Cross-Reference to Related Applications

This application claims priority to Kenya provisional patent application serial number KE/UM/2016/00661, filed 16 ^th June 2016, the entire contents of which are incorporated herein by reference.

Field of the invention

In embodiments, the technical field of the invention is formulations for control of ectoparasites.

Background

Ticks are obligatory bloodsucking arthropods, living on mammals, birds, and occasionally reptiles and amphibians. Although all ticks are obligate blood feeders, 90% of them are specific for particular hosts that normally do not include humans and their livestock. The remaining 10%, however, are of immense concern due largely to their parasitic nature and ability to transmit pathogenic organisms to humans and other vertebrates. They can transmit human diseases such as relapsing fever, Lyme disease, Rocky Mountain spotted fever, tularemia, African tick fever, Colorado tick fever, and several forms of ehrlichiosis as well as dermatoses in humans. Additionally, they are responsible for transmitting livestock and pet diseases, including babesiosis, anaplasmosis and cowdriosis. The ability of ticks to transmit pathogens to livestock has resulted in great economic losses in the subtropical regions of the world.

Current methods used to control ticks depend largely on chemical acaricides such as amitraz, pyrethroids and organophosphates. However, heavy reliance on these acaricides has resulted in toxicological, environmental and non-target concerns. More importantly, ticks have developed resistance to various classes of compounds. Some work has been carried out with pathogens (i.e., bacteria, fungi and nematodes) that naturally contribute to limit tick populations. Most of the studies to control ticks in this way have focused on off-host delivery, e.g., on vegetation, with limited success. Previous formulations, such as in US 2010/0112060, comprise conidia, canola oil, glycerin, oleic acid, Triton-XlOO/Tween, lauryl amide and NaOH to obtain an emulsifiable formulation. Such formulations are not ideal as they have inferior performance. Improved formulations, and methods of their use, for the control of ticks remain a goal of livestock scientists and the ranching industry.

Summary of the invention

In an aspect is a formulation comprising a plant-derived oil, a non-ionic surfactant, a liquid hydrocarbon mixture, and conidia from Metarhizium anisopliae (M. anisopliae). In embodiments:

the liquid hydrocarbon mixture is kerosene;

the non-ionic surfactant is Triton X-100, and the plant-derived oil is canola oil;

the formulation further comprises an acaricide;

the liquid hydrocarbon mixture (e.g., kerosene) is present in an amount of 2.5-5%, the plant-derived oil is present in an amount of 10-15%, the non-ionic surfactant is present in an amount of 1.0-1.5%;

the formulation is suitable for use in controlling ticks

the formulation is suitable as a repellent and/or killing agent against ticks; the conidia are present in an amount of 10 ⁸ -10 ⁹ spores per ml;

the formulation is suitable for use in controlling ticks, and wherein the tick is selected from Rhipicephalus appendiculatus, R. decoloratus, R. pulchelus, Hyalomma truncatum and Amblyomma variegatum;

the formulation comprises conidia produced using diphasic fermentation technology;

the formulation comprises conidia produced via vegetative mycelia production by liquid medium followed by surface conidiation of the mycelia on solid substrate such as rice; and

the formulation is suitable and effective for controlling ticks.

In an aspect is a formulation for controlling ticks, the formulation comprising a plant-derived oil, a non-ionic surfactant, a liquid hydrocarbon mixture, and conidia from M. anisopliae. In an aspect is a formulation comprising a plant-derived oil, a non-ionic surfactant, kerosene, and conidia from M. anisopliae.

In an aspect is a formulation for controlling ticks, the formulation comprising a plant-derived oil, a non-ionic surfactant, kerosene, and conidia from M. anisopliae.

In an aspect is a method for making a formulation as above, the method comprising production of conidia using diphasic fermentation technology, and combining the conidia with the other components in amounts configured to impart a tick controlling property to the formulation.

In an aspect is a method for making a formulation as above, the method comprising production of conidia using diphasic fermentation technology, and combining the conidia with the plant-derived oil, liquid hydrocarbon mixture or kerosene, and non-ionic surfactant in amounts configured to impart a tick controlling property to the formulation.

These and other aspects of the invention will be apparent to one of skill in the art from the description provided herein, including the examples and claims.

Brief Description of the Drawings

Figure 1 provides a graph showing the number of ticks following two applications within 7 days of formulation of conidia of M. anisopliae as described herein and the acaricide Alpha-cypermethrinat Kilgoris. Arrows indicate time of application of treatment.

Figure 2 provides a graph showing the number of ticks following two applications within 7 days of formulation of conidia of M. anisopliae and the acaricide Alpha-cypermethrinat Nguruman. Arrow indicates time of application of treatment.

Figure 3 provides a graph showing the number of ticks acquired by cows after single application of novel formulation as prepared herein, the application being carried out in Somaliland. Arrow indicates days of application of treatment Figure 4 provides a graph showing the number of ticks acquired by camels following single application of novel formulation as prepared herein, the application being carried out in Somaliland. Arrow indicates day of application of treatment. Figure 5 provides a graph showing mortality of the ticks collected from cows after each spray and incubated in the laboratory; mortality ranged between 95-99%. All dead ticks from fungus treatment developed mycosis.

Detailed Description of Various Embodiments

Herein is provided compositions comprising an isolate of M. anisopliae as mycoacaricide for control of ticks. Unlike previous compositions, the current formulations include an effective amount of kerosene.

Unless otherwise specified, all percentages provided herein are weight percentages and are based on the overall formulation including any active ingredients, solvents, surfactants, diluents, or other components. It will be appreciated that volume or mole percentages could alternatively be used to specify the composition amounts herein.

In an aspect is a formulation comprising a plant-derived oil, a non-ionic surfactant, a liquid hydrocarbon mixture, and conidia from M. anisopliae.

In an aspect is a formulation comprising a plant-derived oil, a non-ionic surfactant, kerosene, and conidia from M. anisopliae.

In an aspect is a formulation as described herein, useful for the control of ticks.

In an aspect is a method for making a formulation as described herein, the method comprising combining the components of the formulation in effective amounts such that the formulation is effective for the control of ticks.

In embodiments, the non-ionic surfactant is a polyethylene glycol ether derivative. The polyethylene glycol ether derivative has a polar portion and a non- polar portion joined via an ether linkage. In embodiments the polar portion is an oligomer of polyethylene glycol, having an average number of repeat units within the range of 5-20, or between 5-15. In embodiment the non-polar portion is an alkyl, aryl, alkaryl, or aralkyl moiety, such as an alkylphenyl moiety. A specific example of the non-ionic surfactant is polyethylene glycol tert-octylphenyl ether with an average molecular weight of 625 g/mol, commonly marketed as TRITON® X-100. Mixtures of non-ionic surfactants can also be used as desired and appropriate. The non-ionic surfactant can be present in any suitable amount, where the suitable amount may be any of the values given herein or may be based on the overall composition. Where the suitable amount is based on the overall composition, such amount will be readily determined by one of ordinary skill with nothing more than routine optimization being required. Examples of suitable amounts include an amount in the range of 0.2-8, 0.2-5, 0.5-3, 0.5-2, 1-2, or 1-1.5%. Other examples of suitable amounts include amounts less than or equal to 10, 8, 5, 4, 3, 2, 1.5, or 1.0%, or amounts greater than or equal to 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, or 8%.

In embodiments, the plant-derived oil is from the rapeseed [Brassica napus] plant and any cultivar thereof. For example, a low-acid cultivar of rapeseed, commonly referred to as canola, can be the source of the plant-derived oil. Other plant-derived oils can be used such as other cultivars of rapeseed, sunflower oil, grapeseed oil, safflower oil, or peanut oil, may be used, as well as combinations thereof (e.g., combinations of canola oil with any of the other oils, such as sunflower oil, safflower oil, etc.). The plant-derived oil can be present in any suitable amount, where the suitable amount may be any of the values given herein or may be based on the overall composition. Where the suitable amount is based on the overall composition, such amount will be readily determined by one of ordinary skill with nothing more than routine optimization being required. Examples of suitable amounts include an amount in the range of 3-30, 5-25, 5-20, 10-20, or 10-15%.

Other examples of suitable amounts include amounts less than or equal to 30, 25, 20, 15, 10, or 5%, or amounts greater than or equal to 3, 5, 10, 15, 20, or 25%.

In embodiments the liquid hydrocarbon mixture comprises a distillate of petroleum with a density in the range of 0.78-0.81 g/cm ³ . In embodiments the liquid hydrocarbon mixture comprises a distillate of petroleum with a carbon chains in the range of 10-16 carbon atoms per molecule. In embodiments the liquid hydrocarbon mixture comprises a distillate of petroleum comprising branched alkanes, straight chain alkanes, naphthenes, olefins, and aromatic hydrocarbons. In embodiments the liquid hydrocarbon mixture kerosene, and is the distillate between 150 °C and 275 °C from petroleum. The liquid hydrocarbon mixture (e.g., kerosene) can be present in any suitable amount, where the suitable amount may be any of the values given herein or may be based on the overall composition. Where the suitable amount is based on the overall composition, such amount will be readily determined by one of ordinary skill with nothing more than routine optimization being required. Examples of suitable amounts include an amount in the range of 1-10, 1-8, 2-8, 2-5, 2-3, 2.5-5, or 2.5-3%. Other examples of suitable amounts include amounts less than or equal to 10, 8, 5, 3, or 2.5%, or amounts greater than or equal to 1, 2, 2.5, 3, 5, or 8%. In embodiments, the amount is less than 20%, and in embodiments, the amount is substantially less than 20% (i.e., more than or equal to 50% less than 20%). In embodiments, the amount is less than 10%, or significantly less than 10% (i.e., more than or equal to 50% less than 20%). In an embodiment the liquid hydrocarbon mixture is kerosene. In embodiments, the kerosene is present in an amount of 2.5%. In embodiments, the kerosene is present in an amount of 3%. In embodiments, the kerosene is present in an amount of 3.5%. In embodiments, the kerosene is present in an amount of 4%. In embodiments, the kerosene is present in an amount of 4.5%. In embodiments, the kerosene is present in an amount of 5%.

The formulation comprises conidia from M. anisopliae, as an active agent. The active agent (i.e., conidia from M. anisopliae) can be present in any suitable amount, where the suitable amount may be any of the values given herein or may be based on the intended use of the composition (e.g., location, mode of administration, target to be protected by the formulation, etc.). Where the suitable amount is based on the intended use of the composition, such amount will be readily determined by one of ordinary skill with nothing more than routine optimization being required. Examples of suitable amounts include those in the range of 10 ⁷ -10 ¹⁰ , or 10 ⁸ -10 ⁹ spores per ml.

The formulation may optionally comprise an additional active agent. The additional active agent may be active against ticks or may be active against some other undesirable target such as mites, bedbugs, etc. For example, the formulation may optionally comprise an acaricide such as an ixodicide. Acaricides may be chemicals, examples including amitraz, pyrethroids and organophosphates. In embodiments, the additional active agent is an acaricide and the formulation exhibits synergism, although in other embodiments the formulation exhibits additive activity against ticks. Other optional active agents include antibacterial agents, antifungal agents, insecticides, miticide agents, and the like, examples of which are well known. The additional active agent can be used where the specific activity of the selected active agent is desired. In addition to the foregoing components, it may be necessary or desirable in some cases (depending, for instance, on the particular formulation or method of administration) to incorporate any of a variety of additives, e.g., components that improve delivery, shelf-life and the like, but are non-active or are not necessarily against ticks. Suitable additives include acids, antioxidants, antimicrobials (e.g., as a preservative rather than as an active agent against ticks), buffers, colorants, crystal growth inhibitors, defoaming agents, diluents, emollients, fillers, flavorings, gelling agents, fragrances, lubricants, propellants, thickeners, salts, solvents, surfactants, other chemical stabilizers, or mixtures thereof. Examples of these additives can be found, for example, in M. Ash and I. Ash, Handbook of Pharmaceutical Additives (Hampshire, England: Gower Publishing, 1995), the contents of which are herein incorporated by reference.

In embodiments, the balance of the formulation (i.e., after the plant-derived oil, non-ionic surfactant, liquid hydrocarbon mixture, and conidia from M. anisopliae, and any other optional ingredients as described herein) is comprised of a solvent. In embodiments the solvent is water. For example, a formulation may comprise water in an amount in the range 10-90, 20-90, 30-90, 40-90, 50-90, 60-90, or 70-90%, or any other amount necessary based on the relative amounts of other components. In embodiments the amount of water is greater than or equal to 10, 20, 30, 40, 50, 60, or 70%.

The formulations herein are effective and are configured for controlling ticks. By the term "controlling" and "control" as used herein is meant to include any one or combinations of the following situations: the formula acts as a repellant: the formula acts as a killing agent; the formula causes a modification to a behavior of the tick (e.g., reduces the tick's desire to feed, or changes the feeding behavior of the tick); the formula acts to disrupt the breading process and/or breeding activities of ticks (e.g., causes sterility, kills tick eggs, or the like); and the formula acts to disrupt the growth of ticks. In some such situations, the control is a matter of reducing the degree to which an activity or behavior is observed - e.g., control may include reducing (but not eliminating, such as reducing by 10, 20, 30, 40, 50, 60, 70, 80, or 90%) an observed behavior in ticks. In other such situations, control may be absolute - e.g., ticks exposed to the formulation exhibit complete inability to breed. Where the formulation is a killing agent, the formulation may kill equal to or more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% of all ticks exposed to the formulation. The efficacy of the formulation in killing ticks may remain beyond those ticks that are initially contacted with the formulation. For example, an area treated with the formulation may remain toxic to ticks (i.e., ticks entering the region are killed) for a period of time such as greater than or equal to 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks. Alternatively or in addition, repellency of ticks may be exhibited by the formulations herein. Repellency means that, where the formulation is applied to a space, the number of ticks entering or remaining in that space after a given period of time is reduced compared to a similar area that is not treated with the formulation. Such reduction may be equal to or more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% of the number of ticks observed where the formulation is not in use. For example, a formulation can repel infestation or re-infestation of an animal (or other location) by ticks for a given period of time, such as greater than or equal to 1, 2, 3, or 4 weeks. Without wishing to be bound by theory, it is believed that the liquid hydrocarbon mixture has a repellency effect, and the amount of repellency of the formulation can be adjusted by adjusting the amount of the liquid hydrocarbon mixture. The formulations herein can exhibit combinations of the above types of control, such as acting as a killing agent and as a repellant formulation.

The formulations may exhibit control against ticks for a length of time after administration. The length of time of activity may depend on the exact formulation and other factors (environmental and otherwise), but may in embodiments be within the range of 1-60, 1-45, 1-30 or 1-14 days, or may be greater than or equal to 1, 3, 5, 7, 14, 21, 28, 35, or 45 days. It will be appreciated that the efficacy of the formulation may be reduced over such time periods but these periods are generally provided as those in which the formulation remains sufficiently active to obviate the need for re-administration of the formulation.

The formulation is active in controlling ticks, and this may include control of at least one of the following species of tick: Rhipicephalus appendiculatus, R.

decoloratus, R. pulchelus, R. microplus, Hyalomma truncatum and Amblyomma variegatum, or any combinations thereof.

Methods for preparing the formulations herein are also within the scope of the invention. The chemical components will be of a grade/purity suitable for the intended use, and such suitable grades/purities are readily determined by one of ordinary skill without need for anything more than routine experimentation. In embodiments, conidia are produced using diphasic fermentation technology. For example, conidia are produced via vegetative mycelia production by liquid medium followed by surface conidiation of the mycelia on solid substrate such as rice. In embodiments, preparing the formulation involves combining the conidia with the plant-derived oil, liquid hydrocarbon mixture, and non-ionic surfactant in amounts configured to impart a tick controlling property to the formulation as described herein. In embodiments the order of combining the components is not critical.

In embodiments, the formulation as prepared herein is administered to a location where tick control is desired. Such location may be a location in the environment, a location on an animal, or another location as desired and as suitable. In embodiments, the locations are those that are frequented by livestock,

particularly livestock that is susceptible to being affected by ticks (e.g., cows, goats, sheep, etc.). Locations in the environment include, for example, on plants that are eaten by, or in the vicinity of, livestock or any other animals susceptible to ticks.

Any suitable dosages and regimens of the formulations herein may be used in delivery. Suitable dosages and regimens may be taken from this disclosure or determined for the specific formulation, mode of delivery, and intended use.

Dosages and regimens are readily determined by one of ordinary skill without need for anything more than routine experimentation. For example, the formulation may be administered regularly such as weekly, or every 2 ^nd , 3 ^rd , or 4 ^th week, or may be administered "as needed". Suitable dosages include an amount in the range of 0.5-4, or 1-3 L per animal dosed. For example, an animal (e.g., cow) can be dosed with 0.5, 1, 1.5, 2, 2.5, or 3 L of formulation as described herein. The skilled artisan will appreciate, however, that the dosage will depend on the specifics of the formulation.

Any suitable modes of administration of the formulations herein may be used to deliver the formulation to the desired locations. Suitable modes of delivery may be determined based on the context (formulation, intended use, etc.), and such determination is readily determined by one of ordinary skill without need for anything more than routine experimentation. Examples of suitable modes of administration include spot treatment, spraying, and bulk administration of a liquid or aerosol formulation. Other modes of administration include drip-delivery devices and others known and later discovered. Throughout this disclosure, use of the term "or" is inclusive and not exclusive, unless otherwise indicated expressly or by context. Therefore, herein, "A or B" means "A, B, or both," unless expressly indicated otherwise or indicated otherwise by context. Moreover, "and" is both joint and several, unless otherwise indicated expressly or by context. Therefore, herein, "A and B" means "A and B, jointly or severally," unless expressly indicated otherwise or indicated otherwise by context.

It is to be understood that while the invention has been described in conjunction with examples of specific embodiments thereof, that the foregoing description and the examples that follow are intended to illustrate and not limit the scope of the invention. It will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention, and further that other aspects, advantages and

modifications will be apparent to those skilled in the art to which the invention pertains. The pertinent parts of all publications mentioned herein are incorporated by reference. All combinations of the embodiments described herein are intended to be part of the invention, as if such combinations had been laboriously set forth in this disclosure.

Examples

Example 1

The efficacy of compositions produced according to the disclosure herein was tested in the field at Kilgoris, Transmara District, Kenya. Conidia of M. anisopliae were formulated in 10% canola oil and 2.5% kerosene and applied on cattle. The efficacy was compared to amitraz and emulsifiable formulation of M. anisopliae without kerosene. Two applications of M. anisopliae and acaricide within 7 days resulted in 90.3% reduction of tick populations [R. appendiculatus, R. decoloratus and A variegatum) with the new formulation, 76.8% with M. anisopliae alone while 70.1% with acaricide Alpha-cypermethrin (see FIG. 1). Cattle were released after treatments and returned grazing in open field where they were regularly re- infested. After three weeks (two weeks after the last treatment), fewer number of ticks (47.2 ± 8.8) was recorded in M. anisopliae + kerosene as compared to negative control (338.7 ± 12.8) and alpha-cypermethrin (143.0 ± 19.7) treatments (see FIG. 1). In Figure 1, "CON-H20" represents treatment with water alone; "CON-F" is an emulsifiable water formulation + kerosene; "Ma7-F" is an emulsifiable formulation as prepared according to the disclosure; and "Acaricide" is Alpha-cypermethrin. The arrows indicate dates of treatment.

In the figures, "Ma 7" represents conidia from M. anisopliae, and "Ma 7-F" represents a formulation prepared according to the invention (see, e.g., Example 6 below).

Example 2

Trial was carried in Nguruman, Rift Valley, Kenya, to test the formulations herein and compared the results with Alpha-cypermethrin. A formulation as described herein was prepared and provided protection to cattle for at least two weeks despite re-infestation since animals were released after treatment to graze in tick-infested grass (see FIG. 2). In Figure 2, "CON-H20" represents treatment with water alone; "CON-F" is an emulsifiable water formulation + kerosene; "Ma7-F" is an emulsifiable formulation as prepared according to the disclosure; and "Acaricide" is Alpha-cypermethrin. The arrow indicates dates of treatment.

Example 3

A formulation according to the invention was prepared and had the advantage to reduce the attachment of ticks on the skin of the animal and the same time affects the feeding of ticks that are already attached on the skin. For instance, application of the formulation on rabbit's skin before ticks were allowed to attach resulted in 33.3% of tick attachment as compared to 78.3% in the control. Moreover, 60.0% of ticks were able to engorge in the control while only 20.0% in the fungal formulation. On the other hand, application of fungus on rabbit's skin after ticks have already attached resulted in 23.3% of engorged ticks compared to 90.0% in the control. More eggs were laid by female in the control (4,386) than in fungal formulation (605). Example 4

A formulation according to the disclosure was prepared. The formulation was applied to cattle and camels and shown to prevent infestation of ticks on animals for at least two weeks in two separate examples in Somaliland (see FIGs 3 and 4). In Figures 3 and 4, "CON-H20" is a sample with water alone, and "Ma7-F" represents an emulsifiable formulation according to the disclosure. The arrow indicates dates of treatment.

Example 5

Ticks that were collected at different sampling dates from cows after spray application of novel formulation and incubated in the laboratory succumbed to fungal infection; mortality ranging between 95-99% while mortality in the controls was less that than 3%.

Example 6

A formulation was prepared according to the invention, and comprised: 2.5% kerosene; 15% canola oil; TRITON®-X100 in an amount of 1.5%, and conidia of M. anisopliae in an amount of 10 ⁹ spores/ml, and the balance of water. The formulation was prepared as a bulk liquid and suitable for spot or spray administration.

Example 7

A formulation is prepared according to the invention. Each animal (cow) is sprayed with 2 Liters of formulation (suspension) which is the equivalent to 2.0 x 10 ⁸ conidia/mL. The dosage in the field is determined approximately by mixing 50 grams of conidia of Metarhizium anisopliae in 10 L of water containing 1 L of canola oil, 250 mL of TRITON®-X100 and 250 mL of kerosene. The concentration of one gram of conidia is estimated at 1.0 x 10 ¹⁰ conidia.

Example 8

In Figure 5, there is shown the mortality of ticks collected at different sampling days 1, 5 and 7 and incubated at room temperature following treatment with emulsifiable formulation as prepared herein.