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WO2014097338A1 - Composition utile pour la lutte biologique contre des maladies des abeilles - Google Patents

Composition utile pour la lutte biologique contre des maladies des abeilles Download PDF

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Publication number
WO2014097338A1
WO2014097338A1 PCT/IT2013/000342 IT2013000342W WO2014097338A1 WO 2014097338 A1 WO2014097338 A1 WO 2014097338A1 IT 2013000342 W IT2013000342 W IT 2013000342W WO 2014097338 A1 WO2014097338 A1 WO 2014097338A1
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Prior art keywords
bees
microorganisms
composition
hive
treatment
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Stefano TROVO'
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/163—Sugars; Polysaccharides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/90—Feeding-stuffs specially adapted for particular animals for insects, e.g. bees or silkworms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/113—Acidophilus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/157—Lactis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/165—Paracasei
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/515—Animalis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/517—Bifidum

Definitions

  • the present invention is about the area of the compositions and methods for the biological control of the state of health of the bees in a hive and the prevention of the most common diseases of the bees and various etiological origins, for example , bacteria, fungus, viruses, protozoics and parasites that infest the colony of bees and the hive.
  • compositions and the methods, according to the present invention are useful for the care-taking of the health of the bees and for the prophylaxis of pathologies due to infestations of the ecto parasitas bee Varroa destructor (varroamite disease ), and relative conditions, such as viruses, nosema, American foulbrood, European foulbrood and fungus.
  • the health of the bees depends on many factors, such as protection from different kinds of diseases ( bacterial, viral, parasitic , etc. ..) , the availability of an adequate treatment , the presence of invasive species and environmental changes.
  • Varroa destructor a tiny dust-mite that feeds it self on the bee's hemolinpha, by piercing and sucking through the cuticle .
  • Varroa determines an action that depauperates the resources of the insect , but what even more serious is, the transmission of other viral diseases through the wounds , its exactly this the cause of the great number of mortality within the hive, infesting with diseases such as Virus that Deforme Wings or the Virus of the Chronic Paralysis ( CBPV ).
  • Both syndromes may occur in the same colony, but usually one type predominates .
  • the CBPV may be the final cause of the collapse of the colonies, as a result of the illness of the adult bees , all the same there's no known effective treatments against this type of infection .
  • Nosemiasi Another pathology related with the bees and varroa is Nosemiasi , it's due to two different species of unicellular fungus ( microsporidia ) : Nosema apis and Nosema ceranae .
  • the bees become infected through oral feeding of spores of Nosema .
  • the latter when in the intestine, invade the cells of the mucous and multiply until it destroys the cells causing a malabsorption phenomena .
  • the infected bee will eUminate the spores in the feces making it possible to transmit the disease to other bees through direct contact or through feeding with nectar , pollen, honey , or other infectious materials.
  • N. apis causes gastroenteritis, characterized by forms of diarrhea , easily recognizable by the beekeeper .
  • N. ceranae is a pathogen which was recently isolated on Apis mellifera, causer of a disease characterized by a long latency period where the only symptom is of beehives , also, hardly recognizable by beekeepers , It is identifiable only with a slow and gradual depopulation ofthe bee families, where the birth of new beees aren't enough to compensate for the number of adult bees that die .
  • At present one of the methods of treatment for the control of diseases of the hives is the using of chemical and biological methods.
  • the Chemical methods are those creating major risks to human health , to the environment and also causes major damages to other insects and animals that are not under treatment .
  • Hirsutella thompsonii Fisher and Metarhizium anisopliae can act as agents of a biological control of Varroa mite , in fact, it's been demonstrated to be an efficacious solution on the density of mites in honebee colonies ( LHB Kanga et al. 2002 Journal of Invertebrate Pathology , 81, 175-184 ) .
  • microorganisms have been introduced in beekeeping .
  • These microorganisms or in particular a variety of microorganisms, has been made able to exert a beneficial effect in agriculture and in the breeding of animals ,also to clean up natural environments and to increase the immune defense of human beings and also in beekeeping .
  • the Japanese patent JP62022558 describes a method to help the metabolism of bees, promote physiological activation and further oviposition of the queen bee, and the growth of the bee larva, obtained by mixing a product of acid fermentation of grain (such as rice , wheat, barley, corn ) or beans with a microorganism food for beekeeping.
  • the product of the acid fermentation is added to the normal bee's diet 's in a Minimum quantity equal to 3% .
  • the application of the Korean patent KR830001681 describes a food substitute for honey bees composed by 2 % skimmed milk powder, 0.1 % of powdered egg without fat , O, 8 % of bean powder, 0.6 % sweet potato powder, 0.05% methionine, 0.05 % lysine , 0.05% yeast powder, 0.05% calcium lactate , 0.09 % of active lactobacillus , 93.02 % of sugar.
  • This food helps the health of the the bees as demonstrated by the increase in their weight which reach up to the 12, 5 % of the original one.
  • the practicing of microorganisms for the apiculture sector is also the reason for the application of the patent that describes the Korean KR20100125747, of its nutritional composition containing an active concentrated liquid , and the method of production.
  • the composition is useful to eliminate the necessity of antibiotics and also to promote the process of egg laying.
  • the manufacturing method includes a mixing of : Saccharomyces cerevisiae , albumin powder, fermented soybeans, pollen, propolis , and minerals with a sugary liquid, and fermentation period of 6 months.
  • the sugary liquid is obtained by mixing the active and concentrated liquid with a lactobacillus and SiOs.
  • the European patent EP1135020 instead exploits the properties of one or more inoculate properties of microorganisms which produces one or more active antibiotics against one or more pathogens of bees.
  • antibiotics described there are ones against the Melissococcus pluton and/or Paenibacillus larvae , which is the causative agent of the American foulbrood of hives .
  • the reason of the request of the patent is also related to the method of the treatment or the prophylaxis of disease of the bee hive which foresee the inoculum in order to establish the microbial flora for reparation and/or protection .
  • the aim of the present invention is to provide a new composition useful for the wellbeing of the bee colony and prophylaxis against various pathological conditions which includes the association of more kinds of bacterial microorganisms and the method of transfering it into the hive , establishing the corrected microflora and/or protection.
  • the aim of the invention therefore is the combination where the microorganisms of whose metabolic activity, produces substances that induce a positive effect on the conditions of the bee colony , capable of treating and preventing pathological disorders of the bees.
  • the association which includes the micro-organisms are the following species: Lactobacillus paracasei ssp.
  • Another point of the invention is to provide a method to practice the biological control of the general well-being and diseases of bees in the hive or various causes that infest the hive and the bees in the colony, applying the effective amount, the composition of microorganisms consist of Lactobacillus paracasei ssp. , Bifidobacterium bifidum , Lactobacillus acidophilus, Lactococcus lactis , Bifidobacterium animalis , Lactobacillus thermophilus , Bacillus clausii .
  • Diagram n. 1 shows the percentage change in the number of adult bees in the hives, treated with the composition of microorganisms according to the invention obtained by the estimate of the sixth period of experiment .
  • Diagram n. 2 shows the percentage change in the number of capped brood in the hives treated with the composition of microorganisms according to the invention obtained by the estimation of the sixth period of the experiment .
  • Diagram n.3 shows the percentage change in the number of broods in the hives non operculated treated with the composition of microorganisms according to the invention obtained by the estimation of the sixth period of the experiment .
  • Diagram n. 4 shows the percentage change in the number of eggs in the hives treated with the composition of microorganisms according to the invention obtained by the estimation of the sixth period of the experiment .
  • Diagram n. 5 shows the graphic illustrating the change in the viral load of CBPV expressed in a function of the number of viral RNA molecules identified in bee samples before and after treatment with the composition of microorganisms obtained by the method Real- time PCR.
  • Diagram n. 6 shows the graphic that illustrates the change in the scale of infection of Nosema Cer nae expressed in function of the numbers of RNA molecules of the pathogen identified in bee samples before and after treatment with the composition of microorganisms obtained by Real-time PCR method .
  • the invention proposes an association of microorganisms which has a metabolic activity that is able to induce a positive effect on the health conditions of a colony of bees , capable of treating and preventing pathological disorders of the bees , which comprises of the following microorganisms Lactobacillus paracasei ssp. , Bifidobacterium bifidum , Lactobacillus acidophilus, Lactococcus lactis , Bifidobacterium animalis , Lactobacillus thermophilus , Bacillus clausii .
  • the mentioned association of microorganisms is provided in a sucrose solution at 5%.
  • the microbial species which form the composition according to the invention include a non- spore-forming bacteria Gram-positivi and bacilli which produce endospores .
  • the 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 depends largely by the characteristics of the host. In addition, some species of Gram- positive are commonly used for their role in food fermentation and therefore used in the preparation of human and and animal food . These microorganisms are deliberately introduced in food as breeding primer , food additives (eg , probiotics) and source of additives (for example , enzymes and amino acids )
  • composition according to the present invention is included amongst Gram -positive non- spore forming as follow : Bifidobacterium , Lactobacillus and Lactococcus .
  • the Bifidobacteria are part of the normal intestinal flora of adult humans and are also one of the first genres to colonize the intestines of children, they also live normally in the intestines of animals. Only a limited number of species of bifidobacteria is used in dairy products , in particular acids , such as yogurt and fermented milk drinks . Bifidobacteria belong to the branch of the phylum Actinomycetes Firmicutes . They are non mooving , non-spore forming , and have avariable aspect, usually like curved stick , and often has a branch shape as Y and V. Normally they are strictly anaerobic , although some species and strains tolerate oxygen.
  • the type species is Bifidobacterium bifidum.T e Bifidobacteri are saccharolytic organisms and have the ability to ferment glucose , galactose and fructose. The glucose is fermented by the fructose -6-phosphate in acetic acid and lactic acid. The differences that exist between the various species consist in their ability to ferment different substrates of carbohydrates and alcohols.
  • the combination according to the present invention comprises two types of Bifidobacteria : Bifidobacterium bifidum and Bifidobacterium animalis .
  • lactobacilli is a taxonomic unit of gram -positive non-spore- forrning wide and heterogeneous , comprising a rod shaped lactic acid bacteria .
  • This genus comprises more than 100 different species, with a wide phenotypic, biochemical and physiological variety .
  • Many species of lactobacilli are the important constituents of the normal intestinal flora of humans and animals, even in this case as that of bifidobacteria , their presence and the entity of colonization depends largely by the host.
  • the genus Lactobacillus are divided into three groups: group 1, compalsory homofermentative , group 2 ,optionan heterofermentative group 3 compalsory heterofermentative .
  • group 1 compalsory homofermentative
  • group 2 compalsory homofermentative
  • group 3 compalsory heterofermentative .
  • the phylogenetic taxonomy of the genus is now defined on a molecular basis .
  • Today thanks to the most modern techniques of molecular characterization there are 112 types belonging to the genus Lactobacillus.
  • lactobacilli are used as priming farming in a variety of food fermentations , such as dairy products , fermented meats and , fermented vegetables , sourdough and sausages . So lactobacilli are daily consumed in large quantities in a variety of fermented foods by people of all ages , ethnic groups and health conditions, apparently without negative effects. Apart from their possible involvement in the development of dental caries, lactobacilli are usually considered as non-pathogenic .
  • the specias included according to the invention are, Lactobacillus paracasei ssp , Lactobacillus acidophilus , Lactobacillus thermophilus .
  • lactococci The kind of micro-organisms of lactococci was previously known as N- streptococci.
  • the type Lactococcus lactis (formerly known as Streptococcus lactis ) , which is used in the composition , is a well-known launching organism of dairy products and the members of these species are common elements of the bacterial common in the cheese.
  • the lactococci belong to the phylum Firmicutes , they are coccoidies , non- mobile and metabolizes in a omofermentative, hexose and pentose eterofermentativa manner . They a re mesophilic for which prefer environments where the temperature fluctuates between 0 20° and 40° C , with the temperature usually at 30 0 C or lower .
  • Bacillus clausii is a Gram-positive bacterium whose cells , in stressful environmental conditions , produce endospores ovals that can remain inactive for long periods. As most of the bacteria of the genus Bacillus ,its cane- shaped and grows in groups or colonies . It's an alcalofilo microorganism and lives in alkaline environments , and is found mainly in the soil. One of the characteristics of this organism lies in its ability to produce a particular type of enzymes, the subtilisins , which have a protease activity in the highly alkaline environment .
  • the spores of some logs of B. clausii are used as probiotics , and are able to survive acid environments, a characteristic of the stomach , and to grow and multiply and transform into vegetative forms at the level of the intestine.
  • enzymes from the metabolic products of B. clausii acts improving the microbial balanceing ( eubiosis ) of the human microbiota and protecting it from infections of other bacteria.
  • the spores in fact stick on to the mucosa preventing the adhesion of pathogens, competing with them to use the nutrients and produce antimicrobial substances (immunoglobulins ) blocking the growth.
  • the bacterial logs that make up the composition according to the present invention have been identified, selected and isolated from various microenvironments (ie. soil , marshes , cheese preparations, etc.), through direct observation of the morphology of the cells under a microscope or through the colonies grown on slabs, and then propagated to the appropriate growth conditions , or alternatively , logs obtained from an official collections, types such as the American Type CultureCollection , ATCC ( Rockville, Md. , USA).
  • the bacterial strains were then grown under appropriate conditions in order to obtain liquid cultures in an appropriate growth .
  • the cultures were then diluted 1:100 in a sucrose solution of 5% , and finally combined in quantities from 12 to 16 % of the total volume in a sucrose solution of 5%.
  • the target of the present invention and a composition formed by the combination of selected microorganisms in liquid form , in sucrose solution of 5% in which each strain initially is present in an amount ranging from 12 to 16 % of the total volume , preferably 14 % .
  • the percentages of each log in the together may vary, depending on the specific environmental conditions of each hive , where each log may find more or less a favourable condition for its growth , and therefore could prevail or be limited compared to the other , therefore , after the application of the microorganisms each log can be present in percentages different from 14% .
  • a simple microbial association may be regarded as a single entity , but according to the species from which it is made it interacts as antagonist and as synergistic . Such interactions may influence both the development of a given log as well as its metabolic activity , thereby leading to a completely new and unexpected effect , at times giving a therapeutic combination in the particular field of application.
  • a subject 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 the bees and for the treatment of serious infections of bees, especially of viruses , determined by CBPV .
  • a further object of the present invention is the method based on the use of the microorganisms according to the present invention that finds application in the treatment and prevention of infection of certain pathogens of bees, in particular in the treatment of chronic paralysis of bees and/or of the infection caused by Nosema ceranae .
  • This method helps the treatment of bees , larvae, and other components of the beehive with the comprising composition together with the combination of micro-organisms.
  • the positive effect exerted by the use of the composition can be evaluated as a function of the strength of the families of bees , by the number ofthe adult bees , by the number of capped brood , by the number of un covered broods and the number of eggs in the hive. Moreover, as a parameter indicative of the good condition of the bee colony and the strength of the families of bees in the hive, it's able to assess the impact of Varroa infection , and the consequences of varroamite disease and other health conditions , known and not, as nosema and viruses .
  • the hives must rest on supports, suitable to prevent the access of ants in the diagnostic drawer, then the hives must be supported in containers with water .
  • the bases of anti varroa in the hive must be intact and free of obstructions .
  • it in order to allow a correct assessment of the actual effectiveness of the treatment on the state of health of the hive , it must not be treated with un allowed products or with products of synthesis in block brood
  • Methods of evaluation of the hives include:
  • composition according to the present invention is applied within the hive by any suitable means known to skilled people , preferably , but not exclusively , the composition is applied by a hand dispensing pump .
  • the composition of microorganisms in sucrose 5% is applied directly on each box of silkworm eggs hive, after opening the roof of the hive without pulling up the box of silkworm eggs .
  • the silkworm eggs, the brood as well as the bees are been wet .
  • the composition can also be sprayed on the door entrance of the hive so as to wet the surface which is been crossed by the bees.
  • the number of applications depends on the conditions of the colony and by the ammount of pests and diseases present .
  • the application is repeated at intervals of about 2 to 5 weeks , for a total number of treatments equal to 3 or 4 .
  • the treatments must necessarily be done in the period between spring and late summer.
  • the application is repeated at intervals of approximately 15 days for a period of two months, and then subsequently the administration can be repeated monthly.
  • Obvously skilled personal in this field can easily find various protocols of application (for example, for frequency of application and the effective quantity applied ) which are effective depending on the actual conditions and locations of the hive .
  • the exact amount is the amount of composed according to the invention, able to control , reduce , inhibit, enhance or positively affect of one or more symptoms of the disease or conditions to be treated .
  • composition content of the invention provided only for illustrative purposes without in any way Umiting the invention .
  • the experiment were made on 24 hives of colonies in hives containing frames, with movable antivarroa at the bottom.
  • the 24 hives were divided into three different groups for treatment: the first group of 8 hives ( marked as T ) they were subjected to the administration of an amount equal to 10ml of a composition for each structure covered by bees, every 15 days for four times , for which the hive is subjected to treatment on day 0 , 15 , 30 , and 45 ; a second group of 8 hives ( marked as M ) was subjected to the adrninistration of an amount equal to 5ml of composition for each structure covered by bees following the same temporal profile of administration , and the third group (group C ) consisting of 8 hives were used as a controller and wasn't subjected to any kind of treatment.
  • n° 1 are the results obtained by estimation of the percentage variation relativeto the sixths of adult bees in the experimental period, represented graphically in Figure 1.
  • Chart 2 shows the values obtained by estimation of the sixth concerning the change in the percentage of capped brood experimental period, represented graphically in Figure 2.
  • Chart N° 3 shows the values obtained by estimation of the percentage change in its sixth brood not capped during the period of experimentation, represented graphically in Figure 3.
  • Chart N° 4 shows the values obtained by estimating its sixth the percentage change of eggs during the period of experimentation, represented graphically in Figure 4.
  • the evolution of change in the number of adult bees in group M also reflects the evolution in the same experimental group of the variation in the number of capped brood and non- operculate , as shown in the graphs of charts 2 and 3, which show a greater and significant increase in the number of hatching, followed by treatment with 5ml of composition of microorganisms , and the adininistration of a double amount of composition determines a variation that coould be compared to that of the control group .
  • the evaluation of the strength of the bee family according to the variation of eggs following treatment shows that the composition oil microorganisms are able to determine and limit the loss of eggs compared to the controlled group where the loss is equal to to 70 % . Also in this case the best effectiveness of the treatment undergone by the group M is confirmed by the lesser percentage change in the number of eggs in this group (about 20 % ) compared to the group that received a double amount of the composition , the group T, which shows a decrease in the number of eggs are about 40% .
  • CBPV and N. ceranae and N. apis are responsible for persistent and latent infections which often do not show clinical signs but are responsible for huge loss of bees, final and definite symptom of infection.
  • Using the technique of molecular biology Real - time PCR (Polymerase Chain Reaction)it is possible to amplify a very specific characteristic of the genome of the two pathogens , allowing to reach to a definite diagnosis . Having a quantitative the technique allows to evaluate the viral load of CBPV , quantifying the specific RNA of the species and , more generally , the entity of the infection.
  • the sporulation of the spores allow to extract much more easily the DNA of Nosema spp.
  • vegetative phase of microsporidia is characterized by a very thin cell wall it allows to simplify the protocol of extraction .
  • the germination buffer has the following composition : 75 ml of sodium hydrogen carbonate 1M to pH 6.5 and 75ml of sodium chloride 1M .
  • Two PCR are performed : the first specification for Nosema ceranae and the second specific for Nosema apis .
  • the reaction mixture has the following composition : 5ul Buffer 10X , 0,4 uM 218 MTTOC fOR, 0,4 uM 218 MITOC REV, 0,4mM deoxynucleotide ( dNTP ) 3mM MgCl2 0, 2mg/ml Bovine Serum Albumin , 0 , 1% TritonX -100 , 0,05 U/ul Ampli Gold Taq (5U/ul ) , 5ul of template DNA and H2O DEPC until it reaches the final volume of 50ul .
  • the reaction mixture was prepared using the Ampli Gold Taq Polymerase kit ( Applied. Biosystems , Branch
  • the amplification was carried out using a thermal cycler Gene Amp ® PCR System 9700 (Applied Biosystems , Carlsbad, USA) according to the following protocol :
  • the reaction mixture has the following composition : 5ul Buffer 10X , 0,4uM 3211 API- FOR, 0.4 uM321 APIS -REV , 0, 4mM deoxynucleotide ( dNTP ) , 3mM MgCb , 0, 2mg/ml Bovine Serum Albumin, 0,1% TritonX-100, 0,05 U/ul Ampli Gold Taq ( 5U/ul ) , 5ul of template DNA and DEPC H20 until it reaches the final volume of 50ul .
  • the reaction mixture was performed using the Ampli Gold Taq Polymerase kit ( Applied. Biosystems , Branchburg , New Jersey , USA).
  • the amplification was carried out using a thermal cycler Gene Amp ® PCR System 9700 (Applied Biosystems , Carlsbad, USA) according to the following protocol :
  • the amplified products of the various PCR reactions were visualized by electrophoresis in agarose gel of 1 , 5 % ( p / v) .
  • the quantifications were performed by the method of absolute quantification in which it was used as a standard in which a plasmid was cloned in the DNA sequence of interest .
  • the sequences of the oligonucleotides used in the study were designed on the basis of specific genomic sequences of both the strain Nosema ceranae ( GenBank DQ486027 ) and of the strain Nosema apis ( GenBank U97150 ) .
  • the amplicon obtained from the amplification of the DNA obtained from spores of N. ceranae is a fragment of 142bp that appears to be a specific type .
  • CBPV Probe 5 -FAM-TCAAGAACGAGACCACCGCCAAGTTC -TAMRA- 3 ' .
  • the primers and the sonde were positioned within the RNA of the gene code the RNA 1 - for RNA viral polymerase and amplify a fragment of lOlbp
  • composition according to the present invention has thus demonstrated to be useful for the treatment of viral CBPV , revealing an unexpected ability to cancel the viral load following treatment according to the method described .
  • This feature is very important , because in the first place, there aren't any known effective kind of treatments against this type of virus , usually transported and distributed by the acaro varroa. Often this is the real cause of death or of the depopulation of households, infested with varroa .

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Abstract

L'invention concerne une composition utile utilisée pour la lutte biologique du bien-être des abeilles, et pour, la prophylaxie et le traitement de troubles pathologiques des abeilles conduisant à diverses causes, et en particulier l'état provoqué par le virus de la paralysie des abeilles ou CBPV, comprenant une association spécifique de micro-organismes, et son procédé d'utilisation.
PCT/IT2013/000342 2012-12-11 2013-12-10 Composition utile pour la lutte biologique contre des maladies des abeilles Ceased WO2014097338A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020505067A (ja) * 2017-01-24 2020-02-20 フラグシップ パイオニアリング イノベーションズ ブイ，インコーポレーテッド 農業用組成物及び関連する方法
CN113265350A (zh) * 2021-05-11 2021-08-17 昆明理工大学 一种双歧杆菌w8118及其应用
US11471433B1 (en) 2019-08-01 2022-10-18 Flagship Pioneering Innovations V, Inc. Postbiotic compositions and related methods for agriculture
US11690387B2 (en) 2017-01-24 2023-07-04 Flagship Pioneering Innovations V, Inc. Methods and related compositions for manufacturing food and feed

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR830001681A (ko) 1980-02-22 1983-05-18 챨스 브루넨코 강자성입자를 함유하는 유체정화용 여과장치
JPS6222558B2 (fr) 1981-04-10 1987-05-19 Mitsubishi Electric Corp
EP1135020A2 (fr) 1998-11-26 2001-09-26 UC3 University College Cardiff Consultants Ltd. Traitement contre la loque
EP1405641A2 (fr) * 2002-08-07 2004-04-07 Proge Farm S.r.l. Composition pharmaceutique et/ou nutraceutique comprenant une combination des microorganismes probiotiques non-sporogènes et des spores de Bacillus subtilis et/ou Bacillus clausii
WO2008136730A1 (fr) * 2007-05-03 2008-11-13 Tobias Olofsson Nouvelles bactéries isolées à partir du miel frais ou de la voie de production de miel d'abeilles
KR20100125747A (ko) 2009-05-21 2010-12-01 국동호 벌 영양제
US8053223B2 (en) 2006-11-30 2011-11-08 The United States Of America As Represented By The Secretary Of Agriculture Biocontrol of Varroa mites
WO2011138310A2 (fr) 2010-05-04 2011-11-10 Universita' Degli Studi Di Milano Méthodes de prévention et de lutte contre les infections pathogènes chez les abeilles, et composition associée

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR830001681A (ko) 1980-02-22 1983-05-18 챨스 브루넨코 강자성입자를 함유하는 유체정화용 여과장치
JPS6222558B2 (fr) 1981-04-10 1987-05-19 Mitsubishi Electric Corp
EP1135020A2 (fr) 1998-11-26 2001-09-26 UC3 University College Cardiff Consultants Ltd. Traitement contre la loque
EP1405641A2 (fr) * 2002-08-07 2004-04-07 Proge Farm S.r.l. Composition pharmaceutique et/ou nutraceutique comprenant une combination des microorganismes probiotiques non-sporogènes et des spores de Bacillus subtilis et/ou Bacillus clausii
US8053223B2 (en) 2006-11-30 2011-11-08 The United States Of America As Represented By The Secretary Of Agriculture Biocontrol of Varroa mites
WO2008136730A1 (fr) * 2007-05-03 2008-11-13 Tobias Olofsson Nouvelles bactéries isolées à partir du miel frais ou de la voie de production de miel d'abeilles
KR20100125747A (ko) 2009-05-21 2010-12-01 국동호 벌 영양제
WO2011138310A2 (fr) 2010-05-04 2011-11-10 Universita' Degli Studi Di Milano Méthodes de prévention et de lutte contre les infections pathogènes chez les abeilles, et composition associée

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
ALEJANDRA VÁSQUEZ ET AL: "Symbionts as Major Modulators of Insect Health: Lactic Acid Bacteria and Honeybees", PLOS ONE, vol. 7, no. 3, 1 January 2012 (2012-01-01), pages e33188, XP055039097, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0033188 *
BLANCHARD ET AL.: "Evaluation of a real-time two- step RT -PCR assay for quantitation of Chronic bee paralysis virus ( CBPV ) genomic in experimentally -infected bee tissues and in life stages of a symptomatic colony", JOURNAL OF VIROLOGICAL METHODS, vol. 141, 2007, pages 7 - 13, XP005896659, DOI: doi:10.1016/j.jviromet.2006.11.021
DATABASE WPI Week 201138, Derwent World Patents Index; AN 2010-Q50453, XP002724595 *
EVANS JAY D ET AL: "Bacterial Probiotics induce an immune response in the honey bee (Hymenoptera: Apidae)", JOURNAL OF ECONOMIC ENTOMOLOGY, vol. 97, no. 3, June 2004 (2004-06-01), pages 752 - 756, XP003023503, ISSN: 0022-0493 *
KIMOTO-NIRA HIROMI ET AL: "Survival of a Lactococcus lactis strain varies with its carbohydrate preference under in vitro conditions simulated gastrointestinal tract.", INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY 15 OCT 2010, vol. 143, no. 3, 15 October 2010 (2010-10-15), pages 226 - 229, XP027376894, ISSN: 1879-3460 *
LHB KANGA ET AL., JOURNAL OF INVERTEBRATE PATHOLOGY, vol. 81, 2002, pages 175 - 184
MARTIN-HERNANDEZ R. ET AL.: "Outcome of colonization of Apis mellifera by Nosema ceranae", APPL ENVIRON MICROBIOL, vol. 73, 2007, pages 633 - 8
MUDRONOVA DAGMAR ET AL: "Lactobacillus sp as a potential probiotic for the prevention of Paenibacillus larvae infection in honey bees", JOURNAL OF APICULTURAL RESEARCH, INTERNATIONAL BEE RESEARCH ASSOCIATION, CARDIFF, GB, vol. 50, no. 4, 1 January 2011 (2011-01-01), pages 323 - 324, XP009162786, ISSN: 0021-8839 *
N. APIS; N. CERANAE, JOURNAL OF INVERTEBRATE PATHOLOGY, vol. 103, pages 53 - 58
RIBIERE ET AL.: "Molecular diagnosis of chronic bee paralysis virus infection", APIDOLOGIE, vol. 33, 2002, pages 339 - 351
SABATE D C ET AL: "Inhibition of Paenibacillus larvae and Ascosphaera apis by Bacillus subtilis isolated from honeybee gut and honey samples", RESEARCH IN MICROBIOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 160, no. 3, 1 April 2009 (2009-04-01), pages 193 - 199, XP026143585, ISSN: 0923-2508, [retrieved on 20090407], DOI: 10.1016/J.RESMIC.2009.03.002 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020505067A (ja) * 2017-01-24 2020-02-20 フラグシップ パイオニアリング イノベーションズ ブイ，インコーポレーテッド 農業用組成物及び関連する方法
US11350648B2 (en) 2017-01-24 2022-06-07 Flagship Pioneering Innovations V, Inc. Compositions and related methods for agriculture
US11690387B2 (en) 2017-01-24 2023-07-04 Flagship Pioneering Innovations V, Inc. Methods and related compositions for manufacturing food and feed
US11471433B1 (en) 2019-08-01 2022-10-18 Flagship Pioneering Innovations V, Inc. Postbiotic compositions and related methods for agriculture
CN113265350A (zh) * 2021-05-11 2021-08-17 昆明理工大学 一种双歧杆菌w8118及其应用
CN113265350B (zh) * 2021-05-11 2022-05-06 昆明理工大学 一种双歧杆菌w8118及其应用

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