Anthelminthic composition

Abstract

The present invention relates generally to methods for reducing the number of free- living parasitic helminths, particularly nematodes, on various surfaces by applying compositions comprising calcium nitrate (CN) and / or calcium ammonium nitrate (CAN).

Description

[0001] ANTHELMINTHIC COMPOSITION

[0002] TECHNICAL FIELD

[0003] The present invention relates generally to methods, compositions, and uses for reducing the number of free-living parasitic helminths, particularly nematodes, on forages, grasses and pastures.

[0004] BACKGROUND OF THE INVENTION

[0005] Livestock herds are subject to infection with internal parasites, including the parasitic helminths; roundworms, tapeworms and flukes (i.e., Nematoda, Cestoda and Trematoda) which have either free-living stages or intermediate hosts. Recurring infection with these parasites is a constant challenge to the health of livestock, particularly in young animals. Standard treatments comprise the use of synthetic compounds often called "dewormers", however, the use of such dewormers offers only a short-term solution. Many parasitic helminths are cosmopolitan in distribution and present naturally in pasture and field environments. Therefore, grazing livestock are continually subject to reinfection from parasites present in fields and pastures and require treatment after each new infection event.

[0006] A major drawback related to the routine use of synthetic dewormer compounds, is that overuse can select for parasite strains that are resistant to these compounds (e.g., levamisole, oxfendazole and ivermectin). There are also indications in the literature that deworming compounds may slow the development of immunity to these parasites in young animals and slow the decomposition of manure by impacting negatively on soil biota. Accordingly, there is a need in the art for new methods, compositions, and uses for controlling the populations of free-living parasitic helminths, particular free-living parasitic nematodes, and particularly on pastures or fields, such that infection and reinfection rates of all susceptible animals is reduced.

[0007] It is an object of the invention to provide methods, compositions, and / or uses for controlling free-living parasitic helminths, particularly free-living parasitic nematodes, and / or to go at least some way towards avoiding the above disadvantages in the art and / or that at least provides the public with a useful choice.

[0008] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

[0009] SUMMARY OF THE INVENTION

[0010] In a first aspect, the invention provides a method of controlling free-living stages of parasitic helminths in a treatment area comprising a surface; the method comprising applying a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) to the treatment area at an application rate sufficient to achieve a concentration of at least about 0.05 g / m2of nitrogen on the surface.

[0011] In a second aspect, the invention provides a method of controlling free-living stages of parasitic helminths in a treatment area comprising a surface; the method comprising applying a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) to the treatment area at a rate of 5-60 kg / ha of nitrogen.

[0012] In a third aspect, the invention provides a method of controlling free-living stages of parasitic helminths in a treatment area comprising a plant, wherein the plant comprises a leaf having a leaf surface, and wherein the treatment area has a leaf area index (LAI); the method comprising applying a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) to the treatment area such that the application rate in kg / ha of nitrogen divided by the LAI is at least about 0.8.

[0013] In a further aspect, the invention provides a use of a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) for controlling free-living stages of parasitic helminths in a treatment area comprising a surface; wherein the composition is applied to the treatment area at an application rate sufficient to achieve a concentration of at least about 0.05 g / m2of nitrogen on the surface.

[0014] In a further aspect, the invention provides a use of a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) for controlling free-living stages of parasitic helminths in a treatment area comprising a surface; wherein the composition is applied to the treatment area at a rate of 5-60 kg / ha of nitrogen.

[0015] In a further aspect, the invention provides a use of a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) for controlling free-living stages of parasitic helminths in a treatment area comprising a plant, wherein the plant comprises a leaf having a leaf surface, and wherein the treatment area has a leaf area index (LAI); wherein the composition is applied to the treatment area such that the application rate in kg / ha of nitrogen divided by the leaf area index (LAI) is at least about 0.8

[0016] In a further aspect, the invention provides a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) when used for controlling free-living stages of parasitic helminths in a treatment area comprising a surface; wherein the composition is applied to the treatment area at a rate sufficient to achieve a concentration of at least about 0.05 g / m2on the surface.

[0017] In a further aspect, the invention provides a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) when used for controlling free-living stages of parasitic helminths in a treatment area comprising a surface; wherein the composition is applied to the treatment area at a rate of 5-60 kg / ha of nitrogen.

[0018] In a further aspect, the invention provides a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) when used for controlling free-living stages of parasitic helminths in a treatment area comprising a plant, wherein the plant comprises a leaf having a leaf surface, and wherein the treatment area has a leaf area index (LAI); wherein the composition is applied to the treatment area such that the application rate in kg / ha of nitrogen divided by the leaf area index (LAI) is at least about 0.8. In a further aspect, the invention provides a use of calcium nitrate (CN) or calcium ammonium nitrate (CAN) in the manufacture of a composition for controlling free-living stages of parasitic helminths in a treatment area comprising a surface.

[0019] Any of the following embodiments, alone or in any combination, may apply to any one or more of the above aspects.

[0020] In some embodiments, the composition is applied to the treatment area at a rate sufficient to achieve a concentration of at least about 0.05 g / m2nitrogen on the surface, such as at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,

[0021] 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1,

[0022] 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5,

[0023] 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or at least about 6 g / m2nitrogen, and useful ranges may be selected between any of these values (for example, from 0.05 to 6, from 0.05 to 5.8, from 0.05 to 5.6, from 0.05 to 5.4, from 0.05 to 5.2, from 0.05 to 5, from

[0024] 0.05 to 4.8, from 0.05 to 4.6, from 0.05 to 4.4, from 0.05 to 4.2, from 0.05 to 4, from

[0025] 0.05 to 3.8, from 0.05 to 3.6, from 0.05 to 3.4, from 0.05 to 3.2, from 0.05 to 3, from

[0026] 0.2 to 6, from 0.2 to 5.8, from 0.2 to 5.6, from 0.2 to 5.4, from 0.2 to 5.2, from 0.2 to

[0027] 5, from 0.2 to 4.8, from 0.2 to 4.6, from 0.2 to 4.4, from 0.2 to 4.2, from 0.2 to 4, from 0.2 to 3.8, from 0.2 to 3.6, from 0.2 to 3.4, from 0.2 to 3.2, from 0.2 to 3, from 0.5 to 6, from 0.5 to 5.8, from 0.5 to 5.6, from 0.5 to 5.4, from 0.5 to 5.2, from 0.5 to 5, from 0.5 to 4.8, from 0.5 to 4.6, from 0.5 to 4.4, from 0.5 to 4.2, from 0.5 to 4, from 0.5 to 3.8, from 0.5 to 3.6, from 0.5 to 3.4, from 0.5 to 3.2, from 0.5 to 3, from 1 to 6, from 1 to 5.8, from 1 to 5.6, from 1 to 5.4, from 1 to 5.2, from 1 to 5, from 1 to 4.8, from 1 to 4.6, from 1 to 4.4, from 1 to 4.2, from 1 to 4, from 1 to 3.8, from 1 to

[0028] 3.6, from 1 to 3.4, from 1 to 3.2, from 1 to 3, from 1.2 to 6, from 1.2 to 5.8, from 1.2 to 5.6, from 1.2 to 5.4, from 1.2 to 5.2, from 1.2 to 5, from 1.2 to 4.8, from 1.2 to 4.6 from 1.2 to 4.4, from 1.2 to 4.2, from 1.2 to 4, from 1.2 to 3.8, from 1.2 to 3.6, from

[0029] 1.2 to 3.4, from 1.2 to 3.2, from 1.2 to 3, from 1.4 to 6, from 1.4 to 5.8, from 1.4 to

[0030] 5.6, from 1.4 to 5.4, from 1.4 to 5.2, from 1.4 to 5, from 1.4 to 4.8, from 1.4 to 4.6, from 1.4 to 4.4, from 1.4 to 4.2, from 1.4 to 4, from 1.4 to 3.8, from 1.4 to 3.6, from

[0031] 1.4 to 3.4, from 1.4 to 3.2, from 1.4 to 3, from 1.6 to 6, from 1.6 to 5.8, from 1.6 to

[0032] 5.6, from 1.6 to 5.4, from 1.6 to 5.2, from 1.6 to 5, from 1.6 to 4.8, from 1.6 to 4.6, from 1.6 to 4.4, from 1.6 to 4.2, from 1.6 to 4, from 1.6 to 3.8, from 1.6 to 3.6, from 1.6 to 3.4, from 1.6 to 3.2, from 1.6 to 3, from 1.8 to 6, from 1.8 to 5.8, from 1.8 to

[0033] 5.6, from 1.8 to 5.4, from 1.8 to 5.2, from 1.8 to 5, from 1.8 to 4.8, from 1.8 to 4.6, from 1.8 to 4.4, from 1.8 to 4.2, from 1.8 to 4, from 1.8 to 3.8, from 1.8 to 3.6, from 1.8 to 3.4, from 1.8 to 3.2, from 1.8 to 3, from 2 to 6, from 2 to 5.8, from 2 to 5.6, from 2 to 5.4, from 2 to 5.2, from 2 to 5, from 2 to 4.8, from 2 to 4.6, from 2 to 4.4, from 2 to 4.2, from 2 to 4, from 2 to 3.8, from 2 to 3.6, from 2 to 3.4, from 2 to 3.2, from 2 to 3, from 2.2 to 6, from 2.2 to 5.8, from 2.2 to 5.6, from 2.2 to 5.4, from 2.2 to 5.2, from 2.2 to 5, from 2.2 to 4.8, from 2.2 to 4.6, from 2.2 to 4.4, from 2.2 to 4.2, from 2.2 to 4, from 2.2 to 3.8, from 2.2 to 3.6, from 2.2 to 3.4, from 2.2 to 3.2, from

[0034] 2.2 to 3, from 2.4 to 6, from 2.4 to 5.8, from 2.4 to 5.6, from 2.4 to 5.4, from 2.4 to 5.2, from 2.4 to 5, from 2.4 to 4.8, from 2.4 to 4.6, from 2.4 to 4.4, from 2.4 to 4.2, from 2.4 to 4, from 2.4 to 3.8, from 2.4 to 3.6, from 2.4 to 3.4, from 2.4 to 3.2, from

[0035] 2.4 to 3, from 2.6 to 6, from 2.6 to 5.8, from 2.6 to 5.6, from 2.6 to 5.4, from 2.6 to 5.2, from 2.6 to 5, from 2.6 to 4.8, from 2.6 to 4.6, from 2.6 to 4.4, from 2.6 to 4.2, from 2.6 to 4, from 2.6 to 3.8, from 2.6 to 3.6, from 2.6 to 3.4, from 2.6 to 3.2, or from 2.6 to 3 g / m2nitrogen).

[0036] In some embodiments, the composition is applied at a rate of at least 5 kg / ha of nitrogen, such as at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or at least 60 kg / ha of nitrogen, and useful ranges may be selected between any of these values (for example, from 5 to 60, from 5 to 55, from 5 to 50, from 5 to 45, from 5 to 40, from 5 to 35, from 5 to 30, from 5 to 25, from 5 to 20, from 10 to 60, from 10 to 55, from 10 to 50, from 10 to 45, from 10 to 40, from 10 to 35, from 10 to 30, from 10 to 25, from 10 to 20, from 15 to 60, from 15 to 55, from 15 to 50, from 15 to 45, from 15 to 40, from 15 to 35, from 15 to 30, from 15 to 25, from 15 to 20, from 20 to 60, from 20 to 55, from 20 to 50, from 20 to 45, from 20 to 40, from 20 to 35, from 20 to 30, from 20 to 25, from 25 to 60, from 25 to 55, from 25 to 50, from 25 to 45, from 25 to 40, from 25 to 35, from 25 to 30, from 30 to 60, from 30 to 55, from 30 to 50, from 30 to 45, from 30 to 40, from 30 to 35, from 35 to 60, from 35 to 55, from 35 to 50, from 35 to 45, from 35 to 40, from 40 to 60, from 40 to 55, from 40 to 50, or from 40 to 45 kg / ha of nitrogen).

[0037] In a preferred embodiment, the composition is applied at a rate of 25-60 kg / ha of nitrogen.

[0038] In some embodiments, the treatment area comprises a plant, wherein the plant comprises a leaf having a leaf surface, and wherein the treatment area has a leaf area index (LAI). In some embodiments the composition is applied to the treatment area such that the application rate in kg / ha of nitrogen divided by the leaf area index (LAI) is at least about 0.8, such as at least about 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or at least about 60, and useful ranges may be selected between any of these values (for example, from 0.8 to 60, from 0.8 to 50, from 0.8 to 40, from 0.8 to 30, from 0.8 to 20, from 0.8 to 18, from 0.8 to 16, from 0.8 to 15, from 0.8 to 14, from 0.8 to 13, from 0.8 to 12, from 0.8 to 11, from 0.8 to 10 2.0 to 60, from 2.0 to 50, from 2.0 to 40, from 2.0 to 30, from 2.0 to 20, from 2.0 to 18, from 2.0 to 16, from 2.0 to 15, from 2.0 to 14, from 2.0 to 13, from 2.0 to 12, from 2.0 to 11, from 2.0 to 10, 4.0 to 60, from 4.0 to 50, from 4.0 to 40, from 4.0 to 30, from 4.0 to 20, from 4.0 to 18, from 4.0 to 16, from 4.0 to 15, from 4.0 to 14, from 4.0 to 13, from 4.0 to 12, from 4.0 to 11, from 4.0 to 10, 5.0 to 60, from 5.0 to 50, from 5.0 to 40, from 5.0 to 30, from 5.0 to 20, from 5.0 to 18, from 5.0 to 16, from 5.0 to 15, from 5.0 to 14, from 5.0 to 13, from 5.0 to 12, from 5.0 to 11, from 5.0 to 10, 6.0 to 60, from 6.0 to 50, from 6.0 to 40, from 6.0 to 30, from 6.0 to 20, from 6.0 to 18, from 6.0 to 16, from 6.0 to 15, from 6.0 to 14, from 6.0 to 13, from 6.0 to 12, from 6.0 to 11, or from 6.0 to 10).

[0039] In some embodiments, the composition comprises, consists essentially of, or consists of soluble CAN consisting of calcium nitrate and ammonium nitrate. In some embodiments, the composition comprises, consists essentially or, or consists of calcium nitrate (CN).

[0040] In some embodiments, the composition has a molar ratio of calcium nitrate to ammonium nitrate of at least 1:1, such as at least 2:1, 3:1, 4: 1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1, and useful ranges may be selected between any of these values (for example, from 1:1 to 20:1, from 2:1 to 20:1, from 2:1 to 18:1, from 2:1 to 16:1, from 2:1 to 14:1, from 2:1 to 12:1, from 2:1 to 10:1, from 2:1 to 8:1, from 2:1 to 6:1, from 3:1 to 20:1, from 3:1 to 18:1, from 3:1 to 16:1, from 3:1 to 14:1, from 3:1 to 12:1, from 3:1 to 10:1, from 3:1 to 8:1, from 3:1 to 6:1, from 4:1 to 20:1, from 4:1 to 18:1, from 4:1 to 16:1, from 4:1 to 14:1, from 4:1 to 12:1, from 4:1 to 10:1, from 4:1 to 8:1, from 4:1 to 6:1, from 5:1 to 20:1, from 5:1 to 18:1, from 5:1 to 16:1, from 5:1 to 14: 1, from 5: 1 to 12:1, from

[0041] 5:1 to 10:1, from 5: 1 to 8: 1, or from 5:1 to 6:1). In one embodiment, the composition is in a liquid form.

[0042] In some embodiments, the composition further comprises an agriculturally acceptable carrier, diluent or excipient.

[0043] In some embodiments, the composition further comprises an agriculturally acceptable buffering agent, preferably an agriculturally acceptable buffering agent selected from TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, or MES.

[0044] In some embodiments, the composition further comprises one or more additional active agents selected from the group consisting of anti-scorching agents, osmotic protection agents, nitrification inhibitors, urease inhibitors, pesticides, insecticides, plant and insect growth regulators, trace elements for plants and animals, plant nutrients, soil conditioners, soil nutrients, and bio-control agents. In a preferred embodiment, the composition further comprises an anti-scorching agent, preferably betaine trimethylglycine.

[0045] In one embodiment, the composition is applied using a spray nozzle, preferably a spray nozzle adapted for foliar application.

[0046] In some embodiments, the treatment area comprises, or consists of, a house, garage, shed, farm building, outbuilding, milking shed, barn, or other enclosed or partially enclosed structure where free living parasitic helminths, particularly free living parasitic nematodes are present, or may be present. In some embodiments, the treatment area comprises, or consists of, pasture.

[0047] In some embodiments, the treatment area comprises a plant. In some embodiments, the treatment area comprises animal waste, such as faeces. In some embodiments, the treatment area comprises a plant and / or animal waste, such as faeces. In some embodiments, the surface is a surface of the plant and / or animal waste. In some embodiments, the treatment area comprises a surface that is a plant surface and / or a faeces surface. In some embodiments, the plant comprises a leaf having a leaf surface. In some embodiments, the treatment area comprises a surface that is a leaf surface. In some embodiments, the plant comprises a leaf having a leaf surface and the treatment area comprises a surface that is the leaf surface.

[0048] In some embodiments, the plant is a plant used for grazing and / or harvesting. In some embodiments, the plant is a livestock food plant. In some embodiments, the livestock food plant is a grass plant and / or a legume plant.

[0049] In some embodiments, the method, use, or composition also increases the growth of the plant.

[0050] In some embodiments, the parasitic helminths are mammalian endoparasites. In some embodiments, the parasitic helminths are parasites of canines, felines, bovines, ovines, cervines, equines, caprines, porcines, lagomorphs, rodents, camelids, and / or hominids; preferably parasites of bovines, ovines, cervines, and / or equines. In some embodiments, the parasitic helminths are selected from the group consisting of flukes (trematodes), tapeworms (cestodes) and roundworms (nematodes). In some embodiments, the parasitic helminths are selected from the group consisting of Ancylostomatoidea, Strongyloidea, Trichostrongyloidea, and Metastrongyloidea.

[0051] In some embodiments, the composition reduces the number of live parasitic helminth larvae by at least about 25% in an in vitro faecal assay when applied at a rate of 5000 L / ha, such as less than about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%, and useful ranges may be selected between any of these values (for example, from about 25% to about 100%, from about 25% to about 95%, from about 35% to about 100%, from about 35% to about 95%, from about 45% to about 100%, from about 45% to about 95%, from about 55% to about 100%, from about 55% to about 95%, from about 65% to about 100%, from about 65% to about 95%, from about 75% to about 100%, from about 75% to about 95%, from about 85% to about 100%, from about 85% to about 95%, or from about 95% to about 100%).

[0052] In some embodiments, the composition reduces the number of live parasitic helminth larvae by at least about 25% in an in vitro leaf assay when applied at a rate of 350 L / ha, such as less than about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%, and useful ranges may be selected between any of these values (for example, from about 25% to about 100%, from about 25% to about 95%, from about 35% to about 100%, from about 35% to about 95%, from about 45% to about 100%, from about 45% to about 95%, from about 55% to about 100%, from about 55% to about 95%, from about 65% to about 100%, from about 65% to about 95%, from about 75% to about 100%, from about 75% to about 95%, from about 85% to about 100%, from about 85% to about 95%, or from about 95% to about 100%).

[0053] In some embodiments, the number of parasitic helminths in the treatment area is reduced by at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%, and useful ranges may be selected between any of these values (for example, from about 10% to about 100%, from about 10% to about 80%, from about 10% to about 60%, from about 20% to about 100%, from about 20% to about 80%, from about 20% to about 60%, from about 30% to about 100%, from about 30% to about

[0054] 80%, from about 30% to about 60%, from about 40% to about 100%, from about 40% to about 80%, from about 40% to about 60%, from about 50% to about 100%, from about 50% to about 80%, or from about 50% to about 60%). Preferably, the number of parasitic helminths in the treatment area is reduced by at least about 25%, more preferably at least about 50%.

[0055] In some embodiments, the treatment area comprises soil, and wherein the top 50 mm of the soil comprises fewer than about 4,000,000 parasitic helminths per m3of soil, such as less than about 3500000, 3000000, 2500000, 2000000, 1500000, 1000000, 500000, 400000, 300000, 200000, 100000, 90000, 80000, 70000, 60000, 50000, 40000, 30000, 20000, 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 250, 100, 50, or about 0, and useful ranges may be selected between any of these values (for example, from about 0 to about 4000000, from about 0 to about 1000000, from about 0 to about 50000, from about 0 to about 10000, from about 0 to about 5000, from about 0 to about 1000, from about 1000 to about 4000000, from about 1000 to about 1000000, from about 1000 to about 50000, from about 1000 to about 10000, or from about 1000 to about 5000).

[0056] In some embodiments, the treatment area comprises soil, and wherein the soil comprises, consists essentially of, or consists of sand, silt, pumice, ash, or any combination of any two or more of these.

[0057] In some embodiments, the composition is applied during or between the months of October-March in the Northern Hemisphere or during or between the months of April- September in the Southern Hemisphere; preferably during or between the months of November-February in the Northern Hemisphere or during or between the months of

[0058] May-August in the Southern Hemisphere; more preferably during or between the months of December-February in the Northern Hemisphere or during or between the months of June-August in the Southern Hemisphere.

[0059] In some embodiments, the composition is applied at a time between the Autumnal equinox and the Spring equinox. In some embodiments, the composition is applied at a time that is at most 100 days before or after the Winter solstice, preferably at most 90, 80, 70, 60, 50, 40, or at most 30 days. In some embodiments, the composition is applied during Autumn and / or Winter; preferably during Winter.

[0060] In some embodiments, the composition is applied at a time of year when the mean daily temperature, calculated as (daily maximum temperature + daily minimum temperature) 2, is less than about 20°C, such as less than about 19°C, 18°C, 17°C, 16°C, 15°C, 14°C, 13°C, 12°C, 11°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C, 2°C, 1°C, or about 0°C, and useful ranges may be selected between any of these values (for example, from about 0°C to about 20°C, from about 0°C to about 18°C, from about 0°C to about 16°C, from about 0°C to about 14°C, from about 0°C to about 12°C, from about 0°C to about 10°C, from about 0°C to about 8°C, from about 5°C to about 20°C, from about 5°C to about 18°C, from about 5°C to about 16°C, from about 5°C to about 14°C, from about 5°C to about 12°C, from about 5°C to about 10°C, from about 5°C to about 8°C, from about 10°C to about 20°C, from about 10°C to about 18°C, from about 10°C to about 16°C, from about 10°C to about 14°C, or from about 10°C to about 12°C). Preferably the composition is applied at a time of year when the mean daily temperature, calculated as (daily maximum temperature + daily minimum temperature)

[0061] 2, is less than about less than about 18°C, more preferably less than about 16°C, most preferably less than about 14°C. In some embodiments, the composition is applied at an ambient temperature of less than about 20°C, such as less than about 19°C, 18°C, 17°C, 16°C, 15°C, 14°C, 13°C, 12°C, 11°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C, 2°C, 1°C, 0°C, -1°C, -2°C, -3°C, -4°C, -5°C, -6°C, -7°C, -8°C, -9°C, or less than about -10°C, and useful ranges may be selected between any of these values (for example, from about -10°C to about 20°C, from about -10°C to about 15°C, from about -10°C to about 10°C, from about -10°C to about 5°C, from about -5°C to about 20°C, from about -5°C to about 15°C, from about -5°C to about 10°C, from about -5°C to about 5°C, from about 0°C to about 20°C, from about 0°C to about 15°C, from about 0°C to about 10°C, or from about 0°C to about 5°C).

[0062] In some embodiments, the composition is applied at a time of year when the mean daily relative humidity, calculated as (daily maximum temperature + daily minimum temperature) 2, is less than about 50%, such as less than about 45%, 40%, 35%, or less than about 30%, and useful ranges may be selected between any of these values (for example, from about 30% to about 50%, from about 30% to about 45%, or from about 30% to about 40%).

[0063] In some embodiments, the composition is applied during a drought. In some embodiments, the composition is applied when the daily rainfall, averaged over 1 week, is at least about 5 mm less than the typical daily rainfall for the treatment area, such as at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or at least about 60 mm less, and useful ranges may be selected between any of these values (for example, from about 5 to about 60, from about 5 to about 50, from about 15 to about 60, from about 15 to about 50, from about 25 to about 60, from about 25 to about 50, from about 35 to about 60, or from about 35 to about 50). In some embodiments, the treatment area comprises a plant and the composition is applied after the plant is harvested. In some embodiments, the treatment area comprises, or consists of, pasture and the composition is applied after the pasture is harvested. Preferably, the treatment area is not grazed between the plant or the pasture being harvested and the composition being applied. In some embodiments, the plant or the pasture is harvested for hay or silage. In some embodiments, the treatment area comprises a plant and the composition is applied less than about 24 weeks after the plant is harvested, such as less than about 22, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 1, or about 0 weeks, and useful ranges may be selected between any of these values (For example, from about 0 to about 24, from about 0 to about 18, from about 0 to about 12, or from about 0 to about 6).

[0064] In some embodiments, the treatment area comprises a newly sewn plant. In some embodiments, the composition is applied to a treatment area comprising, or consisting of, newly sewn plants. In some embodiments, the composition is applied after sewing plants in the treatment area.

[0065] In some embodiments, the treatment area (preferably wherein the treatment area comprises a plant) has not been grazed for at least about 2 weeks prior to applying the composition, such as at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or at least about 52 weeks, and useful ranges may be selected between any of these values (for example, from about 2 to about 52, from about 2 to about 42, from about 2 to about 32, from about 2 to about 22, from about 2 to about 12, from about 4 to about 52, from about 4 to about 42, from about 4 to about 32, from about 4 to about 22, from about 4 to about 12, from about 6 to about 52, from about 6 to about 42, from about 6 to about 32, from about 6 to about 22, from about 6 to about 12, from about 8 to about 52, from about 8 to about 42, from about 8 to about 32, from about 8 to about 22, or from about 8 to about 12).

[0066] In some embodiments, the parasitic helminths are selected from the group consisting of Haemonchus, Teladorsagia, Trichostrongylus, and Cooperia. Preferably, the parasitic helminths are selected from the group consisting of Teladorsagia, Trichostrongylus, and Cooperia.

[0067] Various embodiments of the different aspects of the invention as discussed above are also set out below in the detailed description of the invention, but the invention is not limited thereto.

[0068] Other aspects of the invention may become apparent from the following description which is given by way of example only and with reference to the accompanying drawings.

[0069] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0070] This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0071] BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The invention will now be described by way of example only and with reference to the drawings in which:

[0073] Figure 1 shows the nematicidal effect of CAN formulations in an in vitro leaf assay.

[0074] Values shown are the mean (± 95% confidence interval) number of L3 recovered from the leaf (3 days post treatment; 10 replicates per treatment). Percent efficacy compared to water is indicated above the bars. All treatments were adjusted to a pH of approximately 8 using either Ca(OH)2 or dropwise addition of a 28% ammonia solution. Product identifier, formulation components, N content and pH (prior to application) is listed below the x-axis.

[0075] Figure 2 shows the effect of CAN concentration and volume in an in vitro leaf assay.

[0076] Values shown are the mean (± 95% confidence interval) number of L3 recovered from the leaf (3 days post treatment; 10 replicates per treatment). Percent efficacy compared to water is indicated above the bars. Treatment, volume, concentration and total amount of N applied per petri dish are indicated below the x-axis.

[0077] Figure 3 shows the nematicidal effect of soluble CAN on L3 recovered from grass in a tunnel house trial. Values shown are mean (± SEM) number of L3 recovered from herbage (4 days post-treatment; 4 replicates per treatment). Efficacy compared to the water control is listed above each bar. Figure 4 shows the nematicidal effect of soluble CAN (CAN) and calcium nitrate (CN) on L3 recovered from grass in a tunnel house trial. Values shown are the mean (± 95% confidence interval) number of L3 recovered from herbage (4 days post treatment; 5 replicates per treatment). Percent efficacy compared to water is indicated above the bars.

[0078] Figure 5 shows the effect of grass height and volume on soluble CAN formulation efficacy in a conveyor belt trial. Values shown are the mean (± 95% confidence interval) number of L3 recovered from herbage (4 days post treatment; 6 replicates per treatment). Turfs were trimmed at the start of the trial to four different herbage heights (1.5, 4, 6.5 and 10 cm). Turfs for each height were treated with soluble CAN at 0, 214, 379 or 591 L / ha. Turfs receiving no soluble CAN (i.e., 0 L / ha) were used as a control and treated with water at 282 L / ha.

[0079] Figure 6 shows the worm burden of lambs grazed on treated or untreated pasture. Values shown are the mean (± 95% confidence interval) number of worms (adults + L4) recovered from each organ for each group. A significant reduction in worm burden was observed following pasture treatment with soluble CAN. Group 1 and 2 grazed the trial site following a single or double treatment application, respectively. ABO = abomasum; SI = small intestine.

[0080] Figure 7 shows the worm burden by genera. Values shown are the mean (± 95% confidence interval) number of worms recovered by species. Group 1 and 2 grazed the trial site following a single or double treatment application respectively.

[0081] Figure 8 shows the faecal egg count (FEC) of lambs grazed on treated (solid line) or untreated (dashed line) pasture. Values shown are the mean ± 95% confidence interval. Figure 9 shows the faecal egg count (FEC) of lambs grazed on treated (solid line) or untreated (dashed line) pasture. Values shown are the mean ± 95% confidence interval.

[0082] Figure 10 shows the worm burden of lambs grazed on treated (Can) or untreated (Control) pasture by worm genus. Values shown are the mean ± 95% confidence interval. Coop - Cooperia; Tela - Teladorsagia Haem - Haemonchus; Trie - Trichostrongylus .

[0083] Figure 11 shows the worm burden of lambs grazed on treated (Can) or untreated (Control) pasture by worm genus. Values shown are the mean ± 95% confidence interval. Coop - Cooperia; Tela - Teladorsagia Haem - Haemonchus; Trie - Trichostrongylus .

[0084] Figure 12 shows the proportion (±95% CI) of total, Teladorsagia circumcincta (dashed line) and Trichostrongylus colubriformis (solid line) infective stage larvae recovered from soil on previously worm-free plots where faeces containing eggs were placed on the pasture surface at monthly intervals for 13-18 months averaged across three sites in Example 6, Trial 1.

[0085] Figure 13 shows the proportion (±95% CI) of total, Teladorsagia circumcincta (dashed line) and Trichostrongylus colubriformis (solid line) infective stage larvae recovered from soil on previously worm-free plots where faeces containing eggs were placed on the pasture surface at monthly intervals for 13 months averaged across two sites in Example 7, Trial 2.

[0086] Figure 14 shows the proportion (±95% CI) of total, Teladorsagia circumcincta (dashed line) and Trichostrongylus colubriformis (solid line) infective stage larvae recovered from herbage on previously worm free plots when faeces containing eggs were buried in soil at monthly intervals for 13-18 months, averaged across three sites in Example 7, Trial 1.

[0087] Figure 15 shows the mean (±95% CI) number of infective parasite larvae recovered from herbage in a tunnel trial.

[0088] Figure 16 shows the mean (±95% CI) number of infective parasite larvae recovered from herbage in field plots.

[0089] DETAILED DESCRIPTION OF THE INVENTION

[0090] Definitions

[0091] Unless otherwise specified, all technical and scientific terms used herein are to be understood as having the same meanings as is understood by one of ordinary skill in the relevant art to which this disclosure pertains. Examples of definitions of common terms in medicine, molecular biology and biochemistry can be found in: Dictionary of Microbiology and Molecular Biology, Singleton et al., 2ndedition, (1994); The Encyclopedia of Molecular Biology, Kendrew et al. (Eds.), Blackwell Science Ltd., (1994); Molecular Biology and Biotechnology: a Comprehensive Desk Reference, Robert A.

[0092] Meyers (Ed.), VCH Publishers, Inc., (1995); The Dictionary of Cell & Molecular Biology, 4th Edition, Lackie. J (Ed.), Academic Press Inc (2007); and The Oxford Dictionary of Biochemistry and Molecular Biology, 2ndedition, Cammack et al. (Eds.), Oxford University Press Inc. (2006); Nematode parasites of vertebrates: their development and transmission. Anderson, R. C. (2000) University of Guelph, Guelph, Ontario, Canada;

[0093] Greenwood, Norman N.; Earnshaw, Alan (1997). Chemistry of the Elements (2ndEd.).

[0094] Butterworth-Heinemann. It is also believed that practice of the present invention can be performed using standard chemical and biochemical protocols and procedures as known in the art, and as described, for example in the above reference materials, and other commonly available reference materials relevant in the art to which this disclosure pertains, and which are all incorporated by references herein in their entireties.

[0095] The following definitions are presented to better define the present invention and as a guide for those of ordinary skill in the art in the practice of the present invention.

[0096] The term "ambient temperature" as used herein means the temperature of the environment (for example, the air temperature) where the composition is to be applied.

[0097] The term "helminth" as used herein means worm, taking its generally accepted meaning as known in the art. The term "parasitic helminth" as used herein is also used as recognized in the art and means the three groups of medically relevant helminths, Flukes (Trematodes), Tapeworms (Cestodes) and Roundworms (Nematodes) (Medical Microbiology. 4thEd. Baron, S ed., University of Texas Medical Branch at Galveston; 1996; Chap. 86). In some embodiments the free-living stage of a parasitic helminth is a free-living parasitic nematode. Preferably the free-living parasitic nematode is a known parasite of bovine, cervine, ovine, porcine, caprine, equine, and camelids.

[0098] The term "free-living stage of a parasitic helminth" as used herein means any stage of a parasitic helminth outside of the animal. For example, a free-living stage of a parasitic helminth can be an egg or larval (LI, L2, L3) stages of a parasitic helminth, including all larval stages.

[0099] The terms "control" and "controlling" (and similar grammatical constructions) as used herein with regard to free-living parasitic helminths mean that the composition of the invention is effective in reducing the population of the free-living parasitic helminth in an environment treated with the composition as compared to an environment that is not treated, including both the treated environment pre-treatment, and an untreated control environment.

[0100] More specifically, "control" and "controlling", as used herein, mean that the composition is effective in killing the respective free-living parasitic helminth(s), inhibiting its growth, development, movement, and / or survival. Generally, when used for the treatment of an environment, the composition of the invention is applied directly or indirectly to various surfaces present in the environment, but not limited thereto.

[0101] By way of non-limiting example, where the environment is a pasture or field, the composition of the invention may be applied directly or indirectly to the surfaces of any plants, other biological materials including faeces or organisms present in the pasture or field.

[0102] "Pasture(s)" as used herein refers to any area used for grazing, such as a field or paddock and the like, and is intended to include all of the component parts typically found in a pasture as known in the art such as grass, herbage, and forage, but not limited thereto.

[0103] The terms "leaf area index" and "LAI" as used herein refer to a value that describes the amount of leaf area per unit ground. Leaf area index is defined as the ratio of one-sided leaf area per unit ground area (for example, the leaf area in m2divided by the ground area in m2). Leaf area index can be measured using means known in the art.

[0104] "Contact" and "contacting" as used herein mean that the composition of the invention is brought into contact with a free-living parasitic helminth. "Brought into contact" means that the composition is either contacted to the free-living parasitic helminth, or is placed in a treatment environment, for example a leaf surface, such that a free-living parasitic helminth will come into contact with the composition as a matter of course. Preferably the free-living parasitic helminth will come into contact with the composition of the invention by contacting a surface, for example a leaf surface, to which the composition of the invention has been applied.

[0105] The term "anthelmintic" as used herein takes its commonly accepted meaning in the art and refers to a composition that controls parasitic helminths, including free-living parasitic helminths, particularly free-living parasitic nematodes.

[0106] The terms "control environment", "control locus" and a "control treatment" take their ordinary meaning in the art and are used herein to indicate environments, loci and / or treatments from which baseline data is gathered for comparison to data obtained from a corresponding treatment environment or locus, allowing identification of any variation seen between the baseline and the treatment environment and / or locus.

[0107] A level "higher" or "lower" than a control, or a change or deviation from a control in one embodiment is statistically significant. A higher level, lower level, deviation from, or change from a control level or mean control level can be considered to exist if the level differs from the control level by 5% or more, by 10% or more, by 20% or more, or by 50% or more compared to the control level. Statistically significant may alternatively be calculated as P < 0.05, P < 0.01, or P < 0.001. In a further alternative, higher levels, lower levels, deviation, and changes can be determined by recourse to assay reference limits or reference intervals. These can be calculated from intuitive assessment or nonparametric methods. Overall, these methods calculate the 0.025, and 0.975 fractiles as 0.025* (n+1) and 0.975 (n+1). Such methods are well known in the art (Hunt et al., 1997 Clin. Endocrinol. 47:287-296; The Immunoassay Handbook. 3rdedition, ed. David Wild. Elsevier Ltd, 2005). The terms "reduces", "reduced" and "reducing" and other grammatical variations as used herein mean the same thing as "lower".

[0108] A "statistically significant amount" as used herein describes a mathematical measure of difference between groups. The difference is said to be statistically significant if it is greater than what might be expected to happen by chance alone. For example, in some embodiments, a difference is said to be statistically significant if there is less than a 5%, 1%, or 0.1% chance of an equal or greater difference occurring by chance (e.g. a p value < 0.05, < 0.01, or < 0.001).

[0109] The term "weight for weight (w / w)" means the amount of the solute as a percent (by mass) of the total solution.

[0110] As used herein, where a value or integer is indicated as a "%" only with no units appended, the "%" refers to a mass concentration (mass / volume).

[0111] The term "comprising" as used in this specification means "consisting at least in part of". When interpreting statements in this specification that include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as "comprise" and "comprised" are to be interpreted in the same manner.

[0112] The term "consisting essentially of" as used herein means the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0113] The term "consisting of" as used herein means the specified materials or steps of the claimed invention, excluding any element, step, or ingredient not specified in the claim. The inventors believe that they are the first to show that a composition of the invention, comprising or consisting essentially of at least one compound selected from the group consisting of calcium nitrate (CN) and calcium ammonium nitrate (CAN), is effective at controlling free-living parasitic helminths, particularly free-living parasitic nematodes. Specifically, the inventors believe they are the first to identify that a composition of the invention is effective at controlling free-living parasitic helminths, particularly free-living parasitic nematodes when the composition is applied directly to the helminths, or when it is applied directly to a treatment area such as a pasture where the free-living parasitic helminths will come into contact with the composition.

[0114] In particular, the inventors have identified that a composition of the invention can be used to control free-living parasitic helminths in fields and / or pastures by applying the composition of the invention to fields and / or pastures prior to the field and / or pasture being grazed by stock animals.

[0115] Accordingly, in one aspect the invention provides a method of controlling free-living stages of parasitic helminths in a treatment area comprising a surface; the method comprising applying a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) to the treatment area at an application rate sufficient to achieve a concentration of at least about 0.05 g / m2of nitrogen on the surface.

[0116] In another aspect, the invention provides a method of controlling free-living stages of parasitic helminths in a treatment area comprising a surface; the method comprising applying a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) to the treatment area at a rate of 5-60 kgN / ha of nitrogen. In another aspect, the invention provides a method of controlling free-living stages of parasitic helminths in a treatment area comprising a plant, wherein the plant comprises a leaf having a leaf surface, and wherein the treatment area has a leaf area index (LAI); the method comprising applying a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) to the treatment area such that the application rate in kg / ha of nitrogen divided by the LAI is at least about 0.8.

[0117] It will be appreciated that the use of calcium compounds and nitrate compounds is known in some agricultural contexts as soil fertilisers, particularly as sources of macro- (e.g., calcium) and micro- (e.g., selenium) minerals and nitrogen. In such uses, calcium nitrate and / or calcium ammonium nitrate are typically applied as granules and / or are applied to the soil, avoiding leaf contact which can cause scorching. In contrast, the methods of the present invention are not for the purposes of fertilising the soil, although of course some fertilising effect may also occur. Accordingly, in some embodiments the present methods comprise applying a composition in liquid form, and / or comprise applying the composition to the treatment area at an application rate sufficient to achieve a concentration of at least about 0.05 g / m2of nitrogen on the plant surface, such as on a leaf surface, to control free-living parasitic helminths present thereon.

[0118] Compositions

[0119] As the reader will no doubt appreciate, one main point of difference between a composition of the invention that is an anthelmintic and known anthelmintic compositions lies in the external application of the composition of the invention to the environment in which free-living parasitic forms of helminths are found. This is in sharp contrast to anthelmintic compositions know in the art, and to methods of treating parasitic helminths of livestock as known in the art, both of which are directed to internal use in an affected animal.

[0120] The methods described herein comprise applying a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) to a treatment area. Also provided are uses of a composition comprising, consisting essentially of, or consisting of CN or CAN; and compositions comprising, consisting essentially of, or consisting of CN or CAN.

[0121] In some embodiments the composition is a pesticidal composition, preferably an anthelmintic. In some embodiments the anthelmintic is effective at controlling free- living parasitic helminths, preferably free-living parasitic nematodes, upon contact.

[0122] In some embodiments, contact between the anthelmintic and the free-living parasitic helminths occurs in a treatment area, preferably on a leaf surface.

[0123] In some embodiments, the composition comprises, consists essentially of, or consists of calcium nitrate (CN). In some embodiments, the composition comprises, consists essentially of, or consists of calcium ammonium nitrate (CAN).

[0124] "Calcium nitrate" or"CN" as used herein refers to any compound or mixture of compounds that, when dissolved in water, produce Ca2+and NO3 ions. A preferred embodiment of calcium nitrate is the inorganic compound with the chemical formula Ca(NO3)2(H2O)x, whether in its hydrated or anhydrous form. However, it will be appreciated that other compounds or mixtures of compounds may be used to provide sources of calcium and nitrate ions. For example, any mixture of compounds that produces Ca2+and NO3 ions when dissolved in water is considered to be "calcium nitrate" for the purposes of this specification, even if the original compounds did not contain Ca(NOs)2. In a preferred embodiment, the CN has a molar ratio of calcium to nitrate of about 1:2.

[0125] The term "calcium ammonium nitrate" or "CAN" refers to a group of different formulations that comprise calcium, ammonium, and nitrate. For example, one embodiment of CAN is a mixture of calcium carbonate (CaCOs) and ammonium nitrate (NH4NO3).

[0126] It will be appreciated by the person skilled in the art that in fertiliser form, calcium is typically calcium carbonate used in solid form and not solubilised in water for spray application to pasture or grazed crops.

[0127] In a preferred embodiment, the CAN comprises soluble CAN. The term "soluble CAN" refers to a double salt of calcium nitrate (Ca(NOs)2) and ammonium nitrate (NH4NO3). Soluble CAN may be produced by dissolving calcium nitrate and ammonium nitrate in water in an appropriate ratio.

[0128] It will be appreciated that the relative proportion of calcium nitrate to ammonium nitrate may vary. In some embodiments, the soluble CAN has a molar ratio of calcium nitrate to ammonium nitrate of at least 1 : 1, preferably at least 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1 and useful ranges may be selected between any of these ratios (for example, from 1: 1 to 10: 1, from 2: 1 to 10: 1, from 3: 1 to 10: 1, from 4: 1 to 10: 1, from 5: 1 to 10: 1, from 1: 1 to 7: 1, from 2: 1 to 7: 1, from 3: 1 to 7: 1, or from 4: 1 to 7: 1, from 1 : 1 to 5: 1, from 2: 1 to 5: 1, from 3: 1 to 5: 1, or from 4: 1 to 5: 1). Preferably, the soluble CAN has a molar ratio of calcium nitrate to ammonium nitrate of about 5: 1. The CN or CAN may be provided in a number of different forms, for example as a solution or as a solid (e.g., a powder or a granule) suitable for dissolving to form a solution. In some embodiments, the CN or CAN is provided as one or more solids, separate or combined, that can be dissolved to form a CN or CAN solution. For example, CAN may be provided as a combination of calcium nitrate and ammonium nitrate powders, either mixed together or provided separately.

[0129] It will be appreciated that compositions formulated for application to a treatment area, for example to a pasture, are preferably in a liquid form, such as a solution or suspension in a liquid, such as water. In some embodiments, the composition is in liquid form, such as a solution or a suspension. In some embodiments, the composition is an aqueous solution. Alternatively, compositions formulated for application to a treatment area such as pasture may be formulated to be dissolved or suspended in a liquid prior to application. For example, the composition may be formulated as water-soluble or water-dispersible powder or granules.

[0130] In some embodiments, the composition is formulated for direct application or indirect application to a treatment area, such as a pasture. In some embodiment the composition is formulated to be applied as a liquid, slurry, spray, mist, aerosol, micro aerosol, or gel.

[0131] In some embodiments, the composition is formulated for, or is in a form that, provides controlled release of the CN and / or CAN. In some embodiments, the composition is formulated for, or is in a form that, provides controlled dissolution. In some embodiments, controlled release and / or controlled dissolution comprises release of the CN and / or CAN or dissolution over at least 30 days, preferably at least 25 days, preferably at least 20 days, preferably at least 15 days, preferably at least two weeks, preferably at least 10 days, preferably at least one week, preferably at least 5 days, preferably at least 4 days, preferably at least three days, preferably at least two days, preferably release is over at least one day.

[0132] In a preferred embodiment, the CN comprises about 3.57 M calcium nitrate dissolved in water.

[0133] In a preferred embodiment, the soluble CAN comprises about 2.98 M calcium nitrate and about 0.6 M ammonium nitrate, dissolved in water. In some embodiments, the pH is about 5.5-6, the viscosity is about 3.1 mPa.s and / or the surface tension is about 60 mN / m.

[0134] In some embodiments, the composition comprises at least about 10 g / L total nitrogen, such as at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, or at least about 250 g / L total nitrogen, and useful ranges may be selected between any of these values(for example, from 10 to 250, from 10 to 230, from 10 to 210, from 10 to 190, from 10 to 170, from 10 to 150, from 10 to 130, from 10 to 110, from 20 to 250, from 20 to 230, from 20 to 210, from 20 to 190, from 20 to 170, from 20 to 150, from 20 to 130, from 20 to 110, from 30 to 250, from 30 to 230, from 30 to 210, from 30 to 190, from 30 to 170, from 30 to 150, from 30 to 130, from 30 to 110, from 40 to 250, from 40 to 230, from 40 to 210, from 40 to 190, from 40 to 170, from 40 to 150, from 40 to 130, from 40 to 110, from 50 to 250, from 50 to 230, from 50 to 200, from 50 to 190, from 50 to 180, from 50 to 170, from 50 to 160, from 50 to 150, from 50 to 140, from

[0135] 50 to 130, from 50 to 120, from 50 to 110, from 50 to 100, from 60 to 200, from 60 to

[0136] 190, from 60 to 180, from 60 to 170, from 60 to 160, from 60 to 150, from 60 to 140 from 60 to 130, from 60 to 120, from 60 to 110, from 60 to 100, from 70 to 200, from 70 to 190, from 70 to 180, from 70 to 170, from 70 to 160, from 70 to 150, from 70 to 140, from 70 to 130, from 70 to 120, from 70 to 110, from 70 to 100, from 80 to 200, from 80 to 190, from 80 to 180, from 80 to 170, from 80 to 160, from 80 to 150, from 80 to 140, from 80 to 130, from 80 to 120, from 80 to 110, from 80 to 100, from 90 to 200, from 90 to 190, from 90 to 180, from 90 to 170, from 90 to 160, from 90 to 150, from 90 to 140, from 90 to 130, from 90 to 120, from 90 to 110, from 90 to 100, from 100 to 200, from 100 to 190, from 100 to 180, from 100 to 170, from 100 to 160, from 100 to 150, from 100 to 140, from 100 to 130, from 100 to 120, or from 100 to 110 g / L total nitrogen). Preferably, the composition comprises about 100, about 125, or about 150 g / L total nitrogen.

[0137] In some embodiments the composition further comprises a buffering agent, preferably an agriculturally acceptable buffering agent. Preferably the buffering agent is selected from group consisting of TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, and MES.

[0138] In some embodiments, the composition comprises or consists essentially of CN or CAN as described herein and a buffering agent as described herein.

[0139] In some embodiments, the composition comprises or consists of CN or CAN as described herein, a buffering agent as described herein, and a carrier, diluent, or excipient.

[0140] In some embodiments, the composition has a pH of about 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or about 9.5, and useful ranges may be selected between any of these values (for example, from about 4.5 to about 9.5, from about 4.5 to about 9, from about 4.5 to about 8.5, from about 4.5 to about 8, from about 4.5 to about 7.5, from about 5 to about 9.5, from about 5 to about 9, from about 5 to about 8.5, from about 5 to about 8, from about 5 to about 7.5, from about 5.5 to about 9.5, from about 5.5 to about 9, from about 5.5 to about 8.5, from about 5.5 to about 8, from about 5.5 to about 7.5, from about 6 to about 9.5, from about 6 to about 9, from about 6 to about 8.5, from about 6 to about 8, from about 6 to about 7.5, from about 6.5 to about 9.5, from about 6.5 to about 9, from about 6.5 to about 8.5, from about 6.5 to about 8, from about 6.5 to about 7.5, from about 7 to about 9.5, from about 7 to about 9, from about 7 to about 8.5, from about 7 to about 8, from about 7 to about 7.5).

[0141] In some embodiments the composition comprises one or more additional carriers, diluents, or excipients. In some embodiments the additional carriers, diluents or excipients are selected from the group consisting of wetting agents, surfactants, anticaking agents, osmotic protection agents, and dispersing agents.

[0142] In some embodiments the additional carriers, diluents or excipients is, or comprises a surfactant.

[0143] In some embodiments the composition comprises one or more additional active agents. In some embodiments the additional active agent is selected from the group consisting of anti-scorching agents, osmotic protection agents, nitrification inhibitors, pesticides, insecticides, plant and insect growth regulators, trace elements for plants and animals, plant nutrients, bio-control agents, bacteria, fungi, and biostimulants. In one embodiment the anti-scorching agent is betaine trimethylglycine.

[0144] In some embodiments the trace element is selected from the group consisting of Co,

[0145] Se, Mo, I, Cu, Fe, and Zn.

[0146] Parasitic helminths The methods, uses, and compositions described herein are for controlling free-living stages of parasitic helminths.

[0147] In some embodiments, the composition is effective at controlling the egg stage, the larval stage and / or the adult stage of free-living parasitic helminths, particularly free- living parasitic nematodes. In some embodiments, the composition is effective at controlling the first, the second, and / or the third larval stage of free-living parasitic helminths, particularly free-living parasitic nematodes.

[0148] In some embodiments, the composition reduces the number of infective third stage larvae (L3) of the free-living parasitic helminths, particularly of the free-living parasitic nematodes in a treatment area, upon contact, as compared to the number of L3 at a control area.

[0149] In some embodiments, the composition is effective at controlling free-living parasitic helminths, particularly free-living parasitic nematodes that are mammalian endoparasites.

[0150] In some embodiments, the parasitic helminths are mammalian endoparasites. In some embodiments, the parasitic helminths are parasites of a mammal selected from the group consisting of: canines, felines, bovines, ovines, cervines, equines, caprines, porcines, lagomorphs, rodents, camelids and hominids. In a preferred embodiment, the parasitic helminths are parasites of a mammal selected from the group consisting of bovines, ovines, equines, and cervines.

[0151] In some embodiments, the free-living parasitic helminths are members of the

[0152] Nematoda, Cestoda or Trematoda. In some embodiments, the free-living parasitic helminths are Nematoda. In some embodiments the Nematoda are in the superfamily Trichostrongyloidea, preferably the family Cooperiidae or the family Trichostrongylidae. In one embodiment the Nematoda are Ostertagia spp., Teladorsagia spp., Haemonchus spp. and / or Cooperia spp., preferably O. ostertagi, T. circumcincta, H. contortus and / or C. o neo ph ora.

[0153] In some embodiments the Nematoda are selected from the group consisting of members of the order Strongylida (known as the bursate nematodes), particularly members of the superfamilies Ancylostomatoidea (hookworms), Strongyloidea (Strongyles), Trichostrongyloidea (Trichostrongyles) and / or Metastrongyloidea (lungworms). In a particular embodiment the Nematoda are selected from the group consisting of the Strongyloidea and Trichostrongyloidea, particularly members of the Cyathostominae, Ostertagia, Teladorsagia, Haemonchus, Trichostrongylus, Cooperia, and Oesophagostomum.

[0154] Treatment area

[0155] The methods, uses, and compositions described herein are for controlling free-living stages of parasitic helminths in a treatment area comprising at least one plant, the at least one plant comprising a surface.

[0156] In some embodiments, the treatment area comprises, or consists of, grasslands and / or pasture. In some embodiments, the composition is formulated for application to grasslands and / or pasture.

[0157] In some embodiments, the treatment area is an area where free-living parasitic helminths, particularly free-living parasitic nematodes are present, or may be present. In some embodiments, the treatment area is an area where animals, such as livestock, are grazed. In some embodiments, the treatment area is an area where animals, such as livestock, defecate. In some embodiments, the treatment area comprises animal waste, such as faeces.

[0158] In some embodiments, the treatment area is an area where animals that are vulnerable to helminth infection are, or will be, grazed. In some embodiments, the treatment area is an area where animals that at a life stage wherein they have increased vulnerability to helminth infection are, or will be, grazed. This may include, for example, young animals and / or animals in late pregnancy and / or early lactation.

[0159] In some embodiments, the plant comprises a leaf having a leaf surface. In some embodiments, the plant surface is a leaf surface. In some embodiments, the plant surface is a surface on which the parasitic helminths may be found. In some embodiment, method comprises treating a plant surface with the composition.

[0160] In some embodiments, the plant is a plant suitable for grazing or harvesting. In some embodiments, the plant is a crop plant or part thereof. In some embodiments, the plant is a forage, grass, or pasture plant or part thereof. In some embodiment the plant or part thereof is a grass plant, a legume plant, or part thereof.

[0161] In some embodiments, the plant surface comprises free-living parasitic helminths, preferably free-living parasitic nematodes, preferably at least one of the three larval stages of free-living parasitic helminths, preferably of free-living parasitic nematodes.

[0162] In one embodiment the composition upon application to a treatment environment provides from 5 kg / hectare of nitrogen to 60 kg / hectare of nitrogen.

[0163] Methods of controlling free-living parasitic helminths The inventors believe that they are the first to demonstrate a method of controlling free-living parasitic helminths, particularly free-living parasitic nematodes, on pasture using a composition comprising calcium nitrate (CN) and / or calcium ammonium nitrate (CAN). Without wishing to be bound by theory, the inventors believe that through the use of the method disclosed herein, practitioners will be able to reduce frequency and severity of parasitic helminth infections in various domestic and farm animals by controlling the free-living states of relevant parasitic helminths, particularly nematodes.

[0164] Accordingly, in a first aspect, the invention provides a method of controlling free-living stages of parasitic helminths in a treatment area comprising a surface; the method comprising applying a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) to the treatment area at an application rate sufficient to achieve a concentration of at least about 0.05 g / m2of nitrogen on the surface.

[0165] In a second aspect, the invention provides a method of controlling free-living stages of parasitic helminths in a treatment area comprising a surface; the method comprising applying a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) to the treatment area at a rate of 5-60 kg / ha of nitrogen.

[0166] In a third aspect, the invention provides a method of controlling free-living stages of parasitic helminths in a treatment area comprising a plant, wherein the plant comprises a leaf having a leaf surface, and wherein the treatment area has a leaf area index (LAI); the method comprising applying a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) to the treatment area such that the application rate in kg / ha of nitrogen divided by the LAI is at least about 0.8.

[0167] Fertilisers, such as may be used to improve the growth of pasture, are typically applied to the soil rather than to foliage (i.e. leaves), as fertiliser uptake is primarily via the root system of plants. Furthermore, application to foliage is typically avoided as it can cause undesirable discolouration and / or damage due to "scorching", particularly when high concentrations are used. Accordingly, methods of applying fertiliser typically aim to minimise the concentration of fertiliser on plant surfaces, such as leaf surfaces. This is achieved by preferring solid fertiliser formulations (e.g., granules) over liquid formulations, or where liquid formulations are used, preferring application methods such as drip or jet application that minimise plant surface coverage.

[0168] In contrast, without wishing to be bound by theory, it is believed that the methods of the present application achieve control of free-living stages of parasitic helminths by applying the compositions of the invention to plant surfaces, such as leaf surfaces, where the compositions will come into contact with the parasitic helminths.

[0169] Of course, despite not being a primary object of the invention, the compositions of the invention will also provide a source of nitrogen that may promote plant growth.

[0170] Therefore, in some embodiments the method further comprises promoting or increasing the growth of at least one plant in the treatment area.

[0171] The amount of leaf coverage in a treatment area may vary, for example depending on the plant coverage, plant height, species, and leaf area. The leaf area index (LAI) can be a useful measure to account for this variation. Pasture typically has a leaf area index between 1 and 6. In some embodiments the application rate may be determined taking into account the leaf area index of the treatment area, so that an effective concentration can be achieved at the leaf surface. For example, in some embodiments the composition is applied to the treatment area such that the application rate in kg / ha of nitrogen divided by the leaf area index (LAI) is at least about 0.8, such as at least about 0.9, 1.0, 1.1,

[0172] 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or at least about 60, and useful ranges may be selected between any of these values (for example, from 0.8 to 60, from 0.8 to 50, from 0.8 to 40, from 0.8 to 30, from 0.8 to 20, from 0.8 to 18, from 0.8 to 16, from 0.8 to 15, from 0.8 to 14, from 0.8 to 13, from 0.8 to 12, from 0.8 to 11, from 0.8 to 10, 2.0 to 60, from 2.0 to 50, from 2.0 to 40, from 2.0 to 30, from 2.0 to 20, from 2.0 to 18, from 2.0 to 16, from 2.0 to 15, from 2.0 to 14, from 2.0 to 13, from 2.0 to 12, from 2.0 to 11, from 2.0 to 10, 4.0 to 60, from 4.0 to 50, from

[0173] 4.0 to 40, from 4.0 to 30, from 4.0 to 20, from 4.0 to 18, from 4.0 to 16, from 4.0 to

[0174] 15, from 4.0 to 14, from 4.0 to 13, from 4.0 to 12, from 4.0 to 11, from 4.0 to 10, 5.0 to 60, from 5.0 to 50, from 5.0 to 40, from 5.0 to 30, from 5.0 to 20, from 5.0 to 18, from 5.0 to 16, from 5.0 to 15, from 5.0 to 14, from 5.0 to 13, from 5.0 to 12, from 5.0 to 11, from 5.0 to 10, 6.0 to 60, from 6.0 to 50, from 6.0 to 40, from 6.0 to 30, from 6.0 to 20, from 6.0 to 18, from 6.0 to 16, from 6.0 to 15, from 6.0 to 14, from 6.0 to 13, from 6.0 to 12, from 6.0 to 11, or from 6.0 to 10).

[0175] In some embodiments, the composition is applied to the treatment area at a rate sufficient to achieve a concentration of at least about 0.05 g / m2nitrogen on the surface, such as at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,

[0176] 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1

[0177] 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or at least about 6 g / m2nitrogen, and useful ranges may be selected between any of these values (for example, from 0.05 to 6, from 0.05 to 5.8, from 0.05 to 5.6, from 0.05 to 5.4, from 0.05 to 5.2, from 0.05 to 5, from

[0178] 0.05 to 4.8, from 0.05 to 4.6, from 0.05 to 4.4, from 0.05 to 4.2, from 0.05 to 4, from

[0179] 0.05 to 3.8, from 0.05 to 3.6, from 0.05 to 3.4, from 0.05 to 3.2, from 0.05 to 3, from

[0180] 0.2 to 6, from 0.2 to 5.8, from 0.2 to 5.6, from 0.2 to 5.4, from 0.2 to 5.2, from 0.2 to

[0181] 5, from 0.2 to 4.8, from 0.2 to 4.6, from 0.2 to 4.4, from 0.2 to 4.2, from 0.2 to 4, from 0.2 to 3.8, from 0.2 to 3.6, from 0.2 to 3.4, from 0.2 to 3.2, from 0.2 to 3, from 0.5 to 6, from 0.5 to 5.8, from 0.5 to 5.6, from 0.5 to 5.4, from 0.5 to 5.2, from 0.5 to 5, from 0.5 to 4.8, from 0.5 to 4.6, from 0.5 to 4.4, from 0.5 to 4.2, from 0.5 to 4, from 0.5 to 3.8, from 0.5 to 3.6, from 0.5 to 3.4, from 0.5 to 3.2, from 0.5 to 3, from 1 to 6, from 1 to 5.8, from 1 to 5.6, from 1 to 5.4, from 1 to 5.2, from 1 to 5, from 1 to 4.8, from 1 to 4.6, from 1 to 4.4, from 1 to 4.2, from 1 to 4, from 1 to 3.8, from 1 to

[0182] 3.6, from 1 to 3.4, from 1 to 3.2, from 1 to 3, from 1.2 to 6, from 1.2 to 5.8, from 1.2 to 5.6, from 1.2 to 5.4, from 1.2 to 5.2, from 1.2 to 5, from 1.2 to 4.8, from 1.2 to 4.6, from 1.2 to 4.4, from 1.2 to 4.2, from 1.2 to 4, from 1.2 to 3.8, from 1.2 to 3.6, from 1.2 to 3.4, from 1.2 to 3.2, from 1.2 to 3, from 1.4 to 6, from 1.4 to 5.8, from 1.4 to

[0183] 5.6, from 1.4 to 5.4, from 1.4 to 5.2, from 1.4 to 5, from 1.4 to 4.8, from 1.4 to 4.6, from 1.4 to 4.4, from 1.4 to 4.2, from 1.4 to 4, from 1.4 to 3.8, from 1.4 to 3.6, from 1.4 to 3.4, from 1.4 to 3.2, from 1.4 to 3, from 1.6 to 6, from 1.6 to 5.8, from 1.6 to

[0184] 5.6, from 1.6 to 5.4, from 1.6 to 5.2, from 1.6 to 5, from 1.6 to 4.8, from 1.6 to 4.6, from 1.6 to 4.4, from 1.6 to 4.2, from 1.6 to 4, from 1.6 to 3.8, from 1.6 to 3.6, from 1.6 to 3.4, from 1.6 to 3.2, from 1.6 to 3, from 1.8 to 6, from 1.8 to 5.8, from 1.8 to

[0185] 5.6, from 1.8 to 5.4, from 1.8 to 5.2, from 1.8 to 5, from 1.8 to 4.8, from 1.8 to 4.6, from 1.8 to 4.4, from 1.8 to 4.2, from 1.8 to 4, from 1.8 to 3.8, from 1.8 to 3.6, from

[0186] 1.8 to 3.4, from 1.8 to 3.2, from 1.8 to 3, from 2 to 6, from 2 to 5.8, from 2 to 5.6 from 2 to 5.4, from 2 to 5.2, from 2 to 5, from 2 to 4.8, from 2 to 4.6, from 2 to 4.4, from 2 to 4.2, from 2 to 4, from 2 to 3.8, from 2 to 3.6, from 2 to 3.4, from 2 to 3.2, from 2 to 3, from 2.2 to 6, from 2.2 to 5.8, from 2.2 to 5.6, from 2.2 to 5.4, from 2.2 to 5.2, from 2.2 to 5, from 2.2 to 4.8, from 2.2 to 4.6, from 2.2 to 4.4, from 2.2 to 4.2, from 2.2 to 4, from 2.2 to 3.8, from 2.2 to 3.6, from 2.2 to 3.4, from 2.2 to 3.2, from 2.2 to 3, from 2.4 to 6, from 2.4 to 5.8, from 2.4 to 5.6, from 2.4 to 5.4, from 2.4 to 5.2, from 2.4 to 5, from 2.4 to 4.8, from 2.4 to 4.6, from 2.4 to 4.4, from 2.4 to 4.2, from 2.4 to 4, from 2.4 to 3.8, from 2.4 to 3.6, from 2.4 to 3.4, from 2.4 to 3.2, from 2.4 to 3, from 2.6 to 6, from 2.6 to 5.8, from 2.6 to 5.6, from 2.6 to 5.4, from 2.6 to 5.2, from 2.6 to 5, from 2.6 to 4.8, from 2.6 to 4.6, from 2.6 to 4.4, from 2.6 to 4.2, from 2.6 to 4, from 2.6 to 3.8, from 2.6 to 3.6, from 2.6 to 3.4, from 2.6 to 3.2, or from 2.6 to 3 g / m2nitrogen).

[0187] In other embodiments, the application rate does not depend on the leaf area index. For example, in some embodiments, the composition is applied at a rate of at least 5 kg / ha of nitrogen, such as at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,

[0188] 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,

[0189] 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or at least 60 kg / ha of nitrogen, and useful ranges may be selected between any of these values (for example, from 5 to 60, from 5 to 55, from 5 to 50, from 5 to 45, from 5 to 40, from 5 to 35, from 5 to 30, from 5 to 25, from 5 to 20, from 10 to 60, from 10 to 55, from 10 to 50, from 10 to 45, from 10 to 40, from 10 to 35, from 10 to 30, from 10 to 25, from 10 to 20, from 15 to 60, from 15 to 55, from 15 to 50, from 15 to 45, from 15 to 40, from 15 to 35, from 15 to 30, from 15 to 25, from 15 to 20, from 20 to 60, from 20 to 55, from 20 to 50, from 20 to 45, from 20 to 40, from 20 to 35, from 20 to 30, from 20 to 25, from

[0190] 25 to 60, from 25 to 55, from 25 to 50, from 25 to 45, from 25 to 40, from 25 to 35 from 25 to 30, from 30 to 60, from 30 to 55, from 30 to 50, from 30 to 45, from 30 to 40, from 30 to 35, from 35 to 60, from 35 to 55, from 35 to 50, from 35 to 45, from 35 to 40, from 40 to 60, from 40 to 55, from 40 to 50, or from 40 to 45 kg / ha of nitrogen).

[0191] It is known that application of compositions comprising high concentrations of nitrogen to the leaves of plants may cause discolouration or "scorching". Without wishing to be bound by theory, it is believed that in the methods of the present invention, higher concentrations of the compositions of the invention on the leaf surface are likely to correlate with higher efficacy. In some circumstances, it may be desirable to minimise scorching, particularly when using compositions of the invention at high concentrations. It may be desirable, therefore, to apply anti-scorching agents prior to, during, or after application of the compositions of the invention. In some embodiments, the method further comprises applying preferably 2-3 kg / ha of an anti-scorching agent, preferably betaine trimethylglycine. In some embodiments, the anti-scorching agent is applied simultaneously with the compositions of the invention. For example, the anti-scorching agent may be combined with or included in compositions of the invention.

[0192] In some embodiments, applying the composition of the invention to the treatment area comprises contacting one or more surfaces within the treatment area with the composition, for example one or more plant surfaces. In some embodiments the surface(s) are surface(s) in the treatment area where a free-living parasitic helminth, particularly a free-living parasitic nematode, is commonly found, is expected to be found, or where control of free-living parasitic helminths, particularly free-living parasitic nematodes, is desired, for example, a plant surface such as a leaf surface.

[0193] In some embodiments, contacting comprises directly contacting at least one surface within the treatment area with the composition. In some embodiments, applying the composition of the invention to the treatment area comprises contacting at least one free-living parasitic helminth, preferably at least one free-living parasitic nematode, within the treatment area with the composition. In some embodiments, the free-living parasitic helminths, particularly the free-living parasitic nematodes, are present in the treatment area, and more particularly on a surface, for example, a plant surface such as a leaf surface.

[0194] In some embodiments, contacting at least one free-living parasitic helminth, particularly at least one free-living parasitic nematode, with a composition of the invention comprises contacting for a sufficient time to allow the composition of the invention to control the free-living parasitic helminths.

[0195] In some embodiments, applying comprises directly or indirectly applying the composition of the invention to the treatment area. In some embodiments, applying is directly applying. In other embodiments, applying is indirectly applying.

[0196] Applying the composition to the treatment area can be by any suitable means known in the art. For example, in some embodiments the method comprises spraying the composition on or over the treatment area.

[0197] Spraying can be achieved by a wide variety of methods as is known in the art.

[0198] Examples of spraying equipment can include hand sprayers, backpack sprayers, push sprayers, boom sprayers, vehicle-mounted sprayers, and aerial sprayers. In some embodiments, applying the composition to the treatment area can be by an irrigation system (for example, a low rate irrigation system, or a pod irrigation system).

[0199] It will be appreciated that different apparatuses (such as sprayers, nozzles, strainers, etc) are adapted to apply compositions in different manners. For example, apparatuses for the application of fertilisers are typically adapted to maximise application to the soil and minimise application to foliage. This is because fertiliser update is primarily by the root system of plants, and application to foliage may cause undesirable discolouration known as "scorching". In contrast, the compositions of the present invention are preferably applied to plant surfaces, such as leaf surfaces. Accordingly, preferred apparatuses are those designed for foliar application, rather than soil application.

[0200] In some embodiments, spraying uses a nozzle. In some embodiments, spraying uses a strainer. In some embodiments, spraying uses both a nozzle and a strainer.

[0201] It will be appreciated that a wide range of nozzles and strainers may be useful in methods of the invention. Preferred nozzles are those designed for foliar application, for example flat fan spray nozzles or cone spray nozzles.

[0202] The pressure used to apply the compositions of the invention will vary depending on the application system used (such as the nozzle and / or strainer). The skilled person will be able to select suitable pressures depending on their needs, but as a non-limiting example, in some embodiments the pressure is at least about 10 psi, such as at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, or at least about 60 psi, and useful ranges may be selected between any of these values (for example, from about 10 to about 60 psi, from about 10 to about 55 psi, from about 10 to about 50 psi, from about 15 to about 60 psi, from about 15 to about 55 psi, from about 15 to about 50 psi, from about 15 to about 45 psi, from about 20 to about 60 psi, from about 20 to about 55 psi, from about 20 to about 50 psi, from about 20 to about 45 psi, from about 20 to about 40 psi, from about 25 to about 60 psi, from about 25 to about 55 psi, from about 25 to about

[0203] 50 psi, from about 25 to about 45 psi, from about 25 to about 40 psi, from about 30 to about 60 psi, from about 30 to about 55 psi, from about 30 to about 50 psi, from about 30 to about 45 psi, or from about 30 to about 40 psi).

[0204] It will be appreciated that droplet size is dependent on spray system used (for example, the nozzle and / or strainer) and pressure applied. The skilled person will be able to select suitable parameters to adjust the droplet size. Preferably the droplet size should be selected such that it adheres to surfaces (such as a leaf surface) and provides good coverage. In some embodiments, the spray parameters (for example the nozzle, strainer, and / or pressure) are selected to provide coverage of at least about 1% of a plant surface (such as a leaf surface), for example at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or about 100%, and useful ranges may be selected between any of these values (for example, from 1% to 100%, from 1%, to 90%, from 1%, to 80%, from 1%, to 70%, from 1%, to 60%, from 1% to 50%, from 1% to 40%, from 1% to 30%, from 5% to 100%, from 5%, to 90%, from 5%, to 80%, from 5%, to 70%, from 5%, to 60%, from 5% to 50%, from 5% to 40%, from 5% to 30%, from 10% to 100%, from 10%, to 90%, from 10%, to 80%, from 10%, to 70%, from 10%, to 60%, from 10% to 50%, from 10% to 40%, from 10% to 30%, from 20% to 100%, from 20%, to 90%, from 20%, to 80%, from 20%, to 70%, from 20%, to 60%, from 20% to 50%, from 20% to 40%, from 20% to 30%, from 30% to 100%, from 30%, to 90%, from 30%, to 80%, from 30%, to 70%, from 30%, to 60%, from 30% to 50%, from 30% to 40%, from 40% to 100%, from 40% to 90%, from 40% to 80%, from 40% to 70%, from 40% to 60%, from 40% to 50%, from 50% to 100%, from 50% to 90%, from 50% to 80%, from 50% to 70%, or from 50% to 60%).

[0205] Surface coverage can be determined by methods known in the art. The timing of the application of the composition to the pasture will vary depending on a number of factors related to the pasture to be treated. For example, the timing of treatment will vary based on factors that will be familiar to the pasture manager or farmer such as the time of year, level of precipitation, and type of livestock previously and / or currently grazing the pasture, but not limited thereto.

[0206] For example, the compositions and methods of the present invention may be particularly useful to reduce the numbers of parasitic free-living helminths at critical times during an animal's life. This may include, for example, reducing parasite challenge to young animals, or to adult animals at critical times such as late pregnancy and / or early lactation. Selecting the appropriate timing for applying a composition of the invention to a pasture to reduce the numbers of parasitic free-living helminths is believed to be within the skill of a person in the art based on their particular knowledge of the pasture to be treated. For example, application timing may be similar to, or the same as, that for other known anthelmintic compositions. It will be appreciated that the timing of applying compositions of the invention for controlling free-living stages of parasitic helminths may differ from those that would be used for fertilising pasture.

[0207] In some embodiments, the method comprises applying the composition of the invention to the treatment area once. In other embodiments, the method comprises applying the composition of the invention to the treatment area at least two, three, four, five, six, seven, eight, nine, or ten times. In some embodiments, the method comprises applying a composition of the invention at lx, or 2x, or 3x, or 4x, or 5x, or 6x, or 7x, or 8x, or 9x, or lOx, or llx, or 12x or more per year. In some embodiments, the method comprises applying a composition of the invention at least once every 12 months, preferably at least once every 6 months, or at least once every 3 months. In some embodiments, the method comprises applying the composition no more than four times, three times, twice, or once per year. In some embodiments, the method comprises applying the composition no more than once every 3 months, such as no more than once every 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

[0208] One exemplary embodiment of a method of the first, second, and / or third aspects is presented below.

[0209] A composition comprising soluble CAN is prepared by dissolving calcium nitrate and ammonium nitrate in water in sufficient quantities to produce a solution containing 2.98 M calcium nitrate and 0.6 M ammonium nitrate, with a total nitrogen content of ~100 g / L.

[0210] The composition is applied to pasture having a leaf area index (LAI) of 3.5 at a rate of 250 L / ha, giving an application rate of 25 kg / ha of nitrogen, and an application rate / LAI value of ~7.1. The composition is applied by spraying over plant surfaces, for example using a manually pushed spray unit fitted with nozzles using Teejet XR11003 tips.

[0211] Nematode migration

[0212] Infectious parasitic helminth larvae may be present both in soil and on foliage. Parasitic helminth larvae may migrate from soil to foliage and / or from foliage to soil. When larvae are removed from foliage (for example, by treating foliage with anthelminthic compositions), such migration can result in the levels of larvae present on foliage rebounding. This can present a greater challenge when levels and / or rates of migration are high. The rate and level of migration varies with environmental conditions including (but not limited to) temperature and moisture levels. For example, migration levels are typically lower in winter (when temperatures are low) than summer, and sufficiently low humidity reduces or prevents larval migration. Similarly, the rate and level of migration varies depending on the number of larvae present in the soil.

[0213] The compositions and methods described herein may be particularly useful when parasitic helminth larval migration (particularly migration from soil to foliage) is low. This may occur when environmental conditions are not conducive to larval migration, and / or when the soil harbours only a low level of parasitic helminths.

[0214] Environmental conditions that are not conducive to larval migration include low temperatures, such as experienced during winter, and / or low humidity.

[0215] The soil may harbour only a low level of parasitic helminths. This may occur, for example, when the soil type is not conducive to parasitic helminths. Without wishing to be bound by theory, it is believed that poorly drained soils with a narrow particle size range and few pores, such as sand, silt, pumice, or ash, will harbour fewer L3 than soils with higher frequency of macro- and micro-pores. Alternatively or additionally, the soil of pasture that has not been grazed by contaminating livestock for some time will also harbour lower levels of parasitic helminths than that of actively grazed pasture. Farm management practices such as when making hay or silage, will also reduce the levels of parasitic helminths, and newly sewn pastures will also harbour lower levels. Under such conditions, reinfection of foliage from the soil is reduced.

[0216] Accordingly, in some embodiments the treatment area comprises soil, and the top 50 mm of the soil comprises fewer than about 4,000,000 parasitic helminths per m3of soil, preferably fewer than about 1,000,000, more preferably fewer than about 100,000, most preferably fewer than about 10,000.

[0217] The number of parasitic helminths per m3of soil can be determined as described in the Examples, by taking a soil core sample to a depth of 50 mm and counting the number of larvae present.

[0218] In some embodiments, the composition is applied during or between the months of October-March in the Northern Hemisphere or during or between the months of April- September in the Southern Hemisphere; preferably during or between the months of November-February in the Northern Hemisphere or during or between the months of May-August in the Southern Hemisphere; more preferably during or between the months of December-February in the Northern Hemisphere or during or between the months of June-August in the Southern Hemisphere. In some embodiments, the composition is applied during Autumn, Winter, and / or Spring; preferably Autumn and / or Winter; more preferably Winter. In some embodiments, the composition is applied at a time between the Autumnal equinox and the Spring equinox. In some embodiments, the composition is applied at a time that is at most 100 days before or after the Winter solstice, preferably at most 90, 80, 70, 60, 50, 40, or at most 30 days.

[0219] In some embodiments, the composition is applied at a time of year when the mean daily temperature, calculated as (daily maximum temperature + daily minimum temperature) - 2, is less than about 20°C, preferably less than about 18°C, more preferably less than about 16°C, most preferably less than about 14°C.

[0220] The mean daily temperature in degrees Celsius may be determined by measuring the temperature over the course of a day, adding the maximum and minimum temperatures in degrees Celsius, and dividing by two. It will be appreciated that the mean daily temperature will fluctuate over the course of a year, and will generally be lower in Winter and higher in Summer. Accordingly, the mean daily temperature at any given day may also be estimated by plotting the mean daily temperatures over the year.

[0221] In some embodiments, the composition is applied at an ambient temperature of less than about 20°C, preferably less than about 18°C, more preferably less than about 16°C, most preferably less than about 14°C.

[0222] The invention will now be described by way of the following representative methods and examples which are provided to further illustrate the subject matter to which the invention relates. The use of any and all examples, or exemplary language (e.g., "such as" or "including") provided herein, is intended solely for the purposes of better describing the invention. The presence of examples and the use of exemplary language does not limit the scope of the invention as disclosed herein unless specifically otherwise indicated. No language used in the entirety of the disclosure of this application should be interpreted as indicating that any particular element or feature pertaining to the invention and as disclosed herein is essential to the practice of the invention, unless explicitly stated. For example, the skilled worker will be able to modify the quantities of reagents and processing times in the methods and processes as exemplified, as known in the art, according to the inventive concepts disclosed herein. Such modifications are considered to be within the scope of the present invention.

[0223] EXAMPLES

[0224] Experimental Program

[0225] The experimental programme included both laboratory and field / animal studies. The laboratory study investigated the efficacy of compositions of CAN / CN against L3. The field study investigated the efficacy of compositions of CAN / CN, and also whether the actives were effective under field conditions.

[0226] Example 1 — In vitro faecal assay

[0227] Materials and Methods

[0228] The 'standard' formulation used in the examples is described in W02018 / 083611, and is used for comparison with the formulations of the present invention. It consists of 3.57 M ammonium nitrate and 0.027 M calcium hydroxide dissolved in water. This contains 100 g / L of nitrogen, resulting in an application rate of 25 kg N / ha when applied at 250 L / ha. The pH is approximately 6.5-7.5, viscosity is 1.1 mPa.s and surface tension is 53- 68 mN / m.

[0229] The soluble CAN formulation used in this example consists of 2.98 M calcium nitrate and 0.6 M ammonium nitrate dissolved in water. This contains 100 g / L of nitrogen, resulting in an application rate of 25 kg N / ha when applied at 250 L / ha. The pH is approximately 5.5-6, viscosity is approximately 3.1 mPa.s and surface tension is 60 mN / m.

[0230] Five grams of a 50:50 sheep-faeces:vermiculite mix was placed into each petri dish and approximately 2000 L3 from a mixed culture comprising a variety of cattle, sheep and deer parasites added. The mixed culture was obtained by combining pure strains of parasites cultured previously in the laboratory, including Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Cooperia oncophora, Ostertagia leptospicularis and Ostertagia ostertagi. Each petri dish was placed inside a larger dish and surrounded by a water barrier.

[0231] Treatments were made up 1 day prior to application and stored in airtight containers until used. The pH of the formulations was recorded just prior to application. Two mL of product (equivalent to 5000 L / ha) was applied using an atomizer. This high application rate is an artifact of the artificial assay used. The treated surface consists of a dry faecal matter where it is much harder to target the infective larvae compared to herbage.

[0232] Live larvae were extracted three days after treatment using Baermann funnels containing approximately 250 mL of water, a coarse sieve, and a single layer of tissue paper. The petri dishes were emptied and rinsed into the funnels on top of the tissue and left to sit for a minimum of 18 hours (overnight). During this period, any L3 surviving the treatments moved through the tissue and sieve and were concentrated in the base of the funnel from where they were collected in 50 mL tubes. The tubes were left upright to settle overnight in the fridge prior to syphoning to 10 mL and counting the larvae present in chambered slides under a compound microscope.

[0233] For statistical analysis the data were square root transformed to obtain an even spread of residuals and analysed by linear regression modelling using the transformed data (R version 3.6.3). Pairwise comparison with Bonferroni correction, estimated marginal means and 95% confidence intervals were calculated using the predictmeans package.

[0234] Results

[0235] All treatments with the soluble CAN formulations of the invention significantly decreased the number of infective larvae recovered. Specifically, unadjusted soluble CAN, soluble CAN adjusted to pH 8, and soluble CAN with the addition of calcium hydroxide or calcium oxide showed a 77%, 71%, 73%, and 67% reduction respectively in the number of L3 recovered compared to water (p < 0.001 for CAN, CAN at pH 8, and CAN + Ca(OH)2; p < 0.01 for CAN + CaO). The 'standard' formulation (28% ammonium nitrate with 0.1% calcium hydroxide) also showed a significant reduction (95% reduction; p < 0.001). There was no significant difference between the four CAN treatments.

[0236] Discussion

[0237] This example shows that the CAN formulation of the present invention produced a significant reduction in the number of L3 larvae recovered in an in vitro faecal assay.

[0238] Example 2 — Plot trial

[0239] Materials and Methods

[0240] A soluble CAN formulation and the 'standard' formulation were prepared as described in Example 1.

[0241] A predominantly ryegrass - white clover paddock was used to conduct the plot trial. Random pasture plucks (randomly collecting a small handful of grass from various locations in the paddock, and recovery of larvae using the Baermann method) indicated an L3 population of approximately 10,000 L3 / kg dry matter. To increase the larval population on the pasture, two mobs of 42 un-drenched Romney-cross ram hoggets and 49 Romney-cross ewe hoggets (approximately 1 year old) were run on the paddock 4 weeks and 2 weeks prior to the first treatment application, respectively. The paddock was then mown, and 10 replicate blocks were set out, each consisting of 32 plots measuring 2 x 4 m. All blocks were adjacent to one another and located in the same paddock, and each block contained 2 replicates of each treatment x time combination.

[0242] Two weeks after mowing, a subset of the plots was treated (application A). One week after the first treatment the second application round occurred (application B), and one week after the second application, herbage from all plots was sampled. Formulations were made up fresh the day before each application. Formulations were applied at 200 mL of product per plot, equivalent to 250 L / ha, using a manually pushed spray unit. This unit had a 1.5m wide boom at 50 cm high, containing 4 nozzles (fitted with Teejet XR tips) and spraying at a pressure around 22 psi. Treatment applications were performed on a dry day, and on each treatment date pasture coverage was measured using a capacitance-based pasture probe. On each measurement date, five low coverage and five high coverage quadrats (0.09 m2each) were probed and the herbage subsequently cut and dried. These calibration cuts were used to establish a linear equation for each measurement date, to convert the 'corrected meter readings (CMR)' obtained from the probe into dry matter values.

[0243] At the end of the experiment, 3 m2of herbage was cut from each plot to a height of approximately 2-5 cm. The cut herbage was collected, and wet weights recorded. A subsample of approximately 300 g was obtained using the quartering technique and its weight recorded. Larvae were extracted using large Baermann funnels with a course sieve and collected after 18-22 hours on the funnels. Dry matter weights were recorded, and larvae were counted in chambered slides under a compound microscope.

[0244] The data was log transformed to obtain an even spread of residuals and analysed via a generalized linear mixed model fit by maximum likelihood (Laplace approximation) (Ime4 package, R version 3.6.3).

[0245] Results

[0246] Overall, the treatment resulted in a 23% reduction in L3 recovered for the standard formulation (p = 0.32) and a 48% reduction with the soluble CAN formulation (p < 0.05). There was no statistically significant difference in treatment effect between different application times (Application A vs B, p = 0.07) or between single and double applications (p=0.18) (data not shown).

[0247] Discussion

[0248] This Example shows that treatment of pasture with the soluble CAN formulation of the present invention significantly reduces the number of infectious L3 larvae in a field plot trial.

[0249] Example 3 — Leaf assay of CAN

[0250] Materials and Methods

[0251] The 'standard' formulation was as described in Example 1. Soluble CAN formulations were as described in Example 1, with the exception that a range of calcium nitrate to ammonium nitrate ratios, concentrations, and application volumes were tested.

[0252] Maize or winter crop (oat or wheat) leaves were freshly severed from the plant and cut into pieces approximately 5 cm wide. One (maize) or two (winter crop) pieces were placed in a petri dish "face up". Larvae from a mixed culture were added by placing ten drops of 10 pL (each drop containing approximately 200 L3) on top of each leaf. The mixed culture was obtained by combining pure strains of parasites cultured previously in the laboratory, including Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Cooperia oncophora, Ostertagia leptospicularis and Ostertagia ostertagi. Each petri dish was placed inside a larger dish and surrounded by a water barrier. The parasites were left to distribute throughout the petri dish. For the Maize leaves treatments were applied approximately 4-6 hours later, whereas for the winter crop the larvae were left overnight prior to treatment as droplets were maintained for longer on these leaves. Treatments were made up 6-24 hours prior to application and stored in airtight containers until used. Unless otherwise indicated, two sprays of product were applied using an atomizer, equivalent to 200 pL or 350 L / ha.

[0253] Live larvae were extracted three to five days after treatment using Baermann funnels, a coarse sieve, and a single layer of paper tissue. The petri dishes were emptied and rinsed into the funnels on top of the tissue and left to sit for a minimum of 18 hours (overnight). During this period, any L3 surviving the treatments moved through the tissue and sieve and were concentrated in the base of the funnel from where they were collected in 50 mL tubes. The tubes were left upright to settle overnight in the fridge prior to syphoning to 10 mL and counting the larvae present in chambered slides under a compound microscope.

[0254] The data were either square root transformed, log transformed or remained untransformed depending on which showed the best spread of residuals. Analysis was done by linear regression modelling (R version 3.6.3) if no covariates were present. If extraction of the larvae was done over multiple days or on multiple funnel types, this was included in the statistical analysis as a blocking factor, and the data was analysed using a linear mixed model fit by restricted maximum likelihood (REML). For both methods, pairwise analysis with Bonferroni correction, estimated marginal means and 95% confidence intervals were calculated using the predictmeans package.

[0255] Results

[0256] CAN formulations with ratios of calcium nitrate to ammonium nitrate ranging from 1 : 1 to 5: 1 were tested. All treatments with these CAN formulations significantly reduced the number of infective larvae in the in vitro leaf assay (p < 0.001; Figure 1), but no differences were found between treatments. The highest efficacy was obtained with the soluble calcium ammonium nitrate (CAN) solutions (56-77% reduction depending on the calcium nitrate:ammonium nitrate ratio). Treatment with the CAN 5: 1 formulation showed the best efficacy, however, the difference between each of the CAN formulations did not rise to the level of statistical significance. Treatment with all of the CAN formulations also showed numerically higher efficacy than treatment with the 'standard' formulation, but again this did not rise to the level of statistical significance.

[0257] CAN formulations were also tested at different concentrations and application rates. The number of larvae recovered was reduced by 78% (p < 0.01) when soluble CAN (5: 1 ratio, 100 g N / L) was applied at 350 L / ha (Figure 2). To test the effect of concentration and volume, CAN was applied at lx, 1.25x, and 1.5x concentration and at1 / 2 the application rate, i.e. 100, 125, and 150 g / L nitrogen was applied at 175 L / ha. These treatments reduced the L3 numbers by 38%, 51%, and 75%, respectively (Figure 2). The 1.5x treatment significantly reduced the number of L3 recovered compared to the water control (p < 0.01). The lx and 1.25x treatments also showed reductions in the number of L3 recovered, but did not reach the level of statistical significance.

[0258] Discussion

[0259] This Example shows that treatment with soluble CAN formulations of the invention significantly reduced the number of L3 in an in vitro leaf assay at a range of different ratios of calcium nitrate to ammonium nitrate, and at a range of concentrations and application volumes.

[0260] Example 4 — Tunnel house assay

[0261] The soluble CAN formulation was developed based on laboratory assays and had previously undergone very little testing in the field. Tunnel house trials were performed to confirm the efficacy of this product in a more realistic setting before its application in animal trials.

[0262] Materials and Methods

[0263] Soluble CAN and the 'standard' formulations were used as described in example 1.

[0264] Trials 1 and 2

[0265] Pasture turfs were cut out of a parasite free pasture with a predominantly ryegrass - white clover herbage. The turfs were placed in a tunnel house, thoroughly watered, and left to acclimate for a few days. Individual bags were prepared containing 150 g (trial 1) or 250 g (trial 2) sheep faeces supplemented with 40,000 L3 from a mixed culture comprising a variety of cattle, sheep, and deer parasites. The L3 were then left to distribute through the faeces overnight. The herbage on the turfs was cut short (approximately 5 cm length) and the faeces parasite mix was removed from the bag and placed on top of the turfs (one bag per turf). The turfs were left for a week to allow the L3 to move onto the herbage. Watering was done using edge watering.

[0266] Treatments were made up 1 day prior to application and stored in airtight containers until used. Each turf was treated with 4 mL of the formulation (equivalent to approximately 250 L / ha) using an atomizer, and the treatment left on for 5 days. Faeces and grass from each turf were collected separately, and live larvae extracted overnight using Baermann funnels. Any L3 surviving the treatments were collected and counted under a compound microscope. Wet and dry weights of the samples were recorded.

[0267] The data were either square root transformed, log transformed or remained untransformed depending on which showed the best spread of residuals. Analysis was done by linear regression modelling (R version 3.6.3). Pairwise analysis with Bonferroni correction, estimated marginal means and 95% confidence intervals were calculated using the predictmeans package.

[0268] Trial 3

[0269] This trial aimed to investigate the potential effect of herbage height, application rates and the interaction of these two variables on efficacy in reducing L3 numbers on pasture. The herbage heights used in this trial were 1.5, 4, 6.5, and 10 cm with the aim of achieving approximately 600, 1200, 1800, and 2400 kg DM / ha, respectively. The selected application rates were 250, 500, and 750 L / ha.

[0270] A combination of a TJ6011003VS nozzle and a brass strainer with 100 pm stainless steel mesh was selected for this trial. The conveyor belt was calibrated to achieve the correct speed using water: an empty tray was run across the conveyor belt 10 times, with the amount of water collected, measured and the application rate calculated. The speed was then adjusted to achieve each of the three desired application rates. One week prior to the start of the experiment, water sensitive paper (Syngenta) was used to assess spray pattern and coverage for the selected grass heights and application volumes. This was done on spare plots with water as a medium.

[0271] Pasture turfs were cut from a parasite free pasture with a mixed pasture sward. The turfs were placed in a tunnel house and left to acclimate for a few weeks. One day prior to the trial individual bags were prepared containing 80 g sheep faeces supplemented with 30,000 L3 from a mixed culture comprising a variety of cattle, sheep, and deer parasites. The L3 were left to distribute through the faeces overnight after which the faeces parasite mix was placed on top of the turfs, and the bag rinsed over the turf (one bag per turf). The turfs were left for a week to allow the L3 to move onto the herbage.

[0272] Watering was done using edge watering. Formulations for treatment were made up 1 day prior to application and stored in airtight containers until used. Grass heights (five measures per turf) for each of the turfs were recorded just prior to treatment. Using the conveyor belt (with a TJ6011003VS nozzle, the brass strainer with 100 pm stainless steel mesh, and a constant pressure of 30 psi) each turf was treated with either water at 250L / ha or the soluble CAN formulation at 250, 500, or 750 L / ha. At the time of treatment an empty tray was run across the conveyor belt 10 times, and the amount of formulation collected was measured to calculate the application rate for each of the treatments.

[0273] The turfs were left for 4 days after treatment, after which faeces and herbage from each turf were collected separately, and live larvae extracted overnight using Baermann funnels. Any L3 surviving the treatments were collected and counted under a compound microscope. Wet and dry weights of the samples were recorded.

[0274] A generalized linear mixed effects model with Poisson distribution was used in the data analysis. In the model, 'Grass', 'Volume' and their interaction were fixed effects, 'Block', 'Column' and 'Row' were random effects. Predicted means with pairwise comparison were performed on the modelling result, associated p-values were adjusted by Benjamini-Hochberg method. The analysis used the 'predictmeans' and 'Ime4' packages in R 4.0.2.

[0275] Results

[0276] Trial 1

[0277] In this trial treatment with the 'standard' formulation resulted in a 34% reduction in L3 relative to water treatment (Figure 3) while treatment with the soluble CAN formulation resulted in a 66% reduction. As with previous trials, there was limited efficacy against L3 in faeces (data not shown).

[0278] Trial 2

[0279] Treatment with both CAN and CN formulations showed a significant (p < 0.05) reduction in larvae recovered from herbage compared to the water control (Figure 4). There were no other significant differences between treatments. There was no significant difference in L3 recovered from the faeces (data not shown).

[0280] Trial 3

[0281] The trial was set up as a 4 x 4 treatment matrix, investigating four different application rates and four different grass heights at application.

[0282] Prior to the start of the trial, the selection of grass heights and nozzles was tested using spare plots with water as a medium. The spray pattern indicated reducing coverage with increased grass height, as well as increased coverage with increased volumes. The selection of grass heights and application volumes was deemed appropriate, and the trial proceeded as planned.

[0283] Five turfs were randomly allocated for destructive sampling for each of the selected grass heights. These turfs were used to measure grass height, herbage cover, herbage composition and leaf area index at the time of treatment. Turfs trimmed to 1.5, 4, 6.5 and 10 cm at the time of contamination measured on average 4.2 (range 3.3 - 5.4), 6.3 (range 4.8 - 7.4), 9 (range 8.6 - 9.8) and 11.4 (range 10.2 - 13) cm at treatment.

[0284] Pasture cover was respectively 715 (range 236-1005), 1802 (range 1414-2111), 2583

[0285] (range 2264-2905) and 3139 (range 2770-3340) kg DM / ha. The herbage consisted predominantly of ryegrass with presence of clover, dead matter and other species (e.g., buttercup). With higher herbage heights, the relative contribution of grass increased, whereas prevalence of clover decreased (data not shown).

[0286] Turfs selected for treatment were trimmed to 1.5, 4, 6.5 and 10 cm at the time of contamination and measured on average 4.6 (range 3.1 - 6.2), 7.3 (range 5.5 - 9.7), 9.1 (range 7.1 - 11.5) and 12.4 (range 9.6 - 15.4) cm at treatment, respectively. The soluble CAN formulation was applied at 4 different application rates for each of the grass heights.

[0287] Measurement of the exact amount of water and soluble CAN delivered at the three targeted application volumes (250, 500 and 750 L / ha) found that water was applied at 282 L / ha, whereas the amount of CAN applied was 214, 379 and 591 L / ha, respectively. These were considerably lower than anticipated based on the initial calibration with water.

[0288] Following treatment application, low to moderate scorching was observed on the herbage, predominantly on the clover and legumes present in the turfs.

[0289] Application of the soluble CAN formulation at 214, 379 and 591 L / ha reduced the number of larvae recovered compared to water (p < 0.01; Figure 5). While there was no significant difference in efficacy between the two lowest application rates, the highest application rate had a significantly larger effect on reducing L3 numbers recovered. In general, less larvae were recovered from turfs with lower herbage height (p < 0.01; Figure 5). Identification of the larvae to genus level indicated that soluble CAN and overall herbage height significantly reduced Cooperia and Ostertagia spp., but the differences in Haemonchus spp. numbers did not reach significance (data not shown).

[0290] Discussion

[0291] This example shows that treatment with CN and CAN formulations significantly reduced the number of L3 present on grass in a tunnel house trial at a variety of different grass heights and application rates.

[0292] Example 5 — Animal trial

[0293] In this example the worm burden in tracer lambs was used as the measure of pasture infestation with parasite larvae. Young lambs have only minimal immunity against parasites, and parasite establishment in those lambs is driven by the number of viable parasite larvae they ingest from the paddock. Worm counts in tracer animals are considered the most reliable indication of infective larvae on pasture.

[0294] Materials and Methods

[0295] Soluble CAN and the 'standard' formulations were used as described in Example 1.

[0296] Two adjacent ryegrass - white clover paddocks (N1 and N3) grazed with sheep were selected. Sixteen individual plots (5 blocks of three plus one extra pen) of approximately 400 m2were created on these paddocks. Prior to the start of the trial, the site was left to recover under normal management practices i.e. grazed intermittently by sheep for 15 weeks, and a single herbicide application for thistle control. Plots were randomly assigned a treatment while taking the blocks into account. The extra plot was assigned to a water treatment, resulting in a total of six plots treated with water, five with the standard product and five with the soluble CAN. Treatments were applied twice with a 30-day interval between treatments. For each application, formulations were made up 1 day prior to application. On the day of treatment, prior to application, multiple measures were taken. Pasture plucks were collected for each individual plot. Walking in a zigzag pattern across the plot, herbage was randomly plucked to ground level in five locations every five paces. Herbage was collected in a plastic bag and larvae extracted the same day using the Baermann technique. Grass was recovered from the extraction funnels and dried to determine dry matter content.

[0297] Pasture height was measured using a sward stick with a minimum of 10 measurements per plot. Pasture cover for each plot was measured using a capacitance-based pasture probe. In addition, five low coverage and five high coverage quadrats (0.09 m2 each) were probed and the herbage subsequently cut and dried. These calibration cuts were used to establish a linear equation to convert the 'corrected meter readings (CMR)' obtained from the probe into dry matter values. Samples for herbage composition were obtained by taking pasture plucks for each individual plot on each treatment day (prior to product application) after the second group of animals was removed from the trial site. Samples were submitted for botanical analysis and dry matter values for the different herbage species determined.

[0298] Application was achieved using a manually pushed spray unit. This unit had a 1.5m wide boom at 50 cm high and contained 4 nozzles fitted with Teejet XR11003 tips. Formulations for the different treatments were applied in the following order: water - 'standard' formulation - soluble CAN formulation, and the spray unit was cleaned thoroughly between treatments. The total walking time during product application was recorded for each plot and used in conjunction with the calibrated nozzle output per minute to calculate the total volume applied per plot. After arrival the lambs were drenched with commercial anthelmintics to remove any parasite infection present. Lambs were divided into two groups (representing application time 1 and 2) based on weight with the heaviest animals assigned to group 1, and the lightest ones in group 2. They were then housed indoors (group 1) or kept on parasite free pasture prior to being housed indoors (group 2) to maintain their low immunity.

[0299] Approximately one week following treatment application the animals were moved onto the trial site (group 1 following the first and group 2 following the second application). Lambs were randomized across the plots ensuring an even distribution of lamb weights. Each plot was grazed by three tracer lambs for 10-12 days before the animals were moved back indoors. Animals were kept indoors for a further 3 weeks to allow development of larvae into adult worms. Rectal faecal samples were taken on multiple occasions during the establishment period for faecal nematode egg counts (FEC). Eggs were counted using the Parasight system (https: / / www.parasightsystem.com) with a sensitivity of 13 eggs per gram (epg). Slaughter and recovery of adult worms was scheduled on days 32-34 following their entry into the trial site.

[0300] Following slaughter, the abomasum and the first 10m of small intestine of each animal were collected and any remaining fat removed. The contents of each organ were recovered, and they were washed thoroughly using lukewarm water. The combined digesta and washings from each of these organs were made up to volumes of 7 L and two representative 10% aliquots collected.

[0301] One of these aliquots, selected at random, was rinsed through a 38 pm aperture sieve.

[0302] The worms recovered were then counted and identified according to parasite species and sex. In addition, a count of the number of fourth stage larvae (i.e., larvae that had not yet completed development to an adult) present was included.

[0303] A generalized linear mixed effects model with Poisson (or negative binomial) distribution was used for the data analysis. In the model, 'Treatment', 'Organ', 'Time points' and their interaction were the fixed effects; 'Paddock', 'Block' nested within 'Paddock' and 'Plot' nested within 'Block' were the random effects. Predicted means with pairwise comparison was performed on the modelling result, and associated p-values adjusted by Benjamini-Hochberg method. The analysis was done using 'predictmeans' and 'Ime4' in R 4.0.2.

[0304] Prior to the current animal trial, the trial paddock was maintained under intermittent grazing with sheep and an application of herbicide for thistle control. Pasture plucks collected nine days prior to treatment showed an average recovery of 2335 (range 222- 7296) L3 / kg DM. The majority of larvae recovered from pasture were Cooperia (61%) and Ostertagia (23%) spp.

[0305] Results

[0306] Formulations for each treatment were successfully applied on a dry day with limited wind exposure. Calculated average application rates were 259 and 256 L / ha for water, 260 and 255 L / ha for the 'standard' formulation and 411 and 257 for the soluble CAN formulation for the first and second treatment applications, respectively. The pH of the formulations was 7.24 & 7.25 for water, 7.95 & 7.91 for the 'standard' formulation, 5.48 & 5.47 (DO application) and 5.62 (DI application) for the soluble CAN formulation at the first application; 8.9 & 8.1 for water, 8 & 7.89 for the 'standard' formulation, and 5.63 & 5.669 for the soluble CAN formulation at the second application (respectively samples 1 and 2 at each application). Note: During the first application, the equipment malfunctioned and under-applied the soluble CAN formulation. Efforts to fix this issue resulted in an increased pressure, causing the applied volume to overshoot the desired application volume (411 L / ha instead of 250 L / ha) in subsequent plots. To obtain a consistent application volume across the treatment groups, underdosed plots received a "second pass" calculated to obtain a total application volume of 411L / ha.

[0307] Pasture height and cover at the time of application are listed in Table 1. On average herbage was higher (12.5 compared to 10.6 cm) and denser (2427 compared to 1864 kg DM / ha) at the time of the first application.

[0308] Table 1. Pasture height and cover at the time of application.

[0309] Pasture contamination was measured via pasture plucks. The number of larvae recovered from the herbage was approximately 888 L3 / kg DM at the time of the first treatment application. The majority of larvae present were Cooperia parasites, with Ostertagia and Trichostrongylus being the second and third most prevalent genera (Table 2). At the time of second treatment application the number of larvae present on the herbage had decreased to approximately 406 L3 / kg DM (Table 2). This was expected as a result of grazing by the first group of animals, as well as interactions with the environment. At the time of the second application Ostertagia spp were most prevalent, followed by Cooperia spp. and Trichostrongylus spp. (Table 2). A number of parasite larvae could not be identified to genus. They are not listed in the table but were included in the calculations for larval composition (i.e. this is why the sum of the different genus percentages in the table does not equal 100%).

[0310] Table 2. Pasture contamination throughout the trial. Discoloration of pasture compared to the water control was observed following application of the 'standard' and soluble CAN formulations for a few hours to a few days after treatment. This is in line with past observations.

[0311] Treatment with these formulations did not affect pasture cover during the trial.

[0312] However, pasture composition did change significantly following treatment. The percentage grass in the herbage increased following a single application of soluble CAN (p = 0.01). By the end of the trial (i.e., following two treatment applications) the percentage grass increased even further (p < 0.01), with a corresponding decrease in the percentage clover (p < 0.01) compared to pasture treated with water. Application of the 'standard' formulation also resulted in increased prevalence of grasses (p < 0.01) and reduced presence of clover leaf (p = 0.018) by the end of the trial. This increase in grass and reduction in clover in the herbage may be due to the application of N increasing grass vigour while reducing legume competitiveness. The greater scorching observed on legumes may further enlarge the effect of the treatments on herbage composition.

[0313] Prior to the start of the trial, a number of animals in group 2 were removed from the trial and later euthanized due to persistent pneumonia.

[0314] Animal weights were followed throughout the trial. Entering onto the trial site, animals weighed approximately 30 and 34 kg for group 1 and 2, respectively. Weights increased to 33 kg and 36 kg, respectively, by the end of the trial. No differences in liveweight or weight gain were observed between treatment groups (data not shown), indicating that the animals were not negatively impacted by the treatment.

[0315] Worm burden at the time of slaughter confirmed a significant reduction in parasite burden (p = 0.015) in animals grazed on pastures treated with the soluble CAN formulation compared to the water control (Figure 6). In the abomasum and small intestine, the reduction in worm burden was respectively 27% and 35% for animals from group 1, and 33% and 45% for animals from group 2.

[0316] A significant (p < 0.001) reduction in Haemonchus, Teladorsagia, Trichostrongylus, and Cooperia spp. was observed in animals from both groups 1 and 2 grazed on paddocks treated with the soluble CAN formulation (Figure 7). Pasture treatment with the 'standard' formulation resulted in significant (p < 0.001) reductions for Teladorsagia, Trichostrongylus, and Cooperia spp. in animals from both groups 1 and 2 and

[0317] Haemonchus in animals from group 2 (Figure 7). Treatment with the soluble CAN formulation also produced a significantly greater reduction in Teladorsagia, Trichostrongylus, and Cooperia spp. than the 'standard' formulation (p<0.05).

[0318] Discussion

[0319] This Example shows that treatment of pastures with the soluble CAN formulation of the invention resulted in reduced infection rates in animals subsequently grazing these pastures. There was a significant 27-45% reduction in total worm burden in animals grazed on pastures treated with the soluble CAN formulation compared to water treated pasture. Significant reductions were measured in the four most prevalent species / genera of parasites Haemonchus, Teladorsagia, Trichostrongylus, and Cooperia).

[0320] Example 6 — Animal trial

[0321] Materials and Methods

[0322] Two trials were conducted on commercial sheep and beef farms. Pastures were a permanent ryegrass I white clover sward and had been grazed intermittently by sheep (Trial 1) or sheep followed by cattle (Trial 2) in the previous months. No chemicals had been applied to either paddock for at least six months prior to the start of the trial.

[0323] On each farm, sixteen individual plots (in eight blocks of two) of approximately 400 m2were created. The plots were randomly assigned to the control or treatment group taking the blocks into account.

[0324] Plots were treated with soluble CAN (72.2 % w / v Ca(NOs)2.4H2O with 4.8% w / v

[0325] NH4NO3) or water as a single application at 400 L / ha at the start of the trial. Forty-eight lambs (approx. 6 months of age) were used for each trial. Before entering the trial, lambs were treated with a combination anthelmintic, with treatment efficacy confirmed by zero FEC seven days later.

[0326] Approximately one week after treatment of the pastures, the lambs were randomized across the plots to ensure even distribution of lamb weights. Each plot was grazed by three lambs for 12-15 days before the animals were moved to covered facilities for an additional three weeks.

[0327] Data was analyzed for each trial site separately using R4.20 and its packages 'predictmeans' and 'Ime4'. A poisson mixed model (estimated using ML and Nelder-Mead optimizer) was used to model FEC. The model included treatment, sampling date and their interaction as fixed effects, with block as a random effect. Predicted means with pairwise comparison was performed on the modeling result, and associated p-values adjusted by Benjamini-Hochberg method (Benjamini and Hochberg, 1995).

[0328] A poisson mixed model (estimated using ML and Nelder-Mead optimizer) was used to model worm counts. The model included treatment as a fixed effect and block as a random effect and predicted means calculated. Analysis was done by organ for each of the species individually.

[0329] Results

[0330] For Trial 1, there was an overall significant difference in FEC between treatments (p<0.05), with a significant effect of time (p<0.001) and the interaction term was also significant (p<0.001). For four of the five sample dates, lambs which had grazed the CAN treated pastures had significantly lower FECs than those which had grazed the water treated pastures (Figure 8). For Trial 2, the overall treatment effect approached significance (p=0.066). Again, the time effect and the interaction terms were both significant (p<0.001). For the first two sample dates lambs which had grazed the CAN treated pastures had significantly lower FECs than those which had grazed the water treated pastures. However, for the last two samples FECs tended to converge resulting in the differences becoming non-significant (Figure 9).

[0331] For Trial 1, lambs grazed on pastures treated with CAN had significantly lower worm burdens for Cooperia (p<0.001) and Haemonchus (p<0.05), and which approached significance for Teladorsagia (p=0.051; Figure 10).

[0332] For Trial 2, treatment of pastures resulted in reduced worm burdens of grazing lambs for all four of the main species (p<0.05 for Teladorsagia; p<0.01 for Cooperia and Haemonchus; and p<0.001 for the intestinal Trichostrongylus spp.; Figure 11). In addition, there was also a significant reduction in the counts for Trichostrongylus axei (p<0.01), a species was not seen in numbers in the other trials. Mean counts for T. axei were 269 (95% CI 136-533) and 78 (95% CI 39-154) for the water and CAN treatments, respectively.

[0333] Discussion

[0334] This Example shows that treatment of pastures with the soluble CAN formulation of the invention resulted in reduced infection rates in animals subsequently grazing these pastures.

[0335] Example 7 — Migration of helminth larvae In this example, the migration of helminth larvae between the soil and foliage was assessed.

[0336] Materials and Methods

[0337] Data on the presence of L3 in different substrates (faeces, soil and herbage) was collected at monthly intervals in two field plot trials at multiple sites around the North Island of New Zealand, over several years.

[0338] For Trial 1, field plots (Im x Im) were established on parasite-free pastures. Plots were contaminated and destructively harvested at 4-weekly intervals. On each visit, replicate (N = 10) plots were contaminated with faeces containing nematode eggs of Teladorsagia circumcincta and Trichostrongylus colubriformis. These were either deposited on the surface of the herbage or were buried 50-100mm in the soil. Four weeks (28-30 days) later plots were harvested in a series of zones (substrates). Faeces remaining was collected. Herbage within a 200mm circle surrounding the point of deposition was cut close to ground level. The interface zone comprising the remaining herbage root mass and upper soil to a depth of 25mm was removed. A soil core 100mm diameter by 200mm deep was then taken directly below the plot centre, any faeces present within the core was collected separately, before the core was divided into an upper and lower half. Third stage larvae were recovered from all samples by baermannisation and concentrated by sedimentation. Subsamples were taken to allowed estimation of the total number of L3 in each sample, divided into species using the proportion of T. circumcincta and T. colubriformis L3 estimated in each.

[0339] Trial 2 utilised an almost identical methodology to Trial 1. In this trial faeces were only deposited on the herbage. Further, because in Trial 1 the two soil cores taken beneath the deposition sites mostly returned few L3 (large numbers of zero counts) these harvest zones were not sampled in this trial.

[0340] Results

[0341] Faeces placed on surface.

[0342] Of the 75 contaminations (site x month) in which faeces were deposited on the surface, only two resulted in no Teladorsagia larvae being found in the soil, and only three returned no Trichostrongylus larvae from this zone. Statistical analysis indicated high levels of variation between sites (p < 0.001) and months (p < 0.001) and their interaction (p < 0.001).

[0343] For Trial 1, when faeces were deposited on the surface, the proportion of T. colubriformis L3 in soil was highest from April to June (Figure 12). The equivalent data from Trial 2, again showed higher values in the May to August period with one exception of a low value in June (Figure 13). In Trial 1, the proportion of T. circumcinta L3 recovered from soil tended to be higher overall (compared to T. colubriformis) with the highest levels occurring in March, April, May, August and December (Figure 12). Trial 2 also showed higher proportions occurring between April and August (Figure 13).

[0344] Faeces buried in soil.

[0345] Of the 49 contaminations for which faeces were buried below the soil surface, ten resulted in less than 1% of Teladorsagia larvae being recovered on herbage, and 11 resulted in less than 1% of Trichostrongylus larvae. With one exception all of these occurred in the months of June, July or August. For both parasite species the lowest proportions recovered from herbage occurred between April through to August, with the exception of a single higher recovery for both species in July (Figure 14). Discussion

[0346] This Example shows that infectious parasitic helminth larvae can migrate from soil to foliage and from foliage to soil. It also shows that such migration shows seasonal variation.

[0347] Example 8 — Reinfection trial

[0348] In this example, the effect of reinfection of herbage by L3 present in the soil was tested .

[0349] Materials and Methods

[0350] A paddock of approximately 1 ha, sown in a permanent ryegrass I clover sward, was maintained without grazing for 4-5 years. This was the source of the turfs used in this Example.

[0351] The test product used comprised 16.2% w / w total nitrogen, 1.0% w / w ammonium, 15.2% w / w nitrate nitrogen, and 20.2% w / w calcium.

[0352] Tunnel house trial.

[0353] Pasture was mown, allowed to regrow to a uniform height of approximately 50 mm, and turfs were extracted. Turfs were placed in seeding trays in a plastic tunnel house and watered daily from underneath. Turfs were allowed to acclimate for several weeks. Prior to trial commencement, all turfs were trimmed to a uniform 50 mm height and randomly allocated to one of four treatment groups.

[0354] Fresh nematode larvae were acquired by collecting faeces from lambs with positive faecal nematode egg counts on a commercial farm. Faecal samples were mixed and cultured at 23°C for 14 days with regular aeration and moisture maintenance. Infective stage larvae were extracted by baermannisation, concentrated by sedimentation in water and stored at 8°C until required. Prior to application to the turfs, larvae were filtered through a sieve to ensure viability of all applied larvae.

[0355] On Day 0 of the trial, all 40 turfs were contaminated with 25,000 L3 suspended in 8 mL of water by evenly distributing droplets directly onto the herbage. Additionally, half of the turfs (n = 20) received a soil injection of 50,000 L3 suspended in 16 mL of water, evenly administered in a grid pattern across the soil surface. After contamination, all turfs remained undisturbed for 4 days.

[0356] The four treatments comprised:

[0357] 1) herbage-contaminated turfs treated with water,

[0358] 2) herbage-contaminated turfs treated with test product,

[0359] 3) herbage plus soil-contaminated turfs treated with water,

[0360] 4) herbage plus soil-contaminated turfs treated with test product.

[0361] On Day four, treatments were applied by spryaing using a fine droplet-size at a rate equivalent to 400 L / ha. On day nine, two applications of water (simulated rainfall) were applied at rates equivalent to 1.78 mm of rain during the morning and an additional 1.81 mm during the afternoon.

[0362] On day 12 all plots were harvested. All herbage was cut from each turf to a height of 10 mm and placed in prelabelled plastic bags. Larvae were extracted overnight using Baermann funnels and larvae were concentrated by sedimentation in water prior to counting under a binocular microscope. Herbage weights were recorded both wet and dry.

[0363] Statistical analysis The number of larvae recovered per plot was analyzed using a generalized mixed effects model with poisson distribution. The model included treatment (test product v water control), contamination (herbage, herbage + soil) and their interactions as a fixed effect, herbage dry matter as a covariate, and turf position in the tunnel house as random effects. Predicted means were generated for the fixed effects and underwent multiple comparisons, which were performed with adjusted p-values. The statistical analysis was conducted using R version 4.4.1, utilizing the 'predictmeans' and 'glmmTMB' packages.

[0364] Field plot trial.

[0365] An area of pasture was mown to a uniform height of 50 mm. A nine x eight matrix of Im x Im plots was established. Plots within each of the eight rows, were randomly allocated into three replicates of each of three contamination types, a) surface contamination, b) soil contamination, and c) combined surface plus soil contamination.

[0366] Faeces containing eggs were collected on a commercial farm and cultured as described above. The cultured faeces were thoroughly mixed, and the plots were contaminated with the cultured material containing developed L3.

[0367] On day one plots assigned to the surface group were contaminated by placing 110 g (±0.1 g) of the cultured faecal medium at the center of each plot. Plots allocated to the soil group received an equivalent weight of faeces buried below the surface. Plots designated to the combined (surface plus soil) group received both treatments simultaneously.

[0368] On day eight, plots in rows 2-3 and 6-7 were enclosed using sturdy plastic foil barriers to protect adjacent rows and treated i.e., all plots in rows 2, 3, 6 and 7 were treated while plots in rows 1, 4, 5 and 8 remained untreated. The test product was applied using a hand propelled lawn sprayer with a fine droplet-size at a rate of 400 L / ha. The control plots were not sprayed.

[0369] On Day 10, all plots were destructively harvested. Firstly, any faecal material remaining on the surface and combined plots were carefully collected into plastic bags. Then, herbage within a 200 mm metal ring placed over the centre of each plot was cut and collected. The herbage-soil interface, comprising the root mat and upper soil layer, was also collected from within this area. Finally, a soil core, 100 mm diameter x 50 mm depth was extracted from directly beneath the harvested herbage and interface layers.

[0370] Wet weights were recorded for all samples. Larvae were recovered from the faecal and herbage samples using Baermann funnels, while Whitehead trays were used for the interface and soil samples. Larvae were concentrated and cleaned by sedimentation in water prior to counting under a binocular microscope. Finally, all sample material was oven-dried for determination of dry matter.

[0371] Statistical analysis.

[0372] The data was analysed using a generalized mixed effects model with poisson distribution which incorporated fixed effects of Treatment, Contamination and their interactions, random effects for plot location (row and column) and covariates of herbage dry-matter and L3-count. After modelling the data, predicted means were generated for the fixed effects. These predicted means underwent multiple comparisons, which were performed with adjusted p-values using the 'BH' method. The statistical analysis was conducted using R version 4.4.1, utilizing the 'predictmeans' and

[0373] 'glmmTMB' packages. Results

[0374] Tunnel House Trial.

[0375] The main effects of contamination and treatment were both significant, along with their interaction (p < 0.001). The mean number of L3 recovered from the herbage plus soil contaminated turfs (4430) was significantly higher than the mean recovered from the herbage only contaminated turfs (2237; p < 0.01; Figure 15). Treatment with the test product produced a significant reduction in the number of L3 on herbage from a mean of 3443 to 2878 (p < 0.05).

[0376] Field Plot Trial

[0377] There was no difference in the number of L3 recovered from the herbage when faeces were deposited on the surface and when they were buried 50-100 mm in the soil. When faeces were deposited both on the surface and in the soil, the number of L3 recovered from the herbage was almost doubled (p < 0.01) i.e., mean counts of 633, 641 and 1183 for surface, soil, and surface plus soil contaminations, respectively (Figure 16).

[0378] Treatment was also significant (p < 0.05) with treated plots averaging 52% fewer L3 on herbage than the control plots, when averaged across the different contamination types (1126 v 545 for control and treated plots, respectively).

[0379] Larvae recovered from faeces

[0380] Because there were little I no faeces present in the soil cores from the soil and herbage plus soil contaminations, analysis was only possible for those faeces collected off the herbage. Overall, there was a small but significant difference in the number of L3 recovered from faeces between the surface and soil plus surface contaminations (2055 v 1818, respectively). Overall, treatment with the test product had no effect on L3 numbers recovered from faeces (p = 0.617).

[0381] Larvae recovered from the herbage-soil interface

[0382] There were significant differences between each of the contamination types in the number of L3 recovered from the herbage-soil interface (p < 0.01) with the most L3 in the surface plus soil contamination group (401), less in the soil group (204) and least in the surface group (92). There was also a significant difference between the treated and control groups (p = 0.049) with treated plots having more L3 in this zone than the untreated plots (means of 225 and 170 for the treated and control groups, respectively).

[0383] Larvae recovered from the soil core

[0384] The number of L3 recovered from the soil core was significantly lower for surface contamination than for soil and soil plus surface contamination groups (p < 0.01) with mean counts of 5, 1502 and 1317, respectively. There was also a significant effect of treatment with the treated plots having significantly more L3 (mean = 271) compared to the controls (mean = 180) (p < 0.05). The increase in L3 present in the soil may be due to spraying carrying L3 down into the soil.

[0385] Discussion

[0386] This Example shows that treatment of pastures with the soluble CAN formulation of the invention significantly reduce the number of L3 on herbage, even when L3 are present in the soil. Example 9 — Animal trial

[0387] In this example, the effect of a single pre-grazing application of a test product on parasite uptake by young lambs under conditions that reflect realistic commercial management scenarios was tested.

[0388] Materials and Methods

[0389] The test product used comprised 16.2% w / w total nitrogen, 1.0% w / w ammonium, 15.2% w / w nitrate nitrogen, and 20.2% w / w calcium.

[0390] The trial was conducted on a commercial sheep and beef property. Six paddocks, consisting of a perennial ryegrass-white clover pasture mix, were split into two farmlets each, for twelve farmlets in total. Within each paddock, farmlets were randomly assigned to either the test product treatment or the liquid urea control group.

[0391] Prior to the trial, all paddocks were grazed by lambs with patent gastrointestinal nematode infections, to evenly contaminate the pasture with mixed-species parasite eggs. To reduce herbage mass, this was followed by grazing using cattle, targeting a residual herbage mass of 1200-1300 kg DM / ha. The site was then rested to allow pasture growth to reach 1500 kg DM / ha before treatment.

[0392] Ten days prior to the introduction of experimental animals, farmlets were treated with either test product or a liquid urea control. Both products were applied in liquid form using a commercial spray contractor under favourable weather conditions. Application rates were calibrated to deliver 40 kg N per hectare. For the test product, this corresponded to a volume of 400 L / ha. The liquid urea control was applied at a volume providing an equivalent N rate. Following treatment, daily herbage nitrate tests were performed to ensure levels had returned to a safe threshold for grazing. Eight days post-treatment, a total of 196 lambs were selected, tagged, weighed, and randomised by liveweight. The following day (Day 9 post-treatment), all lambs were treated with a broad-spectrum anthelmintic. On Day 10 post-treatment, lambs were assigned to their designated farmlets and remained set-stocked for the duration of the five-week trial period.

[0393] Faecal samples for individual faecal egg counts (FEC) were collected at 4 and 5 weeks following the start of grazing. FECs were determined using the standard McMaster technique (Lyndal-Murphy, M., 1993. Anthelmintic resistance in sheep, in: Corner, L.A., Bagust, T . (Eds.), Australian Standard Diagnostic Techniques for Animal Diseases. CSIRO, Melbourne, Australia, pp. 1-17).

[0394] FEC data were analysed using a generalised linear mixed model (GLMM) with a negative binomial distribution to account for overdispersion. The model included fixed effects for Treatment, Date, and their interaction. Random effects included individual animal ID nested within farmlet, and farmlet nested within paddock, to account for repeated measurements and experimental hierarchy.

[0395] Results

[0396] Lambs grazing on the test product-treated pastures had significantly lower faecal egg counts (FECs) than those in the urea control group at both time points (Table 3; X2(l) = 5.89, p = 0.0152).

[0397] Table 3. Faecal egg counts. Statistical analysis also showed a strong date (time) effect (X2(l) = 112.83, p < 0.001).

[0398] No significant treatment x date interaction (p = 0.393), suggesting the effect of the test product treatment was consistent over time.

[0399] Discussion This Example shows that treatment of pastures with a formulation of the invention resulted in reduced infection rates in animals subsequently grazing these pastures.

[0400] It is not the intention to limit the scope of the invention to the abovementioned examples only. As would be appreciated by a skilled person in the art, many variations are possible without departing from the scope of the invention (as set out in the accompanying claims).

[0401] CONCLUSIONS

[0402] The inventors have surprisingly found that compositions comprising CN or CAN are able to control the free-living stages of parasitic helminths when applied to a leaf surface. The inventors have shown that this effect is present in not only in vitro assays and tunnel house assays, but also that it results in reduced parasite burden in animals when tested in field trials.

Claims

WHAT WE CLAIM IS:

1. A method of controlling free-living stages of parasitic helminths in a treatment area comprising a surface; the method comprising applying a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) to the treatment area at an application rate sufficient to achieve a concentration of at least about 0.05 g / m2of nitrogen on the surface.

2. A method of controlling free-living stages of parasitic helminths in a treatment area comprising a surface; the method comprising applying a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) to the treatment area at a rate of 5-60 kg / ha of nitrogen.

3. A method of controlling free-living stages of parasitic helminths in a treatment area comprising a plant, wherein the plant comprises a leaf having a leaf surface, and wherein the treatment area has a leaf area index (LAI); the method comprising applying a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) to the treatment area such that the application rate in kg / ha of nitrogen divided by the LAI is at least about 0.8.

4. Use of a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) for controlling free-living stages of parasitic helminths in a treatment area comprising a surface; wherein the composition is applied to the treatment area at an application rate sufficient to achieve a concentration of at least about 0.05 g / m2of nitrogen on the surface.

5. Use of a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) for controlling free-living stages of parasitic helminths in a treatment area comprising a surface; wherein the composition is applied to the treatment area at a rate of 5-60 kg / ha of nitrogen.

6. Use of a composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) for controlling free-living stages of parasitic helminths in a treatment area comprising a plant, wherein the plant comprises a leaf having a leaf surface, and wherein the treatment area has a leaf area index (LAI); wherein the composition is applied to the treatment area such that the application rate in kg / ha of nitrogen divided by the leaf area index (LAI) is at least about 0.

87. A composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) when used for controlling free-living stages of parasitic helminths in a treatment area comprising a surface; wherein the composition is applied to the treatment area at a rate sufficient to achieve a concentration of at least about 0.05 g / m2of nitrogen on the surface.

8. A composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) when used for controlling free-living stages of parasitic helminths in a treatment area comprising a surface; wherein the composition is applied to the treatment area at a rate of 5-60 kg / ha of nitrogen.

9. A composition comprising, consisting essentially of, or consisting of calcium nitrate (CN) or calcium ammonium nitrate (CAN) when used for controlling free-living stages of parasitic helminths in a treatment area comprising a plant, wherein the plant comprises a leaf having a leaf surface, and wherein the treatment area has a leaf areaindex (LAI); wherein the composition is applied to the treatment area such that the application rate in kg / ha of nitrogen divided by the leaf area index (LAI) is at least about 0.8.

10. The method, use, or composition of any previous claim, wherein the composition is applied to the treatment area at a rate sufficient to achieve a concentration of from 0.05 to 6.0 g / m2of nitrogen on the surface, preferably from 0.5 to 6.0 g / m2, more preferably from 1.0 to 6.0 g / m2, more preferably from 2.2 to 6.0 g / m2of nitrogen.

11. The method, use, or composition of any previous claim, wherein the composition is applied at a rate of 25-60 kg / ha of nitrogen.

12. The method, use, or composition of any previous claim, wherein the treatment area has a leaf area index (LAI) and wherein the composition is applied to the treatment area such that the application rate in kg / ha of nitrogen divided by the LAI is at least about 0.8, preferably at least about 2.0, more preferably at least about 5.0.

13. The method, use, or composition of any previous claim, wherein the composition comprises soluble CAN consisting of calcium nitrate and ammonium nitrate.

14. The method, use, or composition of claim 13, wherein the composition has a molar ratio of calcium nitrate to ammonium nitrate of at least 1: 1, preferably at least 2: 1, 3: 1, 4: 1, or 5:

115. The method, use, or composition of any previous claim, wherein the composition is in liquid form.

16. The method, use, or composition of any previous claim, wherein the composition further comprises an agriculturally acceptable carrier, diluent or excipient.

17. The method, use, or composition of any previous claim, wherein the composition further comprises an agriculturally acceptable buffering agent.

18. The method, use, or composition of any previous claim, wherein the composition further comprises one or more additional active agents selected from the group consisting of anti-scorching agents, osmotic protection agents, nitrification inhibitors, urease inhibitors, pesticides, insecticides, plant and insect growth regulators, trace elements for plants and animals, plant nutrients, soil conditioners, soil nutrients, and bio-control agents.

19. The method, use, or composition of any previous claim, wherein the composition is applied using a spray nozzle, preferably a spray nozzle adapted for foliar application.

20. The method, use, or composition of any previous claim, wherein the treatment area comprises, or consists of, a house, garage, shed, farm building, outbuilding, milking shed, barn, or other enclosed or partially enclosed structure where free living parasitic helminths, particularly free living parasitic nematodes are present, or may be present.

21. The method, use, or composition of any one of claim 1 to 19, wherein the treatment area comprises, or consists of, pasture.

22. The method, use, or composition of any previous claim, wherein the treatment area comprises a plant and / or animal waste.

23. The method, use, or composition of claim 22, wherein the surface is a surface of the plant and / or animal waste.

24. The method, use, or composition of claim 22 or 23, wherein the plant comprises a leaf having a leaf surface, and wherein the surface is the leaf surface.

25. The method, use, or composition of any one of claims 22 to 24, wherein the plant is a plant used for grazing and / or harvesting.

26. The method, use, or composition of any one of claims 22 to 25, wherein the plant is a livestock food plant.

27. The method, use, or composition of claim 26, wherein the livestock food plant is a grass plant and / or a legume plant.

28. The method, use, or composition of any one of claims 22 to 27, wherein the method, use, or composition also increases the growth of the plant.

29. The method, use, or composition of any previous claim, wherein the parasitic helminths are mammalian endoparasites.

30. The method, use, or composition of any previous claim, wherein the parasitic helminths are parasites of canines, felines, bovines, ovines, cervines, equines, caprines, porcines, lagomorphs, rodents, camelids, and / or hominids; preferably parasites of bovines, ovines, cervines, and / or equines.

31. The method, use, or composition of any previous claim, wherein the parasitic helminths are selected from the group consisting of flukes (trematodes), tapeworms (cestodes) and roundworms (nematodes).

32. The method, use, or composition of any previous claim, wherein the parasitic helminths are selected from the group consisting of Ancylostomatoidea, Strongyloidea,Trichostrongyloidea, and Metastrongyloidea.

33. Use of calcium nitrate (CN) or calcium ammonium nitrate (CAN) in the manufacture of a composition for controlling free-living stages of parasitic helminths in a treatment area comprising a surface.

34. The method, use, or composition of any previous claim, wherein the parasitic helminths are selected from the group consisting of Haemonchus, Teladorsagia, Trichostrongylus, and Cooperia.

35. The method, use, or composition of any previous claim, wherein the number of parasitic helminths in the treatment area is reduced by at least about 10%, preferably at least about 25%, more preferably at least about 50%.

36. The method, use, or composition of any previous claim, wherein the treatment area comprises soil, and wherein the top 50 mm of the soil comprises fewer than about 4,000,000 parasitic helminths per m3of soil, preferably fewer than about 1,000,000, more preferably fewer than about 100,000, most preferably fewer than about 10,000.

37. The method, use, or composition of any previous claim, wherein the treatment area comprises soil, and wherein the soil comprises, consists essentially of, or consists of sand, silt, pumice, ash, or any combination of any two or more of these.

38. The method, use, or composition of any previous claim, wherein the composition is applied during or between the months of October- February in the Northern Hemisphere or during or between the months of April-August in the Southern Hemisphere; preferably during or between the months of December-February in the Northern Hemisphere or during or between the months of June-August in the SouthernHemisphere.

39. The method, use, or composition of any previous claim, wherein the composition is applied at a time of year when the mean daily temperature, calculated as (daily maximum temperature + daily minimum temperature) -? 2, is less than about 20°C, preferably less than about 18°C, more preferably less than about 16°C, most preferably less than about 14°C.

40. The method, use, or composition of any previous claim, wherein the composition is applied at a time of year when the mean daily relative humidity, calculated as (daily maximum temperature + daily minimum temperature) -? 2, is less than about 50%.

41. The method, use, or composition of any previous claim, wherein the composition is applied when the daily rainfall, averaged over 1 week, is at least about 20 mm less than the typical daily rainfall for the treatment area.

42. The method, use, or composition of any previous claim, wherein the treatment area comprises a plant and the composition is applied after the plant is harvested.

43. The method, use, or composition of any previous claim, wherein the treatment area has not been grazed for at least about 4 weeks prior to applying the composition.

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