Abstract:
The aim of the invention is to provide a biocide composition which consists of a commercial natural olive leaf extract ( olea europea ) and a carrier which allows administering the plant extract in the beehive, preferably syrup (50% w/v sucrose dissolution) 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:
[0001]    This invention refers to a biocide composition to inhibit the growth of the  P. larvae  bacteria, both vegetative cells and spores,  Nosema  sp and/or the bacteria of  Melisococcus plutonius.  This composition comprises the combination of a commercial olive leaf extract ( Olea europaea ), whose main component (40%) is a secoiridoid glucoside, oleuropein, in a biocompatible carrier, with the addition of other components not being necessary. 
         [0002]    According to the merits of this invention, the composition can be produced industrially and marketed as a reagent to be used for the following purposes: 
         [0003]    1. Food supplement of  larvae  and  A. mellifera  adult bees. 
         [0004]    2. Preventative and curative control of the infection of  A. Mellifera larvae  by  P. larvae  and  M. plutonius,  as well as the preventative and curing control for the infection of  A. Mellifera  adult bees by  Nosema  spp. 
         [0005]    3. Disinfection of beehive equipment. 
         [0006]    STATE-OF-THE-ART 
         [0007]    The European honeybee ( Apis mellifera ), together with the Asiatic honey bee ( Apis cerana ) are the basis of the honey-making industry (Ellis and Munn, 2005). The two main aspects of economic sustainability for breeding bees are pollination and honey production (Reynaldi et al, 2008). Increasingly, other products of the hives including pollen, propolis, Queen Bees are being sold (Genersh, 2010, Genersch et al, 2010). 
         [0008]    One of the biggest challenges of the honey-making industry is that bees, in their different stages of development, are exposed to a wide range of pathogens (viruses, bacteria, fungi, microsporidia, arthropod parasites, etc.) which weaken the hives&#39; populations (Gilliam et al, 1977). One of the bacterial diseases causing great concern for beekeepers and government agencies around the world is American Foulbrood (Lindstrom I, 2008). Its causal agent is the  Paenibacillus larvae,  a  larvae  sub species, gram positive spore forming bacteria, belonging to the  Bacillus  group (Genersch et al, 2006; Bailey, 1962). The spores (considered as the infective stage of  P. larvae ) are highly resistant to heat and dehydration, can lie dormant for decades (Haseman, 1961), both in beekeeping equipment and in the cells of the hive&#39;s frames. The disease attacks the bees&#39;  larvae  which are infected by ingesting spores, causing their death. 
         [0009]    Currently the procedures that are available to treat the disease range from the most drastic ones, removal of diseased hives and the equipment associated to their handling by incineration, the sterilization of the hives and their contents using chemical products (formaldehyde, chlorine, ethylene oxide), and the prophylactic and therapeutic use of antibiotics. All these treatments have limitations associated to their use, including: i) economic losses due to the destruction of the burnt colonies; ii) generation of resistant strains, antibiotics have shown limited effectiveness in the field (Hansen and Broadsgaard, 1999). In Argentina and the United States, the appearance of strains of  P. larvae  resistant to tetracycline, oxytetracycline and sulfathiazole (Alippi, 1996; Miyagi et al, 2000) has been reported. This is mainly because the  P. larvae  spores are resistant to these and can remain in the hive and the equipment spreading the infection; iii) adverse effects associated to the accumulation of toxic chemicals and of antibiotic waste in the hives (honey and pollen), which affect the quality of the honey produced (Jimenez et al, 2006) and that cause alterations in the bees&#39; behavior, including the abandonment of hives and a reduction of the colony&#39;s total population (Bastos et al, 2008). The latter effect is explained by high mortality in offspring. In contrast, this invention, detailed below—i) inhibits the growth of the bacteria,  Paenibacillus larvae  and  Melissococcus plutonius  and the germination of  P. larvae  spores; ii) inhibits the growth and proliferation of the fungus,  Nosema ceranae;  iii) the composition is stable during the period that it must be used in; iv) the stock composition does not experience major changes through time; v) it is effective for the curative treatment or prophylaxis of the disease; vi) is selective; vii) is harmless for the  larvae  and adult bees; viii) its administration is consistent with the colony&#39;s foraging for food; ix) it can be administered to any component of the hive (for example occupied or unoccupied honeycombs) or to the group of bees itself. That is to say, the invention has clear advantages over treatments used currently, both in terms of its use and its effectiveness and harmlessness. 
         [0010]    This invention is directed to a composition comprising a commercially available olive leaf extract ( Olea europaea ), which includes as its main active ingredient, Oleuropein, a naturally occurring secoiridoid glucoside component, to inhibit the growth of pathogens which affect both  larvae  and adult honeybees given that:
       The olive leaf extract has been shown to have strong in vitro antimicrobial properties against viruses, gram negative and positive bacteria, yeast and parasites, both in animals and in humans. It has also been shown to alter bacterial spore germination delaying the organism&#39;s growth.   The olive leaf extract has phenolic components; the main active phenol component in the olive leaf is Oleuropein and its derivatives, such as hyroxytyrosol and tyrosol, which gives the extract its antimicrobial properties.   It has been shown to be non-toxic in animal experiments (in high doses (1 g/kg body weight) during a week&#39;s treatment), and in cultures of human cell lines (at 1 mg/ml of extract).   It is consumed as a dietary supplement in humans as has numerous cardiovascular benefits, hypoglycemic and antioxidant activity.   Oleuropein is found in high quantities in all the parts of the olive tree, especially in the leaves (6-9% p/p dry leaf).   A Hydroxytyrosol metabolite of Oleuropein has recently been proven to be effective against human pathogens,  Haemophilus influenzae, Salmonella typhi, Vibrio parahaemolyticus  and  Staphylococcus aureus  ( ).   Oleuropein and its derivatives have demonstrated in vitro antimicrobial effectiveness and, therefore, may have application in the treatment of infections of  Apis mellifera larvae.          
 
         [0018]    The only two documents currently available relating to a) antimicrobial activity of commercial olive leaf extract and b) antimicrobial activity of olive leaf extract (obtained with organic solvents) against  P. larvae  are presented and discussed below: 
         [0019]    “Antimicrobial activity of commercial  Olea europaea  (Olive) leaf extract”. Aurelia N. Sudjana et al, 2009: the in vitro antimicrobial activity of a commercial liquid extract of olive leaves was investigated with a guaranteed minimum oleuropein content (4.4 mg/ml) against a wide range of microorganisms;  P. larvae  was not considered. It is shown that the extract does not have a broad spectrum of action, being shown to only be effective against  C. Jejuni, H. Pylori  and  Staphylococcus  spp. Furthermore, in this table, what is described in the aforementioned scientific article versus this invention is compared in detail. 
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                   
                 Sudjana et al, 2009 
                 This invention 
               
               
                   
               
             
             
               
                 Objective 
                 Investigate the antimicrobial 
                 Inhibit the growth of  P.   
               
               
                   
                 activity of a commercial  
                   larvae  and  M.   plutonius   
               
               
                   
                 extract derived from  
                 with a commercial extract  
               
               
                   
                   Olea   europaea  (olive)  
                 of  Olea   europaea  (olive)  
               
               
                   
                 leaves, against a wide range  
                 leaves, whose main active  
               
               
                   
                 of micro-organisms that  
                 ingredient is Oleuropein 
               
               
                   
                 does not include  P.   larvae . 
                   
               
               
                 Type of  
                 Bacterial strains 
                 Bacterial strains 
               
               
                 organisms 
                   
                   
               
               
                 Pathogen 
                 
                   Campylobacter 
                   jejuni 
                 
                 
                   P. 
                   larvae 
                 
               
               
                   
                 
                   Helicobacter 
                   pylory 
                 
                 
                   M. 
                   plutonius 
                 
               
               
                   
                 
                   Staphylococcus 
                   aureus 
                 
                   
               
               
                   
                 and another 30 organisms 
                   
               
               
                 Toxicity test 
                 Was not done 
                 Was done on larvae 
               
               
                 Olive leaf  
                 Liquid commercial contract 
                 Commercial powder extract. 
               
               
                 extract 
                 (guaranteed minimum 
                 Oleuropein (400 mg/ml) 
               
               
                   
                 oleuropein content  
                   
               
               
                   
                 (4.4 mg/ml)) 
                   
               
               
                 Exposure  
                 3 days 
                 5 days 
               
               
                 time 
                   
                   
               
               
                 Outcomes 
                 i) antimicrobial activity 
                 i) antimicrobial activity 
               
               
                   
                 ii) minimum inhibitory 
                 ii) minimum inhibitory 
               
               
                   
                 concentration 
                 concentration 
               
               
                   
                 iii) selectivity 
                 iii) selectivity 
               
               
                 Results 
                 The exposure to the extract 
                 The exposure to the extract 
               
               
                   
                 results in: 
                 results in: 
               
               
                   
                 i) inhibition in the growth  
                 i) in regards to  
               
               
                   
                 of,  C. Jejuni ,  H.   Pylory  and 
                 antimicrobial activity: 
               
               
                   
                   Stapylococcus  spp. 
                 Inhibition of the growth of 
               
               
                   
                 ii) the minimum inhibitory 
                   P.   larvae  vegetative cells 
               
               
                   
                 concentration was ≧50 v/v in 
                 Inhibition of the growth of 
               
               
                   
                 the less susceptible organisms 
                   P. larvae  spores 
               
               
                   
                 and between 0.31 and 0.78  
                 Inhibition of the growth of 
               
               
                   
                 v/v in susceptible organisms. 
                 
                   M. plutonius 
                 
               
               
                   
                 iii) A broad activity spectrum  
                 The minimum inhibitory 
               
               
                   
                 is not shown 
                 concentration was ≧2% w/v  
               
               
                   
                   
                 in the least susceptible  
               
               
                   
                   
                 organisms and ≦0.1% w/v  
               
               
                   
                   
                 in susceptible organisms 
               
               
                   
                   
                 iii) A broad activity  
               
               
                   
                   
                 spectrum is not shown 
               
               
                 Conclusion 
                 i) This is effective only for 
                 The extract inhibits the  
               
               
                   
                   C.   Jejuni ,  H. Pylory  and 
                 growth of  P.   larvae  and  
               
               
                   
                   Staphylococcus  spp 
                   M.   Plutonius . The extract  
               
               
                   
                 ii) the susceptibility data 
                 does not cause the death  
               
               
                   
                 obtained does not allow  
                 of the larvae even over  
               
               
                   
                 stating that the extract is  
                 the concentration that  
               
               
                   
                 effective in therapies of a  
                 has antimicrobial activity  
               
               
                   
                 wide range of diseases 
                 facing the  P.   larvae . 
               
               
                   
               
             
          
         
       
     
         [0020]    “In vitro growth-inhibitory effect of plant-derived extracts and compounds against  Paenibacillus larvae  and their acute oral toxicity to adult honey bees”. Jaroslav Flesar et al, 2010: the antibacterial activity of the extract of raw  olea europaea  leaves was assayed in vitro using the micro-dilution method, the extract was made using methanol:dichloromethane 1:1 and dissolved in DMSO at a concentration of 25.6 mg/ml. The MIC was defined as the lowest dilution which inhibits 80% of the growth compared to the control. The MIC of the olive leaf extract facing  P. larvae  was 256 μg/ml; one of the highest values when compared with another 16 assayed extracts. The olive leaf extract was not included in the toxicity tests for oral consumption in adult bees. This is where said work differs from the present invention in terms of how it is obtained and its composition, an aqueous extract is not used and nor is a commercial extract. Its toxicity is not tested either in  larvae  or adult  A. Mellifera  bees. Furthermore, in the following table, what is described in the aforementioned scientific article versus this invention is compared in more detail. 
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                   
                 Flesar et al, 2010 
                 This invention 
               
               
                   
               
             
             
               
                 Objective 
                 In vitro assay, 26 natural 
                 Inhibit the growth of  P.   
               
               
                   
                 components of several 
                   larvae  and  M.   plutonius   
               
               
                   
                 commercially available 
                 with a commercial extract  
               
               
                   
                 chemical classes (flavonoids, 
                 of  Olea   europaea  (olive)  
               
               
                   
                 terpenoids, alkaloids) and 19 
                 leaves, whose main active  
               
               
                   
                 raw plant extracts (among 
                 ingredient is Oleuropein 
               
               
                   
                 these, the extract of  Olea   
                   
               
               
                   
                   europaea  (olive) leaves), due 
                   
               
               
                   
                 to their antimicrobial activity 
                   
               
               
                   
                 when facing  P.   larvae . 
                   
               
               
                 Type of  
                 Bacterial strains 
                 Bacterial strains 
               
               
                 organisms 
                   
                   
               
               
                 Pathogen 
                 
                   P. 
                   larvae 
                 
                 
                   P. 
                   larvae 
                 
               
               
                   
                   
                 
                   M. 
                   plutonius 
                 
               
               
                 Toxicity test 
                 In adult bees, only with two 
                 On larvae 
               
               
                   
                 extracts. Not done  
                   
               
               
                   
                 with olive leaf extract 
                   
               
               
                 Olive leaf  
                 Collected and dried leaves, 
                 Commercial powder  
               
               
                 extract 
                 methanol-dichloromethane 
                 extract. Ethanol and water  
               
               
                   
                 (1:1) extraction solvents, 
                 extraction solvents 
               
               
                   
                 soluble in DMSO. 
                 water soluble 
               
               
                   
                 concentration of active 
                 Oleuropein (400 mg/ml) 
               
               
                   
                 ingredient is unknown 
                   
               
               
                 Exposure  
                 2 days 
                 5 days 
               
               
                 time 
                   
                   
               
               
                 Outcomes 
                 i) antimicrobial activity 
                 i) antimicrobial activity 
               
               
                   
                 ii) minimum inhibitory 
                 ii) minimum inhibitory 
               
               
                   
                 concentration 
                 concentration 
               
               
                   
                   
                 iii) selectivity 
               
               
                 Results 
                 The exposure to the extract 
                 The exposure to the extract 
               
               
                   
                 results in: 
                 results in: 
               
               
                   
                 i) olive leaf extract was one  
                 i) in regards to antimicrobial 
               
               
                   
                 of the least active facing the 
                 activity: 
               
               
                   
                 vegetative cells of  P.   larvae   
                 Inhibition of the growth of 
               
               
                   
                 ii) the minimum inhibitory 
                   P.   larvae  vegetative cells 
               
               
                   
                 concentration was 256 μg/ml 
                 Inhibition of the growth of 
               
               
                   
                   
                   P.   larvae  spores 
               
               
                   
                   
                 Inhibition of the growth of 
               
               
                   
                   
                 
                   M. 
                   plutonius 
                 
               
               
                   
                   
                 ii) The minimum inhibitory 
               
               
                   
                   
                 concentration of the  P.   larvae   
               
               
                   
                   
                 vegetative cells was 80 μg/ml 
               
               
                   
                   
                 iii) A broad activity  
               
               
                   
                   
                 spectrum is not shown 
               
               
                 Conclusion 
                 i) In vitro effective natural 
                 i) The commercial olive leaf 
               
               
                   
                 products against  P.   larvae   
                 extract inhibits the growth  
               
               
                   
                 vegetative cells were  
                 of  P.   larvae  (vegetative cells  
               
               
                   
                 identified. 
                 and spores) and  M. Plutonius   
               
               
                   
                 ii) the effective extracts were 
                 ii) the extract does not cause 
               
               
                   
                   Humulus   lupulus   L . and  
                 the death of the larvae at the 
               
               
                   
                 
                   Myrtus 
                   communis 
                   L. 
                 
                 same concentration which  
               
               
                   
                   
                 has antimicrobial activity 
               
               
                   
               
             
          
         
       
     
         [0021]    “Compositions and methods for inhibiting a honey bee pathogen infection or controlling a hive infestation” patent W02008060591: a composition is disclosed which prevents or treats pathogen infections or hive infestations using a derivative (an organic acid) of the hop plant ( Humulus lupulus ), an alpha acid, a beta acid or a combination thereof. The effective concentration of this derivative inhibits the growth, the proliferation of bacteria or fungi. In in vitro assays, the composition inhibited  Melissococcus plutonius, Paenibaccilus larvae,  or the fungus,  Ascosphaera apis.  In addition, it kills bacteria and fungi spores. The component does not alter the biological function of the bee. Of the aforementioned compounds, none is shared with this invention to inhibit the growth of  M. Plutonius, P. larvae  and  Nosema  spp. 
         [0022]    “Treatment against bee  larvae  disease” (“Tratamiento contra la enfermedad de las larvas de las abejas”) patent ES2215412T3: The document covers procedures and compositions for the cure and prevention of diseases in honeybees. The composition is an inoculation comprising one or more bacterial microorganisms and a delivery carrier. The procedure consists of inoculating the hive with the aforementioned composition. Consequently, in this patent it is proposed to use a mixture of microorganisms, of which none is shared with the present invention. 
         [0023]    “Method for the control of infestations of honeybee colonies”, patent W0197047193: This invention refers to a method to control several diseases in hives, by applying thereto, an effective amount of an essential oil in a slow release formulation, whereby the term oil includes, but is not limited to oils extractable from plants, or the essential component thereof, monoterpenes such as menthol, geraniol, thymol, myrcene citral, limonene, carene, camphor, eugenol or cineol (eucalyptol), natural oils such as lemon oil, eucalyptus oil or  Brickellia cavanillesii  oil, or organic acids such as formic acid, acetic acid or oxalic acid; the ones preferred most are monoterpenes like thymol or menthol, thymol however is preferred most of all. Consequently, in this patent it is proposed to use compounds, none of which is shared by this invention. Also their effectiveness against  P. larvae, M. Plutonius  and  Nosema  spp is not explained. The term pest refers to any organism that infects the bee colony. 
         [0024]    “Novel composition apiary” patent W02008132524: the composition comprises naturally occurring substances, the aqueous alcoholic extract of herbs ( Thymus vulgaris, Organum vulgare, Junglandis folium, Organum majoranna ) plus the addition of oils such as thymol, cinnamon and anise. Consequently, in this patent it is proposed to use compounds, none of which is shared by the present invention. 
         [0025]    “Compositions and methods for prevention and treatment of diseases associated with honeybees” patent W01997008954: The compositions of this invention are aqueous solutions comprising organic and inorganic acids and a chlorite ion. Optionally, the compositions may further comprise a gelling agent, a colorant and a preservative. Consequently in this patent, it is proposed to use compounds, none of which is shared by the present invention. 
         [0026]    “Method, apparatus and compositions for the prophylaxis and treatment of colony collapse disorder” patent W02013030854 A1: The invention relates to a method for preventing and treating Colony Collapse Disorder (CCD). It refers to a device which automatically delivers a replacement diet or as a natural food whose composition considers aqueous solutions comprising essential oils, none of which is shared by the present invention. 
         [0027]    There is an article about a commercial  Olea europaea  leaf extract; however, its antimicrobial activity against any pathogen of  A. Mellifera  is not proven. “Antimicrobial activity of commercial  Olea europaea  (olive) leaf extract”. Aurelia Sudjana N. et al., 2009. Other articles describe the antimicrobial activity of aqueous extracts and essential oils of plants against  P. larvae, M. Plutonius  and  Nosema ceranae.  None of them consider  Olea europaea.  Some of these articles are mentioned below. 
         [0028]    “Laurel leaf extracts for honeybee pest and disease management: antimicrobial, microsporicidal, and acaricidal activity”. N. Damiani et al., 2013. 
         [0029]    “Antimicrobial activity of Scutia buxifolia against the honey bee pathogen  Paenibacillus larvae ” A. A Bolignon et al, 2013. 
         [0030]    “Antimicrobial activity of Amazonian oils against  Paenibacillus  species”. R. C. Santos et al, 2012. 
         [0031]    In vivo evaluation of antiparasitic activity of plant extracts on  Nosema ceranae  ( Microsporidia ), M. Porrini et al, 2011. 
         [0032]    “Antibacterial activity of water extracts and essential oils of various aromatic plants against  Paenibacillus larvae,  the causative agent of American Foulbrood”. M. J. Gonzalez, J. M. Marioli, 2010. 
         [0033]    “In vitro antibacterial effect of exotic plants essential oils on the honeybee pathogen  Paenibacillus larvae,  causal agent of American foulbrood” S.R. Fuselli et al, 2010. 
         [0034]    Screening of natural compounds for the control of  nosema  disease in honeybees ( Apis mellifera ). L. Maistrello et al, 2008. 
         [0035]    “Short communication. Inhibition of  Paenibacillus larvae  subsp  larvae  by the essential oils of two wild plants and their emulsifying agents” S. R. Fuselli., 2005 
         [0036]    The state of the art described herein reports compositions of herbal extracts and essential oils that do not include olive leaf extract with oleuropein as an active ingredient for the control of bee pathogens. Furthermore, from the patents mentioned only one of them proposes these extracts specifically for use in controlling American foulbrood and/or European foulbrood, so this invention is novel. In addition, the data reported is not sufficient for it to be obvious for an expert in the area, that the use of extract from olive leaves rich in oleuropein achieves controlling bacterial diseases and spores of  P. larvae.  As such, this invention is highly innovative. Finally, the authors of this invention have observed that the use of the composition comprising the extract of olive leaves rich in oleuropein controls the growth of  Melissococcus plutonius, Paenibacillus alvei  (secondary pathogen to the infection by  M. Plutonius ) and  Nosema  spp. 
     
    
     
       DRAWINGS 
         [0037]    The figures described below are proposed with the aim of showing background information to support and serve as an example for the description of the composition; therefore they do not seek to restrict or in no way be considered as limiting the scope of the proposed development. 
           [0038]      FIG. 1 , the invention inhibits the growth of the vegetative cell of  P. larvae    
           [0039]    Vegetative cells were grown in the absence (A) and presence (B) of added extract diffusion disks. At 5 days, by visual inspection under a microscope, the bacterial growth around the paper disk was analyzed on culture plates (n=6). 
           [0040]      FIG. 2 , the invention inhibits the growth of  P. larvae  spores. 
           [0041]    The spores of  P. larvae  were exposed to the medium without extract (A), or to the medium supplemented with extract (B). At 5 days, the growth of colonies on culture plates (n=6) was analyzed by visual inspection. 
           [0042]      FIG. 3 , the invention is not toxic for the  Apis mellifera larvae,  at different concentrations.  Larvae  were exposed to food without extract (diet A) and supplemented food with extract (diets B, C and D). After 5 days incubation, the  larvae  were analyzed by visual inspection and observation under optical microscope (n=2) 
           [0043]      FIG. 4 , the invention inhibits the growth of other pathogenic bacteria of  A. Mellifera. Melissococcus plutonius  and  Paenibacillus alvei  bacteria were exposed to the medium without extract and with extract. At 1, 2 and 5 days of incubation, the colony growth on culture plates was analyzed by visual inspection and under optical microscope (n=2). 
           [0044]      FIG. 5 , the invention does not inhibit the growth of other  Bacillus  and  Paenibacillus  present in the hives. 
           [0045]    The bacteria  B. megaterium, B. subtilis, P. Polymyxa, B. pumilus,  were exposed to the medium without extract and with extract. At 1, 2 and 5 days, the colony growth on culture plates was analyzed by visual inspection and under optical microscope observation and colony growth on culture plates (n=2). 
           [0046]      FIG. 6 , the invention inhibits the proliferation and multiplication of  Nosema ceranae.    
           [0047]    Hives naturally infected with  N. Ceranae  were exposed to the extract. The rate of infection of spores purified from the stomachs of adult bees was analyzed by observation under an optical microscope at 7, 14, 21, 28 and 35 days post application. (n=1) 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0048]    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. 
         [0049]    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. 
         [0050]    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. 
         [0051]    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. 
         [0052]    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. 
         [0053]    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. 
         [0054]    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. 
         [0055]    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). 
         [0056]    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.    
         [0057]    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. 
         [0058]    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. 
         [0059]    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. 
         [0060]    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. 
         [0061]    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. 
         [0062]    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). 
         [0063]    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. 
         [0064]    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. 
         [0065]    The application is also related to a method for controlling pests of bees comprising the steps of: 
         [0066]    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. 
         [0067]    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. 
         [0068]    Preferably step ii) is done by spraying, dipping, impregnation or within the screening process of the frame. 
         [0069]    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. 
         [0070]    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. 
         [0071]    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. 
         [0072]    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. 
       EXAMPLES 
       [0073]    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. 
       Example 1 
     Growth Inhibition of  P. larvae  Vegetative Cells 
       [0074]    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 ( FIG. 1 ) 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. 
       Example 2 
     Growth Inhibition of  P. larvae  Spores 
       [0075]      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 ( FIG. 2 ). 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. 
       Example 3 
     In Vitro Feeding of  Apis mellifera larvae  Using Different Concentrations of Extract 
       [0076]      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 ( FIG. 3 ). The dead  larvae  were identified according to three criteria: elasticity, size, color, food consumption and decomposition. 
       Example 4 
     Growth Inhibition of Other Bacteria, Characteristic to the Hive, in the Presence of Different Concentrations of Extract 
       [0077]    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 ,  FIG. 4 ) and between 0.01 mg/ml to 5 mg/ml (inhibitor concentrations  1  to  8 ,  FIG. 5 ) 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 ( FIGS. 4 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.    
       Example 5 
     The Administration of the Extract Resolves the Infection by  Nosema ceranae  in Adult Honeybees 
       [0078]    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 ( FIG. 6 ). 
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