JAMES, Peter (665 Fairfield Road, Yeerongpilly, Queensland 4105, AU)
| CLAIMS: 1. A method of reducing or preventing ectoparasite infestation in/on an animal comprising administering to the animal a formulation comprising tea tree oil. 2. A method for treating a wound on an animal comprising administering to the animal a formulation comprising tea tree oil. 3. A method according to claim 2 wherein the wound is a result of myiasis (flystrike). 4. A method according to claim 2 wherein the wound is a result of a husbandry practice such as tail docking, castration, mulesing or dehorning. 5. A method according to any one of claims 1 to 4 wherein the formulation is a dust, dip, "pour-on" formulation, "spot-on" formulation or spray. 6. A method according to any one of claims 1 to 5 wherein the formulation comprises an aqueous emulsion of tea tree oil. 7. A method according to claim 6 wherein the formulation comprises an emulsifying agent selected from the group consisting of polysorbate 80 (e.g., Tween®80), an octylphenol ethoxylate (e.g., Triton®), sodium cocoyl isethionate (e.g., Geropon® AS-200), disodium laureth sulfo succinate (e.g., Geropon® SBFA-30), sodium dioctyl sulfo succinate (e.g., Geropon® SS-O-75), sodium methyl cocoyl taurate (e.g., Geropon® TC-270), sodium methyl cocoyl taurate (e.g., Geropon® TC-42), ethoxylated castor oil (e.g., Alkamuls OR/36®), ethoxylated oleic acid (e.g., Alkamuls A) and mixtures thereof. 8. A method according to claim 7 wherein the emulsifying agent is a combination of ethoxylated castor oil and ethoxylated oleic acid (ALK). 9. A method according to claim 8 wherein the formulation comprises ethoxylated castor oil and the ethoxylated oleic acid in a ratio of about 7:3 (v/v). 10. A method according to any one of claims 7 to 9 wherein the formulation comprises about 1 -15 (v/v) emulsifying agent. 11. A method according to any one of claims 1 to 10 wherein the formulation comprises about 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40% or 50% (v/v) tea tree oil. 12. A method according to any one of claims 1 to 11 wherein the formulation comprises ethoxylated castor oil/ethoxylated oleic acid (ALK) at a concentration of about 0.5%, 1%, 2%, 5% or 10% (v/v). 13. A method according to any one of claims 1 to 12 wherein the formulation comprises tea tree oil at a concentration of 1 to 5% (v/v) and ethoxylated castor oil ethoxylated oleic acid (ALK) at a concentration of 1 to 5% (v/v). 14. A method according to any one of claims 1 to 13 wherein the formulation further comprises an ectoparasitical agent selected from the group consisting of organophosphates, carbamates, organochlorines, pyrethroids, formamidines, triazapentadienes, triazinethiones and thioureas. 15. A method according to any one of claims 1 to 14 wherein the ectoparasite is selected from the group consisting of lice, flies, blowflies, or eggs, pupae or larvae thereof. 16. A method according to any one of claims 1 to 15 wherein the animal is a sheep, goat, alpaca, llama, cow, horse, dog, cat or rabbit. 17. A topical formulation effective against ectoparasite infestation of an animal comprising tea tree oil and an emulsifier selected from the group consisting of polysorbate 80 (e.g., Tween®80), an octylphenol ethoxylate (e.g., Triton®), sodium cocoyl isethionate (e.g., Geropon® AS-200), disodium laureth sulfo succinate (e.g., Geropon® SBFA-30), sodium dioctyl sulfo succinate (e.g., Geropon® SS-O-75), sodium methyl cocoyl taurate (e.g., Geropon® TC-270), sodium methyl cocoyl taurate (e.g., Geropon® TC-42), ethoxylated castor oil (e.g., Alkamuls OR/36®), ethoxylated oleic acid (e.g., Alkamuls A) and mixtures thereof. 18. A topical formulation according to claim 17 wherein the emulsifying agent is a combination of ethoxylated castor oil and ethoxylated oleic acid (ALK). 19. A topical formulation according to claim 18 wherein the formulation comprises ethoxylated castor oil and the ethoxylated oleic acid in a ratio of about 7:3 (v/v). 20. A topical formulation according to any one of claims 17 to 19 wherein the formulation comprises about 1%-15% (v/v) emulsifying agent. 21. A topical formulation according to any one of claims 17 to 20 wherein the formulation comprises about 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50% (v/v) tea tree oil. 22. A topical formulation according to any one of claims 17 to 21 wherein the formulation comprises ethoxylated castor oil/ethoxylated oleic acid (ALK) at a concentration of about 0.5%, 1%, 2%, 5% or 10% (v/v). 23. A topical formulation according to any one of claims 17 to 22 wherein the formulation comprises tea tree oil at a concentration of 1 to 5% (v/v) and ethoxylated castor oil/ethoxylated oleic acid (ALK) at a concentration of 1 to 5% (v/v). 24. A topical formulation according to any one of claims 17 to 23 wherein the formulation further comprises an ectoparasitical agent selected from the group consisting of organophosphates, carbamates, organochlorines, pyrethroids, formamidines, triazapentadienes, triazinethiones and thioureas. |
Field of invention
The present invention relates to the treatment of animals to reduce or prevent infection by ectoparasites. The present invention also relates to a method of treating a wound on an animal. The present invention also relates to a topical formulation suitable for use in the methods of the invention.
Background of the invention
Two of the main parasite diseases in sheep are flystrike, caused primarily by the Australian sheep blowfly, Lucilia cuprina and infestation with the sheep louse (Bovicola ovis). Most recent estimates put the cost of these two diseases to the sheep industry at $280m and $123m respectively (Sackett et al. 2006). Control of these two parasites relies significantly on the application of chemical insecticides. However the efficiency control programs has been compromised by development of resistance to these insecticides in both sheep blowflies (Levot 1995, Levot and Sales, 2002) and lice (Levot 1995, James et al. 2008) and there is an ongoing need to minimise chemical residues in sheep products (Pattinson, 1995; Savage, 1998; Williams and Brightling, 1999), reduce occupational exposure to chemicals (Murray et al., 1992; Russell, 1995; Savage, 1998) and avoid environmental contamination (Littlejohn and Melvin, 1991) from chemical parasiticides.
Summary of the invention
The present inventors have now developed a formulation comprising tea tree oil that is effective in reducing or preventing ectoparasite infection in animals.
Accordingly, the present invention provides a method of reducing or preventing ectoparasite infestation in an animal comprising administering to the animal a formulation comprising tea tree oil.
The present invention also provides a method for treating a wound on an animal comprising administering to the animal a formulation comprising tea tree oil. The wound may be the result of myiasis (flystrike) or a husbandry practice such as tail docking, castration, mulesing or dehorning.
By "treating a wound" we mean applying the formulation to the wound to facilitate healing and/or preventing microbial or ectoparasitic infection of the wound. The tea tree oil formulation may be suitably administered in the form of a dust, dip, "pour-on" formulation, "spot-on" formulation or spray comprising a suspension or aqueous emulsion of tea tree oil. Thus, the tea tree oil formulation may be a topical formulation. Administration in the form of prolonged-release boluses or implants is also possible.
In one embodiment the tea tree oil formulation is administered in the form of a formulation or spray comprising an aqueous emulsion of tea tree oil.
In one example, the emulsifying agent has a hydrophilicity-lipophilicity balance of 10-14, such as 11-13, preferably 12. The skilled artisan will understand that the hydrophilic-lipophilic balance of an emulsifier is a measure of the degree to which it is hydrophilic or lipophilic, determined by calculating values for the different regions of the molecule. Methods for determining the hydrophilic-lipophilic balance are described by Griffin (1949) or in Davies (1957).
In one example, the emulsifying agent is selected from the group consisting of polysorbate 80 (e.g., Tween®80), an octylphenol ethoxylate (e.g., Triton®), sodium cocoyl isethionate (e.g., Geropon® AS-200), disodium laureth sulfo succinate (e.g., Geropon® SBFA-30), sodium dioctyl sulfo succinate (e.g., Geropon® SS-O-75), sodium methyl cocoyl taurate (e.g., Geropon® TC-270), sodium methyl cocoyl taurate (e.g., Geropon® TC-42), ethoxylated castor oil (e.g., Alkamuls OR/36®), ethoxylated oleic acid (e.g., Alkamuls A) and mixtures thereof. Preferably, the emulsifying agent is a combination of ethoxylated castor oil and ethoxylated oleic acid. In one example, the ethoxylated castor oil and the ethoxylated oleic acid are combined in a ratio of 1 : 1 or 2: 1 or 3: 1 or 4: 1 or 5: 1 (v/v). In one example, the ethoxylated castor oil and the ethoxylated oleic acid are combined in a ratio of about 7:3 (v/v) (this combination is referred to herein as ALK).
In one example, the formulation comprises about 1%-15% (v/v) emulsifying agent, such as about 1%-10% emulsifying agent, for example about 1%, 2% or 2.5% or 3% or 4% or 5% or 6% or 7% or 8% or 9% or 10% emulsifying agent.
In another embodiment the formulation comprises about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40% or 50% (v/v) tea tree oil.
In another embodiment the formulation comprises ALK at a concentration of about 0.5%, 1%, 2%, 5% or 10% (v/v).
In another embodiment the formulation comprises tea tree oil at a concentration of 1 to 5% (v/v) (i.e., 1%, 2%, 3%, 4% or 5% or any numerical values in between) and ALK at a concentration of 1 to 5% (v/v) (i.e., 1%, 2%, 3%, 4% or 5% or any numerical values in between), in any combination or permutation. The tea tree oil formulation may be administered in combination with conventional ectoparasitical agents such as organophosphates, carbamates, organochlorines, pyrethroids, formamidines, triazapentadienes, triazinethiones or thioureas. In one embodiment the formulation is administered in combination with diazinon, ivermectin, boric acid or cryozamine.
The method of the invention is suitable for reducing or preventing infestation of ectoparasites such as lice, flies, blowflies, or eggs, pupae or larvae thereof.
The method of the invention may be applied to any animal, preferably a mammal such as a sheep, goat, alpaca, llama, cow, horse, dog, cat or rabbit.
The present invention also provides a topical formulation effective against ectoparasite infestation of an animal comprising tea tree oil and an emulsifier selected from the group consisting of polysorbate 80 (e.g., Tween®80), an octylphenol ethoxylate (e.g., Triton®), sodium cocoyl isethionate (e.g., Geropon® AS-200), disodium laureth sulfo succinate (e.g., Geropon® SBFA-30), sodium dioctyl sulfo succinate (e.g., Geropon® SS-O-75), sodium methyl cocoyl taurate (e.g., Geropon® TC-270), sodium methyl cocoyl taurate (e.g., Geropon® TC-42), ethoxylated castor oil (e.g., Alkamuls OR/36®), ethoxylated oleic acid (e.g., Alkamuls A) and mixtures thereof. Preferably, the emulsifying agent is a combination of ethoxylated castor oil and ethoxylated oleic acid. In one example, the ethoxylated castor oil and the ethoxylated oleic acid are combined in a ratio of 1: 1 or 2: 1 or 3: 1 or 4: 1 or 5: 1 (v/v). In one example, the ethoxylated castor oil and the ethoxylated oleic acid are combined in a ratio of about 7:3 (v/v) (this combination is referred to herein as ALK).
In one example, the topical formulation comprises about 1%-15% (v/v) emulsifying agent, such as about 1 - 10% emulsifying agent, for example about 1%, 2% or 2.5% or 3% or 4% or 5% or 6% or 7% or 8% or 9% or 10% emulsifying agent.
In another embodiment the topical formulation comprises about 0.5%, 1%, 2%, 5%, 10%, 20%, 15%, 30%, 40% or 50% (v/v), tea tree oil.
In another embodiment the topical formulation comprises ALK at a concentration of about 0.5%, 1%, 2%, 5% or 10% (v/v).
In another embodiment the topical formulation comprises tea tree oil at a concentration of 1 to 5% (v/v) (i.e., 1%, 2%, 3%, 4% or 5% or any numerical values in between) and ALK at a concentration of 1 to 5% (v/v) (i.e., 1%, 2%, 3%, 4% or 5% or any numerical values in between), in any combination or permutation.
In another embodiment the topical formulation comprises a conventional ectoparasitical agent such as an organophosphate, a carbamate, an organochlorine, a pyrethroid, a formamidine, a triazapentadiene, a triazinethione or a thiourea. In one embodiment the topical formulation further comprises diazinon, ivermectin, boric acid or cryozamine.
The present invention also provides a formulation comprising tea tree oil for use in reducing or preventing ectoparasite infestation in or on an animal.
The present invention also provides a formulation comprising tea tree oil for use in treating a wound on an animal.
Furthermore, the present invention provides the use of a formulation comprising tea tree oil in the manufacture of a medicament for reducing or preventing ectoparasite infestation in or on an animal.
The present invention also provides the use of a formulation comprising tea tree oil in the manufacture of a medicament for treating a wound on an animal.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
As used herein, the term "about", unless stated to the contrary, refers to +/- 20%, more preferably +/- 10%, of the designated value. For the avoidance of doubt, the term "about" followed by a designated value is to be interpreted as also encompassing the exact designated value itself (for example, "about 1" also encompasses 1 exactly).
Brief Description of the Figures Figure 1: Amount of uptake of 5% tea tree oil (TTO)/water formulation into wool with different emulsifiers.
Figure 2: Rate of weight loss from 5% TTO solutions mixed in water with different emulsifiers.
Figure 3: Mortality of first instar L. cuprina larvae with different concentrations of TTO emulsified in bovine serum at 24 h.
Figure 4: Mortality of (a) second instar larvae and (b) third instar larvae at different concentrations of Melaleuca oil in agar-based tests system. Figure 5: Mortality of third instar L. cuprina larvae following dipping in different concentrations of TTO for 1 minute with either 8% Tween 80 or 8% Alkamuls as emulsifiers. Figure 6: Number of dead or live larvae remaining on agar 24 hours after application of insecticide as single active ingredient or in combination with TTO.
Figure 7: Effect of treatment of third instar larvae with insecticide as single active ingredient or in combination with TTO on the number of ecloding adult flies in agar assays: (a) diazinon (1 gL-1), ivermectin (32 mgL-1) and boric acid (25 gL-1) (b) cyromazine (0.1 gL-1 and 1 gL-1).
Figure 8: Effect of immersion of third instar larvae in insecticides with and without tea tree oil on the number of ecloding adult flies: (a) diazinon (1 gL-1), ivermectin (32 mgL-1) and boric acid (25 gL-1) (b) cyromazine (0.1 gL-1 and 1 gL-1).
Figure 9: Effects topical application of tea tree oil on adult L. cuprina. (a) different concentrations in butanone, (b) different amounts of 100% tea tree oil. (6-09). Figure 10: Mortality of L. cuprina females treated topically with and without 1 μΐ of 2% piperonyl butoxide and various concentrations of TTO (Experiment 1).
Figure 11: Mortality of L. cuprina females treated topically with and without 1 μΐ of 2% piperonyl butoxide and various concentrations of TTO (Experiment 2).
Figure 12: Fumigant effect: mortality of adult L. cuprina males and females exposed to 60 μΐ of different concentrations of TTO in acetone following 2 h and 24 h of exposure; (controls exposed to acetone alone). Figure 13: Mortality of adult L. cuprina males and females exposed to different amounts of pure TTO following 2 h and 24 h of exposure; (controls exposed to grape seed oil).
Figure 14: Mean number of flies present and exhibiting oviposition behaviour on control wool preparations baited with sheep liver homogenate - choice test. No flies were recorded on TTO- treated wool. Figure 15: Mean number of flies recorded on control (+ s.e.) and TTO-treated wool preparations baited with sheep liver homogenate - non-choice test. No flies were recorded on TTO- treated wool. Figure 16: Mean (+s.e.) number of flies recorded on the surface of TTO-treated wool or wool treated with emulsifier only at different times after treatment. Bars represent the average of numbers of flies over 6 cages at 24 observation times over 1 day at the times after TTO-treatment shown in the x axis. Figure 17: Mean (+s.e.) number of egg masses recorded on the surface of control wool preparations following one days exposure to flies up to 44 days after treatment. Bars represent the average of numbers of egg masses per cage over 6 cages. No egg masses were recorded on TTO-treated wool. Figure 18: Mean percent mortality of L. cuprina eggs exposed to 60 μΐ of different concentrations of tea tree oil in acetone (control = 60 μΐ acetone).
Figure 19: Proportion of L. cuprina which were killed as pupae, killed as flies during or following eclosion or hatched to flies following exposure of pupae to fumigant action of different concentrations of TTO.
Figure 20: Mortality of lice exposed to cotton squares treated with different concentrations of TTO in butanone. Cotton squares were dried overnight in a fume hood prior to louse exposure.
Figure 21: Mortality of lice exposed to filter paper treated with different concentrations of TTO in grapeseed oil. Oil was allowed to disperse on the filter paper, but papers were not dried prior to addition of lice. Figure 22: Percent mortality of lice at 24 h following wool dipping in different concentrations of TTO emulsified with 5% ALK in two assays.
Figure 23: Percent mortality of lice following wool dipping in different concentrations of TTO emulsified with 1% ALK plus 1% TTO emulsified with 5% ALK. Figure 24: Effect of synergism with % piperonyl butoxide added to different concentrations of TTO (1% ALK) on percent mortality of lice in wool dipping assays.
Figure 25: Mortality of lice exposed to wool treated with different concentrations of TTO oil and dried in a fume hood prior to lice exposure.
Figure 26: Percent mortality (+ s.e.) of lice exposed to fumigant effect of different concentrations of TTO in 8% Tween 80. Figure 27: Mean percent mortality of lice (+ s.e.) in wool fumigation assays. (In this assay the vials were closed with parafilm).
Figure 28: Percent mortality of lice (+ s.e.) in wool fumigation assays (vials not closed with parafilm).
Figure 29: Mortality of lice exposed to the fumigant effects of different concentrations of TTO and the TTO components terpin-4-ol, alpha terpinene and gamma terpinene at 3 and 24 h after exposure. Figure 30: Mortality of lice exposed to the fumigant effects of 5% TTO, 2.15% and 1.5% terpinen-4-ol and some other TTO components at 3 h and after 24 h.
Figure 31 : Percent hatch of lice eggs exposed to 0, 15 or 60 μΐ of TTO for 24 hours. Figure 32: Mean (+ s.e.) for counts of lice per 10 cm fleece parting on sheep in the 6 treatment and control groups at allocation.
Figure 33: Mean (+ s.e.) for counts of lice per 10 cm fleece parting on sheep in the 6 treatment and control groups at allocation in the jetting study.
Figure 34: Mean counts of lice per 10 cm fleece part over 12 weeks in groups of sheep jetted with 1% or 2% TTO or left untreated (control).
Figure 35: Mean wool rubscore in untreated and sheep jetted with 1% TTO (TT1P) and 2% TTO (TT2P) formulation at 6 and 12 weeks after treatment. (Values for two sheep were missing in the TT2P group at 12 weeks). Figure 36: Changes in the rump wool concentrations of some major TTO components over 12 weeks after dipping in 2% TTO.
Figure 37: Changes in concentration of terpinen-4-ol in the shoulder wool of individual sheep dipped in 2% TTO at 1, 3, 6 and 12 weeks post dipping.
Figure 38: Fall in dip volume with progressive dipping of wool samples. (Initial dip volume = 500 ml; each wool sample = 10 g).
Detailed description of the invention
Tea tree oil
Tea tree oil is the volatile essential oil obtained from the Australian paperbark tree Melaleuca alternifolia. This tree is native to the swampy regions of northern New South Wales and southern Queensland on the eastern coast of Australia. Tea tree oil has been shown to contain more than 100 components, the most abundant of which is the monoterpene alcohol terpinen-4-ol (-40%), followed by γ-terpinene (-20%) and a- terpinene (-10%). It has been defined in the art by international standard ISO 4730:2004 ("Oil of Melaleuca, terpinen-4-ol type (Tea Tree oil)"), which specifies levels of 15 different components present in tea tree oil. Amongst its many components, at least terpinen-4-ol is believed to provide a level of antimicrobial activity.
In one example, the tea tree oil comprises the following components:
Component Composition (%) Component Composition (%) terpinen-4-ol 42.4 limonene 1.1
γ-terpinene 20.1 aromadendrene 1.1
a-terpinene 9.0 δ-cadinene 0.8
1,8-cineole 3.7 ledene 0.6
terpinolene 3.2 sabinene 0.3
p-cymene 3.1 globulol 0.2
a-terpineol 3.1 viridiflorol 0.1
a-pinene 2.4 Alternative tea tree oil compositions, comprising different levels of these and other known components of tea tree oil, may also be used.
Treatment or prevention of ectoparasite infestation
The present invention encompasses the treatment or prevention of an infestation by any ectoparasite, for example, ticks, mites, lice, keds, head fly and blow fly and larvae thereof, amongst others. In this regard, domesticated animals, such as cats and dogs, are often infested with one or more of fleas (Ctenocephalides felis, Ctenocephalides sp. and the like), ticks (Rhipicephalus sp., Ixodes sp., Dermacentor sp., Amblyoma sp. and the like), and mites (Demodex sp., Sarcoptes sp., Otodectes sp. and the like) or lice (Trichodectes sp., Cheyletiella sp., Lignonathus sp., and the like).
Fleas are a particular problem since they cause discomfort to the animal and are also vectors of pathogenic agents in animals, such as dog tapeworm (Dipylidium caninum).
Ticks are also harmful to the physical health of an animal. Ticks also release toxins which cause inflammation or paralysis in the host. Occasionally, these toxins are fatal to the host. For example, ticks contribute to a condition known as tickborne fever which is an infectious disease affecting almost all sheep which graze tick- infected pastures. It is caused by the microorganism Cytoecetes phogocytophilia. Ticks also contribute to tick pyaemia, or "cripples". Ticks are also a vector of pathogenic agents. Exemplary diseases which are caused by ticks include borrelioses (Lyme disease caused by Borrelia burgdorferi), babesioses (or piroplasmoses caused by Babesia sp.) and rickettsioses (also known as Rocky Mountain spotted fever).
Mites cause intense irritation to animals, such as sheep, which react by rubbing against fence posts, or the like. This can reduce the quality or yield of wool from the sheep. If no fixed objects are available, they rub against each other spreading the disease further- Mites also cause foot scab, a mange infestation of sheep.
Lice is the common name for over 3000 species of wingless insects of the order Phthiraptera that are obligate ectoparasites of most animals. The most common forms of lice that affect mammals are the sucking lice (suborder Anoplura). Lice can cause mange and can result in considerable fleece damage, reduced wool quality and reduced production levels.
Mites and lice are particularly difficult to combat since there are very few active substances which act on these parasites and they require frequent treatment.
In another example, the ectoparasite infestation may be a myiasis (also known as
"flystrike"). The Calliphoridae family, together with the Sarcophagidae and the Oestridae families, contain the species responsible for the most important myiases of domestic animals and man. Myiasis is the infestation of living animals with the larvae of dipteran flies. Myiasis caused by members of the family Calliphoridae is commonly called "blowfly strike." The "blow" is the laying of the eggs by the fly at or near a strike site. The "strike" is the development of the eggs into maggots and the damage that this development causes at that site. Strikes are classified by the area of the body affected.
Blowfly myiasis primarily affects sheep; however, many other animals may be affected. Major species of blowflies include Lucilia sericata (greenbottles), Phormia terraenovae (blackbottles), Calliphora erythrocephala and C. vomitoria (bluebottles) in Europe. Lucilia cuprina, L. caeser, L. illustris, Phormia regina, Calliphora stygia, C. australis, C. fallax, Chrysomyia albiceps, C. chlorophyga, C. micropogon, and C. rufifacies are major species of blowflies in the tropics and subtropics.
The blowflies that attack sheep fall into two main categories:
(1) Primary flies, which are capable of initiating a strike on living sheep. These include Lucilia and Phormia spp. and some Calliphora spp.
(2) Secondary flies, which cannot initiate a strike, but attack an area already struck or otherwise damaged. They frequently extend the injury, rendering the strike one of great severity. Examples include many Calliphora spp. and, in warmer climates, Chrysomyia spp.
Eggs laid on the wool of sheep by primary flies, under favorable conditions, hatch within 12 hours. The hatched larvae migrate down the wool to the skin where the larvae lacerate the skin with their oral hooks and secrete proteolytic enzymes into the skin to establish the lesion. The larvae feed on the surrounding tissues, grow rapidly, and moult twice before becoming fully mature maggots. The maggots then drop to the ground and develop into mature flies. During the period of larval development, extensive tissue damage occurs, and the strike becomes available for the establishment of secondary infections or, worse, becomes an attractive site in which secondary blowflies may lay their eggs.
The irritation and distress caused by blowfly strikes are extremely debilitating, and sheep can rapidly lose condition. Where death occurs, it is often due to septicaemia. Affected sheep are anorexic, appear dull, and usually stay away from the main flock.
The term "infestation" encompasses the presence of any number of ectoparasites in or on the animal to be treated. The method of the invention may comprise administering the formulation to a wound on an animal. Thus, the present invention also encompasses the treatment of a wound on an animal, comprising administering the formulation of the invention to the animal. Administering the formulation to the animal may prevent or treat an infestation by any ectoparasite, as described herein. Any wound may be treated. For example, a wound in which the animal' s skin has been broken may be treated.
Production of formulations
Exemplary formulations of the invention are emulsions. Processes for preparing an emulsion will be apparent to the skilled artisan. For example, tea tree oil may be combined with an emulsifying agent and an aqueous solution and agitated to thereby produce an emulsion. Optionally, additional aqueous solution (and optionally, emulsifying agent) may be added and the formulation may again be agitated to thereby dilute the tea tree oil.
In one example, the formulation is agitated using a homogenizer. Exemplary homogenizers include pressure jetting homogenizer and the like, an ultrasonic homogenizer and the like, and a homomixer such as a high-rate mixer and the like. Homogenization can be performed under pressure and/or in the presence of heat to enhance emulsification.
Application of formulations
As discussed herein, a formulation of the present disclosure can be applied by any means. In one example, the formulation is applied topically.
An exemplary method for applying a formulation is dipping. In such a method a subject is immersed in a formulation of the disclosure. The head of the subject need not necessarily be immersed, in which case the formulation should be applied to this region by another means. Dipping provides an advantage that the majority of the subject is exposed to the formulation, thereby increasing the likelihood of the formulation providing a therapeutic/prophylactic benefit.
Another exemplary method is jetting, in which the formulation is sprayed onto a subject. The jet or spray may be a hand-held device which is used to manually apply the formulation. Alternatively, the jet is an automatic system, e.g., a run or path comprising multiple sprays along which the subject must travel thereby having the formulation applied. In order that the nature of the present invention may be more closely understood preferred forms thereof will now be described with reference to the following Examples. EXAMPLES
Example 1. Materials and methods
1.1 TTO composition
All TTO used in experiments was extracted from plants with a terpinen-4-ol chemotype (Homer, et al. 2000) from the north eastern region of New South Wales in Australia. The composition of the TTO used in these studies, measured by GCMS (NSW Department of Primary Industries Diagnostic and Analytical Services Laboratory, WoUongbar NSW), together with the acceptable range for TTO under ISO standard 4730, is given in Table 1.
Table 1: Composition of TTO used in the studies.
1.2 Formulation development
TTO is not miscible with water and emulsifiers and carriers were required both for laboratory assays and ultimately for animal application. In these experiments, acetone, grapeseed oil and Tween® 80 and a number of other potential emulsifying agents and combinations were tested. Key characteristics that were required included the ability to form stable emulsions of TTO in water and the ability to readily wet wool (which is covered in water repellent lipid). The rate of evaporation of TTO from the emulsion was also assessed. Without wishing to be bound by theory, it is generally considered that solubilisation is most efficient with emulsifiers with similar HLB (hydrophilicity-lipophilicity balance) to the agent being mixed. The HLB of TTO is 12 and agents with approximately similar HLB values were tested. These included Tween® 80, Triton®, Geropon® and various combinations of Ethoxylated Castor Oil (Alkamuls OR/36®, Rhodia) and Ethoxylated Oleic Acid (Alkamuls A®, Rhodia). Grapeseed oil was also used as a carrier and assessed in combinations with some other agents. For assays with L. cuprina in which TTO was mixed in sheep serum Tween 80 was used as the emulsifier.
To assess the efficiency of wool wetting with different formulations a system similar to that described by Lipson (1976) was used. Briefly the weathered tip was trimmed from about 100 mg of wool to leave about 3 cm in length. The wool was inserted into glass tubing which was glued in position through the screw top lids of plastic vials and then weighed. Test solution was added to the vials so that about 1cm of the tube was immersed when the tops were screwed in place. After about 1 minute the amount of fluid taken up by the wool was determined by reweighing the tube and wool. Stability of solutions was assessed subjectively, noting the degree of separation over time and rate of evaporation was estimated by measuring the reduction in weight of emulsion with time. Some typical results for fluid uptake and evaporation are shown in Figures 1 and 2.
Ethoxylated Castor Oil (Alkamuls OR/36®, Rhodia) and Ethoxylated Oleic Acid (Alkamuls A®, Rhodia; Brenntag Pty Ltd, Notting Hill, Vic) combined in a ratio of about 7:3 (v/v) gave an emulsifier blend (ALK) that produced stable emulsions of TTO in water, did not give unacceptable TTO loss on standing, provided good wetting of wool and was judged more suitable for animal application than some of the other solvents. The Alkamuls mixture (ALK) was used to emulsify TTO in most wool assays and in all animal studies. 1.3 Sheep blowfly
Lucilia cuprina (L. cuprina) used for these experiments were of the LS insecticide- susceptible strain (Kotze et al. 1997) maintained in culture at the Queensland Primary Industries Animal Research Institute laboratories at Yeerongpilly, Queensland.
1.4 Effects against larvae
1.4.1 First instar larvae - serum assay
Assays were conducted using a method adapted from Hughes and Levot (1987). Tea tree oil was diluted in sterile bovine serum containing about 2% yeast and about 0.5% monobasic potassium orthophosphate to give concentrations of between about 0.1%, and about 2%, following preliminary experiments to establish a dose range. Rolled pieces of chromatography paper (about 12 cm x 3 cm) were placed inside flat bottom glass vials (about 5 cm x 1.5 cm diameter) and about one miUilitre aliquots of serum with the desired concentrations of tea tree oil dispensed onto it. Small clumps of about twenty, 1st instar L. cuprina larvae were placed on the top of the treated chromatography paper and the mouth of the vial covered with filter paper secured with a rubber band. Vials were held at about 29°C in a foam rack over water in a plastic container with the lid resting on top, but not airtight. After about 24 h, the larvae were washed into a Petri dish and numbers of live and dead larvae recorded. Four replicate tubes were used for each concentration tested.
1.4.2 Second and third instar larvae - agar assay
An agar based system was developed to test for likely effect in the resolution of strikes without the use of live animals. Meat liver agar (Merck) containing about 5% bovine serum was added in about 10 ml amounts to about 75 ml (about 42 mm diam. x 56 mm height) plastic vials and allowed to set in about a 1 cm layer in the base of each vial. Groups of ten, second instar larvae or feeding third instar larvae (not prepupal) were transferred from liver into each container and about 1 ml of bovine serum added on top of the agar. Each container was covered with gauze and placed in an incubator to allow the "strike" to develop. After four hours the containers were removed from the incubator, checked to ensure that larvae had commenced feeding in the agar, and about 1 ml aliquots of the test formulations applied to the surface of the growth medium. The vials were then covered with nylon mesh, held at about 29°C and about 60% RH and numbers of dead and live larvae recorded at about 24 h and about 48 h. As TTO has potentially beneficial repellent and antibacterial activities it was also assessed against third instar L. cuprina in combination with other insecticidal compounds used in strike treatments. In the first study TTO was emulsified in water at about 2.5% v/v with about 2.5% ALK and boric acid (about 25 gL "1 ), diazinon (about 1 gL _1 ) or ivermectin (about 32 mgL "1 ). The concentrations of diazinon and ivermectin were those used in commercial flystrike treatment formulations. In a second experiment TTO and about 2.5% ALK were mixed with cyromazine at about 0.1 gL "1 and about 1 gL "1 . The control in both experiments was about 2.5% alkamuls in water. Following the application of treatments the vials were placed (uncovered) inside 600 ml round disposable food containers (95 mm diameter and 78 mm height) containing a layer of about 2 cm of vermiculite, which were covered with gauze and placed in an incubator at about 29°C and about 60% RH. This enabled the larvae to vacate the vials if repelled from the agar or when they finished feeding and to pupate in the vermiculite. The number of adult flies that had emerged was recorded after 14 days.
1.4.3 Third instar larvae - dipping assay
Groups of third instar larvae (n = 50 to 100) were removed from liver, placed into mesh bags (about 4 cm x 2 cm) and submerged for about 60 s. in solutions containing the test compounds at appropriate dilution. After removal, the larvae were held on paper towelling within plastic food containers for about 24 h before addition of vermiculite to an approximate depth of about 2 cm to allow pupation to occur. They were held at about 28°C and the number of adult flies that emerged counted after about 14 days. Following preliminary studies to establish dose range, TTO concentrations from about 5% to 50% emulsified in water with about 8% ALK or about 8% Tween80® were tested.
TTO was also tested in combination with diazinon, ivermectin and boric acid and in a separate experiment with cyromazine at the same concentrations as for the agar tests by this method. For these studies TTO and insecticide were emulsified in water with about 2.5% ALK. The control was about 2.5% ALK solution without TTO or insecticide. There were 3 replicates for each treatment in both experiments.
1.4.4 Larval repellence assay
Meat liver agar (Merck) mixed with about 5% bovine serum solution, prepared as described above, was added to eight approximately 90 mm diameter Petri dishes and allowed to set. TTO was mixed into the agar (without emulsifier) at concentrations of about 0.5%, 2% and 5% (two petri dishes for each concentration). Once set, the agar from each dish was sectioned across the diameter and transferred to clean Petri dishes so that each new dish contained one half untreated agar, and one half tea tree oil spiked agar. The control dishes had untreated agar in both halves. Groups of about 15 third instar larvae were added to each dish. Larvae were applied to the half of the dish with TTO agar in two of the four replicates for each concentration and to the untreated agar in the other two. A filter paper cover was secured over each dish with a rubber band and the dishes placed on a bench at about 26°C. The numbers of larvae in each half of the dish were counted after about 2 h and the dishes then transferred to incubator at about 28°C for about a further 22 h. Distribution of the larvae was recorded again and the dishes photographed at about 24 h.
1.5 Effects against adult flies
1.5.1 Toxic effects - topical application
L. cuprina females about 7-10 days old were immobilised with C0 2 and about Ιμΐ of Melaleuca oil diluted in butanone to the required concentration was applied to each fly's thorax with a micropipette. Three replicates of about 20 flies (about 10 male and 10 female) were used for each concentration. Control flies were treated with butanone only. Groups of flies were held at about 27°C in about 540 ml plastic containers with gauze lids, with sugar and water provided. Mortality was assessed at about 24 and about 48 h after treatment. In separate studies, flies were treated with about 0.15, 0.25 or 0.5 Ιμΐ of pure TTO and assessed using similar methods. In these studies control flies were treated with about 0.5 μΐ grape seed oil.
1.5.1.1 Topical application - synergism with piperonyl butoxide (PBO)
Following a report of PBO synergising the effect of plant essential oils in mosquitoes (Waliwitiya et al. 2008), two pilot studies were conducted to indentify potential synergistic effects in L. cuprina. Preliminary studies determined that the maximum sublethal dose of PBO for L. cuprina was about 2% in about 1 μΐ butanone or about 20 μg per fly. This concentration of PBO was used in both experiments.
Female flies were anaesthetised with C0 2 and about Ιμΐ of diluted PBO was applied to the dorsal thorax with a micropipette. The flies were then returned to their holding containers with water and sugar provided and held for about one hour. After this time they were anaesthetised again and treated with about Ιμΐ of the required concentration of TTO in butanone. Cages were held in a temperature and humidity controlled room (about 27°C) between and following fly treatment. In the first experiment the concentrations of TTO tested were about 1%, 5%, 10%, 25% and 50%. There were two replicates of about 20 flies for each concentration and mortality was assessed after about 24 h. In the second experiment about 5%, 15%, 25%, 35% and 50% concentrations of TTO were used. There were three replicates of about 20 flies for each concentration and mortality was assessed after about 24 and about 48 h. Control flies were treated with PBO and butanone.
1.5.2 Fumigant effects
Adult flies were anaesthetised with C0 2 and sorted to groups of about 20 (about 10 male, 10 female) before being transferred to fumigation chambers. Fumigation chambers were constructed using approximately 90 mm glass Petri dish lids containing approximately 55 mm glass Petri dishes in which anaesthetised flies were introduced to the chambers. Glass coverslips were placed outside of the 55 mm dishes onto which a drop of the test concentration was added. Two different types of studies were conducted. One to test TTO presented as different concentrations of TTO in acetone in about 60 μΐ droplets (about 0%, 5%, 25%, 50% and 100%) and in the other pure TTO was presented in different size droplets (about 0μ, Ιμΐ, 3μ1, 6μ1, 15μ1). The dishes were held in an incubator at about 27 °C and mortality recorded after at hourly intervals up to about 6 h after introduction and again after about 24 h. Three replicates were used for each concentration or amount of TTO.
1.5.3 Repellent effects
L. cuprina used in the assays were about 9-11 days old females that had been protein fed with sheeps liver about 4 days after emergence. Dissection and examination of a subsample of females prior to commencement of the assays confirmed that most were gravid. Flies were anaesthetised with C0 2 sorted to groups of about 40, allocated to six approximately 40 x 30 x 30 cm cages with Perspex sides and mesh ends. They were provided with sugar and water and allowed to "acclimatise" overnight before commencement of the assay. Cages were laid out on about a 1.5 m mesh platform with a bank of overhead fluorescent lamps positioned above to provide even lighting and held at about 28°C and about 60% RH. Temperature and humidity were monitored and recorded with a data logger.
Oviposition stimulus was provide by way of a cotton plug, approximately 0.9 cm diameter and 3.7cm length, fully submerged in a mix of about 40 g homogenised liver and about 30 ml water for about 5 minutes. Test wool samples were arranged around the cotton plug and inserted into an approximately 50 ml glass beaker, ensuring the top of the wool and plug were flush with the top of the beaker. This arrangement provided about 2 cm of wool between the plug and sides of the beaker on which flies could alight and stand to oviposit. Two beakers were positioned about 20 cm apart, about 10 cm from each end of the cage. For the choice tests there was one control beaker and one test beaker in each cage arranged in alternate positions in the 6 cages. For the non choice tests there were either two test or two control beakers in each cage.
Test solutions consisted of about 3% TTO emulsified with about 3% Alkamuls solution. Emulsifier without TTO was used as the control. Wool for use in the assays was prepared by immersing about 3 g of wool of about 4-5 cm in length into the test solution for about 30 s. Each sample was then drained of free liquid, allowed to sit for about a further 30 seconds on paper towelling and then squeezed to remove excess liquid. For tests of persistency of the repellent effect enough samples were prepared for the period of the study and held in their beakers in a cupboard at about room temperature until needed. Each preparation were moistened with water sprayed from a plastic spray bottle held about 20 cm away prior to fly exposure to ensure a moist area for flies to lay.
The test preparations were placed in each cage one hour before observations commenced (about 9 am on most days). The number of flies present on the wool in each preparation was recorded at point counts at about 10 min intervals for about an hour at about two hourly intervals until about 4 pm on each day (about 4 periods of 6 fly counts) in the 1 day experiments and in about two 1 h periods of 6 counts beginning at about 9:00 am and 11:00 am in the two experiments to examine effectiveness over longer periods (about 6 days and 6 weeks). In addition, if flies were stationary, backed into a cavity in the fleece and probing with their ovipositor, or seen to be depositing eggs they were recorded as ovipositing. This corresponds to phase (iii) and (iv) in the description of the sequence of events in the oviposition behaviour of Lucilia spp given by Cragg (1956). At the conclusion of observations the preparations were removed from the cages and placed in a freezer to kill the eggs. Numbers of egg masses deposited were counted on the following day. As it was often difficult to identify individual egg masses, a scoring system was also used: 0 = no eggs; 1 = occasional or scattered eggs; 2 = one egg mass; 3 = 2-4 egg masses; 4 = large numbers of eggs (> 5 egg masses). Four different experiments were carried out. In the first two, repellent effects were assessed over one day in choice and non-choice situations. The later two studies examined persistence of the repellent effect in choice tests, the first using 3 cages over 6 days, and the second using 6 cages over 6 weeks. 1.6 Effects on eggs and Pupae
1.6.1 Fumigant effects
Fumigation chambers were constructed using approximately 90 mm glass Petri dish lids containing approximately 55 mm glass Petri dishes to hold either L. cuprina eggs or pupae and about 21 x 21 mm glass coverslips placed outside of the 55 mm dishes onto which about a 60 μΐ drop of TTO diluted to the required concentration in acetone was placed.
Eggs were collected by introducing a fresh piece of liver to a cage of gravid L. cuprina blowflies. Small, freshly collected clumps of eggs (about 40-100) were transferred to moistened about 55 mm diameter filter paper (Whatman No. 1) inserted into the internal Petri dishes. Measured amounts of TTO were applied to the coverslips and the chamber immediately sealed by placing larger Petri dish base on its lid. Chambers were held in an incubator set at about 29 °C and about 75%RH for about 18 h. The number of emerged larvae was determined by counting numbers of larvae and empty egg shells under a dissecting microscope.
For tests against pupae, groups of about 15, approximately four day old pupae were placed on dry filter paper in the bottom of the 55 mm Petri dishes. TTO dilutions were dispensed onto the coverslips and the chamber immediately sealed as above. Two experiments were conducted, the first with concentrations of TTO from about 0 to 15% and the second with concentrations from about 25% to 100%. A treatment with about a 600 μΐ drop of TTO was also included in the second assay. Chambers were held at about 29°C and about 65% RH for about 7 d and the number of unecloded pupae, live flies and dead flies recorded at that time. 1.7 Effects on sheep lice
1.7.1 Treated surface effects
Initially, standard treated surface methods for lousicide susceptibility testing were used (Levot and Hughes 1989). Briefly, cotton squares were treated with test solution and held in place with about 5 cm metal rings. Essential oils were applied with butanone or acetone as carriers and the squares left to dry overnight to avoid physical or solvent effects on the lice.
In order to reduce loss of volatile components a number of modified systems were also tested initially. The most successful of these used TTO diluted in grapeseed oil applied to about 90 mm filter papers. A wt ratio of about 1: 1 oil to filter paper (about 0.57g of oil per paper) gave good dispersion of oil over the paper without leaving excess oil in which lice could become trapped. In one such test, given as an example, groups of about 10 lice were exposed to concentrations of about 0%, 0.1%, 0.5%, 2.5% 10% and 20% TTO applied to about 90 mm filter papers in glass Petri dishes. Lice were added to the test dishes once oil had dispersed on the filter paper and the dishes held uncovered in an incubator at about 36°C and about 65% RH. Dishes were inspected at about 24 h and about 48 h and lice assessed as dead (no response to a stimulus) or alive at each inspection.
1.7.2 Wool Dipping assays
As results of the previous assay types were considered unlikely to give a good indication of effectiveness in a practical context, a method that more closely simulated the situation likely to be present on sheep was developed.
Volumes of about 20 ml of test solutions, with different concentrations of TTO emulsified with ALK were prepared. Amounts of about 400 mg of pesticide free wool were immersed in these solutions in about 50 ml beakers for about 60 seconds and agitated to ensure complete wetting. The wool was then removed and a standard method of shaking seven times with a brisk wrist movement used to remove excess fluid. The treated wool was added to about 28 ml capacity flat bottom glass bottles and at least about 20 adult lice, freshly collected from live sheep added to each bottle. In one experiment with both adults and nymphs about 20 of each stage (about 40 per bottle) were used. Two control groups were used, one with lice in dry untreated wool and one with lice in wool treated with emulsifier without TTO and there were three replicates per treatment or control. Assay tubes were held in an incubator at about 36°C and about 65% relative humidity (RH) and assessed as alive or dead at about 24 and about 48 h after addition of the lice. Tests were also conducted to determine the effect of reducing the percent of emulsifier (ALK) from about 5% to 1% and to assess the effect of including about 200mg/L of the synergist piperonyl butoxide in the dipping emulsion.
1.7.3 Dried wool assays
To assess residual effect of TTO, about 50 mg amounts of pesticide free wool were thoroughly wetted with about 500 μΐ of TTO at the desired concentration solubilised in tap water with about 2% ALK. Control wool was treated with ALK with no TTO. The wool was dried in a fume hood at about room temperature for about 1.5 h. then inserted into about 20 ml disposable flat bottom vials. Twenty lice were added to each and the vials placed in an incubator at about 36°C and about 65% relative humidity. Numbers of live and dead lice assessed at about 24 h and about 48 h. 1.7.4 Fumigant effects against lice
Two methodologies were used for testing fumigant effects:
(a) Fumigation chambers: Chambers were constructed as described above for the fly studies. Test arenas consisted of about 90 mm glass Petri dishes with uncovered about
55 mm glass Petri dish bottoms inside to contain the lice. Lice were counted onto about 55 mm filter papers, added to the small Petri dishes. Measured volumes of test solution were dispensed onto about 21 mm x 21 mm glass coverslips placed within the large dishes but outside of the about 55 mm dishes. Lids were applied to the large dishes which were held in an incubator at about 36°C and about 65% RH. Lice were inspected at about 1 h and about 2 h or 3 h and the numbers of lice that were knocked down or dead and still moving were recorded. The small dishes containing lice were then removed from the larger dishes and returned uncovered to the incubator. After about 24 hours they were re-examined to identify lice that recovered. In early studies of fumigant effect, acetone was used to solubilise TTO whereas in later studies about 8% Tween 80 was also tested.
(b) Wool fumigation method: To determine if the fumes of TTO applied to wool could be effective in killing lice not actually contacted by TTO the following apparatus was used. Glass tubing about 50 mm in length and about 15 mm diameter was glued into the screw tops of about 80 mm x 27 mm diameter glass vials. Wool was weighed to about 400 mg amounts and submerged in test solution for about 30 s, removed and allowed to drain for about 30 s and then added to the glass vials. Lice were contained in about 20 mg of pesticide free wool held the tubes and prevented from contacting the treated wool by nylon mesh secured across the bottom end of the tube. In the first assay the top of the tube was covered with Parafilm® (American National Can, Neenah, WI), but in the second assay, the tubes were left uncovered. Concentrations of TTO between about 0 and 2% emulsified in water with about 1% ALK were tested, with two groups of controls, dry wool, and wool treated with ALK. There were about 20 adult lice in each vial, 3 replicates for each treatment or control and mortality of lice was assessed after about 24 h of exposure.
1.7.5 Effect of TTO components in fumigation assays
Two experiments were carried out using the fumigation chamber method above and with acetone as solvent to investigate the relative effect of different components of TTO. In the first experiment the components tested were terpinen-4-ol (about 2.15%), alpha terpinene (about 0.5%) and gamma terpinene (about 1.045%). TTO at about 7.5%, 5%, 2.5% and 1% of was included to give a basis for comparison. All test substances were solubilised in about 60 μΐ of acetone and the component concentrations equated to that which would be contained in about 5% TTO, which was the minimum level at which about 100% knockdown or death had been achieved in the previous assays. In the second experiment, about 60 μΐ amounts were again used and about 5% TTO was used as the positive control. Terpinen-4-ol was tested at about 2.15% as in the previous experiment, but also at about 1.5% which corresponded to the minimum level of this compound allowable under ISO 4730 specification for TTO, terpinen-4- olchemotype (Table 1). Limonene was tested at about 0.04%, 1,8 cineole at about 0.085%, alpha pinene at about 0.115%, alpha terpineole at about 0.14% and an additional treatment with all of the above components at the specified concentrations, except for terpinen-4-ol was included. Acetone was used as the negative control in all assays. Lice were exposed in the chamber for about 3 h, at which time knockdown/death was assessed. The Petri dish lids were then removed and the lice returned to the incubator to check for recovery.
1.7.6 Lice eggs - dipping studies
To provide louse eggs, wool containing lice was clipped from a heavily infested area on a sheep and placed in an incubator overnight. The next morning the clipped wool was inspected under a dissecting microscope and fibres with eggs that were solid and white and apparently healthy were separated from the rest of the wool. About 40 viable eggs were available.
Separate pesticide-free wool was weighed to about 400 mg lots and treated with about 1% TTO emulsified with about 1% ALK, with about 1% ALK with no TTO, or left untreated. Treatment was conducted by immersing the wool in test fluid for about 30 s and then allowing it to drain for about 30 s. The wool was parted, about 10 eggs placed in the middle and the wool folded back over the eggs. The preparations were placed in about 28 ml McCartney bottles in an incubator at about 36°C and about 65% RH and inspected over about 11 days to allow time for viable eggs to hatch. There was one preparation with about 10 eggs for the TTO/ALK treated wool, two preparations with about 10 eggs each for the ALK treated wool and about a further 10 eggs placed on untreated wool. 1.7.7 Lice eggs -fumigant effects
Lice were collected from a heavily infested sheep and held overnight in the laboratory on wool collected some weeks previously from an infested sheep, but with no live lice present. The wool was inspected the next morning for the presence of newly deposited eggs. Eggs, were carefully selected from the wool, leaving them with wool fibres when attached. About 90 eggs were counted to about nine groups of ten on filter paper in about 50 mm Petri dishes and added to fumigation chambers. Treatments were about 60 μΐ of about 100% TTO, about 15 μΐ of about 100% TTO added to coverslips in the fumigation chamber as previously described, or no TTO. There were three replicates for each treatment and control. Eggs were exposed in the fumigation chamber in an incubator set at about 3636°C and about 65% RH for about 24 h before being transferred to clean dishes, maintained at similar temperature and humidity. Hatching was assessed daily over about 14 days. 1.8 Sheep studies
1.8.1 Dipping
Sheep: Louse counts were conducted on a mob of about 40 adult male castrate Merino sheep, infested with sheep lice (Bovicola ovis) and with bodyweight range of about 50-65 kg. The sheep had been purchased from an infested flock and had been running on pasture at the Centre for Advanced Animal Science at the University of Queensland, Gatton, Qld, Australia for a number of months prior to commencement of the experiment. All sheep were drenched with Coopers Colleague® Broad Spectrum Sheep and Lamb Drench (Coopers Animal Health, 66 Waterloo Road, North Ryde NSW Australia) (about 19 g/L albendazone and about 150 g/L pyraclofos) at rate of about 1 m 1/5 kg bodyweight two weeks before entry to the trial to control endoparasites. Treatment with this product had previously shown no effect on louse burdens. About 18 sheep with moderate to heavy infestations of sheep lice were chosen from these animals and allocated to about six groups of three, balanced for louse counts. Groups of about three were randomly assigned to treatment (or control) with two groups for each treatment (control). The sheep were shorn and groups assigned to outdoor pens about 7.5 m x 7.5 m in dimensions with about 1.3 m high closed sides located outdoors and open to weather, although part covered at one end by about a 1.8 m strip of shade cloth. Sheep were left in the pens to 'acclimatise' until dipping, about 2 weeks after shearing, and remained in the same pens for the duration of the study. Dipping formulations: On the basis of the results from laboratory testing, and with the view to development of an economical product, formulations containing about 1% and about 2% TTO emulsified with about 1% and about 2% ALK respectively were chosen for testing in sheep studies. Methylene blue at about 0.004% was also added to the dipping solution to check the thoroughness of wetting and identify any areas where poor coverage rather than lousicide failure may be responsible for poor results. Laboratory studies showed methylene blue to have no effect on louse survival.
Immersion dipping: Groups of about 3 sheep were held in an open topped cage of about 0.9 m x 0.9 m x 1.4 m and dipped by lowering into about a l m x l m x l.2 m dipping vat, which immersed the bodies of all animals, for about 2 min. The bath was not deep enough to cover the animal's heads, so the wool on the head of each sheep was wetted by pouring over dipping fluid from a small pail. Following dipping, groups were returned to their respective pens where they remained except during inspection for lice or collection of samples. The same dipping fluid was used for both groups of about 3 sheep for each concentration of TTO.
Assessment of louse numbers: All louse inspections were conducted with the sheep standing in a holding crate with widely space horizontal bars. There were two assessors, one on each side of the sheep, for each inspection. Pre-shearing assessment of louse burdens was made by counting the number of adult lice and nymphs in about 12, 10 cm fleece partings on each side of the sheep (about 24 parts). Partings were made in three rows, about 10 cm from the vertebral column, midway between the vertebral column and ventral midline and on the lower woolled areas with one parting on the shoulder/front leg, two on the flanks and one on the rump/backleg along each line. Post shearing assessments were made by inspecting about 40 fleece parts (about 20 on each side). Partings were distributed on the front leg, flanks and backleg as noted above except that there were about four lines from the vertebral column to the lower woolled areas as well as about four parts down the underside of the neck and dewlap on each side. Post dipping inspections were made at about 2, 6, 12 and 20 weeks post treatment.
Residues: A series of samples were collected to determine if residues of TTO components could be detected in the blood tissues and wool of sheep treated with TTO formulation. Tissue samples were collected about 1 week after treatment. Blood samples were collected at about 1, 3 and 6 weeks and wool samples at about 2, 6, 12 and 20 w. For collection of tissue samples the sheep were firmly restrained, a small area on the rump of each sheep closely clipped and a local anaesthetic administered. A small muscle biopsy (< about 2 gm) was removed using forceps. Care was taken to peel back overlaying skin and sample only the underlying tissue. Following collection of the sample at least one stitch was inserted to close the wound and the animals closely supervised until healed. All muscle biopsies were conducted by a registered veterinarian. As no residues were detected in the first lot of samples, no further tissue samples were collected as agreed under our animal ethics approval. Blood samples (about 20 ml) were collected by jugular venipuncture using about 18g hypodermic needles. Once the needle was inserted a small amount of blood was drawn off into one Vacutainer TM, the first Vacutainer was removed from the needle and a second attached. Only the blood from the second Vacutainer was used to minimise the possibility of contamination from skin surface TTO. Wool samples were collected using scissors and animal clippers. About 3 g samples were collected from each of the shoulder, mid back and rump, about 25 cm from the dorsal midline, on each sheep. All samples were stored at about -25°C until processed for analysis.
1.8.2 Jetting
Sheep: About 18 adult male Merino wethers with heavy infestations of lice were managed prior to the experiment as described above were allocated to about six groups of 3 sheep balanced for louse counts and randomly assigned to treatment (or control) There were two groups of about 3 sheep for each treatment (control). Groups were randomly allocated to pens as described for the dipping study. Unfortunately two sheep in one group of the about 2% TTO-treated sheep contracted an unrelated disease (ovine posthitis) and were not present for the final louse count.
Jetting: Jetting formulations containing about 1% and about 2% tea tree oil emulsified with about 1% and about 2% ALK respectively and about 0.004% methylene blue to monitor the thoroughness of wetting were tested. Control sheep remained untreated. Jetting was conducted with a Dutjet® hand jetting wand connected to centrifugal pump run to provide about 500 kpa at the handpiece head. Jetting was conducted slowly in two sweeps of the jetting wand from the poll to the tail along either side of the backline, taking care to allow time for the fluid to pool ahead of the jetting wand and run down through the fleece over the sides of each sheep. The time for the handpiece to deliver about 4L of formulation was measured and an attempt made to deliver this quantity into the fleece of each sheep. Asssessment of louse numbers and fleece derangement: Inspections were conducted and louse numbers assessed at about 2, 6 and 12 weeks after treatment as described for the dipping study. Fleece derangement (signs of biting and rubbing at the fleece) were assessed on both sides of each sheep at about the 6 and 12 week inspections using the scoring system described by James et al. (2007). Briefly, the scores were: 0, no rub; 1, suspect, but not sure; 2, light but obvious rub, fluffy tip or definite pulled fibres at some sites; 3, distinct but dispersed pulled strands (thicker than fluffy); 4, definite patches or areas of pulled strands, less than 20% of the fleece affected, no bare areas; 5, definite patches or areas of pulled strands, greater than 20% of the fleece affected; 6, grossly matted fleece, often with some areas rubbed bare.
Animal ethics: All animal management and experimentation in both the jetting and dipping studies was carried out according to criteria specified under Animal Research Institute Animal Ethics Committee approval SA 2009/02/282.
1.9 'Stripping' study (laboratory)
Stripping occurs when dipping chemicals is more soluble in wool grease than in water and so is removed from the dipping fluid at a faster rate than fall in dip volume, causing a progressive drop in the concentration of the active ingredient as more sheep are dipped. Stripping does not occur with all dipping chemicals. Whether or not dipping fluids "strip" during dipping is an important practical consideration when determining dip mixing procedure. With stripping dips chemical must be added at a higher concentration when topping up than in the initial mix to compensate for reduction in the concentration of dip active. A 'bench top' simulation was designed to determine whether TTO was likely to strip from solution during dipping.
A stock mixture of about 8L of about 1% TTO emulsified with about 1% ALK was mixed and stored in a sealed Winchester at about 5°C. This emulsion was used in all experiments. 'Dipping' was conducted with about 10 g wool samples in about 500 ml beakers. Wool was weighed into about 10 g lots and dipped using a vegetable masher adapted for the purpose. For each dipping the wool was placed in the beaker and immersed for about 20 s during which time the wool was compressed gently against the bottom of the beaker about 3 times. The wool was then compressed in the vegetable masher above the beaker allowing excess fluid to run back into the beaker. Wool samples were then reweighed and the post dipping weight recorded. About 15 samples were dipped sequentially, for each beaker of fluid and samples of dip fluid were taken after each lot of about 3 wool samples had been dipped. Wool and fluid samples were stored at about -25°C until later chemical analysis for TTO components. TTO components were subsequently measured in both dipping samples and wool samples by GCMS as described below. 1.10 GCMS analysis of TTO components
1.10.1 Determination of tea tree oil component residues in tissue and blood
Tissue and blood samples were subsampled by taking an aliquot of about 0.2 mg for tissue or about 2 mL for blood and the subsample then extracted with hexane (about 2mL). The hexane extract was chromatographed on a Gas Chromatograph/Mass Spectrometer (GCMS) with the detector operated in the full scan mode. Components of tea tree oil were identified and quantified by comparison of retention time, mass spectrum and peak area by comparison with authentic tea tree oil and with individual standards. 1.10.2 Determination of tea tree oil components in wool and dipping fluid
Wool samples were subsampled by taking about 200 mg of wool and placing in about a 10 mL headspace vial which was then sealed with a silicon/PTFE septa. The sample was allowed to equilibrate for at least about four hours (usually overnight). The headspace was sampled using a solid phase micro extraction (SPME) fiber (polydimethylsiloxane about 100 μιη) for about 10 minutes. The fiber was desorbed into a Gas Chromatograph inlet at about 200°C for about one minute. The remaining GC conditions were the same as used for liquid extracts, described above. Dipping samples from the stripping study were diluted by about lxlO "4 and analysed by the same method as wool samples.
1.11 Statistical analysis
experiments in this project were of relatively similar design, with a number of experimental subjects (various stages of L. cuprina, B. ovis or sheep) divided amongst treatments, with experiments replicated at least twice and sometimes with repeat measurements at different times. As such, analysis was reasonably similar for most experiments. In most instances, one or two way analysis of variance was conducted, with appropriate transformation to normalise the data where necessary. A repeated measures analysis was conducted for the sheep jetting study. All analyses were carried out using GenStat v l l(Payne et al., 2007). Example 2. Results
2.1 Sheep blowfly
2.1.1 First instar larvae - serum assay
Mortality of 1st instar larvae at different concentrations of TTO is shown in
Figure 3. A concentration of about 0.9% TTO in serum was effective in giving about 100% mortality in this assay and about 1% TTO reliably gave about 100% kill in three other assays (results not shown). 2.1.2 Second and third instar larvae - agar method
TTO at about 2.5% gave about 100% kill of second instar larvae (Figure 4).
2.1.3 Third instar - dipping assay
After dipping in about 50% TTO, about 36 % of larvae with ALK as the emulsifier and about 54% with Tween80 successfully developed to adult flies (Figure 5). There was no significant difference between the two emulsifiers (p>0.05). A number of other similar assays were carried out with different carriers and all achieved similar results (data not shown). 2.1.4 Larval repellence (3rd instar)
Very few larvae were found in the TTO half of the Petri dish, regardless of if they were placed there initially (see Table 2). By about 24 h there was clear evidence of larval activity and feeding in the half of the dish without TTO whereas there was no indication of any larval feeding in the TTO treated agar. Occasional larvae observed on the TTO-treated half were usually on the surface rather than burrowing into the agar and may have been post feeding larvae searching for a site to pupate. In the dishes with untreated TTO in both halves, larvae were found on both sides of the plate and there was clear evidence of larval burrowing and feeding throughout the agar in the dish. Table 2: Percent of larvae found in TTO-treated and untreated halves of meat liver agar in petri dishes after 2 and 24 h (means for 2 dishes). There were 4 dishes with 10 larvae per dish for each concentration (total = 12 dishes) and larvae were initially placed on the treated agar in 2 dishes a and the untreated agar b in the other 2. Time TTO Initial untreated Initial TTO b Total cone. a
Cont c TTO c Cont c TTO c Cont d TTO d
2 h 5% 93.3 6.7 96.7 3.3 95.0 5
2% 100.0 0 100.0 0 100.0 0
0.50% 100.0 0 93.3 6.7 96.7 3.3
24 h 5% 86.7 13.3 90.0 10 88.3 11.7
2% 90.0 10 96.7 3.3 93.3 6.7
0.50% 100.0 0 93.3 6.7 96.7 3.3 c Mean of 2 dishes.
d Mean of 4 dishes. 2.1.5 Formulation of TTO with other insecticides
Agar method (3rd instar)
In the comparison of diazinon, ivermectin and boric acid with and without TTO the repellent effect of TTO against larvae was again evident. Many of the larvae in the treatments with TTO had left the agar when the preparations were first examined at about 24 h (see Figure 6) Most of the larvae remaining on the agar in the treated vials were dead, presumably killed by the toxic action of the insecticides. The numbers of dead larvae remaining on the agar treated with TTO formulations with diazinon and ivermectin were higher than those with TTO/boric acid (p<0.05).
No larvae successfully pupated and emerged as adult flies in the diazinon, ivermectin or boric acid treatments without TTO (see Figure 7a). In the experiment with cyromazine the average mortality was lower in those treatments with TTO (about 62 % and 78% compared to about 84% and 90%, see Figure 7b). Although a greater proportion of flies successfully emerged as adult flies in the treatments containing TTO than with insecticide alone, in no instance more than about 38% emerged, compared to about 100% emergence in the untreated controls.
2.1.6 Dipping assay (3rd instar)
Mortalities were lower in the dipping studies than the agar assays, but as with the agar assays, in most instances addition of TTO to the formulation lowered mortality in comparison with the equivalent formulation without TTO (p<0.05) (Figure 8). The maximum kill of about 83% was observed in the diazinon treatment without TTO. 2.2 Adult flies
2.2.1 Toxic effects - topical application
In this study and in other similar studies (data not presented) with about 1 μΐ of topically applied formulation, at least about 50% TTO was required to give about 100% mortality in treated flies (see Figure 9a). This represents a total amount of about 0.5 μΐ of pure TTO. This was the amount also required when pure TTO was applied (see Figure 9b).
2.2.2 Topical application - synergism with piperonyl butoxide (PBO)
The results from the two experiments conducted with L. cuprina to assess the effect of PBO gave equivocal results. There was no detectable difference between mortality with and without PBO at high and low concentrations of TTO (see Figures 10 and 11). However in the range from about 10% TTO to about 35% TTO there may have been an effect. Mortality was higher in the treatments with PBO than without PBO at about 10% and 25% in experiment 1 and at about 35% in experiment 2 (P<0.05).
2.2.3 Adult flies - Fumigant effects
In the control treatments with acetone in the study with different concentrations of acetone, 2 flies were immobile at the initial 2 h inspection (see Figures 12 and 13), but all were active at inspections at 3 h and 4 h. In the experiment with different quantities of TTO, in the controls all flies were active by the time of the first exposure at about 0.5 h. Although some mortality was seen in some control groups at about 24 h, it was generally low level. There were clear fumigant effects evident against L. cuprina even at low concentrations and amounts of TTO. With about 5% TTO, about 63% of flies were knocked down or dead at about 2 h and this increased to about 95% at about 24 h, while with about 1 μΐ of pure TTO about 67% were knocked down or dead at about 2 h and this increased to about 97% at about 24 h. There also seemed to be a sex effect with more males than females knocked down at low concentrations/amounts of TTO. No flies were seen moving in treatments with concentrations of about 25% TTO or above following about 2 h of exposure or at any time after that. Similarly in the experiments with different quantities of TTO, no flies were seen moving in treatments with about 3 μΐ and above from the first inspection following about 0.5 h of exposure to the last inspection at about 24 h. 2.2.4 Adult flies - repellency
Figure 14 shows the numbers of flies present on wool preparations and which were stationary and probing with their ovipositors or actually ovipositing at about 24 inspections of the control preparations between 9: 15am to 4:05 pm in the first choice test. No flies were seen resting on the TTO preparations at any observation during this period suggesting that TTO-treatment provided a strong repellent effect. Table 3 shows the number of egg masses in the wool of the control preparations at the end of exposure. No eggs were found on the TTO -treated wool. Table 3: Number of L. cuprina egg masses deposited on control and TTO-treated wool - choice test
Egg masses estimated
Cage Treatment # egg masses Score
1 Control 28 4
TTO 0 0
2 Control 22 4
TTO 0 0
3 Control 21 4
TTO 0 0
4 Control 19 4
TTO 0 0
5 Control 23 4
TTO 0 0
6 Control 29 4
TTO 0 0
The non-choice testing confirmed that TTO is a powerful repellent and oviposion deterrent for L. cuprina. Even though the flies were gravid, were provided with a powerful stimulus for both feeding and egg laying and were not given an untreated choice, only on about 35 occasions were flies observed resting on TTO treated wool compared to about 672 times on the control wool, a reduction of about 95%. Flies that did land on the treated wool generally left soon after landing and although oviposition was observed on all control preparations, no ovipositing flies or egg masses were observed on TTO-treated wool. Figure 15 shows the mean number of flies recorded on control and TTO-treated wool preparations examined over 8 hours.
Table 4: Mean number of egg masses deposited on control and TTO- treated wool preparations - non choice test. Egg masses estimated
Cage Treatment # egg masses Score
1 Control 22 4
Control 24 0
2 TTO 0 4
TTO 0 0
3 Control 23 4
Control 24 0
4 TTO 0 4
TTO 0 0
5 Control 16 4
Control 16 0
6 TTO 0 4
TTO 0 0
In the first persistency study, conducted over about 6 days, treatment with the about 3% TTO formulation almost completely repelled flies. Only one fly was counted on TTO-treated wool at any of the observations (day 6) and no egg masses were found on any of the TTO-treated wool preparations.
In the second experiment conducted to examine persistency of effect, some flies were recorded on the wool at all observations from about day 9 and later, although these flies remained on the wool surface for less than a few seconds in most instances. Consistent with earlier studies, no flies were counted on the treated wool at observations on day 1. At about 44 days the number of flies on the TTO preparations significantly increased in comparison to earlier times (p<0.05), although the numbers counted were still much lower than on the control preparations and there were no egg masses deposited on the TTO-treated wool. Figure 16 shows mean number of flies recorded on the surface of TTO-treated wool or wool treated with emulsifier only at different times after treatment. Figure 17 shows mean number of eggs recorded on the surface of control wool preparations following one days exposure to flies up to 44 days after treatment. Table 5: Mean number of flies counted on wool preparations (means across 3 cages, 12 observation times) and egg masses deposited on control and TTO- treated wool preparations up to 6 days after treatment with 3% TTO formulation - choice test. Flies on wool Egg masses
Day Control TTO Control TTO
1 5.7+0.28 0 12.7+2.0 0
2 6.4+0.0.2 0 12.0+1.5 0
3 7.6+0.3 0 13.3+0.7 0
6 5.8+0.6 0.03+0.03 7.0+0.6 0
2.3 Eggs and Pupae
2.3.1 Fumigant effect on eggs
A number of tests were conducted giving reasonably consistent results and confirming a fumigant effect of TTO on L. cuprina eggs. The results from these studies are summarised in Figure 18. Whereas the mortality induced by a concentration of 1% was not significantly different from controls (P>0.05), concentrations of 2.5% and above induced significant mortality. 2.3.2 Fumigant effect on pupae
In the first experiment, exposure of pupae to concentrations of TTO up to about 15% did not significantly (P<0.05) reduce the emergence of flies in comparison to controls and there was no relationship between pupal viability and TTO concentration within this range. In the second experiment where higher concentrations of TTO were used there was a significant reduction in numbers of flies emerging at concentrations of about 75% and above (P<0.05) (Figure 19). However, even with about 60 μΐ of about 100% TTO the direct pupal mortality induced was less than about 40%. Although pupae were not killed directly, most flies exposed to concentrations of about 50% and above died during or soon after emergence. This may be because exposure in the pupal phase weakened the flies and affected their ability to emerge, or that the emerging flies were more susceptible to TTO fumes than pupae and that there was high enough vapour pressure of residual TTO present in the dishes to interrupt the eclosion process or kill the flies directly. Increasing the amount of about 100% TTO to about 600 μΐ markedly increased the direct toxic effect against pupae and no flies emerged from pupae in this group. 2.4 Sheep lice
2.4.1 Laboratory studies: Treated surface assays
Figure 20 shows the results of a typical contact assay conducted by a standard method (Levot and Hughes 1989) with butanone as the solvent for TTO. Clearly this method was not suitable for tests with TTO. Drying overnight may have allowed for the evaporation of some key active components before the lice were exposed and reduced efficacy in comparison with that seen with other assay designs. Better effect was seen in tests with grapeseed oil as the solvent where the papers were not dried overnight before exposure. Careful choice of the quantity of test solution, and using the dishes uncovered allowed immediate exposure of lice without high control mortalities. In this system concentrations of at least about 10% were required to give high louse mortality (Figure 21).
2.4.2 Wool dipping assays
In all wool dipping assays, including a number for which data is not shown, about 1% TTO and concentrations above consistently gave about 100% mortality of lice and was equally effective against nymphs and adult lice (Figures 22 to 24). In early assays TTO was emulsified with about 5% ALK, but assays in which about 1% ALK was tested with the aim of reducing ultimate cost of a formulation, suggested no reduction in efficacy (Figures 23).
Effect of synergism: Although only one preliminary study was carried out, the results from this experiment suggested little effect of PBO at any of the TTO concentrations tested (Figure 24). However, once again dipping wool in about 1% TTO resulted in a complete kill of lice.
2.4.3 Dried wool assay
The results shown in Figure 25 suggest very little residual effect from TTO when the wool was allowed to dry before lice were added. It seems that insecticidal effect is markedly reduced once the wool has dried. In some other assays conducted where wool was allowed to dry before lice were added (results not shown) there was more variability in results and some mortality in wool treated with higher concentrations of TTO. For example in one assay conducted as above, except that wool was dried on a bench top rather than in a fume hood, about 3% and about 7.5%TTO gave about 57% and about 70% mortality compared to about 13% in controls. 2.4.4 Fumigant effects
TTO demonstrated clear fumigant effects against lice. In the assays conducted with TTO either used at about 100% or solubilised in about 60 μΐ acetone, concentrations of about 10% and above gave about 100% mortality of lice (Tables 6 and 7). With about 5% TTO (about 3 μg TTO), although all lice were knocked down at about 1 and 2 h, about 3 lice (about 5%) were able to recover overnight and at about 1% (about 0^g) although most lice were knocked down when inspected at about 1 and 3 h, by about 24 h, about 35% had recovered. Table 6: Percent mortality (+ s. e.) of lice exposed to fumigant effect of different concentrations of TTO in acetone.
Treatment TTO mass (pg) Time after exposure 1
l h 3 h 24 h
Control
(60 μΐ acetone) 0 0 0 2.3 (0.3)
25% (60 μΐ) 15 100 100 100
25% (120 μΐ) 30 100 100 100
50% (60 μΐ) 30 100 100 100
100% (60 μΐ) 60 100 100 100
Table 7: Percent mortality (+ s. e .) of lice exposed to fumigant effect of different concentrations of TTO in acetone.
Treatment TTO mass Time after exposure 1
(μ )
l h 3 h 24 h
Control 0
(60 μΐ acetone) 0 0 0
1 % (60 μΐ) 0.6 11.7 (4.4) 4.4 (6.7) 35.0 (2.9)
5% (60 μΐ) 3 100 100 95.0 (5.0)
10% (60 μΐ) 6 100 100 100
25% (60 μΐ) 15 100 100 100
^ice were exposed to test solution up to 3 h then allowed to recover without exposure to about 24 h. 2 n= 60 per concentration exposed in three replicates of about 20 lice. In a further study with lice exposed to similar amounts of TTO either in about 100% droplets or emulsified at different concentrations in about 60μ1 of Tween80, about 6μg amounts of TTO and above, whether at about 100% or in Tween gave close to about 100% knock down after about 2 h of exposure. However, when they were inspected after removal from the test arenas for about 22 h, a significant proportion had recovered (Figure 26). At about 24 h there was marked difference in effectiveness of similar amounts of TTO presented in different forms with significantly more of the lice exposed to TTO emulsified in Tween recovering (P<0.05) (Figure 26). The difference appeared most marked with lower amounts of TTO. With about 3μg of TTO about 63% more lice recovered in the emulsified TTO treatment than with pure TTO, whereas with about 6 and 15 μg the difference was about 25 %. This may have been because the vapour pressure of TTO was lower with emulsified than pure TTO and sufficient knock down, but not to kill all lice over the two hours of exposure. 2.4.5 Wool fumigation assays
Figure 27 shows the percent mortality in the first assay, with parafilm covering the tube. The large standard error in the controls with ALK was due to about 100% mortality of lice in one replicate. The wool containing the lice in this tube was quite damp to touch and it is thought that this could have caused high mortality in this replicate. Although the wool also felt damp in the other two replicates with ALK but no TTO, mortality in these two replicates was about5% and 15%. This compared to about 100% mortality in the treatments exposed to about 0.5%, 1 % and 2% TTO. In the second assay where the tubes were not sealed with parafilm (Figure 28), no lice died in either the dry wool control or the wool treated with ALK without TTO. Even though the tubes were not sealed, about 98% of lice were killed (1 found alive) in the about 1% TTO treatment and about 73% were dead with about 0.5% TTO.
2.4.6 Effect of TTO components in fumigation assays
In the first experiment about 7.5% and about 5% TTO and terpinen-4-ol at an equivalent concentration to that found in about 5% TTO both gave about 100% knock down at about 3h (Figure 29). When examined at about 24 h, about 10 lice (5 in each of two replicates) had recovered in the about 5% TTO treatment whereas in the terpinen-4-ol treatment only about 3 lice, all in one replicate had recovered. These numbers were not significantly different (P>0.05). Neither alpha terpinene nor gamma terpinene caused mortalities that were significantly different from the control (P>0.05). Results were similar in the second experiment (Figure 30). The percentages knocked down or killed with about 2.15% and about 1.5% terpinen-4-ol were not significantly different to that with about 5% TTO at either about 3h or 24 h, but all three of these gave mortality much higher than in the controls or other components (P<0.05 ). However mortality induced by the other components at about 24 h either individually or in combination (without terpinen-4-ol) was significantly higher than in the control P<0.05).
2.4.7 Effects against eggs
Dipping studies: The results of this study are shown in Table 8. Egg hatch rate was about 70% in the dry wool control and one egg apparently developed fully but died during hatching. In the ALK treated groups, about 60% hatched and about a further 20% showed signs of development. In comparison, none of the eggs in the TTO treated group hatched or showed any signs of development. There appeared to be a physical effect on the chorion or outer layers of the egg in the TTO treated groups, although not in the group treated with ALK only.
Table 8: Hatching % of eggs placed in dry wool, in wool dipped in about 1% ALK and in wool treated with about l%TTO emulsified in water with about 1% ALK.
Number Unhatched
Treatment of eggs Hatched Developed Undeveloped
Dry wool 10 7 1 2
ALK 20 6 2 2
TTO 10 0 0 10
Fumigant effects: Most eggs hatched in the absence of TTO in this assay and this was consistent across replicates. There was a clear effect of TTO on egg viability in comparison to controls (P=0.038), but difference in mortality between the two quantities of TTO tested was not significant (P>0.05), largely because of high variability amongst replicates within the two TTO treatments. In the about 60 μΐ treatment, no eggs hatched in two replicates whereas in the third about 60% of eggs hatched and in the about 15 μΐ treatment , no eggs hatched in one replicate, about 40% in the second and about 80% in the third (Figure 31). TTO is therefore likely to have fumigant effects against B. ovis eggs, in addition to any physical effects it may have. 2.5 Sheep studies
2.5.1 Dipping
Mean and standard errors for pre- shearing counts per part for each group of three sheep are shown in Figure 32. Counts in the different groups ranged from about 11.4 to about 13.7 per part which by convention would be considered heavy infestations of lice (>5 lice per 10 cm fleece part). Despite inspection of 40 parts on each sheep, no lice were found in any of the sheep following dipping in about 1% or about 2% TTO at any of the four post treatment inspections. It appears highly likely that dipping with TTO formulation completely eradicated lice in all treatment groups.
In the control group, all sheep remained infested throughout the study. Numbers of lice fell from a mean of about 13.2 prior to shearing to about 2.4 at about 2 w after shearing. Reduction in lice numbers generally occurs after shearing and is due to both direct removal of lice during shearing and the effect of environmental exposure following fleece clipping. After this time, louse numbers increased rapidly and a heavy infestation (mean count of about 12.2 lice per part) was present at the final inspection about 20 weeks after treatment.
Table 9: Mean louse counts of untreated sheep and sheep immersion dipped in 1% and 2% TTO formulation.
Treatment Time of inspection
Pre- 2 weeks 6 weeks 12 weeks 20 weeks
shearing
Controls 13.2 (3.0) 2.4 (0.7) 2.5 (0.9) 3.7 (1 .8) 12.3 (4.2)
1 % TTO 12.4(3.7) 0.0( 0.0 0.0 0.0
2% TTO 12.4(3.6) 0.0 0.0 0.0 0.0
2.5.2 Jetting
Mean louse counts for the different treatment groups of sheep at allocation are shown in Figure 33. Louse densities varied from about 3.8 to 7.2 per part which by convention would be considered to be moderate (1-5 lice per part) to heavy (> 5 lice per part) infestations. The mean count for the first about 2% TTO treatment group was about 60% higher than the other groups mainly because of a leverage effect of one sheep with very high counts. Louse density on this sheep was about 16.5 per part at the start of the experiment, significantly above the mean of all sheep at the start of the experiment (5.0 per part) and well above the next highest count in the experiment of 10.6 per part on one of the control sheep. It maintained relatively high counts through the experiment (about 3.9 per part at 2w, about 7.9/part at 6 w, and about 12.4 per part at 12 w compared to counts of about 0.8/part, about 2.3/part and about 4.0 per part respectively for the sheep with the next highest counts in the treated groups) and markedly affected results for the about 2% TTO treatment.
Mean counts for the individual groups of 3 within each treatment are shown in Figure 34. Whereas louse numbers increased at each count for the two control groups, they dropped markedly following jetting (about 2 w count) in all treatment groups and then increased at the about 6 w and 12 w counts. Jetting with these concentrations gave maximum reduction in louse numbers of about 94% in comparison to controls (Table 10).
Table 10: Percent reduction in louse counts at 2, 6 and 12 weeks after jetting (based geometric means, calculated with the Henderson- Tilton formula).
Treatment Time after jetting
2 weeks 6 weeks 12 weeks
1 % TTO 93.5 93.9 78.1
2% TTO 93.5 91.1 84.0 a
Based on pre and post shearing counts for the 4 sheep remaining at 12 weeks
The distributions of rubscores in the different groups are shown in Figure 35. Scores were significantly lower in the treated groups than in the controls (P<0.05). Three of the six control sheep had maximum rubscores of about 6 at both about 6 and 12 w, which could be expected to be correlated with significant wool loss and reduction in wool quality. In the TTO-treated groups, no sheep had reached score 6 at 6 w and only one sheep had reached this level byl2 w. 2.5.3 Residues
Tissue samples: Analysis of muscle sub-samples of about 0.2 g collected one week after dipping from the 6 control sheep, the 6 sheep treated with about 1% TTO and 5 of the 6 sheep treated with about 2% TTO found no evidence of the presence of any TTO components. As no residues were found in any sample no further tissue samples were collected, as agreed under the Animal Ethics approval.
Blood samples: GCMS analysis of blood samples collected about 1 and 3 weeks after treatment from all sheep dipped in 1% and 2% TTO formulation found no indication of any TTO components.
Wool samples: Figures 36 and 37 show changes in the relative concentration of some TTO components in the wool of sheep at different times after dipping in about 2% TTO. For most compounds, concentrations were at highest levels at the one week measure and then dropped relatively quickly to close to non-detectable levels by about 12 weeks after treatment. It is notable however that there seemed to be a jump in the level of terpin-4-ol from the 3 week measure where it was quite low to the 6 week measure where it was close to the 1 week levels in most sheep. It is tempting to suggest that this is an experimental artefact. However, the rump and shoulder samples were measured at different times by different operators, the same increase wasn't seen with other compounds and it seemed to occur in all sheep (Figure 37). Although sampling or experimental artefacts cannot be completely ruled out, we believe the increase to be real. The sheep were exposed to about 45 mm of rainfall the day before the samples were collected and this may have had an effect.
2.5.4 'Stripping ' study ( laboratory )
Figure 38 shows the mean fall in volume of TTO formulation in the beakers after each three wool samples were dipped and Table 11 shows the reduction in concentration of TTO components from before dipping commenced until after the last (fifteenth) 10 g sample was dipped, retrieved and drained. At this stage the mean volume of wash had fallen by more than about 60% from about 500 ml to a mean of about 195 (+ 9) ml and the concentration of terpinene-l-ol had fallen by a mean of 49%.
Table 11: Percentage reduction (+ s.e.) in the concentration of some major TTO components in TTO formulation used in a simulated wool dipping study and in control fluid left standing on the bench top under equivalent conditions, but without wool dipping.
Treatment Tea tree oil component
terpinen-4-ol γ-terpinene- OC-terpinene p-cymene
Control
1.9 (±6.4) -1.4 (±2.1) -1.9 (±5.6) n.d.
Dipping
48.6 (±8.0) 54.4 (±0.9) 53.6 (±0.1) 12.5 (±5.3)
3. Conclusions
Sheep blowflies
TTO demonstrated insecticidal effects against all stages of sheep blowfly maggots. Formulations containing 1% TTO reliably gave 100% kill of 1st instar larvae and 2.5% TTO caused mortality of most 2nd and 3rd instar larvae in agar assays. However in experiments where 3rd instars were dipped in TTO formulations for 60 s, even 50% TTO gave only 46% kill. TTO was strongly repellent to blowfly maggots with most rapidly evacuating TTO treated agar. Killing maggots in strike wound is undesirable to avoid septic effects from putrefaction products.
When mixed in formulations with some currently used larvicides TTO appeared to reduce insecticidal effect. In agar studies this was thought to be due to rapid exodus of maggots from the treated agar before a toxic dose of insecticide was acquired, but the reason for similar reduction in effectiveness in larval dipping studies is less certain. TTO also showed insecticidal effects, often by fumigant action against eggs, pupae and adults of L. cuprina.
TTO also had strong repellent effects against adult flies. In laboratory studies with wool treated with 3% TTO complete suppression of egg laying by adult L. cuprina was evident in for up 6 weeks.
With its documented antimicrobial effects and reputed wound healing properties, toxic effects against young larvae and eggs, repellent action against older larvae and repellent effects against gravid female flies it is considered that TTO could be a useful ingredient in flystrike or wound treatments. Sheep lice
In laboratory wool dipping assays, 1% TTO formulation reliably gave 100% kill of lice. The majority of this effect appeared to be due to fumigant effects from the most abundant component of TTO, terpinen-4-ol. There also appeared to be both physical and fumigant effects against sheep lice eggs. In pen studies with sheep shorn two weeks previously, conducted according to Australian Pesticides and Veterinary Medicines Authority guidelines, dipping of sheep in both 1% and 2% TTO formulation appeared to eradicate lice. No lice were found on any of the treated sheep despite careful inspection of at least 40 fleece partings per animal at 2, 6, 12 and 20 weeks after treatment.
In studies of long wool efficacy, jetting (high pressure spraying into the fleece) sheep with 1% and 2% TTO formulations, both formulations reduced louse numbers by 94% in comparison to controls at two weeks after treatment. For the 1% formulation the reductions at 6 and 12 weeks after treatment were 94% and 91% respectively and for the 2% formulation, 78% and 84% respectively. Both formulations significantly reduced wool damage in comparison to controls.
The results of this project indicate significant potential for use of TTO formulations as sheep ectoparasiticides. Both insecticidal and repellent effects were demonstrated against sheep blowflies and sheep studies demonstrated probable eradication of lice when sheep were dipped in 1% and 2% TTO formulations.
Insecticidal and repellent effects against L. cuprina larvae and adult flies also suggest considerable potential for use of TTO in flystrike treatments. These effects, together with the demonstrated antimicrobial and wound healing properties, and the perception of TTO as natural and soothing, also suggests possibilities for more widespread use in general veterinary wound treatments, particularly for treatment and protection of wounds resulting from husbandry practices such as tail docking, castration, mulesing and dehorning.
All publications discussed and/or referenced herein are incorporated herein in their entirety.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.
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