Agricultural compositions and methods

Abstract

The present invention relates to compositions and kits for the treatment or prevention of bacterial diseases in terrestrial plants or animals, comprising a phage and / or an immunogenic substance and an attracting factor for flying insects. The present invention also relates to a method for attracting flying insects to a phage and / or an immunogenic substance. The present invention also relates to the use of flying insects in a method for transferring a phage and / or an immunogenic substance to an animal or a plant, and to a method for treating or preventing diseases in animals and plants using flying insects. The present invention also relates to formulations of phages and / or immunogenic substances for use in the treatment and / or prevention of diseases in animals or plants.

Description

Technical Field

[0001] Field of the Invention The present invention relates to compositions and kits for the treatment or prevention of bacterial diseases in terrestrial plants or animals, comprising phages and / or immunogenic substances and flying insect attractants. The present invention also relates to methods for attracting flying insects to phages and / or immunogenic substances. The present invention also relates to the use of flying insects in methods for transferring phages and / or immunogenic substances to animals or plants, and to methods for treating or preventing diseases in animals and plants using flying insects. The present invention also relates to formulations of phages and / or immunogenic substances for use in the treatment and / or prevention of diseases in animals or plants.

Background Art

[0002] Background Flying insects such as flies are known to transmit diseases caused by bacteria, parasites, and viruses to animal and plant species. Flies pick up and carry microorganisms from surfaces they contact, especially those containing human and animal excrement and decaying organic matter. When a fly then physically contacts an animal or plant, the fly infects the animal or plant host with the microorganisms it is carrying, acting as a mechanical vector for the microorganisms.

[0003] As an example of a fly that is a common vector of bacterial diseases, Musca autumnalis is cited, which is generally referred to as the face fly, is a pest species of cattle and horses, is native to Europe and Central Asia, and is found throughout Canada and most of the United States. Other members of the Muscidae family, including the Australian bush fly (Musca vetustissima) and the buffalo fly (Haematobia exigus), are flies that can act as vectors of bacterial diseases in southern hemisphere regions such as Australia. In the case of the face fly, it emerges from its overwintering sites in the spring and the female lays eggs in fresh cow dung. Depending on the temperature, adult flies live for 20 to 50 days, and from early autumn, the surviving flies prepare for overwintering and the insect burden on cattle and horses is reduced. The face fly can move several kilometers in search of a host, but once a suitable habitat is found, the face fly tends to gather around cattle, especially near watering places.

[0004] Face flies, especially females, feed on the blood, sweat, saliva, nasal mucus, feces, and tears of cattle and horse hosts. Feeding occurs only during the day, which means that face flies leave the cattle at night and return again every morning. During the day in summer, typically, the number of flies per cow's face is observed to be about 5 to 30.

[0005] Generally, the disease in which face flies act as vectors is bovine infectious keratoconjunctivitis (IBK), which is the most common eye disease in cattle and is also known by the aliases New Forest eye and pinkeye. Its main pathogen is the gram-negative bacterium Moraxella bovis, but other microorganisms, including Moraxella catarrhalis, Moraxella bovoculi, and Mycoplasma bovoculi infections, have also been associated with a potential predisposing role in the pathogenicity of IBK.

[0006] Face flies are common carriers of the Moraxella bovis bacterium and are therefore common vectors of IBK. In particular, the surfaces of face flies, including the mouthparts, body, and legs, can be contaminated with Moraxella bovis bacteria that can survive for several hours, but Moraxella bovis bacteria ingested by face flies can survive in the fly's digestive tract for up to three days, meaning that face flies carrying Moraxella bovis bacteria can cause bovine eye infections over several days by ejecting droplets from the foregut and midgut.

[0007] The economic impact of this disease is considerable, and IBK is the most commonly seen condition affecting young female beef and dairy cattle and the second most commonly seen disease in suckling calves over three weeks of age.

[0008] Currently, the most commonly used treatments for IBK are antibiotic drugs delivered by systemic injection, subconjunctival injection, intrapalpebral injection or topical application. Alternatively, preventive measures such as the use of ear tags containing insecticides must be resorted to. However, all of these treatment options are compromised by the pharmacokinetics of drug delivery to the ocular surface and the need for animal handling and restraint to implement therapeutic or preventive measures. Therefore, there remains a need for effective methods to reduce the incidence of bacterial infections in animals, particularly for reducing the incidence of face fly-induced IBK in cattle that do not require animal handling and restraint.

[0009] Cattle tend to spend certain parts of the day rubbing against fence posts, trees, or shrubs, during which they appear to derive pleasure and satisfaction. Observation of this behavior has led to the development of commercially manufactured brushes and body buffers that can be placed in fields and enclosures to encourage this behavior. In particular, head rubbing temporarily dislodges face flies from the cattle's face, where they remain airborne until they find another resting place or return to the face. It would be advantageous if this natural tendency of cattle could be harnessed into a strategy to reduce fly-induced IBK or other bacterial infections in cattle. Other fly species, including the horn fly (Haematobia irritans) and stable fly (Stomoxys calcitrans), are major pests of cattle. These fly species, like the common house fly (Musca domestica) and the blow fly (Calliphora vomitoria), are vectors for the spread of bacteria, and the horn fly in particular has been implicated in the transmission of bacteria, including Staphylococcus species that can cause mastitis in cattle. Therefore, repelling these harmful flies from cattle and eliminating their bacterial load is beneficial from the perspective of cattle health and welfare.

[0010] Another host species susceptible to bacterial diseases carried by flying insects is the banana tree (part of the Musa family). Banana trees are susceptible to several bacterial diseases, including "banana bacterial wilt" caused by the bacterium Xanthomonas campestris and "Moko disease" caused by the bacterium Ralstonia solancearum. In banana plantations, parasitic flies, such as the oriental fruit fly [Bactrocera dorsalis] and wasps, are active and can therefore act as vectors for transporting bacterial diseases to banana trees. Similarly, olive trees [Olea europaea] are susceptible to bacterial diseases caused by bacteria carried by the olive fly [Bactrocera oleae], including Xylella fastidiosa and Pseudomonas savastanoi.

[0011] Bees such as the bumblebee [Bombus terrestris audax] can carry bacterial infections to plant species such as flowers. In particular, many plants are susceptible to bacterial canker diseases caused by one of several strains of Pseudomonas syringae, which has a very wide range of host species. These pathogens can be carried and spread by bees and other flying insects. For example, commercial beehives (bee nests) are commonly used to promote the pollination of fruit-bearing plants such as kiwifruit [Actinidia deliciosa] as a means of increasing fruit yields. However, kiwifruit plants are very susceptible to infections caused by Pseudomonas syringae pv. Actinidiae, a kiwifruit canker pathogen that can be carried by bees. Therefore, it would be highly advantageous if the bacterial load of bees in commercial beehives could be reduced and, as a means of disease prevention, if bees could be enabled to spread phages that are active against this pathogen to plants. Currently, agricultural strategies for reducing bacterial infections in plants rely heavily on artificial chemicals introduced into the soil or applied to plant species, which disrupt the surrounding ecosystem. Therefore, an effective method for reducing the incidence of bacterial infections in plants is still needed.

Brief Description of the Drawings

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[0013] Generalization of the Invention In a first aspect of the invention, there is provided a method of attracting flying insects to a phage and / or an immunogenic substance, comprising (i) combining (bringing together) the phage and / or the immunogenic substance with a flying insect attractant, or (ii) placing the phage or the immunogenic substance within the attraction radius of the flying insect attractant.

[0014] A second aspect of the invention relates to the use of flying insects in a method of transferring a phage and / or an immunogenic substance to a terrestrial animal or plant, wherein the flying insects act as carriers of the phage and / or the immunogenic substance.

[0015] A third aspect of the invention relates to a method of treating or preventing a disease in a terrestrial animal or plant, comprising using flying insects to transfer a phage and / or an immunogenic substance to the animal or plant, wherein the flying insects act as carriers of the phage and / or the immunogenic substance.

[0016] In a fourth aspect of the invention, there is provided a composition for treating or preventing a bacterial disease in a terrestrial plant or animal, comprising (i) a phage and / or an immunogenic substance, and (ii) a flying insect attractant selected from one or more of feces, pheromones, volatile substances, odoriferous substances, substances capable of reflecting or emitting light within the visible electromagnetic spectrum, substances capable of reflecting or emitting electromagnetic radiation outside the visible spectrum, nutrient sources of flying insects, and combinations thereof. Also provided is an article comprising the composition of the invention.

[0017] In a fifth aspect of the present invention, there is provided a kit for the treatment or prevention of a bacterial disease in a terrestrial plant or animal, comprising: (i) a phage and / or an immunogenic substance, and (ii) a flying insect attractant selected from one or more of feces, pheromones, volatile substances, odoriferous substances, substances capable of reflecting or emitting light within the visible electromagnetic spectrum, substances capable of reflecting or emitting electromagnetic radiation outside the visible spectrum, a nutrient source for flying insects, or a resting place to which flying insects are innately attracted (e.g., a beehive in the case of honeybees) and combinations thereof.

[0018] In some embodiments, the present invention involves the use of phages. The use of phages addresses the above problem because phages can act on bacteria that are naturally transported by flying insects, reducing the likelihood that flying insects will introduce infection to terrestrial animals and plants. Additionally, phages can treat or control bacteria that have already infected the target animals and plants, and phages can be transferred by flying insects to uninfected organisms. In other words, flying insects, which typically act as vectors for bacteria that cause disease in terrestrial animals or plants, can become vectors for phages that act on the bacteria that cause disease. This provides a targeted means for reducing the incidence of bacterial infections in terrestrial animals and plants without the need for direct human intervention, which may involve handling, manual manipulation, or restraint.

[0019] In some embodiments, the present invention involves the use of immunogenic substances. The present invention also provides a means for delivering an immunogenic substance to induce an immunogenic response without the need for direct human intervention. This is achieved by combining the immunogenic substance with a flying insect attractant or placing the immunogenic substance within the attraction radius of a flying insect attractant, resulting in the transfer of the immunogenic substance to the flying insect. Subsequent transfer of the immunogenic substance to the target animal or plant provides a targeting means for inducing a defensive response in terrestrial animals and plants without the need for direct human intervention involving handling, manual manipulation, or restraint. It is understood that an immune response by humoral or cellular mechanisms does not occur in plants as it does in animals. Nevertheless, it is widely recognized that plants have an immune system and can induce a defensive response in response to pathogen molecules.

[0020] In a preferred embodiment, the phage and / or immunogenic substance is combined with a flying insect attractant that includes a nutrient source that flying insects tend to feed on, thereby promoting the uptake of the phage or immunogenic substance. The phage or immunogenic substance is then transferred to the target organism during the regurgitation of droplets of the flying insect's digestive tract contents, which occurs as a normal feature of the flying insect's feeding behavior.

[0021] Definitions As used herein, the term "flying insect" means any insect having the ability to fly. Thus, the term "flying insect" includes all insects having one or more pairs of wings.

[0022] As used herein, the noun "fly" means any flying insect characterized by using only one pair of wings for flight. This includes, but is not limited to, dipteran species.

[0023] As used herein, the term "Diptera" means insects of the order Diptera, and the term "dipteran" means a single insect of the order Diptera.

[0024] As used herein, the term "face fly" means the species Musca autumnalis.

[0025] As used herein, the term "Australian bush fly" means the species Musca vetustissima.

[0026] As used herein, the term "buffalo fly" means the species Haematobia exigus.

[0027] As used herein, the term "oriental fruit fly" means the species Bactrocera dorsalis.

[0028] As used herein, the term "olive fruit fly" means the species Bactrocera oleae.

[0029] As used herein, the term "blow fly" means any insect of the species Calliphora vomitoria or Lucilia sericata.

[0030] As used herein, the term "bumblebee" means any insect of the species Bombus terrestris or Bombus terrestris audax.

[0031] As used herein, the term "hoverfly" means any insect of the family Syrphidae.

[0032] As used herein, the term "horn fly" means the species Haematobia irritans.

[0033] As used herein, the term "stable fly" means the species Stomoxys calcitrans.

[0034] As used herein, the term "immunogenic substance" means any substance that can elicit an immune response in a target terrestrial organism.

[0035] As used herein, the term "attractant" means any substance that attracts a target animal. Thus, for example, a "flying insect attractant" means any substance that attracts flying insects. An attractant can be a substance other than a nutrient source, it can be a substance that constitutes or contains a nutrient source, or it can be a place to which an insect is innately attracted, such as a nest, as will be understood.

[0036] As used herein, "terrestrial animals" are animals that live mainly or entirely on land. As used herein, "terrestrial plants" are plants that grow on land, within land, or from land.

[0037] As used herein, the term "comprising" means that at least all of the recited elements must be present, but other elements not recited may also be present.

[0038] In the context of the present invention, "attraction radius" means the range within which a target animal is attracted to an attractant. For example, in the case of an insect attractant that involves the use of an odor to attract insects, the attraction radius is the range within which an insect can sense the odor of the attractant. It will be understood that the attraction radius for a particular attractant and target animal can be determined by routine experimentation. For example, the attraction radius can be determined by placing the attractant in a test chamber under normal atmospheric conditions and incrementally increasing the distance from the attractant and placing the target animal to determine the maximum distance at which the target animal is attracted to the attractant.

[0039] As used herein, the term "combine" (which means "bring together") when referring to a combination of X and Y includes mixing X and Y and contacting X with Y. In some embodiments, the term "combine" means mixing. In some embodiments, the term "combine" means contacting a first component with a second component.

[0040] As used herein, the terms "IBK", "New Forest Eye" and "Pink Eye" all mean the disease bovine infectious keratoconjunctivitis.

[0041] As used herein, the term "virus" in the present invention includes double-stranded or single-stranded RNA or DNA viruses that infect bacterial, plant and / or animal cells.

[0042] The terms "phage" and "bacteriophage" are used interchangeably herein to mean bacteriophage, which is a virus that infects specific bacteria.

[0043] As used herein, the term "substrate" in the present invention is understood to mean any solid phase material to which a virus such as bacteriophage can be immobilized.

[0044] As used herein, the term "about" when applied to one or more values of interest refers to a value similar to the indicated reference value. In some embodiments, the term "about" means a range of values that are within 25% of the reference value in either direction (greater than or less than the reference value). In some embodiments, the term "about" means a value that is within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the indicated reference value (provided that such a numerical value is excluded if it exceeds 100% of the possible values).

[0045] Detailed Description The present invention relates to the use of flying insects in a method of transferring phages and / or immunogenic substances to terrestrial animals or plants, where the flying insects act as carriers of the phages and / or immunogenic substances.

[0046] The present invention also relates to a method for treating or preventing diseases in terrestrial animals or plants, comprising transferring phages and / or immunogenic substances to the animals or plants using flying insects, where the flying insects act as carriers of the phages and / or immunogenic substances. In a preferred embodiment, the method for treating or preventing diseases in terrestrial animals or plants comprises directly transferring phages and / or immunogenic substances to the site of an infection, such as a bacterial infection, on the terrestrial animal or plant.

[0047] The present invention also relates to a method of transferring phages and / or immunogenic substances to flying insects, comprising (i) combining the phages and / or immunogenic substances with a flying insect attractant, or (ii) placing the phages and / or immunogenic substances within the attraction radius of a flying insect attractant.

[0048] In embodiments of the methods and uses of the present invention, the flying insect attractant is preferably a flying insect attractant selected from feces, pheromones, volatile substances, odoriferous substances, substances capable of reflecting or emitting light within the visible electromagnetic spectrum, substances capable of reflecting or emitting electromagnetic radiation outside the visible spectrum, resting places, nutrient sources to which flying insects are innately attracted, or combinations thereof.

[0049] The present invention further relates to a composition for treating or preventing bacterial diseases in terrestrial plants or animals, comprising (i) phages and / or immunogenic substances, and (ii) a flying insect attractant selected from feces, pheromones, volatile substances, odoriferous substances, substances capable of reflecting or emitting light within the visible electromagnetic spectrum, substances capable of reflecting or emitting electromagnetic radiation outside the visible spectrum, nutrient sources for flying insects, and combinations thereof.

[0050] Furthermore, the present invention relates to a kit for the treatment or prevention of bacterial diseases in terrestrial plants or animals, comprising (i) a phage and / or an immunogenic substance, and (ii) a flying insect attractant selected from feces, pheromones, volatile substances, odoriferous substances, substances capable of reflecting or emitting light within the visible electromagnetic spectrum, substances capable of reflecting or emitting electromagnetic radiation outside the visible spectrum, resting places to which flying insects are innately attracted, nutrient sources for flying insects, or combinations thereof.

[0051] In the context of the present invention, "prevention" of a bacterial disease or "preventing" a bacterial disease includes any way in which a phage or immunogenic substance transferred to a flying insect can act against bacteria, whether the bacteria are carried by the insect itself or are present on a terrestrial animal or plant. For example, prevention of a bacterial disease can occur by the action of a phage or immunogenic substance on bacteria present on a flying insect, reducing the bacterial load of the flying insect and preventing the spread of the bacterial disease by the insect. Alternatively, prevention of a bacterial disease can occur when a flying insect contacts a terrestrial animal or plant and deposits a phage or immunogenic substance on the terrestrial animal or plant, preventing a bacterial infection that occurs on the animal or plant.

[0052] In a preferred embodiment, the phage and / or immunogenic substance is an antimicrobial substance. In other preferred embodiments, the phage and / or immunogenic substance comprises a bacteriophage. More preferably, the bacteriophage is an engineered bacteriophage, i.e., a bacteriophage prepared for the methods, compositions, kits and uses of the present invention.

[0053] Bacteriophages are viruses that target specific bacteria and, upon infecting their host bacteria, can multiply via either the lytic or lysogenic pathway. Lytic bacteriophages work by infecting bacteria and using them, which can result in the replication and propagation of the virus. Eventually, after sufficient virus replication, bacterial cell lysis releases a large number of new bacteriophages that can themselves infect neighboring bacteria. Lysogenic bacteriophages work by integrating their DNA into the bacterial chromosome and replicating with the host chromosome, generating new copies of the integrated viral DNA. Advantageously, bacteriophages are specific to the bacteria they target, meaning that, unlike antibiotics with low specificity, they can be used as antibacterial agents in a host without damaging other bacteria in the host.

[0054] It will be understood that bacteriophages having activity against specific bacteria can be isolated by those skilled in the art. For example, bacteriophages can be isolated by first culturing the relevant bacteria and plating them on agar to obtain a growing bacterial lawn. Next, a liquid can be sampled from a suitable source of bacteriophages, including but not limited to animal tissues and secretions, feces, plant material, soil, or water samples. For example, bovine rumen fluid, nasal and tear secretions can be sampled to isolate bacteriophages having activity against Moraxella bovis, a bacterium found in bovine animals, and a liquid can be sampled from porcine feces to isolate bacteriophages having activity against Salmonella enterica, a bacterium found in porcine animals. The bacterial lawn on agar is then exposed to an aliquot of the sampled liquid, and, for samples where bacterial lysis is observed, a bacteriophage lysate can be concentrated therefrom.

[0055] In a preferred embodiment, the bacteriophage is stabilized. For example, as described in EP 1496919 B1, it is stabilized by immobilization on a substrate. This is advantageous because it is ensured that the phage retains its antibacterial activity while its stability is improved compared to free phage.

[0056] In some embodiments, as described in EP 1496919 B1, the bacteriophage is immobilized on a substrate by a covalent bond formed between the substrate and the phage. In a preferred embodiment, the bacteriophage is immobilized via its head while keeping its tail in a free state. This ensures that the bacteriophage retains its activity because the tail of the bacteriophage is involved in the ability of the phage to recognize and infect specific bacteria.

[0057] In a preferred embodiment, the flying insect is a dipteran. In a particularly preferred embodiment, the dipteran is a face fly, an Australian bush fly, a buffalo fly, a stable fly or a horn fly. This is advantageous because these flies are pest species of cattle and horses. Thus, the use of these flies for carrying phages and / or immunogenic substances provides an effective method for targeting these substances to cattle and horse species because the flies transfer the phage or immunogenic substance to cattle and horse species. In a particularly preferred embodiment, the dipteran is a face fly.

[0058] In some embodiments, the phage and / or immunogenic substance has activity against bacteria associated with diseases in terrestrial animals or plants. In preferred embodiments, the phage and / or immunogenic substance has activity against bacteria associated with diseases in terrestrial animals. In preferred embodiments, the terrestrial animal or plant is a terrestrial animal. In particularly preferred embodiments, it is of the bovine or equine species, preferably bovine, such as female bovine or calf. In particularly preferred embodiments, the phage and / or immunogenic substance has activity against pathogens associated with the etiology of bovine infectious keratoconjunctivitis. In these embodiments, the phage and / or immunogenic substance can have activity against one or more of Moraxella bovis, Moraxella catarrhalis, Moraxella bovoculi, and Mycoplasma spp, preferably against Moraxella bovis. It will be understood that while the phage can bring about direct activity against bacteria, the immunogenic substance exerts its effect indirectly by inducing an immune response against the bacteria. Nevertheless, it is possible to say that both have activity against bacterial species, which will be understood by those skilled in the art. Accordingly, the methods, kits, and uses of the present invention provide an efficient way to deliver a phage or immunogenic substance to an animal to combat pathogens that cause disease by natural processes without the need for direct human intervention.

[0059] In some embodiments, the phage and / or immunogenic substance has activity against bacteria associated with diseases in terrestrial plants. In particularly preferred embodiments, the phage and / or immunogenic substance has activity against pathogens associated with the etiology of banana bacterial wilt. In these embodiments, the phage and / or immunogenic substance may have activity against Xanthomonas campestris. In other preferred embodiments, the phage and / or immunogenic substance has activity against pathogens associated with the etiology of moko disease. In these embodiments, the phage and / or immunogenic substance may have activity against Ralstonia solancearum. In some embodiments, the flying insect is a honeybee. In some embodiments, the flying insect is a fruit fly. In other preferred embodiments, the phage and / or immunogenic substance has activity against Xylella fastidiosa, Pseudomonas syringae or Pseudomonas savastanoi. In some embodiments, the flying insect is an olive fly. In another embodiment, the insect is a bee, preferably a bumblebee. Thus, the methods, kits and uses of the present invention provide an efficient way to deliver phages and / or immunogenic substances to plants to counter pathogens that cause disease by natural processes, by taking advantage of the natural behavior of insect species towards plants without the need for direct human intervention.

[0060] The present invention also relates to a method of transferring phages and / or immunogenic substances to flying insects, comprising combining an immunogenic substance with a flying insect attractant or placing the phage and / or immunogenic substance within the attraction radius of a flying insect attractant.

[0061] In some embodiments, the attractant comprises one or more of feces, pheromones, volatile substances, odoriferous substances, substances that can reflect or emit light within the visible electromagnetic spectrum, substances that can reflect or emit electromagnetic radiation outside the visible spectrum, or resting places to which flying insects are innately attracted. Resting places to which flying insects are innately attracted include, but are not limited to, rubbing brushes, nests, and the surfaces of terrestrial plants and animals. For example, flies are innately attracted to the face of cattle, and bees are innately attracted to flowers. It will be understood that a nutrient source under appropriate circumstances can also act as an attractant. Thus, the attractant preferably can comprise a combination of two substances (i.e., one that is a nutrient source and one that is not a nutrient source).

[0062] In preferred embodiments, the composition or kit comprises a flying insect attractant that includes a nutrient source for flying insects, or the method comprises combining (combining) a phage and / or an immunogenic substance with a nutrient source for flying insects. The nutrient source can be sugar, preferably glucose or dry icing sugar. In some embodiments, the combination of the phage and / or immunogenic substance and the nutrient source is provided in liquid form. In some embodiments, the combination of the phage and / or immunogenic substance and the nutrient source is provided in solid form.

[0063] The use of a flying insect attractant that includes a nutrient source is advantageous because it increases the likelihood of ingestion of the phage and / or immunogenic substance by the insect when the insect ingests the nutrient source. This is advantageous because it ensures that the phage and / or immunogenic substance is transferred to the digestive tract of the flying insect. This is particularly advantageous when the flying insect is a fly because the ejection of droplets of digestive tract contents is a normal feature of the feeding activity of flies. As a result, when the fly moves from ingestion of the nutrient source to feeding on an animal or plant host, a particularly high transmission of the phage and / or immunogenic substance to the animal or plant host occurs.

[0064] Preferably, the phage and / or immunogenic substance is combined with a flying insect attractant comprising a nutrient source to which flying insects tend to feed, thereby promoting the uptake of the substance by the flying insects.

[0065] Figure 1 shows an exemplary liquid nutrient source for flies, consisting of a 50:50 mixture of glucose syrup and water. Figures 2a and 2b show exemplary solid nutrient sources for flies, consisting of dry icing sugar. These figures show flies feeding on each of the nutrient sources, and that when a phage and / or immunogenic substance useful for the treatment or control of bacteria causing disease in terrestrial organisms is combined with the nutrient source or present within the attraction radius of the nutrient source, it is shown that it will be picked up by the flies, either by ingestion, inhalation or contamination of the limbs.

[0066] Preferred attractants include feces. The use of feces as an attractant is advantageous because insects come into frequent contact with feces throughout their life cycle. In particular, in the case of insect species that lay eggs in feces, including face flies, contact with feces occurs during each egg-laying cycle. Thus, when feces are used as an attractant, this provides an efficient way to ensure that the immunogenic substance is picked up by the dipterans and then transferred to the plant or animal host as the dipterans move. In a preferred embodiment, the feces include horse feces.

[0067] In particularly preferred embodiments, the feces include bovine feces. This is advantageous for the combined use with phages and / or immunogenic substances having activity against bacteria that cause infections in bovine animals (e.g., cows). Also, this is particularly advantageous for insects that lay eggs in bovine feces. Taking the face fly as an example, since female face flies lay eggs in bovine feces, if the phages and / or immunogenic substances are located inside or on the surface of bovine feces, the likelihood that the face fly will come into contact with the phages and / or immunogenic substances is very high. Also, female face flies have a particularly high rate of feeding on the blood, sweat, saliva, nasal mucus, feces, and tears of bovine and equine hosts. Therefore, a method including bovine feces has a particularly high likelihood of causing efficient transfer of immunogenic substances to bovine hosts by face flies.

[0068] In some embodiments, the phages and / or immunogenic substances are combined with flying insect attractants. This is advantageous, for example, in the case of attractants that include flying insect nutrient sources. Because it ensures that when the flying insects come into contact with the nutrient source or ingest a part of the nutrient source, the flying insects also ingest a part of the phages and / or immunogenic substances.

[0069] In other embodiments, the phages and / or immunogenic substances are placed within the attraction radius of the attractant. This is advantageous in situations where the phages and / or immunogenic substances cannot be combined with the attractant. Because that placement within the attraction radius of the attractant ensures that when the flying insects move towards the attractant, the phages and / or immunogenic substances are transferred to the flying insects.

[0070] The attraction radius will be understood to vary depending on the type, concentration and potency of the attracting factor and what the target animal or plant is. The attraction radius for a particular combination of attracting factor and target animal or plant is determined by routine experimentation. For example, the attraction radius can be determined by placing the attracting factor within a test chamber under normal atmospheric conditions and placing the target animal at increasing distances from the attracting factor to determine the maximum distance at which the target animal is attracted to the attracting factor. An example of a study determining the attraction radius of houseflies using a poultry farm as the attracting factor can be found in Nazni WA et al., Trop Biomed. 2005 Jun;22(1):53-61.

[0071] In some embodiments, the attraction radius can be within about 1 kilometer from the attracting factor. In some embodiments, the attraction radius can be within about 500 meters from the attracting factor. In some embodiments, the attraction radius can be within about 100 meters from the attracting factor. In some embodiments, the attraction radius can be within about 10 meters from the attracting factor. In some embodiments, the attraction radius can be within about 5 meters from the attracting factor. In some embodiments, the attraction radius can be within about 1 meter from the attracting factor.

[0072] It will be understood that the optimal concentration of the phage or immunogenic substance combined with the attracting factor can be determined using challenge studies that examine various concentrations. In some embodiments, the concentration of the phage is greater than 10 2 pfu / ml (i.e., greater than 10 2 pfu / ml), preferably greater than 10 3 pfu / ml, greater than 10 4 pfu / ml, or most preferably greater than 10 6 pfu / ml. In particularly preferred embodiments, the concentration of the phage is about 10 6 pfu / ml.

[0073] In some embodiments, the composition or kit includes two or more phages and / or immunogenic substances, or the use or method includes the use of two or more phages and / or immunogenic substances. In particularly preferred embodiments, the composition includes a combination of two or more phages. The use of combinations of phages and / or immunogenic substances is advantageous because bacteria have evolved numerous defense mechanisms against viruses and are known to develop resistance. Therefore, in order to minimize the impact of such resistance, it is desirable for the formulation to include a cocktail of two or more active substances.

[0074] In some embodiments, the composition of the present invention can be applied to a plastic substrate, which can be attached to a rubbing post for animals such as cows or horses, or placed within the attraction radius of the rubbing post. Alternatively, the plastic substrate can be in the form of a liquid or a liquid glass. If it is a liquid formulation, it can be coated, absorbed, or dispersed in other substances such as feces. In some embodiments, the composition of the present invention can be placed within a housing that protects the composition from external conditions such as weather conditions.

[0075] For example, the plastic substrate is attached to or placed within the attraction radius of a post, tree, or other natural or artificial structure that an animal such as a cow or horse can contact, for example rub against, or that is present in its environment. The structure is preferably a brush or a body buffer.

[0076] This is particularly advantageous in the case of cattle where face flies act as disease vectors. In particular, when a cow rubs its head against other structures such as posts, trees or brushes or body buffers, the face flies are driven away from the cow's face, so the flies remain in the air or seek another resting place until they can return to the cow's face. Thus, when a phage or immunogenic substance having activity against Moraxella bovis is combined with a fly attractant and placed at or near the rubbing location, their uptake by the flies is facilitated. For example, a face rubbing brush incorporating a fly resting place containing a reservoir of phage / immunogenic substance, in the presence or absence of a fly attractant, encourages the fly to land on the reservoir of phage / immunogenic substance when the fly is driven away from the cow's face. The fly then picks up the phage / immunogenic substance and, when the fly returns to the cow's face, transfers them to the cow's face, resulting in the treatment or prevention of bacterial infections such as IBK. This strategy is beneficial because it utilizes the natural behavior of cows and face flies and requires no direct human intervention.

[0077] Alternatively, applying a phage or immunogenic substance directly to a face rubbing brush, in the presence or absence of a flying insect attractant, enables the transfer of the phage / immunogenic substance to the cow's face without direct human intervention. Another way to transfer the phage / immunogenic substance to the cow's face without direct human intervention involves adding the phage / immunogenic substance to the edge of a suitably sized container containing a food source for the cow, in the presence or absence of a flying insect attractant. When the cow licks the food from such a container, the cow's muzzle contacts the edge of the container and the phage / immunogenic substance is transferred to the cow's face. In either of these cases, the flying insect can then transfer the phage / immunogenic substance from the animal's face to the area of the animal being treated (e.g., the eye). This is particularly advantageous when a flying insect attractant is present because it encourages the flying insect to pick up the phage from the cow's face and transfer it to the infection site.

[0078] In some embodiments, the compositions of the present invention can be incorporated into wearable articles for terrestrial animals, such as ear tags for cattle or horses. Alternatively, the compositions of the present invention can be attached directly or indirectly to the outer skin of an animal. In these embodiments, when the terrestrial animal is a grazing animal, the composition is not left in one place but is carried by the terrestrial animal. This is advantageous because the composition moves with the animal and continues to attract flying insects to the animal even as the animal moves. Ear tags are particularly advantageous because the composition is placed in the vicinity of the animal's face, allowing easy access to flying insects (e.g., face flies) attracted to the animal's face.

[0079] In all of the above embodiments, the final form of the composition is such that it allows flying insects to ingest and take up phages and / or immunogenic substances.

[0080] Examples Experiments were conducted to examine the ability of flying insects to transport bacteriophages from a bacteriophage source to a distant infection site. In these proof-of-concept experiments, three different flying insect vectors and two different phage / pathogen systems were used to demonstrate the reproducibility and utility of the concept. More specifically, experiments were conducted using face flies [Musca autumnalis], blowflies [Calliphora vomitoria], and bumblebees [Bombus terrestris audax] combined with a bacteriophage specific for Moraxella bovis or a bacteriophage specific for Salmonella enterica. In the case of tests involving Salmonella enterica, bacteriophages previously isolated against Salmonella enterica serotype Typhimurium SL1344 were used. These Salmonella enterica phages were isolated as follows: An aliquot of liquid collected from pig feces suspended in sterile aqueous buffer was exposed to a Salmonella enterica bacterial lawn growing on agar, and then, after incubation at 37°C for a certain period, plaques in the bacterial lawn were observed.

[0081] In the case of Moraxella bovis, bacteriophages were isolated as follows. Moraxella bovis was purchased from the National Collection of Type Cultures (NCTC 9426) and cultured in Brain Heart Infusion (BHI) supplemented with 5% horse blood. The bacteria were plated on BHI agar supplemented with 5% horse blood, and the plates were incubated at 37°C for 48 hours. Bacteriophages with lytic activity against Moraxella bovis were isolated from bovine rumen fluid and abattoir specimens of bovine nasal and lacrimal secretions in which bacteriophage lysates were concentrated. This was achieved by introducing the lysate onto plates of Moraxella bovis in blood-agar and further incubating, followed by observing bacterial lysis. The addition of triphenyltetrazolium chloride to the blood-agar plates facilitated the visualization of bacterial lysis by the bacteriophage. Plaques were picked for enrichment in Moraxella bovis / BHI broth supplemented with 5% horse blood. Four separate cultures were serially passaged to obtain a clonal population of bacteriophage, which was subjected to transmission electron microscopy (TEM). TEM images of four phages with activity against the isolated Moraxella bovis are shown in Figures 7, 8, 9, and 10. Figures 11a and 11b show clusters generated after serial passage of the isolate that produced the phage in Figure 10. It can be observed that all of the phages in the isolate had the same morphology, consistent with the clonal population after serial passage. Furthermore, it can be observed that each of the isolated phages had various morphologies. The phage in Figure 7 had a typical phage morphology, while the phage in Figure 10 had a significantly different morphology. The phages in Figures 8 and 9 had very similar morphologies to each other.

[0082] Example 1 A model system was established to experimentally examine the ability of flies to transport bacteriophages from a bacteriophage source to a distant infection site. In this model, the infection site was represented by the growth of Moraxella bovis on blood agar or Salmonella enterica on agar, and the flying insects used were face flies [Musca autumnalis] or blowflies [Calliphora vomitoria]. The face flies used in the experiment were widely captured specimens collected near cattle. In the case of blowflies, these were 3-day-old adult flies newly hatched from pupae incubated at 23 °C under a 12:12 light:dark cycle. Bacteriophages with lytic activity against Moraxella bovis or Salmonella enterica were added to a 20% sucrose solution that acts as a flying insect attractant. In the case of the Moraxella bovis experiment, a cocktail of 4 bacteriophages shown in Figures 7-10 was used. The concentration of the bacteriophage with lytic activity against Moraxella bovis was about 10 6 pfu / ml, and the concentration of the bacteriophage with lytic activity against Salmonella enterica was about 10 11 pfu / ml.

[0083] A sealed container containing the bacteriophage in the sucrose solution, 5 adult flies, and an agar plate with pathogen growth placed away from the sucrose solution was prepared. In this way, inside the sealed container, the flies fed on the sucrose solution, ingested the bacteriophage in the process, moved to the infection site on the distant agar plate, and here, the flies were able to feed further.

[0084] In addition, other sealed containers containing the following were prepared as controls.

[0085] 1. Control 1: Contains a 20% sucrose solution without bacteriophage, and contains flies and agar plates located separately with the growth of Moraxella bovis or Salmonella enterica.

[0086] 2. Control 2: Contains bacteriophage in a 20% sucrose solution and an agar plate with the growth of Moraxella bovis or Salmonella enterica located separately, but does not contain flies.

[0087] Table 1 summarizes this experimental setup for each of the Moraxella bovis and Salmonella enterica pathogen experiments.

[0088]

Table 1

[0089] Each sealed container was left at room temperature for 24 hours. Then the agar plates were taken out from each sealed container and analyzed to determine whether there was an effect on the growth of the pathogen.

[0090] Figure 3 shows each of the agar plates at the end of the Moraxella bovis experiment. In the experimental replicates of Control 1 (Figure 3A) and Control 2 (Figure 3B), it can be observed that the Moraxella bovis flora continued to grow without being hindered. In contrast, in the experimental replicate where both bacteriophage in the sucrose solution and flies were present (Figure 3C), areas where the growth of Moraxella bovis disappeared due to bacteriophage-mediated cell lysis were seen. The visualization of this lytic activity was enhanced by the addition of triphenyltetrazolium chloride.

[0091] Figure 4 shows each of the agar plates at the end of the experiment with Salmonella enterica. In the experimental replicates of Control 1 (Figure 4A) and Control 2 (Figure 4B), it can be observed that the bacterial flora of Salmonella enterica continued to grow without hindrance. In contrast, in the experimental replicates where both bacteriophage and flies were present in the sucrose solution (Figure 4C), areas where the growth of Salmonella enterica disappeared due to bacteriophage-mediated cell lysis were seen. The visualization of this lytic activity was enhanced by the addition of triphenyltetrazolium chloride.

[0092] The results of Example 1 demonstrate that flying insects can act as mediators for transferring bacteriophage from one location to another, and thus, in the presence of a bacterial infection, flying insects can be a means to improve the infection at that second location.

[0093] Example 2 A model system was established to experimentally examine the ability of flying insects to transport bacteriophage from a source of bacteriophage to the eyes of cows located at a distance. In this model, the flying insects used were face flies [Musca autumnalis]. The face flies used in the experiment were widely captured and maintained on a 20% sucrose solution under external conditions with a lighting pattern under a 12:12 light:dark cycle before conducting the experiment. The eyes of cows were ex vivo cow eyes derived from a slaughterhouse. A bacteriophage having lytic activity against Salmonella enterica was added to the 20% sucrose solution that acts as a flying insect attractant. The concentration of the bacteriophage having lytic activity against Salmonella enterica was approximately 10 11 pfu / ml.

[0094] A sealed container containing bacteriophage in a sucrose solution, five adult flies, and a bovine eye placed away from the sucrose solution was prepared. In this way, inside the sealed container, the flies were able to feed on the sucrose solution, ingest the bacteriophage in the process, and move towards the bovine eye.

[0095] Also, another sealed container containing the following was prepared as a control.

[0096] 1. Control 1: Contains a 20% sucrose solution without bacteriophage, flies, and an ex vivo bovine eye located away.

[0097] 2. Control 2: Contains bacteriophage in a 20% sucrose solution and an ex vivo bovine eye located away, but does not contain flies.

[0098] Table 2 summarizes this experimental setup.

[0099]

Table 2

[0100] Each sealed container was left at room temperature for 24 hours. Then, the bovine eyes were removed from each sealed container, the surface of the eyes was washed with 1 ml of sterile PBS solution, and 100 μl of it was transferred to the growing bacterial colonies of Salmonella enterica on agar and incubated at 37°C overnight. Then, the agar plates from each eye were analyzed to determine if there was an effect on the growth of the pathogen.

[0101] Figure 5 shows each of the agar plates at the end of the experiment with Moraxella bovis. In the agar plates of Control 1 (Figure 5A) and Control 2 (Figure 5B), the addition of the PBS effluent from the ex vivo bovine eye caused disruption of the pathogen flora, but no bacterial lysis zone was detected. In the experimental replicates where all variables were present (Figure 5C), the addition of the PBS effluent from the ex vivo bovine eye caused disruption of the pathogen flora, and furthermore, lytic activity was seen at the site where the liquid pooled.

[0102] The results of this experiment indicate that flies are capable of transferring bacteriophages from one location to the bovine eye, in which case they have the potential to suppress or eradicate pathogens of susceptible bacterial species.

[0103] Example 3 A model system was established to experimentally investigate the ability of flying insects to transport bacteriophages from a certain bacteriophage source to distantly located plant species. In this model, the flying insects used were bumblebees [Bombus terrestris audax]. The plant species was freshly cut flowers. A bacteriophage having lytic activity against Salmonella enterica was added to a 20% sucrose solution that acts as a flying insect attractant. The concentration of the bacteriophage having lytic activity against Salmonella enterica was about 10 11 pfu / ml.

[0104] A sealed container containing the bacteriophage in the sucrose solution, five bumblebees, and freshly cut flowers placed away from the sucrose solution was prepared. In this way, inside the sealed container, the bees were able to feed on the sucrose solution, ingest the bacteriophage in the process, and move to the flowers.

[0105] In addition, other sealed containers containing the following were prepared as controls.

[0106] 1. Control 1: It contains bees and flowers located at a distance, and contains a 20% sucrose solution without bacteriophage.

[0107] 2. Control 2: It contains bacteriophage in a 20% sucrose solution and flowers located at a distance, but does not contain bees.

[0108] Table 3 summarizes this experimental setup.

[0109]

Table 3

[0110] Each sealed container was left at room temperature for 24 hours. Then the flowers were taken out from each sealed container, washed with 1 ml of sterile PBS solution, and 100 μl of it was transferred to the growing bacterial colony of Salmonella enterica on agar and incubated at 37°C overnight. Then, in order to determine whether there was an effect on the growth of the pathogen, the agar plates from each flower were analyzed.

[0111] Figure 6 shows each of the agar plates at the end of the experiment. In the experimental replicates of Control 1 (Figure 6A) and Control 2 (Figure 6B) of Example 3, the addition of the PBS solution effluent from the flowers did not produce a visible area of bacterial lysis. In contrast, in the experimental replicates where all variables were present (Figure 6C), the addition of the PBS flower effluent to the growing bacterial colony of Salmonella enterica resulted in obvious lytic activity.

[0112] The results of this experiment indicate that vector insects are capable of transferring bacteriophages from one location to plant species, in which case they have the potential to suppress or eradicate pathogens of susceptible bacterial species. Furthermore, the results of this experiment show that bees are good vectors for spreading bacteriophages to flowering plants, which results in beneficial crop protection during plant pollination. Indeed, it would be reasonable to conclude that the addition of bacteriophages with activity against specific pathogens to commercial beehives (bee nests) reduces the presence of those pathogens on plants visited by bees and within the bee population in the nest.

[0113] Example 4 A model system was established to experimentally investigate the ability of flying insects to transport bacteria from a bacterial source to a distant location.

[0114] The bacteria were Moraxella bovis cultured in BHI medium supplemented with 5% horse blood. A portion of this broth was added to sucrose to prepare a 20% solution containing the bacteria. The flying insects used were blowflies [Calliphora vomitoria].

[0115] A sealed container containing the bacteria in the sucrose solution, five blowflies, and a sterile blood-agar plate located away from the sucrose solution was prepared. In this way, within the sealed container, the flies were able to feed on the sucrose solution, ingest the bacteria while doing so, and move to the sterile agar plate.

[0116] Also, as a control, another sealed container was prepared. This contained the bacteria in the sucrose solution and a sterile blood-agar plate located away from the sucrose solution but did not contain blowflies.

[0117]

Table 4

[0118] Each sealed container was left at room temperature for 24 hours. Subsequently, the blood-agar plates were taken out and incubated overnight at 37°C. The blood-agar plates taken out from the sealed containers that did not contain flies showed no bacterial growth. However, the blood-agar plates taken out from the sealed containers that contained flies produced visible bacterial colonies.

[0119] This experiment demonstrates that flies can transfer bacteria from one location to a distant location. Bacteria are generally understood to be immunogenic, and in fact, vaccines can be developed from inactivated or killed bacteria, or from bacterial components such as cell membrane proteins. Therefore, it is suggested that flying insects can transport immunogenic substances from one location to another, and it is generally understood that supplying such substances in combination with fly attractants and vector flying insects can be utilized to spread those immunogenic substances to animals and plants, etc.

[0120] Numbered embodiments of the present disclosure 1. (i) A phage and / or an immunogenic substance, and (ii) A flying insect attractant A composition for the treatment or prevention of bacterial diseases in terrestrial plants or animals, comprising the same.

[0121] 2. (i) Combining (putting together) a phage and / or an immunogenic substance with a flying insect attractant, or (ii) Placing a phage or an immunogenic substance within the attraction radius of a flying insect attractant A method of attracting flying insects to a phage and / or an immunogenic substance, comprising the same.

[0122] 3. The method according to embodiment 2, wherein when a flying insect moves to or contacts an attractant, the phage and / or the immunogenic substance is transferred to the flying insect.

[0123] 4. (i) A phage and / or an immunogenic substance, and (ii) Flying insect attracting factor A kit for the treatment or prevention of bacterial diseases in terrestrial plants or animals, comprising .

[0124] 5. Use of flying insects in a method of transferring phages and / or immunogenic substances to terrestrial animals or plants, wherein the flying insects act as carriers of the phages and / or immunogenic substances.

[0125] 6. Use according to embodiment 5, wherein the phages and / or immunogenic substances have been transferred to the flying insects by the method according to embodiment 2 or 3.

[0126] 7. A method for the treatment or prevention of diseases in terrestrial animals or plants, comprising transferring phages and / or immunogenic substances to the terrestrial animals or plants using flying insects, wherein the flying insects act as carriers of the phages and / or immunogenic substances.

[0127] 8. Method according to embodiment 7, wherein the phages and / or immunogenic substances have been transferred to the flying insects by the method according to embodiment 2 or 3.

[0128] 9. The composition according to embodiment 1, the method according to embodiment 2, 3 or 8, the kit according to embodiment 4, or the use according to embodiment 6, wherein the flying insect attracting factor comprises a flying insect nutrient source.

[0129] 10. The composition, method, kit or use according to embodiment 9, wherein the flying insect attracting factor comprises a flying insect nutrient source combined with a second substance.

[0130] 11. The composition according to embodiment 1, 9 or 10, the method according to embodiment 2, 3, 7, 8, 9 or 10, the kit according to embodiment 4, 9 or 10, or the use according to embodiment 5, 6, 9 or 10, wherein the phages and / or immunogenic substances have activity against bacteria associated with diseases in terrestrial animals or plants.

[0131] 12. The composition or kit according to any one of the preceding embodiments, wherein the composition comprises two or more immunogenic substances and / or bacteriophages, preferably two or more bacteriophages.

[0132] 13. The method or use according to any one of the preceding embodiments, wherein two or more immunogenic substances and / or bacteriophages are used.

[0133] 14. The composition or kit according to any one of the preceding embodiments, which comprises a bacteriophage, or the method or use according to any one of the preceding embodiments, wherein a bacteriophage is used.

[0134] 15. The composition, method, kit or use according to embodiment 14, wherein the bacteriophage is stabilized by immobilization on a substrate, preferably, the bacteriophage is immobilized via its head.

[0135] 16. The composition, method, kit or use according to any one of the preceding embodiments, wherein the immunogenic substance comprises a denatured bacterial antigen.

[0136] 17. The composition, method, kit or use according to any one of the preceding embodiments, wherein the flying insect is a fly, preferably a dipteran, preferably, the flying insect is a face fly, an Australian bush fly or a buffalo fly, most preferably a face fly.

[0137] 18. The composition, method, kit or use according to any one of the preceding embodiments, wherein the flying insect is a wasp, a fruit fly or an olive fly.

[0138] 19. The composition, method, kit or use according to any one of the preceding embodiments, wherein the phage and / or immunogenic substance has activity against bacteria associated with diseases in terrestrial animals, preferably, here, the animal is a bovine or equine animal, more preferably a bovine animal.

[0139] 20. The composition, method, kit or use according to any one of the preceding embodiments, wherein the phage and / or immunogenic substance has activity against bacteria associated with diseases in terrestrial plants, preferably wherein the plant is a banana tree or an olive tree.

[0140] 21. The composition, method, kit or use according to any one of the preceding embodiments, wherein the phage and / or immunogenic substance has activity against a pathogen involved in the etiology of bovine infectious keratoconjunctivitis. 22. The composition, method, kit or use according to any one of the preceding embodiments, wherein the phage and / or immunogenic substance has activity against Moraxella bovis.

[0141] 23. The composition, method, kit or use according to any one of the preceding embodiments, wherein the phage and / or immunogenic substance has activity against Moraxella catarrhalis, Moraxella bovoculi, Mycoplasma spp, Xanthomonas campestris, Ralstonia solancearum, Xylella fastidiosa and / or Pseudomonas savastanoi.

[0142] 24. The composition, method, kit or use according to any one of the preceding embodiments, wherein a flying insect attractant is present, preferably wherein the attractant comprises one or more of feces, pheromones, volatile substances, odoriferous substances, substances capable of reflecting or emitting light within the visible electromagnetic spectrum, substances capable of reflecting or emitting electromagnetic radiation outside the visible spectrum, or resting places to which flying insects are innately attracted.

[0143] 25. The composition, method, kit or use according to any one of the preceding embodiments, wherein the attracting agent comprises one or more natural and / or synthetic substances, preferably one or more substances selected from pheromones, volatile substances and odoriferous substances, preferably volatile substances.

[0144] 26. The composition, method, kit or use according to any one of the preceding embodiments, wherein the attracting agent comprises a substance that reflects or emits light within the visible electromagnetic spectrum, or a substance that reflects or emits electromagnetic radiation outside the visible spectrum.

[0145] 27. The composition, method, kit or use according to any one of the preceding embodiments, wherein the flying insect attracting agent comprises a flying insect nutrient source, wherein the nutrient source is sugar.

[0146] 28. The composition, method, kit or use according to any one of the preceding embodiments, wherein the attracting agent and the phage and / or immunogenic substance are present in solid or liquid form.

[0147] 29. The composition, method, kit or use according to any one of the preceding embodiments, wherein the attracting agent comprises feces, preferably bovine feces.

[0148] 30. The composition, method, kit or use according to any one of the preceding embodiments, wherein the phage and / or immunogenic substance is mixed with the attracting agent.

[0149] 31. An article comprising the composition according to any one of the preceding embodiments.

[0150] 32. The article according to embodiment 31, wherein the article is wearable by a terrestrial animal.

[0151] 33. The article according to embodiment 32, wherein the article is an ear tag for a cow or a horse.

[0152] 34. A method of applying the composition according to any one of the preceding embodiments to the skin of a terrestrial animal.

[0153] This disclosure provides exemplary embodiments of the present invention and is not intended to be limiting. It will be understood that various other modifications and variations of the present invention are possible.

Claims

**Claim 1** (i) combining a phage and / or an immunogenic substance with an attractant for flying insects, or (ii) placing a phage and / or an immunogenic substance within the attraction radius of an attractant for flying insects A method of transferring a phage and / or an immunogenic substance to flying insects, comprising attracting flying insects to the phage and / or the immunogenic substance, wherein the phage and / or the immunogenic substance is transferred to the flying insects when the flying insects move towards or come into contact with the attractant. **Claim 2** Use of flying insects in a method of transferring a phage and / or an immunogenic substance to a terrestrial animal or plant, wherein the flying insects act as carriers of the phage and / or the immunogenic substance. **Claim 3** Use according to claim 2, wherein the phage and / or the immunogenic substance has been transferred to the flying insects by the method according to claim 1. **Claim 4** A method for treating or preventing a disease in a terrestrial animal or plant, comprising transferring an effective amount of a phage and / or an immunogenic substance to the terrestrial animal or plant using flying insects, wherein the flying insects act as carriers of the phage and / or the immunogenic substance. **Claim 5** The method according to claim 4, wherein the phage and / or the immunogenic substance has been transferred to the flying insects by the method according to claim 1. **Claim 6** (i) a phage and / or an immunogenic substance, and (ii) an attractant for flying insects selected from feces, preferably bovine feces, pheromones, volatile substances, odoriferous substances, substances capable of reflecting or emitting light within the visible electromagnetic spectrum, substances capable of reflecting or emitting electromagnetic radiation outside the visible spectrum, nutrient sources for flying insects, and combinations thereof A composition for treating or preventing a bacterial disease in a terrestrial plant or animal. **Claim 7** (i) a phage and / or an immunogenic substance, and (ii) an attractant for flying insects selected from feces, preferably bovine feces, pheromones, volatile substances, odoriferous substances, substances capable of reflecting or emitting light within the visible electromagnetic spectrum, substances capable of reflecting or emitting electromagnetic radiation outside the visible spectrum, natural resting places to which flying insects are attracted, nutrient sources for flying insects, and combinations thereof A kit for treating or preventing a bacterial disease in a terrestrial plant or animal. **Claim 8** The flying insect attracting factor contains a flying insect nutrient source, and optionally, the nutrient source may be sugar. Preferably, the flying insect attracting factor contains a flying insect nutrient source combined with a second substance. The composition according to claim 6, the method according to claim 1 or 6, the kit according to claim 7, or the use according to claim 2 or 3.

9. The phage and / or immunogenic substance has activity against bacteria associated with diseases in terrestrial animals or plants. The composition according to claim 6 or 8, the method according to claim 1, 5 or 6, the kit according to claim 7 or 8, or the use according to claim 2, 3 or 8.

10. The composition or kit according to any one of the preceding claims, wherein the composition or kit contains two or more immunogenic substances and / or bacteriophages, preferably one or more bacteriophages.

11. The method or use according to any one of the preceding claims, using two or more immunogenic substances and / or one or more bacteriophages.

12. The composition or kit according to any one of the preceding claims, or the method or use according to any one of the preceding claims, containing a bacteriophage, wherein the bacteriophage is used, preferably, the bacteriophage is stabilized by immobilization on a substrate, preferably, the bacteriophage is immobilized via its head. Composition or kit, or method or use.

13. The composition, method, kit or use according to any one of the preceding claims, wherein the immunogenic substance contains a bacterial antigen.

14. The flying insect is a fly, preferably a dipteran, preferably, the flying insect is a face fly, an Australian bush fly, a buffalo fly, a stable fly or a house fly, most preferably a face fly, or the flying insect is a wasp, an oriental fruit fly or an olive fruit fly, a flower fly, a black fly or a bee. The composition, method, kit or use according to any one of the preceding claims.

15. The phage and / or immunogenic substance has activity against bacteria associated with diseases in terrestrial animals, preferably where the animal is a bovine or equine animal, more preferably a bovine animal, and / or the phage and / or immunogenic substance has activity against bacteria associated with diseases in terrestrial plants, preferably where the plant is a fruit-bearing plant, most preferably where the plant is a banana tree, olive tree or kiwifruit plant, and / or the phage and / or immunogenic substance has activity against pathogens involved in the etiology of bovine infectious keratoconjunctivitis, and / or the phage and / or immunogenic substance has activity against Moraxella bovis, and / or the phage and / or immunogenic substance has activity against Moraxella catarrhalis, Moraxella bovoculi, Mycoplasma spp, Xanthomonas campestris, Ralstonia solanacearum, Xylella fastidiosa, Pseudomonas savastanoi and / or Pseudomonas syringae, The composition, method, kit or use according to any one of the preceding claims.

16. The flying insect attractant comprises one or more of feces, preferably bovine feces, pheromones, volatile substances, odoriferous substances, substances capable of reflecting or emitting light within the visible electromagnetic spectrum, substances capable of reflecting or emitting electromagnetic radiation outside the visible spectrum, resting places to which flying insects are innately attracted or nutritional sources of flying insects. The method or use according to any one of the preceding claims.

17. The attractant and the phage and / or immunogenic substance are present in solid or liquid form, and / or the phage and / or immunogenic substance is mixed with the attractant. The composition, method, kit or use according to any one of the preceding claims.

18. Preferably, the article is wearable by a terrestrial animal, most preferably, the article is an ear tag, and optionally, it may be an ear tag of a cow or a horse. An article comprising the composition according to any one of the preceding claims.

19. A method of applying the composition according to any one of the preceding claims to the skin of a terrestrial animal.

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