American Foulbrood In Honey Bees Research Articles

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

A taxonomic study was performed on Gram-stain-positive, catalase-negative and regular rod-shaped bacterial strains R4B(T) and R4C, isolated from the stomachs of honeybees. 16S rRNA gene sequence analysis revealed that the phylogenetic position of the novel strains was within the genus Lactobacillus; the highest sequence similarity to R4B(T) was shown by Lactobacillus acidophilus BCRC 10695(T) (93.6 %). Lower sequence similarities were found to other obligately homofermentative lactobacilli. A PCR-DGGE method could detect the sequence of the 16S rRNA gene of strain R4B(T) at different developmental stages of honeybees occurring in two different locations in the Czech Republic. The distinctiveness of the strains from other lactobacilli was also confirmed by analysis of sequences of other phylogenetic markers applicable to the taxonomy of the genus Lactobacillus, ribotyping and rep-PCR analysis. The DNA G+C content of strain R4B(T) was 41.3 mol%. The predominant cellular fatty acids of strain R4B(T) were C18 : 1ω9c, summed C19 : 1ω6c/C19 : 0 cyclo ω10c, C16 : 0, summed C18 : 1ω7c/C18 : 1ω6c and summed C16 : 1ω7c/C16 : 1ω6c. The major polar lipids of strain R4B(T) were glycolipids, lipids and phospholipids. Phenotypic and phylogenetic characteristics also confirmed the independent status of the strains at the species level. Interestingly, strain R4B(T) was able to inhibit growth in vitro of Paenibacillus larvae subsp. larvae (causal agent of American foulbrood in honeybees) and Melissococcus plutonius (causal agent of European foulbrood). The name Lactobacillus apis sp. nov. is proposed for this novel taxon; the type strain is R4B(T) ( = CCM 8403(T) = LMG 26964(T)).

Identification and Functional Analysis of the S-Layer Protein SplA of Paenibacillus larvae, the Causative Agent of American Foulbrood of Honey Bees

The Gram-positive, spore-forming bacterium Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a globally occurring, deathly epizootic of honey bee brood. AFB outbreaks are predominantly caused by two genotypes of P. larvae, ERIC I and ERIC II, with P. larvae ERIC II being the more virulent genotype on larval level. Recently, comparative proteome analyses have revealed that P. larvae ERIC II but not ERIC I might harbour a functional S-layer protein, named SplA. We here determine the genomic sequence of splA in both genotypes and demonstrate by in vitro self-assembly studies of recombinant and purified SplA protein in combination with electron-microscopy that SplA is a true S-layer protein self-assembling into a square 2D lattice. The existence of a functional S-layer protein is novel for this bacterial species. For elucidating the biological function of P. larvae SplA, a genetic system for disruption of gene expression in this important honey bee pathogen was developed. Subsequent analyses of in vivo biological functions of SplA were based on comparing a wild-type strain of P. larvae ERIC II with the newly constructed splA-knockout mutant of this strain. Differences in cell and colony morphology suggest that SplA is a shape-determining factor. Marked differences between P. larvae ERIC II wild-type and mutant cells with regard to (i) adhesion to primary pupal midgut cells and (ii) larval mortality as measured in exposure bioassays corroborate the assumption that the S-layer of P. larvae ERIC II is an important virulence factor. Since SplA is the first functionally proven virulence factor for this species, our data extend the knowledge of the molecular differences between these two genotypes of P. larvae and contribute to explaining the observed differences in virulence. These results present an immense advancement in our understanding of P. larvae pathogenesis.

Heterologous expression of green fluorescent protein in Paenibacillus larvae, the causative agent of American Foulbrood of honey bees

We aimed at expressing heterologous proteins in Paenibacillus larvae, the causative agent of American Foulbrood of honey bees, as a prerequisite for future studies on the molecular pathogenesis of P. larvae infections. For this purpose, we established a protocol for the transformation of the plasmid pAD43-25 carrying a functional GFP gene sequence (gfpmut3a) into different P. larvae strains representing the two most relevant P. larvae genotypes ERIC I and ERIC II. We determined the optimal field strength for electroporation and the optimal regeneration time after transformation. Stable GFP expression could be detected in the mutants during their entire life cycles and even after sporulation and re-germination. This method is suitable not only for the expression of GFP in P. larvae but also for the expression of heterologous proteins or GFP-tagged proteins in P. larvae. Mutants can be used for infection assays because GFP expression remained stable after sporulation and re-germination. This method provides the first true molecular tool for P. larvae and, therefore, is an immense advancement from what we had previously at our hands for the study of P. larvae pathogenesis.

Synergistic effect of surfactin from Bacillus subtilis C4 and Achyrocline satureioides extracts on the viability of Paenibacillus larvae

The aim of this work was to determine the in vitro effect of the mixture between the lipopeptide surfactin, synthesized by Bacillus subtilis C4 (strain isolated from honey) and the most active vegetal extract from Achyrocline satureioides, a traditional medicinal plant, on local strains of Paenibacillus larvae, the agent of American Foulbrood in honeybees. Five P. larvae strains isolated in Córdoba, Argentina, were phenotypically characterized. These and 12 other P. larvae strains from different regions of Argentina were analysed. The antimicrobial activities of the essential oil, hexane (HE) and benzene extracts from A. satureioides were assessed against P. larvae and the HE showed the highest anti-P. larvae activity. A combination of the biosurfactant surfactin, produced by B. subtilis C4, and the HE of A. satureioides revealed a synergistic action on P. larvae. The effective surfactin concentration in the mixture decreased from 32 to 1 μg ml(-1) and the HE concentration from 32 to 4 μg ml(-1), values similar or equal to minimal inhibitory concentrations observed for oxytetracycline. The fractional inhibitory concentration index confirmed synergism in 4 strains and partial synergism in one strain. The combination of surfactin synthesized by B. subtilis C4 and the HE from A. satureioides could be a natural alternative to help beekeepers to combat the American foulbrood agent P. larvae.

Paenibacillus larvaeBacteremia in Injection Drug Users

Paenibacillus larvae causes American foulbrood in honey bees. We describe P. larvae bacteremia in 5 injection drug users who had self-injected honey-prepared methadone proven to contain P. larvae spores. That such preparations may be contaminated with spores of this organism is not well known among pharmacists, physicians, and addicts.

American Foulbrood in honeybees and its causative agent, Paenibacillus larvae

After more than a century of American Foulbrood (AFB) research, this fatal brood infection is still among the most deleterious bee diseases. Its etiological agent is the Gram-positive, spore-forming bacterium Paenibacillus larvae. Huge progress has been made, especially in the last 20 years, in the understanding of the disease and of the underlying host-pathogen interactions. This review will place these recent developments in the study of American Foulbrood and of P. larvae into the general context of AFB research.

Diagnosis of American foulbrood in honey bees: a synthesis and proposed analytical protocols

Worldwide, American foulbrood (AFB) is the most devastating bacterial disease of the honey bee (Apis mellifera). Because the distinction between AFB and powdery scale disease is no longer considered valid, the pathogenic agent has recently been reclassified as one species Paenibacillus larvae, eliminating the subspecies designations Paenibacillus larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens. The creamy or dark brown, glue-like larval remains of infected larvae continue to provide the most obvious clinical symptom of AFB, although it is not conclusive. Several sensitive and selective culture media are available for isolation of this spore-forming bacterium, with the type of samples that may be utilized for detection of the organism being further expanded. PCR methods for identification and genotyping of the pathogen have now been extensively developed. Nevertheless, biochemical profiling, bacteriophage sensitivity, immunotechniques and microscopy of suspect bacterial strains are entirely adequate for routine identification purposes.

Reclassification, genotypes and virulence ofPaenibacillus larvae, the etiological agent of American foulbrood in honeybees – a review

L'agent de la loque americaine (AFB) a ete classe au cours des temps comme Bacillus larvae, Paenibacillus larvae et Paenibacillus larvae larvae. En meme temps une bacterie qui tuait les larves et les dessechait en croutes poudreuses etait caracterisee comme Bacillus pulvifaciens, Paenibacillus pulvifaciens et Paenibacillus larvae pulvifasciens. L'etude moleculaire des souches types et des souches de references des deux sous-especes et des souches de terrain a montre qu'il s'agissait d'une seule espece, Paenibacillus larvae. L'analyse par PCR-repetitive a l'aide de quatre amorces ERIC a permis de differencier quatre genotypes. Les tests d'infection experimentale n'ont montre aucune difference de pathologie entre les deux sous-especes. Toutes les souches etudiees etaient pathogenes pour les larves et les decomposaient en une masse filante qui se dessechait en une croute dure. En Allemagne seuls deux des quatre genotypes de P. larvae existent, comme le montre l'analyse par PCR-rep : il s'agit des genotypes ERIC I et ERIC II. On les differencie en combinant les amorces BOX AIR et MBO REP1. Cette methode permet de scinder encore plus le groupe ERIC I en deux. En Allemagne on trouve principalement les genotypes BOX/MBO Ab, ab et αβ. Le groupe ERIC II est identique au genotype BOX/MBO AB. Les differences entre les deux groupes sont principalement phenotypiques. Le genotype AB/ERIC Il presente une pigmentation orange sur gelose au sang de mouton et il est le seul a pouvoir metaboliser le fructose et le psi-cose. Les genotypes du groupe ERIC I se comportent exactement de facon opposee; en outre le genotype AB/ERIC 1 est le seul a posseder un plasmide. Les differences de virulence de P. larvae ont ete etudiees a l'aide d'infections experimentales. La CL 50 , i.e. la concentration en spores pour laquelle 50 % des larves exposees meurent d'AFB, est specifique a la souche et varie d'un facteur de 10. On a trouve des differences dans le deroulement de la loque americaine specifiques a la souche. Il est remarquable qu'environ 5 % des larves infectees par le genotype AB/ERIC Il etaient encore en vie apres l'operculation, tandis qu'environ 75 % de celles infectees par les autres genotypes survivaient a l'operculation. Puisque les larves, qui sont moribondes ou mortes avant l'operculation, ont tres vraisemblablement ete eliminees par les ouvrieres, pour un meme nombre d'insectes infectes il faut s'attendre a des symptomes cliniques plus faibles avec le genotype AB/ERIC II qu'avec les autres genotypes.

Antimicrobial activity of some Argentinean wild plant essential oils against Paenibacillus larvae larvae, causal agent of American foulbrood (AFB)

SummaryFive essential oils from Argentinan wild plants were tested in vitro for antimicrobial activity against different strains of Paenibacillus larvae larvae, causal agent of American foulbrood in honey bees, Apis mellifera L. The species selected from which to extract the essential oils were aguaribay (Schinus molle var. areira L), Andean thyme (Acantholippia seriphioides A. Gray), peperine (Mintosthachys mollis H.B.K. Gris), camomile (Tagetes minuta L.) and pennyroyal (Lippia turbinata Griseb.). Oils were extracted from different parts of the plants by steam distillation. Minimal inhibitory concentration (MIC) and minimal bactericide concentration (MBC) were evaluated on Mueller-Hinton broth and on MYPGP agar respectively, by the tube dilution method. MIC and MBC minimum and maximum values obtained were 200–250 mgL−1 and 200–300 mgL−1 for Andean thyme and 800–1000 mgL−1 and 850–1100 mgL−1 for pennyroyal, respectively. The other oils had intermediate MIC and MBC values between the formers mentioned. Highly significant differences between MIC and MBC values for the essential oils tested were found. Tukey's mean separation test indicated that Andean thyme (A. seriphioides) essential oil was different from the other four essential oils at α<0.01. Andean thyme proved to be the most effective in vitro essential oil against P. larvae larvae. Therefore, it may be a promising natural alternative to traditional antibiotics, meriting further studies on its field applicability for AFB control.

Evidence for intra-colonial genetic variance in resistance to American foulbrood of honey bees ( Apis mellifera): further support for the parasite/pathogen hypothesis for the evolution of polyandry.

Explanations for the evolution of multiple mating by social insect (particularly honey bee) queens have been frequently sought. An important hypothesis is that multiple mating is adaptive because it increases intracolonial genetic diversity and thereby reduces the likelihood that parasites or pathogens will catastrophically infect a colony. We tested one assumption of this model: that honey bee worker patrilines should differ in disease resistance. We used American foulbrood (caused by the bacterium Paenibacillus larvae) as a model pathogen. We found that patrilines within colonies do indeed vary in their resistance to this disease.

American foulbrood in honey bees

In recent years, one of the major problems hampering the development of apiculture is the disease American foulbrood (AFB). In this article, AFB is reviewed in detail, including its detection and diagnosis, prevention and control. Paenibacillus larvae larvae, the causative agent of the disease, is also examined. Products of P. I. larvae culture are mentioned, particularly the antibiotic substances and proteases.

Comparative study of tylosin, erythromycin and oxytetracycline to control American foulbrood of honey bees

SUMMARYThe efficiency of tylosin and erythromycin was evaluated and compared to oxytetracycline hydrochloride and Terramycin for the control of American foulbrood (AFB) (Paenibacillus larvae larvae) in infected honey bee colonies. Oral acute toxicity tests for adult bees of all antibiotics were determined by calculations of LD50 values at 24 h, 48 h and 72 h, using dimethoate as reference compound. All the antibiotics tested showed no toxicity for adult bees, with LD50 values greater than 100 μg per bee. The times of degradation in honey were analysed for tylosin and erythromycin through HPLC and bioassays, respectively. Erythromycin, as ethyl succinate, was not effective for the control of AFB at any of the tested doses and its degradation time in honey was about 35–40 days. On the other hand, tylosin, as tartrate, was highly effective for the control of AFB, and also suppressed disease signs for one year after treatment. Its degradation time in honey was about 60 days. AFB was effectively controlled by oxytetracycline hydrochloride and Terramycin but in all cases, colonies exhibited disease recurrence from five to 10 months after treatment. Tylosin tartrate is a valuable alternative for controlling AFB in apiaries in Argentina.

Growth of Paenibacillus larvae, the causative agent of American foulbrood in honey bees and Bacillus popilliae, the causative agent of milky disease in beetle larvae in lepidopteran cell cultures

AbstractTNM‐FH Lepidopteran insect cell culture medium containing 10% fetal bovine serum (FBS), while allowing limited vegetative growth of Paenibacillus larvae (wild‐type strain), the causative agent of American foulbrood, contained no viable vegetative cells upon subculture, nor were any heat resistant spores produced in this medium alone. However, TNM‐FH medium cotaining embryonic or midgut cells from Trichoplusia ni, hemocytes from Estigmene acrea, ovarian and embryonic cells from Spodoptera frugiperda, embryonic cells from Plutella xylostella, Spodoptera exigua and Pseudaletia unipuncta or ovarian cells from Lymantria dispar, supported both heavy vegetative cell growth and moderate production of heat resistant spores. EX‐CELL 405 serum‐free insect cell culture medium alone appeared to contain the appropriate nutrients required for both vegetative growth and sporulation of P. larvae. However, in the presence of embryonic cells from T. ni, limited vegetative growth occurred and the P. larvae cells appeared to die off. This was confirmed by the fact that no colony growth occurred upon subculture, nor were any heat resistant spores detected. This was true also in the presence of fat body cells from T. ni, except that a limited number of spores (4,000/ml) were detected in the form of cology‐forming units (CFU) on plates following heating to 80°C for 20 minutes. In a parallel study with a wild‐type strain of Bacillus popilliae, vegetative cells grew only in TNM‐FH medium in the presence of mid‐gut BTI‐Tn‐MG and ovarian (Tn‐368) cells of T. ni. No heat resistant spores, however, were detected in any of the cultures. When BTI‐Tn‐MG and Tn‐368 cells were further challenged with four variant cultures of B. popilliae, vegetative growth and limited sporulation were achieved. The BTI‐Tn‐MG cell line in TNM‐FH medium produced as many as 12,000 spores/ml after 21 days in culture.

Media for the detection of Bacillus larvae spores in honey

AbstractBacillus larvae, the causative agent of American foulbrood in honey bees completes its life cycle of germination, outgrowth and sporulation in young honey bee larvae by killing them and often bringing about the destruction of the entire hive. While B. larvae germinates and outgrows on complex organic media in vitro, the literature suggests, for reasons that are not at all clear, that a relatively large number of spores of B. larvae are required to yield each visible colony (colony forming units, CFU) on media. Various researchers have reported that from 16 to 3,000 or more spores of B. larvae are required to yield a single colony on an agar plate. HANSEN in Denmark designed a useful method of spreading approximately 80 mg of honey directly on the surface of a PETRI plate containing “J” agar medium to determine if B. larvae spores are present in the honey. In the present study, selected media were tested for the ability to recover B. larvae spores in honeys in the form of visible colonies (CFU) using HANSEN's strek method. A modification of a medium (TMYGP) developed by DINGMAN and STAHLY, (T‐HCL‐YGP agar), recovered considerably more viable B. larvae spores in the form of visible colonies (CFU) than HANSEN's “J” medium. When “J” medium was fortified with 0.1% sodium pyruvate, it was comparable to modified T‐HCL‐YGP medium in its recovery of B. larvae spores. Brain heart infusion agar (BHIA) with the addition of thiamine recovered more spores in the form of viable colonies than did “J” medium but it was not as efficient as T‐HCL‐YGP medium. Serial dilution from 100 to 10,000 times of weighed samples of honey with deionized water led to higher spore counts (CFU per g honey) than that indicated by undiluted honeys plated at 80 mg levels directly onto the surface of media by the HANSEN procedure.

Properties of clear plaque mutants of the Bacillus larvae bacteriophages PBL0.5 and PBL2

Two temperate bacteriophages, designated PBL0.5 and PBL2, were obtained from two independently isolated strains of Bacillus larvae. Clear plaque mutants of both phages were isolated and characterized. By a number of different criteria the phages were identical. These phages are not ideal for prevention of American foulbrood of honey bees, because they are temperate phages. They are potentially useful for epidemiological studies.

A study on the possible utilization of immunodiffusion and immunofluorescence techniques as the diagnostic methods for American foulbrood of honeybees ( Apis mellifera)

Four types of antisera were obtained from rabbits hyperimmunized with either spores or vegetative rods from two strains of the American foulbrood pathogen, Bacillus larvae. The specificity and sensitivity of these antisera were tested with immunofluorescence and immunodiffusion methods. No cross-reactions were observed between the antisera and other different species of Bacillus or different genera of bacteria. The specificity was not found between the antisera and two strains of B. larvae although stronger fluorescent intensity was observed between the antiserum and its corresponding strain of antigen in the immunofluorescence tests. Eight samples of 1- to 2-day-old larvae, 3- to 4-day-old larvae, decayed tissue, and dry remain, collected from eight infected colonies, were tested against antisera by the immunofluorescence and the immunodiffusion methods. The results indicated that both methods are sensitive and specific for making diagnosis of field samples of American foulbrood of honey bees.

Resistance to American foulbrood in honey bees: XII. Persistence of viable Bacillus larvae spores in the feces of adults permitted flight

Adult honey bees of two colonies given one feeding of Bacillus larvae spores egested them in their feces over a period extending more than 2 months in one population. Spore egestion began 1 day after spore ingestion, and reached a maximum on the 2nd and the 6th day. Spores that passed through the alimentary tract of adult bees remained viable and incited American foulbrood disease when fed to susceptible honey bee larvae.

Resistance to American foulbrood in honey bees. XI: Fate of Bacillus larvae spores ingested by adults

Ingestion of large numbers of Bacillus larvae spores over various lengths of time by individually caged adult honey bees, Apis mellifera, of three different genetic stocks did not produce observable pathological effects. Dissection and microscopical examination revealed spores abundantly present in the alimentary canal, but no vegetative rods were found in either the canal (with two possible exceptions) or the hemolymph. Recovery of spores from the three major gut regions of adult worker bees showed that 4 min after spore ingestion the main spore concentration was located in the foregut. After 40 min a majority of the spores were in the midgut where they remained for more than 400 min. By the 4,000 min interval most spores had passed into the hindgut. Spores remained viable in the alimentary canal for a period of at least 40,000 min. Viability was demonstrated through the growth of B. larvae colonies in solid yeast-starch medium. When fed to young honey bee larvae, these spores proved to be infectious and caused American foulbrood disease.

Resistance to American foulbrood in honey bees IX. Effects of honey-bee larval food on the growth and viability of Bacillus larvae

Experiments were designed to learn whether there are any differences between honey-bee ( Apis mellifera) larval food from American-foulbrood-resistant and American-foulbrood-susceptible lines of bees in regard to antimicrobial action on Bacillus larvae, the pathogenic agent of American foulbrood, cultured in vitro. To test for inhibition of germination, spores were exposed to various concentrations of brood food from both genetic lines in culture medium, heat-shocked to kill vegetable cells, and then plated for germination of those spores which were inhibited from germinating. Results indicated that brood food, from an American-foulbrood-resistant line (Brown) of bees was more effective in inhibiting spore germination than brood food from a disease-susceptible line (Van Scoy). Tests for effects of brood food on vegetable cells involved their exposure for different periods of time to various concentrations of the food in an aqueous medium. It was found that brood food from the resistant line was more effective in reducing the number of viable vegetable cells than brood food from the susceptible line.

Resistance to American foulbrood in honey bees X. Comparative mortality of queen, worker, and drone larvae

Queen, worker, and drone honey-bee ( Apis mellifera) larvae of similar ages and genotypes were given, in their food, a constant dosage of water-suspended Bacillus larvae spores, or water only, in 10 experimental replications. Pooled data from all replications showed that spore treatment resulted in about 93% mortality of female larvae reared as queens, 82% mortality of those reared as workers, and 68% mortality of the male (drone) larvae. Control treatment mortality was less than 5% for each caste. Chi square analyses of mortality associated with control treatments showed no significant differences between any two of the three castes, whereas mortality differences between castes following spore treatments were highly significant and are interpreted as resulting from differences in resistance. These differences in resistance are attributed to differences in food supplied by adult bees to the larvae.