Patent Application: US-201515115485-A

Abstract:
the aim of the invention is to provide a biocide composition which consists of a commercial natural olive leaf extract and a carrier which allows administering the plant extract in the beehive , preferably syrup to inhibit the growth of the paenibacillus larvae bacteria in all the phases of its biological cycle , both in its vegetative state and as a spore , along with melissococcus plutomius and / or nosema spp . achieving a curative treatment or the prophylaxis of the disease in the apis mellifera larvae and adult bees . the active substance is preferably oleuropein , a secoiridoid glucoside , naturally present in plants of the oleaceae family , although in lower concentrations .

Description:
the invention is related to a biocide composition for controlling pests affecting honeybees . the composition comprises a water soluble olea europaea extract and a carrier . the preferred concentration of the extract in the biocide composition is in a range of concentration chosen from 0 . 001 to 40 mg / ml , 0 . 05 to 30 mg / ml , 0 . 05 to 0 . 08 mg / ml , 0 . 1 to 2 mg / ml , 2 to 10 mg / ml , 2 to 20 mg / ml and 2 to 40 mg / ml . preferably , the range of concentrations of the extract in the biocide composition is 0 . 01 to 20 mg / ml . the concentration ranges which the biocide composition presents are important as they cover various uses for bee pest control . some of these ranges can be used to control pests in their vegetative state , paenibacillus larvae or melissococcus plutonius ; others however , can be used to control the states of spores , p . larvae or nosema spp . moreover , the biocide composition of this invention comprises an extract concentration range for use as a disinfectant . the concentrations of growth inhibition in the spore state are vital for the control of diseases ; spores are the most aggressive infective stage of the american foulbrood or nosemosis disease and thus directly affect the dynamics of the honeybee population in the beehive , causing losses in the honey production phase . as there is no effective treatment , outside the honey production season , both the hive &# 39 ; s frames and box drawers , contaminated with spores , are stored until next season without being disinfected , becoming the source of infection at the start of the new production season , hence the relevant biocide treatment with a composition with a concentration range capable of removing bacteria along with fungal spores . the concentration range in the composition can also be expressed not referring to the extract but to the main metabolite , oleuropeia . in that case , the composition has a concentration range for oleuropein of 0 . 0004 to 16 mg / ml , 0 . 02 to 12 mg / ml , from 0 . 02 to 0 . 032 mg / ml , 0 . 04 to 0 . 8 mg / ml , 0 . 8 to 4 mg / ml , 0 . 8 to 8 mg / ml and 0 . 8 to 16 mg / ml . despite these calculations of the composition &# 39 ; s concentration based on the major metabolite of the water soluble olea europaea extract , it is important to stress the possible synergistic importance and biocide activity within the composition of the invention with the aqueous extract of the olea europaea &# 39 ; s leaves , which can have other metabolites present in lower proportions within the extract . the biocide composition of the invention has the ability to control pests caused by paenibacillus larvae ( american foulbrood ) either in their vegetative state or in the spore state . the composition is also capable of controlling melissococcus plutonius ( european foulbrood ). in addition , it has been shown that the biocide composition of the invention is able to control the fungus , nosema spp . the biocide composition of the invention comprises a carrier with the necessary properties to give the composition the ability to be delivered in the hive , the hive &# 39 ; s frames , the hive frame &# 39 ; s screen and / or in the hive &# 39 ; s feeder . this carrier of the composition can be a syrup , a wax , be liquid , powder or be a solid substrate . in a preferred embodiment of the invention the carrier is a syrup . this syrup consists of sucrose at a concentration in the ranges chosen from 10 to 600 % w / v , 10 - 500 % w / v , 20 to 400 % w / v , 30 to 400 % w / v , 30 to 200 % w / v , 50 - 100 % w / v . the biocide composition also consists of one or more other biocides selected from one or more biocides from the group of : a synthetic organic biocide , a natural organic biocide and an inorganic biocide present in lower proportions within the extract . from the additional biocides , these are chosen from one or more biocides from the group of : soybean essential oil , mugwort essential oil , eucalyptus oil , lemon oil , anise essential oil , cinnamon essential oil , black pepper essential oil , soapbark essential oil , rosemary essential oil , thyme essential oil , winter savory essential oil , celery essential oil , basil essential oil , bergamot essential oil , onion essential oil , cardamom essential oil , coriander essential oil , cypress essential oil , clove essential oil , cumin essential oil , turmeric essential oil , juniper essential oil , tarragon essential oil , geranium essential oil , fennel essential oil , frankincense essential oil , jasmine essential oil , laurel essential oil , mandarin essential oil , lemon balm essential oil , mint essential oil , oregano essential oil , rosemary essential oil , sage essential oil , sandalwood essential oil , thyme essential oil , turpentine essential oil , vanilla essential oil , valerian essential oil , verbena essential oil , carrot essential oil , garlic essential oil and combinations thereof . the additional biocides in the composition used are selected from one or more biocides from the group of : soybean extract , mugwort extract , eucalyptus extract , lemon extract , anise extract , cinnamon extract , black pepper extract , soapbark extract , rosemary extract , thyme extract , winter savory extract , celery extract , basil extract , bergamot extract , onion extract , cardamom extract , coriander extract , cypress extract , clove extract , cumin extract , turmeric extract , juniper extract , tarragon extract , geranium extract , fennel extract , frankincense extract , jasmine extract , laurel extract , mandarin extract , lemon balm extract , mint extract , oregano extract , rosemary extract , sage extract , sandalwood extract , thyme extract , turpentine extract , vanilla extract , valerian extract , verbena extract , carrot extract , garlic extract and combinations thereof . the additional biocide present in the composition may be chosen from one or more , which control pests of honeybees selected from the following ectoparasites and endoparasites : mites , such as varroa , aethina tumida , acarapis woodi , tropilaelaps clareae , t . koenigerum , fungi like ascosphaera apis , aspergillus , protozoa like malpighamoeba mellificae , bacteria such as pseudomonas auriginosa , viruses like the sacbrood bee virus ( sbv ), deformed wing virus ( dwv ), kashmir bee virus ( kbv ), acute bee paralysis virus ( abpv ), black queen cell virus ( bqcv ), chronic bee paralysis virus ( cbpv ) or the israeli acute paralysis virus ( iapv ). one of the advantages of this invention is that the biocide composition has selectivity since it does not inhibit the growth , proliferation or survival of other bacteria characteristic of the hive . the proposed solution does not inhibit the growth , proliferation or survival of bacillus megaterium , bacillus subtilis , bacillus pumilus or paenibacillus polymyxa . another major advantage is that the composition of the invention contains concentrations of biocide that do not present adverse effects during any of the bee &# 39 ; s stages of development . the invention has no adverse effects during the larval development or on the adult bee . the proposed solution comprises the use of a water soluble olea europaea leaf extract in a concentration range of 0 . 001 to 40 mg / ml , 0 . 05 to 30 mg / ml , 0 . 05 to 0 . 08 mg / ml , 0 . 1 to 2 mg / ml , 2 to 10 mg / ml , 2 to 20 mg / ml and 2 to 40 mg / ml and is a carrier . the concentration range used in the composition can also be expressed , not in reference to the extract but referring to the main metabolite oleuropein . preferably , the invention is related to the use of the biocide composition which comprises oleuropein as the main metabolite in a range of concentration which runs from 0 . 0004 to 16 mg / ml , 0 . 02 to 12 mg / ml , from 0 . 02 to 0 . 032 mg / ml , 0 . 04 to 0 . 8 mg / ml , 0 . 8 to 4 mg / ml , 0 . 8 to 8 mg / ml and 0 . 8 to 16 mg / ml . some of these ranges can be used to control pests in their vegetative state , paenibacillus larvae or melissococcus plutonius ; others however , can be used to control the states of spores , p . larvae or nosema spp . moreover , the invention proposes the use of the biocide composition of the present invention as a disinfectant . the invention is also related with the use of the biocide composition with the features described above comprising a carrier with the necessary properties that allow the composition to be used on the hive , on the hive &# 39 ; s frames , on the hive frame &# 39 ; s screens and / or on the hive &# 39 ; s feeder . this carrier of the composition used can be a syrup , a wax , be liquid , powder or be a solid substrate . preferably the carrier used in the composition is a syrup . this syrup consists of sucrose at a concentration in the ranges chosen from 10 to 600 % w / v , 10 - 500 % w / v , 20 to 400 % w / v , 30 to 400 % w / v , 30 to 200 % w / v and 50 - 100 % w / v . the use of the invention may also comprise in the biocide composition consisting of the water soluble olea europaea and a carrier , one or more other biocides selected from one or more biocides of the group of : a synthetic organic biocide , a natural organic biocide and an inorganic biocide present in lower proportions in the extract . additional biocides in the composition used are chosen from one or more biocides of the group of : soybean essential oil , mugwort essential oil , eucalyptus oil , lemon oil , anise essential oil , cinnamon essential oil , black pepper essential oil , soapbark essential oil , rosemary essential oil , thyme essential oil , winter savory essential oil , celery essential oil , basil essential oil , bergamot essential oil , onion essential oil , cardamom essential oil , coriander essential oil , cypress essential oil , clove essential oil , cumin essential oil , turmeric essential oil , juniper essential oil , tarragon essential oil , geranium essential oil , fennel essential oil , frankincense essential oil , jasmine essential oil , laurel essential oil , mandarin essential oil , lemon balm essential oil , mint essential oil , oregano essential oil , rosemary essential oil , sage essential oil , sandalwood essential oil , thyme essential oil , turpentine essential oil , vanilla essential oil , valerian essential oil , verbena essential oil , carrot essential oil , garlic essential oil and combinations thereof . additional biocides in the composition used are chosen from one or more biocides from the group of : soybean extract , mugwort extract , eucalyptus extract , lemon extract , anise extract , cinnamon extract , black pepper extract , soapbark extract , rosemary extract , thyme extract , savory extract , celery extract , basil extract , bergamot extract , onion extract , cardamom extract , coriander extract , cypress extract , clove extract , cumin extract , turmeric extract , juniper extract , tarragon extract , geranium extract , fennel extract , frankincense extract , jasmine extract , laurel extract , mandarin extract , lemon balm extract , mint extract , oregano extract , rosemary extract , sage extract , sandalwood extract , thyme extract , turpentine extract , vanilla extract , valerian extract , verbena extract , carrot extract , garlic extract and combinations thereof . the invention includes the use of an additional biocide which controls honeybee diseases , selected from the following ectoparasites and endoparasites : mites such as varroa , aethina tumida , acarapis woodi , tropilaelaps clareae , t . koenigerum , fungi like ascosphaera apis , aspergillus , protozoa like malpighamoeba mellificae , bacteria like pseudomonas auriginosa , virus such as the sacbrood bee virus ( sbv ), deformed wing virus ( dwv ), kashmir bee virus ( kbv ), acute bee paralysis virus ( abpv ), black queen cell virus ( bqcv ), chronic bee paralysis virus ( cbpv ) or the israeli acute paralysis virus ( iapv ). the proposed use in the invention has the advantage of presenting selectivity in its biocide effect , it does not inhibit the growth , proliferation or survival of the bacteria characteristic of the hive . the proposed solution does not inhibit the growth , proliferation or survival of bacillus megaterium , bacillus subtilis , bacillus pumilus or paenibacillus polymyxa . the invention can be used to spray , bathe and impregnate frame screens with the biocide composition on the hive , on the hive &# 39 ; s frames , the frame &# 39 ; s screen and / or the hive &# 39 ; s feeder . the invention consists in providing or preparing a biocide composition comprising an olea europaea extract in water in a concentration range of 0 . 01 to 20 mg / ml and a carrier . the application is also related to a method for controlling pests of bees comprising the steps of : i ) preparing or providing a biocide composition comprising a water soluble olea europaea extract in a concentration range of 0 . 01 to 20 mg / ml and a carrier . ii ) incorporating olea europaea extract to the hive &# 39 ; s breeding chamber feeder , to the hive , to the hive frame &# 39 ; s screen and / or to the hive &# 39 ; s frame . preferably step ii ) is done by spraying , dipping , impregnation or within the screening process of the frame . the proposed solution comprises incorporating olea europaea extract to the hive &# 39 ; s breeding chamber feeder and / or in the screening of the hive &# 39 ; s frame . the proposed solution is incorporated by spraying , dipping and impregnation . the motive of this invention includes an apparatus which has impregnated the biocide composition . the apparatus of the invention is a hive , is a hive frame , is the screen of the hive &# 39 ; s frame , is the hive &# 39 ; s breeding chamber feeder . the proposed invention also includes a kit comprising the biocide composition , as well as other components such as other biocides or user instructions . preferably , in addition to the biocide composition , the kit comprises user instructions for the biocide composition of this invention for controlling honeybee infections . the invention considers the biocide composition as part of a kit . the proposed solution also has as part of the kit , user instructions of the biocide composition . the examples listed below are illustrative and are only incorporated to further the understanding of the specification and are not meant to limit in any way the scope of the claims requested . the p . larvae vegetative cells were seeded at a density of 4 × 10 6 in a j solid culture medium , a paper disk ( diffusion disk ) was placed in the central area of each culture plate on p . larvae sown , with 10 μl of the carrier ( without extract ) or 10 μl of extract ( 50 mg / ml ) and these were kept under microaerophilic conditions , 0 2 / c0 2 5 %/ 5 - 10 % the direct observation of an inhibition halo around the diffusion disk considered growth inhibition . the inhibition zone was also observed under a microscope . at 5 days , the growth of p . larvae was evaluated . the inhibition halo on the plate which contained extract ( fig1 ) was observed . while on the plate that did not contain extract , the growth of p . larvae was not inhibited . therefore , it was shown that the invention inhibits the growth of p . larvae vegetative cells . p . larvae spores were seeded at a density of 200 spores per culture plate in a solid j culture medium without extract and in a j medium with 6 mg / ml of extract and were kept under microaerophilic conditions 0 2 / c0 2 5 %/ 5 - 10 % at 37 c . growth inhibition was considered with the direct observation of the absence or reduction in the number of colonies of p . larvae on the plate with extract compared to the plate without extract . at 5 days , the growth of p . larvae was evaluated . absence of colonies on the plate with extract was observed ( fig2 ). however , on the plate containing no extract , p . larvae colonies were observed . thus , the invention is shown to be effective in inhibiting the growth of p . larvae spores . in vitro feeding of apis mellifera larvae using different concentrations of extract larvae 12 - 24 hours after birth were fed with 10 μl of artificial diet , diet a ( 12 - 18 % glucose , 12 - 18 % sucrose , 2 - 4 % yeast extract , 50 % royal jelly ) or with 10 μl of artificial diet supplemented with extract at a final concentration range of 3 mg / ml ( diet b ), 5 mg / ml ( diet c ) and 10 mg / ml ( diet d ). twenty four larvae in each condition were used to calculate the survival % from day 1 to 6 of incubation ( fig3 ). the dead larvae were identified according to three criteria : elasticity , size , color , food consumption and decomposition . growth inhibition of other bacteria , characteristic to the hive , in the presence of different concentrations of extract bacteria p . larvae , m . plutonius , paenibacillus alvei , bacillus megaterium , bacillus subtilis , bacillus pumilus and paenibacillus polymyxa were seeded at a density of 1 × 10 6 bacteria per culture plate and p . larvae spores at a density of 200 spores per culture plate in culture j or solid lb medium with extract and without extract in a range of concentration between 0 . 01 mg / ml and 25 mg / ml ( inhibitor concentrations 1 to 12 , fig4 ) and between 0 . 01 mg / ml to 5 mg / ml ( inhibitor concentrations 1 to 8 , fig5 ) in conditions similar to example 2 or in anaerophyl 10 % c0 2 at 35 ° c . between day 1 and day 5 in incubation , it was evaluated whether the extract inhibited bacterial growth . all bacteria grew in the medium without extract . the absence of p . larvae , m . plutonius and p . alvei colonies was only observed in plates with extract ; the invention is shown to be effective in a range of concentrations ( fig4 and 5 ). these results allow concluding that the invention is applicable for other diseases that also attack the larvae of a . mellifera , like european foulbrood whose causal agent is m . plutonius and p . alvei as a secondary pathogen to the infection by m . plutonius . the administration of the extract resolves the infection by nosema ceranae in adult honeybees the extract ( 10 mg / ml ) was administered to hives infected naturally with spores of n . ceranae at an infection rate of 7 × 10 5 spores / bee . the administration is performed once every 10 days . the infection rate was determined every 7 days . it was noted that at 7 days after the first administration , the rate of infection was reduced by 71 % to ( 2 × 10 6 spores / bee ), reaching a reduction of 99 % ( 5 × 10 3 spores / bee ) at 35 days post administration ( fig6 ). albo , g . n ., e . cerimele , m . s . re , m . de giusti , and a . m . alippi . logue americana : ensayos de campo para evaluar la efectividad de algunos aceites esenciales . vida apicola ( 2001 ) 108 : 41 - 46 . alippi , a . m ., j . a . ringuelet , e . l . cerimele , m . s . re , and c . p . henning . antimicrobial activity of some essential oils against paenibacillus larvae , the causal agent of american foulbrood disease . journal of herbs , spices and medicinal plants ( 1996 ) 4 : 9 - 16 . antunez , k ., j . harriet , l . gende , m . maggi , m . eguaras , and p . zunino . efficacy of natural propolis extract in the control of american foulbrood . vet . microbiol . 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