ITRM20120635A1 - "USEFUL COMPOSITION FOR BIOLOGICAL CHECK OF BEE DISEASES" - Google Patents

Description

â € œCOMPOSITION USEFUL FOR BIOLOGICAL CONTROL

OF BEE DISEASESâ €.

DESCRIPTION

Field of application

In its most general aspect, the present invention relates to the field of compositions and methods for the biological control of the health of bees in a hive and the prevention of the most common apiary diseases of various etiological origins, for example, bacteria, fungi , viruses, protozoa, and parasites that infest the bee colony and the hive. The composition and the method according to the present invention are useful for maintaining the good health conditions of bees and for the prophylaxis of pathologies due to the infestations of the Varroa destructor bee ectoparasite (varroatosis or varroasis), and related pathologies , such as virosis, nosema, American foulbrood, European foulbrood, fungi.

Known technique

In the last ten years various health problems have affected the beekeeping sector in various countries around the world. In general, an increase in bee mortality has been repeatedly reported in recent years. This has raised great concern in the reference sector for the important environmental and economic consequences.

The health of bees is linked to numerous factors such as diseases of various kinds (bacterial, viral, parasitic, etc.), the availability of adequate treatments, the presence of invasive species and alterations in the environment.

Those who are dedicated to beekeeping, both amateur and professional, have come across Varroa at least once in their life. Unfortunately most of the apiaries are infested with it and every year, regularly, it is necessary to resort to preventive or therapeutic measures to deal with a problem that is anything but mild.

Responsible for the disease, known as varroasi or varroatosis, is Varroa destructor, a small mite that feeds on the bee's hemolymph. By piercing the cuticle of the bee with its buccal apparatus and sucking the hemolymph, varroa determines a depleting action of the insect's resources, but even more serious is the transmission of other viral diseases through the wounds caused ; it would be the latter that cause the high mortality rate inside the hive by transmitting diseases such as the Deformed Wings Virus or the Chronic Paralysis Virus (CBPV). Hives with heavy varroa infestations are doomed to death, precisely because bees become more vulnerable to other diseases. A large infestation of these mites leads to the death of the bee colony, usually between the end of autumn and the beginning of spring. The parasite, in addition to debilitating adult bees, also attacks the larvae, leading to the birth of wingless or deformed insects. The varroa mite is the parasite with the most pronounced economic impact in the beekeeping industry. Completely eliminating the mite from the breeding is practically impossible, but a series of precautions and preventive treatments allow to contain mortality and avoid risky losses.

The parasitic syndrome caused by the varroa mite, and the related diseases, is now endemic in all hives where the parasite is present and does not allow any definitive cure, both for the difficulty in the total eradication of the parasite from the single family, and for the very fast phenomenon of drift re-infestation.

Related to varroa is the infection of the chronic bee paralysis virus (CBPV), endemic in most apiaries, the virus multiplies in bee tissues causing two types of syndromes. In a first form of the syndrome, the bee presents an abnormal tremor of the wings and of the body. Bees cannot fly and often crawl on the ground or on plant stems. The wings are partly spread or dislocated. Affected crawling bees can be present in large numbers in the hive where they tend to huddle together on the top or top bars of the hive. They may have a swollen abdomen due to distension of the honeycomb sac.

Bees with the second type of CBPV syndrome are able to fly, but their bodies are almost completely hairless. They appear dark or black and appear smaller. They too have a relatively large abdomen. A few days after the onset of the infection the tremor begins, they stop flying and shortly afterwards they die.

Both syndromes can occur in the same colony but usually one type is predominant. CBPV may be the ultimate cause of colony collapse from other adult bee diseases, yet there are currently no known effective treatments for this type of infection.

Another pathology affecting bees and correlated with varroa is Nosemiasis, due to two different species of unicellular fungi (microsporidia): Nosema apis and Nosema ceranae. Bees become infected by taking nosema spores orally. The latter, once arrived in the intestine, invade the cells of the mucosa and here they multiply until they are destroyed, leading to malabsorption phenomena. The infected bee will eliminate the spores with the faeces, making it possible to transmit the disease to other bees through direct contact, or through feeding with nectar, pollen, honey or other infected material. While N. apis causes gastrointestinal forms in bees characterized by diarrheal phenomena easily recognizable by the beekeeper.

N. ceranae is a recently isolated pathogen on Apis mellifera, responsible for a disease characterized by a long latency period whose only symptom affecting the hives, which is difficult to recognize by beekeepers, is identifiable with a slow and progressive depopulation of families, where nascent bees are unable to compensate for the number of adult bees that die.

Currently, chemical and biological methods are used among the most effective treatment methods for the control of beehive diseases.

Chemical methods, while posing the greatest risks to human health, and causing the greatest damage to the environment due to side effects against other insects and animals not directly recipients of the treatment, are the most widely used.

Commonly used synthetic chemicals are pyrethroids (Fluvalinate) and organophosphates (Coumaphons). Both, really effective only with brood block, have the big limit of the contamination of wax and honey; therefore more recently there is a tendency to prefer the use of chemical formulations of natural origin or based on:

· Oxalic acid, dripped, sprayed or vaporized;

· Formic acid, evaporated;

· Lactic acid, sprayed;

· Essential oils, such as thymol.

We have tried to overcome the significant disadvantages of chemical control against pathogens in the hive with the use of biopesticides and in particular entomopathogenic fungi. In Europe preparations based on Bacillus thuringiensis are used against the larva of Galleria mellonella, also called honey camola.

Some species of entomopathogenic fungi, including Beauveria bassiana, are able to infect the Varroa mite and are therefore exploited for the biological control of the beehive parasite. The US patent US 8,053,223 relates to an innovative formulation based on a pure culture of a new strain, that is identified and isolated for the first time, of the Beauveria bassiana mushroom which, in addition to a vector usable in agriculture, was particularly useful in controlling Varroa destructor infestations.

Also other species of pathogenic fungi such as Hirsutella thompsonii Fisher and Metarhizium anisopliae can act as biological control agents of the Varroa mite, in fact, they have been shown to be able to affect the density of mites in bee colonies (Kanga L.H.B. et al. , 2002, Journal of Invertebrate Pathology, 81, 175-184).

The use of microorganisms has recently been introduced in beekeeping. These are microorganisms, or particular associations of microorganisms, able to exert a beneficial effect in agriculture, animal breeding, to clean up natural environments and also to increase the immune defense of human beings, which have found useful application in beekeeping.

The Japanese patent application JP62022558 describes a method to assist the metabolism of bees, promote the physiological activation and further oviposition of the queen bee and the growth of the bee larva, obtained by mixing the product of the acid fermentation of a cereal (such as rice, wheat, barley, maize) or beans with a microorganism and beekeeping food. The product of acid fermentation is added to the normal apiary diet in a quantity equal to 3%.

Korean Patent Application KR830001681 describes a honey bee substitute food consisting of 2% skim milk powder, 0.1% fat-free egg powder, 0.8% bean powder, 0.6% sweet potato powder, 0.05% methionine, 0.05% lysine, 0.05% yeast powder, 0.05% calcium lactate, 0.09% active lactobacillus, 93.02% of sugar. The food thus formulated promotes the well-being of bees as demonstrated by their weight gain which can reach up to 12.5% of the original one.

The application of microorganisms to the beekeeping sector is also the subject of Korean patent application KR20100125747 which describes a nutrient composition for bees containing a concentrated and active liquid, and the related production method. The composition is useful to eliminate the need for antibiotics and to promote the egg-laying process. The manufacturing method involves a mixing phase of: Saccharomyces cerevisiae, albumin, fermented soy powder, pollen, propolis, and minerals with liquid sugar, and a fermentation phase lasting 6 months. The sugary liquid is obtained in turn by mixing the concentrated and active liquid with a lactobacillus and SiO3.

The European patent EP1135020 instead exploits the properties of an inoculum of one or more species of microorganisms that produce one or more antibiotics active against one or more bee pathogens. Among the antibiotics described are those against Melissococcus pluton and / or Paenibacillus larvae, the causative agent of the American plague of hives. The subject of the patent application is also the relative method for the treatment or prophylaxis of bee disease which involves inoculation in the hive in order to establish the repairing and / or protective microbial flora.

Also the international patent application WO 2011/138310 relates to a probiotic mixture useful for the protection of the health of bees, and more specifically for the protection against pathologies with bacterial determination, such as those caused by Paenibacillus larvae.

It must be said that the application of combinations of particularly stable, reproducible and effective non-pathogenic bacterial cultures represents a biological method to control bee infestations and diseases that heavily affect the health of the hive, particularly attractive to beekeepers. , as they represent an economical method and more respectful of environmental conditions than the chemical agents and biopesticides commonly used based on types of organisms such as viruses, nematodes, and pathogenic fungi and bacteria.

Although various forms of biological control of beehive diseases and colony welfare are currently available, the need for further products and methods based on the particular association of microorganisms, whose metabolic products induce a positive effect on the conditions of the bee colony, contribute to preventing the onset of pathological conditions that afflict the colony itself and are able to reduce the presence of pathogens in the bee colony, in particular of those primary determinants of infectious diseases endemics for which effective treatments are not currently available, for example CBPV, especially without leaving any residue in the hive.

Summary of the invention

The task of the present invention is to provide a new composition useful for the well-being of the bee colony and the prophylaxis against various pathological conditions which includes the association of several species of bacterial microorganisms and a means of transfer to the bees. Hive such that it can favor the establishment in the hive of a corrective and / or protective microflora.

Therefore, the subject of the invention is an association of microorganisms whose metabolic activity produces substances that induce a positive effect on the conditions of the bee colony, capable of treating and preventing the pathological diseases of bees, which association includes microorganisms of the following species: Lactobacillus paracasei ssp., Bifidobacterium bifidum, Lactobacillus acidophilus, Lactococcus lactis, Bifidobacterium animalis, Lactobacillus termophilus, Bacillus clausii. Said association of microorganisms is supplied in a 5% sucrose solution.

It is still an object of the invention to provide a method for exercising the biological control of the general welfare of bees and of the diseases of the hive of various etiologies that infest the hive and the colony of bees that reside there, applying, in quantity effective, the composition of microorganisms including Lactobacillus paracasei ssp., Bifidobacterium bifidum, Lactobacillus acidophilus, Lactococcus lactis, Bifidobacterium animalis, Lactobacillus termophilus, Bacillus clausii.

Further characteristics and advantages of the invention are better highlighted by the attached tables of drawings which summarize, by way of non-limiting example only, the results obtained during the experimentation period.

Brief description of the figures

Figure 1 shows the graph showing the percentage variation in the number of adult bees in the hives subjected to treatment with the composition of microorganisms according to the invention obtained by estimating the sixths in the experimentation period.

Figure 2 shows the graph showing the percentage variation in the number of capped broods in the hives subjected to treatment with the composition of microorganisms according to the invention obtained by estimating the sixths in the experimentation period.

Figure 3 shows the graph showing the percentage variation in the number of uncapped broods in the hives subjected to treatment with the composition of microorganisms according to the invention obtained by estimating the sixths in the experimentation period.

Figure 4 shows the graph showing the percentage variation in the number of eggs in the hives subjected to treatment with the composition of microorganisms according to the invention obtained by estimating the sixths in the experimentation period.

Figure 5 shows the graph illustrating the variation in the viral load of CBPV expressed as a function of the number of viral RNA molecules identified in the bee samples before and after treatment with the composition of microorganisms obtained by the Real-time PCR method.

Figure 6 shows the graph illustrating the variation in the extent of Nosema ceranae infection expressed as a function of the number of pathogen RNA molecules identified in bee samples before and after treatment with the microorganism composition obtained by Real-time PCR method.

Detailed description of the invention

According to the invention, an association of microorganisms is proposed whose metabolic activity is able to induce a positive effect on the health conditions of a colony of bees, capable of treating and preventing the pathological diseases of bees, which includes microorganisms of the following species Lactobacillus paracasei ssp., Bifidobacterium bifidum, Lactobacillus acidophilus, Lactococcus lactis, Bifidobacterium animalis, Lactobacillus termophilus, Bacillus clausii.

Said association of microorganisms is supplied in a 5% sucrose solution.

The microbial species that make up the composition according to the invention include non-spore-forming Gram-positive bacteria and endospore-producing bacilli.

Non-spore-forming Gram-positive bacteria are normal inhabitants of the digestive tract of humans and animals, although their presence and the extent of colonization largely depend on the characteristics of the host. Furthermore, some species of Gram-positive bacteria are commonly used for their role in food fermentation therefore they are used in the preparation of food and feed. These microorganisms are deliberately introduced into food as a trigger culture, food additives (e.g., probiotics), and source of additives (e.g., enzymes and amino acids)

The composition according to the present invention includes among the non-spore-forming Gram-positive bacteria the following genera: Bifidobacterium, Lactobacillus and Lactococcus.

Bifidobacteria are part of the normal intestinal flora of adult humans and are also one of the first genera to colonize the intestines of children, they also normally live in the intestines of animals. Only a limited number of bifidobacteria species are used in dairy products, particularly acids, such as yoghurt and fermented milk drinks. Bifidobacteria belong to the Actinomycetes branch of the phylum Firmicutes. They are not motile, do not form spores, and have a variable appearance, generally curved rod, and often branched with a Y and V shape. They are normally strictly anaerobic, although some species and strains tolerate oxygen. The type species is Bifidobacterium bifidum. Bifidobacteria are saccharolytic organisms and have the ability to ferment glucose, galactose and fructose. Glucose is fermented via fructose-6-phosphate into acetic and lactic acid. The differences between the various species consist in their ability to ferment different substrates of carbohydrates and alcohols.

The association according to the present invention includes two species of bifidobacteria: Bifidobacterium bifidum and Bifidobacterium animalis.

The genus of lactobacilli is a large and heterogeneous taxonomic unit of non-spore-forming gram-positive bacteria, including rod-shaped lactic acid bacteria. This genus includes more than 100 different species, with a great variety of phenotypic, biochemical and physiological characteristics. Many species of lactobacilli are the important constituents of the normal intestinal flora of humans and animals, also in this case as for bifidobacteria, their presence and the extent of colonization largely depends on the host.

Classically, the genus Lactobacillus is divided into three groups: group 1, obligate homofermentants, group 2, facultative heterofermentants and group 3 obligate heterofermentants. The phylogenetic taxonomy of the genus is now defined on a molecular basis. Currently, thanks to the most modern molecular characterization techniques, 112 species belonging to the genus Lactobacillus are recognized.

The characteristics and habitat of most lactobacilli species are well known. Some of the species have a long history of safe use in industrial and agricultural applications. Lactobacilli are used as trigger cultures in a variety of food fermentations, such as dairy products, fermented meats and hams, fermented vegetables, sourdough and sausages. As a result, lactobacilli are consumed daily in large quantities in a variety of fermented foods by people of all ages, ethnicities and health conditions, apparently with no ill effects. Aside from their possible involvement in the development of dental caries, lactobacilli are generally considered non-pathogenic.

The following species are present in the association according to the invention: Lactobacillus paracasei ssp, Lactobacillus acidophilus, Lactobacillus termophilus.

The genus of lactococcal microorganisms was formerly known as N-streptococci. The type species, Lactococcus lactis, (formerly Streptococcus lactis), used in the composition, is a known starter organism of dairy products and members of this species are a common element of the bacterial communities of cheese.

Lactococci belong to the Firmicutes phylum, are coccoid, non-motile and metabolize hexoses in a homofermentative way and pentoses in a heterofermentative way. They are mesophiles for which they prefer environments in which the temperature oscillates between 20 and 40 ° C, with an optimal temperature generally at 30 ºC or lower.

Another species of microorganisms present in the association useful for the welfare of bees according to the present invention is Bacillus clausii. Unlike the other microorganisms used in the composition according to the invention and described up to now, Bacillus clausii is a Grampositive bacterium whose cells, under stressful environmental conditions, produce oval endospores which can remain dormant for prolonged periods. Like most bacteria of the Bacillus genus, it is rod-shaped and grows in clusters or colonies. It is an alkalophilic microorganism and lives in alkaline or basic environments, it is found mainly in the soil. One of the characteristics of this microorganism lies in its ability to produce a particular type of enzymes, the subtilisins, which have protease activity in a highly alkaline environment.

The spores of some strains of B. clausii are used as probiotics, and are able to survive the acidic environments, characteristic of the stomach, and to grow and multiply and transform into vegetative forms in the intestine.

Once in the intestine, the enzymes produced by the metabolic activity of B. clausii work by improving the microbial balance (eubiosis) of the human microbiota and protecting it from infection by other bacteria. In fact, the spores adhere to the mucosa preventing the adhesion of pathogens, compete with them for the use of nutrients and produce antimicrobial substances (immunoglobulins), inhibiting their growth.

Several clinical studies have in fact highlighted the efficacy and tolerability of B. clausii spores in case of alterations (dysbiosis) of the human microbiota, meteorism, diarrhea and abdominal pain.

The bacterial strains that make up the composition according to the present invention have been identified, selected and isolated from various microenvironments (i.e. soil, swamps, dairy preparations, etc.), by direct observation of the morphology of the cells themselves under a microscope or of the colonies grown on a plate, and then propagated under the appropriate growing conditions; alternatively, the strains were obtained from official collections such as the American Type Culture Collection, ATCC (Rockville, Md, USA).

The bacterial strains were then grown under appropriate conditions in order to obtain liquid cultures in an appropriate growth medium. The cultures were then diluted 1: 100 in 5% sucrose solution and finally combined each in a ratio of 12 to 16% of the total volume in a 5% sucrose solution.

Therefore, the object of the present invention is a composition consisting of the selected association of microorganisms in liquid form, in a 5% sucrose solution, in which each strain is initially present in quantities ranging from 12 to 16% of the total volume, preferably the 14%. Once applied to the hive, the percentages of each strain in the association can vary according to the specific environmental conditions of each hive, where each strain of the association can find conditions more or less favorable to its own development, and therefore prevail or be limited compared to the others, so that, after application in the association of microorganisms, each strain can be present in a percentage other than 14%. For simplicity, a microbial association can be considered as a single entity, but the species from which it is constituted interact in both antagonistic and synergistic ways. These interactions can influence both the development of a given strain and its metabolic activity, thus determining a completely new and unexpected, sometimes therapeutic, effect of the association in the particular field of application.

The object of the present invention is therefore also a composition consisting of a particular association of microorganisms that provides a useful tool for the biological control of the general well-being of bees and for the treatment of serious infections affecting bees, in particular viroses, such as that determined by CBPV.

A further object of the present invention is the method based on the use of the association of microorganisms according to the present invention which finds application in the treatment and prevention of infections of some bee pathogens, in particular in the treatment of chronic paralysis of bees and / or the infection caused by Nosema ceranae. This method involves the treatment of bees, larvae, hive and other components related to the hive with the composition including the association of microorganisms.

Specific experiments and observations conducted in order to verify the positive effect exerted on the colony of bees have shown that the composition has a high potential in protecting adult bees and larvae through multiple mechanisms of action. The treatment resulted in a high level of protection in the colony against external stress agents and a considerable improvement in the well-being of the bees in the treated colony and the total elimination of the presence of CBPV viral particles. It cannot be excluded that with a similar mechanism of action, positive effects can also be obtained against other viroses, American foulbrood and European foulbrood which very often afflicts apiaries. Specific investigations are ongoing and preliminary data are very encouraging.

The positive effect exerted by the use of the composition can be evaluated according to the strength of the bee families, that is to say the number of adult bees, the number of capped broods, the number of uncapped broods and the number of eggs in the € ™ beehive. Furthermore, as an indicative parameter of the good condition of the bee colony and the strength of the bee families, it is possible to evaluate the incidence in the hive of varroa infection, and of the consequences of varroatosis, and other related and not, like nosema and virosis.

For this purpose, the hives must be placed on suitable supports to prevent the ants from accessing the diagnostic drawer, therefore the hives must be supported in containers with water. The anti varroa funds of the hives must be intact and free from occlusions. Furthermore, in order to allow a correct evaluation of the effective effectiveness of the treatment on the state of health of the hive, it must not have been treated with products that are not allowed or in any case with synthetic products in brood block.

The hives evaluation criteria include:

- assessment of the level of varroa infestation at the start of the test and after honey extraction;

- assessment of the level of infection of pathogens closely related to varroa (Nosema ceranae and virosis) at the start of the test and after honey extraction;

- evaluation of the quantity of honey produced; - estimate of the strength of the families at the start of the trial and after honey extraction.

The composition according to the present invention is applied inside the hive by any suitable means known to the skilled in the art, preferably, but not limited to, the composition is applied by hand pump. In this mode of application, the 5% sucrose composition of microorganisms is applied directly to each frame of the hive, after having opened the roof of the hive without removing the frame itself. In this way, the bees and broods present are also wetted with the frame. Preferably, the composition can also be sprayed on the entrance door of the hive in order to wet the surface crossed by the bees.

The number of applications depends on the general conditions of the colony and the severity of the infestations and pathologies present. Typically, the application is repeated at intervals of about 2-5 weeks, for a total number of treatments equal to 3-4. The treatments must necessarily be carried out in the period between spring and late summer. Preferably, the application is repeated at intervals of about 15 days for a period of two months and then the administration can be repeated on a monthly basis. Obviously the person skilled in the sector can easily find different application protocols (for example for frequency of application and effective quantity applied) which are more effective according to the real conditions and locations of the hive.

The effective amount is the amount of composition according to the invention that is able to control, decrease, inhibit, improve or positively influence one or more symptoms of the disease or condition to be treated.

Experimental procedure

Below are reported operational examples of use of the composition object of the invention, provided solely for illustrative purposes without limiting the invention in any way.

The effect of the invention-specific composition of microorganisms against bee pathogens and on bee health conditions were investigated both in vivo and in vitro. The main phases of the investigation were conducted as described below.

1. Estimation of the strength of bee families

The field tests were carried out from the beginning of May 2012 to the end of June 2012 for a total of 60 days in an apiary located in the province of Rome. The experimentation involved 24 hives on colonies in hives containing frames, equipped with a movable antivarroa bottom.

At the beginning of the experimentation, the bees were not affected by any pathology.

At the beginning of the experimentation (day 0) all the hives were subjected to the sixth estimate of the number of adult bees, of capped broods, of uncapped broods, and of the number of eggs.

The 24 hives were divided into three different treatment groups: a first group of 8 hives (indicated with T) was subjected to the administration of a quantity equal to 10ml of composition for each frame covered by bees every 15 days for four times , for which the hive is subjected to the treatment on days 0, 15, 30, and 45; a second group consisting of 8 hives (indicated with M) was subjected to the administration of a quantity equal to 5ml of composition for each frame covered by bees following the same time profile of administration; the third group (group C) consisting of 8 hives was used as a control and was not subjected to any treatment.

After the honey extraction carried out on day 60 after the start of the experimentation, all the hives were again subjected to the sixth estimate relative to the number of adult bees, of capped broods, of non-capped broods, of eggs.

Table 1 shows the values obtained by estimating the sixths relating to the percentage variation of adult bees in the experimentation period, graphically represented in Figure 1.

Table 1 - Percentage variation of adult bees GROUP M GROUP T GROUP C

Pre treatment 100 100 100 Post treatment 205.45 102.81 122.02

Table 2 shows the values obtained by estimating the sixths relating to the percentage change of capped brood in the experimentation period, graphically represented in Figure 2.

Table 2 - Percentage variation of capped brood

GROUP M GROUP T GROUP C

Pre treatment 100 100 100 Post treatment 148.29 45.72 41.68

Table 3 shows the values obtained by estimating the sixths relating to the percentage change in uncapped brood in the experimentation period, graphically represented in Figure 3.

Table 3 - Percentage variation of uncapped brood

GROUP M GROUP T GROUP C

Pre treatment 100 100 100 Post treatment 207.23 56.27 32.93

Finally, table 4 shows the values obtained by estimating the sixths relating to the percentage variation of eggs in the experimentation period, graphically represented in Figure 4.

Table 4 - Percentage variation of eggs

GROUP M GROUP T GROUP C Pre-treatment 100 100 100 Post-treatment 81.19 55.09 26.97 As regards the evaluation of the positive effect on the general conditions of the bee families being tested, in the graph of Figure 1 Ã ̈ It is possible to observe how the treatment according to the previously described time profile with 5 ml of composition (group M) determines, two months after the first administration, a significant increase in the number of adult bees, while the treatment with a double quantity ( group T) of composition leaves practically unchanged the number of bees and at a level comparable to that of the control group that did not receive the treatment. The trend of the variation in the number of adult bees in group M also reflects the trend in the same experimental group of the variation in the number of capped and non-capped broods, as shown in the graphs of Figures 2 and 3 which show a greater and significant increase in the number of broods following treatment with 5ml of composition of microorganisms; the administration of a double quantity of composition determines a variation comparable to that of the control group.

The evaluation of the strength of the bee family as a function of the variation of eggs following the treatment shows that the composition of microorganisms in general is able to determine a limitation in the loss of eggs compared to the control group where the loss is equal to 70%. Also in this case the best efficacy of the treatment undergone by group M is confirmed by the lower percentage variation in the number of eggs in this group (about 20%) compared to the group that received a double quantity of composition, group T which has a decrease in the number of eggs of about 40%.

2. Detection of the CBPV virus and of the genetic material of Nosema ceranae and Nosema apis spores in foraging bees.

During the experimentation, a sampling of bees was conducted for the detection of nosemiasis and virosis by searching for the CBPV virus and the genetic material of the spores of Nosema ceranae and Nosema apis in foraging bees from the hives subjected to the study, an evaluation of the extent of infestation before and after treatment with the mixture of microorganisms

CBPV and N. ceranae and N. apis are responsible for latent and persistent infections which often do not show clinical signs but are responsible for huge losses of bees, the last and definitive symptom of the infection. Using the Real time-PCR (Polymerase Chain Reaction) molecular biology technique it is possible to amplify a very specific and characteristic part of the genome of the two pathogens, allowing to arrive at a certain diagnosis. Being quantitative, the technique allows to evaluate the viral load of CBPV, quantifying the specific RNA of the species and, more generally, the extent of the infection.

However, the quantitative PCR method was preceded by an investigation using traditional (qualitative) PCR.

Preparation of the homogenate and sporulation

The sporulation of the spores makes it easier to extract the DNA of Nosema spp. given that the vegetative phase of microsporidia is characterized by a much less complex cell wall which allows to simplify the extraction protocol.

In order to obtain the spores of Nosema ceranae and Nosema apis, about 10-15 bees were collected to which 10 ml of 1X PBS were added. 2ml of bee homogenate thus obtained were then collected by means of a tip and placed in a 2ml eppendorf tube.

The homogenate samples were centrifuged at 13,000rpm, at room temperature for 10 minutes. At the end of the centrifugation, the supernatant was delicately eliminated and the precipitate was weighed on a precision balance in such a way as to add the right amount of reagents indispensable for the sporulation of the spores. To the precipitate obtained, generally weighing between 35 and 50 mg, 40µl of germination buffer was added and it was immediately incubated for 15 minutes at 37 ° C and stirred at 300rpm. The germination buffer has the following composition: 75ml of 1M sodium hydrogen carbonate at pH 6.5 and 75ml of 1M sodium chloride. Once prepared, the Germination Buffer is filtered with a 0.22Î1⁄4m filter and stored at room temperature.

At the end of the incubation period, 180Î1⁄4l of lysozyme [10mg / ml] was added to the precipitate, mixed gently and left to incubate at 37 ° C for 30 minutes.

DNA extraction from the vegetative stage

The commercial QIAamp <®> DNA Blood Mini Kit (Qiagen) was used for DNA extraction following the manufacturer's instructions.

Traditional PCR reaction

Two PCRs are performed: the first specific for Nosema ceranae and the second specific for Nosema apis.

1) Search for Nosema ceranae by PCR

The reaction was carried out following the protocol indicated in Martìn-Hernà ndez R. et al., 2007 (Outcome of colonization of Apis mellifera by Nosema ceranae. Appl Environ Microbiol. 73: 6331-8).

The following pair of oligonucleotides was used as a trigger for the amplification reaction:

- 218MITOC-FOR: 5â € ™ -CGGCGACGATGTGATATGAAAATATTAA-3â € ™; - 218MITOC-REV: 5â € ™ -CCCGGTCATTCTCAAACAAAAAACCG-3â € ™, these oligonucleotides allow the amplification of a portion of 218-219 bp inside the main 16S subunit of ribosomal RNA (16S rRNA), the reaction mixture has the following composition: 5Î1⁄4l Buffer 10X, 0.4 Î1⁄4M 218 MITOC FOR, 0.4 Î1⁄4M 218 MITOC REV, 0.4mM deoxynucleotides (dNTP), 3mM MgCl2, 0.2mg / ml Bovine Serum Albumin, 0.1% TritonX-100, 0.05U / Î1⁄4l Ampli Gold Taq (5U / Î1⁄4l), 5Î1⁄4l of template DNA and H2O-DEPC until the final volume of 50Î1⁄4l is reached. The reaction mix was prepared using Ampli Gold Taq Polymerase kit (Applied Biosystems, Branchburg, New Jersey, USA).

Amplification is performed using the Gene Amp® PCR System 9700 thermal cycler (Applied Biosystems, Carlsbad, USA) according to the following protocol:

- 94 ° C for 10 minutes;

- 35 cycles consisting of 94 ° C for 1 minute, 61.8 ° C for 1 minute, 72 ° C for 1 minute;

- 72 ° C for 10 minutes.

2) Search for Nosema apis by PCR

(Martìn-Hernà ndez R. et al., 2007)

The following pair of oligonucleotides was used as a trigger for the amplification reaction:

- 321APIS-FOR: 5â € ™ -GGGGGCATGTCTTTGACGTACTATGTA-3â € ™; - 321APIS-REV: 5â € ™ -GGGGGGCGTTTAAAATGTGAAACAACTATG-3â € ™, these primers allow the amplification of a portion of 321 bp inside the main 16S subunit of ribosomal RNA (16S rRNA), the reaction mixture has the following composition: 5Î1⁄4l Buffer 10X, 0.4 Î1⁄4M 321 APIS-FOR, 0.4 Î1⁄4M321 APIS-REV, 0.4mM deoxynucleotides (dNTP), 3mM MgCl2, 0.2mg / ml Bovine Serum Albumin, 0 , 1% TritonX-100, 0.05U / Î1⁄4l Ampli Gold Taq (5U / Î1⁄4l), 5Î1⁄4l of template DNA and H2O-DEPC until the final volume of 50Î1⁄4l is reached. The reaction mix was made using Ampli Gold Taq Polymerase kit (Applied Biosystems, Branchburg, New Jersey, USA).

Amplification is performed using the Gene Amp® PCR System 9700 thermal cycler (Applied Biosystems, Carlsbad, USA) according to the following protocol:

- 94 ° C for 10 minutes;

- 35 cycles consisting of 94 ° C for 1 minute, 61.8 ° C for 1 minute, 72 ° C for 1 minute;

- 72 ° C for 10 minutes.

The amplifications of the various PCR reactions were visualized by electrophoresis in 1.5% (w / v) agarose gel.

Quantitative Real time-PCR assay.

The quantifications were carried out with the absolute quantification method in which a plasmid in which the DNA sequence of interest was cloned was used as a standard.

The N. ceranae detection assay by real time PCR was performed according to the protocol described in Bourgeois et al. 2010 (Genetic detection and quantification of N. apis and N. ceranae in the honey bee. Journal of Invertebrate Pathology. 103: 53-58), using the following set of oligonucleotides: N. ceranae CRA Forw: 5â € ™ -AAGAGTGAGACCTATCAGCTAGTTG- 3â € ™; N. ceranae CRA Rev: 5â € ™ -CCGTCTCTCAGGCTCCTTCTC-3â € ™;

N. ceranae CRA Probe: 5â € ™ -JOE-ACCGTTACCCGTCACAGCCTTGTT-3â € ™ -BHQ-1.

The sequences of the oligonucleotides used in the study were designed on the basis of the specific genomic sequences of both the Nosema ceranae (GenBank DQ486027) and Nosema apis (GenBank U97150) strains. The amplicon obtained from the amplification of the DNA obtained from the spores of N. ceranae is a 142bp fragment which appears to be species specific.

The CBPV detection assay by real time PCR was performed according to the protocol described in Blanchard et al. 2007, (Evaluation of a real-time two-step RT-PCR assay for quantitation of Chronic bee paralysis virus (CBPV) genomic in experimentallyinfected bee tissues and in life stages of a symptomatic colony, Journal of Virological Methods 141: 7-13) , using the following set of oligonucleotides:

CBPV Forward: 5â € ™ -CGCAAGTACGCCTTGATAAAGAAC-3â € ™;

CBPV Revesre: 5â € ™ -ACTACTAGAAACTCGTCGCTTCG-3â € ™;

CBPV Probe: 5â € ™ -FAM-TCAAGAACGAGACCGCCAAGTTC-TAMRA-3â € ™.

The primers and the probe were positioned inside the RNA 1 gene that codes for the viral dependent RNA-polymerase-RNA and amplify a 101bp fragment (Ribià ̈re et al. 2002 Molecular diagnosis of chronic bee paralysis virus infection. Apidologie . 33: 339-351).

The primer sequences used for the quantification of the viral load of CBPV and the extent of Nosema ceranae infection are summarized in Table 5.

Table 5

Agent Primers Amplicon Pathogen Ref

CBPV CBPV Forward: 5â € ™ -CGCAAGTACGCCTTGATAAAGAAC-3â € ™; 101 bp Ribià ̈re et CBPV Revere: 5â € ™ -ACTACTAGAAACTCGTCGCTTCG-3â € ™; to the. 2002 CBPV Probe: 5â € ™ -FAM-TCAAGAACGAGACCGCCAAGTTC-TAMRA-3â € ™

Nosema N. ceranae CRA Forw: 5â € ™ -AAGAGTGAGACCTATCAGCTAGTTG- 142 bp DQ486027 ceranae 3â € ™;

N. ceranae CRA Rev: 5â € ™ -CCGTCTCTCAGGCTCCTTCTC-3â € ™;

N. ceranae CRA Probe: 5â € ™ -JOE-ACCGTTACCCGTCACAGCCTTGTT-3â € ™ -BHQ-1

The colonies of bees from the 24 hives involved in the experiment described in point 1 were then tested for the presence and extent of the infection of CBPV and Nosema ceranae on the basis of the quantity of genomic molecules of the two pathogens detected by Real Time PCR.

The analysis of the quantitative data obtained showed that indeed during the experimentation period the hives tested were contaminated by the microsporidium responsible for nosemiasis and by the virus of chronic paralysis of bees. The data also showed that the treatment with the association of microorganisms according to the present invention is able to exert a significant effect on N. ceranae infection (Figure 6). The treatment effect on CBPV infection is consistent and significant in the two groups that received the treatment compared to the control group, with the difference that in group T the treatment with 10 ml of composition keeps the The extent of the infection, as the viral load remains substantially unchanged before and after treatment, while in group M the treatment with 5ml of composition is able to reset the viral load, which is not the case in the control group that where the viral load during the experimental period increases by a factor of 10 <4> (Figure 5).

The composition according to the present invention has therefore proved to be useful for the treatment of viral infection of CBPV, revealing an unexpected ability to cancel the viral load following the treatment according to the described method. This property is very important, first of all because there are currently no known effective treatments against this type of virus, usually carried and spread by the varroa mite, and often the real cause of depopulation or death of varroa-infested families.

In the light of the above results, it is therefore important to underline that it is possible to optimize the effect on the health of bees by suitably regulating and modulating the quantity of microorganism composition that must be applied, as well as the number of applications.

The results obtained regarding the infestation of varroa and other varroa-related pathogens are currently being analyzed by the Applicant, even in these cases the preliminary data are very comforting and demonstrate a considerable reduction in the extent of infection. in the experimental groups that received the administration of the composition of microorganisms according to the invention.

Claims (8)

CLAIMS 1. Composition useful for the biological control of the well-being of bees, for the prophylaxis and treatment of pathological diseases of bees of various etiology, characterized by the fact of including an association of microorganisms including: Lactobacillus paracasei ssp., Bifidobacterium bifidum, Lactobacillus acidophilus, Lactococcus lactis, Bifidobacterium animalis, Lactobacillus termophilus, Bacillus clausii in 5% sucrose solution. 2. Composition of microorganisms according to claim 1 in 5% sucrose solution in which each bacterial strain is initially present in a quantity ranging from 12 to 16% of the total volume, preferably each bacterial strain is present in the ratio of 14% of the total volume. 3. Composition of microorganisms according to claims 1 and 2 useful for the treatment of viral infection of bees caused by the chronic bee paralysis virus or CBPV. 4. Composition of microorganisms according to claims 1 and 2 useful for the treatment of the infection caused by Nosema ceranae. 5. Method for the biological control of the well-being of bees, for the prophylaxis and treatment of pathological diseases of bees of various etiology, characterized by the fact of using the association of microorganisms of claim 1 and 2 by dispensing within the € ™ beehive. 6. Method for the treatment of bee viral infection caused by the chronic bee paralysis virus or CBPV, characterized by using the association of microorganisms of claim 1 and 2 by dispensing inside the hive. 7. Method for the treatment of the infection caused by Nosema ceranae, characterized by using the association of microorganisms of claim 1 and 2 by dispensing inside the hive. 8. Method as in claims 5 and 6 characterized by the fact that the dispensing takes place by applying the composition directly on each frame of the hive, after having opened the roof of the hive without extracting the frame itself, wetting the bees and broods present, preferably the composition is also sprayed on the entrance door of the hive in order to wet the surface crossed by the bees.