Effect on the Antioxidant Properties of Native Chilean Endemic Honeys Treated with Ionizing Radiation to Remove American Foulbrood Spores.

In Chile, honey is produced from several native species with interesting biological properties. Accordingly, those attributes are present in Chilean honeys owing to the presence of phenolic compounds inherited from specific floral sources. In recent years, the exported volume of Chilean honeys has been increased, reaching new markets with demanding regulations directed toward the fulfilment of consumers' expectations. Accordingly, there are countries with special requirements referring to Paenibacillus larvae spore-free honeys. This microorganism is the pathogen responsible for American foulbrood disease in beehives; however, antibiotics are not allowed when an apiary tests positive for P. larvae. On the other hand, it is mandatory to have an accurate method to remove the potential presence of spores in bee products intended for export. Exposure to ionizing radiation can be an efficient way to achieve this goal. In this work, 54 honey samples harvested from northern, central and southern Chile were analyzed for physicochemical patterns, total phenols, antioxidant activity and antiradical activity. Honeys with and without spores were exposed to ionizing radiation at three levels of intensity. Afterwards, the presence of spores and the effect on phenol bioavailability, antiradical activity and antioxidant activity were measured again. This research presents results showing a positive correlation between the percentage of prevalence of native endemic species in the set of honeys analyzed and the capacity to resist this process, without altering their natural attributes determined before irradiation treatments.

Probing the interaction of Paenibacillus larvae bacteriophage as a biological agent to control the american foulbrood disease in honeybee.

American foulbrood (AFB) is a harmful honeybee disease primarily caused by Paenibacillus larvae. The study aims to isolate and identify the AFB causative agent P. larvae and their specific phages to use as a new biological method for AFB disease control. Eight apiaries were inspected for AFB infections. Symptoms of diseased brood comb, were odd brood cells with soft brown decayed brood amongst healthy brood, were identified in the field and demonstrated the prevalence of AFB in every apiary. Three P. larvae isolates were identified using traditional techniques using a 452-bp PCR amplicon specific to the bacterial 16SrRNA gene and was compared between Paenibacillus isolates. Additionally, specific phages of P. larvae strains were applied to examine their efficiency in reducing the infection rate under the apiary condition. The infection rate was reduced to approximately 94.6 to 100 % through the application of a phage mixture, as opposed to 20 to 85.7 % when each phage was administered individually or 78.6 to 88.9 % when antibiotic treatment was implemented. Histological studies on phage-treated bee larvae revealed some cells regaining normal shape, with prominent nuclei and microvilli. The gastrointestinal tract showed normal longitudinal and circular muscles, unlike bee larvae treated with bacterial strains with abnormal and destroyed tissues, as shown by the basement membrane surrounding the mid-gut epithelium. Phage techniques exhibited promise in resolving the issue of AFB in honeybees due to their ease of application, comparatively lower cost, and practicality for beekeepers in terms of laboratory preparation.

Core proteome mediated subtractive approach for the identification of potential therapeutic drug target against the honeybee pathogen Paenibacillus larvae.

Background & Objectives: American foulbrood (AFB), caused by the highly virulent, spore-forming bacterium Paenibacillus larvae, poses a significant threat to honey bee brood. The widespread use of antibiotics not only fails to effectively combat the disease but also raises concerns regarding honey safety. The current computational study was attempted to identify a novel therapeutic drug target against P. larvae, a causative agent of American foulbrood disease in honey bee. Methods: We investigated effective novel drug targets through a comprehensive in silico pan-proteome and hierarchal subtractive sequence analysis. In total, 14 strains of P. larvae genomes were used to identify core genes. Subsequently, the core proteome was systematically narrowed down to a single protein predicted as the potential drug target. Alphafold software was then employed to predict the 3D structure of the potential drug target. Structural docking was carried out between a library of phytochemicals derived from traditional Chinese flora (n > 36,000) and the potential receptor using Autodock tool 1.5.6. Finally, molecular dynamics (MD) simulation study was conducted using GROMACS to assess the stability of the best-docked ligand. Results: Proteome mining led to the identification of Ketoacyl-ACP synthase III as a highly promising therapeutic target, making it a prime candidate for inhibitor screening. The subsequent virtual screening and MD simulation analyses further affirmed the selection of ZINC95910054 as a potent inhibitor, with the lowest binding energy. This finding presents significant promise in the battle against P. larvae. Conclusions: Computer aided drug design provides a novel approach for managing American foulbrood in honey bee populations, potentially mitigating its detrimental effects on both bee colonies and the honey industry.

Effects of Disinfectants on Bacterium Paenibacillus larvae in Laboratory Conditions.

American foulbrood is an infectious disease of the honeybee brood that causes multiple types of damage to beekeeping. The causative agent of the disease is the bacterium Paenibacillus larvae, which forms resistant infective spores and is viable for decades. After the eradication measures have been implemented, in cases of clinically visible disease, it is necessary to conduct effective final disinfections of equipment and tools. This study aimed to determine the effect of ten commercially available and commonly used disinfectants on certified strains of P. larvae under laboratory conditions, as well as to compare the obtained results among individual genotypes of P. larvae. Selected products were tested by determining the zone of inhibition using an agar diffusion test, a suspension test for viable bacteria, a surface disinfectant test, and a sporicidal effect in the suspension test. Incidin OxyFoam S and Sekusept Aktiv are both effective against all examined genotypes of P. larvae. Despadac and Despadac Secure have a bactericidal effect, but their sporocidal effect is not as satisfactory as that of Genox. Genoll does not exhibit a sporicidal effect, and Ecocide S at 1%, Bee protect H forte, and Bee protect F did not exhibit a satisfactory sporocidal effect. Additionally, EM® PROBIOTIC FOR BEES did not exhibit any bactericidal effect. The effective application of control measures and proper application of final disinfection can reduce the reoccurrence of visible clinical signs of disease, whereas methods of early diagnosis can significantly reduce the incidence of the disease.

Simultaneous PCR detection of Paenibacillus larvae targeting insertion sequence IS256 and Melissococcus plutonius targeting pMP1 plasmid from hive specimens.

Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)-American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I-IV (strain DSM 7030-ERIC I, DSM 25430-ERIC II, LMG 16252-ERIC III, DSM 3615-ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius.

Spent coffee grounds extract: antimicrobial activity against Paenibacillus larvae and its effect on the expression of antimicrobial peptides in Apis mellifera.

In recent years, natural alternatives have been sought for the control of beekeeping pathologies; in the case of American Foulbrood (AFB) disease, the use of synthetic antibiotics was prohibited due to honey contamination and the generation of resistant bacteria. The significant increase in population growth worldwide has led to great concern about the production of large amounts of waste, including those from agribusiness. Among the most important beverages consumed is coffee, generating thousands of tons of waste called spent coffee grounds (SCG). The SCG is a source of many bioactive compounds with known antimicrobial activity. The aims of the present work were: (1) to obtain and chemically analyse by HPLC of SCG extracts (SCGE), (2) to analyse the antimicrobial activity of SCGE against vegetative form of Paenibacillus larvae (the causal agent of AFB), (3) to evaluate the toxicity in bees of SCGE and (4) to analyse the effect of the extracts on the expression of various genes of the immune system of bees. SCGs have a high content of phenolic compounds, and the caffeine concentration was of 0.3%. The MIC value obtained was 166.667 µg/mL; the extract was not toxic to bees, and interestingly, overexpression of abaecin and hymenoptaecin peptides was observed. Thus, SCGE represents a promising alternative for application in the control of American Foulbrood and as a possible dietary supplement to strengthen the immune system of honeybees. Therefore, the concept of circular bio-economy could be applied from the coffee industry to the beekeeping industry.

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)

Anti-Bacterial Activity of Four Distinct Propolis Extracts against P. larvae and M. plutonius; Etiological Agent of American and European Foulbrood Disease of Honeybees.

Four different propolis samples obtained from different regions of Iran were evaluated for their antibacterial effects against the bacterial agents responsible for two important honeybee diseases. Paenibacillus larvae (P. larvae) and Melissococcus plutonius (M. plutonius), as the etiological agents of American foulbrood (AFB) and European foulbrood (EFB) diseases, were subjected to propolis ethanolic extracts in the agar well diffusion assay. The minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) of the antibacterial effects of the samples against the two indicator organisms were determined by the microdilution technique using different concentrations of the propolis extracts. Finally, the synergistic antibacterial actions of the mixed propolis samples were determined, and their MIC and MBC values were recorded. A two-way analysis of variance was used to evaluate correlations among the diameters of the inhibition zones, the bacterial agents, and the propolis extracts. Based on our results, three of the propolis samples showed significant antibacterial effects against P. larvae and M. plutonius during the agar well diffusion assay. Furthermore, the antibacterial capacity of the propolis samples, when mixed in equal proportions, was significantly enhanced, as indicated by the obtained MIC and MBC values. Approximately, 0.02 mg/mL of mixed propolis samples was required for inhibiting the growth of both pathogens. A direct correlation was observed between propolis concentrations and their antibacterial activity. The results of the study are conclusive of the significant antibacterial actions of Iranian propolis samples against the etiological agents of the mentioned honeybee diseases, suggesting their probable use as a safe biological agent to control AFB and EFB diseases.

Oxytetracycline-resistant Paenibacillus larvae identified in commercial beekeeping operations in Saskatchewan using pooled honey sampling.

American foulbrood (AFB) is an infectious disease of honey bee brood caused by the endospore-forming bacterium Paenibacillus larvae. P. larvae spores are resilient in the environment, thus colonies with clinical signs of AFB are often destroyed by burning to eradicate the causative agent. To prevent outbreaks of AFB, oxytetracycline metaphylaxis is widely used in North America, resulting in sustained selective pressure for oxytetracycline resistance in P. larvae. To determine if antimicrobial resistance (AMR) is present among P. larvae isolates from commercial beekeeping operations in Saskatchewan, Canada, we performed antimicrobial susceptibility testing of 718 P. larvae samples cultured from pooled, extracted honey collected from 52 beekeepers over a 2-y period, 2019 and 2020. We found that 65 of 718 (9%) P. larvae samples collected from 8 beekeepers were resistant to oxytetracycline with minimum inhibitory concentration (MIC) values of 64-256 µg/mL. Eight of 718 (1%) samples from 4 beekeepers had intermediate resistance to oxytetracycline (MIC: 4-8 µg/mL). Susceptibility testing for tylosin and lincomycin indicated that P. larvae in Saskatchewan continue to be susceptible to these antimicrobials (tylosin MIC: <1 µg/mL, lincomycin MIC: ≤2 µg/mL). Most oxytetracycline-resistant P. larvae samples were identified in northeastern Saskatchewan. Whole-genome sequence analysis identified the P. larvae-specific plasmid pMA67 with tetracycline-resistance gene tet(L) in 9 of 11 oxytetracycline-resistant P. larvae isolates sequenced. Our results highlight the advantage of using pooled, extracted honey as a surveillance tool for monitoring AMR in P. larvae.

Probiotic candidates for controlling Paenibacillus larvae, a causative agent of American foulbrood disease in honey bee.

BACKGROUND: American foulbrood (AFB) disease caused by Paenibacillus larvae is dangerous, and threatens beekeeping. The eco-friendly treatment method using probiotics is expected to be the prospective method for controlling this pathogen in honey bees. Therefore, this study investigated the bacterial species that have antimicrobial activity against P. larvae. RESULTS: Overall, 67 strains of the gut microbiome were isolated and identified in three phyla; the isolates had the following prevalence rates: Firmicutes 41/67 (61.19%), Actinobacteria 24/67 (35.82%), and Proteobacteria 2/67 (2.99%). Antimicrobial properties against P. larvae on agar plates were seen in 20 isolates of the genus Lactobacillus, Firmicutes phylum. Six representative strains from each species (L. apis HSY8_B25, L. panisapium PKH2_L3, L. melliventris HSY3_B5, L. kimbladii AHS3_B36, L. kullabergensis OMG2_B25, and L. mellis OMG2_B33) with the largest inhibition zones on agar plates were selected for in vitro larvae rearing challenges. The results showed that three isolates (L. apis HSY8_B25, L. panisapium PKH2_L3, and L. melliventris HSY3_B5) had the potential to be probiotic candidates with the properties of safety to larvae, inhibition against P. larvae in infected larvae, and high adhesion ability. CONCLUSIONS: Overall, 20 strains of the genus Lactobacillus with antimicrobial properties against P. larvae were identified in this study. Three representative strains from different species (L. apis HSY8_B25, L. panisapium PKH2_L3, and L. melliventris HSY3_B5) were evaluated to be potential probiotic candidates and were selected for probiotic development for the prevention of AFB. Importantly, the species L. panisapium isolated from larvae was identified with antimicrobial activity for the first time in this study.

Genomic analysis of the population structure of Paenibacillus larvae in New Zealand.

New Zealand is a remote country in the South Pacific Ocean. The isolation and relatively late arrival of humans into New Zealand has meant there is a recorded history of the introduction of domestic species. Honey bees (Apis mellifera) were introduced to New Zealand in 1839, and the disease American foulbrood was subsequently found in the 1870s. Paenibacillus larvae, the causative agent of American foulbrood, has been genome sequenced in other countries. We sequenced the genomes of P. larvae obtained from 164 New Zealand apiaries where American foulbrood was identified in symptomatic hives during visual inspection. Multi-locus sequencing typing (MLST) revealed the dominant sequence type to be ST18, with this clonal cluster accounting for 90.2% of isolates. Only two other sequence types (with variants) were identified, ST5 and ST23. ST23 was only observed in the Otago area, whereas ST5 was limited to two geographically non-contiguous areas. The sequence types are all from the enterobacterial repetitive intergenic consensus I (ERIC I) genogroup. The ST18 and ST5 from New Zealand and international P. larvae all clustered by sequence type. Based on core genome MLST and SNP analysis, localized regional clusters were observed within New Zealand, but some closely related genomes were also geographically dispersed, presumably due to hive movements by beekeepers.

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.

A Comparison of the Antibacterial Efficacy of Carbohydrate Lipid-like (Thio)Ether, Sulfone, and Ester Derivatives against Paenibacillus larvae.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), the most serious bacterial disease affecting developing honeybee larvae and pupas. In this study, a library of 24 (thio)glycosides, glycosyl sulfones, 6-O-esters, and ethers derived from d-mannose, d-glucose, and d-galactose having C10 or C12 alkyl chain were evaluated for their antibacterial efficacy against two P. larvae strains. The efficacy of the tested compounds determined as minimal inhibitory concentrations (MICs) varied greatly. Generally, dodecyl derivatives were found to be more potent than their decylated analogs. Thioglycosides were more efficient than glycosides and sulfones. The activity of the 6-O-ether derivatives was higher than that of their ester counterparts. Seven derivatives with dodecyl chain linked (thio)glycosidically or etherically at C-6 showed high efficacy against both P. larvae strains (MICs ranged from 12.5 µM to 50 µM). Their efficacies were similar or much higher than those of selected reference compounds known to be active against P. larvae-lauric acid, monolaurin, and honeybee larval food components, 10-hydroxy-2-decenoic acid, and sebacic acid (MICs ranged from 25 µM to 6400 µM). The high efficacies of these seven derivatives suggest that they could increase the anti-P. larvae activity of larval food and improve the resistance of larvae to AFB disease through their application to honeybee colonies.

Beneficial bacteria as biocontrol agents for American foulbrood disease in honey bees (Apis mellifera).

American foulbrood (AFB) is a cosmopolitan bacterial disease that affects honey bee (Apis mellifera) larvae and causes great economic losses in apiculture. Currently, no satisfactory methods are available for AFB treatment mainly due to the difficulties to eradicate the tenacious spores produced by the etiological agent of AFB, Paenibacillus larvae (Bacillales, Paenibacillaceae). This present review focused on the beneficial bacteria that displayed antagonistic activities against P. larvae and demonstrated potential in AFB control. Emphases were placed on commensal bacteria (genus Bacillus and lactic acid bacteria in particular) in the alimentary tract of honey bees. The probiotic roles lactic acid bacteria play in combating the pathogenic P. larvae and the limitations referring to the application of these beneficial bacteria were addressed.

American Foulbrood-Old and Always New Challenge.

American foulbrood (AFB) is exclusively an infectious disease of honey bee larvae (Apis mellifera) and their subspecies that is spread easily and rapidly and is often present in apiaries. Due to the resistance and pathogenicity of the bacterial causative agent of the disease, which has considerable epizootiological and economic significance for beekeeping, AFB was classified as a highly dangerous, infectious animal disease by the World Organization for Animal Health (WOAH). Considering the severity of the infection, a frequent occurrence, rapid and easy spread, epizooty and enzooty are common. We tried to present an overview of the latest information related to AFB through several chapters. In addition to the latest data on the etiology of the causative agent, the most important elements of the clinical signs of the disease are also listed. Along with an overview of classic microbiological and the latest molecular methods of diagnosis, we also discuss AFB treatment from its differential diagnostic aspect. We hope that through demonstrating the mentioned preventive measures and measures of good beekeeping practice, the review will contribute to the preservation of the health of bees and therefore the overall biodiversity of the planet.

Monitoring of Paenibacillus larvae in Lower Austria through DNA-Based Detection without De-Sporulation: 2018 to 2022.

American foulbrood is caused by the spore-forming Paenibacillus larvae. Although the disease effects honey bee larvae, it threatens the entire colony. Clinical signs of the disease are seen at a very late stage of the disease and bee colonies are often beyond saving. Therefore, through active monitoring based on screening, an infection can be detected early and bee colonies can be protected with hygiene measures. As a result, the pressure to spread in an area remains low. The cultural and molecular biological detection of P. larvae is usually preceded by spore germination before detection. In this study, we compared the results of two methods, the culture detection and RT-PCR detection of DNA directly isolated from spores. Samples of honey and cells with honey surrounding the brood were used in a five-year voluntary monitoring program in a western part of Lower Austria. DNA-extraction from spores to speed up detection involved one chemical and two enzymatic steps before mechanical bashing-beat separation and additional lysis. The results are comparable to culture-based methods, but with a large time advantage. Within the voluntary monitoring program, the proportion of bee colonies without the detection of P. larvae was high (2018: 91.9%, 2019: 72.09%, 2020: 74.6%, 2021: 81.35%, 2022: 84.5%), and in most P. larvae-positive bee colonies, only a very low spore content was detected. Nevertheless, two bee colonies in one apiary with clinical signs of disease had to be eradicated.

Trans-generational immune priming against American Foulbrood does not affect the performance of honeybee colonies.

Honeybees are major pollinators for our food crops, but at the same time they face many stressors all over the world. One of the major threats to honeybee health are bacterial diseases, the most severe of which is the American Foulbrood (AFB). Recently a trans-generational vaccination approach against AFB has been proposed, showing strong potential in protecting the colonies from AFB outbreaks. Yet, what remains unstudied is whether the priming of the colony has any undesired side-effects. It is widely accepted that immune function is often a trade-off against other life-history traits, hence immune priming could have an effect on the colony performance. In this experiment we set up 48 hives, half of them with primed queens and half of them as controls. The hives were placed in six apiaries, located as pair of apiaries in three regions. Through a 2-year study we monitored the hives and measured their health and performance. We measured hive weight and frame contents such as brood amount, worker numbers, and honey yield. We studied the prevalence of the most common honeybee pathogens in the hives and expression of relevant immune genes in the offspring at larval stage. No effect of trans-generational immune priming on any of the hive parameters was found. Instead, we did find other factors contributing on various hive performance parameters. Interestingly not only time but also the region, although only 10 km apart from each other, had an effect on the performance and health of the colonies, suggesting that the local environment plays an important role in hive performance. Our results suggest that exploiting the trans-generational priming could serve as a safe tool in fighting the AFB in apiaries.

Genomic analysis of Paenibacillus larvae isolates from the Czech Republic and the neighbouring regions of Slovakia.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybee larvae. In the Czech Republic, two large infested regions were recognised. This study aimed to analyse P. larvae strains occurring in the Czech Republic in the years 2016-2017 and to characterise the genetic structure of their population with the use of Enterobacterial Repetitive Intergenic Consensus genotyping (ERIC), multilocus sequence typing (MLST) and whole genome sequence (WGS) analysis. The results were complemented by the analysis of isolates collected in the year 2018 in areas of Slovakia located near the Czechia-Slovakia border. ERIC genotyping revealed that 78.9% of tested isolates belonged to the ERIC II genotype and 21.1% to ERIC I genotype. MLST showed six sequence types with ST10 and ST11 being the most frequent among isolates. Within six isolates we found discrepancies in correlations between MLST and ERIC genotypes. The use of MLST and WGS analysis of isolates revealed that each of the large infested geographic regions had its own dominating P. larvae strains. We assume that these strains represented primary sources of infection in the affected areas. In addition, the sporadic presence of strains identified by core genome analysis as genetically related was unveiled in geographically distant regions suggesting possible human-mediated transmission of AFB.

A Comparison of Different Matrices for the Laboratory Diagnosis of the Epizootic American Foulbrood of Honey Bees.

American Foulbrood (AFB) of honey bees caused by the spore-forming bacterium Paenibacillus larvae is a notifiable epizootic in most countries. Authorities often consider a rigorous eradication policy the only sustainable control measure. However, early diagnosis of infected but not yet diseased colonies opens up the possibility of ridding these colonies of P. larvae spores by the shook swarm method, thus preventing colony destruction by AFB or official control orders. Therefore, surveillance of bee colonies for P. larvae infection followed by appropriate sanitary measures is a very important intervention to control AFB. For the detection of P. larvae spores in infected colonies, samples of brood comb honey, adult bees, or hive debris are commonly used. We here present our results from a comparative study on the suitability of these matrices in reliably and correctly detecting P. larvae spores contained in these matrices. Based on the sensitivity and limit of detection of P. larvae spores in samples from hive debris, adult bees, and brood comb honey, we conclude that the latter two are equally well-suited for AFB surveillance programs. Hive debris samples should only be used when it is not possible to collect honey or adult bee samples from brood combs.

Biocontrol potential of Apilactobacillus kunkeei EIR/BG-1 against infectious diseases in honey bees (Apis mellifera L.).

The significant reduction of honey bee colonies due to the various infectious agents highlights the need for the development of new alternatives and integrated management strategies to keep a hive strong and healthy. The main purpose of this study was to develop an environmentally and friendly microbial feed supplements to prevent honey bee mortality and keep the bee colony population healthy and productive. For this aim, Apilactobacillus kunkeei EIR/BG-1 isolated from honey bee gut microbiota was evaluated for its preventive role against American Foulbrood disease and nosemosis. To test the ability of the strain EIR/BG-1 for suppressing Paenibacillus larvae growth under in vitro conditions, the agar well diffusion method was used and viable cells of the strain EIR/BG-1 inhibited the growth of P. larvae with an efficient inhibition zone (24 ± 0.8 mm) similar to tetracycline antibiotic (30 µg). To determine the preventive role of the strain EIR/BG-1 on infection progression, its viable cells were applied against nosemosis in a laboratory experimental setting. Our results showed that prophylactic supplementation of Al. kunkeei EIR/BG-1 (106 cfu/bee) significantly reduced the spore load (66 ± 6.1%). Besides, gene expression of antimicrobial peptides in gut tissue has been up-regulated and infected midgut epithelium integrity and peritrophic membrane production were improved. In conclusion, our findings suggest that prophylactic supplementation of Al. kunkeei EIR/BG-1 as a natural strategy may enhance the honey bee's response when challenged by pathogens. Field applications towards gaining a better understanding of its biocontrol role will be the main goal of our future researches.