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PubMed2006

Reclassification of Paenibacillus larvae subsp. pulvifaciens and Paenibacillus larvae subsp. larvae as Paenibacillus larvae without subspecies differentiation.

A polyphasic taxonomic study of the two subspecies of Paenibacillus larvae, Paenibacillus larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens, supported the reclassification of the subspecies into one species, Paenibacillus larvae, without subspecies separation. Our conclusions are based on the analysis of six reference strains of P. larvae subsp. pulvifaciens and three reference strains and 44 field isolates of P. larvae. subsp. larvae. The latter originated from brood or honey of clinically diseased honey bee colonies or from honey of both clinically diseased and asymptomatic colonies from Sweden, Finland and Germany. Colony and spore morphology, as well as the metabolism of mannitol and salicin, did not allow a clear identification of the two subspecies and SDS-PAGE of whole-cell proteins did not support the subspecies differentiation. For genomic fingerprinting, repetitive element-PCR fingerprinting using ERIC primers and PFGE of bacterial DNA were performed. The latter method is a high-resolution DNA fingerprinting method proven to be superior to most other methods for biochemical and molecular typing and has not previously been used to characterize P. larvae. ERIC-PCR identified four different genotypes, while PFGE revealed two main clusters. One cluster included most of the P. larvae subsp. larvae field isolates, as well as all P. larvae subsp. pulvifaciens reference strains. The other cluster comprised the pigmented variants of P. larvae subsp. larvae. 16S rRNA gene sequences were determined for some strains. Finally, exposure bioassays demonstrated that reference strains of P. larvae subsp. pulvifaciens were pathogenic for honey bee larvae, producing symptoms similar to reference strains of P. larvae subsp. larvae. In comparison with the type strain for P. larvae subsp. larvae, ATCC 9545T, the P. larvae subsp. pulvifaciens strains tested were even more virulent, since they showed a shorter LT100. An emended description of the species is given.

Animals↗

PubMed2003

Diverse origins of tetracycline resistance in the honey bee bacterial pathogen Paenibacillus larvae.

Paenibacillus larvae is the causative agent of the important honey bee larval disease American Foulbrood (AFB). This pathogen has been treated in bee colonies by a single registered antibiotic, oxytetracycline (OTC), for fifty years. Recently, widespread resistance to OTC has been reported. In this study, the degree of antibiotic resistance was contrasted with DNA sequence variation for 125 P. larvae isolates collected in North America. Resistance was uncorrelated with bacterial haplotype, suggesting either that resistance has evolved multiple times in P. larvae or that resistance involves recent horizontal transfer via a non-genomic (e.g., plasmid or conjugal transposon) route. The recency of OTC resistance in P. larvae across this broad survey area underscores the need to manage foulbrood infections carefully and to monitor populations for resistance.

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PubMed2004

Proposal to reclassify Paenibacillus larvae subsp. pulvifaciens DSM 3615 (ATCC 49843) as Paenibacillus larvae subsp. larvae. Results of a comparative biochemical and genetic study.

The bacterial pathogen Paenibacillus larvae subsp. larvae (P. l. larvae), is the etiological agent of American foulbrood, an extremely contagious and disastrous disease of honeybee brood. In case of American foulbrood the destruction of infected colonies is often considered the only workable control measure. Therefore, the ability to diagnose this disease properly is important to prevent unnecessary economic loss to beekeepers. The development of suitable methods for the early and reliable detection of P. l. larvae is hampered by the fact that the two subspecies of Paenibacillus larvae, P. l. larvae and Paenibacillus larvae subsp. pulvifaciens (P. l. pulvifaciens), seem to be indistinguishable by cultural characteristics as well as by PCR protocols. Here we present an extensive analysis of several P. larvae reference strains. We employed conventional culture techniques, morphological and biochemical identification, PCR-based methods and sequencing of the 16S rDNA. We found indeed that P. l. pulvifaciens strain DSM 3615 is indistinguishable from P. l. larvae (DSM 7030). We did not face any problems to discriminate between P. l. larvae and P. l. pulvifaciens strains DSM 8442 and DSM 8443. Therefore, classification of DSM 3615 as type strain of P. l. pulvifaciens seems not to be justified. We propose to reclassify this strain as P. l. larvae. Former problems in differentiating the two subspecies might have arisen from this misclassification. PCR-based methods as well as appropriate biochemical identification systems provide a reliable means for the discrimination between the two subspecies P. l. larvae and P. l. pulvifaciens.

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PubMed1999

In situ localization of heat-shock and histone proteins in honey-bee (Apis mellifera l.) larvae infected with Paenibacillus larvae.

The immunohistochemical localization of the heat shock proteins (Hsp70 and Hsp90) and histone protein in healthy and Paenibacillus larvae infected honeybee (Apis mellifera L.) larvae has been studied. Hsp70 was found in the nuclei and the cytoplasm of infected midgut, salivary gland cells and haemocytes, but not in uninfected larvae. Hsp90 was localized in both infected and uninfected cells. Exposed histone proteins were localized in the nuclei of dying uninfected cells undergoing programmed cell death. The distribution of histone protein in uninfected cells of midgut, salivary gland, and other tissues was nuclear and indicative of normal programmed cell death at levels between 1 and 5%. After applying histone protein antibodies to P. larvae infected honeybee larvae, the DAB based reaction product was located in the nuclei or immediate surroundings of all larval cells. The Hsp70, Hsp90 and histone protein distribution patterns are discussed in relation to the morphological, cytochemical and immunocytochemical characteristics of programmed cell death and pathological necrosis. Results produced by methyl green-pyronin staining confirm an elevation of RNA levels in normal programmed cell death and a reduced staining for RNA in necrotic infected cells.

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PubMed2000

Histochemical characterization of cell death in honeybee larvae midgut after treatment with Paenibacillus larvae, Amitraz and Oxytetracycline.

A number of techniques were employed to assess cell death induced in honeybee larvae midgut after per os inoculation of bacterium Paenibacillus larvae var. larvae, the causative agent of American foulbrood disease, and separately with acaricide Amitraz and antibiotic Oxytetracycline. In honeybee larvae exposed to Amitraz, which demonstrates both necrosis and apoptosis, cell death was found in 82% of midgut columnar and in 50% of regenerative epithelial cells, 24 h after treatment. Cell death reduced to 36% in the epithelial cells, 48 h after treatment. In Oxytetracycline-treated larvae, cell death was identified in 40% of midgut epithelial cells, 24 h after inoculation and increased to 55% over the next 24 h. In Paenibacillus -infected larvae, all midgut epithelial cells died. Using ApopTag (Oncor) to label the multiple DNA ends generated by DNA fragmentation showed programmed cell death in 49% of columnar midgut cells 24 h after Amitraz application. Cell death was reduced to 9% over the next 24 h. Our data indicate that cell death could be identified and quantified in situ, using TUNEL techniques. This study also shows that the acaricide Amitraz is a trigger for programmed cell death in the midgut epithelial cells of honeybee larvae, unlike Paenibacillus which induces necrosis only. The data show that immunohistochemical methods are useful for studying in situ tissue pathology, and indicate possibilities for monitoring the effects of infective and chemical environmental stressors on cell death in honeybee larvae tissue.

Adrenergic alpha-Agonists↗

PubMed2004

Transcriptional immune responses by honey bee larvae during invasion by the bacterial pathogen, Paenibacillus larvae.

Honey bee larvae are highly susceptible to the bacterial pathogen Paenibacillus larvae only during the first instar of larval development. Transcript levels were measured for genes encoding two antimicrobial peptides, abaecin and defensin, as well as for two candidates in the immune response cascade (PGRP-LD and masquerade) in control larvae and larvae exposed to the pathogen. Transcripts for all four are present throughout development. This suggests that other physiological or dietary factors may better explain the age-based change in vulnerability to this pathogen. One of these genes, abaecin, shows significant up-regulation 24 h following oral inoculation with P. larvae, precisely when the bacterium surmounts the midgut epithelia of bees. Expression of both antimicrobial peptides varied by 1000-fold across different nestmate bees, indicating an allelic component to their expression. The implications of these results for current hypotheses related to disease tolerance in social insects are discussed, along with implications for breeding bees resistant to this important disease.

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PubMed2004

A PCR-based method that permits specific detection of Paenibacillus larvae subsp. larvae, the cause of American Foulbrood of honey bees, at the subspecies level.

AIMS: A reliable procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American Foulbrood disease of honey bees (Apis mellifera L.) based on the polymerase chain reaction (PCR) and subspecies - specific primers is described. METHODS AND RESULTS: By using ERIC-PCR, an amplicon of ca 970 bp was found among P. l. larvae strains but not in other closely related species. Based on the nucleotide sequence data of this amplicon, we designed the pair of oligonucleotides KAT 1 and KAT 2, which were assayed as primers in a PCR reaction. A PCR amplicon of the expected size ca 550 bp was only found in P. l. larvae strains. CONCLUSIONS: This PCR assay provides a specific detection for P. l. larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed PCR assay is highly specific because can differentiate Paenibacillus larvae subsp. larvae from the closely related Paenibacillus larvae subsp. pulvifaciens. The technique can be directly used to detect presence or absence of P. l. larvae spores in honey bee brood samples and contaminated honeys.

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PubMed2004

Biochemical characterization of different genotypes of Paenibacillus larvae subsp. larvae, a honey bee bacterial pathogen.

Paenibacillus larvae subsp. larvae (P. l. larvae) is the aetiological agent of American foulbrood (AFB), the most virulent bacterial disease of honey bee brood worldwide. In many countries AFB is a notifiable disease since it is highly contagious, in most cases incurable and able to kill affected colonies. Genotyping of field isolates of P. l. larvae revealed at least four genotypes (AB, Ab, ab and alpha B) present in Germany which are genotypically different from the reference strain DSM 7030. Therefore, based on these data, five different genotypes of P. l. larvae are now identified with genotype AB standing out with a characteristic brown-orange and circled two-coloured colony morphology. Analysing the metabolic profiles of three German genotypes (AB, Ab and ab) as well as of the reference strain using the Biolog system, a characteristic biochemical fingerprint could be obtained for each strain. Cluster analysis showed that while genotypes Ab, ab and the reference strain DSM 7030 are rather similar, genotype AB is clearly different from the others. Analysis of all isolates for plasmid DNA revealed two different plasmids present only in isolates belonging to genotype AB. Therefore, genotype AB is remarkable in all aspects analysed so far. Future analysis will show whether or not these differences will expand to differences in virulence.

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PubMed2002

Differentiation of Paenibacillus larvae subsp. larvae, the cause of American foulbrood of honeybees, by using PCR and restriction fragment analysis of genes encoding 16S rRNA.

A rapid procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American foulbrood (AFB) disease of honeybees (Apis mellifera L.), based on PCR and restriction fragment analysis of the 16S rRNA genes (rDNA) is described. Eighty-six bacterial strains belonging to 39 species of the genera Paenibacillus, Bacillus, Brevibacillus, and Virgibacillus were characterized. Amplified rDNA was digested with seven restriction endonucleases. The combined data from restriction analysis enabled us to distinguish 35 profiles. Cluster analysis revealed that P. larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens formed a group with about 90% similarity; however, the P. larvae subsp. larvae restriction fragment length polymorphism pattern produced by endonuclease HaeIII was found to be unique and distinguishable among other closely related bacteria. This pattern was associated with DNA extracted directly from honeybee brood samples showing positive AFB clinical signs that yielded the restriction profile characteristic of P. larvae subsp. larvae, while no amplification product was obtained from healthy larvae. The method described here is particularly useful because of the short time required to carry it out and because it allows the differentiation of P. larvae subsp. larvae-infected larvae from all other species found in apiarian sources.

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PubMed2006

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.

The bacterium Paenibacillus larvae, the causative agent of American foulbrood disease of honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of antagonic aerobic spore-forming bacteria isolated from honey samples and other apiarian sources were evaluated. Each isolate was screened against one strain of Paenibacillus larvae (ATCC 9545) by using a perpendicular streak technique. Ten randomly selected bacterial strains from the group that showed the best antagonistic effect to P. larvae ATCC 9545 were selected for further study. These were identified as Bacillus subtilis (m351), B. pumilus (m350), B. licheniformis (m347), B. cereus (mv33), B. cereus (m387), B. cereus (m6c), B. megaterium (m404), Brevibacillus laterosporus (BLAT169), B. laterosporus (BLAT170), and B. laterosporus (BLAT171). The antagonistic strains were tested against 17 P. larvae strains from different geographical origins by means of a spot test in wells. The analysis of variance and posterior comparison of means by Tukey method (P < 0.01) showed that the best antagonists were B. megaterium (m404), B. licheniformis (m347), B. cereus (m6c), B. cereus (mv33), and B. cereus (m387).

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PubMed2026

Comparative genomics and phenotypic divergence of ERIC I and ERIC II genotypes of Paenibacillus larvae, the causative agent of American Foulbrood disease.

Honeybees of the species Apis mellifera are important pollinators of crops and wild plants. Paenibacillus larvae, a spore-forming bacterium, is a problematic pathogen that causes American foulbrood (AFB) in honeybee larvae worldwide. In many countries, AFB is a notifiable disease, requiring the destruction of diseased colonies, resulting in economic loss that impacts beekeeping and agriculture. Disease onset starts with larval ingestion of P. larvae spores, which germinate into growing cells that proliferate in the larval gut, leading to larval death and eventually bee colony collapse. As infection progresses, P. larvae produce spores, reinitiating the disease cycle. Thus, growth, sporulation and germination underlie AFB. In this study, using various microbiological assays, quantitative cell biology methods, transmission electron microscopy and genomics, we sought to identify genetic and phenotypic characteristics associated with the predominant ERIC I and ERIC II genotypes of P. larvae during growth, sporulation and germination. Extending previous findings, our data identify genetic differences between ERIC I and ERIC II strains and some genetic variation between strains of the same ERIC type. Furthermore, we describe significant differences in cellular morphology during growth, differences in spore envelope structure and differences in germination efficiency between ERIC I and ERIC II genotypes. Collectively, our findings improve understanding of P. larvae biology and provide a foundation for developing genotype-specific disease management strategies for AFB.

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PubMed2001

Adult honeybee's resistance against Paenibacillus larvae larvae, the causative agent of the American foulbrood.

American foulbrood is a widespread disease of honeybee larvae caused by the spore-forming bacterium Paenibacillus larvae subsp. larvae. Spores represent the infectious stage; when ingested by a larva they germinate in the midgut. The rod-shaped vegetative forms penetrate the larva's intestinal tissue and start multiplying rapidly, which finally kills the larva. Spores fed to adult honeybees, however, do not harm the bees. We investigated this phenomenon. Specifically, we studied the influence of the adult honeybee midgut on the vegetative growth and on the germination of spores of P. larvae larvae. We focused on two groups of adult workers that are likely to have large numbers of spores in their gastrointestinal tracts in infected colonies: middle-aged bees, which are known to remove or cannibalize dead larvae and clean brood cells, and winterbees, which do not have frequent chances to defecate. We found that midgut extract from winterbees and worker-aged bees of different colonies almost completely inhibited the growth of the vegetative stage of P. larvae larvae and suppressed the germination of spores. The inhibiting substance or substances from the adult midgut are very temperature stable: they still show about 60% of their growth-inhibiting capacity against this bacterium after 15 min at 125 degrees C. We established a method to test growth-inhibiting factors against P. larvae larvae in vitro.

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PubMed2005

Strain- and genotype-specific differences in virulence of Paenibacillus larvae subsp. larvae, a bacterial pathogen causing American foulbrood disease in honeybees.

Virulence variations of Paenibacillus larvae subsp. larvae, the causative agent of American foulbrood disease of honeybees, were investigated by analysis of 16 field isolates of this pathogen, belonging to three previously characterized genotypes, as well as the type strain (ATCC 9545) of P. larvae subsp. larvae, with exposure bioassays. We demonstrated that the strain-specific 50% lethal concentrations varied within an order of magnitude and that differences in amount of time for the pathogen to kill 100% of the infected hosts (LT100) correlated with genotype. One genotype killed rather quickly, with a mean LT100 of 7.8 +/- 1.7 days postinfection, while the other genotypes acted more slowly, with mean LT100s of 11.2 +/- 0.8 and 11.6 +/- 0.6 days postinfection.

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PubMed2003

Development and evaluation of PCR assays for the detection of Paenibacillus larvae in honey samples: comparison with isolation and biochemical characterization.

PCR assays were developed for the direct detection of Paenibacillus larvae in honey samples and compared with isolation and biochemical characterization procedures. Different primer pairs, designed from the 16S rRNA and the metalloproteinase precursor gene regions, and different DNA extraction methods were tested and compared. The sensitivity of the reactions was evaluated by serial dilutions of DNA extracts obtained from P. larvae cultures. The specificity of the primers was assessed by analyzing related Paenibacillus and Bacillus strains isolated from honey. The PCR assays also amplified these related bacteria, but at lower sensitivity. In the next step, the PCR assays were applied to contaminated honey and other bee products originating from 15 countries. Lysozyme treatment followed by proteinase K digestion was determined to be the best DNA extraction method for P. larvae spores. The most sensitive primer pair detected P. larvae in 18 of 23 contaminated honey samples, as well as in pollen, wax, and brood. Honey specimens containing saprophyte bacilli and paenibacilli, but not P. larvae, were PCR negative. Although the isolation and biochemical identification method (BioLog) showed higher sensitivity and specificity, PCR proved to be a valuable technique for large-scale screening of honey samples for American foulbrood, especially considering its rapidity and moderate costs.

Animals↗

PubMed2003

Rapid detection of Paenibacillus larvae from honey and hive samples with a novel nested PCR protocol.

The bacterial pathogen Paenibacillus larvae is the causative agent of American foulbrood disease in honeybees (Apis mellifera). A touchdown nested PCR protocol was developed to detect the presence of P. larvae spores directly in honey and hive samples. This approach allows early discovery of the bacteria even at concentrations below pathogenic levels, opening the door to new prophylactic approaches against American foulbrood and real-time epidemiological studies.

Bacillus↗

PubMed1999

A PCR detection method for rapid identification of Paenibacillus larvae.

American foulbrood is a disease of larval honeybees (Apis mellifera) caused by the bacterium Paenibacillus larvae. Over the years attempts have been made to develop a selective medium for the detection of P. larvae spores from honey samples. The most successful of these is a semiselective medium containing nalidixic acid and pipermedic acid. Although this medium allows the growth of P. larvae and prevents the growth of most other bacterial species, the false-positive colonies that grow on it prevent the rapid confirmation of the presence of P. larvae. Here we describe a PCR detection method which can be used on the colonies that grow on this semiselective medium and thereby allows the rapid confirmation of the presence of P. larvae. The PCR primers were designed on the basis of the 16S rRNA gene of P. larvae and selectively amplify a 973-bp amplicon. The PCR amplicon was confirmed as originating from P. larvae by sequencing in both directions. Detection was specific for P. larvae, and the primers did not hybridize with DNA from closely related bacterial species.

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PubMedDate not supplied

Paenibacillus larvae larvae spores in honey samples from Uruguay: a nationwide survey.

American foulbrood is a severe bacterial disease affecting larvae of the honeybee Apis mellifera and it is caused by Paenibacillus larvae larvae. The disease is present worldwide and cases have been reported in almost all the beekeeping regions of the five continents. During 2001 and 2002 we carried out a nationwide study to assess the presence and amount of P. l. larvae spores in honey samples from Uruguay, combining classic bacteriological, and molecular approaches. The distribution of P. l. larvae spores in honey of the whole country showed a clear pattern and may provide useful data for a control and prevention strategy of American foulbrood.

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PubMed2001

Disinfection of wooden structures contaminated with Paenibacillus larvae subsp. larvae spores.

AIMS: The aim of the study is to examine the disinfection of wood contaminated with Paenibacillus larvae subsp. larvae spores, in order to find a practical decontamination method for hive materials. METHODS AND RESULTS: The number of viable spores recovered after the treatment, on the surface by swabbing, and in the deeper parts of the wood by scraping, was used to test the efficiency of the disinfection. Our results indicate that chemical disinfection is only complete when high concentrations (> 50%) of the disinfectant are used. Heat treatment in general was found to be very effective. The scorching of wood was not satisfactory as it only killed spores at the surface. CONCLUSION: Complete disinfection is only possible with some heat treatments or by using high concentrations of chemical disinfectants. SIGNIFICANCE AND IMPACT OF THE STUDY: This study puts forward some methods that can provide complete decontamination, which is necessary for an effective control of American foulbrood disease.

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