AU2003231685A1 - Biological pest control methods and compositions - Google Patents

Description

Regulation 3.2

AUSTRALIA

PATENTS ACT, 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name of Applicant: Actual Inventor: Address for service in Australia: Invention Title: THE HORTICULTURE AND FOOD RESEARCH INSTITUTE OF NEW ZEALAND LIMITED CHRISTIAN JOHN COOK A J PARK, Level 11, 60 Marcus Clarke Street, Canberra ACT 2601, Australia BIOLOGICAL PEST CONTROL METHODS AND

COMPOSITIONS

The following statement is a full description of this invention. including the best method of performing it known to us.

-1- (followed by page 1 a) 76684_LDOC BIOLOGICAL PEST CONTROL METHODS AND COMPOSITIONS TECHNICAL FIELD The present invention relates to methods to increase the efficacy and acceptability of control methods. Also provided are baits and adjuvants useful in these methods.

BACKGROUND

Pest animal species are responsible for significant enivironmental damage, as well as having deleterious effects on human activities such as forestry, horticulture, and agriculture. Animal species cover a plethora of both vertebrate and invertebrate species.

Damage is caused by the pests in many different ways including: competing for food with farmed animals; damaging indigenous flora, especially through overgrazing; destroying indigenous animal species through predation; damaging agricultural, horticultural and silvicultural crops through grazing; and acting as vectors for infectious agents.

Effective control of pests is an expensive and ongoing problem for which new and more effective solutions are constantly being sought.

Baiting programmues are commonly employed to control a wide variety of biological pests.

Typically, the bait will comprise a substance which an animal finds palatable or attractive and a toxic substance or an active substance otherwise having an effect on the animal, for example, a contraceptive agent.

A common problem associated with bait programmes is the development in an animal population of bait aversion. Aversion develops when the animals consume a sub-lethal dose of the toxin, or other substance, through numerous mechanisms and then associates subsequent ill effects with the bait and/or toxin or active substance. As a result, the animal avoids the consumption of that bait. Techniques for addressing bait aversion are known in the art and discussed in the present applicants New Zealand Patent No. 260302.

NZ 260302 addresses bait aversion in animals by disrupting the associative learning, process using glutamate antagonists or agonists to block or disrupt the glutamate neurotransmitter pathway. The purpose of disrupting the learning process is to ensure that animals consuming sublethal doses of poisons on first contact with a bait will return for.

further feeding.

It is not the intention of the present invention to address the problem of bait aversion.

la- Rather, the problems which the present invention seeks to address are the need for more effective and acceptable methods of controlling, and in particular poisoning, target animal species and for improved baits for use in such methods.

One way of increasing the efficacy of a baiting programme is to control the consumption of bait by targeted animals so that an effective dose of active substance, or lethalfdose of toxin, is more likely to be delivered on first contact. Focussing on consumption of baits, animals which sample unknown foodstuffs may not ingest sufficient toxins (or other active substance) at a first sampling for it to be effective, even with known foodstuffs this may be the case. Incorporating high levels of active substances, including toxins, into the bait so that the first sampling is more effective is one option for addressing this. However, this can be expensive and dangerous. Moreover, if the first sampling is not effective bait aversion may develop such that a particular bait will no longer be effective against the targeted animal. Instead, it will avoid the bait which it associates with illness or discomfort or other adverse effects.

A further problem with control of some animals is the low density of animals per area of control region. This can make options for control such as bait stations or hunting difficult and economically unviable.

Animal welfare concerns are also of considerable public importance. There is an expectation that methods used in the biological pest control will not only be quick but also "humane". That is, any control techniques employed should ensure that the suffering of the animal is minimal and of short duration. Baits currently employed in poisoning programmn~es have resulted in many deleterious effects on the poisoned animal such as hyperresponsiveness to light and sound, ataxia, convulsions, and considerable gastric discomfort. The length of kill time can be as long as days to weeks depending on the level of toxin consumed.

It is an object of the present invention to provide pest control methods and compositions which go at least some way towards addressing the foregoing problems or which at least provides the public with a use ful choice.

SUMMARY OF THE INVENTION Accordingly, in one aspect, the present invention provides a method of attracting a targeted animal species to a bait, the method comprising the use of an attractant substance, in or near the bait, which attractant substance includes at least one of the following: one or more of dodecyl acetate or its chemical analogues; -3- (ii) one or more of oxytocin or its chemical analogues; (iii) one or more of gamma-amino-4-butvric acid (GABA) or its analogues; and (iv) one or more mimetics of(i)-(iii) above.

According to a further aspect of the invention, there is provided a bait which includes at least one of(i) to (iv) above.

In a further aspect, the present invention provides a bait adjuvant which includes at least one of(i) to (iv) above.

In a further aspect, the present invention provides a method of attracting an animal to a bait, the method comprising the inclusion in the bait of at least one of(i) to (iv) above.

In a further aspect, the present invention relates to the use of at least one of to (iv) above to attract targeted animals to a bait or bait site.

Preferably, the mimetic substances are selected from the group including barbituates, alcohol based substances, and benzodiazepines, as well as similar mimetics that may influence GABA or related transmission.

In another aspect, the present invention provides a method of controlling uptake of a bait by an animal, the method comprising the use of a bait adjuvant, in or near the bait, which comprises at least one of the following: one or more of neuropeptide Y or its analogues; one or more neuropeptide Y antagonists; one or more of leptin or its analogues; one or more leptin antagonists; one or more of cholecystokinin or its analogues; one or more cholecystokinin antagonists; one or more of serotonin or its analogues; one or more serotonin antagonists or re-uptake inhibiting factors; one or more serotonin releasing factors; and one or more mimetics above.

In a further aspect, there is provided a bait which includes at least one of to j) above, with the proviso that the bait does not comprise or above alone, or combinations thereof alone.

-4- In a further aspect, there is provided a bait adjuvant which includes at least one of to above.

In a further aspect, there is provided a method for controlling uptake of a bait by an animal, the method comprising providing in or near the bait at least one of to above.

In a still further aspect there is provided a method of controlling uptake of a bait by an animal, the method comprising administering to said animal at least one of(a) to above.

In a further aspect, there is provided a bait adjuvant for altering the stress response in an animal which bait includes at least one or more stress response altering substances.

According to a still further aspect of the invention, there is provided a method of altering the stress response in an animal, said method comprising the use of one or more stress response altering substances in or near the animal's bait.

In a further aspect, there is provided a method of altering the stress response in an animal, said method comprising the inclusion in the animal's bait of one or more corticosterones, or analogues or antagonists thereof.

In a further aspect of the invention, there is provided a method of altering the stress response in an animal, said method comprising administering to said animal one or more stress response altering substances.

Preferably, the stress response altering substance comprises one or more corticosterones, or analogues or antagonists thereof A further method of the invention provided is a method for re-attracting bait shy animals to bait, the method comprising employing a combination of two or more methods, baits or adjuvants of the invention referenced above.

Accordingly, in a further aspect, the present invention provides a complex bait or bait adjuvant comprising at least two compounds selected from the following groups A. B and

C:

A. one or more of dodecyl acetate or its chemical analogues; (ii) one or more of oxytocin or its chemical analogues; (iii) one or more of gamma-amino-4-butyric acid (GABA) or its analogues; and (iv) one or more mimetics of above; B. (a) (b) (c) (d) (e) (0 (g) (h) (i) Cj) one or more of neuropeptide Y or its analogues; one or more neuropeptide Y antagonists; one or more of leptin or its analogues; one or more leptin antagonists; one or more of cholecystokinin or its analogues; one or more cholecystokinin antagonists; one or more of serotonin or its analogues; one or more serotonin antagonists or re-uptake inhibiting factors; one or more serotonin releasing factors; and one or more mimetics of above.

C. one or more corticosterones, or analogues or antagonists thereof provided that when only two compounds are selected they are selected from different groups.

In a further aspect, the present invention provides a method of re-attracting bait shy animals to bait, the method comprising the use of a complex bait or adjuvant above.

In a further aspect, the present invention provides a bait or bait adjuvant comprising: A. (i) (ii) (iii) (iv) B. (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) one or more of dodecyl acetate or its chemical analogues; one or more of oxytocin or its chemical analogues; one or more of gamma-amino-4-butyric acid (GABA) or its analogues; one or more mimetics of above; one or more ofneuropeptide Y or its analogues; one or more neuropeptide Y antagonists; one or more of leptin or its analogues; one or more leptin antagonists; one or more of cholecystokinin or its analogues; one or more cholecystokinin antagonists; one or more of serotonin or its analogues; one or more serotonin antagonists or re-uptake inhibiting factors; one or more serotonin releasing factors; and one or more mimetics above.

-6- C. one or more corticosterones, or analogues or antagonists thereof provided that when only two compounds are selected they are selected from different groups.

Preferably, the bait or adjuvant includes at least three compounds, with at least one compound being selected from each of groups A, B and C above.

The baits and bait adjuvants of the invention preferably also include a lipid membrane transfer facilitator.

Accordingly, the present invention also provides a method for increasing the speed of delivery of an active substance to one or more organs, of a targeted animal, the method comprising administering to said animal an active substance together with a lipid membrane transfer facilitator.

In a further aspect, the present invention is directed to the use of lipid membrane transfer facilitators to increase the speed of delivery of an active substance to one or more organs of a targeted animal. A preferred lipid membrane transfer facilitator is a pyrrolopyrimidine. The lipid membrane transfer facilitators may also be employed in known feedstuffs and baits.

Also provided is a method of the invention as above, further comprising the use of a mechanism for facilitating the uptake of an active substance into a target area in an animal.

Preferably, the target area is a vulnerable organ or the central nervous system. The brain is particularly suited for targeting. Brain target areas include the nucleus solitarius (NTS) and striatum.

Also provided is a method of the invention as defined above further comprising the use of a mechanism for increasing the speed of uptake of an active substance. The mechanism may be mechanical or chemical. The mechanism is preferably designed to stimulate the face or oral cavity of an animal.

According to a further aspect of the invention, there is provided a method of baiting an animal, the method comprising administering to said animal an active substance formulated for administration to a targeted area. Preferably, the target area is as defined -7above.

Also provided therefore is an active substance formulation comprising an active substance and at least one of the following: one or more of dodecyl acetate or its chemical analosues; (ii) one or more of oxytocin or its chemical analogues; (iii) one or more of gamma-amino-4-butyric acid (GABA) or its analogues; (iv) one or more mimetics of above; and optionally one or more lipid membrane transfer facilitators.

Preferably all of or mimetics thereof are present in the formulation.

In a preferred embodiment, the active substance is a toxin.

In a further aspect. the present invention provides a toxin formulation comprising to (iv) and optionally above.

Accordingly, in an even further aspect of the present invention, there is provided a method of poisoning an animal, the method comprising administering to said animal a toxin formulated to target the striatum and/or NTS brain areas of the animal.

According to an even further aspect of the present invention, there is provided a method of effectuating a rapid death in an animal, said method comprising administering to said animal a toxin formulated to target the stratum and/or NTS brain areas of the animal.

In a further aspect, the present invention provides a method lessening the symptoms of poisoning or severity of the symptoms, in an animal, the method comprising administering in addition to a toxin, an adjuvant or bait of the invention.

In a still further aspect, the present invention provides a method of lessening the symptoms of poisoning or severity of the symptoms, in an animal, the method comprising the use of an adjuvant or bait of the invention near a bait or toxin but separate therefrom.

Preferred compositions for lessening the symptoms or severity of the symptoms exhibited by a poisoned animal include at least one or more of the following: one or more anti-emetics; and/or one or more anxiolvtics, In another aspect, the invention further comprises a method for preventing or treating poisoning in an animal, the method comprising administering one or more antidotes which can prevent damages to other areas of the brain by their actions on pathways in the brain activated by toxins.

A preferred anti-emetic is a serotonergic antagonist and a preferred anxiolytic is a cholecystokinin antagonist.

The methods of the invention may be further enhanced by the use of a pre-baiting step which comprises administering to the targeted animal, baits free of an active substance to be administered later, but otherwise having the same attributes as the bait.

Accordingly, in a further aspect there is provided a method of the invention which includes as a pre-step administering to a targeted animal species an active substance-free bait of the invention.

The methods of the invention may also alternatively be enhanced or supplemented by the use of two or more, but preferably two, adjuvants or baits at the same time and at the same site.

In one embodiment, both a bait and an active substance-free bait of the invention are present. Preferably, the bait is a bait of the invention.

Accordingly, in a further aspect there is provided a method of the invention which further comprises the simultaneous use of a bait and an active substance-free bait at a bait site.

Desirably, the bait is a bait of the invention.

The methods of the present invention may also be used in combination with existing bait aversion reversing techniques to re-attract and control bait shy animals.

Preferred aspects of the present invention will now be further described in relation to the accompanying drawings in which: Figure 1: Demonstrates the attractant ability, in terms of visits to a source, of oxytocin and/or GABA and their effect in re-attracting bait shy animals to a bait-shy material. Data present as meant +S.E.M from repeat measure Anova. a: p 0.01 (control); b: p <0.01 (control)I c: p 0.01 (bait shy); d: p 0.05 (bs oxytonin) and GABA alone).

-9- Figure 2: Illustrates attractant ability of dodecyl acetate (DA) on field rats with or without oxytocin and GABA in the field at two sites (S 1 and S2) before and after the development of bait shyness to the bait material.

Figure 3: Demonstrates the ability of dodecyl acetate (da) in combination with oxytocin to attract to a particular site in a two site choice procedure (site 1, site Readings represent day l(dl), day 2 (d2) and overall mean (4 days).

Figure 4: Exhibits the effect of different bait additives on increasing consumption of a bait (expressed as a of the normal feed level). NPY neuropeptide, Y 5 HTA serotonin antagonist, CCKA cholecystokinin antagonist, GLUTA glutamate antagonist, CONT control substance.

Figure 5: Represents a similar experiment to that expressed in Figure 4 but for possums as a species. In addition the effects of modulating stress are shown through the use of a cortisol antagonist.

Figure 6: Demonstrates the effect of manipulating stress levels via a cortisol antagonist RU 38486 on consumption of bait and bait-poisons and on fatalities to bait-poisons in possums LD50 a lethal dose 50% level.

Figure 7: Illustrates the effects of feed additives in curtailing consumption of a feed.

CCK cholecystokinin.

Figure 8: Demonstrates the enhancement of toxic effect with the addition of a pyrrolopyrimidine Cumulative animal death was increased within 3 time intervals 6, 12, 24 hours after ingestion.

Figures 9 Are flow diagrams illustrating a single and two step procedure for greater and 10: field kill effectiveness.

Figure 11: Is a bar graph illustrating the effectiveness of glutamate/NOS antagonists in preventing 1080 toxin damage.

Figure 12: Is a bar graph illustrating the effectiveness of GABA antagonists in preventing 1080 toxin damage.

Figure 13: Is a bar graph illustrating the effectiveness of salicylate in preventing 1080 toxin damage.

Figure 14: Is a bar graph illustrating the effectiveness of glutamate/NOS antagonists and GABA agonists in preventing 1080 toxin damage.

Figure 15: Changes in concentration (pmol/1) GABA and glutamate in the somatosensory cortex following probe insertion h) and saline administrations (0 Similar changes where seen for the cerebellum and striatum. Data present as mean S.E.M.

Figure 16: Changes in glutamate (pmol/1) in different brain areas following oral administration of 1080 and saline (0 Data are mean S.E.M.

10 Figure 17: Figure 18: Figure 19: Figure 20: Figure 2 1: Figure 22: Figure 23: Figure 24: Figure 25: Changes in GABA (gmol~l) in different brain areas following oral administration of 1080 and saline (0 Data are mean S.E.M.

Is modified Latin Square testing of the independent variables: the trial substances (one substance per feeder, two per trial). Shadowing indicates substance versus substance, Is a bar graph illustrating the effects of attractants; and repellants on feed consumed. The feed consumed per day was calculated from total consumption in trial for that substance divided by days that substance was presented. Data presented as mean and S.E.M.

Is a bar graph illustrating the effects of attractants and repellants on- eating occurrences of substances. Total occurrences for each substance in trial for that substance divided by days that substance was presented are given. Data presented as mean and S.E.M.

Is a bar graph illustrating the effects of attractants and repellants on animals numbers per day. Total numbers of animals in a 10 metre radius per day are shown. Individuals were not identified so may be represented more than once. Data presented as mean and S.E.M.

Is a diagram depicting the layout of deer study site.

Is a bar graph showing hay consumed from feeders associated with a particular attractant or repellant compound. Errors are S.E.Ms.

Is a bar graph showing number of occasions a deer appeared to feed from a feeder associated with a particular compound. Errors are S.iE.Ms.

Is a bar graph showing the number of occasions when a deer approached a feeder associated with a particular compound. Errors are S.E.Ms.

Is a bar graph showing ten minute focal scan results: Number of individuals standing within 10 m of a feeder associated with a particular compound.

DESCRIPTION OF THE INVENTION invention relates generally to methods for increasing the efficacy and of baits for use in biological pest control. Target biological pests include Figure 26:

DETAILED

The present acceptability marsupials, birds, mammals, insects, arthropods, amphibians. and reptiles. The term animal" is used generically to encompass all such pests. Of the mammal groups particular targets include rabbits, hares, possums, mice, and rats. A particular target 3 5 marsupial is possums.

The success of any animal baiting campaign depends for its effectiveness upon targeted animals consuming an. effective dose of an active substance, or a lethal dose of toxin in 11particular. To achieve this result it is necessary to both attract targeted animals to a bait and to ensure that they' consume an effective or lethal dose of active substance or toxin as appropriate. The use of attractant substances to draw targeted animals to a bait is known in the art. For example, the use of compounds such as colour lures, pheromones, sweeteners, and aromatic agents has been proposed.

The present applicant has identified a particularly effective group of attractant substances comprising the following: one or more of dodecyl acetate or its chemical analogues; (ii) one or more of oxytocin or its chemical analogues; and (iii) one or more of gamma-amino-4-butyric acid (GABA) or its analogues; and (iv) one or more mimetics of(i)-(iii) above.

Dodecyl acetate has proved particularly useful. It appears to act as a long distance attractant and has been shown to act as such for elephants Combinations of two or more attractant substances are specifically contemplated. A preferred combination of attractant substances comprises GABA and oxytocin, and optionally dodecyl acetate.

Butyric acid groups have been purported to have attractant properties Oxytocin appears to have a social facilitating effect causing animals to group together. GABA appears to have a similar effect.

The present applicants have demonstrated that oxytocin and dodecyl acetate are potent attractants in rabbits, rats and possumes in up to a 2 km attractant range incorporated herein by reference).

Based on the identification of these attractants, the applicants have devised a method for attracting a targeted animal species to bait, the method comprising the use of an attractant substance, in or near the bait, which attractant substance includes at least one of the attractant substances set out above.

The use of attractants help to address the problem of low density of animal per area of control region. Encouraging an increased number of animals to a set focus point markedly assists in control programmes.

Further provided are baits which include at least one of the attractant substances set out above.

-12- In a further aspect, the present invention provides a bait adjuvant which includes at least one of the attractant substances set out above.

Also provided is a method of attracting a targeted animal species to bait, the method comprising the inclusion in the bait of at least one of the attractant substances from the group set out above.

The term "mimetic" substance is used generally herein to refer to those substances which have the same or substantially the same activity and effect as those substances identified as above. These mimetic substances may comprise substances which stimulate the same pathways within the animal's physiology (particularly neural pathways), ot within the social communication network of the animal species, as Preferably. the mimetic substances are selected from the group including barbituates, alcohol based substances and benzodiazcpines (such as benzodiazepan), as well as similar mimetics that may influence GABA or related transmission.

The term "adjuvant" is used herein to refer to a compound per se or a composition formulated for use in or near bait. The adjuvant may increase the effectiveness, assist in attracting animals to, or assist in increasing the uptake or consumption of', the bait.

For example, in the bait designed as attractants, adjuvants may include substances which alter the lifetime of the attractants, their volatility, distance over which the attractant is effective, and availability to the senses of smell or taste of a targeted animal species. Slow release formulations are also contemplated. This may be achieved using chemical agents or mechanical devices such as are known in the art. For example, a slow releasing dialysate type mechanism. Adjuvants can be similarly used with respect to the uptake and consumption control compositions.

The term "analogue" used herein includes acceptable salts, derivatives, and isomers of the chemical compound in question as may be known in the art.

The term "bait" refers broadly to any material which is suitable for use in making an active substance available in the environment of a targeted pest species, or for administration to a targeted pest species. Preferably, the bait is an edible material or feedstuff.

In terms of the delivery of the bait to an animal, any suitable delivery method known in 13 the art may be employed. This may comprise self-administration or administration by mechanical devices or humans. Some methods of administration contemplated include oral, topical, nasal and parenteral methods of administration, but are not limited thereto.

It is presently considered that oral ingestion by an animal is the most feasible and effective maimer of administering bait and adjuvants of the invention to a targeted animal species.

Many suitable consumable baits are known in the art. Conventional baits include for example, vegetable, meats and commercial pastes and feedstuffs such as hay, meal and silage, but are not limited thereto. Preferably, the bait selected will be a bait particularly attractive for consumption by the targeted animal.

However, other methods of administration are also possible. For example, in terms of the use of mechanical devices, this may comprise injection via a mechanism in which a needle or sharp object at a bait station can prick the animal and introduce an effective dose of the active substance or toxin. Topical administration may comprise selIf-app li cation via scratching or rubbing of the animal against a mechanical surface to which the compound is applied. Similarly, sprays or vapours which are topically administered or inhaled are further possibilities. The optimal method of administration can be selected depending on the toxin to be administered and the bait to which it is to be incorporated.

The levels of the active substances and toxins present in the adjuvants or baits will vary according to the bait composition or feedstuff to be consumed by the targeted animal species. An active substance or toxin may comprise anywhere from 0.000 1 to 99.99% of a composition. Preferred ranges are from 0.1 to 50% and more preferably 1 to Where small amounts are being consumed or otherwise administered, then a higher concentration of active substance will be required. Where smaller baits are to be consumed, an increase in the concentration of the active substance is feasible.

In determining an appropriate amount or dose of active substance, a number of factors are considered including but not limited to size, age, and health of individuals and populations to be targeted, responses, compound to be administered, mode of administration, bioavailability characteristics of preparation, use of other agzents and other relevant circumstances. An effective amount of a compound is expected to vary from about 0.0 1 milligrams per kilogram of body weight to about 1000 mg/kg. Preferred amounts are expected to vary from 0.5 to 100 mg/kg, and preferably I mg to 50 mg/kg. Appropriate amounts can be readily calculated by a skilled worker.

Any bait compositions known in the art may be employed, for example, baits with a -14graduated concentration of active ingredient there through rather than uniformly dispersed therein. Baits or feedstuffs with active agents sprayed there over is one method of achieving this result.

Active substances which may be targeted to a pest species include toxins, contraceptives, vaccines, repellants and anti-infective agents, but are not limited thereto. Preferably, the active substance is a toxin.

Toxins suitable for use herein include sodium fluoroacetate (1080), warfarin, arsenic, cyanide, strychnine, brodificoum, cholecalciferol and similar new generation anticoagulants and coagulants, but are not limited thereto. Any suitable toxins known in the art may be employed.

It will also be appreciated that in addition to the compositions proposed above, various other substances may be included in the bait compositions and adjuvants. including other attractants, flavour enhancers, substances increasing palatability of the bait, masking agents which can cover any unpleasant taste or smells associated with the bait compositions, repellants directed against non-targeted animal species, preservatives, lubricants, antioxidants, buffers and the like. The use of acceptable carriers and diluents in the baits and adjuvants is also contemplated.

A presently preferred attractant is decanolactone or its analogues, or functional equivalent.

The applicants have found that while decanolactone has variable attractant properties on its own, it surprisingly potentiates the effects of other attractants particularly DA and/or oxytocin-GAGA above what would be expected for the attractants alone. Accordingly, in one aspect decanolactone is also used in the baits, adjuvants and methods of the invention.

The baits may also be formulated to include compositions which have a calming effect on the animal. Some appropriate dietary compositions are contemplated in NZ 329084 filed 29 October 1997, and incorporated herein by reference and annexed.

In formulating the bait compositions, it should also be borne in mind that there are substances which can inhibit the effectiveness of the active ingredients of the compositions of the present invention. This may be by reacting with or competing with the active in2redients, thereby reducing their effectiveness. Generally, substances compatible with bait compositions can be determined by review of manufacture data sheets from specific active ingredients to determine which substances are to be avoided in bait compositions and adjuvants of the invention.

The term "bait site" is used herein to refer broadly to anywhere bait is located for access by a targeted animal species. This may include man-made bait stations.

A significant factor in the efficacy of baiting campaigns is the level of uptake of an active substance by a targeted pest. For convenience, the following discussion focuses on consumption of toxins but is not limited thereto.

It is necessary to ensure that a full, and in the case of a toxin, lethal dose is delivered to a targeted animal on first contact. This prevents the development of bait aversion, and reduces costs associated with repeat baiting campaigns.

The present applicants have now found that the following groups of compounds: neuropeptide Y or its analogues; neuropeptide Y antagonists; leptin or its analogues; leptin antagonists; cholecystokinin or its analogues; cholecystokinin antagonists; serotonin or its analogues; serotonin antagonists or re-uptake inhibiting factors; or one or more serotonin releasing factors may be used in controlling uptake of a bait by an animal.

The term "controlling uptake" is used herein to refer to either increasing or decreasing uptake of the bait by the animal to ensure that an effective dose of active substance is delivered. Uptake generally comprises consumption but as discussed above is not limited thereto. The substances identified above may be used to increase or decrease consumption as required. Increasing consumption may be required to ensure that a full dose of active substance is consumed. Decreasing consumption may be required where animals consume more active substance than is needed. Decreasing consumption in this case reduces wastage and in long life field delivery setups may extend the unattended field life.

Accordingly, in a further aspect, the present invention provides a method of controlling uptake of a bait by an animal, the method comprising the use of a bait adjuvant in or near the bait, which adjuvant comprises at least one of the following: one or more of neuropeptide Y or its analogues; -16one or more neuropeptide Y antagonists; one or more of leptin or its analogues; one or more leptin antagonists; one or more of cholecystokinin or its analogues; one or more cholecystokinin antagonists; one or more of serotonin or its analogues; one or more serotonin antagonists or re-uptake inhibiting factors; one or more serotonin releasing factors; and one or more mimetics above.

Further provided is a bait which includes at least one of the group of compounds set out above. Similarly, provided is a bait adjuvant which includes at least one of(a)-(j) set out above.

In the case of baits per se generally, the bait will not comprise alone or combinations thereof alone.

In a still further aspect, there is provided a method of controlling uptake of a bait by a targeted animal, the method comprising the inclusion in the bait of at least one of above.

The compounds identified above have been found to be particularly effective in increasing consumption of bait. The bait adjuvant need not be included in the bait but plays an effective role if placed in proximity to the bait, for example, at the same bait site.

The degree of proximity of the bait to the bait adjuvant to be effective when not incorporated in the bait will depend on the animal being targeted, the bait being used, and the type and concentration of active substances therein and in the adjuvant. Optimum levels of proximity can be readily determined by skilled workers in this field.

Compounds are known in the art. Many preferred compounds are discussed in NZ 260302 which is incorporated herein by reference.

Of the neuropeptide Y compounds, the preferred peptide is [Leu", Pro 3 4 Neuropeptide Y. Preferred neuropeptide Y antagonists include [D-Thr 3 2 neuropeptide Y, Fragments 18-36 neuropeptide Y, and Methyl-Tyr neuropeptide Y, but are not limited thereto.

Cholecystokinin is a recognised gastrin releasing peptide. A preferred cholecystokinin is -17- CCK-8. Cholecystokinin antagonists preferred for use in the invention include proglumide, proglumide sodium salt, N-methyl-D-glucamine salt, PD142,898, PD 135,138, benzotript, RB21 1, C1988, L365260 Lorglumide sodium salt.

Serotonin is a well known neurotransmitter. Preferred antagonists include ketanserin, cinanserin, MDL-72222, LY-278, 584, maleate, metergoline and methysergide maleate.

Examples of re-uptake inhibiting factors include fluoxetine, fluvoxamine and paroxetine.

Serotonin releasing factors include fenfluramines and amphetamines such as 4methylthioamphetamine.

Leptin is the protein encoded by the obese (ob) gene and acts on the central nervous system. Any known leptin peptides Tyr-leptin) and antagonists may be used herein.

It is recognised that many of the above substance have been proposed for use in feral pest control via their contribution to the process of aversion formation and reversal. The above methods, baits and adjuvants of the present invention are not intended to address the issue of preventing or reversing aversion, but rather their use in the control of uptake of active substances in biological pests. There is no evidence or suggestion in the prior art that these compounds can be used to control the uptake of baits, and in particular, the consumption of feedstuffs.

The levels of compounds to for use in the invention may be varied in a similar manner as for the active substance according to size, age, health, etc. of the animal population. An effective amount of a compound is expected to vary from about 0.01 milligrams per kilogram of body weight to about 1000 mg/kg. Preferred amounts are expected to vary from 0.5 to 100 mg/kg, and preferably 1 mg to 50 mg/kg.

A further important element in the effectiveness of an animal baiting campaign is being able to alter the stress response in an animal.

The applicants have determined that one way of altering the stress response of an animal is to provide a bait adjuvant in a palatable form in proximity to a bait. The use of a stress modulating adjuvant in proximity to the bait can be used to increase uptake of a bait, thereby avoiding the subsequent development of aversion. Such a mechanism for preventing the development of aversion has not been previously suggested.

-18- Accordingly. in one aspect the present invention provides a bait adjuvant for altering the stress response in an animal which bait includes one or more corticosterones, or analogues, or antagonists thereof.

One corticosterone antagonist contemplated for use include RU38486 (mifepristone) and RU 28318. Levels of corticosterone antagonist used in the invention vary as for to (j) above.

Similarly, the present invention provides a method for altering the stress response in an animal, said method comprising the use of one or more stress response altering substances in or near the animal bait.

In a further aspect. there is provided a method of altering the stress response in an animal.

said method comprising the inclusion in the animal bait of one or more stress response altering substances.

The present invention also provides a method of altering the stress response in an animal, said method comprising administering to said animal one or more stress response altering substances.

Preferably, the stress response altering substances are selected from corticosterone compounds, analogues or antagonists identified above.

In a further aspect, the invention provides a method for re-attracting bait shy animals to bait, the method comprising employing a combination of two or more methods, baits or adjuvants of the invention as referenced above.

Similarly, the stress response altering bait adjuvant identified above can be used in reattracting bait shy animals to bait.

Accordingly, in a further aspect, the present invention provides a complex bait or adjuvant comprising at least two compounds selected from the following groups A, B and C: A. one or more of dodecyl acetate or its chemical analogues; (ii) one or more of oxytocin or its chemical analogues; (iii) one or more of gamma-amino-4-butyric acid (GABA) or its analogues; (iv) one or more mimetics of above: 19- B. (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) one or more of neuropeptide Y or its analogues; one or more neuropeptide Y antagonists; one or more of leptin or its analogues; one or more leptin antagonists; one or more of cholecystokinin or its analogues; one or more cholecystokinin antagonists; one or more of serotonin or its analogues; one or more serotonin antagonists or re-uptake inhibiting factors; one or more serotonin releasing factors; and one or more mimetics of above.

C. one or more corticosterones, or analogues or antagonists thereof; provided that when only two compounds are selected they are selected from different groups.

These later baits and adjuvants are suited for use in re-attracting bait shy animals to bait.

Accordingly, in a further aspect, the present invention provides a method of reattracting bait-shy animals to a bait, the method comprising administering at least two compounds of A, B and C as above.

Preferably, the baits or adjuvant includes at least three compounds, with at least one compound being selected from each of groups A, B and C.

Another important element in the effectiveness of animal treatment campaigns is the speed at which compositions act on the targeted animal species. In the case of baiting campaigns using toxins, animal welfare concerns dictate that the time between consumption of a toxin and death should be as short as possible and as painless as possible. At least, efforts should be made to minimise animal suffering.

In order to increase the efficiency of delivery of bait toxins, it is desirable to use baits wherein the transfer of toxins to targeted organs or the Central Nervous System (CNS) is facilitated. The applicants have found that this can be achieved using Iipid membrane transfer facilitators.

The term "lipid membrane transfer facilitator" unless otherwise defined, refers to a compound facilitating transfer of active substances across lipid membranes. Their general effect is to facilitate transfer of active substances into target organs or the CNS, and desirably at higher concentrations than would otherwise often be possible, and typically within a shorter time span. In this case, preferably the substance to be administered is a bait toxin and the main target organ is the brain.

Accordingly, in a further aspect the present invention provides baits and adjuvants, including those of the invention, which further comprise a lipid membrane transfer facilitator.

The use of lipid membrane transfer facilitators to speed the delivery of an active substance to target organs of an animal is also contemplated.

Accordingly, also provided is a method for increasing the speed of delivery of an active substance to one or more organs of a targeted animal, the method comprising administering to said animal an active substance together with a lipid membrane transfer facilitator.

It will be appreciated that these facilitators may also be included in known baits to enhance transfer of active substances to targeted organs.

The preferred lipid transfer membrane facilitators of the invention comprise lipid penetrating antioxidant compounds such as pyrrolopyrimidines and analogues thereof but are not limited thereto. A preferred pyrrolopyrimidine is U-101033E. Examples of pyrrolopyrimidines useful in the present invention are given in Hall Ed. et al., 1995 Acta NeuroChir, 66: 107-113, and Andreous P et al., 1997 J. Neuro Science Res., 47: 650-654.

Also included within the group are derivatives of pyrrolopyrimidines all exhibiting useful transfer properties, and including salts and isomers thereof. The lipid transfer membrane facilitators may be provided as adjuvants, that is, in the form of the compound per se or formulated into baits. In such cases, the compounds may be conjugated to components of these baits. Combinations of selected lipid transfer membrane facilitators, as well as combinations of conjugated forms, are also contemplated for use.

The efficacy of administration of an active substance to an animal can be increased by targeting the active substance to a vulnerable area in the animal. Vulnerable target areas include organs and the Central Nervous System (CNS). The brain is a particularly suitable target.

-21 The applicants have also ascertained that the nucleus tractus solitarius (NTS) and striatum are areas of the brain which show particular vulnerability to toxin damage contributing to the lethal effects of toxins. Uptake of toxins into the NTS in particular can be achieved through activating or stimulating the trigeminal system.

Other methods for increasing the speed of toxin uptake into vulnerable target organs, particularly the brain therefore also include the use of stimulant mechanisms, more particularly those aimed at stimulating the trigeminal system. These mechanisms may be mechanical or chemical in nature. Examples of ways in which the mechanisms may be used, is in the stimulation of the face or oral cavity of an animal. The stimulation may consist of, for example, irritation or cooling. These actions activate the trigeminal system facilitating uptake of substances in the NTS brain area.

Suitable mechanical stimulant devices include sprays, fans, brushes and the like. A spray associated device that delivers a cold spray onto the face of an animal is desirable.

Chemical irritants and coolants are also contemplated for use.

Also provided in relation to these findings is an active substance formulation comprising an active substance and at least one of the following: one or more of dodecyl acetate or its chemical analogues; (ii) one or more of oxytocin or its chemical analogues; (iii) one or more of gamma-amino-4-butyric acid (GABA) or its analogues; (iv) one or more mimetics of above; and optionally one or more lipid membrane transfer facilitators.

A combination of at least one of each of is preferred.

Also forming an aspect of the present invention is a bait including such an active substance formulation.

In a further aspect, the present invention provides a method of poisoning an animal, the method comprising administering to said animal a toxin formulated to target the stratum and/or NTS brain areas of a targeted animal species.

Also provided is a method of effectuating rapid death in an animal, the method comprising administering to said animal a toxin formulated to target the striatum and/or NTS brain areas of a targeted animal species.

-22- Also contemplated for use are one or more antidotes which can prevent damage by their actions on pathways in the brain activated by toxins. Their use in the event of accidental poisoning is particularly contemplated.

Accordingly, in a further aspect, the present invention provides a method of preventing or treating poisoning in an animal, the method comprising administering one or more antidotes which can prevent damage to other areas of the brain by their actions on pathways in the brain activated by toxins. In particular, the antidotes may be used to reduce specific tissue damage and the attendant side effects arising therefrom. Suitable antidotes are given below. Administration of two or more antidotes may be sequential or simultaneous.

Antidotes contemplated for use in the present invention include: GABA agonists; (II) glutamate antagonists; (III) nitric oxide and nitric oxide synthetase antagonists and inhibitors; (IV) salicylic acid and similar non-steroidal anti-inflammatory agents; opioid agonists; (VI) steroidal hormone mimics; and (VII) optionally one or more lipid membrane transfer facilitators.

Any antidotes of types to (VI) such as are known in the art may be used. Examples of such antagonists and anti-inflammatory agents are dextromethorphan, phaclofen, bicuculline, D-AP5, APH, APV, kynurenic acid, CNQX disodium, L-N 5

NG-

Monomethyl-L-arginine acetate, L-NOARG and the like. Examples of opioid agonists include DAMAGO (D-Ala 2 N-Me Phe 4 Gly-01]-enkephalin) and GR 89696 dichlorophenyl)acetyl]-3-(1-pyrrolidinyl methyl)-1-piperazine carboxyl acid methyl ester fumurate). Examples of steroidal hormone mimics include 3-estradiol and progesterone sulphate. Appropriate levels of antidote may be selected by a skilled worker.

Poisoning results in a raft of noxious sensations and perceptions in the poisoned animal.

Symptoms include hypersensitivity to light and/or sound, convulsions, ataxia (meaning herein a maintained tilting of the head or body or a tendency to fall over), increased water intake, drop in feed intake, balling (lying in a ball while awake), pacing, grooming or scratching of the abdomen. All of these symptoms may be reduced by the administration of the adjuvant or bait of the invention.

Accordingly, in a further aspect, the present invention provides a method of lessening the 23 symptoms of poisoning or the severity of the symptoms in an animal, the method comprising administering in addition to a toxin, an adjuvant or bait of the invention.

Preferably, the bait or adjuvant of the invention includes one or more anxiolytic and/or antiernetics. Any suitable anxiolytic and antiemnetic compounds known in the art may be used. For example, one or more serotonergic antagonists to reduce gastric discomfort, and/or one or more cholecystokinin antagonists to reduce anxiety associated with the toxin. Any of the seroronergic antagonists and cholecystoki nin antagonists set out above may be used.

The methods of the invention may be further enhanced by the use of a pre-baiting step which comprises administering to a targeted animal species an active substance-free baits otherwise having the same attributes as baits to be used in later baiting programmes.

Accordingly, in a further aspect. there is provided a method of the invention which method includes as a pre-step administering to a targeted animal species an active substance-free bait.

These methods of the invention are usefully illustrated in Figure 9 which provides a flow diagram of the methods with reference to the consumption of toxins.

The methods of the invention may alternatively be enhanced or supplemented by the use of two or more, but preferably two, adjuvants or baits at the same time, at the sane site.

In one embodiment- one bait is a bait of the invention, or as is otherwise known in the art, and the other active substance-free bait or adjuvant of the invention corresponding thereto.

Accordingly, in a fulrther aspect, the present invention provides a method of the invention which fthffer comprises the simultaneous use of a bait and an active substance-free form of that bait at a bait site. Preferably, the active substance in this method is a toxin.

These methods of the invention are also illustrated in Figure 10 which provides a flow diagram of the methods with reference to the consumption of toxins.

The rationale behind the pre-baiting step and use of two or more baits at a bait station may be explained in the following way with reference to toxin containing baits for animal consumption. Initially, toxin-free baits are made available to the target animals.

Desirably, the baits employed are the attractant baits of the invention and/or baits formulated to control uptake according to the invention. These baits should have the same 24 attributes as the baits to be provided later.

The rationale is that the animals will consume these baits and not develop an aversion.

If the baits prove palatable and rewarding, this will increase the animals consumption and future exposure. While not being bound by this, it is believed that the use of methods of the invention and related baits to control consumption will cause the animals to consume greater levels of these baits on initial consumption, thereby further increasing the animals classification of the bait compositions as safe and rewarding whilst the use of the attractant methods and baits will cause a greater number of animals to be exposed to the baits. All those baits, including the toxins, are then made available. The animals should recognise the baits as preferred and therefore consume large volumes of 'the bait increasing chances of receiving a lethal dose of poison.

This is again exaggerated by the use of the method of controlling consumption and use of the method for attracting animals to the bait compositions.

The use of two baits at a site wherein both baits are baits formulated to control consumption according to the invention are preferred to allow quick classification of the bait as being safe, while baits additionally comprising the attractant adjuvant of the invention may be used to attract more animals.

One bait composition would then be altered to include a toxin or replaced with a toxincontaining bait of the invention. The other would not be changed. If the targeted animal species had other mechanisms that made them stop eating the toxin-containing baits then they may be more likely to continue eating the toxin-free bait compositions. This would increase the probability that they would consume enough of the control consumption baits including more of the toxin-containing bait. The benefits of the two bait system with only one containing the toxin is the probability of the other "unknown" mechanisms interfering with consumption to an effect level, could be avoided. If a sublethal dose is consumed, 3 0 the use of the consumption controlling composition should prevent subsequent aversion developing, and the animal should consume the baits again on further exposure and maintain a susceptibility to poisoning.

The present invention will now be fther described with reference to the following examples which are provided by way of illustration only.

EXAMPLE 1 Effects of GABA and oxvtocin on Investigative Behaviour rats were housed in a 2 m range home cage for 4 hours per experiment. A feed source was provided at one end of this cage and the number of visits per hour to this feeder were recorded. The feeder provided ad libitum feed. Associated with this feeder various potential attractant substances in an oil based emulsion: oxytocin, gamma amino-4-butyric acid or a mix of these two or a control substance (ascorbic acid) all at the concentration of 1 mg/ml. The attractant properties of both GABA and oxytocin were high as compared to ascorbic acid. Both these effects could be prevented by administering GABA antagonists to the animal or blocking their smelling capacity by dabbing vicks vapourrub onto their noses. Bait shy animals showed little investigative behaviour towards a feed to which they had developed a shyness, however, investigation was encouraged by oxytocin and GABA being present. The results are shown in Figure 1.

EXAMPLE 2 Effects of Dodecyl Acetate (DA) Oxvtocin and GABA on Investigation of Feed Sources at two Sites in Field (wildl Rats Two feed sources (1 m Im open trays of grain) were set up 1 km apart un a farm paddock. Number of visits from wild rats was then measured over time. GABA and oxytocin were made up in DA at 1 mg/ml and associated with the feeders either as open liquids (which could be tasted) or enclosed liquids (which could be smelled but not tasted). GABA, oxytocin in DA proved attractant either as a smell alone or as a taste/smell. In animals that were bait shy, developed by associating sublethal 1080 with the feed for 4 days, the presence of GABA, oxytocin and DA proved reattractant. The results are shown in Figure 2.

EXAMPLE 3 Effects of Additives on Consumption in Rats 100 caged rats were exposed to a feed of Cocopops at a set amount (exceeding consumption) for 3 days and normal feeding established. Different drugs were then associated with the feed at different dosages and effects on consumption measured. The drugs, their dosages and effects are displayed in Figure 4.

NPY neuropeptide Y; a serotonin antagonist ketanserin; CCKA a cholecystokinin antagonist; -26- GLUTA glutamate antagonist (dextromethorphan).

EXAMPLE 4 Effects of Additives on Consumption in Possums 100 caged possums were exposed to a feed of Cocopops at a set amount (exceeding consumption) for 3 days and normal feeding established. Different drugs were then associated with the feed at different dosages and effects on consumption measured. The drugs, their dosages and effects are displayed in Figure NPY neuropeptide Y; a serotonin antagonist ketanserin; CCKA a cholecystokinin antagonist; GLUTA glutamate antagonist (dextromethorphan).

EXAMPLE Modulation of Corticosteroid Response in possums Alters Response to 1080 The consumption of 12 caged possums of a set feed was observed for 5 days with associated administration of a corticosteroid antagonist RU 38486 at 1 mg/kg liveweight.

The animals were then divided into two groups of 6 and exposed to the same familiar feed associated with a sublethal amount of 1080. Consumption patterns, and consumption with repoisoning were then measured for 4 episodes. Animals were then exposed to a higher dose of 1080 and of animals consuming a lethal amount was measured. The results are shown in Figure 6.

EXAMPLE 6 Effects of Drinkin2 Fluid Additives on Amount Drunk 32 rats were exposed to a drinking solution of sucrose in water and the amount drunk in the first 1 hour recorded. Animal had received 3 days prior familiarisation with this fluid and received the fluid at 0800hr each day. The sucrose fluid then had added to it one of ascorbic acid (control), cholecystokinin (cck), a serotonin agonist (paroxetine) or leptin at different dosages. Effects of these additives was then measured. The results are shown in Figure 7.

-27- EXAMPLE 7 Effects of Oxvtocin and Dodecvl Acetate In a field trial, two feeders, containing pellets, were positioned on a farm 2 km apart from each other. Numbers of rabbits visiting each were counted per night, and averaged over time. Following a 14 day initial period, one site was then laced with the dodecyl acetate analogue and numbers averaged over 14 days. This site location was the reversed. As can be seen in Figure 3 and Table 1 below, the presence of the dodecyl acetate analogues significantly increased the number of rabbits visiting a feeder site.

Table 1: Attractant properties of dodecyl acetate analogues Treatment Feeder only Feeder plus dodecyl acetate analogue number of animals visits observed* Site 1 Day 1 75 192' Site I Day 1 118 238' Site I Mean SE 115 ±5 209± 11' of 14 days Site 2 Day 1 85 176' Site 2 Day 2 184 241' Site 2 Mean SE of 155 8 235 8' 14 days Observations were made from 1800 to 0400h per day Indicates a significant (p<0.01) difference using student t-test comparison between mean (2 tailed) EXAMPLE 8 Effect of Sodium Monofluoroacetate (1080) on Neuroloeical Function Methods Animals Animals, sixty male rats (Rattus norvegicus, liveweights 300-450g) were housed individually in cages and maintained in a light:dark cycle environment of 12 h: 12 h at a -28temperature of 20 C. Animals were given fresh feed daily consisting of 60 g standard cereal pellets (Diet 86, Sharpes Grain and Seeds Ltd, Lower Hutt, New Zealand) and 250 ml of water. Consumption was measured every 24 h. Animals were fasted for 12 h prior to experimentation. All procedures were approved by the Ruakura Animal Ethics Committee.

Surgery Ten days prior to the start of experimentation animals were anaesthetised using a mixture of zolazepam/tiletamine hydrochloride (Zoletil, Techver Lab, New Zealand) and 2% xylazine (Rompun, Bayer, Germany) both administered intramuscularly at 0.1 mg/kg liveweight. A further 0.3 ml of lignocaine hydrochloride (Xylocaine Astra,NSW, Australia) was injected into the scalp around surgical area. Antibiotics (Depomycin, Mycofarin, Boxmeer, Holland) were administered intramuscularly (0.4 ml) as a prophylactic measure 6 h prior to surgery and on the two days immediately postsurgery.

After anaesthetic induction, the head was shaved, swabbed with 70% ethanol and covered with an iodinated sterile drape. An incision was made 1 to 2 cm in length, through the skin and scalp and skull exposed and cleaned. The bone suture landmarks, bregma and lambda were used to stereotaxically identify: somatosensory cortex, striatum, purkinje cell layer of the cerebellum, and the nucleus tractus solitarius. In each animal sterile guide cannulae (25 gauge polypropylene catheter) were implanted into two of the four areas minutes per area). A stainless steel screw was fixed into the skull behind the cannulae to provide a counter balance, and screw and cannulae were sealed in place with dental cement (Paladur, Heraeus, Wehrheim, Germany). The skin was sutured around the implant and a sterile obturator placed in each cannula to maintain its potency. Surgical procedure was similar to that previously described [17].

Prophylactic postsurgical analgesia, 100mg soluble aspirin (Solptin, Reckitt and Colman, New Zealand), was administered orally twice daily for three days after surgery.

Experimentation Ten days after surgery obturators were removed and microdialysis probes ME.C-P2, Applied Neuroscience, London, NW1 6DT, UK) fitted to sit 2 mm below the end of each guide cannula. The microdialysis probes were perfused with an artificial cerebrospinal fluid (ACSF) mixture at a rate or 2gl/min driven by a syringe pump (MS16A, Graseby Medical, UK) as has previously been described Collections were made on a min sample basis (10pl per sample). Samples were analysed off-line using HPLC techniques as previously described The microdialysis probes contained -29a chlorided silver wire and electroencephalograms (EEG) of the animal were derived from the potential between this and an Ag/AgCl reference electrode placed onto the animal's skull. The potential between the electrode in the probe and the reference was amplified using high-impedance pre-amplifier (NL 102G, Neurolog System. Digitimer, Ltd.

Welwyn Garden City, UK) and the AC component in the 1-30 Hz window amplified to provide EEG. Linear spectra of consecutive EEG data sections (4s) were computed using Fast Fourier transformation and for each 4s epoch the average amplitude of the EEG linear spectrum computed and displayed. These methods have previously been described [18], The microdialysis probes also contained an associated cannula that allowed direct introduction of pharmacological agents into the neural area from which sampling was made. All tubing and wires were suspended from the top of the cage to the animal's head and animals were tethered allowing them relatively free movement within a cage while measurements were made.

Samples were collected for 2.5h and then animals were given either two oral administrations each of 1 ml 0.9% sterile saline via a syringe (40 animals) or a perfusion via the cannula associated with the microdialysis probe (20 animals).

Administrations were given over a 10 min period. Measurements were then continued for another 18 hours. At the end of that time, microdialysis probes were removed and obturators replaced.

The experimental sequence was repeated 3 d later with all animals receiving an oral administration of 1 ml sterile saline containing 3.5 mg/kg liveweight 1080. In addition, animals received a further oral administration of 1 ml sterile saline alone and animals this containing one of: I. glutamate receptor and nitric oxide synthase (the enzyme responsible for generating nitric oxide) antagonists 2-amino-5-phosphonopentanoic acid Kynurenic acid, L-nitro arginine methyl ester and 6-cyano-7-nitroquinoxaline-2,3dione disodium (CNQX disodium), each at 5mg/kg liveweight.

II. GABA receptor agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride (THIP HCC) and allopregnanolone both at 10mg/kg liveweight.

III. I and II at 3.4 mg kg liveweight each.

IV. sodium salicylate at 20 mg/kg liveweight.

The remaining 20 animals received, in addition to the 1080, a microdialysis associated cannula perfusion of lOpl (over 10 min) sterile saline containing one of: I. glutamate receptor and nitric oxide synthase antagonists as above at 5 pg each.

II. GABA receptor agonist as above at 10 pg each.

III. mix of I and II above at 3.4 gg each.

IV. sodium salicylate at 20 gg.

All drugs were obtained from Research and Biochemicals Incorporated (One Stratmore RD, Natick, MA 01760-2447, USA).

Dosages chosen were based on previous studies in other models [21], [23] and on preliminary pilot trials (unpublished data).

Behavioural Observations Both focal and episodic behavioural observations were made every 5 min following 1080 or control saline administrations. Every 20 min an experimenter shone a torch light at the animal's eye and lightly clicked fingers to close to the animal. This was not performed if the animal was asleep. Animals were familiarised to this procedure for 14 d prior to experimentation, with the process repeated five times per day on a random basis.

EEG was monitored continuously and any incidence of ataxia, wet dog shakes, convulsive-like activity including clonic or tonic-like movements recorded. Shallowed or laboured breathing were also recorded. With severe respiratory depression, accompanied by subjective unconsciousness, ausculation was used to determine heart beat rate.

Prior to and at the end of experimentation, all microdialysis were calibrated in vitro as has been previously described Surviving animals were euthanised humanely, 24 h after 1080 ingestion, and postmortem histology was used to calibrate microdialysis position using methods previously described [24].

Results and Discussion Microdialysis probes showed in vitro recoveries of between 20 and 30%, with individual probes varying less than 10%. Stability was maintained from before to after experimentation and postmortem histology indicated all probes to be within 1 mm of their target areas.

Non-O180 Measures Both glutamate and GABA showed a fall in extracellular levels over the first 30 min to 2.2 1.8 and 1.6 1.0. These levels then maintained a reasonable stability. Oral -31 administrations of saline had a small effect on glutamate levels in the somatosensory cortex, striatum and cerebellum but not NTS. This effect was short lived (less than mmin) and could be induced by handling the animals alone. Basal levels of both glutamate and GABA then maintained a reasonable stability over the next 18 h. and were similar in all four brain areas (Figure Probe insertion disrupts neural function slightly, and a transient increase in extracellular neurotransmitters accompanied by DC and potassium ion shifts have been previously reported, Usually these appear to settle back to basal levels within 15-30 mins, and comparative observations were obtained herein. Glutamate is recognised as a neurotransmitter involved in alert behaviours [25] and its increase in certain brain areas with the handling associated with transient increased in both glutamate and GABA that returned to baselines within 5-10 mins. These transient increases may result from a similar type of neural area disruption as seen with initial probe insertion. Probe insertion and perfusions were not associated with any obvious changes in subjective behaviour nor in the EEG amplitude which remained between 10 and 30 pV for the duration of measurement.

Animals showed no evidence of convulsive activity, ataxia, nor of respiratory depression.

Animals also showed no hyper-responsiveness to either light or sound. Normal response consisted of an initial slight head turning and alerting followed by sniffing around the torch or hand.

1080 Measures Glutamate and GABA levels showed similar profiles in the first 2.5h to those seen in the non-1080 measures. Similar responses were also seen to the oral administration procedures. Within 60 min of 1080 administration both glutamate and GABA had risen significantly (p<0.01) within the somatosensory cortex. This increase lasted for 60-180 min after which time levels gradually (over 120 min) returned to baselines. During the period of 60-180 min after 1080 administration animals tended to "cage pace" a repetitive moving up and down the cage (defined by a frequency of at least three times per minute for at least 1 min), and showed a hyper-responsiveness to both light and noise. In hyper-responsiveness the animals tended to shy or move away quickly from the source and/or display a jump-like "startle" response.

Between 120 and 300 min after 1980 administration, levels of glutamate dramatically rose in the striatum, and a maintained high level was seen for a further 240 min. GABA also rose with a slight time lag (20-60 min) with respect to glutamate. Levels of glutamate and -32- GABA also rose in the cerebellum within a similar time frame to that seen in the striatumn after 1080 administration.

During this time period, animals showed occasional convulsions, as indicated by large magnitude amplitude increases in the EEG, and occasional clonic-tonic motor activity and "Wet dog shakes" as judged subjectively. Some ataxia was also observed. Studies using direct application of fluorocitrate into the brain have shown that striaral areas are particularly susceptible to the generation of spreading depression flowing application fIll and this is somewhat supported by the observations herein.

Glutamate and GABA levels rose in NTS between 300 and 600 min after 1080 administration and fell off in magnitude over a fuirther 180-240 min period. During this time,. respiratory depression, occasional heart rate change and death were observed. Some seizures were seen during this time and these often appeared subjectively to be anoxic related because they were usually preceded by signs of respiratory depression, and occasionally associated with animal death. Figures 16 and 17, and Table 2 below, sumnmarise this data.

-33- Table 2: Percentage of animals exhibiting neurological dysfunctions following oral administrations and time range at which these occurred (min) after administration Saline 1080 1080 plus 1080 plus 1080 plus 1080 plus plus Glut/NOS Glut/NOS GABA Salicylate Saline antagonist antagonist agonist n=5 and GABA agonist Hyper- 0 90% 60% 60% 80% 100% responsive' 120-240 120-300 120-240 min 140-280 120-240 min min mmin nun convulsions 2 0 80% 40% 40% 60% 240-420 240-420 260-500 min 260-400 240-500 min min min min ataxia 3 0 70% 80% 60% 80% 100% 180-320 220-360 180-300 min 160-300 220-340 min min min min death 4 0 75% 60% 60% 80% Percentage of animals in each group is displayed as administration at which symptoms were observed.

is the upper and lower range of time after oral SHyper-responsiveness to both a light source (penlight torch) and a sound (experimenter lightly clicking fingers). Hyper-responsiveness was defined, subjectively, by a shying away from the source, startle-like response or greater. Animals were only tested if awake. Normal animals moved their heads slightly and then tended to investigate the source.

SConvulsions were assessed by a doubling or greater of the EEG amplitude or subjective assessment ofclonic-tonic like movements. Convulsions associated with death were not included.

3 Ataxia was defined by a maintained tilting of head or body or a tendency to fall over.

4 Death within 18 h of administration.

A total of 60% of the animals in this group displayed one or more seizures and 80% of animals died within 18 h of 1080 administration. Although the dose used was less than LD80, animals were fasted prior to administration and this may have increased the fatality rate and reduced duration to time of death.

1080 plus oral administration of antagonists/agonists Oral administration of glutamate and nitric oxide synthase antagonists had no effect on either glutamate or GABA levels. However, convulsions and fatalities were slightly (p<0.05) reduced in this group. The addition of GABA agonists decreased, slightly, -34glutamate levels and further reduced convulsions and fatalities. GABA agonists alone had some effect on glutamate levels and on convulsions (p<0.05) but not on fatalities. Sodium salicylate had no effects. Table 2 summarises this data.

1080 plus brain administration of antagonists/agonists Administration of glutamate and NOS antagonists into different brain areas had no effect on either glutamate or GABA levels. However, administration into the somatosensory cortex reduced hyper-responsiveness to both light and sound and reduced incidence of cage pacing Administration into the striatum reduced convulsions, ataxia, and fatalities (p<0.05) but had no effect upon light/sound hyper-responsiveness not cage pacing activity. Administration into NTS also reduced convulsions, reduced respiratory depression, and fatalities Administration into the cerebellum had no obvious effects. GABA administration gave similar results although there was also an accompanying reduction in glutamate levels. GABA alone did not appear as effective at preventing fatalities or respiratory depression as glutamate/NOS antagonism. Coadministration of GABA agonists and glutamate/NOS antagonists again exhibited similar effects and was slightly more efficacious than glutamate/NOS antagonism alone, particularly at reducing hyper-responsiveness and death when administered into the cortex or NTS respectively. Administration of sodium salicylate had effect (p<0.05) only in the NTS where administration reduced convulsions, respiratory depression and fatalities.

Figures 11 to 14 summarise this data.

The glutamate/NOS antagonists and GABA were chosen because of their proven reduction ofhypoxic-ischemic type damage in other models [23].

This study strongly suggests that glutamate excess may underlie many of the neurological dysfunctions associated with 1080 poisoning. It may also be a contributor to ultimate death. Triggering of NO via glutamate activation may also be a potential contributor although in this study effects of glutamate were not separated out from those of NO. Both glutamate and NO appear to contribute to convulsions as these are reduced by their antagonism. In cyanide toxicity, both glutamate and NO (via peroxidation) contribute to neural damage and convulsive states and this may be a common pathway for many energy deprivational insults that affect the CNS. Release of GABA appears to slightly lag in time glutamate and may be an attempt to control both glutamate levels and ensuing convulsive states. Potentiation of GABA, via agonists, not only reduced convulsions but also amount of glutamate released. A CNS area-time, after ingestion, relationship was seen that correlated with observed neurological behaviour. Glutamate in the somatosensory cortex was associated with hyper-responsiveness to both light and sound.

This hyper-responsiveness was reduced by glutamate antagonists suggesting causal involvement of glutamate. However, altering the effects of glutamate had no influence on convulsions or fatalities which were observed as later events. Glutamate changes in both the striatum and cerebellum correlated in time with both observations of convulsions and ataxia. Glutamate antagonism in the striatum reduced incidence of both of these and of fatalities while in the cerebellum it appeared to have no effect. Changes in the cerebellum, in glutamate, may be acting on receptors unaffected by the glutamate antagonists although this seems slightly unlikely as a broad receptor spectrum coverage was assured by the antagonist selection. Alternatively, glutamate change may only represent a marker of response, rather than effect, in the cerebellum. This does not belie a role of the cerebellum in neurological dysfunction but suggests this is not due to excess glutamate.

Changes in the striatum correlated to fatalities despite the fact that fatalities occurred considerably after the changes seen in the striatum. This may mean that damage to the striatum occurs after a delay period or that the striatum feeds onwards in a damage additive manner to another area, that experiences damage at a latter time.

Alternatively, reduction of convulsions as occurs with pharmalogical manipulation of the stnriatum may reduce later incidence of death. Glutamate peaks in the NTS were seen at a time correlative to respiratory depression and death. Antagonism of glutamate in the NTS was most effective at preventing death, suggesting that this may be a final pathway in the end sequence. The NTS is well known for its roles in cardiorespiratory control [26].

Interestingly, in animals treated with glutamate antagonists in the NTS, that survived, no evidence of lasting neurological dysfunction was seen suggesting that changes in other areas were not of sufficient magnitude to disallow recovery. This is in contrast to cyanide toxicity where survival is often associated with lasting neurological dysfunction [28], However, higher doses of 1080 may cause lasting damage in survivors. This remains to be tested.

Sodium salicylate was efficacious at preventing death when administered into the NTS, although had little effect in other areas. While glutamate contributes to neurodegeneration so does accompanying inflammation. Salicylate appears protective to this later mechanism It may be that part of the final pathway to death lies in inflammation of the NTS neurones and salicylate acts to reduce this. As there was no lasting neurological dysfunction in surviving animals, other areas of the brain affected by 1080 may not have experienced any substantial degree of inflammation. This would be consistent with a lack of apparent effect of salicylate in these other areas. Why the NTS should be more susceptible remains unknown but offers an avenue for exploration of toxin effect.

36 Glutamate and GABA release may be a generalised response to 1080 toxicity. Certainly, it was seen in all areas measured, and in the case of the cerebellum appeared only as a marker rather than effector of any neurological change observed. However, glutamate does appear to have a time separation in terms of appearance in different areas, and at least in some areas its manipulation can influence specific neurological manifestation. Its appearance may relate to spread of 1080 through the CNS or site specific vulnerability.

Manipulation of both glutamate receptor activation and possible CNS area inflammation at the central level reduced some of the toxin effects of 1080 but oral administration was less effective. This may relate to the titre of drug reaching specific CNS areas in the appropriate window of time. Measuring specific neurotransmitter changes in conscious animals and correlating these, via pharmacological manipulation, with neurological behaviour changes offers and approach to understanding toxic effects on the central nervous system.

In summary, 1080 initiates excitotoxin-like reactions in a number of CNS areas that correlate with its neurological manifestations. This provides a platform for constructing a neural basis for understanding 1080 toxicity. In addition, neuronal inflammation appears contributive to final death following 1080 ingestion.

EXAMPLE 9 Effects of Serotonerzic and Cho lecvstokinin _Anta onists on Res ~onse Patterns in Rats to Sodium Monofluoroacetate Rats, (20 males, body weights 300-400 g) were housed in separate cages and fed a standard cereal pellet mix (Diet 86, Sharpes Grain and Seeds Limited, Lower Hutt, NZ) at 60 g per day and 250 ml of water. Consumptions were recorded every 24 h. Animals were subject to a constant 21 0 C environment and a 12 h.:12 h light/dark cycle. Fresh feed and water were provided at the start of the dark cycle, and during this cycle the room was lightly lit via two desk lamps with 15 W bulbs (to allow behavioural observation).

Following 14 d of this the animals were divided into two groups of 10. One group received an oral administration of 1 ml 0.9% sterile saline per animal, in the other group each animal received the saline containing 1080 at 2.5 mg t kg liveweight. Administration was via a 1 ml syringe and took place immediately prior to provision of fresh feed. This was repeated 10 d later each group received two saline and two 10 80 treatments over d period). Following administrations animals were intensely monitored using both focal and episodic (every 5 minutes per animal) observations. Every 20 minutes each animal was exposed to a noise (clicking of experimenter's fingers) and to a light source -37- (hand-held penlight torch). It has previously been reported that rats demonstrate a period of hypersensitivity to light and sound following 1080 ingestion [33].

Following 1080 administration 10 animals in one group (two in the first administration, one in the second) were euthanised because they displayed convulsive-like activity between 4 and 6 h after administration. In the second group two animals (one in each administration trial) were euthanised for the same reason. Replacement animals were used to maintain an n=10 in each group. Replacement animals showed no significant differences from other treatment group counterparts. Animals administered 1080 exhibited six behavioural characteristics, other than convulsions, at a significantly higher level of expression to when these same animals were administered saline (Table 3).

-38- Table 3: Occurrence of behaviour after oral administration of 1080 or saline in rats r Occurrence or Frequency of Behaviour 1080 Saline 1080 Saline Hypersensitivity Light (ii) Sound Grooming or Scratching Abdomen Cage Pacing Lying in a ball while awake Total Water Intake (ml/24h) Total Feed Intake (g/24h) Convulsive-like behaviour I All animals* All animals* 32 7 (1st hour)* 41 ±6 (2nd hour)* 49 6 (3rd hour)* 13 ±4 (Ist hour)* ±5 (2nd hour)* 7 6 (3rd hour)* 15 3 (2nd hour)* 19 4 (3rd hour)* 22 3 (4th hour)* 90 12* 7±8 5 animals* (occurred between 4 and hours after 1080 administration) No animals No animals 8 4 (1st hour) 6 3 (2nd hour) 7 ±5 (3rd hour) 2 ±1 (1st hour) 3 2 (2nd hour) 2 3 (3rd hour) 2 3 (2nd hour) 0 (2nd hour) 3 1 (4th hour) 40± 8 40 ±9 No animals Data are presented as mean SD. Data were summed for all animals within the treatment group within the defined time irrespective of administration sequence as analysis showed this to have no significant effects.

Hypersensitivity to sound or light was defined by the animal actively moving away or cringing from the source or showing a jumping startle-like response. Animals were awake at time of testing and experimenter's hand lowered slowing close to the animal before turning on the torch or clicking fingers.

Cage pacing was defined by repetitive movement (more than 3 times in a sequence) up and down the cage during a single observation not exceeding 2 minutes.

Convulsive-like behaviour was defined subjectively by the presence of either tonic or clonic-like movements and apparent loss of consciousness. Awakeness while lying in a ball was judged by eyes being opened. Other behaviours are self descriptive.

indicates significant difference (p<0.01) between 1080 and saline administration.

-39- Repeating of trials, with 10 d breaks, had no effect upon this pattern, suggesting a stable response to both 1080 and saline.

Following a six week break, during which the animals received normal feeding of pellets, the 1080 and saline administration trials were repeated. However, in addition animals received either: 1 ml of 0.9% saline containing 5mg/kg liveweight of 1-methyl-N-(8methyl-8-azabicyclo 1H-indazole-carboxamide maleate (LY-278,584), a specific 5-HT 3 antagonist or (ii) 1 ml of 0.9% saline containing 2 mg/kg liveweight of PD135,158 N-methyl-D-glucamine, a specific cholecystokinin (B subtype) antagonist.

Both of these were administered orally at the same time as saline or 1080 and the dosages chosen based on effectiveness in other animal models at preventing nausea or anxiety[31],[32]. All animals thus received each additional treatment associated with each of the saline or 1080 administrations in a random sequence. Both the serotonergic antagonist and the cholecystokinin antagonist had effects on observed behaviours following 1080, but not saline, administration. Sequence of drug presentation had no effect. Serotonergic antagonist treatment associated with 1080 administration reduced significantly (p<0.01) the amount of grooming and scratching of the abdomen, lying awake in a ball, and increased the amount of feed consumed in a 24 h period as compared to 1080 administration alone. It is believed that these behaviours may represent signs of nausea and gastric discomfort, relieved by the serotonergic antagonist. The serotonergic antagonist chosen has been reported as having some anti-nausea and strong anti-emetic properties and had no other obvious effects on behaviours in the saline treated group.

Cholescystokinin antagonists co-administered with 1080 reduced significantly (p<0.01) the amount of cage pacing and (p<0.05) grooming and scratching of abdomen (Table 4) Table 4: Changes in behaviour after oral administration of 1080 and either serotonergic or cholecystokinin antagonists Occurrence or Frequency of Behaviour 1080 Serotonergic 1080 Antagonist Cholecystokinin Antagonist Hypersensitivity All animals All animals Light All animals All animals (ii) Sound Grooming or Scratching 10 4 (1st hour)* 21 3 (1st hour)# Abdomen 20 6 (2nd hour)* 18 3 (2nd hour)* 14 3 (3rd hour)* 20 3 (3rd hour)* Cage Pacing 10 3 (1st hour) 5 3 (1st hour)# 14 4 (2nd hour) 2 1 (2nd hour) 6 4 (3rd hour) 3 2 (3rd hour) Lying in a ball while awake 4 2 (1st hour)* 12 3 (1st hour) 6 1 (2nd hour)* 14 4 (2nd hour) 8 3 (3rd hour)* 19 4 (3rd hour) Total Water Intake (ml/24h) 85 8 92 4 Total Feed Intake (g/24h) 28 8* 13 Convulsive-like behaviour 1 animal 1 animal Data are presented as mean SD. Data were summed for all animals within the treatment group within the defined time irrespective of administration sequence as analysis showed this to have no significant effects.

Hypersensitivity to sound or light was defined by the animal actively moving away or cringing from the source or showing a jumping startle-like response. Animals were awake at time of testing and experimenter's hand lowered slowing close to the animal before turning on the torch or clicking fingers.

Cage pacing was defined by repetitive movement (more than 3 times in a sequence) up and down the cage during a single observation not exceeding 2 minutes.

Convulsive-like behaviour was defined subjectively by the presence of either tonic or clonic-like movements and apparent loss of consciousness. Awakeness while lying in a ball was judged by eyes being opened. Other behaviours are self descriptive.

indicates significant difference (p<0.05) to 1080 alone.

indicates significant difference (p<0.01) to 1080 alone.

-41- Cholecystokinin is thought to mediate a number of gastric sensations [34] and it is conceivable that antagonism of this at central levels could reduce any gastric discomfort following 1080 administration. As the antagonist chosen was preferential to B receptor subtypes, which are mainly located in the central nervous system, it would seem most likely that any effects were mediated centrally rather than systemically. Alternatively, these behaviours could represent anxiety and be relieved by the anxiolytic properties of the cholecystokinin antagonist. Cage pacing may represent a generalised anxiety and thus be reduced by the anxiolytic properties of the cholecystokinin antagonist. The antagonist chosen has reported properties of both anxiolysis and mild gastric relief Interestingly, hypersensitivity to both light and sound were not reduced by cholecystokinin treatment.

This suggests they may not be a product of generalised anxiety. Two animals, one in each antagonist group, demonstrated convulsions and were euthanised. Although this is a significant reduction, compared to the non-antagonist groups, it is likely to reflect statistical variance around the 1080 dose effect rather than on treatment.

The observations that an anti-nausea and emetic agents and an anxiolytic agent reduced the behaviour observed, by extrapolation, suggests that they respectively represent some form of gastric upset and anxiety. Further, it suggests that if this is the case, the bait additives such as serotonergic or cholecystokinin antagonists can reduce these welfare concerns.

It will be further appreciated by those persons skilled in the art that the present description is provided by way of example only and the scope of invention is not limited thereto.

EXAMPLE Attractant Effects of Dodecvl Acetate. Oxvtocin. Decanolactone. and GABA Methods and Materials Animals A flock of 60 Angora goats were used for the duration of the trial.

The animals were kept in one of three paddocks in a grazing rotation schedule. The paddocks consisted of: 4.5, 3.5 and 3 acres of open land, and water was available ad libitum. Animals were individually tagged. Trials were undertaken in the months of September, November and December. Animals were individually weighed each week bar one (where normal farm practice prevented this).

-42- Procedures Two feeders were placed in the observation paddock. These feeders were Im x Im in size and contained lucerne chafe as the feed source. Associated with each feeder was a small container (adapted from a plastic jar) containing a Whatman 70 mm filter paper to which approximately 2 ml of the test attractant substance was added. This was topped up with ml daily.

New containers were used for each compound and each week of trial and at all times during handling latex gloves were worn to avoid cross contamination.

Feeders were positioned approximately 250m apart in a line and observers (one for each feeder) positioned 5- 10m away from each feeder (observable by the goats). The areas in which the feeders were positioned were chosen on the basis of previous work showing these areas were frequented regularly by the goats throughout the day.

In the first week feeders each had one small container with the filter papers wetted by a ethanol solution (control).

Immediately prior to 0900h, on each morning, each feeder was filled with 2 buckets of chafe that was weighted. Observations were made between 0900 and 11 00h, 1300 and 1500h and 1600h to 1800h. At the end of each observation period the remaining chafe was weighed and removed. This was returned immediately prior to the next observation period. If the first allotment of food was consumed during the day, it was immediately replaced.

This procedure was repeated Monday to Friday on the first week of the trial.

Behavioural Observations During the feed provision periods, behavioural observations were made: Continuous sampling of all animals approaching the feeders through the observation periods this included: Number of animals approaching the feeders.

Number of feeding occurrences at the feeders.

Identification numbers of animals at the feeders.

Interactions between animals and feeders.

Amount of time animals were present at the feeders.

-43- An episodic scan of the flock's activity was made every 30 min as to where and what individual animals were doing.

Instantaneous scans were made every 10m radius of the feeders. These procedures were then repeated for a further six weeks with the plastic containers in each feeder containing one of the putative attractants per week, for the entire week. Another trial of 4 weeks duration, on a different farm, in the same manner was performed on the animals at a separate date. As baseline data from this did not show any differences those four weeks have been included in this preliminary report. The plastic containers were transferred between feeders in between the morning and afternoon sessions such that if a particular container was associated with feeder 1 in the morning it was positioned in feeder 2 for the afternoon and the next morning and back to feeder I in the subsequent afternoon. Individual observers stayed with a feeder through the week but rotated amongst feeders on a weekly basis.

Substances were put into the plastic containers using a double-blind basis. Codes were not revealed until after data collection.

Substances used were as follows: Formaldehyde sulphate (FS) 1% Dodecyl acetate (DA) 1% Oxytocin plus gamma -4-butyric acid (OX-GA) 1% Decanolactone (Dec) 1% DA plus Ox-GABA DA plus DC Ox-GABA plus DC Control ethanol in water) All substances were made up in the control substance.

A modified form of a Latin square design as depicted in Figure 18 was used to compare the various substances against others. The independent variables were thus -44the substances and the dependent variables: the observers, the feeders, the feed consumed, number of visitations, animals within a 1 Om radius of the feeders and the individual animals.

Multivariate analysis of variance (MANOVA) was first performed across the two substance factors for each week. Further analysis (RM-ANOVA and either student t-test or Mann-Whitney Rank Sums) were then performed on individual dependent variables.

Results and Discussions Dependent variables were summed for each tested substance or substance mix individually in two ways: the first the absolute total sum, the second as a percentage of total per week of substance present (relative normalisation). As this made no statistical difference, only absolute sums, corrected to a daily basis, are presented.

Observer identity had no significant effect. Feeder position had a small significant (p<0.057) effect but because of rotation of substances between feeders this did not bias results. Individual animal had a significant (p<0.05) effect (see below).

The mixture of oxytocin-GABA and decanolactone had the greatest effect (p <0.01) in increasing consumption, increasing animals visitation and increasing the number of animals in a 10m radius of feeders containing this substance compared to the control substance.

The descending order of statistically significant increased effect (as compared to control) in the remaining substances was: Dodecyl acetate plus decanolactone oxytocin-GABA Dodecyl acetate oxytocin-GABA plus dodecyl acetate Decanolactone control. Significance was taken as p<0.05.

The last trial substance formaldehyde sulphate significantly reduced (p 0.0 1) the parameters of consumption, visitation and animals in a 10m radius as compared to the control substance. Figures 2,3 and 4 summarise this data.

Decanolactone did have observation periods where it appeared to be highly significant. However, it was also highly variable, reducing its statistical power.

Interestingly, when in combination with either DA or Ox-GABA it appeared to potentiate their effects.

Ox-GABA and DA were both attractant, however the individual animals 'apparently' attracted by them differed slightly.

DA attracted heavier weight animals, and their kids, and subjectively (not enough data collected to be objective) these animals aggressively displaced any lighter weight animals that investigated the associated feeder.

Ox-GAB A also attracted heavier animals but lighter animals were also present.

Subjectively these lighter animals did not appear to be aggressively displacedOx- (IABA and particularly Ox-GABA plus Dec attracted the greatest number of animals.

However, this was due to the presence of the lighter weight animals. With DA, or its combinations, a greater number of heavier weight animals were present compared to the absence of DA.

When the number of animals in each weight category was taken into account lightweight and heavy-weight animals seemed to have greater access than middle-weight animals.

This is an interesting observation. A current explanation, by which the applicant in no way wishes to be bound, is that sexual mate choice often is associated with aggression and territorial dominance. One could speculate that in the goats, the heavier individuals were more dominant Continuing on that line the middle-weight may pose more of a displacement threat then the light-weight animals. Submissive animals are often tolerated by dominant animals.

Ox-GABA on the other hand appears linked to social calmness and facilitation of grouping. This could account for the greater mix of different weight animals and the apparent "subjective" lack of aggression as compared to the DA containing mixtures.

-46- Formaldehyde sulphate has been suggested, in other species to be a highly effective repellent. It followed a similar pattern in the goats with an equal reduction in both heavier and lighter weight animals.

Summary Two of the tested compounds show high preference, and probably attractant, qualities for farmed goats. The two compounds do, however, differ in the type of individual animals they attract. Dodecyl acetate appears to attract heavier weight animals while Oxytocin-GABA attracts both heavier and lighter weight animals.

A third compound decanolactone has variable attractant properties on its own but does potentiate the effects of DA and/or oxytocin-GABA.

Formaldehyde Sulphate repels goats of all weights. This may be useful for limited area grazing control.

EXAMPLE 11 Attractant Effects of Dodecvl Acetate and Oxvtocin The object of this experiment was to test the effects of two potential attractants, dodecyl acetate and oxytocin, and a potential repellent, formaldehyde sulphate, on fanned red deer (Cervus elaphus).

Methods and Materials Subjects Eighty-one farmed red deer (I stag, 45 hinds, and 35 fawns of unknown sex) were habituated for one week to the presence of two supplementary feeders in their paddock (paddock area 5 Ha). The feeders (Im lone x im wide x 0.2m high) contained a known mass of hay and were placed in the paddock each morning (placement as per Figure 22). Following the final observations for a given day, the feeders were removed. Water and feed (pasture) were available ad libitwn.

-47 Procedure Four compounds were tested: 1% Formaldehyde sulphate 1% oxytocin (OX), 1% dodecyl acetate and a control ethanol in water).

Experimental compounds were presented by soaking a 2 ml aliquot into filter paper (Whatman 70 mm) and were associated with each feeder by placing them in a small plastic container (adapted from a GladeTM fragrance dispenser) situated in the centre of each feeder. Experimental compounds were replaced daily.

Pairs of compounds were tested by placing one in feeder 1 and the other in feeder 2.

After 2 days the compounds were swapped to the opposite feeder where *they remained for a further 3 days. Potential attractants were tested against one another and against the control. The potential repellent was tested against the control only.

Observations were made through binoculars from a distance of 600 m. Three different activities were recorded: The number of occasions deer appeared to feed from a feeder (Eating occurrences), the number of occasions a deer approached a within 10 m of a feeder (Approaches) and the result of a focal scan (which was conducted every 10 minutes). The focal scan recorded the number of deer within mn of a feeder and was intended to reveal any latency to aggregate or associate near a feeder. The quantity of hay consumed was measured daily.

The trial was conducted between 18 January and 5 March when peak daytime temperatures were very hot (up to 32 0 C in the shade). Data collected during the familiarisation week suggested the subjects were relatively inactive between 1 100 h and 1700 h and tended to habitate the shaded areas around the edge of the paddock.

Due to this factor, observations were conducted between 0600 h and 1100 h and again between l700 hand 2O00h.

This experiment was conducted as a double blind procedure.

Result Hay consumption: Very little hay was consumed during this study and thus, this result should be -48 interpreted cautiously. More hay was consumed from feeders associated with DA and OX than feeders associated wit controls (Figure 23), this result was only significant however for DA An apparent lower quantity of hay consumed from feeders associated with ES was not significant.

Eating Occurrences: The apparent differences in the incidences of eating (Figure 24) were not significant except between ES and all other compounds This result took into account a significant effect between feeders (P<0.00 1) for this variable.

Approaches: There was no significant difference in the number of approaches to a feeder revealed by the ANOVA (P=0.259) although visually there appeared to be slightly more approaches to feeders associated with DA and OX compared with controls (Figure Deer within 10 m of a feeder: S ignificantly more deer stood within 10 m o f feeders associated with DA (F igure 2 6) than controls (P<0.0 Discussion This trial goes some way to replicating the earlier work on goats using a deer model.

While the trends are similar the statistical significance of the present results are not.

That these apparent differences were not significant is due in part to the variability of the data resulting in relatively large standard errors.

The lack of statistical significance may reflect a true result no real difference) or could be a function of several variables that logistically could not be incorporated into the experimental design on this occasion. The presence of these variables highlights the difficulties of working with large species in (relatively) uncontrolled environments. Several of the hinds had fawns at hoof and were lactating, some were not pregnant, while others were pregnant and gave birth during the trial. While it would seem reasonable that such a situation could occur in the wild, such situations are not ideal in an experimental environment and may confound results. Lactating -49females for instance may produce greater oxytocin in their brains and thus, may be less responsive to an external oxytocin stimuli. If this did prove to be the case this would not necessarily preclude oxytocin-based attractants from being used but rather may highlight particular time of the year when they could be most effective.

EXAMPLE 12 Experiment demonstrating increased uptake of a drug from systemic circulation into brain tissue by the use of a pyrrolopyrimidine U- 101033E Amount Amount brain Amount brain Amount brain Amount brain systemic tissue no tissue with tissue with tissue with APH (glutamate pyrrolopyrimidine pyrrolopyrimidine pyrrolopyrimidine pyrrolopyrimidine antagonist) added lOng/ml added 100ng/ml added 1000ng/ml mg/mi mg/m 8 g/mi 8 mg/m [50 mg/m 7 mg/ml g19 mg/ml 25 mg/ml 3§ mg/ml 100 mg/mi 11 mg/ml 45 mg/ml 79 mg/mi 8 melml Experiment demonstrating the conjugation of a drug to a pyrrolopyrimide can increase lipid transfer across the blood brain barrier. Two groups of rats each n=10, subjected to LD80 of 1080. Group 2 also received 1 mg/kg of pyrrolopyrimidine (UE 101033F). Measurements of APH were made at 30 min after oral delivery from systemic circulation and from two brain sites.

In the absence of pyrrolopyrimidine approximately 10% of the drug in the systemic circulation equilibrated into brain tissues. Adding increasing dosages of pyrrolopyrimidine increased this.

It will be further appreciated by those persons skilled in the art that the present description is provided by way of example only and that the scope of the invention is not limited thereto.

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Claims (48)

1. A bait or bait adjuvant which includes at least one of the following attractant substances: one or more of dodecyl acetate or its chemical analogues; (ii) one or more of oxytocin or its chemical analogues; (iii) one or more of gamma-amino-4-butyric acid (GABA) or its analogues; and (iv) one or more mimetics of above.

2. A method of attracting a targeted animal species to a bait, the method comprising the use of an attractant substance, in or near the bait, which attractant substance includes at least one of the following: one or more of dodecyl acetate or its chemical analogues; (ii) one or more of oxytocin or its chemical analogues; (iii) one or more ofgamma-amino-4-butyric acid (GABA) or its analogues; and (iv) one or more mimetics of above.

3. A method of attracting an animal to a bait, the method comprising the inclusion in the bait of at least one of to (iv) as set forth in claim 1.

4. The use of at least one of to (iv) as set forth in claim 1 to attract targeted animals to a bait or bait site.

A method of controlling uptake of a bait by an animal, the method comprising the use of a bait adjuvant, in or near the bait, which comprises at least one of the following: one or more of neuropeptide Y or its analogues; one or more neuropeptide Y antagonists; one or more of leptin or its analogues; one or more leptin antagonists; one or more of cholecystokinin or its analogues; one or more cholecystokinin antagonists; one or more of serotonin or its analogues; one or more serotonin antagonists or re-uptake inhibiting factors; one or more serotonin releasing factors; and one or more mimetics of to above, with the proviso that and are not used alone or in combinations thereof alone.

6. A bait which includes at least one of to as set forth in claim 5, with the proviso that the bait does not comprise or alone or in combinations thereof 54 alone.

7. A bait adjuvant which includes at least one of to as set forth in claim with the proviso that the bait adjuvant does not comprise or alone or in combinations thereof alone.

8. A method for controlling uptake of a bait by an animal, the method comprising providing in or near the bait at least one of to 0) as set forth in claim with the proviso that (Q or are not used alone or in combinations thereof alone.

9. A method of controlling uptake of a bait by an animal, the method comprising administering to said animal at least one of to as set forth in-claim wit the proviso that or are not used alone or in combinations thereof alone.

10. A bait adjuvant for altering the stress response in an animal which bait includes at least one or more stress response altering substances.

11. A method of altering the stress response in an animal, said method comprising the use of one or more stress response altering substances in or near the animal's bait.

12. A method of altering the stress response in an animal, said method comprising the inclusion in the animal's bait of one or more corticosterones, or analogues or antagonists thereof.

13. A method of altering the stress response in an animal, said method comprising administering to said animal one or more stress response altering substances.

14. A method according to any one of claims 11I to 13 or an adjuvant according to claim 10 wherein the stress response altering substance comprises one or more Corti costerones, or analogues or antagonists thereof.

A method for re-attracting bait shy animals to bait, the method comprising the use of one or more baits or adjuvants selected from the baits and adjuvants of claims 1, 6,

16. A complex bait adjuvant comprising at least two compounds selected from the following groups A, B and C: A. (i) (ii) (iii) (iv) B. (a) (b) (C) (c) (d) (e) (f) (g) (h) (i) (J) one or more of dodecyl acetate or its chemical analogues; one or more of oxytocin or its chemical analogues; one or more ofgamma-amino-4-butyric acid (GABA) or its analogues; one or more mimetics of above; one or more ofneuropeptide Y or its analogues; one or more neuropeptide Y antagonists; one or more ofleptin or its analogues; one or more leptin antagonists; one or more of cholecystokinin or its analogues; one or more cholecystokinin antagonists; one or more of serotonin or its analogues; one or more serotonin antagonists or re-uptake inhibiting factors; one or more serotonin releasing factors; and one or more mimetics above; C. one or more corticosterones, or analogues or antagonists thereof provided that when only two compounds are selected they are selected from different groups.

17. A bait or adjuvant according to claim 16 which includes at least three compounds, with at least one compound being selected from each of groups A, B and C above.

18. A bait or adjuvant according to any one of claims 1, 16 and 17 which further comprises oxytocin and GABA and optionally dodecyl acetate.

19. A bait or adjuvant according to any one of claims 1 and 16 to 18 which further comprises decanolactone.

A bait or bait adjuvant according to any one of claims 1,6, 7 and 16 to 19 which further includes a lipid membrane transfer facilitator.

21. A bait or bait adjuvant according to claim 20 wherein the facilitator is a pyrrolopyrimidine. 56

22. A method of re-attracting bait-shy animals to bait the method comprising the use of a complex bait or adjuvant according to any one of claims 16 to 21.

23. A method for increasing the speed of delivery of an active substance to one or more organs, of a targeted animal, the method comprising administering to said animal an active substance together with a lipid membrane transfer facilitator, which facilitator is a pyrrolopyrimidine.

24. A use of a pyrrolopyrimidine to increase the speed of delivery of an active substance to one or more organs of a targeted animal.

A method according to any one of claims 2, 3, 5, 8, 11 to 15 and 23, further comprising the use of a mechanism for facilitating the uptake of an active substance into a target area mn an animal.

26. A method according to claim 25 wherein the target area is a vulnerable organ or the central nervous system.

27. A method according to claim 26 wherein the vulnerable organ is the brain.

28. A method according to claim 27 wherein the nucleus solitarius (NTS) and/or striatum of the brain are targeted.

29. A method according to any one of claims 2, 3, 5, 8, 11 to 15, 23 and 2 5 to 28, further comprising the use of a mechanism for increasing the speed of uptake of an active substance.

A method according to claim 29 wherein the mechanism is mechanical or chemical.

31. A method according to claim 29 or claim 30 wherein the mechanism is designed to stimulate the face or oral cavity of an animal.

32. A method of baiting or poisoning an animal, the method comprising administering to said animal an active substance formulated to target the nucleus solitarius (NTS) and/or striatumn in the brain of the animal. -57-

33. following: (i) (ii) (iii) (iv) (v) An active substance formulation comprising a toxin and at least one of the one or more of dodecyl acetate or its chemical analogues; one or more of oxytocin or its chemical analogues; one or more of gamma-amino-4-butyric acid (GABA) or its analogues; one or more mimetics of(i)-(iii) above; and optionally one or more lipid membrane transfer facilitators.

34. A formulation according to claim 33 wherein all of or mimetics thereof are present in the formulation.

A method of effectuating a rapid death in an animal, said method comprising administering to said animal a toxin formulated to target the striatum and/or NTS brain areas of the animal.

36. A method of lessening the symptoms of poisoning or severity of the symptoms, in an animal, the method comprising administering in addition to a toxin an adjuvant or bait according to any one of claims 1, 6, 7, 10 and 16 to 21.

37. A method of lessening the symptoms of poisoning or severity of the symptoms, in an animal, the method comprising the use of an adjuvant or bait according to any one of claims 1, 6, 7, 10 and 16 to 21 near a bait or toxin but separate therefrom.

38. A method according to claim 35 further comprising administering at least one or more of the following: one or more anti-emetics; and/or (ii) one or more anxiolytics.

39. antagonist. antagonist.

A method according to claim 38 wherein the anti-emetic is a serotonergic A method according to claim 39 wherein the anxiolytic is a cholecystokinin

41. A bait according to any one of claims 1, 6, 7, 10 and 16 to 21 or a formulation according to claim 33 or claim 34 further comprising at least one or more of the following: -58- one or more anti-emetics; and/or one or more anxiolytics.

42. A bait according to claim 41 wherein the anti-emetic is a serotonergic antagonist.

43. A bait according to claim 42 wherein the anxiolytic is a cholecystokinin antagonist.

44. A method according to any one of claims 2, 5, 8, 9, 11, 15, 23, 25 to 32 and to 40 which includes as a pre-step administering to a targeted animal species an active substance-free bait according to any one of claims 1, 6, 16 to 21 and 41 to 43 or formulation according to claim 33 or claim 34.

45. A method according to any one of claims 2, 5, 8, 11, 15, 23, 25 to 32 arnd 3 5 to 40 wherein two or more adjuvants or baits are used at the same time and at the same site.

46. A method according to claim 45 wherein both a bait and an active substance-free bait are present.

47. A method according to claim 45 wherein the bait is a bait according to any one of claims 1, 6, 7, 10 and 16 to 21 or a formulation according to claim 33 or claim 34.

48. A method according to any one of claims 2, 5, 8, 11, 15, 23, 25 to 32 and to 40 which further comprises the simultaneous use of a bait and an active substance- free bait at a bait site. THE HORTICULTURE AND FOOD RESEAR T CH INSTITUTE OF NEW ZEALAND LIMI1TED 7 August 2003