PREVENTION OF DISEASES IN HONEYBEES

Abstract

Feeding bees a composition containing at least one oxidizing agent (e.g., peroxide), optionally also containing other components such as an oxidizing agent activator or a viscosifying agent, to treat against, reduce and/or limit the spread of bacterial, virus, and fungi infestation in bees and/or beehives.

Claims

1. A method of reducing pathogen infections in bees and beehives comprising of feeding bees a composition comprised of at least one oxidizing agent.

2. The method of claim 1, wherein the composition is comprised of at least one peroxide as an oxidizing agent.

3. The method of claim 1, wherein the composition comprises at least one peroxide selected from the group consisting of hydrogen peroxide, peroxyacids, peroxycarbonates, urea hydrogen peroxide, perborate compounds, and combinations thereof.

4. The method of claim 2, wherein the peroxide composition is comprised of from about 0.001% to about 50%, from about 0.01% to about 5%, or from 0.1% to about 3% by weight peroxide.

5. The method of claim 1, wherein the composition is additionally comprised of at least one peroxide activator.

6. The method of claim 5, wherein the at least one peroxide activator comprises, consists essentially of or consists of at least one peroxide activator selected from the group consisting of metal-containing peroxide activators, carbonate salts and combinations thereof.

7. The method of claim 5, wherein the composition is comprised of from about 0.001% to about 20% by weight or from about 0.001% to about 5% by weight peroxide activator.

8. The method of claim 1, wherein the composition further comprises at least one thickener selected from the group consisting of viscosifying agents, gelling agents and mixtures thereof.

9. The method of claim 8, wherein the composition is comprised of from about 0.01% to about 10.0% by weight or from about 0.1% to about 5.0% by weight in total of said thickener.

10. The method of claim 1 wherein the pathogens are selected from the group consisting of the American Foulbrood (AFB) disease, Chalkbrood disease and Nosema disease.

11. The method of claim 1 wherein the pathogen is selected from the group consisting of American foulbrood (AFB), and European foulbrood (EFB); fungal diseases selected from the group consisting of Chalkbrood, Stonebrood, and Nosema; and viral diseases selected from the group consisting of Cripaviridae, Chronic bee paralysis virus, Dicistroviridae, Acute bee paralysis virus, Israeli acute paralysis virus, Kashmir bee virus, Black queen cell virus, Cloudy wing virus, Sacbrood virus; Iflaviridae—Deformed wing virus, Kakugo virus; Iridoviridae—Invertebrate iridescent virus type 6, Secoviridae—Tobacco ringspot virus, and Lake Sinai virus.

12. The method of claim 1 wherein the pathogen is bacteria selected from the group consisting of Melissococcus plutonius, Paenibacillus larvae, Spiroplasma apis, S. melliferum, Pseudomonas aeruginosa, Achromobacter euridice, Enterococcus faecalis, Paenibacillus alvei, and Brevibacillus laterosporus.

13. The method of claim 1 wherein the pathogen is fungi selected from the group consisting of Nosema apis, Nosema ceranae, Ascosphaera apis, and Aspergillus spp.

14. The method of claim 1 wherein the pathogen is responsible for a disease is a virus.

15. The method of claim 1 wherein the pathogen is a virus selected from the group consisting of Israeli acute paralysis virus, acute bee paralysis virus, Kashmir bee virus, black queen cell virus, deformed wing virus/Kakugo virus, Varroa destructor virus, sacbrood virus slow bee paralysis virus, chronic bee paralysis virus and Lake Sinai virus.

16. The method of claim 1 wherein the pathogen is selected from the group consisting of bacteria spores, fungi spores or yeast.

17. The method of claim 1 wherein the pathogen is selected from the group consisting of Varroa mites (Varroa destructor or Varroa jacobsoni), Acarine (tracheal) mites (Acarapis woodi), small hive beetles (Aethina tumida), wax moths (Galleria mellonella), and tropilaelaps (Tropilaelaps clareae and T. mercedesae)

18. The method of claim 1 wherein said composition is a bee-ingestible composition in liquid form.

19. The method of claim 1 wherein said composition is a bee-ingestible composition in solid form.

20. The method of claim 1 wherein said composition contains protein.

21. The method of claim 19 wherein said composition is based on pollen and/or soy patties.

22. The method of claim 1 wherein said composition is a bee-ingestible composition based on sucrose solution.

23. The method of claim 1 wherein said composition is a bee-ingestible composition based on corn syrup solution.

24. The method of claim 1 wherein said composition is a bee-ingestible composition that further comprises a carbohydrate.

25. The method of claim 1 wherein said composition is a bee-ingestible composition that further comprises a sugar supplement.

26. The method of claim 1 wherein the composition is administered to the bees via a device selected from the group consisting of strip, controlled release strip, tablet, reservoir, polymer disc, evaporation device, fiber, tube, polymeric block, membrane, pellet, and microcapillary comprising the composition.

Description

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

[0048] Dispensing of hydrogen peroxide to the hive can be done by introducing the compound in the composition used in supplemental feeding of honeybees, which is common practice. Honeybees can be fed various foodstuffs to supplement inadequate supplies of pollen or honey. In early spring before pollen and nectar are available or at other times of the year when these materials are in short supply, supplementary feeding may help the colony survive or make it more populous and productive. In practice, beekeepers feed their bees supplemental foods to develop and maintain colonies with optimum populations. The hydrogen peroxide compound is added at the desired concentration to the composition to be fed to the bees. The composition can be a liquid, gel or solid. As an alternative, the composition can be administered to the bees by being dispensed from a variety of forms or devices. The devices can include a strip, controlled release strip, tablet, reservoir, polymer disc, evaporation device, fiber, tube, polymeric block, membrane, pellet, and microcapillary.

[0049] A common foodstuff for honeybees is a mixture of water and sugar, a sucrose solution. A typical water/sugar mixture comprises a ratio of one part of sugar to one part of water, measured by weight (known as 1:1). A denser syrup of two parts of sugar to one part of water (known as 2:1) may also be used. Generally, 1:1 syrup is used to supplement honey stores, stimulate colonies to rear brood and encourage drawing of comb foundation particularly in spring. The stronger syrup is used to provide food when honey stores in the hive are low. Measuring the sugar and water by weight or volume is all right, as there is no need to be 100% exact about the sugar concentration. Similar foodstuff solutions for bees can be based upon corn syrup.

[0050] The composition of the present invention can also be a component of a solid bee foodstuff such as pollen and/or soy patties, with or without added proteins, carbohydrates or sugar supplements.

[0051] The composition used with the present invention includes at least one oxidizing agent, which may be any organic or inorganic compound capable of treating against any bacterial, viruses, fungi, and protozoa infestation. Oxidizing agents can be selected from a variety of peroxides. For example inorganic and organic peroxides such as but not limited to hydrogen peroxide or peroxide generating compounds such as percarboxylic acids (e.g., peracetic acid), peroxycarbonates, urea hydrogen peroxide, perborate compounds, as well as similar compounds and/or a combination of such compounds. The concentration of oxidizing agent in the composition may be between 0.001 and 50%, or between 0.01 and 5%, or between 0.1 and 3% by weight in a foodstuff, preferably a sucrose solution.

[0052] The composition can be gelled or otherwise increased in viscosity for better handling of the composition using at least one viscosifying agent, which may be an inorganic viscosifying agent or an organic viscosifying agent or a combination of viscosifying agents. Examples of suitable viscosifying agents include, for example, modified silicas (e.g., the silicas sold by Evonik under the brand names Aerosil® and Sipernat®), high molecular weight crosslinked polyacrylic acid polymers (e.g., the polymers sold by Lubrizol under the brand name Carbopol®), xanthan gums (e.g., the xanthan gums sold by CP Kelco under the brand name Kelzan®) and other such gums (guar gums, alginates and the like), polyol and polyether glycol compounds such as glycerol, polyethylene glycol, polypropylene glycol, and all other viscosifying or gelling agents that are compatible with the other components of the composition and preferably nontoxic to bees and environmentally friendly (e.g., non-persistent and/or biodegradable). The viscosifying agent and/or gelling agent may, in certain embodiments of the invention, act as a thixotropic agent. In various embodiments of the invention, the viscosifying or gelling agent concentration in the composition is between 0.01% and 20% by weight or between 0.1% and 5% by weight.

[0053] The order and manner in which the above-described components of the composition are combined and formulated are not believed to be particularly limited. Methods and techniques known in the art may be adapted and modified as appropriate, depending upon the types and relative amounts of the components selected for use. In certain embodiments, the composition may be a one-part formulation, having sufficient physical and chemical stability to permit storage at normal conditions (e.g., in drums, tanks or other containers at room temperature) for extended periods of time. Such formulations may then be directly utilized in accordance with any of the procedures described herein. In other embodiments, the composition may be provided as a multi-part formulation, particularly where certain components of the composition are reactive with each other and it is desired to avoid such reaction until the composition is to be fed to bees. For example, where the composition comprises both a peroxide and a peroxide activator, the peroxide activator may undesirably react prematurely with the peroxide or otherwise transform the peroxide such that the effectiveness of the composition. In such cases, the composition may comprise two parts which are stored separately, wherein one part comprises the peroxide (and optionally one or more components of the composition other than the peroxide activator) and a second part comprises the peroxide activator (and optionally one or more components of the composition other than the peroxide). The two parts are then combined, in the desired proportions, to obtain the composition, either shortly before use.

[0054] The composition can be activated by the presence of an activator for the oxidizing agent, i.e., a substance that assists in catalyzing or otherwise promoting disinfecting against any bacterial, viruses, fungi, protozoa and mite infestation. For example, the activator may convert the oxidizing agent into a more reactive substance, e.g., a substance better able to disinfect against any bacterial, viruses, fungi, protozoa and mite infestation. In certain embodiments of the invention, the oxidizing agent formulated into the composition may be regarded as an oxidizing agent precursor, which by itself has little or no impact but which is transformed in situ to a reactive oxidizing agent through interaction with an activator as described herein. Post-addition of an activator to a composition in accordance with the present invention may be practiced. The activator may be added to the composition right before use of the composition. Activators can be, for example, metal-containing substances such iron oxide (Fenton reaction), carbonate compounds (e.g., salts of carbonic acid such as potassium or sodium bicarbonate), a more complex activator such as Fe-TAML (tetra-amido macrocyclic ligand) developed at Carnegie Mellon or any activator or combination of activators that are known to induce the formation of free radical compounds under various conditions (including the use of UV light). The concentration of activator in the composition may be, for example, between 0.001% and 20% by weight or between 0.001% and 5% by weight.

[0055] Within this specification, embodiments have been described in a way which enables a clear and concise specification to be written, but it is intended and will be appreciated that embodiments may be variously combined or separated without departing from the invention. For example, it will be appreciated that all preferred features described herein are applicable to all aspects of the invention described herein.

[0056] In some embodiments, the invention herein can be construed as excluding any element or process step that does not materially affect the basic and novel characteristics of the composition or the method for using the composition. Additionally, in some embodiments, the invention can be construed as excluding any element or process step not specified herein.

EXAMPLES

Example 1: Stability of Hydrogen Peroxide in Sugar Solution

[0057] Hydrogen peroxide, Peroxal 50% CLG (available from Arkema Inc.) was used to create concentrations of 0.1% and 1.0% hydrogen peroxide in 50% (w/w) sugar (sucrose) solution. Experiments were done at ambient temperature. No degradation of the hydrogen peroxide was observed over 7 weeks.

TABLE-US-00001 TABLE 1 Hydrogen peroxide stability in sugar solution H2O2 Jan. 10, 2017 Jan. 20, 2017 Feb. 6, 2017 Mar. 2, 2017 Conc. (time 0) (10 days) (27 days) (51 days) 0.1% 0.134 0.134 0.134 0.130 1.0% 1.077 1.089 1.058 1.089

[0058] The data in Table 1 shows that hydrogen peroxide is stable in a sugar solution that could be used as a bee food.

Example 2: Determination of the Oral Toxicity of Formulated Hydrogen Peroxide for the Honeybee (Apis mellifera)

[0059] The twenty-four and forty-eight hour oral lethal concentration (LC.sub.50) of formulated hydrogen peroxide (HP) was determined for one-week and two-week old honeybee (Apis mellifera) workers fed a series of solutions of HP diluted in 2 Molar (M) sucrose.

[0060] Five hydrogen peroxide (HP) solutions at 4.7, 3.7, 2.7, 1.7, 0.7 M HP in 2 Molar (M) sucrose, and a 0.00 M HP 2M sucrose solution were prepared using 35% food grade hydrogen peroxide, molecular grade sucrose (Sigma Aldrich, USA), and molecular grade water (Sigma Aldrich, USA).

[0061] Approximately 200 bees were collected into several wooden cages, supplied with ad libitum HP-free 2M sucrose solution, water, and BeePro® supplemented pollen patty. Experimental trials commenced at seven and fourteen days after newly emerged honeybees were placed in cages.

[0062] Non-adjusted mortality data (mean±standard error (se)) are provided in Table 2.

TABLE-US-00002 TABLE 2 Mean (±standard error) mortality of one-week and two-week old honeybees after feeding on hydrogen peroxide-2M sucrose solutions for either 24 or 48 hours. Mean (±se) Mortality Concentration Week 1 Week 1 Week 2 Week 2 H.sub.2O.sub.2 (Molar) 24 Hour 48 Hour 24 Hour 48 Hour 0  0.75 (0.16)  0.87 (0.40)  1.13 (0.30)  1.38 (0.53) 0.7  3.38 (1.38)  9.63 (2.23)  7.00 (2.19) 12.75 (1.96) 1.7 10.25 (1.42) 18.13 (1.06) 12.00 (2.32) 16.25 (1.50) 2.7 15.63 (1.19) 19.88 (1.63) 14.13 (2.12) 19.50 (1.00) 3.7 15.25 (1.70) 21.50 (0.96) 16.63 (1.92) 20.75 (0.53) 4.7 15.75 (1.53) 21.38 (0.99) 17.88 (1.51) 20.13 (1.36)

[0063] Table 3 gives LC.sub.50 values for each assessment point predicted from Probit analyses using login concentrations, LC.sub.50 hydrogen peroxide concentrations calculated from predicted values, and corresponding percent hydrogen peroxide (using MW=34 g/mol for H.sub.2O.sub.2, and p=1.25 g/ml for 2M sucrose solution).

[0064] The twenty-four hour mortality was significantly lower than the 48-hour mortality for both the one-week (Z=3.41, P<0.001) and two-week old honeybees (Z=2.57, P=0.010) (Table 2). However, mortality did not differ significantly between one-week and two-week old honeybees at either the 24 or 48-hour assessment points.

[0065] The twenty-four hour LC.sub.50 values were greater than the 48-hour values for both the one-week and two-week old honeybees (Table 3). This was likely due to HP-free sucrose solution remaining in their crops (social stomach of honeybees) even after six hours of starvation prior to beginning trials. Honeybee workers have a high metabolic rate, but also have the ability to share food (via trophallaxis) among nest mates. After forty-eight hours, however, all HP-free sucrose solution was depleted from worker crops and honeybees then fed directly on HP-containing sucrose solutions.

TABLE-US-00003 TABLE 3 Mean (±standard error) log.sub.10 lethal concentrations of hydrogen peroxide resulting in 50% mortality (LC.sub.50) of one- week and two-week old honeybees feeding on hydrogen peroxide-2M sucrose solutions for 24 or 48 hours. Predicted LC.sub.50 values in Molar hydrogen peroxide were calculated by taking the inverse of log.sub.10 data. Predicted LC.sub.50 Predicted LC.sub.50 Predicted LC.sub.50 Log.sub.10 Molar (±se) (Molar) (Percent) Week 1-24 Hour  0.29 (0.02) 1.95 (0.09) 5.30 (0.25) Week 1-48 Hour −0.10 (0.03) 0.79 (0.05) 2.15 (0.14) Week 2-24 Hour  0.15 (0.03) 1.41 (0.10) 3.84 (0.28) Week 2-48 Hour −0.25 (0.06) 0.56 (0.08) 1.52 (0.22)

[0066] The LC.sub.50 values of two-week old honeybees were slightly lower than those of one-week old honeybees for both the 24 and 48-hour assessment points (Table 2). This is likely due to an increase in sucrose consumption of honeybees as they age. Young adult (nurse) honey bees consume more protein than carbohydrates for the first week post-emergence, but then rapidly increase sucrose consumption after this point.

[0067] The no observed effect concentration (NOEC) could not be determined from the data; honey bee mortality observed for all tested concentrations were significantly greater than mortality of honeybees in control group (Wilcoxon multiple comparisons: χ.sup.2=113.5; df=5; P<0.001). Following this, an EC.sub.5 was calculated by solving the cubic (sigmoidal) model for ‘x’ given an intercept term constrained to zero (concentration) and a ‘y’ value of 0.05. Model solutions and calculated EC.sub.5 (as % H.sub.2O.sub.2) are provided in Table 4.

TABLE-US-00004 TABLE 4 Solutions to cubic models for ECx estimations. Values of X.sub.3 (bold) are best estimates of EC.sub.5 concentration (in Molar hydrogen peroxide), and were used to calculate EC.sub.5 in percent H.sub.2O.sub.2. Solutions for ‘x’ (Molar) Assessment point X.sub.1 X.sub.2 X.sub.3 EC.sub.5 (Percent) Week 1-24 Hour 2.78 −5.36 0.21 0.57 Week 1-48 Hour 2.69 −4.58 0.15 0.41 Week 2-24 Hour 3.23 −5.03 0.20 0.54 Week 2-48 Hour 2.61 −4.20 0.15 0.41

[0068] Although the invention is illustrated and described herein with reference to specific embodiments, the invention is not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the invention.