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.     

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.     

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).     

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.     

Ribosomal protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for phylogenety-based subspecies resolution of Bifidobacterium longum

The taxonomic positions of the subspecies of Bifidobacterium longum (B. longum subsp. longum, subsp. infantis, and subsp. suis) have been controversial. A current proposal is that the former two species “B. infantis” and “B. suis” be unified with B. longum and all three reclassified as three subspecies. To test this proposal, ribosomal protein profiling as observed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied to the classification of 17 strains of B. longum, including three subspecies. Among 41 different kinds of ribosomal proteins selected as biomarkers whose masses were calculated from their amino acid sequences, 31-41 ribosomal proteins were observed in sample strains with the same masses as the references. The high matching rate indicates high conservation of ribosomal proteins within the sample strains, and therefore strongly supports the unification of the former species. However, the masses of some ribosomal proteins varied within species. The phylogenetic tree constructed from the profiles of ribosomal proteins matched the references, showing a clear cluster of the subsp. longum and the subsp. infantis strains. This result supports the proposal to reclassify B. longum into subsp. longum and subsp. infantis. The subsp. suis strains formed an individual sub-cluster within the infantis cluster. However, their ribosomal proteins have both characters of longum and infantis types. This result suggests that the taxonomic position of the subsp. suis should be reconsidered.     

Bacteria in the gut of Japanese honeybee, Apis cerana japonica, and their antagonistic effect against Paenibacillus larvae, the causal agent of American foulbrood

We assessed the complexity of bacterial communities occurring in the digestive tract of the Japanese honeybee, Apis cerana japonica, using histological and 16S rRNA gene sequence analyzes. Both Gram-positive and -negative bacteria were observed, and the number of gut bacteria was higher in old larvae compared with young larvae. A total of 35 clones were obtained by a culture-dependent method, and 16S rRNA gene sequence analysis revealed that the bacterial population in the gut of Japanese honeybee was diverse, including the phyla firmicutes, actinobacteria, and alpha-, beta-, and gammaproteobacteria. Further investigation by in vitro inhibition assays was carried out to determine the ability of an isolate to inhibit Paenibacillus larvae, the causal agent of American foulbrood. Out of 35 isolates, seven showed strong inhibitory activity against P. larvae. Most of the antagonistic bacteria belonged to Bacillus species, suggesting that the bacterial isolates obtained in this study appear to be potential candidates for the biological control of P. larvae.     

Detection of Paenibacillus larvae subspecies larvae spores in naturally infected bee larvae and artificially contaminated honey by PCR

American foulbrood (AFB), a severe bacterial disease of honeybee brood, has recently been found in Uruguayan apiaries. Detection of the causative agent, Paenibacillus larvae subspecies larvae, is a very important concern in order to prevent disease dissemination and decrease of honey production. Since spores are the infective forms of this pathogen, in the present work we report the use of polymerase chain reaction (PCR) to detect P. l. subsp. larvae spores from in vitro cultures, larvae with clinical symptoms and experimentally contaminated honey. The set of primers was designed based on the published P. l. subsp. larvae 16S rRNA gene. Using this approach we could amplify the pathogen DNA and obtain a great sensitivity and a notable specificity. Detection limit for spore suspension was a 10^–2 dilution of template DNA obtained from 32 spores, as determined by plate count. For artificially contaminated honey, we could detect the PCR product at a 10^–3 dilution of template DNA obtained from 170 spores. In addition, when PCR conditions were set to improve specificity, we were able to amplify P. l. subsp. larvae DNA selectively and no cross-reactions were observed with a variety of related bacterial species, including P. l. subsp. pulvifaciens. Since spore detection is very important to confirm the presence of the disease, this method provides a reliable diagnosis of AFB from infected larvae and contaminated honey in a few hours.     

American foulbrood and African honey bees (Hymenoptera: Apidae)

We have taken samples of honey from individual beekeepers (N = 64), and of domestic (N = 35) and imported honey (N = 15) retailed in supermarkets in several sub-Saharan countries and cultivated these samples for Paenibacillus larvae subsp. larvae Heyndrickx et al. causing American foulbrood in honey bee colonies. The results are compared with samples of similar backgrounds and treated the same way but collected in Sweden (N = 35). No P. larvae subsp. larvae spores were found in any honey produced in Africa south of the Sahara although honey imported into this region frequently contains the pathogen. Swedish honey frequently contains P. larvae subsp. larvae spores although the general level of visibly infected bee colonies is low (roughly 0.5%). The results suggest that large parts of Africa may be free from American foulbrood. Behavioral studies (hygienic behavior) on Apis mellifera subsp. scutellata Lepeletier in Zimbabwe suggest that hygienic behavior of African bees could influence the apparent low level, or even absence of American foulbrood in large parts of Africa.     

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.     

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.     

Low-Molecular-Weight Metabolites Secreted by Paenibacillus larvae as Potential Virulence Factors of American Foulbrood

The spore-forming bacterium Paenibacillus larvae causes a severe and highly infective bee disease, American foulbrood (AFB). Despite the large economic losses induced by AFB, the virulence factors produced by P. larvae are as yet unknown. To identify such virulence factors, we experimentally infected young, susceptible larvae of the honeybee, Apis mellifera carnica, with different P. larvae isolates. Honeybee larvae were reared in vitro in 24-well plates in the laboratory after isolation from the brood comb. We identified genotype-specific differences in the etiopathology of AFB between the tested isolates of P. larvae, which were revealed by differences in the median lethal times. Furthermore, we confirmed that extracts of P. larvae cultures contain low-molecular-weight compounds, which are toxic to honeybee larvae. Our data indicate that P. larvae secretes metabolites into the medium with a potent honeybee toxic activity pointing to a novel pathogenic factor(s) of P. larvae. Genome mining of P. larvae subsp. larvae BRL-230010 led to the identification of several biosynthesis gene clusters putatively involved in natural product biosynthesis, highlighting the potential of P. larvae to produce such compounds.     

Species distribution of staphylococci from small wild mammals

A total of 197 isolates of Staphylococcus from small wild animals (insectivores and rodents) were identified by partial sequencing of the rpoB and dnaJ genes. Among the identified isolates the predominant species was S. succinus (28%), followed by S. xylosus (20.8%) and S. stepanovicii (18.3%). The other 14 Staphylococcus species were occasionally isolated. PCR-RFLP of the rpoB gene digested by Hpy8I was a fast and simple method to distinguish the two subspecies of S. succinus. More than 90% of the 55 S. succinus strains isolated belonged to S. succinus subsp. casei and only 9% to S. succinus subsp. succinus. Moreover, the present study describes the first ever isolation of S. fleurettii from healthy animals.     

Molecular and phenotypic characterization of Vibrio aestuarianus subsp. francensis subsp. nov., a pathogen of the oyster Crassostrea gigas

Eleven Vibrio isolates invading the hemolymph of live and moribund oysters (Crassostrea gigas) collected in the field and from a hatchery in France, were characterized by a polyphasic approach. Phylogenetic analysis of 16S rRNA, gyrB and toxR genes indicated high homogeneity between these strains and the Vibrio aestuarianus type strain (ATCC35048(T)), and confirmed previous 16S rRNA analysis. In contrast, DNA:DNA hybridization was from 61% to 100%, while phenotypic characters and virulence tests showed a large diversity between the strains. Nevertheless, several common characters allowed the isolates to be distinguished from the reference strain. On the basis of several distinct phenotypic characteristics, it is proposed to establish two subspecies within the V. aestuarianus spp. group, V. aestuarianus subsp. aestuarianus [D. Tison, R. Seidler, Vibrio aestuarianus: a new species from estuarine waters and shellfish, Int. J. Syst. Bacteriol. (1983) 699-702] and V. aestuarianus subsp. francensis for these French isolates. The characters that differentiate the new strains from V. aestuarianus subsp. aestuarianus(T) are virulence (positive for 63% of the isolates) and 12:0 fatty acid content. The colonies were smaller and uncoloured, whereas no growth occurred at 35 degrees C or on TCBS, and the strains did not utilize several substrates, including L-serine, alpha-cyclodextrin, D-mannitol, alpha-glycyl-L-aspartic acid, L-threonine and glucose-1-phosphate.     

In vitro study of the antimicrobial activity of Brazilian propolis against Paenibacillus larvae

The honey bee disease American foulbrood (AFB) is a serious problem since its causative agent (Paenibacillus larvae) has become increasingly resistant to conventional antibiotics. The objective of this study was to investigate the in vitro activity of propolis collected from various states of Brazil against P. larvae. Propolis is derived from plant resins collected by honey bees (Apis mellifera) and is globally known for its antimicrobial properties and particularly valued in tropical regions. Tests on the activity of propolis against P. larvae were conducted both in Brazil and Minnesota, USA using two resistance assay methods that measured zones of growth inhibition due to treatment exposure. The propolis extracts from the various states of Brazil showed significant inhibition of P. larvae. Clear dose responses were found for individual propolis extracts, particularly between the concentrations of 1.7 and 0.12 mg propolis/treatment disk, but the source of the propolis, rather than the concentration, may be more influential in determining overall activity. Two of the three tested antibiotics (tylosin and terramycin) exhibited a greater level of inhibition compared to most of the Brazilian samples, which could be due to the low concentrations of active compounds present in the propolis extracts. Additionally, the majority of the Brazilian propolis samples were more effective than the few collected in MN, USA. Due to the evolution of resistance of P. larvae to conventional antibiotic treatments, this research is an important first step in identifying possible new active compounds to treat AFB in honey bee colonies.     

Complete genome sequence of Polynucleobacter necessarius subsp. asymbioticus type strain (QLW-P1DMWA-1(T))

Polynucleobacter necessarius subsp. asymbioticus strain QLW-P1DMWA-1(T) is a planktonic freshwater bacterium affiliated with the family Burkholderiaceae (class Betaproteobacteria). This strain is of interest because it represents a subspecies with cosmopolitan and ubiquitous distribution in standing freshwater systems. The 16S-23S ITS genotype represented by the sequenced strain comprised on average more than 10% of bacterioplankton in its home habitat. While all strains of the subspecies P. necessarius asymbioticus are free-living freshwater bacteria, strains belonging to the only other subspecies, P. necessarius subsp. necessarius are obligate endosymbionts of the ciliate Euplotes aediculatus. The two subspecies of P. necessarius are the instances of two closely related subspecies that differ in their lifestyle (free-living vs. obligate endosymbiont), and they are the only members of the genus Polynucleobacter with completely sequenced genomes. Here we describe the features of P. necessarius subsp. asymbioticus, together with the complete genome sequence and annotation. The 2,159,490 bp long chromosome with a total of 2,088 protein-coding and 48 RNA genes is the first completed genome sequence of the genus Polynucleobacter to be published and was sequenced as part of the DOE Joint Genome Institute Community Sequencing Program 2006.     

The possible role of Varroa destructor in the spreading of American foulbrood among apiaries

The aim of this investigation was to establish whether Varroa destructor can play a role in the transmission of Paenibacillus larvae larvae spores from infected to healthy bee colonies. Mites, collected from an Apis mellifera carnica colony heavily infected with American foulbrood and treated with Apistan, were suspended in distilled water and treated in three different ways:homogenizing, shaking and stirring, or sonication. The resulting fluid samples were transferred onto selective agar medium. All culture plates showed colonies that could be identified as P.l. larvae. In view of the numbers of spores they can carry, it is concluded that mites may transmit American foulbrood from infected to healthy bee colonies.     

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.     

蜜蜂幼虫粪肠球菌的分离与鉴定

在对患有欧洲幼虫腐臭病的蜜蜂幼虫进行细菌分离与培养时,获得1株未知菌株。从分离培养特性、形态学、生理生化特性和分子生物学等方面对该菌株进行了鉴定,得知该菌株为欧洲幼虫腐臭病的次生菌——粪肠球菌,属于肠球菌属,并暂定名为Enterococcus faecalis FB102。同时,得知根据该菌的分离培养特性可与欧洲幼虫腐臭病病原区分,并且将其单独接种蜜蜂幼虫后未能使幼虫患病。根据粪肠球菌较欧洲幼虫腐臭病病原易于分离培养的特性,得知通过对粪肠球菌的鉴定可以简化欧洲幼虫腐臭病的诊断过程,而且推测该菌株可能对人体健康有一定的影响。     

Pathogenic effects of bacteria isolated from larvae of Hylesia metabus Crammer (Lepidoptera: Saturniidae)

Hylesia metabus larvae are susceptible to several pathogens indigenous to the area in which they are found. Some larvae show symptoms characteristic of bacterial infection; they become flaccid and lethargic, and show a marked loss of appetite. We isolated and identified 29 bacterial strains from live, dead and experimentally infected H. metabus larvae, and evaluated their pathogenic activity. The bacteria which caused mortality in the larvae were: Pseudomonas aeruginosa (60-93.3%), Proteus vulgaris (20%), Alcaligenes faecalis, Planococcus sp. and Bacillus megaterium (10%), at doses of 3-4 x 10(7). Although P. aeruginosa is a well-known insect pathogen, this is the first report of its pathogenic activity on H. metabus. The potential risk to humans and low virulence make it unlikely that P. aeruginosa could be used in an augmentative biological control programme. However its natural incidence may be enhanced using parasites and predators of H. metabus as carriers.     

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.