1. Introduction
American foulbrood (AFB) [1] and European foulbrood (EFB) [2] are severe infectious diseases in honey bees and have been known since the 18th century. Both occur worldwide as sporadic outbreaks [3,4]. The causative agent of AFB is the Gram-positive, spore-forming bacterium Paenibacillus larvae (P. larvae). Endospores are the infectious form of P. larvae [4] and are responsible for its long, persistent survival in the environment. EFB is caused by the bacterium Melissococcus plutonius, a Gram-positive, lanceolate coccus, sometimes pleomorphic and rod-like, that is normally associated with other microorganisms and most commonly with Paenibacillus alvei, Brevibacillus laterosporus and Enterococcus faecalis that contribute to the course of the disease [3].
The World Organization for Animal Health (WOAH—formerly OIE) includes AFB and EFB on the list of notifiable diseases. AFB and EFB can represent a serious threat to honey bee health and welfare, veterinary public health worldwide and safe international trade with honey bees and their products [5]. European Union animal health legislation (regulation 429/2016 and regulation EU 2018/1882) classifies AFB as category D+E, foreseeing measures to prevent its spread or movement between member states. According to EU legislation, AFB must be monitored within the member states.
Several methods have been tested to control AFB and EFB over the last few decades: the destruction of colonies [6], shook swarm [7], antibiotic treatment alone [8] or antibiotic treatment in association with shook swarm [9].
With regards to the destruction of the infected beehives by incineration [6], this practice does not require the use of antimicrobials, but the method of killing the bees (preferably using organic and low environmental impact compounds and no pesticides) must be chosen carefully to avoid contamination. Burning organic materials and plastic hive gear (e.g., feeders and foundations) can also have great environmental impact, especially in cases of great outbreaks. Incineration is unfortunately not respectful of the bees from an animal welfare point of view, and the cost of replacing the colonies can be high due to destruction of entire hives and other beekeeping equipment. However, incineration is the best method to eliminate spores from beekeeping operations.
Shook swarm is a beekeeping technique in which all the honey bees are shaken off the infected frames and placed into a new clean hive with new comb foundations [9,10]. Replacing all combs from the brood box reduces the infection level by removing the spores. Old, infected combs should be destroyed by incineration. It is very important to always carefully disinfect all beekeeping equipment (boxes, boards, frames, queen excluders, feeders, etc.) and objects used for the manipulation of infected hives (hive tools, gloves, suit, etc.). Possible methods used vary according to the substrate to disinfect.
Oxytetracycline hydrochloride (OTC) has been used in Europe since 1967 to treat honey bee colonies [7,11] adopting several administration methods [8,9].
The antimicrobials oxytetracycline, tylosin tartrate, fumagillin, metronidazole, sulfadimethoxine and trimethoprim are authorised for bacterial and fungal honey bee diseases in different countries worldwide [12]. In the US, Tylovet (tylosin tartare) and Lincomix (lincomycin) are registered for AFB control, while Terramycin (oxytetracycline hydrochloride) and Pennox 50 (oxytetracycline) are available for the control of both foulbroods [13]. In Canada, Foul Brood Mix, Oxytet-25-S and Oxysol 62.5 (oxytetracycline), Tylan Soluble 60 (tylosin) and Lincomix (lincomycin) are commercially available [14].
So far, even if there are no registered antibiotics for honey bees in the EU, OTC can be used for honey bees in an off-label manner through the cascade principle (EU directive 2001/82, regulation EU 2019/6), considering a maximum residue limit (MRL) of 100 ppb (regulation EU 37/2010 and regulation EU 2018/470). Even though antibiotics are prohibited in beekeeping in the EU, the European Food Safety Authority (EFSA) and different studies [15,16,17,18,19,20] report findings of antibiotic residues in honey. For example, between 1% and 7% of honey samples produced in the EU were positive for antibiotic residues [15,16,17,18,19,20].
Antibiotic treatment, when authorized, may be the choice for conventional beekeeping in the early stages of the disease in strong colonies with high prevalence in apiaries. Unfortunately, relapses are frequently reported [6,7,8,9].
Beekeepers should be aware that antibiotics cannot replace good practices. Good beekeeping practices (GBPs) are integrative activities applied to obtain optimal health for humans, honey bees and the environment [21].
In this study, an innovative integrated approach was performed in all infected colonies: the partial shook swarm method in combination with an OTC treatment administered in flat small containers (jar caps) one day after the shook swarm.
The “partial shook swarm” technique [22] consists of the substitution of all the brood combs (both capped and uncapped) with new foundations, while combs with pollen and honey are left in place. This technique allows colonies to regain their strength quicker in comparison to a “full shook swarm”, which implies the substitution of all combs. Removed combs were destroyed by incineration.
The aim of this study was to evaluate a new, integrative approach for controlling AFB and EFB, while increasing the efficacy of chemical treatments (OTC). We combined chemical treatments with biotechnical methods, specifically per os OTC administration and partial shook swarm for symptomatic colonies, as a possible alternative to beehive destruction according to FAO Integrated Pest Management (IPM) [23]. This approach consists of a “pest management system that, in the socioeconomic context of farming systems, the associated environment and pest population dynamics, utilizes all suitable techniques in a compatible manner, as possible solution to maintain the pest population levels below those causing economic injury”. Its goal is to apply the smallest amount of potentially damaging substances to animals, combined with the implementation of cultural practices, with a view to minimizing hazards for human beings and the environment [23].
Efficacy, relapses, performances of colonies after the treatment and the consumption of OTC syrup were monitored.
2. Materials and Methods
The study was performed in Blera (Viterbo Province—Central Italy), as a collaboration among the local veterinary officers (ASL/VT), the official laboratory responsible for animal health (Istituto Zooprofilattico Sperimentale del Lazio e della Toscana “M. Aleandri”) (IZSLT) and the local beekeepers’ association (Apituscia).
Field trials began at the end of March 2016, after the presence of symptoms in 19 colonies affected with AFB and 11 colonies affected with EFB was confirmed by veterinary services with the support of the IZSLT [24,25]. All colonies were placed in a quarantine apiary. All infected colonies were treated with the partial shook swarm method. Immediately after the shook swarm, the number of colonies that lost the queen during the procedure was verified.
The day after the shook swarm, OTC treatment was carried out using an innovative and very simple technique to administer OTC sucrose solution by means of jar caps (Figure 1). Specifically, 180 mL of sucrose solution (1:1) and 0.576 g of OTC (Unione Commerciale Lombarda S.p.A, Brescia, Italy—A.I.C. 102781010) were poured into 6 jar caps, each containing 30 mL of medicated syrup. The jar caps were then placed on the top of the frames in the brood box of each hive weekly (Figure 1) for five consecutive weeks. The consumption of antibiotic solutions was measured after each week of treatment. In total, 2.88 g of OTC were given to each colony.
The colonies were visually inspected for clinical symptoms every two weeks from the beginning of the experiment until March 2018, meaning they were closely monitored for two consecutive years to evaluate the performances of the colonies and relapses.
Performances were considered in terms of honey harvested from the treated hives by measuring the amount of honey collected into supers at the end of the beekeeping season.
The colonies that were clinically positive to foulbroods were sampled, and the suspicious larval samples collected were cultured for the isolation and characterization of P. larvae or M. plutonius isolates following methods reported in the OIE manual of diagnostic tests and vaccines for terrestrial animals.
The strength of the colonies before and after the 5th week since the beginning of treatment was evaluated by counting the number of frames occupied by bees [26].
To verify differences in colony strength before and after treatment, and among AFB and EFB groups, the Wilcoxon signed rank test and Mann–Whitney tests were applied. To relate the syrup consumption rate and strength of the colonies, the Pearson correlation coefficient was calculated [27]. Statistical analyses were performed using XLSTAT™ software (Addinsoft, Paris, France).
3. Results
Three out of thirty queens during the shook swarm procedure were lost. In total, OTC treatment was administered in 27 colonies.
Different rates of consumption among colonies were observed. In fact, not all the colonies consumed all the OTC solution that was offered weekly with the jar caps. The colonies consumed the antibiotic solution according to their respective strength: strong colonies (more than 5.5 frames covered by adult bees with 6 in the AFB group and 5 in the EFB group) consumed all the amount of OTC (2.88 g) administered, whereas the weaker colonies did not consume all the administered syrup. Mean consumption for the AFB group was 444.21 ± 219.53 mL (n = 18) of syrup (=1.42 ± 0.70 g of OTC/hive), while for the EFB group it was 497.23 ± 191.69 mL (n = 9) of syrup (=1.59 ± 0.61 g of OTC/hive); the overall (AFB+EFB) mean consumption was 455 mL (+/−204 mL) (n = 27) (=1.48 g of OTC/hive). No statistically significant differences (expected value = 104.500; variance (U) = 532.830; p = 0.341) were observed when comparing consumption in the AFB and EFB groups.
The observed increase in the adult bee population (+8.95% in AFB and +7.72 in EFB) was statistically significant for both groups (the AFB group: expected value = 95.000, variance (V) = 546.250, p ≤ 0.0001; the EFB group: expected value 27.500, variance (V) = 75.625, p-value 0.002) (Figure 2).
The sucrose OTC solution consumption was related to the strength of the colonies before treatment: a linear correlation between the two variables (p-values < 0.0001; R² = 0.448; Figure 3) for both the AFB and EFB groups was found.
All colonies (AFB and EFB) completely recovered from symptoms of the diseases. However, in the AFB group, four relapses (22.2%) were detected: two cases after 5 months from the end of the treatment and two more cases after 6 months. In the EFB group, no relapses (0%) were observed.
Six colonies (22.2% of the total), four infected with AFB and two infected by EFB, produced a total of 70 kg of honey. These six colonies were able to recover and produce honey in the same beekeeping season in which the integrated treatment was carried out, which is in contrast with the other colonies of the quarantine apiary that did not produce honey.
4. Discussion
Several approaches for antibiotic treatment of colonies are described within the scientific literature; however, they are not always effective and may lead to antibiotic residues or to the spread of the disease due to the persistence of the pathogens after treatments (e.g., spores of P. larvae in the case of AFB).
In this study, we tested a new way of administration of the antibiotic (OTC), giving it per os to the adult bees in a more precise way using jar caps (Figure 1) one day after the shook swarm. Jar caps seemed to be a handy (cheap and not labour intensive) solution for administering OTC to the bees. We developed an “integrated” approach to control AFB and EFB: the antibiotic treatment administered in association with a beekeeping technique (partial shook swarm).
The antibiotics commonly administered to control AFB or EFB are OTC [8,10,28], sulfonamides [29], tylosin [8] and streptomycin [30]. The antibiotics can be given to the bees by feeding them with a sucrose antibiotic solution that is directly trickled onto the empty combs and/or onto the adult bees [9]. Other methods available in the literature include dusting icing sugar mixed with the antibiotic [10], using antibiotic paper packs [28] or ‘extender patty’ consisting of sugar, vegetable shortening and the active principles [31]. Antibiotics applied with these methods could be responsible for the contamination of the beehive content, for example, wood and/or stored honey [10]. Moreover, with these techniques of application, antibiotics may also lead to high mortality of larvae due to direct contact [32] or relapses [8] after treatment. Administering OTC per os ensures direct consumption of the antibiotic, while avoiding dripping on frames and internal parts of the hive. Previous studies [9] showed that OTC treatment in combination with shook swarm could also be successful in colonies in which more than 50% of the brood is infected. Nevertheless, it is fundamental to consider the risk of residues in hive products and the selection of antibiotic resistant strains as clearly evidenced in a similar study [33].
Concerning the results on shook swarm efficacy to control AFB and EFB, they are extremely variable. For example, a high mortality of colonies is reported [34], but high efficacy in reduction of the levels of the infection [35,36] is also documented. We modified the shook swarm technique by leaving frames with stored pollen and honey from the brood box due to a lack of forage in nature.
Combining antibiotic treatment with the shook swarm guaranteed a strong initial reduction of high pathogen loads thanks to the removal of infected combs as the highest levels of pathogen causing AFB and EFB are generally found in the nest combs [37]. The risk of residues could be reduced carrying out the shook swarm technique immediately after antibiotic administration.
Independent of the strength of the colonies during the partial shook swarm procedure, three queens out of thirty (10%) were lost due to the specific risks related to this beekeeping technique. Queen losses could be strongly reduced by handling frames more carefully during the shook swarm procedure, or by finding and caging the queen before shaking the bees off the frames.
The total amount of active ingredient consumed by each colony was on average 1.42 g for AFB and 1.59 g for EFB, i.e., higher than 1 g, which is suggested in the literature for effective EFB treatment [9,10]. In order to achieve this consumption rate and to enable the weaker colonies to consume a sufficient amount of OTC [9,10], it was necessary to offer higher amounts of antibiotics to the bees. The consumption of the active principle seems to be an aspect difficult to manage as it differs greatly from colony to colony, depending mostly on their strength.
Regarding the number of relapses, we found a recurrence rate of 0% for EFB and 22.2% for AFB. In the study by Waite and colleagues [9], 4.8% of relapses were observed after treating colonies infected with EFB using OTC and shook swarm. The total absence of relapses in the 2nd year of observations suggests that the adopted protocol enables control of the diseases.
A complete shook swarm could, in fact, guarantee a reduction in the number of relapses, especially for AFB. However, the strength of the colonies is a key parameter to consider as we observed that colonies with fewer than 2.5 frames covered by bees (approx. 5825 bees) were too weak; therefore, they were at risk of not surviving the shook swarm. Although this kind of management is not well accepted by professional beekeepers because it causes them to lose one honey season, in their economic analysis, Pernal and colleagues [21] showed that shaking is 24% more profitable than using package bees.
An increase in the strength of the colonies before and 5 weeks after the shook swarm was observed (Figure 3). We can conclude that partial shook swarm in association with OTC treatment, if applied in favourable environmental conditions (enough pollen and nectar flow), could be considered as a protocol that does not show any side effects related to colony weakening or mortality.
A correlation between strength and consumption of syrup with OTC was established (p-values < 0.0001; R² = 0.448, Figure 2) in our study. According to our results, based on the concentration of OTC in the sucrose solution administered, it is possible to conclude that hives with more than 3.5 frames occupied by the bees (approx. 8155 bees) could easily consume enough antibiotic solution needed to control the disease (1 g of OTC). Thus far, no specific correlation between strength of a colony and treatment efficacy has been established in the literature.
It is known that OTC may have a bactericidal effect on M. plutonius and a bacteriostatic effect on P. larvae, but with this protocol, the goal of the antibiotic treatment was not to “sterilize” colonies, but to reduce the infection, increasing the chance of recovery after the shook swarm technique. Moreover, the antibiotic treatment can be justified only in cases of high prevalence of the disease and in cases of favourable environmental conditions. Finally, the administration of OTC should be always performed by veterinarians to guarantee appropriate use of the antibiotic at the apiary level.
In our study, the shook swarm technique in combination with OTC treatment administered per os (using jar caps) proved to be completely effective to control EFB. On the other hand, in the cases of AFB, 22.2% relapsed in the first year after treatment. In any case, our approach towards AFB management consisting of the combination of shook swarm and OTC treatment resulted in a greater efficacy than just OTC treatment, whereas Alippi and colleagues [8] registered relapses in all the treated colonies.
Although we had no data to compare the amounts of honey produced in the same area from healthy colonies, the positive outcome was that 22.2% of the treated colonies were productive in the same beekeeping season.
Underlying the importance of applying this protocol only when the bees can collect pollen and nectar (in the active season), this study has clearly shown that OTC treatments in association with partial shook swarm could be completely successful against EFB and only partially (100−22.2 = 77.8%) against AFB.
Moreover, the obtained data show that the OTC treatment in association with shook swarm is effective when the AFB and EFB infected colonies are still strong enough to guarantee therapeutic dose consumption. The experimental results achieved may also contribute to widen the discussion among beekeepers, veterinarians, and legislators and authorize OTC in beekeeping under strict veterinarian supervision. This approach may increase the numbers of honey samples monitored for antibiotic residues, while offering legal and supervised solutions for beekeepers, thus providing better food safety for all consumers of hive products.
5. Conclusions
The integrated treatment of EFB with OTC and partial shook swarm removes clinical cases of EFB allowing colonies to also be productive in the same beekeeping season.
Further studies should be performed to assess the development of antimicrobial resistance and to reduce residues in hive products.
Conceptualization, G.F. and M.P.; methodology, G.F., M.M., J.B. and L.G.; software, M.P.; validation, J.B., L.G. and G.F.; formal analysis, M.P.; investigation, M.M., J.B., L.G., L.F., M.P. and E.B.; data curation, M.P.; writing—original draft preparation, J.B. and M.M.; writing—review and editing, M.P., G.F., M.M. and V.M.; visualization, M.P.; supervision, G.F.; project administration, G.F. All authors have read and agreed to the published version of the manuscript.
Not applicable.
Not applicable.
The authors would like to thank Francesco Tolomei, the Beekeepers Association Apituscia, and Andrea Piunno for their collaboration. Moreover, we would also like to thank to Riccardo Jannoni-Sebastianini from Apimondia for his suggestions in writing this paper.
The authors declare no conflict of interest.
Footnotes
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Enlarge this image.Figure 1. The new technique to administer OTC sucrose solution to the colonies using jar caps on the top of the frames.
Enlarge this image.Figure 2. Box plot of colony strength before and after treatment in the AFB and EFB groups. + sign is the mean value.
Enlarge this image.Figure 3. Linear correlation between the number of frames covered by adult honey bees before treatment and OTC syrup consumption (both groups are included in the figure). p-value < 0.0001; R² = 0.448.
References
1. White, G.F. The Bacteria of the Apiary: With Special Reference to Bee Diseases; Government Printing Office: Washington, DC, USA, 1906.
2. White, G.F. The Cause of European Foulbrood; Circular No. 157 US Department of Agriculture Bureau of Entomology: Washington, DC, USA, 1912.
3. Forsgren, E. European Foulbrood in Honey Bees. J. Invertebr. Pathol.; 2010; 103, pp. S5-S9. [DOI: https://dx.doi.org/10.1016/j.jip.2009.06.016]
4. Genersch, E. American Foulbrood in Honeybees and Its Causative Agent, Paenibacillus Larvae. J. Invertebr. Pathol.; 2010; 103, pp. S10-S19. [DOI: https://dx.doi.org/10.1016/j.jip.2009.06.015]
5. OIE Terrestrial Animal Health Code. Available online: https://www.woah.org/ (accessed on 5 October 2022).
6. Roetschi, A.; Berthoud, H.; Kuhn, R.; Imdorf, A. Infection Rate Based on Quantitative Real-Time PCR of Melissococcus Plutonius, the Causal Agent of European Foulbrood, in Honeybee Colonies before and after Apiary Sanitation. Apidologie; 2008; 39, pp. 362-371. [DOI: https://dx.doi.org/10.1051/apido:200819]
7. Budge, G.E.; Barrett, B.; Jones, B.; Pietravalle, S.; Marris, G.; Chantawannakul, P.; Thwaites, R.; Hall, J.; Cuthbertson, A.G.S.; Brown, M.A. The Occurrence of Melissococcus Plutonius in Healthy Colonies of Apis Mellifera and the Efficacy of European Foulbrood Control Measures. J. Invertebr. Pathol.; 2010; 105, pp. 164-170. [DOI: https://dx.doi.org/10.1016/j.jip.2010.06.004] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20600088]
8. Alippi, A.M.; Albo, G.N.; Leniz, D.; Rivera, I.; Zanelli, M.L.; Roca, A.E. Comparative Study of Tylosin, Erythromycin and Oxytetracycline to Control American Foulbrood of Honey Bees. J. Apic. Res.; 1999; 38, pp. 149-158. [DOI: https://dx.doi.org/10.1080/00218839.1999.11101005]
9. Waite, R.J.; Brown, M.A.; Thompson, H.M.; Bew, M.H. Controlling European Foulbrood with the Shook Swarm Method and Oxytetracycline in the UK. Apidologie; 2003; 34, pp. 569-575. [DOI: https://dx.doi.org/10.1051/apido:2003052]
10. Pernal, S.F.; Albright, R.L.; Melathopoulos, A.P. Evaluation of the Shaking Technique for the Economic Management of American Foulbrood Disease of Honey Bees (Hymenoptera: Apidae). J. Econ. Entomol.; 2008; 101, pp. 1095-1104. [DOI: https://dx.doi.org/10.1093/jee/101.4.1095]
11. Thompson, H.M.; Waite, R.J.; Wilkins, S.; Brown, M.A.; Bigwood, T.; Shaw, M.; Ridgway, C.; Sharman, M. Effects of Shook Swarm and Supplementary Feeding on Oxytetracycline Levels in Honey Extracted from Treated Colonies. Apidologie; 2006; 37, pp. 51-57. [DOI: https://dx.doi.org/10.1051/apido:2005058]
12. Food and Agriculture Organization of the United NationsWorld Health Organization. Discussion Paper on Veterinary Drugs in Honey Production (Prepared by the CX/RVDF Electronic Working Group on Honey). Proceedings of the Joint FAO/WHO Food Standards Programme. Codex Committee on Residues of Veterinary Drugs in Foods, Nineteenth Session; Burlington, VT, USA, 30 August–3 September 2010.
13. American Veterinary Medical Association. The Veterinarian’s Role in Honey Bee Health. Available online: https://www.avma.org (accessed on 5 October 2022).
14. Rhéal Lafrenière Manitoba Agriculture 204–545 University Crescent, Winnipeg, MB R3T 5S6. Recommendations for Administering Antibiotics and Acaricides to Honey Bees. 2018; Available online: https://www.gov.mb.ca (accessed on 10 October 2022).
15. Bogdanov, S.; Fluri, P. Honigqualität Und Antibiotikarückstände. Schweiz. Bienenztg.; 2000; 123, pp. 407-410.
16. Reybroeck, W. Residues of Antibiotics and Sulphonamides in Honey on the Belgian Market. Group; 2002; 200, 50.
17. Wallner, K. Sulfonamide-Residues in German Honey, the Actual Situation. Apidologie; 2003; 34, 485.
18. Baggio, A.; Gallina, A.; Benetti, C.; Dainese, N.; Manzinello, C.; Piro, R.; Mutinelli, F. Antibacterial Residues in Italian and Imported Honey. Proceedings of the First European Conference of Apidology; Udine, Italy, 19–23 September 2004; 147.
19.
20.
21. Rivera-Gomis, J.; Bubnic, J.; Ribarits, A.; Moosbeckhofer, R.; Alber, O.; Kozmus, P.; Jannoni-Sebastianini, R.; Haefeker, W.; Köglberger, H.; MI, S.S. Good Farming Practices in Apiculture. Rev. Sci. Tech. Off. Int. Epizoot.; 2020; 38, pp. 879-890. [DOI: https://dx.doi.org/10.20506/rst.38.3.3032]
22. Pietropaoli, M.; Ribarits, A.; Moosbeckhofer, R.; Koglberger, H.; Alber, O.; Gregorc, A.; Smodis Skerl, M.I.; Presern, J.; Bubnic, J.; Necati Muz, M. et al. Biosecurity Measures in European Beekeeping. Rev. Sci. Tech.; 2021; 39, pp. 725-735. [DOI: https://dx.doi.org/10.20506/rst.39.3.3174]
23. FAO Integrated Pest Management. Available online: http://www.fao.org/agriculture/crops/core-themes/theme/pests/ipm/en/ (accessed on 6 November 2021).
24. OIE. Chapter 3.2.2. American Foulbrood of Honey Bees (Infection of Honey Bees with Paenibacillus Larvae). OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Mammals, Birds and Bees); World Organization for Animal Health: Paris, France, 2018; Volume 1, pp. 719-735.
25. OIE. Chapter 3.2.3. European Foulbrood of Honey Bees (Infection of Honey Bees with Melissococcus Plutonius). OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Mammals, Birds and Bees); World Organization for Animal Health: Paris, France, 2018; Volume 1, pp. 736-743.
26. Delaplane, K.S.; van der Steen, J.; Guzman-Novoa, E. Standard Methods for Estimating Strength Parameters of Apis mellifera Colonies. J. Apic. Res.; 2013; 52, pp. 1-12. [DOI: https://dx.doi.org/10.3896/IBRA.1.52.4.12]
27. Pearson, K. LIII. On Lines and Planes of Closest Fit to Systems of Points in Space. Lond. Edinb. Dublin Philos. Mag. J. Sci.; 1901; 2, pp. 559-572. [DOI: https://dx.doi.org/10.1080/14786440109462720]
28. Wilson, W.T.; Elliott, J.R.; Hitchcock, J.D. Treatment of American Foulbrood with Antibiotic Extender Patties and Antibiotic Paper Packs. Am. Bee J.; 1973; 113, pp. 341-344.
29. Eckert, J.E. Use of Sulfa Drugs in Treatment of American Foulbrood Disease of Honeybees. J. Econ. Entomol.; 1947; 40, pp. 41-44. [DOI: https://dx.doi.org/10.1093/jee/40.1.41]
30. Mutinelli, F. Practical Application of Antibacterial Drugs for the Control of Honey Bee Diseases. J. Invert. Pathol.; 1996; 67, pp. 65-71.
31. Kochansky, J. Analysis of Oxytetracycline in Extender Patties. Apidologie; 2000; 31, pp. 517-524. [DOI: https://dx.doi.org/10.1051/apido:2000103]
32. Thompson, H.M.; Waite, R.J.; Wilkins, S.; Brown, M.A.; Bigwood, T.; Shaw, M.; Ridgway, C.; Sharman, M. Effects of European Foulbrood Treatment Regime on Oxytetracycline Levels in Honey Extracted from Treated Honeybee (Apis Mellifera) Colonies and Toxicity to Brood. Food Addit. Contam.; 2005; 22, pp. 573-578. [DOI: https://dx.doi.org/10.1080/02652030500089986]
33. Mosca, M.; Giannetti, L.; Franco, A.; Iurescia, M.; Milito, M.; Pietropaoli, M.; Leto, A.; di Ruggiero, C.; Mezher, Z.; Palazzetti, M. et al. Impact of Oxytetracycline on Apis Mellifera Colonies: Preliminary Results on Residues and Antibiotic Resistance. J. Apic. Sci.; 2022; 66, pp. 159-170. [DOI: https://dx.doi.org/10.2478/jas-2022-0013]
34. Hornitzky, M.A.Z. Controlling American Foulbrood: Assessing Effectiveness of Shaking Bees and Antibiotic Therapy Strategies: A Report for the Rural Industries Research and Development Corporation; RIRDC: Kingston, Australia, 2001; ISBN 064258270X
35. Knox, D.A.; Shimanuki, H.; Caron, D.M. Ethylene Oxide plus Oxytetracycline for the Control of American Foulbrood in Honey Bees. J. Econ. Entomol.; 1976; 69, pp. 606-608. [DOI: https://dx.doi.org/10.1093/jee/69.5.606]
36. Del Hoyo, M.L.; Basualdo, M.; Lorenzo, A.; Palacio, M.A.; Rodriguez, E.M.; Bedascarrasbure, E. Effect of Shaking Honey Bee Colonies Affected by American Foulbrood on Paenibacillus Larvae Larvae Spore Loads. J. Apic. Res.; 2001; 40, pp. 65-69. [DOI: https://dx.doi.org/10.1080/00218839.2001.11101053]
37. Gilliam, M.; Valentine, D.K. Bacteria Isolated from the Intestinal Contents of Foraging Worker Honey Bees, Apis Mellifera: The Genus Bacillus. J. Invertebr. Pathol.; 1976; 28, pp. 275-276. [DOI: https://dx.doi.org/10.1016/0022-2011(76)90137-3]
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Details
; Giannetti, Luigi 1 ; Fortugno, Luca 3 ; Pietropaoli, Marco 1
; Manara, Veronica 1 ; Bonerba, Elisabetta 4
; Formato, Giovanni 1
1 Istituto Zooprofilattico Sperimentale del Lazio e della Toscana “M. Aleandri”, via Appia Nuova 1411, 00178 Rome, Italy
2 Agricultural Institute of Slovenia, Hacquetova ulica 17, 1000 Ljubljana, Slovenia
3 Servizi Veterinari ASL di Viterbo, via D. Alighieri 100, 01027 Montefiascone, Italy
4 Department of Veterinary Medicine, Food Safety Unit, University of Bari, Str. Prov. Casamassima Km 3, 70010 Valenzano, Italy