Composition and method for treatment of brood infected with bee foul brood

FIELD: apiculture, in particular, controlling of bee diseases through the use of biological methods.

SUBSTANCE: composition is adapted for treatment or prophylaxis of American, European foul brood and flakiness and consists of weakened or avirulent bacteria and carrier. Method for treatment or prophylaxis of said diseases involves introducing said composition into beehive. Also, inoculate comprising weakened or avirulent microorganisms and used as active substance for treatment of bee diseases is disclosed. Method allows utilization of antibiotics to be avoided.

EFFECT: increased efficiency in biological controlling of bee diseases and reduced expenses for effectuating said method.

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The present invention relates to methods and compositions used for the treatment and prevention of diseases of honey bees. In particular, the present invention relates to methods and compositions used for the biological control of microbiological contamination of the brood of honey bees - grilcom.

Background of invention

Honey bees (genus Apis), such as the European honey bee (Apis mellifera) and the Eastern honey bee (Apis cerana), for a very long time have been used to obtain the honey. A.mellifera A.cerana and accumulate large quantities of easily accessible honey and can be entered into a swarm of bees with mobile containers (or hives). In connection with the above mentioned types of bees operated around the world for hundreds of years and is currently based on them the large and complex agricultural industry. In the context of the present description, the term "honey bee" (or simply the bee) refers to the true honey bees of the genus Apis, in particular to A. mellifera and A.cerana.

The development of beekeeping (agricultural areas) is complicated by the fact that bees are exposed to many diseases. These diseases include pathological processes caused by viruses, bacteria, fungi, microspores, protozoa, Paris the political ticks, as well as other insects and nematodes. Two bacterial diseases are ubiquitous and cause General concern as professionals involved directly in agricultural production, and those who are called to control this industry, namely affecting brood American foulbrood American foulbrood and European foulbrood European foulbrood) (often referred to in abbreviated form as just foulbrood).

American foulbrood (AFB, AG) is a disease that affects larvae, which almost always kills them on stage predskolni, while larvae acquire a brownish color, begin to rot and produce a characteristic smell of fish glue. The disease causes spore-forming Gram-positive Bacillus Paenibaciilus larvae, the ssp. larvae. The spores of this bacteria can reside for decades in a state of rest, polluting stored beekeeping equipment, but such equipment is beginning to re-use or does it appear bees from infected colonies, the disease begins to develop. Bacilli infect Roy through the mouth and release during sporoobrazovanie stable proteolytic enzymes, causing the death and ultimately the dissolution of the larvae to the colloidal state of the glue.

European foulbrood (EFB, ΜG) of t is made a condition caused by not forming spores, Gram-positive bacterium Melissococcus pluton. This bacterium causes sporadic outbreaks of EZRA, which lead to the death of bee brood, without this education Liaodong colloidal material.

Currently, there are several treatment methods and AG, and EH, although each of them is not free from disadvantages. Perhaps the most effective, albeit too radical form of treatment is the controlled burning of the colony, and related equipment, which were used for breeding. However, this destructive measure cannot be considered as a preferred method of combat for obvious economic reasons, unless there is the possibility of choice.

Often by shaking trying to separate the adult bees from infected colonies by transferring them into new cells, but this method is unreliable, and, in addition, applies only in laboratory conditions.

Another method of treatment involves sterilization of hives and their contents using the appropriate sterilizing chemicals, such as formalin, chlorine and ethylene oxide. However, the use of such chemicals can make and honey, and pollen in combs toxic to bees or to give satam brittle. In addition, the efficiency of such helices is their substances from the point of view of treatment of hypertension and IT is not unique and varies depending on the circumstances.

An alternative form of treatment involves use of antibiotics for treatment of diseased colonies. Causing bacteria AH, we can influence the antibiotic oxytetracycline, usually in the form of a commercial preparation of Terramicina. However, in the UK in accordance with the decree on measures in connection with diseases of bees (1982) are required to apply the combustion in the event of an outbreak of AG. Such a policy pursued for decades, affected the low level of occurrence of AZR in the UK, whereas in some countries, such as USA or Australia wide dissemination of preventive treatment with tetracycline has resulted in subclinical spread of the disease, which, in turn, is associated with the appearance of dependence on antibiotics and the possibility of tetracyclineinducible strain Paenibacillus larvae subsp. larvae (which can cause catastrophic loss of bee colonies). In addition, antibiotics have no effect on spores contaminating cells and equipment, the use of which may lead to spread of infection between the colonies and thus to increase dependence on their regular use.

In the UK to combat mild forms of hypertension tetracycline applied, whereas in severe cases, and repeated infections are practicing the burning. The use of tetracycline for the prevention or treatment of acute cases, IT is undesirable due to the risk of development of resistance and the possibility that the suppression of IT under the cover of the may hide low level of AG and in this connection there is the chance of developing tetracycline dependencies.

The other major problem arising from the use of tetracycline for the treatment of hypertension and IT is connected with presence of undesirable residues of tetracycline in honey, and the appearance of such residues is related to good health and a number of other things that you expect from a honey of his fans and advocates.

Thus, there remains an urgent need to develop a method of treatment of diseases of bees in General, and in the case of American and European hnilica, including, in order to avoid problems associated with the use of tetracycline and related costs arising from the burning.

A brief description of the invention

It is now known that some microorganisms can prevent the development of infections caused by pathogens of bees, and to eliminate the steady-state infection in infected colonies of bees. These microorganisms can produce one or more antibiotic(s) or may compete with the pathogen(s) of bees, thus preventing infection and promoting its elimination. Some of these microorganisms can be in a natural condition detected in the hive and the honey bee.

So, first of all it was found that bees diseases can easily, safely, and effectively controlled by biological method that avoids the problems associated with the use of antibiotic drugs and additional financial costs that accompany the use of burning and the corresponding destruction.

Thus, the present invention relates to compositions used for the treatment or prevention of diseases of bees, with the specified composition includes the inoculate and appropriate agricultural media used in agricultural practice for the delivery of a specified inoculum in the hive, so in order to install the microflora with therapeutic and/or protective effect.

Another object of the present invention is the processing method for the treatment or prevention of diseases of bees, including the introduction of inoculum in the hive, in order to create a flora for therapeutic and/or protective effect.

In the context of the present description, the term "inoculum" is used in its usual meaning to refer to the drug containing microorganisms capable of installing, create the appropriate microflora after insulinopenia. So, in the context of this pisaniferry "inoculate" includes one or more microorganism(s), present in sufficient quantity and in such metabolic/physical condition that can set microflora after their introduction into the hive or colony of bees.

Used in the context of the present description, the term "antibiotic" refers to any substance (such as bacteriocins or secondary metabolite), able to inhibit the pathogenic activity of one or more of bee pathogens (for example, by suppressing their growth).

In the context of the present description, the term "hive" is used to refer to any of a wide variety of containers used by beekeepers for the maintenance of colonies of honey bees, care and collection produced their honey. For example, the term refers in the context of the present description, among other things, covers, boxes, frames, satam (which is usually a wax matrix, tiled bees inside the framework) or other components of the hives, either singly or in the aggregate).

The hive may contain or not contain bee colony. For example, in the case of applying the present invention with the preventive purpose, you can enter the inoculum in the empty hive. In such embodiments of the invention, the inoculum can be at rest until, as it will spread, and to stimulate its growth under the action of swarming bees, so thatin this case the protective microflora develops under the action of the colony. Alternatively, this flora may (at least partially) to develop in the absence of swarming bees due to the stimulation of growth of the inoculated microorganisms inside the hive using any suitable technique (e.g., by introduction of suitable nutrients and/or substrates). Thus, the microflora with therapeutic and/or protective action may be installed in the presence or in the absence of the impact of population swarming bees.

In the framework of the present invention can be used in any way acceptable for agricultural use media if it is suitable from the point of view of delivery specified inoculum in the hive, so that it ultimately could be established microflora with therapeutic and/or protective effect (in the presence or in the absence of the impact of population swarming bees). Preferably, the specified media is a media that works in the colony of bees taking into account the activity of the bees to collect honey and nutrition, the inoculum being delivered, as soon as selected by the bees (in the case of introducing its corresponding delivering agent will be posted within the hive and/or colonies with subsequent secretion (for example, through Podporozhye glands of larvae in the feeding process), excretion or release.</>

The inoculum can be delivered as a single dose or as multiple smaller doses, administered at certain intervals. Depending on the delivery medium specified the inoculum can be delivered to any component of the hive (e.g. unoccupied satam) or swarm clusters of bees (for example, in the case of selection of a group of bees bucket and spray them with inoculum outside of the hive with the subsequent return to the hive).

The size of the inoculum, the appropriate dosage and the type used for delivery of media can vary depending on such factors as the nature of power colony of bees, nature of microorganisms that must be delivered, the mode of treatment (prophylactic or therapeutic/curative intent), the age of the colony, the size and structure of the hive, geographical, climatic and seasonal conditions, and the nature of the disease to be treated or prevented and the severity of infection. The specialists with the average level of skill in the art can easily determine the optimal parameters for such routine procedures and to prevent possible errors.

In beekeeping there are many used to deliver media that can be used for feeding of wintering bees and for the introduction of known antibiotics. Preferably the mi in the framework of the present invention are the so-called bee candy, syrups, special liquid forms for veterinary use, powder or paste.

In the context of the present description, the term "bee candy" is used to define any of a variety of solid pellets or briquettes, comprising primarily of solid sugar. Especially preferred for use within the present invention are sucrose candy or sweets of fillers, including a mixture of sucrose and fat (e.g., vegetable oil).

The syrups used in the framework of the present invention preferably include sugar and water. Especially preferred are 50% (weight/volume) solution of sucrose.

Powders preferably include powdered sugar. They may be introduced in honeycomb, but also more generally be applied to all internal surfaces of the hive or swarm directly on a bunch of bees.

The paste used in the present invention, can be a substitute for pollen. The term "substitute pollen is used in the art to refer to any means of many songs in the functional and/or chemical behaviour can simulate pollen (used by bees as the main source of protein).

Sugar for use as a carrier for delivery according to the present invention provided is employed, preferably a sucrose but it can also be used for any other composition, which is functionally and/or chemically able to simulate nectar (used by bees as the main source of non-protein food).

The used inoculum according to the present invention includes one or more microorganism(s). Specified(s) organism(s) may(gut) to prevent the development of infections caused by pathogens of bees, and may(may) to promote the elimination of already established infections in colonies patients bees (for example, by producing one or more antibiotics or by competing with the pathogen(s) of bees and thus impeding their development or contributing to the elimination of infections). Some of the microorganisms can occur naturally in the hive and the honey bee.

In the case where the microorganism(s) produce(s) one or more antibiotics, active(s) against one or more pathogens of bees specified(s) antibiotic(s) preferably exhibits(comply) with activity against Melissococcus pluton and/or Paenibacillus larvae subsp. our band. The preferred(s) is(are) antibiotic(s)active(s) and against Melissococcus pluton, and against Paenibacillus larvae subsp. our band. Antibiotics may have a bacteriostatic or bacteriolytic action, and preferably they are bacteriolytic efficiency is so

Especially preferred are the inoculum, which include one or more microorganisms that produce antibiotics(and) against Melissococcus pluton and against Paenibacillus larvae subsp. larvae found in the cells of Paenibacillus larvae subsp. pulvifaciens, or their homologues.

In the context of the present description, the term "homologue" (in relation to the antibiotic) defines an antibiotic that is functionally and/or chemically similar to the standard antibiotic produced by Paenibacillus larvae subsp. pulvifaciens, but it is produced by another organism. Such homologues (and the organisms that they produce) can be identified by structural analysis of the standard(s) of antibiotic(s) and/or when using screening programmes, based on the detection of activism against Melissococcus pluton and/or Paenibacillus larvae subsp. our band.

Antibiotic(s)produced(s) organism(s) to use in the framework of the present invention can be characterized by the activity profile, which includes including activity against Escherichia coli, Salmonella typhimurium, Pseudomonas putida, Pseudomonas aerugenosa, Micrococcus luteus. Bacillus cereus, Bacillus subtilis, Paenibacillus alvei, Paenibacillus larvae subsp. larvae, Paenibacillus apiarius, Listeria monocytogenes, Staphylococcus aureus, Yersinia enterocolitica and Melissococcus pluton.

In preferred embodiments of the present invention is specified(s) organism(s) include(s) bacteria is. Especially preferred are spore-forming bacteria that produce endospores, and in this case, the inoculum preferably contains bacterial endospores (optional in combination with vegetative cells).

In the case of use in the composition of the inoculum of bacterial endospores are supplied in the form of washed or concentrated preparations of spores. These drugs can be obtained using any of the methods from the large number of known microbiological procedures. Typical methods include growing vegetative cells to stationary phase (induction sporoobrazovanie) in liquid medium and then separating them by centrifugation from the spent medium. Such variants are used as the basis of the method of high resistance of bacterial endospore, which allows to obtain an extremely stable composition, inexpensive to manufacture and does not create problems for storage.

Bacterial inoculum preferably includes species or sub-species Paenibacillus (as described by Heyndrickx et alia (1996), Int. J. Syst. Bacteriol. 46(4), 988-1003.

Particularly preferred is a subspecies of Paenibacillus larvae, for example, Paenibacillus larvae subsp. pulvifaciens or weakened, or avirulent strain of Paenibacillus larvae subsp. our band. Other acceptable bacteria can be identified by scanning the natural isolates among declassifier the bathrooms isolates of Paenibacillus species, demonstrating the need antibiotic activity (but are not pathogenic).

Paenibaciltus larvae subsp. pulvifaciens produces antibiotics, active against pathogens Paenibacillus larvae subsp. larvae, Paenibacillus alvei, and also against Melissococcus pluton, and thus this subspecies (or its derivatives) can be used as the basis for creating compositions intended for the prevention and/or treatment of both American and European hnilica.

Weakened or avirulent strains of Paenibacillus larvae subsp. larvae produce antibiotics, active against Melissococcus pluton, as well as against Paenibacillus alvei, and therefore these strains (and their derivatives) can be used as the basis for creating compositions intended for the prevention and/or treatment including European hnilica and diseases mediated infection P.alvei.

Alternatively, using genetic engineering techniques can be created by other microorganisms (especially bacteria)in order to function as host cells for expression of the antibiotic(s), as defined above.

Bacteria used according to the present invention may contain one or more mutations that change the virulence and/or the ability to form antibiotics and/or lead to weakened or avirulent strain. Such options, the ant is particularly preferred in the case of the invention, when the inoculum is a Paenibacillus larvae subsp. pulvifaciens, because some strains of this bacteria cause moderate in severity of the disease known as powdery cheshuichatoe.

Weakened or avirulent strains can be easily obtained by repeating subcultivation in the laboratory, using methods of mutagenesis/screening or when carrying out site-directed mutagenesis virulence determinants.

Disease of bees, which can be treated or prevented in accordance with the present invention, in the typical case is a disease associated with a bacterial infection (e.g., disease caused by Paenibacillus larvae subsp. larvae, Paenibacillus larvae subsp. pulvifaciens, Paenibacillus alvei or Melissococcus pluton).

In especially preferred embodiments of the invention, the disease of bees is a foulbrood (e.g., American or European foulbrood). Alternative or in addition, the disease can be a powdery cheshuichatoe.

Another object of the present invention is weakened or avirulent strain Paenibacillus (e.g., Paenibacillus larvae). Especially preferred are weakened or avirulent strains of Paenibacillus larvae subsp. pulvifaciens, Paenibacillus larvae subsp. larvae or Paenibacillus alvei.

Weakened or avirulent strains according to this is the overarching invention, above, can be used to treat or prevent infections of bees. In particular, it is revealed that these strains can be used as inoculum using the compositions and methods according to the present invention defined above.

Another object of the present invention is the use of inoculum in the manufacture of a medicinal product intended for the treatment or prevention of infection of bees. In a specific aspect of the present invention, the drug is a preferred composition according to the present invention (defined above).

The present invention relates also to the antibiotic(s)allocated(th) of the above microorganisms, and to the use of this antibiotic(s) in the treatment or prevention of the above diseases of bees. Such antibiotic drugs can be administered in the same manner as that previously used antibiotics (also listed above).

Further, the present invention is described with reference to the examples below illustrate non-limiting examples of its implementation.

Example 1

Sensitivity to the antibiotic from .larvae (zone purification)

Conduct biological tests for the detection of antibiotic action Paenibacillus larvae var. pulvifaciens in a large number the of mikroorganizmov. These tests are carried out in accordance with the standard method in Petri dishes containing growth medium, in which inoculant test microorganism. The disk containing Paenibacillus larvae subsp. pulvifaciens (PLP), placed in the centre of Petri dishes and appreciate the growth of the microorganism. The relative sizes of the zones of purification formed around the discs indicated in the table below.

From the results shown in the table, it is seen that Paenibacillus larvae var. pulvifacens effective in destroying pathogenic strain of Paenibacillus larvae var. larvae (PLL), participating in the development of American hnilica (AG), and Paenibacillus alvei, which is also found in hives infected grilcom. In addition, the results demonstrate the presence of antibiotic activity against other bacteria, while the activity against Gram-positive bacteria is higher than the activity against Gram-negative bacteria.

Were investigated three strains PLP, each of which, as shown, destroys a wide range PLL strains originating from different geographical regions, such as the United Kingdom, Slovenia, Australia and New Zealand. While none of the PLL does not affect the PLP.

All three strains Paenibacilli determining the pathogenicity for bees, produce antibiotics, which to some extent explain their impact on bees. So, the material that is infected AG, usually present in the form monocultures. If IT is M.pluton represents the main body, present in large clumps of cells in the guts of bee brood. After the death of brood created conditions conducive seems, development P.alvei, and these organisms become dominant, suppressing M.pluton to such an extent that he can hardly be detected in a dead brood, even though he was dominant and already overgrown culture at an early stage is zabolevaniya. In terms of the antibiotics, the tests show that P.alvei destroys a wide range of bacteria, including M.pluton, but except .larvae (as PLP and PLL). PLL destroys P. alvei, M.pluton and a whole lot of other strains, but has no effect on the PLP. PLP destroys all (in the context of beekeeping), although all three strains, yeast (S.cerevisiae) and Micrococcus luteus show relative resistance to it.

Example 2

Competitive experiments

For additional information regarding the mechanism that destroys the effects of PLP on PLL, were conducted competition experiments with insulinopenia in a rich environment PLP and PLL next to each other. It was shown that both strains grow in the form of plaques and in a few days PLL begins to back on the side closest to the PLP. A few days later, on the side closest to the PLP, remains only a faint ring of a growing culture. Observation of the cells under the microscope by the deviation of its growth has revealed the presence of "shadow cells, protoplasts and other damaged cells; however, samples of lysed part of the PLL plaques showed the presence of fragments of cells. Thus, the experiments showed that the antibiotic produced PLP has likely cellulolyticus, but not a bacteriostatic effect.

Example 3

The mechanism that destroys the impact that the Oia PLP on PLL and other pathogenic strains Paenibacilli

The inventors found that the effect of PLP on PLL (the bacteria that causes American foulbrood) is related to the bacteriocins, which begins to act on the early growth phase and continues its influence throughout the entire cycle. The bacteriocins is a specific remedy against PLL bacteriolytic action. A wide range of bacteria that are listed in the table are not sensitive to bacteriocins, although some species, such as B.subtilis, apparently, this sensitivity have. The authors characterized bacteriocins as temperature-sensitive (temperature 100°C for 2 minutes destroys it) and sensitive to proteinase to the antibiotic.

At the beginning of sporoobrazovanie a broad spectrum of antibacterial activity. This activity is characterized by resistance in the above-described conditions. Not restricting yourself to a specific theory, it should be noted that this activity is called antibiotic(them) of the compound(s). Extract antibiotic after heat exposure did not inhibit PLL, so we can assume that only the bacteriocins responsible for this inhibitory activity detected in mixtures.

Example 4

Composition And

Apicultural composition

Are the liquid composition when the dispersion of the inoculum PLP dispute in sugar syrup, with the holding of 50% sucrose in water. The above composition is used as a food Supplement for bees, which can be placed in the hive or beside it.

Composition B

The composition in the form of bee candy

Get a solid tablet or briquette (known in beekeeping as bee candy) during pressing of sugar, which distributed the inoculate PLP.

Composition

Composition powder

Composition powder obtained when grinding sugar to a fine powder and then mix it with the powder inoculum PLP. The powder can be used for applying to honeycomb inside the hive or can be introduced directly into the swarming bunch of bees.

Composition G

Sprayable composition

Liquid spray composition obtained when the dispersion of the inoculum PLP in the water. This composition can be used for spraying the inner surface of the hive or the honeycomb, or can be used to spray directly on the swarming bunch of bees.

The above examples are given solely for the purpose of illustration and in no way should be construed as limiting in any the present invention.

1. Composition for treatment or prevention of diseases of bees, such as American or European foulbrood or mealy cheshuichatoe, characterized in that the composition includes:

(a) inoculum containing one or more microorganisms, representing bacteria such as Paenibacillus larvae subsp. pulvifaciens or attenuated or avirulent Paenibacillus larvae subsp. larvae or P. alvei;

(b) used in beekeeping media is a means for delivery of the inoculum into the hive to hive installed microflora with therapeutic and/or protective effect.

2. The composition according to claim 1, characterized in that used in beekeeping media for delivery choose from the following list:

(a) bee candy;

(b) syrup;

(C) liquid veterinary drug;

(g) powder;

(d) the paste.

3. The composition according to claim 2 (a), characterized in that the specified bee candy represents:

(a) sugar candy or

(b) the candy including sugar and fat filler.

4. The composition according to claim 2 (b), characterized in that the syrup includes sugar and water.

5. The composition according to item 2 (d), characterized in that the powder comprises sugar.

6. The composition according to item 2 (d), characterized in that the paste is a pollen substitute.

7. The composition according to claim 1, characterized in that hive contains a colony of bees.

8. The composition according to claim 1, characterized in that one or more pathogens Paenibacillus larvae subsp. pulvifaciens, or attenuated or avirulent Paenibacillus larvae subsp. larae, or P. alvei produce one or more corresponding antibiotics, active against one or more pathogens of bees that cause American or European foulbrood or mealy cheshuichatoe.

9. The composition according to claim 8, characterized in that the(s) antibiotic(s) shows(s) activity against Melissococcus pluton and/or Paenibacillus larvae subsp. our band.

10. The composition according to claim 8, characterized in that the(s) antibiotic(s) is(are) a bacteriolytic(s) antibiotic(s).

11. The composition according to claim 8, characterized in that the(s) antibiotic(s) is(are) an antibiotic(s)active(s) against Melissococcus pluton and/or Paenibacillus larvae subsp. larvae found(s) in Paenibacillus larvae subsp. pulvifaciens, or their homologues.

12. The composition of claim 8, wherein the specified(s) antibiotic(s) shows(s) activity against Escherichia coli, Salmonella typhimurium, Pseudomonas putida, Pseudomonas aeruginosa, Micrococcus luteus, Bacillus cereus, Bacillus subtilis, Paenibacillus alvei, Paenibacillus larvae subsp, larvae, Paenibacillus apiarius, Listeria monocytogenes, Staphylococcus aureus, Yersinia enterocolitica and Melissococcus pluton.

13. The composition of claim 1, characterized in that these bacteria include bacterial endospores.

14. The composition according to item 13, wherein the specified bacterial endospores are washed and/or concentrated endospores.

15. The composition of claim 1, characterized in that these bacteria get with m the methods of genetic engineering for the expression of antibiotic(s), specific(s) in item 11.

16. The composition according to claim 1, characterized in that these bacteria

(a) contain one or more mutations that change the virulence and/or the ability to form antibiotics, or

(b) include weakened or avirulent strain.

17. The composition according to claim 1, characterized in that the American or European foulbrood or mealy cheshuichatoe called Paenibacillus larvae subsp. larvae, Paenibacillus alvei or Melissococcus pluton.

18. The method of treatment or prophylaxis of American or European hnilica or the powdery cheshuichatoe bees, including insulinopenia hive composition defined in claim 1, for the establishment of microflora with therapeutic and/or protective effect.

19. The inoculum containing one or more pathogens Paenibacillus larvae subsp. pulvifaciens, or attenuated or avirulent Paenibacillus larvae subsp. larvae, or Paenibacillus alvei and used as an active ingredient of a medicinal product for the treatment and/or prophylaxis of American or European hnilica or the powdery cheshuichatoe from bees, where the medicinal product is a composition according to claim 1.