Probiotics and postbiotics: a promising prophylactic measure for American foulbrood and European foulbrood diseases of honey bees

American foulbrood (AFB) and European foulbrood (EFB) are the two most important honey bee brood diseases which impose heavy economic losses to the apiculture industry worldwide by reducing bee population and honey production. Treatment with antibiotics has led to the emergence of antibiotic-resistant strains, calling for alternative safe treatment procedures that could control these diseases. Honey bee gut microbiota is known to affect the overall health of honey bees by enhancing their resistance to a number of diseases via modulation of the immune response and production of different antimicrobial metabolites. The majority of these gut resident bacteria are identified as probiotic bacteria and secure the health of these tiny insects. In the present review, we highlighted the significance of the honey bee gut microbial community and their probiotic potency for the prevention of AFB and EFB diseases in honey bees.

Here, we present a retrospective study on honey bee brood diseases in the USA. The Bee Disease Diagnostic Laboratory (BDDL) in the USDA-ARS Beltsville Bee Research Laboratory received a total of 4790 brood samples between 2015 and 2022 from U.S. State Apiary Inspectors and beekeepers. Samples from 49 states were analyzed by microscopy for the presence and prevalence of two bacteria, Melissococcus plutonius and Paenibacillus larvae, causing European foulbrood (EFB) and American foulbrood (AFB) diseases, respectively. Samples that tested positive for AFB were cultured and subjected to antibiotic susceptibility tests via the agar disc diffusion method to determine their resistance to tetracycline (Terramycin®) and tylosin (Tylan®) antibiotics. A comprehensive data analysis was conducted at multiple levels, including state, month, year, nationwide, and climate region. Among the 49 states examined, EFB was identified in 44 states, while AFB was found in 31 states. Infection levels vary across states, ranging from 0% to 63.6% for EFB and from 0% to 54.8% for AFB. The national average of EFB (19.2%) was significantly (p < 0.001) higher than AFB (8.6%). Contrary to AFB, significant yearly increases (p < 0.001) were identified for EFB from 2015 to 2022. Furthermore, significant monthly variations were recorded for both brood diseases, with the highest occurrence of EFB observed from April to July. States with the lowest infection rates of both diseases were NV, ND, MS, AK, and AZ. The South U.S. climate region, which comprises six states, had the highest and lowest EFB and ABF infections, respectively. On a nationwide scale, the AFB resistance to tetracycline (38%) was significantly (p < 0.001) higher than the resistance to tylosin (27%).

The balance of the gut microbiome is important for the honey bee’s growth and development, immune function and defense against pathogens. The use of a beneficial bacteria-based strategy for the prevention and biocontrol of American foulbrood (AFB) and European foulbrood (EFB) diseases in honey bees offers interesting prospects. Lactic acid bacteria (LAB) are common inhabitants of the gastrointestinal tract of the honey bee. Among LABs associated with bee gut microbiota, Lactiplantibacillus plantarum (previously Lactobacillus plantarum) and Apilactobacillus kunkeei (formerly classified as Lactobacillus kunkeei) are two of the most abundant species. In this study, four Lactiplantibacillus plantarum strains and four Apilactobacillus kunkeei strains, isolated from the gastrointestinal tract of honey bee (Apis mellifera L.) were selected for their in vitro inhibition ability of Paenibacillus larvae ATCC 9545 and Melissococccus plutonius ATCC 35311. In addition, these LABs have been characterized through some biochemical and functional characteristics: cell surface properties (hydrophobicity and auto-aggregation), carbohydrates assimilation and enzymatic activities. The antimicrobial, biochemical and cell surface properties of these LABs have been functional to their candidature as potential probiotics in beekeeping and for the biocontrol of AFB and EFB diseases.

American foulbrood (AFB) and European foulbrood (EFB) are the two most important bacterial diseases that affect honey bee brood. The aim of this study was to evaluate the effects of an integrated treatment of AFB and EFB in naturally infected colonies in spring, using the beekeeping technique partial shook swarm combined with oxytetracycline treatment. The following parameters were assessed: strength of the colonies, consumption of antibiotic solution, queen losses, honey production and relapses. An increase in strength was observed in both (AFB and EFB) groups. Mean syrup consumption in the AFB colonies was 444.2 mL, while in the EFB colonies it was 497.3 mL. Linear correlation between the strength of the colonies and consumption (p-values &lt; 0.0001; R² = 0.448) was observed. Three queens out of thirty (10%) were lost during the shook swarm procedure: one in the AFB group (5.3%) and two in the EFB group (18.2%). The results highlighted four clinical relapses (22.2%) of AFB, and no relapse (0%) of EFB after two years of observation. Six colonies (four in the AFB group and two in the EFB group) produced honey in the same beekeeping season that the shook swarm and antibiotic treatment were performed. Partial shook swarm is a good alternative to colony destruction, especially for EFB, and provides productive colonies during the foraging season.

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.

The article presents materials on the study of the distribution and manifestation of the main parasitosis in bees in the Krasnodar Territory in the period from 2019 to 2021. The studies were carried out in the farms of the Krasnodar Territory and in the Kropotkin Regional Veterinary Laboratory. For experimental studies, bees of the Caucasian breed Apis mellifera carnica were used. In laboratory studies, various methods were used: clinical, epizootological, bacteriological, pathomorphological, serological, luminescent and light microscopy. As a result of research during the study period, in 2019, out of 2010 bee colonies from different regions of the Krasnodar Territory, positive samples were found for colibacillosis 4, citrobacteriosis 17 and proteus infection 2. In 2020, out of 2010 bee colonies from different regions, 10 positive samples were found, of which 2 samples for colibacillosis, 5 tests for citrobacteriosis, 2 tests for proteus infection, one for hafniosis. In the study of bee colonies from different regions in 2021, out of 1500 samples of pathological material for various infectious diseases, a positive test for colibacillosis 2 samples, citrobacteriosis 6, proteus infection 2, hafniosis 1, nosematosis 108 samples was detected. In general, in the Krasnodar Territory, infection with varroosis averages 6.9%, in different regions from 4 to 9%. The defeat by American and European foulbrood was 3.5–4.4%. It has been established that it was varroosis that was one of the factors in the further development of American and European foulbrood of bees and that the development of these diseases proceeds mainly associatively with severe consequences. As a result, it was established that various diseases of bees are recorded in the Krasnodar Territory, among the most dangerous parasitosis varroosis, American and European foulbrood were found, more often they occur in an associative form, as well as ascospherosis, acarapidosis, braulosis, nosematosis. In the beekeeping farms of the Caucasus, Starominsk and Korenovsky districts in the Krasnodar Territory, the causative agents of varroosis were isolated mite Varroa jacobsoni, American foulbrood Bacillus larvae and European foulbrood Streptococcus pluton. The main clinical and pathoanatomical signs in the associative disease of bees with varroosis, American and European foulbrood: death of pupae, damage mainly to drone brood, deformity or absence of wings, uneven egg laying by the queen, damage to printed and open brood of bees, death and damage to larvae at the age of 3-4 and 8-9 days, the weakening of bee colonies and often their complete death.

From 1984 to 2015, the Bee Disease Diagnostic Lab at the USDA-ARS Beltsville Bee Research Laboratory (MD, USA) analyzed 66,056 samples submitted for disease diagnosis, comprising 35,883 adult bees and 30,173 brood samples collected from symptomatic colonies nationwide. This dataset provided valuable insights into honey bee disease dynamics over three decades. Adult bee samples were screened for Nosema spp. and tracheal mite (Acarapis woodi). Brood samples were microscopically analyzed for the presence of both Paenibacillus larvae and Melissococcus plutonius, the causative agents of American foulbrood (AFB) and European foulbrood (EFB) diseases, respectively. Antibiotic resistance was tested in AFB-positive samples (n = 6,785) for tetracycline and tylosin. Longitudinal analysis revealed significant (p < 0.001) inter-state and seasonal differences in disease prevalence. AFB was significantly more prevalent nationwide (44.71%) compared to EFB (10.01%), with a negative correlation between the two diseases (R = -0.4, p < 0.01). P. larvae resistance to tetracycline and tylosin declined significantly (p < 0.001) in later years, with national resistance averages of 42.52% and 27.78%, respectively. Additionally, positive correlations were recorded between AFB prevalence and P. larvae resistance to both antibiotics. Nosema spp. prevalence ranged from 0% to 77.9% across states, with a national average of 24.09% and significant seasonal variations (p < 0.001). From 2008 to 2015, Nosema infection rates increased significantly, contrasting with a marked decline in tracheal mite prevalence since 2007 (national average of 12.48%), which was negatively correlated with Nosema infection (R = -0.3, p < 0.05). This study provides unprecedented longitudinal insights into honey bee disease dynamics in the United States, highlighting the significant emergence of EFB and Nosema as threats from 2008 to 2015 and a drastic reduction in tracheal mite prevalence. These findings underscore the need for continued monitoring and adaptive management strategies to protect honey bee health and ensure sustainable pollination services.

Aim: To determine the species composition, relative abundance and status of honey bee pests and diseases in South Ari district of South Omo Zone of Southern Western Ethiopia.&#x0D; Study Design: Four major honey producing peasant associations (PAs) representing diverse ecological zones were selected purposively, while the sub-localities, villages and beekeepers were selected randomly using a nested design.&#x0D; Place and Duration of Study: Survey was conducted between 1, August – 30, December 2019 with the interval of one month in major honey producing peasant associations representing diverse ecological zones of Ari district of South Western Ethiopia.&#x0D; Methodology: The assessment on abundance and status was made from of half kilogram of fresh and dry honey comb samples that were taken from 720 farmer’s hives of four peasant associations using taxonomic key of books related to arthropod pests and diseases of honey bee.&#x0D; Results: Five species of predators and parasitoids belonging to four orders were recorded. Of these 76.63% accounts for small hive beetles, followed ants (38.31%) and wasps (19.92%). Besides, six type’s diseases of honey bee caused by different pathogens were recorded. Of which, Chalk brood, other virosis, European foulbrood and American foulbrood, respectively were the most frequently occurring. In terms of the status, the diseases of honey bee were in the following orders, i.e., Chalk brood &gt; other virosis &gt; European foulbrood &gt; American foulbrood &gt; Sac Brood Virus, while their pests were in the following orders, i.e., Small hive beetles &gt; Ants &gt; Wasps &gt; Wax moths &gt; Spiders.&#x0D; Conclusion: Arthropod pests and diseases were the most important constraints of honey colony and honey production in the study areas. Thus, it is important to design and implement effective management strategies to reduce loss of honey colony and honey production in the study area.

SUMMARY“European foul brood” has been initiated in healthy nuclei by feeding either the young larvae directly or the bees naturally infected material. The disease thus produced varied from a mild transient infection to a serious form depending upon whether the inoculation had been made early or late in the brood‐rearing season. In one experiment in which a nucleus very strong in bees was employed no disease resulted.“European foul brood” has also been induced in healthy nuclei by suspending in them combs containing artificially infected larvae in which disease had been produced by feeding them pure cultures of Streptococcus apis or Bacillus alvei and then starving them under conditions favouring the growth of the bacteria. The susceptibility of nuclei to infection by this means seems to be governed by approximately the same conditions as pertain to inoculations made with material obtained from natural sources. The disease thus induced ultimately becomes a mixed bacterial infection of the brood.Attempts to cause disease by feeding the bees or larvae (without starving them) relatively large numbers of S. apis or B. alvei organisms have as yet proved unsuccessful. Whether this is due to the fact that these bacteria become attenuated with respect to virulence by culturing them on artificial media, or that decomposing brood acts as a vehicle and that the relative inoculum is greater by this method, remains to be determined.The failure to produce “European foul brood” by feeding sterile Pasteur‐Chamberland L 2 or L 3 filtrates prepared from naturally infected larvae to the bees or larvae of healthy nuclei, either with or without bacteria, may be taken as strong evidence in support of the view that a filterable virus is in no way implicated as an etiological agent in this type of disease. This conclusion is strengthened by the success which has attended the use of pure cultures of the bacteria associated with this disease employing the “starved larvae” technique described.The introduction of queen bees from nuclei affected with “European foul brood” into healthy queenless nuclei has not caused any transmission of the disease under the conditions of the experiments.Two species of S. apis Maassen have been isolated from affected larvae taken from several different cases of “European foul brood”; one of these hydrolyses both casein and gelatine, the other does not. In other respects these species appear to be identical.The etiology of so‐called “European foul brood” is discussed in detail, and it is suggested from the evidence submitted in this and other papers that it may not be a single disease with one well‐defined etiological agent, as is American foul brood, but is, perhaps, a non‐specific mixed bacterial infection of the brood of bees, especially of the brood of weak colonies. This conclusion must be regarded as temporary pending further investigation.My thanks are due to Mr D. M. T. Morland and to Mr A. Rolt, whose generous advice and assistance in connexion with the apiary work greatly facilitated the carrying out of the practical experiments.

American Foulbrood and European Foulbrood diseases of honeybees were examined in 725 beehives from 23 apiaries located in the South Marmara Region of Turkey. We determined that 19 apiaries were infected and the suspected clinical signs of foulbrood diseases were investigated in 102 beehives by PCR and cultural method. Broods and combs from colonies with suspected clinical symptoms of foulbrood diseases were collected and cultured for bacteriological examination. All of the specimens contaminated with bacteriae and 37 species of bacteriae were isolated such as Stapylococcus epidermidis, Bacillus subtilis, Corynebacterium jeikum, Corynebacterium pseudotuberculosis, Bacillus spp. All of these bacteria are related to human, animal and environmental origins. In this study, Paenibacillus larvae by PCR amplifying the 973-bp region PL1 and PL2 with 1f, Melissococcus plutonius amplifying the 973bp region EFB-F and EFB-R gene were amplified. American Foulbrood causative agent Paenibacillus larvae and European Foulbrood causative agent Melissococcus plutonius were not detected in any sample examined by PCR and cultural methods. On the other hand, Paenibacillus alvei that is a seconder agent to European Foulbrood was found in two samples by cultural methods. In conclusion, the results showed that P. larvae and M. plutonius are not present in South Marmara Region. In this study, human, animal and environment originated agents were isolated.

Antimicrobial activity of camphor tree silver nano-particles against foulbrood diseases and finding out new strain of Serratia marcescens via DGGE-PCR, as a secondary infection on honeybee larvae.

Beekeepers in Colombia are using almost exclusively Africanized hybrids which are considered to be more resistant to diseases and climatic conditions than European-derived honey bee populations. There are no reports of losses of larval and adult bee populations in the country due to infectious diseases. However, there is no official diagnostic system to detect the presence of pathogenic bacteria for Apis mellifera, such as Melissococcus plutonius and Paenibacillus larvae causing European foulbrood and American foulbrood respectively. To assess their presence and prevalence in a large sample sets, representatives of beekeeping populations in the country, we conducted a study on 491 of 5400 registered hives located in three regions in Colombia, analyzing larvae samples with conventional and molecular real-time PCR and molecular taxonomy techniques. Out of the two bacteria targeted, only M. plutonius was detected in 7 of the 491(1.4%) larval samples in all three regions in this sampling campaign. There was no evidence of symptoms consistent with European or American foulbrood. Therefore we are updating and confirming the low prevalence of M. plutonius and the no detection of P. larvae in an extensive sampling survey of Africanized bees at three different regions of Colombia. Our results are consistent with the view that africanized hybrid bees are more more resistant to diseases.

Bacterial diseases, such as American Foulbrood (AFB) and European Foulbrood (EFB), are known to have catastrophic effects on honey bees (if left to spread, can wipe out entire colonies), leading to severe financial losses in the beekeeping industry. The aim of this study was to evaluate the pharmacological properties of methanol extract and its fractions (ethyl acetate, hexane, water) derived from Dicranum scoparium Hedw., which could be utilized as a potential drug to prevent the bacterial diseases (AFB and EFB) affecting the honey bees. For this purpose, crude methanol extract and ethyl acetate/hexane/water fractions were prepared from the aerial part of D. scoparium, collected from Trabzon province. Bio-guided fractionation of the extract and its fractions led to the first-time isolation of five compounds. The structure of all compounds was elucidated by nuclear magnetic resonance (NMR) spectroscopy, ultraviolet (UV) spectral analysis, Fourier-transform infrared spectroscopy (FT-IR), liquid chromatography quadrupole time-of-flight mass spectroscopy (LC-QToF-MS), and by comparison of their NMR data with that of literature. The analysis of these compounds revealed significant antibacterial and sporicidal activities against bacteria causing larval diseases in honey bees. The antibacterial activity of these compounds ranged from 0.6 to 60 μg/mL against AFB and EFB causing bacteria. Therefore, the natural raw extract and fractions of D. scoparium could be used as potential therapeutic agents against bacterial agents affecting honey bees.

Aim. To investigate the microbiota of honeycombs with affected bee brood. Methods. Visual, immunochro- matographic, cultural-morphological, biochemical, electron-microscopic methods were used to isolate and previously identify a number of microorganisms. Results. 10 samples of honeycombs with sealed brood were studied. The following agents of bee diseases were isolated and previously identifi ed: Paenibacillus larvae – American foulbrood; Вasillus paraalvei – parafoulbrood; Melissococcus pluton – European foulbrood. Con- clusions. The number of cocci, spore-forming coli and yeasts, found in the samples of honeycombs, commonly represent normal microfl ora of bees, but their number increases signifi cantly in case of viral and bacterial infections.

Given the increasing interest in alternative methods of prevention and biological control of bacterial diseases in honey bees, the development of new approaches based on probiotic bacteria for honey bee growth, immune function and defense is important. Honey bees have a rich microbiota that varies with seasonal and geographic changes, developmental stages, diet, and social lifestyle. Nevertheless, lactic acid bacteria are an integral part of the honey bee microbiota. In this study, strains of Apilactobacillus, Leuconostoc and Enterococcus isolated from healthy honey bees were identified and their usefulness as an alternative product to control and prevent American and European foulbrood was investigated. The strains were identified by cultural and molecular analysis. The antimicrobial activities against Paenibacillus larvae and Paenibacillus alvei were tested by agar plug and well diffusion method. The inhibitory effect of the obtained 72-h cell-free supernatants on Paenibacillus larvae spore germination was determined by the Bioscreen C method. It turned out that the strains whose prophylactic effect against Paenibacillus larvae spores in larvae was tested were not largely able to prevent infection. Apilactobacillus and Leuconostoc strains did not affect the health of the larvae, and infection was somewhat clinically eliminated in larvae colonized by Apilactobacillus and Enterococcus. The fact that the strains exhibit potent antimicrobial activity and do not reduce larval viability and live weight is a promising finding with regard to their use for treatment, particularly in the early stages of American foulbrood infection.