome comprising pathogenic/environmental bacteria following the removal of core bacterial sequences; varroosis levels were considered in the statistical analysis. No correlation was observed between AFB status and varroosis level, but AFB influenced the worker bee bacterial community, primarily the pathogenic/environmental bacteria. There was no significant difference in the relative abundance of P. larvae between the AFB1 and AFB0 colonies, but we did observe a 9-fold increase in P. larvae abundance in AFB2 relative to the abundance in AFB1. The relative sequence numbers of Citrobacter freundii and Hafnia alvei were higher in AFB2 and AFB1 than in AFB0, whereas Enterococcus faecalis, Klebsiella oxytoca, Spiroplasma melliferum and Morganella morganii were more abundant in AFB0 and AFB1 than in AFB2.

Biological Role of Paenilarvins, Iturin-Like Lipopeptide Secondary Metabolites Produced by the Honey Bee Pathogen Paenibacillus larvae.

The Gram-positive bacterium Paenibacillus larvae (P. larvae) is the causative agent of a deadly honey bee brood disease called American Foulbrood (AFB). AFB is a notifiable epizootic in most countries and, hence, P. larvae is of considerable relevance for veterinarians and apiculturists alike. Over the last decade, much progress has been made in the understanding of the (patho)biology of P. larvae. Recently, several non-ribosomally produced peptides (NRP) and peptide/polyketide (NRP/PK) hybrids produced by P. larvae were identified. Among these NRPs were iturin-like lipopeptides, the paenilarvins A-C. Iturins are known to exhibit strong anti-fungal activity; for some iturins, cytotoxic activity towards mammalian erythrocytes and human cancer cell lines are described. We here present our results on the analysis of the natural function of the paenilarvins during pathogenesis of P. larvae infections. We demonstrated production of paenilarvins in infected larvae. However, we could neither demonstrate cytotoxicity of paenilarvins towards cultured insect cells nor towards larvae in feeding assays. Accordingly, exposure bioassays performed with larvae infected by wild-type P. larvae and a knockout mutant of P. larvae lacking production of paenilarvins did not substantiate a role for the paenilarvins as virulence factor. Further experiments are necessary to analyze the relevance of the paenilarvins' anti-fungal activity for P. larvae infections in the presence of fungal competitors in the larval midgut or cadaver.

Evaluation of antimicrobial activity of glycerol monolaurate nanocapsules against American foulbrood disease agent and toxicity on bees.

The American Foulbrood Disease (AFB) is a fatal larval bee infection. The etiologic agent is the bacterium Paenibacillus larvae. The treatment involves incineration of all contaminated materials, leading to high losses. The Glycerol Monolaurate (GML) is a known antimicrobial potential compound, however its use is reduced due to its low solubility in water and high melting point. The nanoencapsulation of some drugs offers several advantages like improved stability and solubility in water. The present study aimed to evaluate the antimicrobial activity against P. larvae and the toxicity in bees of GML nanoparticles. The nanocapsules were produced and presented mean diameter of 210 nm, polydispersity index of 0.044, and zeta potential of -23.4 mV demonstrating the acceptable values to predict a stable system. The microdilution assay showed that it is necessary 142 and 285 µg/mL of GML nanocapsules to obtain a bacteriostatic and bactericidal effect respectively. The time-kill curve showed the controlled release of compound, exterminating the microorganism after 24 h. The GML nanocapsules were able to kill the spore form of Paenibacillus larvae while the GML do not cause any effect. The assay in bees showed that the GML has a high toxicity while the GML nanoparticles showed a decrease on toxic effects. Concluding, the formulation shows positive results in the action to combat AFB besides not causing damage to bees.

Genetic diversity confers colony-level benefits due to individual immunity.

Several costs and benefits arise as a consequence of eusociality and group-living. With increasing group size, spread of disease among nest-mates poses selective pressure on both individual immunity and group-level mechanisms of disease resistance (social immunity). Another factor known to influence colony-level expression of disease is intracolony genetic diversity, which in honeybees (Apis mellifera) is a direct function of the number of mates of the queen. Colonies headed by queens with higher mating numbers have less variable infections of decreased intensity, though the underlying mechanisms remain unclear. By pathogen-challenging larvae in vitro, we decoupled larval immune response from mechanisms of social immunity. Our results show that baseline immunity and degree of immune response do not vary with genetic diversity. However, intracolony variance in antimicrobial peptide production after pathogen challenge decreases with increasing genetic diversity. This reduction in variability of the larval immune response could drive the mitigation of disease observed in genetically diverse colonies.