Multiple Locus Variable number of tandem repeat Analysis : a molecular genotyping tool for Paenibacillus larvae

American Foulbrood, caused by Paenibacillus larvae, is the most severe bacterial disease of honey bees (Apis mellifera). To perform genotyping of P.larvae in an epidemiological context, there is a need of a fast and cheap method with a high resolution. Here, we propose Multiple Locus Variable number of tandem repeat Analysis (MLVA). MLVA has been used for typing a collection of 209 P.larvae strains from which 23 different MLVA types could be identified. Moreover, the developed methodology not only permits the identification of the four Enterobacterial Repetitive Intergenic Consensus (ERIC) genotypes, but allows also a discriminatory subdivision of the most dominant ERIC type I and ERIC type II genotypes. A biogeographical study has been conducted showing a significant correlation between MLVA genotype and the geographical region where it was isolated

Complete Genome Sequences of Paenibacillus Larvae Phages BN12, Dragolir, Kiel007, Leyra, Likha, Pagassa, PBL1c, and Tadhana

We present here the complete genomes of eight phages that infect Paenibacillus larvae, the causative agent of American foulbrood in honeybees. Phage PBL1c was originally isolated in 1984 from a P. larvae lysogen, while the remaining phages were isolated in 2014 from bee debris, honeycomb, and lysogens from three states in the USA

First detection of Paenibacillus larvae the causative agent of American Foulbrood in a Ugandan honeybee colony

Paenibacillus larvae is a highly contagious and often lethal widely distributed pathogen of honeybees, Apis mellifera but has not been reported in eastern Africa to date. We investigated the presence of P. larvae in the eastern and western highland agro-ecological zones of Uganda by collecting brood and honey samples from 67 honeybee colonies in two sampling occasions and cultivated them for P. larvae. Also, 8 honeys imported and locally retailed in Uganda were sampled and cultivated for P. larvae. Our aim was to establish the presence and distribution of P. larvae in honeybee populations in the two highland agro-ecological zones of Uganda and to determine if honeys that were locally retailed contained this lethal pathogen. One honeybee colony without clinical symptoms for P. larvae in an apiary located in a protected area of the western highlands of Uganda was found positive for P. larvae. The strain of this P. larvae was genotyped and found to be ERIC I. In order to compare its virulence with P. larvae reference strains, in vitro infection experiments were conducted with carniolan honeybee larvae from the research laboratory at Ghent University, Belgium. The results show that the virulence of the P. larvae strain found in Uganda was at least equally high. The epidemiological implication of the presence of P. larvae in a protected area is discussed

Distribution and transmission of American foulbrood in honey bees

The distribution of Paenibacillus larvae spores, the causative agent of American foulbrood, was studied on three different levels in the honey bee system; the apiary level, the colony level and the individual honey bee level. The increased understanding of spore distribution has been used to give recommendations regarding sampling of adult honey bees. The vertical transmission of P. larvae spores through natural swarms has been described for the first time and artificial swarming as a method for control of American foulbrood have been evaluated. The results demonstrated that there is no practical difference in spore load between supers and brood chambers, and that the spore load in samples of adult honey bees on the different levels correspond to the clinical disease status of the colony. The study on individual bees showed that spores are unequally distributed among the bees and that as more bees get contaminated each positive bee also contains more spores. This may present a problem when sampling from colonies with low levels of clinical disease, although the study on colony and apiary level showed no false negatives. A model for calculating the number of bees that needs to be sampled to detect P. larvae in a composite sample of adult bees, given certain detection levels and proportions of positive honey bees in the sample, was developed The swarm study demonstrated vertical transmission of P. larvae spores. Furthermore, the artificial swarm study showed that single and double shaking are equally effective treatment methods, and that the original disease status is of little importance for the spore load decrease

Optimal concentration of organic solvents to be used in the broth microdilution method to determine the antimicrobial activity of natural products against Paenibacillus larvae

American Foulbrood (AFB) is a bacterial disease, caused by Paenibacillus larvae, that affects honeybees (Apis mellifera). Alternative strategies to control AFB are based on the treatment of the beehives with antimicrobial natural substances such as extracts, essential oils and/or pure compounds from plants, honey by-products, bacteria and moulds. The broth microdilution method is currently one of the most widely used methods to determine the minimum inhibitory concentration (MIC) of a substance. In this regard, the fact that most natural products, due to their lipophilic nature, must be dissolved in organic solvents or their aqueous mixtures is an issue of major concern because the organic solvent becomes part of the dilution in the incubation medium, and therefore, can interfere with bacterial viability depending on its nature and concentration. A systematic study was carried out to determine by the broth microdilution method the MIC and the maximum non inhibitory concentration (MNIC) against P. larvae of the most common organic solvents used to extract or dissolve natural products, i.e. ethanol, methanol, acetonitrile, n-butanol, dimethylsulfoxide, and acidified hydromethanolic solutions. From the MIC and MNIC for each organic solvent, recommended maximum concentrations in contact with P. larvae were established: DMSO 5% (v/v), acetonitrile 7.5% (v/v), ethanol 7.5% (v/v), methanol 12% (v/v), n-butanol 1% (v/v), and methanol-water-acetic acid (1.25:98.71:0.04, v/v/v).Fil: Cugnata, Noelia Melina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biología; ArgentinaFil: Guaspari, Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biología; ArgentinaFil: Pellegrini, María Celeste. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biología; ArgentinaFil: Fuselli, Sandra Rosa. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biología; ArgentinaFil: Alonso Salces, Rosa Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Biología; Argentin

Comparative Genomics of 9 Novel Paenibacillus Larvae Bacteriophages

American Foulbrood Disease, caused by the bacterium Paenibacillus larvae, is one of the most destructive diseases of the honeybee, Apis mellifera. Our group recently published the sequences of 9 new phages with the ability to infect and lyse P. larvae. Here, we characterize the genomes of these P. larvae phages, compare them to each other and to other sequenced P. larvae phages, and putatively identify protein function. The phage genomes are 38–45 kb in size and contain 68–86 genes, most of which appear to be unique to P. larvae phages. We classify P. larvae phages into 2 main clusters and one singleton based on nucleotide sequence identity. Three of the new phages show sequence similarity to other sequenced P. larvae phages, while the remaining 6 do not. We identified functions for roughly half of the P. larvae phage proteins, including structural, assembly, host lysis, DNA replication/metabolism, regulatory, and host-related functions. Structural and assembly proteins are highly conserved among our phages and are located at the start of the genome. DNA replication/metabolism, regulatory, and host-related proteins are located in the middle and end of the genome, and are not conserved, with many of these genes found in some of our phages but not others. All nine phages code for a conserved N-acetylmuramoyl-L-alanine amidase. Comparative analysis showed the phages use the “cohesive ends with 30 overhang” DNA packaging strategy. This work is the first in-depth study of P. larvae phage genomics, and serves as a marker for future work in this area

Genomic analysis of 48 paenibacillus larvae bacteriophages

Indexación: Scopus.Funding: Research at UNLV was funded by National Institute of General Medical Sciences grant GM103440 (NV INBRE), the UNLV School of Life Sciences, and the UNLV College of Sciences. E.C.-N. was funded by CONICYT-FONDECYT de iniciación en la investigación 11160905. Research at BYU was funded by the BYU Microbiology & Molecular Biology Department, and private donations through LDS Philanthropies.The antibiotic-resistant bacterium Paenibacillus larvae is the causative agent of American foulbrood (AFB), currently the most destructive bacterial disease in honeybees. Phages that infect P. larvae were isolated as early as the 1950s, but it is only in recent years that P. larvae phage genomes have been sequenced and annotated. In this study we analyze the genomes of all 48 currently sequenced P. larvae phage genomes and classify them into four clusters and a singleton. The majority of P. larvae phage genomes are in the 38–45 kbp range and use the cohesive ends (cos) DNA-packaging strategy, while a minority have genomes in the 50–55 kbp range that use the direct terminal repeat (DTR) DNA-packaging strategy. The DTR phages form a distinct cluster, while the cos phages form three clusters and a singleton. Putative functions were identified for about half of all phage proteins. Structural and assembly proteins are located at the front of the genome and tend to be conserved within clusters, whereas regulatory and replication proteins are located in the middle and rear of the genome and are not conserved, even within clusters. All P. larvae phage genomes contain a conserved N-acetylmuramoyl-L-alanine amidase that serves as an endolysin. © 2018 by the authors. Licensee MDPI, Basel, Switzerland.https://www.mdpi.com/1999-4915/10/7/37

Modelling the spread of American foulbrood in honeybees

We investigate the spread of American foulbrood (AFB), a disease caused by the bacterium Paenibacillus larvae, that affects bees and can be extremely damaging to beehives. Our dataset comes from an inspection period carried out during an AFB epidemic of honeybee colonies on the island of Jersey during the summer of 2010. The data include the number of hives of honeybees, location and owner of honeybee apiaries across the island. We use a spatial SIR model with an underlying owner network to simulate the epidemic and characterize the epidemic using a Markov chain Monte Carlo (MCMC) scheme to determine model parameters and infection times (including undetected ‘occult’ infections). Likely methods of infection spread can be inferred from the analysis, with both distance- and owner-based transmissions being found to contribute to the spread of AFB. The results of the MCMC are corroborated by simulating the epidemic using a stochastic SIR model, resulting in aggregate levels of infection that are comparable to the data. We use this stochastic SIR model to simulate the impact of different control strategies on controlling the epidemic. It is found that earlier inspections result in smaller epidemics and a higher likelihood of AFB extinction

Isolation, characterization, and genetic diversity of Paenibacillus larvae from AFB suspected specimens in the Central and Eastern Black Sea Regions

American foulbrood is an important bacterial disease affecting the larvae of honey bees (Apis mellifera L.) caused by Paenibacillus larvae. Due to easy transmission of disease and the ability of bacteria to create spores, it is a bacterium resistant to both physical and chemical conditions. The study aims to isolate, perform microbiological analyses, and determine biochemical properties and genotypes P. larvae strains from AFB samples collected from Turkey's Central and Eastern Black Sea regions. An isolation study was conducted on adult bees, larvae, honey, and primary honeycomb samples from suspected colonies in the regions under study. After the purification of bacterial isolates from samples, P. larvae strains were identified using biochemical and molecular methods. The genetic diversity and ERIC types of P. larvae isolates were determined by rep-PCR DNA genotyping using BOX A1R and MBO REP1 primers and multiplex-PCR. A phylogenetic tree of P. larvae strains was constructed in the study. All P. larvae isolates were determined as ERIC I type. According to the rep-PCR results of P. larvae strains, 15 of the 28 isolates were Ab genotype (54%), 7 (25%) Aβ genotype, 4 (14%) AB genotype, 1 (3.5%) αB genotype, and 1 (3.5%) ab genotype. From an epidemiological viewpoint, it was determined that Ab and Aβ genotypes were widely distributed, while other genotypes (AB, αB, and ab) showed less spread. The results of the study will guide researchers in taking relevant measures to prevent and control American foulbrood. © 2023, The Author(s), under exclusive licence to Plant Science and Biodiversity Centre, Slovak Academy of Sciences (SAS), Institute of Zoology, Slovak Academy of Sciences (SAS), Institute of Molecular Biology, Slovak Academy of Sciences (SAS)

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.Facultad de Ciencias Agrarias y ForestalesCentro de Investigaciones de Fitopatologí