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Field of the invention

In its most general aspect, the present invention relates to compositions and methods for the biological control of the well-being of hive bees, and in particular relates to the treatment and prevention of the most widespread bee diseases due to microbiological etiology, such as those caused by bacteria and fungi infesting the colony of bees and the hive. The composition and the method according to the present invention are useful for the maintenance of the bees good health and for the prophylaxis and the treatment of bacterial and fungal infections, and in particular of Nosema disease and of pathologies due to infestations of other concomitant agents.

Background of the invention

Maintaining the good condition of the beekeeping sector is obviously important not only for the production of honey and other bee products, but also for the more wide action on pollination of agricultural crops and entomophilous plant species. In recent years an increase in bee mortality has been repeatedly reported and several health problems have affected the beekeeping sector in different countries worldwide. This raised great concern for significant environmental and economic consequences. It is quite evident, therefore, the need to counteract this beekeeping impoverishment through targeted measures, considering that the main causes are pathologies affecting the hive and the not always correct use of agro-drugs. Therefore, it is necessary to focus the activity on new strategies, able to fight bee diseases with low environmental impact substances.

Recently, in beekeeping the use of microorganisms, or particular groups of microorganisms, able to exert a beneficial effect to increase the well-being and the resistance of bees against various pathogens, to increase the bee immunological defences has been introduced. In some cases the microbial associations have been shown to be very effective therapeutic agents that have found application in beekeeping.

The patent application JP19980047329 describes a method for the treatment of health problems due to bee mites and a method for improving the bees immune system by supplementing bee feed with a mixture of Bacillus subtilis, Lactobacillus lactis and Lactobacillus bifidus and bifidobacteria and sugars. The patent application DE 4215534 describes the use of a composition, applied in a method of treatment of varroa, nosema and in general to diseases affecting bees, comprising the administration of a mixture comprising Lactobacillus helveticus and Lactobacillus acidophilus, lactose and inorganic salts.

The patent application RM 2012A000635 describes a composition comprising the association of microorganisms of the species: Lactobacillus paracasei ssp., Bifidobacterium bifidum, Lactobacillus acidophilus _r Lactococcus lactis _r Bifidobacterium animalis , Lactobacillus thermophilus , Bacillus clausii , in 5% sucrose solution. The composition has been shown to induce a positive effect on the bees health and to be particularly useful for the treatment of serious infections such as those determined by CBPV virus infection and/or by Nosema ceranae infections. Kaznowski et al . have investigated the effects of probiotic supplementation on the content of intestinal microflora and chemical composition of worker bees {Apis mellifera) , in particular the authors have tested two probiotic mixtures used as supplements to pollens substitute in feeding honey bees; the advantageous effect deriving from probiotic supplementation has been evaluated in terms of bee survival, higher dry mass and crude fat level in comparison to bees fed with pollen substitute only from 14 and 2 days treatment. One of the two tested probiotic mixture contains the following bacterial species: Pediococcus acidilactici (10%), Lactobacillus acididophilus (35%), Enterococcus faecium (5%) and Bifidobacterium bifidum (50%), the second one contains Lactobacillus acididophilus (37,5%), Lactobacillus delbrueckii subspecies bulgaricus (25%), and Bifidobacterium bifidum (50%) . The study has demonstrated that adding either probiotic mixtures to pollen substituted improved general bee conditions and well-being, as expressed by greater content of dry mass and crude fat content, furthermore, the probiotics favoured the bee survival, but no effect against specific pathologies has been thought.

The international patent application WO 2014/170233 discloses a mixture of Lactobacillus buchneri , Lactobacillus plantarum and Lactococcus lacti used in a method for improving milk-quality in milk producing animals, more specifically the document teaches feeding cows with fermented silage produced by inoculating perennial ryegrass with Lactobacillus plantarum CH6072 (DSM16568), Lactococcus lactis 0224 (DSM11037), L . buchneri Lbl819 (DSM22501), and optionally also Lactococcus lactis (NCIMB 30117) .

The US patent US 6,403,084 Bl disclose a mixture of Lactobacillus buchneri and Lactobacillus plantarum for the use as silage inoculants and the method of treating animal feed or silage with the silage inoculants. More specifically the document teaches to combine specific bacterial species in the proper ratio to provide both an adequate fermentation of silage or animal feed as well as an enhanced aerobic stability upon exposure of the silage or feed to air. The silage inoculant is a combination of at least one viable strain of the homofermentive lactic acid bacteria Lactobacillus plantarum and at least one viable strain of the heterofermentive lactic acid bacteria Lactobacillus buchneri or Lactobacillus brevis. In some embodiments, the silage inoculant will comprise at least 2 to 10 strains of homofermenter and/or heterofermenter . Exemplary strains of L. plantarum include at least one of LP286, LP287, LP329, LP346, LP347, or functional mutants thereof. Exemplary strains of L . buchneri include LN1391, LN4637, LN4750, or functional mutants thereof. The silage inoculant optionally comprises at least one viable strain of Enterococcus faecium, such as but not limited to, strains EF301, EF202, or functional mutants thereof. The number of viable homofermentive bacteria and heterofermentive bacteria in the inoculant are present in a ratio of from about 1:5 to about 1:15. In some embodiments the ratio is about: 1:6 to 1:14, 1:7 to 1:13, 1:8 to 1:12, 1:9 to 1:11, or 1:10.

The US patent application US 2006/046292 describe the effect of inoculation with an association of lactic acid bacteria (L. plantarum 7109, L. paracasei tolerans LC3200, L. buchneri LN4017) on the forage to feed lambs.

The use of combinations of non-pathogenic bacteria cultures, particularly stable, reproducible and effective, represents a biological method to control pests and diseases of bees which seriously affect the health of the hive, especially appealing to beekeepers, as it is cost-effective and more respectful of the environment than the chemical agents and biopesticides.

Generally, bee diseases are divided into brood diseases and diseases of the adult, including also fungal diseases; there are also some diseases, eg. the acariosis, damaging both bee broods and adult bees. Nosema disease affects adult bees, it is caused by unicellular fungi belonging to the class of Microsporidia, Nosematidi Family, Gender Nosema. Two different species of Nosema affect bees: Nosema apis and Nosema ceranae, responsible for two different pathology forms.

Nosema apis is responsible for the "classic" gastro ^¬ enteric disease, whereas Nosema ceranae has been reported more recently in bees as the causative agent of a disease form characterized by slow depopulation of the colony until its death. Both N. apis and N. ceranae appear at the optical microscope in the form of spores, morphologically indistinguishable between the two species: a differential diagnosis can be performed only through genome analysis of the spores. Nosema apis lives and spreads in the epithelial cells of the digestive tract of adult bees. It affects the larval stages. Unlikely the queen bee is concerned by the disease, however, when the queen is sick, after a few weeks she ceases to lay eggs. Spores in excreta are, directly or indirectly, ingested by adult bees, germinate in the intestinal lumen and penetrate the intestinal cells where they develop and multiply by eating the cell cytoplasm up to burst the invaded intestinal cell. Transmission occurs primarily by fecal-oral route. The spores propagating the disease are expelled in the feces and, once outside, they spread by ingestion to other bees that in turn become infected. After the contact with N. apis, in the bee colony the first symptoms appear, such as diarrhea, the young nurses become unable to secrete the royal jelly, while gatherer bees reduce their activity, until it stops completely. A slow depopulation of the colony occurs, the work decreases. Bees are no longer able to fly. Finally, the hive is smeared by diarrhea, and on the bottom of the hive, dead bees with the swollen and soft abdomen appear. The establishment of such severe conditions favours the simultaneous onset of other diseases, such as amoebiase or viruses, exacerbates the feature of Nosema disease. In temperate zones the disease begins to be manifest usually in late winter, the overt symptoms are seen in April-May.

The typical events correlated to the disease caused by Nosema ceranae infections instead are beehives depopulation and bees death, diarrhea is not a typical symptom, furthermore, the disease appears in different time periods than the classic one, infection levels vary greatly during the year. Contact with the microsporidium occurs via the digestive system, e.g. through honey contaminated by spores. Once in the intestinal lumen, the spores germinate and spread to epithelial cells wherein they live and breed. N. ceranae spores of are very resistant, thus facilitating the re-infection of the colonies and the recurrence of the disease after a long time.

For most of these infections are not currently available really valid remedies. Only Nosema microsporidium is very sensitive to the antibiotic fumagillin, but it requires the issuance of special licence to be used in the bee hive.

Furthermore, in case of infection, the prognosis is often fatal because the onset is almost always unnoticed and symptoms manifest in a too advanced stage. Generally, colonies affected do not heal spontaneously and if a particularly severe disease form occurs, or weak families are affected, the hive destruction is advised. After killing the bees, hives sterilized and destroyed the combs, materials can be retrieved, but infected honey absolutely has not to be used to feed other bees, resulting in considerable economic loss.

Therefore, a preventive action against the onset of infection is definitely the strategy to be followed. Preventing infection by the proper conduct of the apiary, taking care of the location, preferring sunny and poorly ventilated areas, with the correct orientation of the hives.

However, it is quite evident that following the good beekeeping practices that normally should be adopted in the apiary is not sufficient to prevent the emergence and spread of new foci of infection, therefore, it is strongly felt the need to develop new compositions for the biological control of hives Nosema disease.

Summary of the Invention

The aim of the present invention is to provide a composition enabling a treatment against hive diseases induced by bacterial and fungal infections, against fungal diseases affecting the brood, such as stone brood {Aspergillus flavus) and chalk brood (Ascosphera apis) , against diseases induced by Nosema apis and Nosema cerana, and bacterial diseases such as American foul brood and European foul brood that very often affect apiaries.

Furthermore, the composition according to the invention determines on bee families a stimulating and reinforcing effect, and reduce sensitivity to other concomitant pathogens.

The composition according to the invention comprises the association of specific bacterial strains hence promoting the establishment in the hive of a corrective and protective microflora whose metabolic activity produces substances that induce a positive effect on the conditions of the bee colony.

The composition comprising the association of microorganisms according to the invention enables to limit the reduction in brood that naturally and physiologically occurs in the hive at the end of summer season.

The composition according to the invention comprises the biological association of viable microorganisms of the species Lactobacillus buchneri and viable microorganisms of at least one specie selected from the group of: Pediococcus pentosaceus, Lactococcus lactis, and Lactobacillus plantarum, and prebiotic agents .

It is still an object of the invention to provide a method to exert the biological control of diseases of the hive and the general well-being of the bees colony, by applying, in an effective amount, the composition comprising the association of viable microorganisms of the species Lactobacillus buchneri and viable microorganisms of at least one species selected from the group: Pediococcus pentosaceus _r Lactobacillus plantarum and Lactococcus lactis, and prebiotic agents.

Further characteristics and advantages of the invention are more readily apparent from the accompanying drawings which summarize the results obtained in the experimental trial.

Brief description of figures

Figure 1 shows the diagram illustrating the percentage of change in the number of adult bees in beehives subjected to treatment with the composition of microorganisms according to the invention obtained by the estimation of sixths;

Figure 2 shows the diagram illustrating the percentage of change in the number of broods, without specifying whether these are operculated or not, in the hives subjected to treatment with the composition of microorganisms according to the invention obtained by estimating sixths;

Figure 3 shows the diagram illustrating the percentage of change in the compactness of the brood in the hives subjected to treatment with the composition of microorganisms according to the invention obtained by the estimation of sixths;

Figure 4 shows the diagram illustrating the number of dead bees found in the hives seven days after the first administration of the composition of microorganisms according to the invention;

Figure 5 shows the diagram illustrating the average weight of the supers placed above the hive for each experimental group;

Figure 6 shows a diagram illustrating the change in the Nosema spp . infection level expressed as a function of the number of spores per bee, before and after treatment with the composition of microorganisms according to the invention;

Figure 7 shows the diagram illustrating the percentage change in the entity of the infection from Nosema spp. expressed as a function of the number of spores per bee, before and after treatment with the composition of microorganisms according to the invention.

Detailed Description of the invention

It has been found that some microorganisms exert a probiotic effect, both on larvae and adult bees, having a therapeutic and immunostimulant effect. Consequently, they of great interest for the preventive action and the control of infections on the hive by pathogens .

Indeed, in the development of a therapeutic biological composition the choice of the particular bacterial strain to be used is crucial to determine the effect sought. Although it is now widely known and documented that certain types of lactobacilli are essential to maintain a state of optimal health in humans and in animal species, it is also known that the physiological and therapeutic properties of lactobacilli vary depending on the specific features of each strain. It has also been shown that there are considerable differences between different strains of L. acidophilus, and that the same strain, grown in different environments and conditions, shows different properties (Bhatia, V. 1991. Growth optimization of L. acidophilus in whey. MS Thesis, Univ. of Nebraska, Lincoln) . In addition, in the determination of the final effect it should be taken in account also the particular interactive action between the different probiotic species that constitute the composition. The combination of more and different species provides a very complex metabolic profile, wherein each element, or species, contributes in a specific way by producing a set of biomolecules that overall determine the positive effect on the bees health conditions.

Such special contributing aspects are of paramount importance and have been carefully considered in the selection of strains of the bacterial association characterizing the composition according to the invention and its therapeutic or prophylactic properties .

The invention provides a biological composition comprising a specific association of viable microorganisms, prebiotic agents, sugars, salts and minerals, one or more excipients and carriers.

The specific association of viable microorganisms comprises microorganisms of the species Lactobacillus buchneri and microorganisms of at least one species selected from the group of: Pediococcus pentosaceus, Lactobacillus plantarum and Lactococcus lactis.

That particular association of microorganisms identified performs metabolic activities that overall are able to:

induce a positive effect on the health of a colony of bees,

- limit the reduction in brood which naturally and physiologically occurs in the hive at the end of the summer season,

- treat and prevent pathological disorders of bees caused by unicellular fungi belonging to the genus Nosema, and related infections.

Thus, in a first embodiment particularly preferred of the invention the microorganism association comprises bacteria of the species Lactobacillus buchneri and Lactococcus lactis. In another embodiment particularly preferred of the invention the microorganism association comprises bacteria of the species Lactobacillus buchneri and Pediococcus pentosaceus .

In a further embodiment particularly preferred of the invention the microorganisms association comprises bacteria of the species Lactobacillus buchneri,. Pediococcus pentosaceus and Lactococcus lactis.

Another particularly preferred embodiment of the invention provides in the microorganism association the presence of probiotic bacteria of the species Lactobacillus buchneri, Lactobacillus plantarum and Pediococcus pentosaceus .

The bacterial strains of the association in the composition of the present invention have been selected for their metabolic properties and for their probiotic activity, which have been found highly effective against the onset of Nosema disease and related diseases thereof. In fact, the use of the combination of the mentioned bacterial strains is designed to strengthen the bees immune system, resulting in a preventive and curative treatment of infections .

The bacterial strains of the specific association according to the invention are obtained from official collections such as DSM, CCM, NCIMB collections.

The composition comprising the association of viable microorganisms of the species Lactobacillus buchneri and viable microorganism of at least one species selected from the group of: Pediococcus pentosaceus, Lactobacillus plantarum and Lactococcus lactis, further comprises: sugars, salts and minerals, prebiotic agents, at least one or more carriers and excipients. In particularly preferred embodiments of the invention the composition comprises inulin and/or fructooligosaccharides (FOS), silica, maltodextrin, levulose, sucrose, sodium chloride.

The composition is obtained by mixing the lyophilized preparation of the bacterial strains of the biological association of microorganisms, together with the other provided components.

Overall, in the composition the microorganisms are at a concentration ranging between l-2xl0 ⁹ and l-2xl0 ¹³ cfu/gram, preferably microorganisms are at a concentration of 1-2X10 ¹¹ cfu/gram, and in particular L. buchneri is present in amount of at least 2xl0 ¹⁰ cfu/gram; the amount of the microorganisms of the other bacterial strains in the composition is varying between 1x10 and 2x10 . Preferably, bacteria of the strain Pediococcus pentosaceus are present in the composition at a concentration of at least lxlO ⁷ cfu/gram, bacteria of the strains Lactobacillus plantarum and Lactococcus lactis are present in the composition at a concentration of at least 2xl0 ¹⁰ cfu/gram.

By way of example it is provided below the formulation of a particularly preferred embodiment of the composition according to the invention: Table 1.

The composition according to the invention may be in either liquid or dry form, however in a preferred embodiments it is in dry form wherein the viable bacteria are lyophilized. Lyophilisation is performedaccording to standard techniques known to the skilled persons.

The composition according to the invention is in solid form, such as capsules, tablets, granules, powder. Preferably, the composition according to the invention is in the form of powders to be dissolved in water before use, i.e. before administration to the hive. It is thus presented as a product packaged in sachets containing 10 g of the composition containing the optimum amount for the complete treatment of 20 hives. A further object of the present invention is the use of the composition comprising the described association of microorganisms, prebiotic agents, salts and minerals, and one or more excipients and carriers, in the treatment and prevention of infection caused by certain pathogens of bees, in particular in the treatment of the infection caused by unicellular fungi like Nosema.

This use involves the administration of the composition to bees, larvae, beehive and other components related to the hive. A feature particularly advantageous from the use of the composition according to the invention is the fact that it can be used in the hive even in the presence of supers.

In particular, the method based on the use of the composition according to the invention comprises the following steps:

- to dissolve 10 g of composition according to the invention in 2 litres of water directly into a sealable container, close the container and mix thoroughly;

to let stand for 3 - 12 hours at ambient temperature ;

- to mix slightly before each use.

to dispense the composition solution by spraying, by syringe, by inoculation into the feeder and in any other method known to the skilled persons, adapted to bring the solution in contact with the bees, .

The dissolved solution can be used within 7 days if stored tightly closed and in a cool, dry place.

In the case of dispensing the composition according to the invention by spraying, about 10 ml of solution prepared as above described, are sprayed either on each frame hive in the brood nest without extracting the frames, or directly on honeycombs, after opening the roof and the crown board.

In the case of dispensing the composition according to the invention by a syringe, about 10 ml of solution prepared as above described are spread over each frame hive in the brood nest without extracting the frames, after opening the roof and the crown board.

In the case of dispensing by inoculums in feeder, about 80-100 ml of the solution prepared as above described are placed directly in the feeder, or added directly to a nourishment syrup introduced in the beehive feeder.

The method based on the use of the composition according to the invention for the treatment of bacterial and fungal diseases affecting the hive, the Nosema disease, and for the strengthening of the bee family and for increasing the compactness of brood requires to perform 3-4 treatments:

1 - before the beginning of spring (start-up treatment) ;

2 - late spring (recall treatment) ;

3 - in the summer (anti-stress treatment) ;

4 - late summer (cleaning-up treatment) .

The composition according to the invention can also be used in winter as feeding administrated by feeder, possibly also together with a syrup.

Specific experiments and observations carried out in order to assess the positive effect on the bees colony have shown that the composition according to the invention has a high potential in protecting adult and larvae bees by multiple mechanisms of action. The treatment resulted in a high protection in the bees colony against the external stress and a considerable improvement of the well-being of the treated bees and a considerable containment of Nosema spp . infections. However, specific investigations are underway and preliminary results are very encouraging about the effect of the composition according to the invention and the relative method of use against other fungal diseases affecting the brood, such as stone brood {Aspergillus flavus) and chalk brood (Ascosphera apis) , bacterial diseases such as American foul brood and European foul brood that very often affect apiaries .

Therefore, the present invention provides an alternative technical solution for the treatment of bacterial and fungal bee diseases; in particular the composition according to the invention and the relative method of use has resulted to be effective in the treatment of Nosema disease and of other concomitant pathologies establishing with the disease, compared to those already present in the state of the art, such as the composition for the treatment of varroa, Nosema disease, and in general other diseases affecting bees, that combine different types of lactobacilli in the presence of sugar.

Specific experiments and observations conducted in order to verify the effectiveness of the composition according to the invention and its method of use on the bees colony have shown that the composition has a high potential in protecting adult bees and larvae by multiple mechanisms of action. The treatment resulted in the colony a high protection against the external stress and a considerable improvement of the well- being of the colony of bees treated and the considerable reduction of the presence of the pathogen responsible for Nosema disease.

The positive effect deriving from the use of the composition was determined according to the strength of families of bees, through the evaluation of parameters indicative of the good condition of the bee colony and strength of bee colonies, by the estimate of the sixth, the discovery of the diarrhea phenomena, by the assessment of the brood compactness, bee mortality, amount of honey produced and evaluation of infection by Nosema spp.

Experimental Procedure

The effect of the composition against bee disorders established by microbial pathogens and more generally on the health of the bees was investigated by in vivo experiments carried out from July 2014 to September 2014 in an apiary located in the territory of Lazio region, Italy.

The bees colony of the apiary under observation has been administered every 14 days for a total of 4 pulses with the composition according to the invention comprising a probiotic mixture constituted by the mixture of lyophilized microorganisms of the species Lactobacillus buchneri , Pediococcus pentosaceus and Lactococcus lactis with total concentration of 2x1ο ¹¹ cfu/g, prebiotic agents, sugars, salts and minerals, one or more excipients and carriers, as reported in table 1 of this description. The effectiveness of the treatment with said composition was evaluated by the following parameters and in accordance with the timetable indicated:

Evaluation of the strength of the hives by estimating the sixth to highlight the acute toxicity of adult bees and brood (without specifying whether concerning ovum/brood uncap or sealed) :

• time 0: pre-treatment;

• time 1: at the end of the last treatment;

• time 2: one month after the last treatment.

Discovery of diarrheal phenomena detected on the flight board and in the nest

• time 0: pre-treatment;

• time 1: day 2 after treatment;

• time 2: every time the probiotic composition is administered;

• time 3: at the end of the last treatment.

Evaluation of the compactness of the brood

For the purpose a 0 to 5 rating scale was used:

score = 1 corresponds to a number of broods <75%;

score = 2 corresponds to a number of broods in the range 75 - 85%;

score = 3 corresponds to a number of broods in the range 85 - 90%;

score = 4 corresponds to a number of broods in the range 90 - 95%;

score = 5 corresponds to a number of broods >95%.

• time 0: pre-treatment;

• time 1: at the end of the last treatment;

• time 2: one month after the end of the last treatment ; • time 3: every time the probiotic composition is administered .

Evaluation of the bees mortality in underbasket cages

• During the first week of treatment

After each administration any clinical symptoms "unexpected" or "abnormal" or other side effects, as well as the number of frame covered by bees were recorded

Evaluation of the infection rating by Nosema spp. in a sample of pooled field bees for each experimental group

• time 0: pre-treatment ;

• time 1: at the end of the last treatment.

Evaluation of the amount of honey produced by the experimental groups

At the beginning of the experimentation a supers to be entirely built (frames with foundations) was placed onto the hives, and at the end of the test supers were weighed .

Four experimental groups were analysed:

1. Group PRO MIN which received the lowest recommended dose of composition comprising viable bacteria in amount equal to lxlO ⁶ cfu per ml of composition solution, corresponding to 10 g of the lyophilized composition according to the invention dissolved in 1 1 of water.

Number of treated hives: 8

2. Group PRO MAX which received a dose of composition comprising viable bacteria in amount equal to 3xl0 ⁶ cfu per ml of composition solution, corresponding to 3x10 g of the lyophilized composition according to the invention dissolved in 1 1 of water. Number of treated hives: 8

3. Group PRO 10 which received a dose of probiotic composition comprising viable bacteria in amount equal to 3xl0 ⁷ cfu per ml of composition solution, corresponding to 30x10 g of the lyophilized composition according to the invention dissolved in 1 1 of water.

Number of treated hives: 8

4. Control Group treated with the same solution no probiotic bacteria.

Number of treated hives: 8

Honeycomb covered with bees of each experimental group were sprayed with 10 ml of the relative solution comprising the association of viable bacteria of the strains Lactobacillus buchneri , Pediococcus pentosaceus and Lactococcus lactis, or of the control solution with no bacteria. The honeycombs have received an administration every 14 days.

The strength of a hive is evaluated through the number of adult worker, the extension of the brood and the amount of stocks.

The evaluation of the number of adult worker is particularly difficult. A good estimate can be obtained by weighing the hive, alternatively by taking a picture of honeycomb showing the bees on it.

The amount of brood and stocks are quantified by counting the cells.

These procedures are time consuming and needs to be performed by expert personnel; to expedite the operations it is possible to use honeycombs calibrated photographs or defined surface area with known number of bees and/or cells. Such surface usually are elliptic or rectangular areas corresponding to 1/6 of honeycomb surface (Accorti M. Numerical evaluation of adult Apis mellifera: variations and changes to the method of the sixth. Beekeeping No. 01 - Ed. Inst. Experimental Agricultural Zoology Florence, Rome 1985) .

Results :

The amount of adult bees detected after treatment with the tested solutions comprising the association of Lactobacillus buchneri , Pediococcus pentosaceus and Lactococcus lactis sprayed on each honeycomb was decreased in all experimental groups compared to the initial estimate of the sixths, as shown in Figure 1 (l=time 0, 2=time 1, 2=time 2) .

The amount of brood after treatment with the tested solutions comprising the association of Lactobacillus buchneri, Pediococcus pentosaceus and Lactococcus lactis sprayed on each honeycomb was decreased in all experimental groups compared to the initial estimate of the sixths, as shown in Figure 2 (l=time 0, 2=time 1 , 2=time 2 ) ..

The analysis of the results obtained in the experiment shows that the compactness of brood increased in all experimental groups except for the control group, hence demonstrating that treatment with the solution according to the invention is effective and improves the general conditions of the bee colony without causing toxicities (Figure 3) (l=time 0, 2=time 1, 2=time 2 ) . In order to highlight acute toxicity in the treated bees family the mortality in underbasket cages was assessed. Underbasket cages are devices placed in front of the hive to collect and retain the dead bees that scavenger bees carry outside the hive, allowing the researcher to count and sample them. As shown in Figure 4 the average number of dead bees found in the underbasket cages in the first 7 days of treatment shows no acute toxicity affected the treated bees.

The number of dead queens during the trial amounted respectively to 3 in PRO MIN group, 4 in PRO MAX group, 3 in PRO 10X group and 3 in the CONTROL group. Orphans hives were excluded from the evaluation of the strength of families.

During the entire experiment period of time no diarrheal phenomena were discovered in the tested hives .

Figure 5 shows that the amount of honey produced in the experimental groups is lower compared to the control group according to the average weight of the supers .

Evaluation of infection by Nosema through quantification of Nosema spp. spores in bees

To assess the level of infestation of bee families by Nosema spp. a quantitative analysis of the spores found in samples of bees collected in the field was carried out.

Sampling was carried at the beginning of trial (beginning July 2014) and 14 days after the last administration of the product (end of August 2014) . At least 10 bees for each experimental group were collected directly from the flight board. The bee abdomens were withdrawn and placed in sterile bags with filter, an amount of sterile distilled water equal to the amount by weight of abdomens used was added. The samples were stored at -20 °C ± 5 °C for at least 24 hours. Subsequently, the analysis was conducted as described below.

Half amount of distilled water added to collect the spores spread on the walls of the bag was removed and stored in a test tube, the remaining sample was homogenized for 2 minutes (Stomacher - Seward Ltd) . The homogenate was poured in a 15 ml polystyrene conical bottom Falcon test tube; using previously stored distilled water spores spread on the walls of the bag were harvested and joined to the sample in the Falcon tube. The tube was centrifuged for 6 minutes at 800 rpm. The supernatant was discarded and distilled water was added until reaching 3 ml volume wherein the spores were resuspended. The centrifugation was further repeated 3 times so as to create a suspension of spores of Nosema spp . 85% pure (Fries I. et al . 2013. Standard methods for Nosema research. Journal of Apicultural Research 52 (1) .

Ten μΐ of the so obtained suspension were placed in the Burker chamber and used to determine the number of spores in the samples and for the statistical analysis .

Conclusions

The experimental results have shown that the probiotic composition and method of treatment of Nosema disease according to the invention do not cause acute toxicity in adult bees, as evidenced by the counts of dead bees found in the underbasket cages. Neither long-term toxicity, or evidence of queen bees mortality, have been highlighted when the maximum dose of probiotic composition, compared to the control group, was administrated .

During the trial no phenomena of diarrhea or other adverse phenomena, such as swarming, were discovered. The amount of adult bees in the hive was reduced in all treated groups compared to control, but the variation was not statistically significant.

At the end of the treatments the amount of brood was increased in all treated groups compared to the control, whereas the amount of honey produced in the hives was not statistically different from the control group .

The estimate of the compactness of the brood has shown that in all treated groups, there has been an increase in the firmness of the brood over the initial conditions. The PRO MIN group showed an increase of 7.6%, in the PRO MAX group the increase was 18.6%, a 6.3% increase occurred in the PRO 10X group, the group CONTROL instead showed a slight decrease in compactness of brood equal to 1% (Fig . 3) . So, at any dose, the treatment with the composition according to the invention determines an increase in the compactness of brood compared to the untreated control group, hence it determines an increase of the vitality of the bee family.

The increase in the compactness of the brood occurred in groups PRO MIN and PRO MAX groups are not due to chance, but due to the administration of the composition that, at all doses tested, acts strengthening the brood and, hence, the health of the entire family, making it stronger against potential pathogens that could infest.

In order to understand the obtained data it should be taken into account that approaching the fall season a natural and physiological decrease of brood occurs, so that as seen from the third column of each experimental group in the diagram of Fig. 3, one month after the end of last treatment (time 2), the variation in the compactness of the brood tends to decrease returning to values close to the initial ones; in the control this decrease is more pronounced. Hence, it can be concluded that the composition according to the invention administered between July and August acts on the treated bees by slowing the natural reduction in the total amount of brood at the end of the summer.

The results also clearly showed that treatment with the composition according to the invention is able to reduce in the treated hives the amount of Nosema spp . compared to an increase found in the control.

In conclusion, the composition administrated according to the described method provides a greater compactness of the brood and has shown to limit the Nosema disease .

Furthermore, the results provide insights about the dosage of the composition allowing to obtain the best performance in term of well-being of bees. As evidenced by the analysed parameters, the most effective dosage was found in the experimental PRO MAX group (3xl0 ⁶ cfu/ml), whereas the most effective dose to treat Nosema spp . infections resulted to be that received by the experimental PRO lOx group (3xl0 ⁷ cfu/ml) .

In order to achieve the best performance spring is the best period of time to administer the composition according to the invention.

Further tests on a larger number of hives are currently undergoing aimed at further confirm the therapeutic efficacy against the pathogenic fungus of the genus Nosema.