Background to the Invention Farm animals such as lambs, weaners and sheep may typically be infected by a wide variety of parasites. Such parasites include Haetnonconchus spp., Ostertagia spp., <BR> <BR> Erichostrongylus spp., Cooperia spp., Nematodirus spp., Cabertia spp., <BR> <BR> <BR> Oesophagostomum spp., Trichuris spp., Strongyloides spp., Bunostomum spp., Oestrus spp., Dictyocaulus spp., Fasciola spp. and Monezia spp. Specific examples of these parasites are set out in Table 1.

For a variety of reasons, there is an increasing number of such parasites that have developed resistance to available drug treatments. Moreover, because of the infective nature and ready transmission from animal to animal, the presence of resistant parasites will rapidly spread to infect a substantial number, if not all, of the animals in a flock or herd. One means by which such infection will rapidly spread is where new animals in which the presence of drug resistance is known or suspected are to be introduced onto a property.

There are a variety of drug substances that are used to treat parasitic infections.

Amongst these broad groups of substances are macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles. Unfortunately, many of the parasites mentioned in Table 1 have developed resistance to these substances.

Although the problem of resistance has been tackled through the development of new substances, the time to develop, evaluate and demonstrate efficacy of such substances is substantial and expensive. Moreover for the reasons that resistance has developed against existing substances it is very likely that resistance will occur in relation to these new substances.

Summary of the Invention Rather then tackling the problem of resistance through the development of new substances, the present inventors have found that it is possible to circumvent resistance by combining specific classes of anthelmintics. The efficacy of this combination arises out of the finding that the combination is synergistic.

Accordingly, the present invention provides in a first aspect, a synergistic anthelmintically effective composition consisting of at least one compound selected from each of the following groups: macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles and a therapeutically acceptable carrier.

Table 1 Parasite Species Species Common Name Comments Haemonchus contortus Barber's pole worm includes inhibited L4 stage Haemonchus placei Large stomach worm Ostertagia circumcincta Small brown stomach includes inhibited L4 stage worm Trichostrongylus axei Stomach hair worm Trichostrongylus colubriformis Trichostrongylus vitrinus Black scour worm Coopesia curtcel Cooperia oncophera Small intestinal worm Nematodirus spathiger Nematodirus Thin-necked intestinal worm Clzcabertia ovina Large mouthed bowel worm Oesophagostomum Nodule worm columbianum -a Oesophagostomum Large bowel worm venulosum Trichuris ovis Whip worm Strongyloides papillosus Intestinal threadworm Bunostomum spp Hookworm Oestrus ovis Dictyocaulus viviparus Large lungworm Fasciola hepatica Monezia Includes head and segments

In a second aspect, the present invention provides a method for treating parasitic infections in an animal, comprising administering to the animal, a synergistic anthelmintically effective amount of a composition which consists of at least one compound selected from each of the following groups : macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles and a therapeutically acceptable carrier.

In a third aspect, the present invention further provides the use of a synergistic anthelmintically effective amount of a composition which consists of at least one compound selected from each of the following groups: macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles and a therapeutically acceptable carrier in the treatment of a parasitic infection in 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.

The aforementioned treatments may be desirably administered to animals prior to introduction to a land area so as to prevent the land area from becoming infested with parasites which may or may not be resistant to one or more compounds selected from the groups consisting of macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles. Typically, animals such as sheep, will be isolated for at least 2 days after treatment before being placed on pasture.

Alternatively, animals may be treated at any time, as appropriate, particularly when it is suspected that the animal may be carrying at least one parasite which is resistant to at least one of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles.

The compositions of this invention have application where the parasites are resistant to known drug treatments. In particular, the compositions are effective in situations where parasites are resistant to at least one of each of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles. Preferably, the compositions are effective in situations where parasites are resistant to at least two of each of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles. More preferably, the compositions are effective in situations where parasites are resistant to at least three of each of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles. Most preferably, the compositions are effective in situations where parasites are resistant to all of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles.

In use, a preferred indication is the treatment of stock to eliminate adult gastro- intestinal worms and liver fluke. Typically, treatment results in the clearance of >95% of total worm count including worms resistant to at least one of each of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles.

Compositions of this invention include at least one compound selected from each of the groups: macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles.

Representative examples of compounds from each of these group are set out in Table 2.

Table 2 Compounds Macrocyclic lactone Benzimidazole Salicylanilide Imidazothiazole abamectin albendazole closantel levamisole ivermectin fenbendazole niclosamide pyrantel pamoate doramectin thiabendazole butamisole moxidectin oxfenbendazole cydectin fenbantel milbenycin mebendazole parbendazole flubendazole oxibendazole carbendazole

Of these combinations which include at least abamectin from the macrocylic lactone group together with one compound from each of the other three groups ; at least albendazole from the benzimidazole group together with one compound from each of the other three groups; closantel together with one compound from each of the other three groups and levamisole together with one compound from each of the other three groups are each preferred. Particularly preferred is the specific combination of abamectin, albendazole, closantel and levamisole. Most preferably, the levamisole is used in the form of a water soluble salt such as the hydrochloride.

The therapeutically active compounds used in the invention are preferably incorporated into formulations in the range of concentrations as follows (g/L) macrocylic lactones: 0.1-20. 0 g/L, preferably 0.5-1. 5 g/L benzimidazoles : 1-100 g/L, preferably 18-30 g/L salicylanilides: 1-100 g/L, preferably 30-50 g/L imidazothiazoles : 1-100 g/L, preferably 30-50 g/L Although drenches are preferred dosage forms for the compositions of this invention, a number of alternative compositions may be used. These pour-on transdermals, slow release boluses for rumenal deposition and injectable formulations.

Each dosage form requires a therapeutically effective carrier. In the case of drenches, typically a formulation will include a solvent system for the macrocylic lactones, one or more dispersing and suspending agents for the benzimidazoles and salicylanilides, one or more surfactants, one or more preservatives, a buffering system and water as a carrier.

The solvent system for the macrocyclic lactones includes at least one solvent selected from the group consisting of polyethylene glycol, tetraglycol, ethanol, benzyl alcohol and propylene glycol.

The dispersing and suspending agents for the benzimidazoles and salicylanilides include at least one selected from the group consissting of : glyceryl palmitostearate, bentonite, colloidal silica, xanthan gum and polymeric pyrrolidones.

Surfactants that may be used include polysorbate 80 and ethoxylated castor oil.

A variety of buffer systems may be used, particularly phosphate buffers based on combinations of varying amounts of monobasic and dibasic sodium phosphate to achieve the desired pH.

The compositions of the invention are effective when used in a variety of animals. For example, sheep, goats, ruminants (including cattle) and camelids.

Modes for Carrying Out the Invention In order to better understand the nature of the invention, a number of examples will now be described as follows: Example 1 Material Supplier Grade Amount (g) Avermectin Haiman Technical 94% 1. 0 Albendazole Pacific Resource USP24 25. 0 Closantel Pacific Resource Technical 98% 37.5 Levamisole Hydrochloride Pacific Resource BP 1998 40. 0 Tetraglycol AGRAR Food Grade 400. 0 Benzyl alcohol APS BP1998 80. 0 Keltrol F (Xanthan Gum) USP24 10.0 Rhone-Poulenc Phenonip (Phenoxyethanol) Food 10.0 Bronson& Jacobs Glyceryl Palmitostearate (Precirol ATO 5) USP24 5.0 Bronson & Jacobs Veegum Regular (Magnesium USP24 Aluminium Silicate) RT Vanderbilt Co. PVP29/30 (Ethenyl-2-USP24 pyrrolidinone homopolymer) ISP PVP C15 ISP USP24 Polyethylene glycol (PEG 2000) BASF USP24- Cremophor (ethoxylated Castor USP24 Oil) BASF Propylene Glycol USP24 Sodium phosphate monobasic Bronson & Jacobs Technical Sodium phosphate dibasic Bronson & Jacobs Technical Polysorbate 80 Bronson & Jacobs USP24 Cab-O-Sil M5 (Colloidal Silicon USP24 Dioxide) Cabot Corp Water qs llitre Example 2 Material Supplier Grade Amount (g) Avermectin Haiman Technical 94% 0.8 Albendazole Pacific Resource USP24 19. 0 Closantel Pacific Resource Technical 98% 30.0 Levamisole Hydrochloride Pacific Resource BP 1998 35.5 Glycerol formal 30. 0 Tetraglycol AGRAR Food Grade- Ethanol 20. 0 Benzyl alcohol APS BP1998 80. 0 Keltrol F (Xanthan Gum) USP24 10. 0 Rhone-Poulenc Phenonip (Phenoxyethanol) Food Bronson& Jacobs Potassium sorbate 10.0 Glyceryl Palmitostearate (Precirol ATO 5) USP24- Bronson & Jacobls Veegum Regular (Magnesium USP24 50.0 Aluminium Silicate) RT Vanderbilt Co PVP29/30 (Ethenyl-2-USP24 50. 0 pyrrolidinone homopolymer) ISP PVP C15 ISP USP24 Polyethylene glycol (PEG 2000) BASF USP24- Cremophor (ethoxylated Castor USP24 - Oil) BASF Propylene Glycol USP24 - Sodium phosphate monobasic Bronson & Jacobs Technical- Sodium phosphate dibasic Bronson & Jacobs Technical Polysorbate 80 Bronson & Jacobs USP24- Cab-O-Sil M5 (Colloidal Silicon USP24 - Dioxide) Cabot Corp Water s llitre Example 3 Material Supplier Grade Amount (g) Avermectin Haiman Technical 94% 1. 0 Albendazole Pacific Resource USP24 25. 0 Closantel Pacific Resource Technical 98% 37.5 Levamisole Hydrochloride Pacific Resource BP 1998 40. 0 Tetraglycol AGRAR Food Grade - Benzyl alcohol APS BP1998 20. 0 Keltrol F (Xanthan Gum) USP24- Rhone-Poulenc Phenonip (Phenoxyethanol) Food 20.0 Bronson& Jacobs Glyceryl Palmitostearate (Precirol ATO 5) USP24 Bronson & Jacobs Bentonite 20. 0 Veegum Regular (Magnesium USP24 Aluminium Silicate) RT Vanderbilt Co. PVP29/30 (Ethenyl-2-USP24 pyrrolidinone homopolymer) ISP PVP C15 ISP USP24 100. 0 Polyethylene glycol (PEG 2000) BASF USP24 Cremophor (ethoxylated Castor USP24 80.0 Oil) BASF Propylene Glycol USP24- Sodium phsophate monobasic Bronson & Jacobs Technical - Sodium phosphate dibasic Bronson & Jacobs Technical - Polysorbate 80 Bronson & Jacobs USP24 Cab-O-Sil M5 (Colloidal Silicon USP24 Dioxide) Cabot Corp Water qs 1 litre Example 4 Material Supplier Grade Amount (g) Avermectin Haiman Technical 94% 1.0 Albendazole Pacific Resource USP24 25. 0 Closantel Pacific Resource Technical 98% 37.5 Levamisole Hydrochloride Pacific Resource BP 1998 40. 0 Tetraglycol AGRAR Food Grade Benzyl alcohol APS BP1998 80. 0 Keltrol F (Xanthan Gum) USP24 Rhone-Poulenc Phenonip (Phenoxyethanol) Food Bronson& Jacobs Glyceryl Pahnitostearate (Precirol ATO 5) USP24 Bronson & Jacobs Veegum Regular (Magnesium USP24 Aluminium Silicate) RT Vanderbilt Co. PVP29/30 (Ethenyl-2-USP24 pyrrolidinone homopolymer) ISP PVP C15 ISP USP24 Polyethylene glycol (PEG 2000) BASF USP24 - Cremophor (ethoxylated Castor USP24 Oil) BASF Propylene Glycol USP24 300.0 Sodium phosphate Bronston & Jacobs Technical 9.0 monobasic Sodium phosphate dibasic Bronson & Jacobs Technical 1. 0 Polysorbate 80 Bronson & Jacobs USP24 200. 0 Cab-O-Sil M5 (Colloidal Silicon USP24 200.0 Dioxide) Cabot Corp Water qs 1 litre

Example 4 was prepared as follows: 1. Dissolve avermectin in benzyl alcohol and propylene glycol.

2. Add polysorbate 80 to step 1.

3. Add water to the solution from step 2 and mix until homogeneous.

4. Dissolve sodium phosphate dibasic and sodium phosphate monobasic in the solution from step 3.

5. Add closantel, albendazole and levamisole hydrochloride. Mix until fully dispersed.

6. Add Cab-O-Sil M5 to the suspension and homogenise until the thicken agent fully hydrated.

Based on this disclosure, the person skilled in the art would appreciate the general approach to be taken in preparing the compositions of this invention.

In order evaluate the efficacy of the compositions of the invention, a number of trials were conducted using Example 4 as above.

Trial RD0201-H002 : A critical pen sacrifice study evaluating the therapeutic efficacy of a combination abamectin, levamisole hydrochloride, albendazole and closantel anthelmintic formulation against resistant strains of Haemonchus contortus, TrichostroMlus colubriformis and Teladorsagia circumcincta in sheep.

This study was conducted from the 25th of February to the 29th of August, 2002, with the animal phase conducted from the 7th May to the 27th June 2002. Suitable sheep (18) were relocated to the University of New England Animal House Facility on the 7th May 2002 were weighed, identified with individually numbered ear tags and treated with twice the recommended dose rate of Ivomec (Liquid for Sheep, Merial Australia Pty Ltd), to remove any residual worm burden.

On 22nd May 2002 (Day-26) faecal samples were collected from each trial animal to confirm individual zero faecal egg counts. Later that day trial sheep were infected with approximately 5000 Haemonchus contorts (macrocyclic lactone and closantel resistant strains), 6000 Trichostrongylus colubriformis (levamisole hydrochloride and albendazole resistant strains) and 5000 Osteratagia eircumoineta (macrocyclic and albendazole resistant strains) infective larvae.

Faecal samples were collected from each sheep on 14th June 2002 (Day-3) and individual faecal egg counts were conducted. Animals were ranked on the basis of decreasing faecal egg counts and blocked into eight blocks each of two animals and randomly allocated to the treatment groups from these blocks. The 16 animals with the highest counts were selected for inclusion in the trial and the two animals with the lowest faecal, egg counts were selected as spare animals.

On 17th June 2002 (Day 0) all trial animals were weighed, faecal sampled and animals in Group 2 were treated as follows. Animals were weighed and dosed according to individual live weight as outlined in Table 3.

Table 3: Dosage regime Do§6'rate Active. T _~ Group 1 Untreated control-- Group 2 Example 4 1 mL/5 kg 37. 5 g/L closantel g/L abamectin 40 g/L levamisole hydrochloride 25 g/L albendazole The 18 trial sheep (including the 2 spare animals) were sacrificed on 27th June 2002 (Day 10) for collection of faecal samples, abomasal and small intestine contents.

Individual faecal egg counts, treatment group coprocultures and total worm counts were conducted for calculation of treatment efficacies.

Drenchrites (CSIRO Research-Horizon Technology 1996) was performed between the lOth July and the 23rd August 2002 to clarify that strains of Trichostroiaggylzcs colubriformis used were resistant to levamisole hydrochloride and albendazole and, Osteratagia circumcinctcl were resistant to albendazole.

Faecal samples were collected according to standard procedures and submitted to the Veterinary Health Research parasitological laboratory. Individual strongyle faecal egg counts and group bulk coproculture for larval differentiation were carried out.

Gastrointestinal tracts were recovered according to standard procedures and following gut washing were submitted to the parasitological laboratory. Individual total worm counts were conducted and results are summarised in the accompanying tables, 4-10 and figures 1-4.

Table 4: Group mean strongyle faecal egg counts. Group Treatrnent Arithnitic ans 1 9 2 2 . Geo'm. 1 2 Example4 8 1. 82 Day 01 Means with different superscripts within the same column are significantly different at p<0.05 Table 5: Percentage reduction of strongyle species (based on group mean strongyle faecal egg count data) Cn=oup xnent. , y D GL, OUP ent 2Example 2 Example 2 Example 4 >99.

Table 6: Group mean abomasal total Worm Counts Gr nt |Group Y... a.. _. AithnieiMecrii 1Control1093006. 6 1913. 3 24. 3 2 Geomet7lcMeans 1 2 Ex. u Note: Example 4 = abamectin / closantel /albendazole /levamisole hydrochloride<BR> aHaemonchus species; b Ostertagia species; d imm-immature ; cL4=fourth larval stage<BR> 1Means with differnt superscripts within the same column are significantly differnet at p<0.05 Table 7: Percentage rewduction of abomasal worms (based on Total Worm Counts) b Group Treaunertt (adult - Day . ic. aiea 2 9% >99. 9% >99. 9% >99. 9% >99. 9% I, | 2 10 9 >99. 9% >99. 9% >99. 9% >99.

31angaHaemonchus species; bOstertagia species; dimm-immature; cL4-fourth larval stage<BR> Table 8: Groupe mean small intestinal Total Worm Counts |Group C&O ) , r ul . Lts}. ihr2z rea. _. 1 1 6. 7 2 Example 4 10 00 1 Control 1 2 Treatatrichostrongyle species: cNematodirus species; dimm=immature; cCooperia species<BR> 1Means with different superscripts within the same column are significantly different at p<0.05 Table 9: Percentage reduction of small intestinal worms (based on Total Worm Counts) <jro cA < , rithmetic ccs 2 Geometric 2 TreataTrichostrongyle species; dimm-immature;<BR> Talbe 10: Larval differentiation results following group bulk corproculture- Larvae as a % of the total number counted. l Day 'Moc/KM e&e epp. . pp pp Day-3 AIJ, ày Day U Day 0 Example

Conclusion: Excellent control (>99.9% reduction) of a mixed gastrointestinal strongyle burden as assessed by geometric faecal egg counts was achieved by the use of the Example 4 formulation at the conclusion of the trial (Day 10),.

Excellent control (>99.9% reduction) was achieved by the Example 4 formulation against the major nematodes, macrocyclic lactone and closantel resistant strains of Haemonchus spp. (adult and immature stages-geometric means), macrocyclic and albendazole resistant strains Ostertagia spp. (adult, immature and L4 stages-geometric means) and levamisole hydrochloride and albendazole resistant strains of Trichostrongylus spp (adult and immature stages-geometric means) as assessed by geometric total worm counts.

Trial JUA1240r : A property faecal egg count reduction study evaluating the therapeutic efficacy of the Example 4 formulation against field strains of mixed nematode population of either Haemonchus contorts Trichostrongylus colubriformis and/or Teladorsagia circumcincta in sheep.

This study was conducted from the 16 May 2002 to the 20 August 2002 with the animal phasel conducted between the 1st July 2002 to the 17th July 2002 and animal phase 2 between 5th September 2002 to the 4th October 2002. A trial site was sought, containing a mob of Merino sheep that were known to be harbouring resistant strains of nematodes (including either closantel resistant and/or macrocyclic lactone resistant Haemonchus species, as well as either benzimidazole resistant and/or levamisole resistant Trichostrongylus colubriformis and/or Teladorsagia circumcincta.) Pre-trial monitoring of the site confirmed that the intended trial animals carried a nematode burden of greater than 400 eggs/gram. A group coproculture was performed on these prospective trial sheep to establish the genera present.

On Day-3 of the trial, a mob of approximately 300 Merino ewes was mustered into a set of sheep yards. Eighty ewes were identified with uniquely numbered eartags and faecal sampled as they presented in the race. The faecal samples were returned to Veterinary Health Research for individual faecal egg counts and a bulk coproculture.

The sixty animals with the highest strongyle faecal egg counts, as determined by the Day-3 faecal egg counts, were selected for inclusion in the trial. These sheep were allocated to one (1) of six (6) treatment groups, on the basis of their faecal egg counts, such that each group had a similar arithmetic group mean faecal egg count.

On Day 0 (treatment day), each animal was weighed and treated according to the treatment schedule outlined in Table 11. Clinical observations were conducted one hour post-treatment to determine whether any adverse reactions had occurred in relation to treatments. None were detected.

Table 11: Treatment table (phase 1) .'lE'reatment''Pormulation''.- |'Gröup 1 2 10 3 Ivomec (Liquid for Sheep, 1 10 Merial Pty Ltd) 4 Sustain (Dover Laboratories 1 10 Pty Ltd) 5 Youngs Levamisole (Youngs 1 mL/4 kg 10 Animal Health Pty Ltd) 6 Valbazen (Coopers Animal 1 10 Health) : ; ;-""'-. Dosage regimen, Number,,, ofThe trial concluded on Day 13 when faecal samples were collected and returned to the Veterinary Health Research Laboratory for individual faecal egg counts and group coprocultures. The entire mob was administered an effective broad-spectrum anthelmintic to remove any existing worm burden.

The aim of this field study was to study and evaluate under field conditions, the therapeutic efficacy of Example 4 when administered to sheep that are known to be harbouring resistant strains of nematodes. The selected trial site was known to harbour closantel resistant Haemonchus contorts. This however was not confirmed during the initial phase of the study as a full dose of closantel was administered (as stated in the

protocol). Standard industry practice for diagnosis of closantel resistance in the field involves either the administration of a full dose of closantel and sequential sampling of treated sheep over three to six weeks post treatment, or alternatively administration of a 1/3 dose and sampling at 10 to 14 days post treatment.

A second faecal egg count reduction study was conducted after consultation with the Study Sponsor to confirm the closantel resistance status at the trial site, "Kelvin East".

The second phase of the study involved two groups of sheep each consisting of ten animals. Ten random faecal samples were collected prior to treatment from the mob of wethers to confirm a nematode burden of greater than 400 eggs/gram, and a group coproculture that confirmed a very high percentage (91 %) of HaemoncAus co7jtortus were present. On Treatment Day (Day 0), individual faecal samples were collected from twenty animals as they presented in the race. These animals were weighed and weights recorded and treatments administered in accordance to the treatment regime (detailed in Table 12). Faecal samples were returned to Veterinary Health Research for individual faecal egg counts and group coprocultures. Animals were observed post treatment for adverse reactions. None were detected.

Table 12: Treatment table (phase 2) 'Treatiiant.''Formiation-sage Group I I Untreated control-10 2 (Dover Laboratories 1 10 Pty The second phase of this trial concluded on Day 11, with the collection of individual faecal samples from all animals. These samples were returned to Veterinary Health Research for individual faecal egg counts and group coprocultures.

Faecal samples for phase 1 were collected during pre trial monitoring, (Day-3), at treatment (Day 0) and at the conclusion of the trial (Day 13) and for phase 2 at treatment (Day 0) and at the conclusion of the trial (Day 11). Results from faecal egg counts, larval differentiation and calculated treatment efficacies are summarised in the accompanying tables 13-24 and figures 5 and 6. Note that in figures 5 and 6,"Jurox" refers to example 4.

Table 13: Pre trial monitoring results. Date nsmecal ecal count pg), 01 July 2002 (Day-3) 900 320-1880 Group Table 14: Group arithmetic mean faecal egg counts and body weights at Day 0. s Group TreaXent''f' 4-iL cunt 1 Untreated control 9841 49. 11 2 3 4 Sustain 5841 5 6 Valbazen 32S1 48. 251 (3roup'meán faecAl egE up meán body w. eititl1 Means in the same column with different superscripts are significantly different at p<0. 05 Table 15: Group arithmetic-and geometric mean faecal egg counts (epg). l Pte-tnal r-. . Arithmeti'c . <4A/McMga'.' 1 controls 892 2 P 3 omee 0 4Sustain924584"160"' I 5 Levamisole l 6 Geom --ometrc an 1 1 2 5 3 I 4 4 5 6 Valbazen'818. 9 179. 14. 9 ; X Pre-ti Dw-&verbar Dåy ; ; 0- iE) ay 13 2 X > l1 Means in the same column with different superscripts are significantly different at p<0. 05 Table 16: Overall percentage efficacy calculated using arithmetic and geometric group mean faecal egg counts. Group-Trleatniont 2 2 4 3 9 4 9 i 5 19 I 6 Valbazen 89. 76 _,. i J 2. 87 3 9 I 4 5 4 I 6 Valbazen EfficTable 17: Percentages of nematode types present pre-trial, Day 0 and Day 13. : a 'rtmet ,, . y p iii lo ''zcl, : : :. l¢'eI .-lo., oC ? es alal. Larae C : aunted Pre-trial All-94 Day 2 Example4 83 5 8 2 2 100 3 91 3 100 Sustain 5 6 Valbazen 82 8 6-4 Day 13 94 2 1 100 2 3 4 5 6 lHaem - Haemonchus, Trich - Trichostrongylus, Tel _ Teladorsagia, Coop - Cooperia, Oes - Oesophagostomum<BR> Table 18 : Efficiacies calculated for each nomatode genus. | Treatment 2 Example >99. 9% 3 >99. 9% >99. 9% >99. 9% na 4 9%-ve value-ve value na na 15 >99. 9% 20. 48% 92. 78% na na 16 >99. 9%-ve value-ve value na (JrouHaem - Haemonchus, Trich - Trichostronglyus, Tel - Teladorsagia, Coop - Cooperia, Oes - Oesophagostomum<BR> na - not assessed Phase 2 Table 19: Treatment day monitoring results. Mean, X A. A 23 Sept 0) 1428 80-3000 Table 20: Group arithmetic mean faecal egg counts and body weights at Day 0. Gro'Treatniectt jBggGnmp''""''dy 1 1 controls 16281 2 Sustain 11228'150-21

?. Group 1 Means in the same column with different superscripts are significantly different at p<0. 05 Table 21 : Group arithmetic and geometric mean faecal egg counts. Group Treatment Day 0 Day 11 Arithmetic Means Untreated controls 1628. 0' 3008. 01 2 Sustain 1228. 01 2088. 01 Geometric Means 1 Untreated controls 1237.7 2041.1 2 Sustain 940.0 1511.9 1 Means in the same column with different superscripts are significantly different at p<0. 05 Table 22: Overall percentage efficacy (arithmetic and geometric means) Peicentage -.. UP je 2 'Geoinetric J 2 1/3 Sustain 25. 9 EfficTable 23: Nematoe populatioin % - Pre-trial, Day 0 and Day 11 (based on faecal culture and larval differnetiation). Group Day ., I Pre-trial All-91 100 Day 2 1/3 Sustain 7 2 100 Day 13 1 2 ; enuHaem - Haemonchus, Trich - Trichostrongylus, Tel - Teldrosagia, Coop - Cooperia, Oes - Oesophagostomum<BR> Table 24: Individual nematode efficacies. L 2 na na P aneHaem - Haemonchus, Trich - Trichostrongylus, Tel _ Teladorsagia, Coop - Cooperia, Oes - Oesophagostomum<BR> na - not assessed<BR> -ve - negative Conclusion: The second faecal egg count reduction test was to confirm the presence closantel resistant Haemonchus at the trial site. This was achieved by administering a one third dose of closantel to a group of ten (10) animals and the addition of another group of ten (10) animals retained as untreated controls. The use of a one third dose of closantel is standard industry practice for diagnosis of closantel resistance in the field.

Reduced efficacy of closantel was observed against Haemonchus, confirming. The presence of closantel resistant Haemonchus at the trial site.

The inclusion of the levamisole and benzimidazole groups confirmed the resistance status of Trichostrongylus.

Excellent efficacy (> 99.0%) was attained by the Example 4 formulation against a mixed gastrointestinal population including closantel resistant Haemonchus as well as levamisole and benzimidazole resistant Trichostrongylus.

Trial JUA1273r : A property faecal egg count reduction study evaluating the therapeutic efficacy of the Example 4 formulation against field strains of mixed nematode populations, including closantel resistant strains of Haemonchus contortus in sheep.

This study was conducted from the 5 th of September 2002 to the 23rd of October 2002, with the animal phase conducted between 10th of September 2002 and the lSt of October 2002. Routine monitoring of a trial site known to harbour closantel resistant strains of Haemonchus contortus was conducted to identify a suitably infected group of sheep. Pre-trial monitoring confirmed that one group of sheep (270 Merino hoggets) was suitably infected with a high burden of Haemonchus contortus.

On Day-2 of the trial individual faecal samples were collected from ninety (90) potential trial sheep and individual strongyle faecal egg counts performed. Trial sheep had already been identified using uniquely numbered ear tags as part of standard farming practice at the trial site. From the ninety (90) potential trial sheep sixty (60) sheep were selected and allocated (according to individual strongyle faecal egg counts) to six (6) groups of ten (10) sheep each, such that each group had a similar group arithmetic mean strongyle faecal egg count and range of faecal egg counts within the group.

On Day 0 of the trial (luth September 2002) selected trial sheep were weighed (see figure 7 for arithmetic mean body weights and note that the treatment"Jurox"refers to the treatment with Example 4), the weights recorded and individual faecal samples collected for individual strongyle faecal egg counts. Trial sheep in Group 2 were treated according to individual body weight with the test formulation, trial sheep in Groups 3-6 were treated with the respective reference formulation and trial sheep in Group 1 were retained untreated as negative controls. Groups 2,3, 5 and 6 were treated at the recommended dose rate for each active, while sheep in Group 4 were treated at one third the normal closantel dose rate, to determine and demonstrate the presence of closantel resistance (Reference : Rolfe PF; Fourth International Congress for Sheep Veterinarians 1997, pg 55). Sheep were observed in the immediate post-treatment period for adverse reactions (none were observed). Individual strongyle faecal egg counts and group bulk coprocultures for larval differentiation were subsequently performed on the samples collected.

Trial sheep were returned to the sheep yards on Day 13 of the trial (1"October 2002) and individual faecal samples again collected. All trial sheep received a single therapeutic dose of Rycozole (Dl due to animal welfare concerns. Individual strongyle faecal egg counts and group bulk coprocultures for larval differentiation were subsequently performed on the samples collected.

Treatment efficacies were then calculated using group arithmetic and geometric strongyle faecal egg counts for the major strongyle species present (see figures 8 and 9 and note that the treatment"Jurox"refers to treatment with Example 4).

'Rycozole Oral Anthelmintic for Sheep and Cattle, Novartis Animal Health Australasia Pty Ltd Table 25: Treatment table. Group nbe - . -_--_-ctive onsstiitaat o. 'T. a lume X (nA) 10 2 10 Example 4 closantel 37. 5 mg/mL, FS489 1 closantel 7. 5 mg/kg, abamectin 1. 0 mg/mL, abamectin 0. 2 mg/kg, albendazole albendazole 5. 0 mg/kg, levamisole hydrochloride 40 levamisole hydrochloride 8 mg/mL 3 0. 8 mg/mL ivermectin 0. 2 4 10 113 closantel 37. 5 mg/mL 13146 1 closantel 2. 5 mg/kg 5 10 Levamisole levamisole hydrochloride 32 8 mg/mLmg/kg 610 mL/4 kg 4. 75 mg/kg NTTalbe 26: Group arithmetic mean, maximum and miniumum strongyle faecal egg counts and standard deviations following allocation. Group Treatnent t ii l mu l azzt a-2 Icrzclard eviation : , , : E CcukT." W2 s/, an' v 1Untreated1404. 2 4 3Ivomec@1408. 9 1/3 1436. 0 7 5Levamisole 3 6 Valbazen 8 2680520737.

1° Table 27: Treaemtn details. Cxrocp'1''tx° 3 ,'aa.-I. co °Ioa, an b. srd (kg)'Vonme MC ... 1 2 4 FS489 24. 91 5. 0 94 3Ivomec@ 24. 61 5. 9 4 1. 6je714. 15 5 mL/4 kg 5. 7 94 6 : 1Means within the same column with the same superscript are not significantly different at p<0.05<BR> Faecal samples were collected during pre trial monitoring, on Day-2 for allocatino purposes, at treatment (Day 0) and at the conclusion<BR> of the trial (Day 13). Results from faecal egg counts, larval differentiation and calculated treatment efficacies are summarised in the<BR> accompanying tables.<BR> <P>Table 28: Pre trial monitoring results Date of Sampling S.., vCount Ra ram) 10 10 Umup'Table 29: Group Arithmetic and Geometric Mean strongyle faecal egg counts during the trial (excluding Nematodirus spp.) -z Group j gg aecal , , otxiit al (pre.-Tlia) Day et^ g"ntlF''ty ! ti 1 : 1 1360. 01 2-Example4 1432. 0' 1536. 0' 0. 03 3Ivomec@ 51983 1408. 0 4 13146 1436. 01 Sustain 5 6053 1400. 0'1784. @ 6Valbazen@ V03790/2 1400. 0 204. 012 0--= 1 2 4 FS489 1253. 7' 1365. 0' 0. 0 3Ivomec@ 1180. 6' 1065. 0' -, 4 13146 1390. ' Sustain 5 6053 1263. 0'1483. @ 6Valbazen@ 176. 1'2 s = 1,2,3Means within the same column with the same superscript are not significantly differnet at p<0.05 Table 30: Larval differentiation results from group bulk coprocultures. 121, 'cvt' ^-°. Da_ . . jt ra . 'lrsr, c-c. pp, z . , SPP. L 10-Sep-02-9 -0 1 2 4 100% 0% 0% 0% 0% 100 19-Sep-02 0% 0% 0% 100 4 100% 0% 0% 0% 0% 100 5Levamisole@ 0% 0% 0% 0% 100 6 Valbazen@ 00J00 f 1 Untreated 100% 0% 0% 0% 2 4 0% 0% 0% 0% 0% 100 01-Oct-02 13 3Ivomec@ 89% 3% 0% 8% 0% 100 4 0% 0% 2% 0% 100 5Levamisole@ 0% 0% 6 97% 3% 0% 0% 0% 100 ° k= aHaemonchus spp. , bTrichostrongylus spp., cCooperia spp., dOesophagostomum spp.

Table 31 : Overall treatment efficacies, against all strongyle species (apart from Nematodirus spp.) (3mup hNo. Dayl3 aou-P, Atiflimetic Means 2 9% 3 6% 4 6% 5 6 0% g Geo 2 3 2% 4 2% 5Levamisole@6053>99. 6 4% I Table 32: Treatment efficacies against Haemonchus Table 32: Treatment efficacies against Haemoncus contortus. Group, acy , . . , " 2 9% 3 I 4 0% 5 8% 6Valbazen@V03790/285. 185. zu =s 6 2 amp 3Ivomec@5193397. 4 5 6Valbazen@V03790/284.

Conclusion: Excellent efficacy (greater than 99.9% based on group arithmetic and geometric means and larval differentiation results) was attained by the Example 4 formulation against a gastrointestinal strongyle population consisting almost exclusively of Haemonchus contortus.

Efficaces attained by the comparison formulations against this strain of Haemonchus contortus ranged from 85. 5% for the albendazole formulation (Valbazen@) through 96. 1% for the ivermectin formulation (Ivomec) to 99.8% for the levamisole formulation (Levamisole@), based on group arithmetic mean faecal egg counts and larval differentiation. Efficaces attained against this strain based on geometric mean faecal egg counts and larval differentiation were 84.9%, 97. 5% and >99.9% for these formulations respectively. These results indicate that this strain is moderately resistant to white drenches (benzimidazoles) and fully susceptible to levamisoles, with a slight but non-significant reduction in efficacy for ivermectin. Ivermectin was 96. 1% (arithmetic) and 97.5% (geometric) efficacious, which establishes that this strain could not be defined as macrocyclic lactone resistant at present.

Treatment with a 1/3 dose of closantel resulted in a treatment efficacy of 83.0% based on arithmetic group mean faecal egg counts and a treatment efficacy of 86.8% based on geometric group mean faecal egg counts, confirming the presence of moderate closantel resistance by this Haemonchus strain. Insufficient numbers of other gastrointestinal strongyles (Nematodirus, Teolodorsagia and Trichostrongylus species) were present to draw any conclusions about efficacy of the test formulation against these strains.

Trial JUA1270r : A property faecal egg count reduction study evaluating the therapeutic efficacy of the Example 4 formulation against field strains of mixed nematode populations, including macrogyclic lactone resistant strains of Haemonchus contortus in sheep in sheep.

This study was conducted from the 5 of September 2002 to the 7th of November 2002, with the animal phase conducted between 1 lth and 25th of October 2002. Routine monitoring of a trial site known to harbour macrocyclic lactone resistant strains of Haemoizchus contorts was conducted to identify a suitably infected group of sheep.

Pre-trial monitoring confirmed that one group of sheep (approximately 200 Merino wether hoggets) was suitably infected with a high burden of Haeynoyzclaus contortus.

On Day 0 of the trial, ninety six (96) sheep were randomly selected from a larger mob as they appeared in the sheep handling facility, weighed (see figure 10 for arithmetic mean body weights and note that the treatment"Jurox"refers to treatment with example 4) and individual faecal samples collected for subsequent individual strongyle faecal egg counts and group bulk coprocultures. Sheep had been previously allocated to six (6) treatment groups, one (1) of eleven (11) sheep to act as untreated (negative) controls and five (5) groups of seventeen (17) sheep, to be treated with the test formulation and a range of registered reference formulations. Trial sheep in Group 1 were retained untreated, while sheep in Groups 2-6 were treated according to individual body weight with the test and reference formulations. Sheep were observed in the immediate post-treatment period for adverse reactions (none were observed). Trial sheep were then returned to their parent flock and maintained in open grazing paddocks.

On Day 13 of the trial sheep were returned to the sheep handling facilities. Individual faecal samples were collected from trial sheep and individual strongyle faecal egg counts and group bulk coprocultures for larval differentiation were subsequently performed on the samples collected.

Treatment efficacies were then calculated using group arithmetic and geometric strongyle faecal egg counts for the major strongyle species present (see figures 11 and 12 and note that the treatment"Jurox"refers to treatment with Example 4).

Table 33: Treaemtn table. oup rer . s vc cve . rsttt. ent l. lme Rate X,'S sleep Untreated------ 2 17 Example 4 closantel 37. 5 mg/mL, RD0006 1 closantel 7. 5 mg/kg, abamectin 1. 0 mg/mL, abamectin 0. 2 mg/kg, albendazole albendazole 5. 0 mg/kg, levamisole hydrochloride levamisole hydrochloride 8 40 3 mL/4 kg ivermectin 0. 2 mg/kg 4 17 Sustain 13146 1 2. 5 mg/kg 5 17 Levamisole levamisole hydrochloride 789su2 mL/4 kg levamisole hydrochloride 8 Gold@ 617 19 mg/mL V03790/2 1 mL/4 kg I ; Table 34: Group arithmetic mean, maximum and miniumum strongyle faecal egg counts and standard deviations following allocation. t. arz f. aCLjJ , r > ! i f ! = 1. , f (. n. t"d'a ? Cidt, , t , s'1. '-c'. .. : ; . I. 2n'tI.. l1 r sd4lcrd 1. J..'VIl. lti erow f ... .. , gs/gt'am)'/'. . 1 2 2 4 690. 01 640. 6 3Ivomec@1068. 4280. 0 7 4 9 5 Levamisole 708. 2 0 0. 0 744. 8 Goldg 6 n 668. 2' 5 I » tTable 35: Treatment details. Crroup v. v, ."94i. t ay cs'"azn . . c . au . ziaiste 0 ' ) /,'" '''-.. Dose 1 Untreated 2 4 RD0006 28. 81 1 5. 76 97 3 7. 13 96 4 13146 29. 2 1 5. 84 4. 95 5 Levamisole 7895V2 28. 7. 19 7. 3 3. 96 Gold@ 6 38 7. 96 T1Means within the same column with the same superscript are not significantly differnet at p<0.05<BR> Table 36: Pre trial monitoring results Date egg ! 10 10 September 2002 40 40-760 of SaTable 37: Group Arithmetic and Geometric Means strongyle faecal egg counts during the trial. -bsH -roup , . .- '. l 'c. cnt. 1ah'. 1 at l A 1 2 0006 3Ivomec@ 4 5 Levamisole 7895V2 708. 2i Gold 6 lba_zont !--- : mens' Untreateå 1Untreated 376. 1 673. 0" 2 4 RD0006 Ivomec 4Sustain@ 5 330. 8$-141. Golds 6 w1,2,3Means within the same column with the same superscript arenot significantly different at p<0.05 Table 38: Larval differentiation results from group bulk corprocultures. d !'it's137 t't' I z... _. c. zb ' 1,. , la. la.''bt1 . v oun. SPP-SI) 4-Oct-02-7 7 1 100% 0% 0% 0% 0% 100 2 4 100% 0% 0% 0% 0% 100 11-Oct-02 0 3Ivomec@ 100% 0% 0% 0% 0% 100 4 0% 0% 0% 0% 100 5 Levamisole 100% 0% 0% 0% 0% 100 Gold@ 6 1 100% 0 2Example4 100% 0 25-Oct-02 14 3 100% 0 4 5 Levamisole 100% 0 0 0 0 100 Gold@ 6 100% 10 i aHaemonchus spp. , bTrichostrongylus spp., cCooperia supp., dOesophagosomum spp.

Table 39: Treatment efficacies against Haemonchus contortus. Omup -ati'.."''- _ A 2 8% 3Ivomec@51983Negative 4, 9% SLevamisole 0% 6 2% Geometric Mean.-, 2 7% 3 4Sustain@1314665. 3% S 9% 6 Valbazen

Conclusion: Efficacies attained by the formulations against this strain of Haemonchus contorts for the ivermectin formulation (Ivomec@), the albendazole formulation (Valbazen), the closantel formulation (full dose Sustain), the levamisole formulation (Levamisole Gold) and for the test formulation, based on group geometric mean faecal egg counts and larval differentiation were negative, 3.0%, 65.3%, 78.9% and >95% respectively.

While moderate efficacies were attained by the reference closantel formulation, a full (label) dose rate of this formulation was used in this case. In instances of moderate closantel resistance efficacies are still usually >95% for a full dose, with a reduction in initial efficacy only evident at a I/3 normal dose rate. This particular strain is therefore severely resistant to closantel. These results indicate that this strain also has severe resistance to white drenches (benzimidazoles) and macrocyclic lactones and moderate resistance to levamisole. This strain of Haemonchus contortus is, therefore, moderately to severely resistant to all four drug families. Unexpectedly, the Example 4 formulation containing an example of all four of these families resulted in effective treatment of the infestation.

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 spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.