AU2021107570A4 - Probiotic compositions - Google Patents

Abstract

The invention relates to a composition for treating and/or inhibiting an infestation of a subject by an ectoparasite. The composition comprises bacterial organisms which are active against the ectoparasite and bacterial organisms which exhibit chitinase activity. These two types of bacterial organisms may be different. The invention also provides a method for treating and/or inhibiting an infestation of a subject by an ectoparasite.

Description

PROBIOT IC COMPOSITIONS

Field

[0001] The invention relates to probiotic compositions and methods of their use.

Background

[0002] This application claims priority from Australian provisional patent application no. 2020901107 filed on 8 April 2020, the entire contents of which are incorporated herein by reference.

[0003] A multitude of species of ectoparasitic mites, flies, ticks, lice and fleas impact the economic success of agricultural farming industry in many areas of the world. These ectoparasites may cause disease, spread disease, lower production yields, adversely affect animal welfare and/or reduce value, whilst also increasing costs, especially through costs of control. Failure to control ectoparasites is a significant hurdle for the agricultural industry to overcome in order to improve quality, value, animal welfare and sustainability.

[0004] Currently there are limited options for controlling such pests. Agrichemicals are the most prevalent option today for treating ectoparasite infestations. However, resistance to such chemicals is increasing worldwide, with reducing pesticide efficacy over time and few if any new agrichemical classes under development to replace the current cohort. Vaccines are still not a viable option, even after considerable research effort over the past few decades. Hence, a new method is needed to combat ectoparasite infestations and to protect animals from subsequent infestations.

[0005] Veterinary probiotics have previously been used to combat internal parasites in animals. Over 25 such products are available, claiming to modulate harmful and potentially pathogenic bacterial populations in the gastro-intestinal system. Indeed, it is internal use with which probiotics are most commonly associated. There is no literature precedent for the use of probiotics for the skin surface of animals.

[0006] The ecosystem on the animal skin commonly comprises a layer of discarded skin cells, cellular debris and secretions such as sweat, mucous or other fluids and dust that accumulate on the skin. This ecosystem is commonly protected by hair, fur, wool or feathers in which the natural saprophytic skin microbiota exist. While the composition of this ecosystem will vary between food-producing animal species, breeds and strains, the microbiome is largely comprised of species belonging to the Proteobacteria,Firmicutes,Bacteroidetes, Actinobacteria, Cyanobacteria,and Fusobacteria.

[0007] While this microbiome may in some instances provide a level of protection from infection by bacterial and fungal pathogens, and against infestation by ectoparasites, companion and food-producing host animals are nonetheless subject to infection and infestation and control of such infection and/or infestation is a hitherto unmet need.

[0008] The invention herein disclosed includes a composition that is capable of controlling infestation of companion and food-producing animals by ectoparasites.

Summary of the Invention

[0009] In a first aspect of the invention there is provided a composition for treating and/or inhibiting an infestation of a subject by an ectoparasite, said composition comprising: a) bacterial organisms which are active against the ectoparasite; and b) bacterial organisms which exhibit chitinase activity.

[00010] In the present specification, "bacterial organisms" may refer to bacterial spores, viable bacteria or to a combination of spores and viable bacteria. Thus, for example, where the composition is said to comprise bacterial organisms which are active against the ectoparasite, the composition may comprise viable bacteria which are active against the ectoparasite, spores of such bacteria or both viable bacteria which are active against the ectoparasite and spores thereof

[00011] There may be separate bacterial organisms which are active against the ectoparasite and which exhibit chitinase activity, or there may be a single type of bacterial organism which exhibits both activities. Thus a) and b) may be different, or they may be the same.

[00012] The following options may be used in conjunction with the first aspect, either individually or in any suitable combination.

[00013] The composition may additionally comprise: c) bacterial organisms capable of stimulating the immune system of the subject. Bacterial organisms c) may be different to a) and or to b), or may be the same as one or other thereof

[00014] At least one of a) and b) may be isolated from, or may be found in, soil or dust, or may be isolated from, or may be found in, a plant, a rock or sand. In the event that bacterial organisms c) are also present in the composition, these may be isolated from, or may be found in, soil or dust, or a plant, a rock or sand. In this context, "found in" indicates that the organisms occur naturally in the specified matrix. Thus, for example, if organisms a) are found in soil, they occur naturally in soil.

[00015] Each of the bacterial organisms may have been cultured so as to exhibit the required activity. In this context, and throughout this specification, "the bacterial organisms" refers to bacterial organisms a) and b) (ie. bacterial organisms that are active against the ectoparasite and bacterial organisms which exhibit chitinase activity respectively) and, if present, bacterial organisms c) (i.e. bacterial organisms that are capable of stimulating the immune system of the subject).

[00016] The composition may be formulated as a liquid suitable for external application to the subject. It may be formulated as a spray-on, dip, a pour-on, a rub-on or a spot-on. It may be formulated as a paint. It may be oil-based or it may be aqueous. Alternatively, it may be formulated as a powder or a dust.

[00017] The composition may additionally comprise an organic solid and/or an inorganic solid. Each of these, independently, may be in the form of a powder. The inorganic solid may be, for example, diatomaceous earth, silica, clay, talc or some other inorganic diluent generally regarded as safe (GRAS), or may be some combination of such solids. The organic solid may be, for example, lactose and/or milk powder, another organic polymeric or monomeric diuent, cellulose, starch, protein solid, lactose, whey, glucose, xylose, an amino acids or a fat, or may be some combination of such solids. It may be a sorbent material, i.e. it may be adsorbent and/or absorbent.

[00018] The infestation to be treated and/or inhibited may be selected from the group consisting of ticks (acari), mites, lice, fleas, biting flies and other insects. It may be a tick infestation. It may be an infestation of cattle tick Rhipicephalus (Boophilus) microplus (Australis).

[00019] The composition may additionally comprise one or more micronutrients, trace element minerals, amino acids, sugars and/or vitamins. It may additionally comprise at least one additive selected from the group consisting of an emulsifier, a spreading agent, an antioxidant and a growth medium. It may comprise a prebiotic.

[00020] The composition may consist essentially of, or may consist of; microbial organisms and GRAS substances.

[00021] The bacterial organisms (ie. bacterial organisms a), b) and, if present, c)) may each, independently, be present in the composition in a concentration of from 102 to 1014 cfu/g. In the event that the bacterial organisms comprise both viable bacteria and spores thereof, the concentration should be taken to be the sum of the concentrations of the bacteria and the spores thereof

[00022] In some forms, the only bacterial organisms present in the composition are a) type and b) type bacterial organisms, and optionally c) type bacterial organisms. Thus the composition may consist essentially of a) type and b) type bacterial organisms, and non-bacterial matter such as carriers and fillers. It may consist essentially of a) type bacterial organisms, b) type bacterial organisms, c) type bacterial organisms, and non-bacterial matter such as carriers and fillers.

[00023] In one embodiment there is provided a composition for treating and/or inhibiting an infestation of a subject by an ectoparasite, said composition comprising: a) bacterial organisms which are active against the ectoparasite; b) bacterial organisms which exhibit chitinase activity; and c) bacterial organisms capable of stimulating the immune system of the subject. In this embodiment, a), b) and c) may all be different.

[00024] In another embodiment there is provided a composition for treating and/or inhibiting an infestation of a subject by an ectoparasite, said composition comprising: a) bacterial organisms which are active against the ectoparasite; b) bacterial organisms which exhibit chitinase activity; and c) bacterial organisms capable of stimulating the immune system of the subject, vegetable oil (e.g. olive oil) and diatomaceous earth. In this embodiment, a), b) and c) may all be different.

[00025] In another embodiment there is provided a composition for treating and/or inhibiting an infestation of a subject by an ectoparasite, said composition consisting essentially of a) bacterial organisms which are active against the ectoparasite; b) bacterial organisms which exhibit chitinase activity; and GRAS components. In this embodiment, a) and b) may be different.

[00026] In another embodiment there is provided a composition for treating and/or inhibiting an infestation of a subject by an ectoparasite, said composition consisting essentially of a) bacterial organisms which are active against the ectoparasite; b) bacterial organisms which exhibit chitinase activity; c) bacterial organisms capable of stimulating the immune system of the subject; and GRAS components. In this embodiment, a), b) and c) may all be different.

[00027] In a second aspect of the invention there is provided a method for treating and/or inhibiting an infestation of a subject by an ectoparasite, said method comprising applying to at least a portion of an external surface of the subject: a) bacterial organisms which are active against the ectoparasite; and b) bacterial organisms which exhibit chitinase activity. In this aspect, a) and b) may be different or they may be the same.

[00028] The following options may be used in conjunction with the second aspect, either individually or in any suitable combination.

[00029] The method may additionally comprise applying to said external surface: c) bacterial organisms capable of stimulating the immune system of the subject. Bacterial organisms c) may be different to a) and or to b), or may be the same as one or other thereof

[00030] The bacterial organisms (ie. bacterial organisms a) and b) and, if present, c)) may be applied simultaneously. They may be formulated together in a composition and the composition may be applied to the subject. The method may comprise the step of isolating the bacterial organisms. It may comprise culturing the bacterial organisms separately. The method may comprise the step of formulating the bacterial organisms into one or more compositions suitable for application to the external surface of the subject. The steps of isolating, culturing and formulating, if performed, may be performed before the step of applying.

[00031] The step of applying may comprise one or more of spraying, jetting, dipping, pouring, spotting, painting, rubbing and dusting. It may comprise a combination of any two or more of these.

[00032] The ectoparasite may be selected from the group consisting of ticks, mites, lice, fleas and biting flies. It may be ticks. It may be cattle tick Rhipicephalus (Boophilus) microplus (Australis).

[00033] In an embodiment there is provided a method for treating and/or inhibiting an infestation of a subject by an ectoparasite, said method comprising applying to at least a portion of an external surface of the subject: a) bacterial organisms which are active against the ectoparasite; b) bacterial organisms which exhibit chitinase activity; and c) bacterial organisms capable of stimulating the immune system of the subject. In this embodiment, bacterial organisms a), b) and c) may all be different.

[00034] In another embodiment there is provided a method for treating and/or inhibiting an infestation of a subject by an ectoparasite, said method comprising applying to at least a portion of an external surface of the subject a composition according to the first aspect.

[00035] In another embodiment there is provided a method for treating and/or inhibiting an infestation of a subject by an ectoparasite, said method comprising separately culturing: a) bacterial organisms which are active against the ectoparasite; b) bacterial organisms which exhibit chitinase activity and optionally c) bacterial organisms capable of stimulating the immune system of the subject; formulating the bacterial organisms into a composition and applying the composition to at least a portion of an external surface of the subject. In this embodiment, bacterial organisms a), b) and, if used, c) may all be different.

[00036] The bacterial organisms may be applied to the subject once only in the lifetime of the subject. Alternatively, they may be applied to the subject periodically.

[00037] In a third aspect of the invention there is provided use of bacterial organisms for treating and/or inhibiting an infestation of a subject by an ectoparasite, said bacterial organisms being: a) bacterial organisms which are active against the ectoparasite; and b) bacterial organisms which exhibit chitinase activity. Bacterial organisms a) and b) may be different or they may be the same.

[00038] The use may also comprise use of c) bacterial organisms capable of stimulating the immune system of the subject. Bacterial organism c) may be different to one or both of bacterial organisms a) and b).

[00039] In an embodiment the use comprises use of a composition according to the first aspect for treating and/or inhibiting an infestation of a subject by an ectoparasite.

[00040] In a fourth aspect of the invention there is provided use of bacterial organisms for the manufacture of a medicament effective in treating and/or inhibiting an infestation of a subject by an ectoparasite, said bacterial organisms being: a) bacterial organisms which are active against the ectoparasite; and b) bacterial organisms which exhibit chitinase activity. Bacterial organisms a) and b) may be different. The subject may be a non-human subject. It may be a non-human mammal or a bird. It may be a companion animal or may be a livestock animal.

[00041] The use of the fourth aspect may also comprise use of c) bacterial organism capable of stimulating the immune system of the subject. Bacterial organism c) may be different to one or both of bacterial organisms a) and b).

[00042] In an embodiment there is provided use of bacterial organisms for the manufacture of a composition according to the first aspect, said bacterial organisms being: a) bacterial organisms which are active against the ectoparasite; b) bacterial organisms which exhibit chitinase activity; and optionally c) bacterial organisms capable of stimulating the immune system of the subject. In this embodiment, bacterial organisms a), b) and, if used, c) may all be different.

[00043] In a fifth aspect of the invention there is provided a process for making a composition for treating and/or inhibiting an infestation of a subject by an ectoparasite, said process comprising combining a) bacterial organisms which are active against the ectoparasite; and b) bacterial organisms which exhibit chitinase activity. Bacterial organisms a) and b) may be different.

[00044] The following options may be used in conjunction with the fifth aspect, either individually or in any suitable combination.

[00045] The process may comprise the step of culturing the bacterial organisms a) and b) so as to exhibit the required activity, said culturing being conducted before the step of combining.

[00046] The step of combining may additionally comprise combining c) bacterial organism capable of stimulating the immune system of the subject with bacterial organisms a) and b). Bacterial organism c) may be different to one or both of bacterial organisms a) and b). It may comprise combining the bacterial organisms a) and b) with a carrier. The carrier may be aqueous or it may be an oil.

[00047] The process may additionally comprise combining the bacterial organisms a) and b) with an organic solid and/or an inorganic solid.

Brief Description of Drawings

[00048] Preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying figures, wherein:

[00049] Fig. 1 shows plates in the chitinase activity test of Example 2.

Definitions

[00050] The term "ectoparasite" as used herein refers to a parasite that lives entirely or at least for part of the time, on an external surface of a host organism.

[00051] The term "GRAS substance" as used herein refers to substances on the GRAS list. The GRAS list is the list provided by the US FDA of substances which are Generally Regarded As Safe (https-//www.fda.gov/food/food-ingredients-packaging/generally-recognized-safe-gras).

[00052] The term "non-pathogenic" as used herein refers to a microorganism that may colonise a host species commensally, adventitiously or symbiotically, but does not cause disease in the host, even if it may be capable of causing disease in another species.

[00053] The term "probiotic" in reference to a composition indicates the presence of microorganisms, particularly bacteria, which are beneficial to the subject to which the composition is administered or applied. In general probiotic compositions will contain only non pathogenic microorganisms.

[00054] The term "bacterial organism" as used herein encompasses bacterial spores and the viable bacteria which develop therefrom. Where mention is made of bacterial organisms which have a particular property (e.g. exhibiting chitinase activity), this may refer to the spores having that property, or to the viable bacteria having that property, or to both the spores and the viable bacteria having that property.

[00055] As used herein, the term "comprising" means "including". Variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings. As used herein, the terms "including" and "comprising" are non-exclusive. As used herein, the terms "including" and "comprising" do not imply that the specified integer(s) represent a major part of the whole. The term "consists essentially of' means that the specified integers are the only integers intentionally present, although other integers, generally minor, may incidentally be present. The term "consists of' means that the specified integers are the only integers present.

[00056] The term "may" as used herein encompasses both positive and negative options. Thus, for example, the phrase "A may be B" encompasses the alternatives "A is B" and "A is not B".

Description of Embodiments

[00057] The present invention relates to a composition for the treatment and/or inhibition of an ectoparasitic infestation on a subject and to a method for treating and/or inhibiting such infestations. The infestation may be caused by any pest that resides, for at least a portion of its lifecycle, on an external surface of a subject (i.e. an ectoparasite). The composition of the present invention may be a probiotic, which comprises bacteria beneficial to the subject. The inventors have surprisingly found that applying bacterial organisms with a particular combination ofproperties can treat, prevent or inhibit infestation by ectoparasites.

[00058] The combination of features which has been found to be effective in such treatment is: a) activity against the ectoparasite; and b) chitinase activity.

[00059] A third feature which may also be beneficial is c) immune stimulatory activity.

[00060] For convenience, in this specification, the term "a) type bacterial organisms" refers to bacterial organisms exhibiting activity against the ectoparasite and "a) type activity" refers to activity against the ectoparasite. Corresponding definitions apply to b) type bacterial organisms and activity (chitinase activity) and c) type bacterial organisms and activity (immune stimulatory activity).

[00061] It will be understood that these features may be exhibited by a single strain of bacterial organism, or a particular strain of bacterial organism may exhibit two or even all three of these features. Thus in some embodiments the composition of the invention will comprise a single bacterial strain, whereas in other embodiments there may be two or more strains. The composition of the present invention comprises a) type and b) type bacterial organisms. These may be the same, or they may be different. In the case that they are different, there are two types of bacterial organisms, one having a) type activity and the other having b) type activity. In some embodiments, the a) type bacterial organisms have both a) and b) type activity, as well as there being b) type bacterial organisms (having b) type activity), and/or the b) type bacterial organisms may have both a) type and b) type activity, provided that the a) type bacterial organisms (having a) type activity) are also present. Similarly, if c) type bacterial organisms are present (having c) type activity), they may also have a) type and/or b) type activity provided that separate a) type and b) type bacterial organisms are also present. Thus if a) and b) type bacterial organisms are present, there are at least two types of bacterial organism, at least one of which has, at least, a) type activity and at least one of which has, at least, b) type activity.

[00062] It will also be understood that there may be more than one strain of bacterial organism which exhibit a particular activity. For example there may be two or more a) type bacterial organisms present. In most compositions according to the invention, there will be no more than different strains of bacterial organism, commonly 1to 10, 3 to 10, 1 to 5, or 5 to 10 strains.

[00063] The method of the invention relates to provision of bacterial organisms to the outer surface, e.g. skin, fur, hide or feathers (or any two or more of these), of the subject to be treated. Similar considerations apply equally to the method of the invention as are described above for the composition in respect of the nature of the bacterial organisms. According to the method, it is possible to apply the different bacterial organisms simultaneously in a single composition, simultaneously in more than one composition (e.g. one composition having a) type bacterial organisms, a separate composition having b) type bacterial organisms and optionally a third composition having c) type bacterial organisms), or even non-concurrent application of more than one composition. In this last case, it is preferably that the applications be within a relatively short period of time, e.g. within 1 hour, or within 30, 20 or 10 minutes.

[00064] The requirement for activity against the ectoparasite (i.e. a) type activity) is in order to kill or at least prevent proliferation of the ectoparasite. The requirement for chitinase activity (ie.

b) type activity) provides the ability to disable organisms having chitin as a structural component. Many ectoparasites have chitin as a component of their exoskeleton. Therefore chitinase-active bacteria can attack many of the ectoparasites targeted by the present invention. Further, immune stimulatory activity (ie. c) type activity) provides the ability to stimulate the subject's own immune system to act against invading ectoparasite and may therefore also be beneficial. Whereas the stimulation of an immune response is well known against internal microorganisms, its use externally is less well recognised. The inventors have now found that the combination of the separate activities a) and b), and optionally also c), outlined above, can provide effective treatment of ectoparasitic infestations as well as inhibiting or preventing them. It is thought that application of bacterial organisms having the activities specified earlier can lead to colonisation of the skin, feathers, fur and/or hide by these bacterial organisms. In doing so, it is thought that they can stimulate the subject's natural immune response and can also directly inhibit the ectoparasites. The presence of those bacterial organisms can therefore deter infestation. It is further thought that this colonisation is capable of providing long lasting inhibition of the ectoparasites as the a) type, b) type and optionally c) type bacterial organisms may remain on the subject for a long period.

[00065] The bacterial organisms in the composition of the invention are preferably non pathogenic to the subject to which the bacterial organisms are applied. Some or all of the bacterial organisms may be isolated from soil, or a soil-based material, such as dust. They may occur naturally in that environment. They may be grown in a suitable growth medium and under suitable growth conditions to enable the required activities to be evident before they are formulated in the composition. As will be described in greater detail below, and with reference to the examples, it is believed that the bacterial organisms colonise the external surface of a subject and together effect the treatment of and resistance to, ectoparasite infestations on the treated subject.

[00066] One or more of the bacterial organisms of the invention may be saprophytic. As these use non-living tissue and other organic matter for sustenance, they are generally non-pathogenic to healthy animals and birds. They may however proliferate on the outer surface of an animal or bird, using dead skin and other detritus that are naturally present on that surface as nutrients.

[00067] At least one strain of bacterial organism in the present invention is a) type, ie. it is active against the ectoparasite. By "active", it is meant that the bacteria selectively contribute, in some way, to reducing, removing, preventing and/or inhibiting infestation by the ectoparasite without harming the host subject. The activity of the bacterial organisms may be due to the production of a compound that repels the ectoparasite. As used herein, 'repels' means to alter the local environment of the external surface of the subject, in a manner that is non-lethal to the ectoparasite, but which discourages the ectoparasite from remaining on the host and/or from infesting the external surface of the subject in the first instance. For example, the bacterial organisms may make the local environment of the external surface hostile to the ectoparasite, resulting in the ectoparasite leaving the surface of the subject, and/or resisting further infestations due to the hostile environment. The a) type bacteria may comprise at least one Bacillus species, for example B. cereus, B. subtilis, B. thuringiensis or B. pumilus. It may comprise at least one Lactobacillus species, for example L. lactis, L.plantarum, L. paracasei, or L. brevis. It may comprise at least one Streptomyces species, for example, S. aureofaciens, S. griseus, S. lydicus, S. mutabilis, S. olivaceus. . It may contain at least one Amycolatopis species, for example A. australiensis,A. lactamadurans,A. deserti, A. methanolica, A. albidoflavus. It may contain at least one Micromonospora species, for example M auranatiaca,M echinospora, M globose, M Nigra, M. purpurea. The activity of the a) type bacterial organisms may be due to a disruption to the lifecycle of the ectoparasite, which, whilst not necessarily lethal to the ectoparasite, results in the disruption and ultimate failure of the infestation. Such disruption may be caused by inhibiting the ectoparasite from maturing by halting its lifecycle at a particular stage. For instance, the ectoparasite may be restricted from progressing past the egg, larvae or nymph stages, resulting in the repression of breeding adults. The lifecycle may be disrupted by sterilising or inhibiting the breeding of adult ectoparasites, leading to the inhibition of egg laying. It may reduce the production or viability of subsequent life cycle stages such as eggs and thus reduce subsequent infestation from the environment where the animal lives.

[00068] The activity of the a) type bacterial organisms may derive from elimination of the ectoparasite through secreting one or more compound that is deleterious to the ectoparasite, but does not harm the host subject. The elimination may be caused by lethally removing ectoparasites at all lifecycle stages or by debilitating ectoparasites at only specific lifecycle stages. The a) type bacterial organisms may act lethally only towards the ectoparasite causing the infestation on the animal, without harming the host or the endemic microflora of the host animal.

[00069] The active compound released by the a) type bacterial organisms may be an organic molecule, e.g. an organic acid, an ester, an amine, or an alcohol. The organic molecule may be produced and secreted by the bacterial organisms, or it may be a metabolite or waste product. The organic molecule may be a small molecule (e.g. it may be less than 900 daltons in molecular weight). The compound may be a peptide. It may be an oligopeptide of about 2 to about 5 residues in length (i.e., it may be 2, 3, 4 or 5 residues in length), or it may be a polypeptide of about 6 to about 50 residues in length, such as about 6 to 40, 6 to 30, 6 to 20, 10 to 50, 20 to 50, to 50, 40 to 50, 30 to 40, 25 to 50, (ie., about 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 residues) or it may be a peptide of greater than 50 residues in length (ie., greater than about 55, , 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200 or more residues). The compound may be a bioinorganic complex comprising at least one transition metal (e.g., Cr, Mn, Fe, Co, Ni, Cu, Zn, Au, Ag, Cd) bound (e.g., complexed) to at least one organic molecule. It may be a protein or enzyme that exhibits esterase, chitinase amidase or protease activity. The compound may block development of the ectoparasite, impair feeding and/or reduce the integrity of the ectoparasite's exoskeleton, or it may act as a repellent to the ectoparasite.

[00070] The ectoparasite to be treated and/or repelled by a composition of the present invention may be known to be commercially damaging to companion animals and/or agriculturally important host animal species and/or birds. The ectoparasite may be a tick, a mite, a louse, a flea or a biting fly. When the ectoparasite is a tick, it may be an Amblyomma spp., a Boophilus spp., a Dermacentorspp., a Haemaphysalis spp., an Ixodes spp., an Ornithodoros spp., and Otobius spp. or a Rhipicephalus spp. For example, the ectoparasite may be a bush tick (e.g., Haemaphysalis longicornis), a paralysis tick (e.g., Ixodes holocyclus), or a cattle tick (e.g., Rhipicephalus (Boophilus) microplus (australis). The ectoparasite may be a mite that is a Chorioptes spp., a Demodex spp., Psorergates,Dermanyssus, a Psoroptes spp. or a Sarcoptes spp. For example, it may be a chorioptic mite (e.g., Chorioptes bovis), a demodectic mite (e.g., Demodex bovis), a psoroptic mite (e.g., Psoroptes communis, var. bovis) or an itch mite (e.g., Sarcoptes scabiei). The ectoparasite may be a louse that is a Damaliniaspp., a Haematopinus spp., a Linognathus spp. or a Solenopotes spp. Bovicola spp. For example, it may be a red louse / biting louse (e.g., Damalinia (Bovicola) bovis), a short-nosed cattle louse (e.g., Haematopinuseurysternus), a long-nosed or blue cattle louse (e.g., Linognathus vituli), or a little blue sucking louse / hairy cattle louse (e.g., Solenopotes capillatus). The ectoparasite may be a biting fly that is a Chrysomya spp., a Dermatobia spp., a Hypoderma spp. or a Haematobia spp. Stomoxys For example, it may be a cattle screw-worm / old world screw-worm fly (e.g., Chrysomyia bezziana), a tropical warble fly (e.g. Dermatobiahominis), an Ox warble / cattle grub (e.g., Hypoderma bovis) or a buffalo fly (e.g., Haematobiairritans exigua). Particularly important ectoparasites that may be targeted by the present invention include cattle tick Rhipicephalus (Boophilus) microplus (Australis) and buffalo fly Haematobiairritans exigua. Strike flies that may be targeted include Lucilia, Calliphoraspp. Fleas that may be targeted include Ctenocephalides, Echidophagaspp.

[00071] The a) type bacterial organisms of the present invention may be a species commonly found in soil, for example in soil in Northern Australia. In preparing a composition as herein disclosed, the a) type bacterial organisms may be identified in, and/or isolated from, a soil sample. The bacterial organisms isolated from the soil samples may be cultivated before being screened for relevant activity. The screening of the bacteria against the ectoparasite may be carried out in a variety of ways, depending on the mechanism of action of the bacterial organisms against the ectoparasite. For example, an extract of the bacterial organisms may be applied to a life cycle stage of the ectoparasite to determine inhibitory effect. Alternatively the extract may be tested for the presence ofknown anti-ectoparasitic compounds for example avermectin or ivermectin.

[00072] Once isolated, the a) type bacterial organisms may be further grown in a culture medium to produce more bacterial organisms and/or to enhance their anti-ectoparasitic properties. The culture medium and other culturing conditions (temperature, liquid vs. solid culture medium, supplements) may be such as to enhance the activity of the bacterial organisms against the ectoparasite, e.g. to enhance production of anti-ectoparasitic metabolites or to enhance properties or activity of the bacterial organisms. Suitable conditions may be readily determined by routine screening of the culturing conditions.

[00073] The bacteria isolated from the soil samples may be cultivated before being screened for anti-ectoparasitic activity and/or production of anti-ectoparasitic compound(s), for example small molecules, peptides, proteins etc. In some instances, experience will have shown that the bacterial species have relevant activity and in such instances, screening may not be necessary. The screening of the bacteria against the ectoparasite may be carried out by an in vitro assay, or by an in vivo assay.

[00074] Compositions of the present invention also comprise b) type bacterial organisms, which exhibit chitinase activity. These spores should not negatively impact on the anti-ectoparasitic activity of the type a) bacterial organisms. The effect of the b) type bacterial organisms may be caused by a direct attack on the ectoparasite and may therefore complement the a) type activity. As the cell wall of common ectoparasites comprises chitin, the chitinase activity of the b) type bacterial organisms is thought to be harmful to the ectoparasites at one or more stages of their lifecycle. Chitinase activity ofbacterial organisms may be determined by inoculating plates containing chitin with the organisms and checking for clearing zones surrounding the organisms. An example of such a method is described in detail in Example 2. Commonly b) type bacterial organisms are strains of Streptomyces, although other types of bacterial organisms may also exhibit chitinase activity and may therefore be useful as b) type bacterial organisms. These may be identified as described above.

[00075] Compositions of the present invention may also comprise c) type bacterial organisms that stimulate the immune system of the target animal to which the composition has been applied, preferably without being harmful or pathogenic to the target animal. As noted above, this may in some instances be the same strain of bacteria as the a) and/or b) type bacterial organisms, although commonly the a), b) and (if present) c) type bacterial organisms will be different strains. Without being bound to theory, it is believed that the role of the c) type bacterial organisms is to heighten the immune response of the subject, acting to weaken the ectoparasite on the external surface of the subject or at least working in concert with the a) and b) types of bacterial organisms described above to repel the ectoparasite. The immune response may be an innate response (ie., a non-specific response, such as mast-cell activation) or it may be an adaptive response (ie., mediated by lymphocytes).

The mechanism by which the c) type bacterial organisms stimulates the immune system of the subject to which the composition is applied may be a specific response by an element of the adaptive immune system (i.e., mediated by T cells, B cells or other lymphocytes) which elicits a response in the subject towards a specific antigen or it may be a non-specific response mediated by an element of the innate immune system (ie., mast cells) which elicits a general immune response in the subject. The effect of the immune stimulation, whether specific or non-specific, is to weaken the ectoparasite and/or make the ectoparasite more susceptible to the effects of the anti-ectoparasitic bacteria. The c) type bacterial organisms may be effective in weakening the ectoparasite by eliciting an immune response on or at the external surface (e.g., skin) of the subject locally to where the composition is applied to the external surface of the subject. For example, it may cause inflammation at the site of the application of the composition, which when applied to a local site of infestation, may stimulate normal inflammatory processes and hence reduce the ability or efficiency by which biting or blood-sucking ectoparasites can feed. The inflammation may be caused by locally activating the mast cells present in the skin and surrounding tissues of the target animal to which the composition has been applied. The c) type bacterial organisms may increase the expression of natural anti-ectoparasitic agents in the skin of the subject, effectively enhancing the innate immune barrier of the subject. The cell walls of bacteria commonly possess the ability to invoke an immunological response. It is noted that this forms the foundation of adjuvants which are derived from bacterial cell walls. There are examples of immune-stimulators from Streptomyces and other bacteria. For example, Streptomyces olivaceogriseus sp. nov. and S. violaceus produce the immune stimulant FK-156 (JournalofAntibiotics 35: 1293-1299 (1982)) and Lactobacillusplantarum strains produce Pyro-F, Pyro-W, Pyro-L and Pyro-I which are immunomodulators (JournalofAntibiotics 67: 85 88 (2014)). Methods for identifying suitable bacterial organisms as type c), i.e. as immune stimulators, are well known to those skilled in the art and are described in the above-referenced publications and in publications referenced therein, all of which are incorporated herein by reference. Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia or elsewhere.

[00076] The c) type bacterial organisms may be present in the endemic hide microflora of breeds and/or herds of animals that are naturally resistant to at least one ectoparasite, and/or which is known or identified as eliciting an immune response in the skin of the subject. The skilled person familiar with common methods for determining immune system activation would be aware of other appropriate screening and assaying methods which could be applied to evaluate potential species of bacterial organisms for desired bioactivity.

[00077] Each of the a) type, b) type and c) type bacterial organisms may, independently, be Actinobacteria. They may be Actinomycetes. They may be members of the genus Streptomyces or allied genera. Each of the a) type, b) type and c) type bacterial organisms may, independently, be not Bacillus. Each of the a) type, b) type and c) type bacterial organisms may, independently, be gram positive.

Composition and Formulation

[00078] The compositions to which the present invention relates are formulated so that the beneficial bacterial organisms can be applied to, and successfully colonise, the subject on which they are applied. They may be probiotic compositions. The compositions are formulated for external application to the subject. In other words, the bacterial organisms of such compositions are not intended to be ingested. The bacterial organisms may be unsuitable for sustainably colonising the intestinal tract of the subject. Rather, the compositions described herein are designed to be applied externally to the subject, and for the beneficial effects of the bacterial organisms to be localised to the skin and surrounding areas. It is envisioned that the bacterial organisms, being the a) type, b) type and optionally also c) type, are capable of forming colonies on an external surface of the subject, and sustainably or temporarily forming a part of the microbiome of that subject.

[00079] The composition, being for external application, may be in any suitable physical form. It may be a liquid composition or may be a solid composition. For example, it may be a powder or a dust, or it may be a solution, an emulsion, a dispersion or a suspension. When formulated for application, the composition may be as a spray-on preparation, a paint, a dust, a pour-on, a spot-on, a rub-on, a powder, a cream, a lotion, an adhesive patch, dip or any other suitable form for application to at least a portion of an external surface of the subject. In some instances, the composition is an unstable suspension or a suspension of limited stability. In this case it may be necessary to agitate the composition, or otherwise resuspend settled solids in the composition, prior to and/or during application.

[00080] The composition may comprise a liquid carrier. This may be an oil. The oil may be a mineral oil or may be a vegetable oil. It may for example be olive oil. Alternatively the liquid carrier may be aqueous. It may comprise one or more salts or other additives. These may for example serve to achieve a desired pH for the composition. They may comprise buffers.

[00081] The composition may comprise one or more solids. These may be in the form of powders or dusts. They may act as fillers or carriers. They may be organic or may be inorganic or both organic and inorganic solids may be present. These may include diatomaceous earth (DE), talc, silica or other mineral fillers. It is thought that the presence of DE, talc, silica or other mineral fillers may allow bacterial organisms of the composition to penetrate into the particles so as to protect them. The composition may also comprise organic solids such as lactose, milk powder etc. These may provide a nutrient source for the bacteria during storage of the composition. In some embodiments, all components of the composition with the exception of the bacterial organisms thereof may be selected from the GRAS list.

[00082] In some embodiments the composition of the invention may comprise one or more acaricides, or one or more insect or acari repellents, or both. These may serve to supplement the effect of the bacterial organisms which are active against the ectoparasite and those with chitinase activity. They may provide an initial reduction of the ectoparasite popoulation, allowing time for the bacterial organisms of the invention to colonise the subject so as to provide longer term protection. Suitable chemical acaricides include:

Arsenicals: arsenic trioxide, potassium arsenite, dihydro-1, 3, 2,-dithiarsenol-2yl mercaptoacetic acid;

Organochlorines: benzenehexachloride, dichlorodiphenyltrichloroethane (DDT), lindane, aldrin, dieldrin, toxaphene, endosulphan, methoxychlor, hexachlorocyclohexane;

Organophosphates: coumaphos, chlorfenvinphos, diazinon, parathion, malathion, diaxanthion, oxinothiophos;

Carbamates: carbaryl, aldicarb, carbofuran, ethienocarb, fenobuacrb, oxamyl, propoxur; Formamidines: amitraz, chlordimeform, clenpyrin, chloromethiuron;

Pyrethroids: cypermethrin, permethrin, deltamethrin;

Macrocyclic lactones: avermectin, ivermectin, eprinomectin, selamectin, doramectin, abamectin, milbemycins;

Phenylpyrazoles: fipronil, pyriprole;

Spinosyns: Spinosad (mixture of spinosyn A and D), spinetoram;

Insect growth regulators: azadirachtin, methoprene, diflubenzuron, fluazuron and

Isoxazolines: afoxolaner, fluralaner, sarolaner, lotilaner, CPD I.

[00083] Additionally or alternatively to chemical acaricides, the composition may comprise a plant acaricide. As plants are susceptible to certain ectoparasistes that can also affect animals, some have developed the ability to produce acaricides and/or insect or acari repellents. Suitable such substances are described, for example, in "Pesticidal plants as a possible alternative to synthetic acaricides in tick control: A systematic review and meta-analysis." Adenubi, .T. et al Industrial Crops & Products 123 (2018) 779-806. The contents of this publication are incorporated herein in their entirety by reference.

[00084] Additionally or alternatively to either the chemical or plant basedmaterials described above, the composition may comprise an insect or acari repellent, ie. a substance that is not necessarily toxic to the ectoparasite but which discourages the ectoparasite from infecting a host treated with the repellent. Suitable such repellents include N,N-diethyl-meta-toluamide (DEET), 1-piperidinecarboxylic acid 2-(2-hydroxyethyl)-1-methylpropylester (Picaridin), para-menthane 3,8-diol (PMD), ethyl butylactyloaminopropionate (IR3535), 1S,2S-2-methylpiperidinyl-3 cyclohexene-1-carboxamide (SS220), racemic 2-methyl piperidinyl-3-cyclohexene-1 carboxamide (A13-37220).

[00085] Suitable doses, and hence suitable concentrations in the compositions of the invention, are well documented and may be obtained in many instances from the supplier of the acaricide or repellent or from the open literature.

[00086] The process of making the composition of the invention may therefore include the step of adding one or more acaricides, or one or more insect or acari repellents, or both, either during the formulation of the composition or thereafter.

[00087] In general, for an oil based composition the content of the abovementioned solids (i.e. non-bacterial matter other than the carrier) is from about 1 to about 5% w/v, or from about 1 to 3, 2 to 5 or 2 to 4%, e.g. about 1, 2, 3, 4 or 5%. For an aqueous based composition it may be from about I to about 10% w/v, or about I to 5, 1 to 2, 2 to 10, 5 to 10 or 3 to 8%, e.g. about 1, 2, 3, 4, , 6, 7, 8, 9 or 10%. For solid compositions or rubs, itmay be from about 60 to about 95%w/w, or about 60 to 90, 60 to 80, 60 to 70, 70 to 95, 80 to 95, 90 to 95, 70 to 90, 80 to 90 or 70 to 80%, e.g. about 60, 65, 70, 75, 80, 85, 90 or 95% w/w.

[00088] The composition for application to an external surface of the subject, may be formulated so that at least a portion of the applied composition comes into direct contact with the skin of the subject, or it may be formulated so that the composition is applied predominantly to the hair, feathers or hide of the subject, with the bacterial organisms being transported to the skin thereafter.

[00089] The composition, when formulated for application to a subject, may further comprise at least one pharmaceutical excipient. This may be selected from a buffer, a salt, a solvent, a preservative, a spreading agent, an oil, a dispersing agent, a surfactant, an emulsifier, a chelating agent, a viscosity modifier, a desiccant, an antioxidant, a bacterial growth medium, water or any other suitable, non-active additives useful in formulating a composition for administration to a subject, or it may be a combination of any two or more of these.

[00090] The composition may also comprise at least one prebiotic component, such as a nutrient (e.g. malt extract, corn syrup, flour, yeast extract, vitamin) or other materials used to support the culturing of organisms, or other substance. In this context, a prebiotic component is one that encourages growth of the bacterial organisms. For example, a growth medium may act as a prebiotic substance. Prebiotic components are known in the art to promote the growth of probiotic bacteria, and to assist in the establishment of colonies. The use of prebiotic compounds and substances in the formulation of compositions described herein may beneficially assist the applied bacterial organisms to be included in the microbiome of the subject and form a sustainable colony.

[00091] The compositions of the present invention may comprise between about 1% and about % by weight of bacterial organisms, in which the bacterial organisms is the combination of a) and b) type bacterial organisms, as well as the c) type bacterial organisms when present. For example, the bacterial load may account for between about 1% and about 25% of the total weight of the composition, orbetween about 1% and 20%,2% and 20%,3% and 18%, 4% and 17%, % and 2 5 %, 15% and 25% or 5% to 10%, e.g., about 1%, 2%,3%, 4%,5%,6%, 7%,8%, 9%, %, 11%, 12%,l1 3 %,l14 %,15%,l1 6 %, 1 7 %,l18%,19%,20%, 21%, 2 2 %, 2 3 %, 2 4 % or 25%. It may be less than 1%. Each strain of bacterial organisms may, independently or in total, be present at a level of from about 102 to about 1014 cfu/g (colony forming units per gram), or about

102to to2, 102to1010, 106, 106 to 101, 106 to 107, 107 to 109, 10 to 109, 107 to 108, 109 to

1014, 10 to 1014, 10 to 1014 or 106 to 1012 cfu/g, e.g. about 102, 5*102, 10', 5*103, 104, 5*104, ', 5*105,106, 5*106, 107, 5*107, 10', 5*10, 109, 5*109, 10, 5*101, 101, 5*101, 1012, 5*1012

", 5*10" or 1014 cfu/g, although in some instances one or more bacterial species may be present at less than about 102cfu/g or greater than about 1014cfu/g.

[00092] The composition may be formulated so that the bacterial organisms are effective for a minimum amount of time. For example, the formulation when applied to the subject may be effective in treating and/or preventing an ectoparasite infestation for at least 1 month, or at least 12 months, or at least 5 years, or more than five years, e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 years. It may be for the life of the subject to which it is applied.

[00093] The composition may comprise different species of bacterial organisms. The composition may be formulated so that there are multiple (i.e., more than one) different a) type bacterial organisms and/or multiple (ie., more than one) b) type bacterial organisms and optionally also multiple (i.e., more than one) c) type bacterial organisms. However, the composition should be formulated such that the bacterial load remains stable, insofar as the different species of bacterial organisms are able to remain viable in close proximity to each other in the composition.

[00094] The composition may be formulated for application to an animal or a bird. The animal may be a non-human mammal. The non-human animal may be a livestock animal, such as a cow, goat, sheep, pig, llama, alpaca, horse or buffalo, or a companion animal (cat, dog, reptile). The bird may be a poultry bird or an aviary bird. It may for example be a chicken, a duck, a pheasant, a goose, a turkey, a dove, a pigeon, a budgerigar or some other bird.

Method of Treatment and/or Inhibition

[00095] The invention also relates to a method for treating a subject suffering an ectoparastic infestation and for inhibiting or preventing such an infestation. This method comprises applying, to at least a portion of the external surface of a subject, at least one type of a) type bacterial organisms and at least one type of b) type bacterial organisms and optionally also at least one type of c) type bacterial organisms. In this method, the at least one type of a) type bacterial organisms may be different to the at least one type of b) type bacterial organisms, which may be different to the at least one type of c) type bacterial organisms (if present). It is believed that, when bacterial organisms having the separate activities specified herein, the effect of the a) type bacterial organisms is enhanced by the chitinase activity of the b) type bacterial organisms, and that the stimulated immune response of the subject due to the c) type bacterial organisms, if present, can further enhance the anti-ectoparasitic activity of the a) and b) type bacterial organisms. The composition applied in this method may be as hereinbefore described. It may be as defined in any of the claims set out hereafter.

[00096] In this method, the a) type bacterial organisms may be applied simultaneously with the b) type bacterial organisms and optionally also the c) type bacterial organisms, or they may be applied at different times (ie., separately). The different types of bacterial organism may be formulated together into a single composition, or they may be formulated separately into different compositions for independent application, either concurrently or at different times.

[00097] When applied separately, the different types of bacterial organism may be applied in any desired order, or they may be applied concurrently. It is considered that, in order for the combination to be effective, the different types of bacterial organisms should be applied to the same portions of the external surface of the subject e.g., applied in the same, or roughly the same, part of the external surface. This ensures that the different types of bacterial organisms colonise the same region of the external surface.

[00098] In the event that the bacterial organisms are not formulated into a single composition, the formulation of each of the bacterial organisms compositions (i.e., the composition comprising the a), b) and optionally c) type bacterial organisms) may each independently be formulated for application in any suitable physical form. For example, each composition may independently be a powder, a dust, a solution, an emulsion, a dispersion or a suspension. Each composition may independently be applied as a spray-on, a dip, a paint, a dust, a pour-on, a spot on, a rub-on, a powder, a cream, a lotion, or any other suitable form for application. Thus for example a composition may be applied by immersing a substantial portion of the subject in the composition, or it may be applied by painting the composition onto the subject (either manually or by having the animal pass through a brush or roller having the composition thereon) or by spotting the composition onto the subject or by any other suitable composition.

[00099] Each composition may be the same physical type, or they may be different. For example, each composition may be a powder formulated to be a dust, or one composition may be an emulsion suitable for use as a spray-on and another composition may be a powder suitable for use as a dust. Each of these compositions may independently further comprise one or more of the pharmaceutical and/or veterinary excipients as described herein. The excipients may be GRAS. There may be no non-GRAS excipients.

[000100] The method may comprise a further step of isolating the at least one a) type bacterial organisms from a dust or soil sample prior to formulating it into a composition. The isolated bacterial organisms may be cultivated for use in the method of the present invention. The identification of an appropriately active species of bacterial organisms from a soil or dust sample may include subjecting the isolated bacterial organisms to an in vitro assay to measure anti ectoparasitic activity.

[000101] The ectoparasite infestation of the subject is, by definition, located primarily on an external surface of the subject. Most commonly, this external surface is the skin or hide of a subject which is typically mammalian or the feathers of a subject which is avian. Hence, treatments for ectoparasite infestations can be applied directly to the infested surface of a subject in order to eliminate the ectoparasitic infestation, without requiring absorption of the anti ectoparasitic agent into the systemic blood supply of the treated subject. The method of treating an ectoparasite infestation as described herein works on this basis, as the bacterial organisms, formulated for external application to the subject, includes spores of a), b) and optionally c) type bacterial organisms with activity against an ectoparasite that infests the subject. The activity of the bacterial organisms, when applied to at least a portion of the external surface of the subject, is thought to occur entirely, or at least in the majority, on the epidermis of the subject. The a), b) and optionally c) type bacterial organisms, or anti-ectoparasitic compounds that may be released therefrom, need not enter the subject in order to be effective, as the ectoparasite is generally located on the outside of the subject. Instead, the different types of bacterial organisms, irrespective of the mechanism of action against the ectoparasite, generally remain of the external surface of the subject to treat and/or prevent an ectoparasite infestation.

[000102] External surfaces of humans and non-human animals are capable of supporting and sustaining microflora that can be beneficial to the host upon which the colonies form. Hence, it is envisioned that when bacterial organisms are applied to the external surface of a subject via the methods described above, a lasting anti-ectoparasitic effect may be provided to the treated subject if the bacterial organisms are sustainably introduced into the external microbiota of the subject. This lasting effect may effectively result in the prevention of an infestation of the subject by that ectoparasite so long as the bacterial organisms colonised an external surface of the subject. This protection would be expected to be enhanced if the c) type bacterial organisms of the applied composition, or some other immunostimulatory bacterial species, was co colonising the surface of the subject, alongside the a) and b) type bacterial organisms.

[000103] These methods of treatment and/or inhibition with at least one type of a) type spore require co-administration to the subject with at least one type of b) type spore as well as optionally at least one type of c) type spore.

[000104] The methods of the present invention may require subsequent applications after the first application to a portion of an external surface of the subject, or may only require a single application in the lifetime of the subject. In methods where multiple applications are required to provide adequate treatment and/or prevention of ectoparasite infestation for the subject, the time between applications may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 1, 2, 3, 4, 5 or more than 5 years. For instance, the composition may be required to be applied every 6 months to ensure adequate protection of the subject from ectoparasite infestation. The time between applications may be related to the longevity of the bacterial colonies on the external surface of the subject, or the time between applications may be to establish colonization. The time between applications may be regular, (e.g., every 6 months), or they may be irregular (e.g., following a dose of antibiotics, the probiotic composition may need to be reapplied). It may fit with normal handling times used by farmers for other purposes, e.g. drafting, tagging, treatment.

[000105] The methods may be carried out (i.e., the application of the composition) on a subject that is a mammal or a bird. The subject may be a livestock animal, such as a cow, ox, goat, sheep, pig, llama, alpaca, horse or buffalo, or a bird, e.g. chicken, duck, goose, emu, or a companion animal, e.g. a dog or a cat. The subject may in some instances be a human.

Method of Identifying Active Bacterial Species, Culture Conditions

[000106] As described above, compositions of the present invention require the presence bacterial organisms of at least one species that displays anti-ectoparasitic activity against an ectoparasite. The a) type bacterial organisms may be isolated from a source of such bacteria, such as a soil or dust sample. Once isolated, the activity of these bacteria against the ectoparasite must be determined to ensure an effective composition can be produced.

[000107] To determine the activity of an isolated a) type bacterial species, any suitable in vivo or in vitro method can be used. For example, one such method comprises analysing an extract of the bacterial organisms to determine the presence or absence of known anti-ectoparasitic compounds such as avermectins. Alternatively, the target ectoparasite may be exposed to a range of concentrations of the bacterial organisms to determine an effective concentration of the bacterial organisms. Prior to formulating a composition for administration to a subject, the bacterial organisms may be cultured to enhance their a) type activity. For example a routine variation of conditions (e.g. temperature, substrate, additives) may be used to determine an optimum set of culture conditions. A similar process may be employed to identify suitable b) type and, if desired, c) type bacterial species and to identify suitable culturing conditions for enhancing their activity.

[000108] As noted earlier, in some instances, the same strain of bacterial organism may be used for a combination of two or all of a), b) and c) type activities. Inthis instance, the above protocols may be used for identifying suitable strains and for determining suitable culture conditions.

Examples

[000109] Bacterial organisms with relevant activity are identified via a high throughput screen of primary extracts from the applicant's culture collection. Promising candidates are optimised using over 250 distinct isolation strategies, including antibiotic regimens, media selection, incubation conditions and phage selection.

Example 1: Isolation of the microbes

[000110] Streptomyces and related genera were isolated from 300 northern Australian soil samples using conditions known to favour the growth of the majority of actinomycetes present (ISP2 medium, 28°C) while inhibiting the growth of bacteria (naladixic acid, 4 tg/ml) and fngi (nystatin and cycloheximide both at 100 pg/ml). A soil sample (300 x 15 g) was individually dispensed to 300 mL plastic jars followed by the addition of 150 mL of sterile water with vigorous shaking to prepare 10-1 dilutions. 10-2 dilutions were prepared by aseptically transferring 1 mL of 10-1 dilution to 9 mL sterile water in 20 mL vials. 1 mL transfers of the 10-2 dilutions were transferred with the addition of 9 ml sterile water to produce the 10-3 dilution.

This was repeated to give the 10-4 dilution. lOuL sub-samples of the 10-3 and 10-4 samples were applied to individual wells of a 96-well microtitre plate (Technoplas@) containing 0.2 mL of ISP2 agar containing naladixic acid, nystatin and cycloheximide at 4, 100, 100 ug/mL, respectively, and the plates incubated at 28°C for 10 days. After this time wells containing individual colonies (9,840 colonies selected) were transplanted into adjacent wells in fresh microtitre plates containing 0.2 mL ISP2 agar with no naladixic acid, nystatin or cycloheximide and allowed to grow at 28 C for 10 days.

[000111] The mature colonies were extracted with 150 uL methanol and gently shaken. 6 x 10 uL was transferred into microtitre plates to test for activity against a nematode, an insect, an acari, bacteria and two fungi according to methods present in the scientific and patent literature.

[000112] The number of cultures with active extracts for each bioassay are collated in the table below.

Table 1: Activity of extracts derived from Streptomyces and related genera in 6 bioassays

Bioassay Phyla Actives* %

Haemonchus contortus Nematode 504 5.1% Lucilia cuprina Insect 243 2.5% Dermanyssus gallinae Acari 90 0.9% Bacillus subtilis Bacteria 1,597 16% Saccharomyces cerevisiae Fungus 1,335 14% Candida albicans Fungus 1,114 11% Total cultures 9,840 Active cultures* 3,013 Active 31% *An active culture is one where an extract of the culture was active in one or more bioassays

[000113] Overall, 3,013 cultures showed activity in one or more bioassays. A total of 323 cultures showing activity against either an insect or an acari were selected as candidates for probiotic evaluation in in Examples 2, 3 and 4.

Example 2: Bioassay for Chitinase Activity

[000114] Colloidal chitin powder was prepared according to the published method of Hsu and Lockwood (Powdered chitin agar as a selective medium for enumeration of actinomycetes in water and soil. Hus S.C. & Lockwood J.L. Applied Microbiology 1975, 29: 422-426) with minor modifications. Thus, lOg Sigma chitin (Product code: C7170 Batch: BN#84H175) was slowly added to 150mL hydrochloric acid (32% v/v) with stirring for two hours. The resulting brown solution was filtered through a glass fiber plug to remove any large undigested chitin. The filtrate solution was added dropwise to 5L of vigorously stirred cold (4 °C) water, precipitating a colloidal chitin solution. Colloidal chitin was collected by filtration on a Whatman #41 paper under vacuum. The solid was returned to a fresh 5L of vigorously stirred cold (4 °C) water and the step repeated. The colloidal chitin was freeze-dried to provide powdered colloidal chitin suitable for use in the detection of chitinase-producing microbes.

[000115] Chitin plates were prepared using freeze-dried colloidal chitin 4g; yeast extract 4g; malt extract 10g; glucose 4g and bacteriological agar 20g made up to 1 L with deionised water. The suspension was autoclaved at 121°C for 30 minutes. On cooling to 60°C, 15 mL of stirred suspension was dispensed in 9.5 cm diameter Petrie plates and allowed to cool prior to use.

[000116] Sterile loops were used to transfer sample spores from individual growing Streptomyces cultures maintained on ISP2 agar previously incubated at 28°C for a minimum of 7 days. Typically, nine Streptomyces strains were inoculated in a single plate. The plates were then incubated at 28 °C for 7 days and inspected for clearing zones around the growing Streptomyces colonies (Fig. 1).

[000117] Those cultures producing chitinase give a pronounced clearing zone and are recorded as 'Active'. Failure to demonstrate a clearing is recorded as 'Inactive'. For active cultures the section diameter of the clearing zone is used to rank potency; the larger the diameter, the more potent and desirable the chitinase activity. Results for a typical set of Streptomyces under inspection are shown in Table 2 and photograph of the experiment presented as Figure 1.

Table 2: Results of chitinase activity tests

Position Strain Chitin activity Inhibition Zone 1 136998 Active 3mm. Completely clear, defined edges. 2 RA16637 Active 1mm. Completely clear, defined edges. 3 130882 Inactive No zone 4 152257 Active 1.5mm. Opaque zone, blurred edges. 5 138420 Active 2mm. Completely clear, defined edges. 6 115760 Active 2mm. Completely clear, defined edges. 7 137265 Active 2mm. Completely clear, defined edges. 8 130420 Inactive No zone

9 115088 Active 1nm. Completely clear, defined edges. 10 136761 Inactive No zone. 11 137567 Active 1mm. Completely clear, defined edges. 12 150526 Inactive No zone

[000118] Of the 159 Streptomyces cultures investigated for chitinase activity, 84 were found to be active with clearing zones of0.5 up to 5 mm.

Example 3: Tick Activity

[000119] The acaricidal activity of extracts and chemicals against the cattle tick Rhipicephalus (Boophilus) microplus (Australis) was assessed using strain NRFS 3839 by applying tick larvae to paper treated with the sample (tick packet test). (A method for measuring the acaricide susceptibility of the cattle tick Boophilus microplus (Can.) is described in Stone B., Haydock K., Bull. Entomol. Res. 1962, 53 (03), 563-578).

[000120] 670plof each sample was applied to the test papers. Approximately 100 tick larvae were applied to each prepared/treated paper which was folded to form a packet. Packets were incubated for 24 hours at 27°C and 85% relative humidity. The percentage of larvae alive/dead was recorded. Of a total of 57 Streptomyces strains tested, 5 cultures were shown to be active in the packet test.

Table 3: Selected Streptomyces packet test results

Code Conc. (mg/mL) % Mortality Comment 130995 10 100 138458 10 36 Many moribund 191872 10 73 RA20317 10 57 RA20321 10 73

Example 4: Buffalo Fly Activity

[000121] The insecticidal activity of extracts and chemicals against one strain of buffalo fly (Haematobiaexigua ESP Strain) was assessed by applying 0.5 pL of sample to the dorsal thorax of five adult buffalo flies. Each group of 5 flies was held in 300 mL paper cups with a clear lid and blood-soaked cotton wick, incubated at 27°C / 70% relative humidity for 48 hours. Mortality was assessed at 24 and 48 hours. Flies were counted as dead if no movement was seen, and morbid if they were unable to fly or walk. Ivermectin was used as the positive control. The results of selected Streptomyces strains are reported in the table below. Of a total of 57 Streptomyces strains tested, 20 cultures were shown to produce extracts that were active in the in vitro buffalo fly test.

Table 4: In vitro buffalo fly test results with selected Streptomyces strains

Conc. Flies Dead Affected Dead Affected Dead Affected Code (mg/mL) added 2hrs 2hrs* 24hrs 24hrs 48hrs 48hrs 153818 10 5 2 2 2 2 4 0 RA20321 10 5 1 3 3 2 5 0 RA58 10 5 4 1 4 1 5 0 RA20317 10 5 0 2 3 1 3 1 138458 10 5 3 1 5 0 5 0 Control Methanol 0 5 0 0 0 0 0 0 Ivermectin 0.1 5 5 0 5 0 5 0 *Affected= flies walking on base of container but do not fly

Example 5: Microbial Fermentation on Grain

[000122] The Streptomyces strain cultures selected to have the desired activity were recovered from -80 °C storage onto 2 plates of ISP2 agar per culture and incubated at 28 °C for 7 days. Each recovery plate was diced into 1 x 1 cm squares and stirred in 200mL sterile water in sterile 250mL Erlenmeyer flask to prepare a spore suspension for inoculation. 40 x 250mL Erlenmeyer flasks, each containing 35 g jasmine rice hydrated with 25 mL water and autoclaved (121 °C for min), were then inoculated with 10 mL spore suspension per flask. The flasks were incubated at 28°C for 10 days, by which time the cultures reached maximum spore productivity. The ferment was stored at 10 °C until ready for formulation. The process was repeated for all available strains of Streptomyces with chitinase (Example 2), acaricidal (Example 3) and insecticidal (Example 4) activity.

Example 6 :Microbial Fermentation on Liquid

[000123] Streptomyces strain MST-191872 recovered from storage as a 7-day old (see Example ) agar plate was diced and then transferred to 4 x 250ml conical flasks containing 60mL of liquid media and stirred for 3 days. The liquid inoculum was drawn up into 50mL syringes and transferred into a fermenter containing 10 L of sterile ISP2 media. Fermentation conditions of temperature (28°C), agitation at 250 to 750 rpm with an air flow of 0.2 to 1.0 volume per minute (vvm) were applied to maintain a 30% D02 setpoint.

[000124] When oxygen consumption had ceased at Day 5, the fermentation was terminated and transferred into 1.5L centrifuge tubes and centrifuged 8,000 rpm to pellet the mycelia and spores. The clarified supernatant was decanted and the mycelia and spores freeze dried. The process was repeated for all available strains of Streptomyces with chitinase (Example 2), acaricidal (Example 3) and insecticidal (Example 4) activity.

Example 7: Oil Formulation

[000125] Each of5 Streptomyces strains (MST-191872, MST-153053, MST-153818, MST RA19993 and MST-AS6250) fermented on rice grain (Example 5) were separately recovered from storage at 10 C and processed to powders. 500g fermented rice of each Streptomyces strain was transferred using a sterilised scoop into a 5L baffled flask with IL sterile 0.2% carrageenan solution and shaken on rotary platform shaker at 90 rpm for 1 hour. A sterilised glass funnel was lined with sterile gauze folded into 2-ply to filter the spore suspension slowly into a sterile bottle.

[000126] Skim milk powder (35 g) was transferred to sterilised freeze-drying jars to which was added 50 mL of filtered spore suspension jar and stirred to prepare a smooth paste. The paste was gradually diluted by addition of a further 300 mL of filtered spore suspension. Covered jars were placed in a -80°C freezer overnight and then freeze dried to a white powder. The process was repeated for all selected Streptomyces strains.

[000127] The number of spores was counted based on the number of colony forming units (cfu) using a 10-4 to 10-8 dilution of the freeze-dried powder. Thus, 1 g was added to 10 ml of sterile water to prepare a homogenous suspension then serially diluted to 1 in 10 dilution. A 100 uL aliquot of each dilution was applied to an ISP2 plate, spread evenly across the plate and incubated at 28°C. At Day 3 the individual colonies were counted and the number of colonies per gram of powder calculated.

[000128] For each Streptomyces strain the amount of powder containing 3 x 10 cfu (generally 0.5 to 10 g of powder) was weighed and pooled to give a mixed spore powder. To the mixed powder was added 150 g of diatomaceous earth and the solids diluted with 3L olive oil to give a cfu concentration of approximately 1 x 108 cft/mL for each culture to give the final probiotic oil based formulation. Organism activities are summarized in Examples 2, 3 and 4 above.

Example 8: Aqueous Formulation

[000129] 500g fermented jasmine rice (Example 5) of individual cultures was added to IL sterile water in 5L baffled flasks and shaken on a rotary shaker at 90 rpm for 2 hours to dislodge spores and mycelia. The spore suspension was filtered into sterile beakers to remove large particulates. The cfu was calculated (see Example 7) and the spore suspension diluted with sterile water (if required) to give 1 x 108cft/mL which was then stored at10°C. Aqueous suspensions of the desired cultures were combined for final aqueous formulation. Organism activities are summarized in Examples 2, 3 and 4 above and in Table 5 below.

Table 5: Activitiy of bacterial organisms in aqueous formulation

Cultures Tick active Buffalo fly Chitinase Origins 191872 Y Y Y The Gums, Qld RA20321 Y Y N Timber Creek, NT RA20317 Y Y Y Victoria river, NT RA19993 N Y Y Townsville, Qld 153818 N Y Y Halls Creek, WA 189959 N N Y Injune, Qld 153053 N N Y Katherine, NT 152257 N Y Y Oenpelli, NT 152218 N Y Y Oenpelli, NT

Example 9: Field Trial

[000130] The trial was designed to assess the efficacy of the oil formulation (Example 7) against natural infestation of cattle tick (Rhipicephalus australis). The trial protocol followed the

WAAVP recommendations (World Association for the Advancement of Veterinary Parasitology (WAAVP) guideline: Holdsworth, PA, Kemp, D, Green, P, Peter, RJ, De Bruin, C, Jonsson, NN, Letonja, T, Rehbein, S & Vercruysse, J 2006, 'World Associationfor the Advancement of Veterinary Parasitology(WAAVP) guidelines for evaluatingthe efficacy ofacaricides against ticks) and was approved by an appropriate animal ethics committee. The cattle were maintained on their farm of origin in the region of the Atherton Tablelands, Queensland, Australia in spring 2019. Forty cattle carrying more than 20 ticks (single side count) were enrolled. They were predominantly Bos taurus breed, less than 120 kg and were grazing on pastures infected with cattle tick. The cattle were divided into 3 groups: Group 1, untreated control; Group 2, placebo control (oil treatment only); Group 3, probiotic microbe oil suspension. The mean numbers of engorged adult female ticks on each animal were counted 4 and 2 days prior to treatment and the tick numbers averaged and ranked. Following ranking, individual cattle were randomly allocated in a ratio of 1:12 to Groups 1 (10 cattle), 2 (10 cattle) and 3 (20 cattle), respectively. Adult tick counts were performed weekly to 26 days and then on day 41. On days 0, 6, 13, 20 and 26 all 20 animals in Group 3 were treated with (5 x 108) spores that included five Streptomyces isolates using diatomaceous earth as a carrier and suspended in olive oil (Example 5). The treatments were applied along the backline in a volume of100ml. The control animals (Groups 1 and 2) and treated animals (Group3) were maintained under similar conditions in separate paddocks from day 0 to the end of the trial.

[000131] No adverse effects were observed for any of the animals over the 45 days of this trial. Specifically, there were no local application site adverse effects.

[000132] Tick counts for the two control groups were not significantly different and were combined for the purposes of analysis. The group mean (and standard error) tick numbers are tabulated below (Table 6). On days 26 and 41 of the trial the tick burdens on the animals that had received applications of the microbial mixture were statistically and clinically significantly lower than on control animals.

Table 6: Tick counts

Group Day -1 Day 13 Day 20 Day 41 Untreated^ 28.1+4.3* 19.4+4.0 34+6.2 50.5+8.3 Treated 23.6+2.8 22.4+3.9 21.6+5.1 14.9+2.7 ^ Group n = 20 animals, * Mean and standard error of adult tick numbers

Example 10: Field trial

[000133] The trial was designed to assess the efficacy of the aqueous formulation using the protocol and conditions (ie. treatment schedule and outcome measures) described in Example 9. Cattle were grazed and under natural challenge with Rhipicephalus australis (Australian cattle tick). Values are group mean counts of engorged adult female tick on one side of cattle treated by spraying preparations onto both flanks. Doses were 200 ml of a microbial mixture compared with placebo and untreated animals. The preparation contained 9 Streptomyces isolates present at 108 spores per ml (final volume). Spores were diluted in ground rice (culture medium) diatomaceous earth and water. Animals were treated on days, 0, 6, 13, 20, 27 and tick counts performed as indicated in Table 7.

Table 7: Tick counts

Days for start of trial -1 7 13 20 27 28A

Group 1 (n=10) 54.2 21.1 17.8 27.2 73.3 1

Untreated controls

Group 2 (n=10) 55.7 36.3 24.1 32.6 129.6 3

Placebo control carrier only (100ml olive oil)

Group 3 (n=20) 50,0 60.1 11.8* 19.2 26.4* 0

Treated with Microbial culture

* indicates significant differences (P<0.05) between Group 3 and controls.

A indicates the number of animals that were treated with chemical acaricide as a 'rescue

treatment' due to tick burdens exceeding 150 per side.

[000134] From Table 7 it may be seen that after sufficient time for the bacteria present in the inventive composition to colonise the hide of the cattle (in this example 13 days), a substantial reduction in tick counts was observed, as well as a substantial reduction in the number of cattle that needed separate treatment for tick infestation

Claims (5)

1. A composition for treating and/or inhibiting an infestation of a subject by an ectoparasite, said composition comprising: a) bacterial organisms which are active against the ectoparasite; and b) bacterial organisms which exhibit chitinase activity; wherein a) and b) are different strains.

2. The composition according to claim 1 wherein a) are Actinomycetes.

3. A method for treating and/or inhibiting an infestation of a subject by an ectoparasite, said method comprising applying to at least a portion of an external surface of the subject: a) bacterial organisms which are active against the ectoparasite; and b) bacterial organisms which exhibit chitinase activity; wherein a) and b) are different strains.

4. Use of bacterial organisms for the manufacture of a medicament effective in treating and/or inhibiting an infestation of a subject by an ectoparasite, said bacterial organisms being: a) bacterial organisms which are active against the ectoparasite; and b) bacterial organisms which exhibit chitinase activity; wherein a) and b) are different strains.

5. A process for making a composition for treating and/or inhibiting an infestation of a subject by an ectoparasite, said process comprising combining: a) bacterial organisms which are active against the ectoparasite; and b) bacterial organisms which exhibit chitinase activity; wherein a) and b) are different strains.

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