GB2550559A

GB2550559A - Methods, systems and apparatus for control of parasite infestation in aquatic animals

Info

Publication number: GB2550559A
Application number: GB1608666.2A
Authority: GB
Inventors: Cambell Paul; Conneely Michael
Original assignee: University of Dundee
Current assignee: University of Dundee
Priority date: 2016-05-17
Filing date: 2016-05-17
Publication date: 2017-11-29
Anticipated expiration: 2036-05-17
Also published as: CA3062821A1; GB2550559B; CL2018003244A1; NO347719B1; WO2017199019A1; GB201608666D0; NO20181621A1

Abstract

A method of providing bubbles formed in a liquid medium on or near the surface of an animal, and exposing the bubbles to sound waves so as to induce resonance and asymmetric collapse of the bubbles. The method can be to reduce parasite infestation on an aquatic animal or a non-therapeutic method for improving the condition, appearance, meat quality or growth rate of an aquatic animal. The apparatus is also provided, as well as a composition comprising water for use in the method of treating a parasitic infestation. In another embodiment is a method for the delivery of molecules to an aquatic animal comprising stabilising the bubbles by providing a film at the gas-liquid interface or by inclusion of surfactants in the bulk of the liquid, wherein the film or liquid comprises molecules for delivery to the animal or parasite. Ultrasound exposure parameters are adapted to induce collapse of the bubbles and allow molecular delivery to the aquatic animal or to cells of the aquatic animal. Preferably the animal is a fish such as a salmon, and the parasite is non-pathogenic and comprises one or more of Lepeophtheirus salmonis, Caligus clemensi and Caligus rogercresseyi.

Description

METHODS, SYSTEMS AND APPARATUS FOR CONTROL OF PARASITE INFESTATION IN AQUATIC ANIMALS

Field of the Invention

The present invention relates to methods, systems and apparatus for controlling parasite infestation in aquatic animals, for example fish. In particular, the invention relates to the treatment of parasite infestation on the surface of aquatic animals using sound waves.

Background of the Invention

Aquatic animals, for example fish, are frequently subject to infestations of parasites which cause limited damage in small numbers but may cause significant physical damage, increase the risk of pathogen infection and may ultimately cause death of the animal if populations increase. This is particularly likely to happen in confined environments where the fish population is very dense. For example, in the case of salmon farming, parasite infestation such as sea lice {Lepeophtheirus salmonis) and amoeba {Neoparamoeba perurans) - the latter of which leads to a potentially fatal condition, amoebic gill disease (AGD) - are a major problem that has a very significant commercial impact as it negatively affects the quality of the product and demands expensive and increasingly ineffective treatment solutions.

Various chemical, pharmaceutical, biological and physical approaches have been proposed for treating infested fish. Examples include the application of drugs (either inbath or in-feed treatments): vaccination; co-hosting with wrasse 'cleaner-fish' (which forage lice directly off the salmon skin); interaction with lasers; temporary exposure to heated (30-34°C) ambient water and also mechanical scraping. All of these methods have problems to some degree: bath treatments, such as with organophosphates, pyrethroids or topical disinfectants like hydrogen peroxide cause obvious environmental and animal toxicity concerns, and are difficult to implement since the concentration of drugs is difficult to control; the treatment often requires fish crowding which stresses the fish; and prevention of re-infestation is difficult since it requires the effective treatment of the entire fish population over a short period of time. In-feed treatments, such as with avermectins or parasite growth inhibitors, are easier to administer and pose less environmental risk, but are a concern in terms of toxicity both for the fish and for the consumer. Mechanical or thermal treatments are devoid of environmental and toxicity risks, but are labour intensive, potentially harmful / stressful to the fish, and may undermine the quality of the final product.

Accordingly, there is a need for new methods to efficiently, ethically, safely and cheaply control parasite infestation in aquatic animals, and fish in particular.

Summary of the invention

In one aspect, the invention relates to the control (e.g. the reduction) of parasite infestation in aquatic animals, in particular by inducing the collapse of gas-filled bubbles at or near the surface of the animal or parasite in order to kill (either directly or indirectly) or dislodge the parasites. In another aspect, the invention relates to methods for delivery of molecules, particularly small molecules for e.g. vaccination or therapy, to a target aquatic animal. In one aspect the invention relates to methods for the control of parasite infestation in aquatic animals in combination with the delivery of molecules to the aquatic animals. The methods of the invention may also target free-swimming / floating parasites at various developmental life-stages in order to control parasite densities in the local environment. The invention is particularly advantageous for the control of parasite infestations in fish populations, such as control of sea lice infestations in farmed salmon populations. In some aspects the parasites are non-pathogenic and the methods of the invention are non-therapeutic. In one aspect, the invention relates to a non-therapeutic method for improving the condition, appearance, meat quality or growth rate of an aquatic animal. In another aspect the invention relates to apparatus for use in controlling parasite infestations in aquatic animals and/or for the simultaneous or sequential delivery of molecules to the target aquatic animals. In another aspect the invention relates to a method for the treatment of a parasitic infestation of an aquatic animal. In yet another aspect, the invention relates to a composition comprising water and/or a gas (such as oxygen or air) for use in a method for the treatment of a parasitic infestation of an aquatic animal.

According to a first aspect, the invention provides a method of reducing parasite infestation of aquatic animals which includes the steps of a) providing bubbles on or near the surface of the animal or parasite; and b) exposing the local environment surrounding the animal or parasite to sound waves, wherein the sound waves induce resonance and asymmetric collapse of the bubbles. Advantageously, this method achieves parasite removal without the need for chemical treatments that present environmental, toxicological or ethical concerns, or abrasive mechanical treatments that may injure the animal. The surface of the aquatic animal having a parasite infestation is therefore at least partially in contact with water of other appropriate liquid medium in order that the bubbles are formed of air (or other gas) encapsulated in the liquid medium.

In preferred embodiments, the intensity of the sound waves is selected so that the collapsing of the bubbles generates a liquid (water) jet that has sufficient energy to kill parasites on the surface of the animal. In some embodiments, the sound waves may instead or in addition stun, disable or dislodge parasites. In embodiments, the intensity of the sound waves may be such that the collapsing of the bubbles creates a laminar radial flow on the surface of an aquatic animal that is sufficient to shear off a parasite. Advantageously, the intensity and/or duration of exposure may be selected to avoid significant damage to the host aquatic creature. Fine tuning of the sound waves characteristics allow to strike a balance between creating a jet that has sufficient energy to damage or remove the parasites yet low enough energy to avoid damaging or stressing the animal.

In embodiments, exposing the local environment surrounding the animal to sound waves may comprise exposing the environment to longer term continuous wave ultrasound leading to the development of a sustained microstreaming current flow around a resonant bubble, or ensemble of bubbles. Advantageously, this may generate a local hydrodynamic environment that is disruptive to parasite attachment and could lead to their most gentle removal.

In some embodiments, the sound intensity and duration of exposure may be selected to: a) kill parasites by formation of an energetic liquid jet upon asymmetric collapse of the bubbles, using a high intensity and short pulse train; b) remove parasites from the surface of the animal by creating a jet impact with radial flow upon asymmetric collapse of the bubbles, using an intermediate intensity short pulse train; c) remove parasites from the surface of the animal by creating a microstreaming shear flow using low to intermediate intensity with comparatively long to intermediate exposure periods. In some embodiments, a high intensity and short pulse train may have a peak negative pressure in the MPa range, such as e.g. between 1 and 3 MPa, and a duration in the microseconds range, for example, between 1 and 1000 microseconds. In such embodiments, the mechanical index of the ultrasound (see below) may be above 0.5. In some embodiments, an intermediate intensity short pulse train may have a peak negative pressure in the hundreds of kPa, such as e.g. between 100 kPa and 1000 kPa, and a duration in the milliseconds range, for example, between 1 ms and 1000 ms. In such embodiments, the mechanical index of the ultrasound may be between 0.1 and 0.5. In some embodiments, a low intensity ultrasound may have a peak negative pressure in the kPa range, such as e.g. between 10 and 100 kPa, and a duration in the seconds range, for example, between 1 and 10 s. In such embodiments, the mechanical index of the ultrasound may be under 0.1. In some embodiments, the driving frequency for the sound field may be selected to modify the radial response of the targeted bubbles. Advantageously, the frequency, intensity, and duration of the sound field, all of which are hardware controllable, may be modulated to force distinct bubble responses. Control of these variables provides options for treatment protocols, and in effect allows a practical solution to be developed that leads to delousing / treatment yet preserves the quality of the final product that may be derived from the animal, and ensures an ethical approach to parasite control.

In embodiments of the invention, the ideal frequency of the sound waves may be determined using the Minnaert resonance formula:

where ro is the average or median radius of the bubbles, y is the polytropic coefficient, po is the ambient pressure and p is the density of the liquid. Thus, once a bubble target size is determined, the driving sound frequency at which optimal quality factors may be developed can be calculated.

In some embodiments, the sound waves frequency may be between 20 and 100 kHz. Setting the sound waves frequency according to the expected size distribution of the placed bubbles allows to maximise the proportion of bubbles that will enter into resonance and collapse, and as a result enhances the efficiency of the process. Low ultrasound frequencies may be especially useful for generating bubbles in-situ, but this may require higher sound intensity fields in order to generate cavities in the local liquid (e g. water), which may have detrimental effects on the treated animals. Use of low frequencies also promotes higher transmission of the sound waves so that larger volumes may be treated, and the throughput of treated animals may be enhanced. However, such fields may suitably be spatially contained so that the wider marine environment is not affected unduly.

In some embodiments, the bubbles may be created by injection of gas in the liquid medium, preferably in the vicinity of an aquatic animal. In some embodiments, the bubbles may be created by locally flowed release of preformed bubbles. Advantageously the bubbles have a specific diameter, or set of different, but defined, diameters. Advantageously, the diameter or set of diameters of preformed bubbles may be selected to induce a well-defined response from the population of bubbles to driving sound fields, wherein the driving sound field may have multiple drive frequencies. Advantageously, acoustically active bubbly flows may be directed towards preferential locations of parasite infestation on the animal.

In some embodiments, acoustically active bubbles may be created in a macroscopic water jet used to shower the animal partially exposed at the air-water interface. This has the advantage that the macroscopic water jet containing the bubbly flow may self-guide the ultrasound driving field via the acoustical equivalent process to total internal reflection. In such embodiments, the ultrasound energy may not dissipate beyond the isolated flow and threshold energy levels for removing parasites can be minimised so long as the liquid flow is uninterrupted. Direct bubble injection by local gas release at specified rates, or the locally flowed release of preformed bubbles which may be forced to coalesce to some preferred size in-situ, has the desired effect of allowing control of the size of the bubbles and may be used to maximise the probability that bubbles will be placed / positioned at locations where their collapse will produce the desired effect upon the parasite.

In alternative embodiments, the bubbles may be placed by bringing at least a portion of the animal into contact with air and entraining bubbles at the surface of the animal or parasites upon re-immersion. For example, bringing at least a portion of the animal into contact with air may be achieved by lifting or forcing the aquatic animals out of the liquid. In some embodiments this may be achieved conveniently during moving of fish stock to and from well-boats. Such embodiments are particularly advantageous from an economical point of view since they make use of existing infrastructure and require only adjustment in flow protocol in the well-boat delivery conduit.

In particularly advantageous embodiments of the invention, removed parasites may be collected by use of filters in the (recirculated) water.

In some embodiments, the aquatic animal is a fish, such as a salmon. Where the fish is a salmon, the parasites may comprise one or more Lepeophtheirus or Caligus species, in particular one or more of Lepeophtheirus salmonis, Caligus clemensi and Caligus rogercresseyi. Sea lice are a major economical concern for the fish farming industry, and L.saimonis infestations are both common and regularly devastating in the salmon farming industry. Keeping control of these infestations is particularly challenging especially in conditions where it is essential that the food (fish) production chain complies with stringent environmental and ethical criteria.

In advantageous embodiments, the bubbles may be stabilised within the liquid, such as by creating a film at the gas-liquid interface or the inclusion of suitable surfactants to the (bulk) liquid. For example, the film may comprise a surfactant, preferably a lipid compound, or a polymer. Bubble stabilisation in this manner helps prevent bubbles from rapidly dissolving in the liquid and allows an enhanced time period during which bubbles may be targeted by sound waves. Use of a lipid is particularly advantageous since many non-toxic, readily available and inexpensive lipids are available, which may serve to increase the half-life of bubbles on the surface of the animal.

In some embodiments, the film may comprise ligands to target the bubbles to the parasites. In some embodiments, the film may additionally comprise other molecules of interest for delivery to the animal and/or the parasite. For example, lipid films may comprise therapeutic agents such as vaccination products of a pharmaceutic or nucleic acid nature. Advantageously, such molecules may be convected directly into the target animals during bubble collapse and/or the bubble-based parasite removal process. The film may instead or in addition comprise specific [biojchemical / pharmaceutical agents to achieve parasite lethality. In such embodiments, two or more different exposure protocols for the driving sound field may be implemented in order to convect therapeutic agents or other desired molecules directly into the animal or the parasite. The targetable nature of the bubble-based delivery process has the advantage that relatively little reagent is required compared to present treatment approaches.

According to a second aspect, the invention provides an apparatus and/or system for reducing parasite infestation of aquatic animals, wherein the apparatus comprises: a mechanism for placing bubbles on or near the surface of the animal or parasite; and a mechanism for exposing the animal or parasite to sound waves, wherein the sound waves induce resonance and collapsing of the bubbles. Such an apparatus may be readily integrated with existing fish farming infrastructures, and advantageously may require little specifically adapted equipment.

In some embodiments, the mechanism for placing bubbles comprises means for bringing at least a portion of the animal into contact with air or other appropriate gas such that bubbles are entrained at the surface of the animal or parasites upon re-immersion in liquid. In some embodiments, the means for bringing at least a portion of the animal into contact with air or gas may comprise a mechanism for lifting or forcing the aquatic animals at least partially out of the (bulk) liquid at the air-liquid interface. Beneficially, such embodiments may for example be implemented by means of cages where the floor and/or whole cage can be lifted so as to bring the animals into contact with air; or may be implemented during transfer of the animals to well-boats. Such mechanisms and systems are already known for other purposes in the fish farming industry.

In embodiments, the mechanism for placing bubbles may comprise apparatus for injection of bubbles of controlled size into the liquid, preferably in the vicinity of the aquatic animals. Advantageously, bubble injection may be targeted to preferential locations of parasite infestation on the animal.

In some embodiments, the sound waves may be targeted to the animal, more specifically to the bubbles entrained on the animal’s surface, or still more specifically targeted to preferential locations of parasite infestation on the animal.

Injection and sound waves targeting have the desired effect of optimising the location of bubbles collapse in order to maximise the efficiency of parasite removal.

In some embodiments, the apparatus may comprise an effector zone where the sound waves are applied. In some convenient embodiments, the effector zone may comprise a channel through which an aquatic creature can move (or be passaged), specifically between two sub-zones. In preferred embodiments, bubbles may be injected in the first sub-zone. Alternatively, or in addition, bubbles may be placed on the surface of the animal or parasites prior to entry in the effector zone.

Creation of a dedicated effector zone may have the beneficial effect of limiting the costs of the treatment system by avoiding the need to provide infrastructure to create and/or activate bubbles in large volumes of liquid, and instead focuses bubble and ultrasound generation to a region of the apparatus where its effect may be more easily controlled. Such a mechanism may also improve the efficiency and convenience of the parasite removal / inactivation system by avoiding relatively non-specific bubble placement and sound wave exposure. Implementation of such effector zones could be implemented within the infrastructure of well boats, or in conventional (or novel) marine-based pen environments.

In embodiments, the apparatus may further comprise a mechanism for detecting the presence of an animal in an effector zone. Advantageously, sound waves may be generated in the effector zone upon detection of an animal entering or within the effector zone. Similarly, bubbles may be injected in the effector zone, upon detection of an animal entering or within the effector zone. Preferably, bubbles are targeted towards the animal, or towards preferential locations of parasite infestation on the animal. The addition of a detector guarantees that resources are only spent to generate and activate bubbles when an animal is present, and further, can allow fine control over the timing of bubble placement and sound wave generation according to factors such as bubble half-life, animal location and/or traveling speed.

In some embodiments of the apparatus, the bubbles are stabilised in the liquid. This may be achieved by creating a film at the gas-liquid interface. The film may comprise a surfactant, advantageously a lipid compound. Bubble stabilisation can increase the lifetime of a bubble in the liquid, and hence increase the likelihood of bubbles being present on the surface of the animal or parasite when the sound waves are applied. This is particularly advantageous, e.g. if the fish have to be directed towards a specific treatment zone in which sound waves are applied.

In preferred embodiments, the sound waves frequency is selected using the formula:

where Γο is the average or median radius of the bubbles, γ is the polytropic coefficient, po is the ambient pressure and p is the density of the liquid. Conveniently, the sound waves frequency may be between 20 and 100 kHz, according to the average or median bubble size. In some embodiments, sound waves of frequency within the range 100 kHz to 1 MHz may be used instead or in addition. This may be useful, for example, if specific delivery of therapeutics warrants this, and where pre-formed bubble radii have been tailored to ensure a resonant (or off-resonant if desired) response that ultimately leads to jet formation, or some alternative hydrodynamic effect that leads to dislodgement of the parasite. Multi-frequency approaches driving bubbles of specific radii may be advantageous in achieving delousing as one specific result; for delivery of therapeutics to the fish as a further specific result; and for injecting lethal or nullifying agents into attached lice as a further specific result.

In some embodiments, the sound waves are applied at an intensity that is sufficient for the collapsing of the bubbles to generate a jet of liquid that may kill, disable, damage, dislodge or stun parasites; and/or may generate a radial flow, or alternatively microstreaming flow, on the surface of the animal that can dislodge parasites. Preferably, the pressure may be low enough that the collapsing of bubbles does not significantly damage the surface of the aquatic animal.

Fine tuning of the characteristics of the sound waves generated by e.g. ultrasound transducers, can be readily achieved following the teaching of the present application by the skilled person according to requirements. This can help to maximise the efficiency of the systems and methods described herein, while preserving the ethical and economic benefits of the invention.

In another aspect the invention also relates to a method of treating an aquatic animal with a parasite infestation. In some embodiments the method comprises: a), placing providing bubbles in a liquid on or near the surface of the animal or parasite; and b). exposing the local environment surrounding the animal or parasite to sound waves, wherein the sound waves induce resonance and asymmetric collapse of the bubbles. The methods of treatment according to this aspect may comprise any of the embodiments of the methods, systems and apparatuses for reducing parasite infestation as described herein.

In another aspect, the invention provides a non-therapeutic method for improving the condition, appearance, meat quality or growth rate of an aquatic animal, the method comprising the steps of: a) providing bubbles in a liquid medium on or near the surface of the animal; and b) exposing the bubbles to sound waves so as to induce resonance and asymmetric collapse of the bubbles. The non-therapeutic method of this aspect may comprise any of the embodiments of the methods, systems and apparatuses as described herein.

In one aspect, there is also provided a method for the delivery of molecules to an aquatic animal, the method comprising: a) providing bubbles in a liquid on or near the surface of the animal or a parasite on the surface of the animal; b) stabilising the bubbles by providing a film at the gas-liquid interface or by inclusion of surfactants in the bulk of the liquid, wherein the film or liquid comprises molecules for delivery to the animal or parasite; and c) exposing the local environment surrounding the animal or parasite to sound waves, wherein the sound waves induce resonance and asymmetric collapse of the bubbles; and the ultrasound exposure parameters are adapted to allow molecular delivery to the aquatic animal or to cells of the aquatic animal. The methods for delivery of a molecule to a target marine animal according to this aspect may comprise any of the embodiments of the methods, systems and apparatuses for reducing parasite infestation as described herein.

In another aspect of the invention, there is provided a composition comprising water for use in a method for the treatment of a parasitic infestation of an aquatic animal, the method comprising the steps of: a) providing bubbles in a liquid on or near the surface of the animal or parasite; and b) exposing the local environment surrounding the animal or parasite to sound waves, wherein the sound waves induce resonance and asymmetric collapse of the bubbles. The composition for use according to this aspect of the invention may further comprise a gas, such as air or oxygen. The composition for use according to this aspect of the invention may further comprise any feature of a method, apparatus or system as described in connection with any other aspect or embodiment of the invention disclosed herein.

In particular, it should be appreciated that the methods described herein need not be therapeutic and, therefore, in some distinct aspects and embodiments the methods of the invention do not encompass methods for the treatment of animals. In this regard, in some embodiments the parasites are not detrimental to animal viability and are not pathogenic or linked to any illness. In such embodiments, the methods of the invention can be useful in improving animal quality (appearance, texture and/or flavour etc.), condition and/or growth rate for food production purposes. In some embodiments, therefore, the methods of the invention further comprise the selection of an aquatic animal for harvesting / sacrifice.

Brief Description of the Drawings

One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

Figure 1 illustrates a vector field model of bubble collapse caused by sound waves close to a surface: (a) development of a fast moving liquid jet through a collapsing cavity; and (b) jet ‘touchdown’ with developing radial outflow.

Figure 2 shows high-speed images of a lipid microbubble in water cavitating in response to an ultrasound field.

Figure 3 is a schematic representation of an ultrasound treatment system according to an embodiment of the invention: (a) illustrating a submerged system to target activated bubble flow; and b) illustrating an open air system to target activated bubble flow.

Figure 4 is a schematic representation of an ultrasound treatment system according to an embodiment of the invention: (a) illustrating a side view of a fish swimming through a tunnel defining a treatment zone and activating sensing devices; and (b) illustrating a front end view of the fish in the treatment zone with ultrasound devices activated.

Figure 5 is a schematic representation of a suitable bubble placement on parasites according to an embodiment of the invention.

Figure 6 shows an example of the use of ultrasound activated bubbles to lyse a cell.

Figure 7 shows an example of the use of ultrasound activated bubbles to clear a biofilm.

Figure 8 shows an example of the use of ultrasound activated bubbles for molecular delivery.

Detailed Description of the Invention

Although the invention will be described by way of examples, it will be appreciated by a person skilled in the art that the invention could be modified to take many alternative forms without departing from the spirit and scope of the invention as defined in the appended claims.

Control of parasite infestation and treatments

In some aspects and embodiments the present invention is directed to the control of parasite infestation, such as to methods for reducing parasite infestation in aquatic animals, e.g. by killing, inactivating and/or removing parasites from the surface of an aquatic animal or by the eradication of the parasites at other stages in their life cycle, such as the transitory or migratory movement through the marine environment at large. In other aspects and embodiments the invention is directed to the treatment of aquatic animals. Particularly in this regard, the invention is directed to the treatment of surface infections or infestations, and most particularly to the treatment of parasitic infestations. In some aspects and embodiments the treatments are non-therapeutic and provide solely cosmetic or economic benefits through improvements in growth rate, food quality and general fish condition. For example, in some aspects and embodiments the parasite infestations to be ‘treated’ are completely non-pathogenic; and in some aspects and embodiments the parasites are not present on the fish, but rather are in the environment of the fish. In the latter case, the methods and systems of the invention are preventative of infestation.

By “control of parasite infestation” or “treatment” it is meant to encompass an act which is achieve any one or more of cure, alleviate, remove, reduce or lessen the symptoms of, or prevent or reduce the possibility of contracting the infection or infestation. Benefits may be judged by visual inspection or any other form of test to determine whether the animal is in better condition, faster growing, more hygienic, healthy, or viable as a result of the methods or treatments of the invention. Desirably an infection or infestation is eliminated, or the size, severity or visibility of the infestation or infection is reduced or in any other way alleviated compared to the level of infection or infestation immediately prior to treatment. In some embodiments an infestation is prevented.

It will be appreciated that for the purposes of this invention it is not necessary that an infection or infestation is entirely eliminated; rather it can be sufficient that the severity is reduced to within a tolerable threshold. For example, infection or infestation levels may suitably be maintained at acceptable levels by one or repeated applications or treatments of an animal or group of animals. The skilled person can determine what an ‘acceptable’ level will be on a case by case basis: by way of example, such standards may be linked to the occurrence of specific numbers of lice within a specified population of salmon. Thus, the invention encompasses the one-time application or treatment by the methods of the invention to an animal (e.g. immediately before removal of the animal from its habitat, farm or natural environment for harvesting or sacrifice) or repeated applications / treatments of an animal or group of animals at the farm or natural environment.

Aquatic animals

The present invention is directed to aquatic animals that may be susceptible to parasitic infections or infestations. In particular, the invention is directed towards aquatic animals that may be farmed or otherwise harvested from their natural or artificial habitat, especially for the food / produce market.

Suitable aquatic animals may therefore include fishes, molluscs and crabs.

Suitable fish include, for example, carp, bream, roach, salmon, sturgeon, silver carp, trout, perch, cod, haddock, catfish, sea bass and any other farmed or captured fish. A particularly suitable fish is any from the family Salmonidae, which include salmon, trout, char, grayling and whitefish. Preferred salmon include Atlantic salmon and Pacific salmon, such as Atlantic salmon {Salmo salar Linnaeus), Chinook salmon {Oncorhynchus tshawytscha), Chum salmon {Oncorhynchus keta), Coho salmon {Oncorhynchus kisutch), Pink salmon {Oncorhynchus gorbuscha), Sockeye salmon {Oncorhynchus nerka) and Masu salmon {Oncorhynchus masou). Other suitable salmon may include Danube salmon {Hucho hucho), Australian salmon {Arripis trutta), Hawaiian salmon {Elagatis bipinnulata) and Indian salmon {Eleutheronema tetradactylum).

Parasite infestation

Parasites are organisms that take advantage of and/or live at the expense of a host organism, in (endoparasites) or on (ectoparasites) the host for at least a portion of their life cycle. The term is primarily used to refer to macroscopic organisms such as helminths, ticks, flees, lice etc. Parasites do not typically kill their host, and in fact parasite infestation may have a purely cosmetically detrimental effect. In other embodiments, a parasite infestation may reduce the host’s biological fitness and cause various pathologies. Like any other animal, aquatic animals, and fish in particular, can suffer endo- or ectoparasite infections, both in the wild and in farmed populations. The present invention is of course most appropriately for the control or treatment of ectoparasites, although it is again noted that such ectoparasites need not necessarily be attached to the host animal at the moment of performing the methods and treatments disclosed herein, i.e. by way of sound wave exposure, in order to exercise some control over their levels of presence in the local environment.

Any surface parasite infestation of an aquatic animal may be controlled or treated by the methods and systems of the invention, such as myxozoan parasites, e.g. Henneguya salminicola and sea lice.

Sea lice infestation, particularly involving Lepeophtheirus and Caligus species is a common problem in both farmed and wild fish populations. For example, Lepeophtheirus salmonis, Caligus clemensi and Caligus rogercresseyi are common salmon parasites. Sea lice are ectoparasites that feed on mucous, blood and skin of the fish and latch onto the skin of the fish during some of its life cycle. Exceptionally high concentrations of salmon lice may be observed in highly populated salmon farms, commonly causing the death of juvenile salmon. Lepeophtheirus salmonis is the most common and well known sea lice, and a major source of concern for the salmon farming industry.

Bubble collapse

When sinusoidally varying pressure fields, such as those associated with the passage of sound waves in a liquid, are applied to bubbles within the bulk of a liquid, they can react in a linear or non-linear fashion, depending on the pressure intensity (or more specifically, the mechanical index (Ml), which is the ratio of the ultrasound peak negative pressure and square root of the centre frequency of the beam). If larger pressures are used, a non-linear response is elicited whereby bubbles expand then collapse catastrophically. As a result, shock waves may be generated locally at high pressures, and vigorous shear flows may be established at intermediate pressures if the ultrasound pulse train is maintained over time.

The acoustic resonance frequency f of a single bubble in a liquid may be determined using Minnaert’s formula:

where ro is the radius of the bubble, γ is the polytropic coefficient, po is the ambient pressure and p is the density of the liquid. Exposure to (ultras)sound at or around the same frequency as the bubble resonant frequency may then be used to excite the bubble, whereupon its subsequent radial evolution over time is well described by the Rayleigh-Plesset equation:

where PB(t) is the pressure inside the bubble, Px>(t) is the external driving pressure, p/. is the density of the liquid environment, R(t) is the radius of the bubble, ulIS the kinematic viscosity of the liquid and S is the surface tension of the bubble. If the symmetry of the expanding bubble’s environment is disturbed by the presence of nearby solids, then radial collapse becomes non-spherical and an energetic liquid ‘jet’ naturally directed towards the closest rigid surface plane is formed (see Figure 1). Figure 2 shows highspeed images of a lipid microbubble in water cavitation in response to an ultrasound field. The ultrasound is activated in the second frame, upon which the bubble begins to inflate during the first low pressure half cycle. The inflated bubble then rapidly collapses asymmetrically, such that “jetting” directed towards the nearest solid boundary (top of each frame) occurs, as can be seen in the fourth and fifth frames. Such jets are energetic and penetrative and may be exploited to puncture even surfaces exhibiting relatively high stiffness, in the MPa regime.

At high sound field pressures, the 'water hammer' pressure Pwh of the emergent liquid jet is given by the following equation:

Pwh = P C Vy which may be sufficient to puncture a surface, or e.g. kill a parasite. At intermediate pressures (i.e. pressures insufficient to puncture the surface), a high shear radial laminar outflow across the surface occurs, which may be sufficient to e.g. damage and/or dislodge parasites (e.g. see Figure 1b). As the person skilled in the art would understand, the sound pressure and sound intensity of a sound wave are directly related and the terms may be used interchangeably, for example when referring to a sound wave that has a sound intensity or pressure sufficient to generate a desired effect.

Note that the pressure that must be applied to achieve a jetting effect is linked directly to the spatial distance of a bubble relative to the target surface. Therefore, in some embodiments the bubbles may be placed directly on or at a suitable distance from the surface of the animal or parasite, as further described below: for example, using ligands and/or a hose.

The concentration of bubbles may also have an influence on the outcome of the treatment, such that in some embodiments bubble concentration may be optimised to achieve a desired level of parasite removal while causing no or very low levels of damage to the animal. In some embodiments, the bubble concentration may be optimised to minimise the scattering of the incident ultrasound beam while maximising the parasite removal effect. For example, the optimal bubble concentration may be determined empirically as a function of ultrasound frequency, pressure and exposure time.

Use of bubble collapse In the control of parasite infestation

In accordance with the invention, the bubble collapse phenomenon may be applied to the control of parasite infestation in aquatic animals. Without limitation and by way of explanation, this may mean that if bubbles are developed on or near a parasite on an animal and subjected to ultrasound of the correct frequency and pressure, then a jet formed as a result of a collapsing bubble may have sufficient energy to penetrate the body of the parasite, but not so much energy as to damage the infested aquatic creature. Alternatively or in combination, a collapse induced laminar radial flow on the surface of the aquatic animal, or a bubble induced localised microstreaming process, may be used to shear off parasites from their anchor points on the aquatic animal.

Therefore, as a result of the jet formed upon collapse of the bubbles, parasites may be damaged (i.e. by piercing the cuticle, crushing the parasite etc.) or subject to a force that is sufficient to dislodge them from their anchor point on the skin of the aquatic animal, and/or to flush off any e.g. eggs that may have been deposited on the animal’s skin.

Bubble placement

Bubbles within the bulk of a liquid may, for example, be created through a process known as cavitation, where a liquid subjected to low pressures (below the vapour pressure), may be driven to change phase and form bubbles within the bulk of the liquid. However, as this requires large pressures, it is also possible to “seed” (e.g. by injection or otherwise) bubbles in the liquid.

Creation of bubbles in the vicinity of parasites may be effected in several ways. In the simplest approach, the animals may be lifted or forced to the air/water interface so that their surface is exposed to air before being re-submerged. This will entrain air bubbles on the surface of the animal, and particularly to defects on the surface, e.g. a “defect” may be an attached parasite or collection of parasites. In some embodiments, such bubbles may be analysed as to their size distribution, in order to tune the ultrasound frequency according to the formula above, or to allow for a frequency sweep across the target bubbles' resonance frequencies.

Alternatively (or additionally), bubbles may be injected in the vicinity of the animals. Advantageously, these bubbles can be of a controlled (e.g. predetermined) size or range or sizes. In preferred embodiments, bubble injection is targeted at the animal so as to contact the animal. This advantageously allows placing a plurality of bubbles on the surface of the animal or the parasite(s). In some preferred embodiments, bubbles are placed on the parasites.

In some embodiments, the distance between the bubble and the surface of the animal or parasite may be controlled and/or the location of the bubble relative to the animal or parasite. For example, bubbles may be coated with a ligand, and optionally a spacer as required (see below), such as e.g. a polyethylene glycol (PEG) linker chain with a biotin terminal group to attach a chosen ligand. In some embodiments, one or more ligands may be used. In some embodiments, ligands may comprise compounds / agents that specifically interact with or bind to the surface of the parasite or animal - suitably a receptor on the surface or the organism. This can provide beneficial effects in that the distance between bubble and parasite / animal can be controlled and/or the bubble can be targeted to preferential positions on the animal, such as parasite locations.

Advantageously, the use of pre-seeded bubbles (i.e. placed rather than generated by cavitation) allows the system to work at relatively low pressures such that any indiscriminate damage that might otherwise occur to the fish population is reduced or eliminated.

Furthermore, pre-seeded bubbles may be preferentially placed, i.e. generated in contact with or targeted towards desirable areas of the aquatic animal, e.g. areas on the surface of the animal that are particularly susceptible to parasite infestations. Such areas may depend on the host and parasite, and knowledge of the life cycle of the parasite and associated preferential locations of attachment on the host is particularly advantageous in optimising bubble placement. The optimal bubble placement location(s) may be simply determined by inspection of the infested animal. For example, parasites may preferentially be found on areas of the animal that are less exposed to the environment, such as e.g. behind the fins in the case of fish. In particular, any part of a fish that is exposed to low hydrodynamic drag may be particularly relevant. In some embodiments, bubbles may be flowed towards the gill area. Such embodiments may be particularly advantageous when amoebic gill disease is a concern.

In some embodiments, in order to achieve bubble placement at preferred locations, bubbles may be provided and flowed directly towards target sites via flexible conduits that maintain acoustically active bubbly flow.

For example, the distance between the bubble and the surface of the animal or parasite may be controlled by flowing bubbles directly at the desired distance from the animal or parasite, such as, for example, using a hose that is directly in contact with the body of the animal. In such embodiments, the bubbles may be formed close to the hose outlet by optical (lasing), electrochemical or ultrasonic means, or may be formed by direct aeration with a gas of choice. Forming the bubbles close to the hose outlet can be beneficial in avoiding unwanted scattering of ultrasound waves. When gases are used to form the bubbles, a gas would advantageously be chosen so as to avoid or reduce the risks of toxicity to the animal.

Figure 3a shows an embodiment in which a flexible sheath 300 of high acoustic impedance in comparison to the external material (e.g. bulk liquid, commonly water) is used to guide the bubbly flow to target structures and direct the ultrasound field energy. An ultrasound transducer 302 is connected to the flexible sheath guide and activates bubbles flowing into the flexible sheath guide 300 via a bubble injector port 304. The apparatus of Figure 3a may be entirely or partially submerged in the bulk liquid. In some embodiments, the bubbles may include ligands to target the parasites (see further below), such as for example ligands that target the mechanoreceptors on the louse cuticle. Advantageously, such embodiments may increase the probability for bubble attachment. Thus, the apparatus of the invention may comprise a flexible sheath defining a fluid flow path for bubbles and sound waves in liquid (i.e. a bubbly flow) towards the target animal, and particularly towards preferential locations on the target animal. In some embodiments, the flexible sheath 300 may comprise a water (or salt water, if applicable) resistant durable polymer. In preferred embodiments, the polymer is translucent and/or treatable to protect against biofouling. For example, medical grade polymers such as silicones, polyurethanes, polyesters, polyvinylchlorides and polyamides may be used.

Figure 3b shows an embodiment in which the fish stock to be treated can be partially removed from the bulk liquid and exposed to air (for example by raising it to a shallow water-air interface), and the fish 'showered' with a directed and acoustically active bubbly flow. In such embodiments, the high acoustic impedance difference between the open flow bubbly liquid 306 and the external air replaces the flexible sheath in guiding the ultrasound generated by the ultrasound transducer 302. The bubbly flow may then be showered onto the fish.

Bubble stabilisation

Air bubbles dissolve relatively quickly in water (i.e. in the order of a few seconds, depending e.g. on their size). It can be beneficial in the methods, systems and treatments of the invention to extend the life of bubbles in liquid / water. Any appropriate mechanism / component may be used in order to prolong the half-life of bubbles in a liquid. For example, liquid additives may be used to stabilise an appropriate gas/liquid interface. In some embodiments, a “film” may be created at the air-water interface such that entrained air bubbles are encapsulated in the film. Advantageously, this film may be made of a liquid that creates an interaction tension with the bulk of the liquid. Surfactants, i.e. compounds that lower the surface tension of the liquid can be used. Preferably the film material can be less permeable to the gas in the bubble than the bulk liquid. In some embodiments, the core gas itself may be selected to have a relatively high molecular weight and therefore reduced potential for dissolution in bulk liquid. For example, a thin polymer or preferably a lipid film may be used. In some embodiments, the film material may comprise one or more lipids, and the properties of the film may be varied depending on the identity and relative proportions of the lipids in the film. In some embodiments, the film material may comprise phospholipids. In preferred embodiments, one or more non-toxic lipids may be used. In some embodiments, the film material may comprise polymers, such as proteins and peptides. In some embodiments, the film material may comprise albumin and/or chemically modified or cross-linked albumins. Such a system has the advantage of allowing more time between bubble seeding and collapse induction stages, such as to allow time for the animals to be moved to an area where ultrasounds are applied.

Additional properties of this film material may be desirable, such as a higher propensity to adhere to the parasite (for example via a ligand-receptor strategy) or the surface of the animal, a low toxicity, an ease of sourcing (i.e. cheap and readily available), and of subsequent separation from the bulk of the liquid. This list is non-exhaustive and the ideal film material that may be used can be selected accordingly to preferences and/or the circumstances of the use. In preferred embodiments, a non-toxic material may be used. In some advantageous embodiments, one or more food-grade lipids may be used.

Bubble size

As outlined above, the optimal ultrasound frequency to use in the treatments, methods and systems of the invention will typically depend on the size distribution of the bubbles present in the liquid. Bubble size distribution may be determined by inspection, such as e.g. when bubbles are placed by entrainment on the surface of the animal or parasite, or by assessment via ultrasound imaging, by which the echogenic properties of attached bubbles can be detected. In such cases, a suitable statistical parameter of the bubble size distribution may be used to tune the ultrasound frequency that will cause a large proportion of bubbles to resonate, such as e.g. the average or median bubble size. In some embodiments, the operational band-width of the transducer may be electronically swept through a range of frequencies within which the target bubble population can be acoustically excited.

In some embodiments, it may be advantageous to target bubbles of a size other than the statistically most likely one, such as e.g. when it is more convenient to use a specific ultrasound frequency, or where bubble placement and/or stability is size-specific.

In some embodiments, bubbles of a controlled or predetermined size may be specifically created, i.e. injected into the liquid. In some embodiments, the size may be chosen depending on e g. the desired ultrasound frequency to use. In preferred embodiments, bubbles of a controlled size are created by injection of gas into the liquid wherein the gas injection outlet has the appropriate diameter.

In preferred embodiments, bubbles will have a size distribution centred around about 5-12 micrometres for molecular delivery, towards millimetre-sizes for lethal jet-induced lysis of parasites, or between these ranges to damage or dislodge parasites (as calculated by the median or average bubble size, or any other adequate statistical metric of distribution location, such as filtered / truncated or weighted averages, geometric, harmonic or arithmetic means, etc.). Suitable characteristic values will be immediately apparent to the person skilled in the art.

Ultrasound generation

Ultrasounds are sound waves with frequencies higher than the upper audible limit of human hearing. While this is commonly defined as sounds at frequencies greater than 20 kHz, the term is used loosely here to refer to sound waves of appropriate frequency to induce resonance and collapsing of the bubbles created above.

Considering the expected bubble size distributions outlined above, such frequencies are conveniently above 10 kHz, although particularly advantageous embodiments of the invention make use of frequencies above 20 kHz, preferably between 20 and 2 MHz. It is to be understood that depending on the circumstances, sound frequencies between 10 kHz and 100 kHz, 10 kHz and 200 kHz, 10 kHz and 300 kHz, 10 kHz and 500 kHz, 10 kHz and 1 MHz, 20 kHz and 200 kHz, 20 kHz and 300 kHz, 20 kHz and 500 kHz, 20 kHz and 1 MHz, 30 kHz and 100 kHz, 30 kHz and 200 kHz, 30 kHz and 300 kHz, 30 kHz and 500 kHz, 30 kHz and 1 MHz, 50 kHz and 100 kHz, 50 kHz and 200 kHz, 50 kHz and 300 kHz, 50 kHz and 500 kHz or 50 kHz and 2 MHz may advantageously be used. In particular, ultrasound frequency and pressure may be selectively chosen, preferably within the 20 kHz to 2 MHz range, depending on the specific purpose such as molecular delivery, lysis of parasites, damaging or dislodging of parasites.

Suitable ultrasound generating apparatus will be known to the person skilled in the art. For example, ultrasound may be generated by ultrasonic transducers that convert alternating electrical current into sound waves. Transducers may be driven at specific (centre) frequencies, at harmonics thereof, or be programmed to deliver a frequency sweep across a range of target frequencies within which some proportion of the exposed population of bubbles will be responsive (resonant).

Apparatus configuration

The apparatus according to aspects of the invention comprises a mechanism, apparatus or means for providing and/or placing bubbles at the surface of an aquatic animal or on the surface of a parasite thereon; and a mechanism, apparatus or means for applying ultrasound to the bubbles. These means may be in operation simultaneously in the same area of the apparatus, simultaneously in different areas of the apparatus, in alternation or succession in the same area of the apparatus, or in alternation or in succession in different areas of the apparatus. For example, the apparatus may comprise a bubble placement zone and an ultrasound zone, wherein the zones can be physically separate or can be sub-zones of the same physical ‘effector’ zone.

The bubble placement zone (or region) or step in the method or system of the invention may include means for bringing at least a portion of the surface of the animal into contact with an air-liquid interface. A convenient mechanism may be by use of e.g. an immersed or partially immersed cage that can be raised in / out of the liquid, or wherein at least the floor of the cage is lifted. However, it should be appreciated that any mechanism for lifting aquatic animals at least partially out of the bulk of the liquid, either individually or in bulk is envisaged.

Alternatively or in combination with the above, the bubble placement zone may comprise gas injectors that create bubbles in the liquid. These bubbles may be randomly created in the bulk of the liquid or in some embodiments may be targeted towards the animal.

Where the animal, e.g. a fish, is relatively mobile, targeting of bubbles towards and/or onto the animal may be realised for example through use of a constricted volume through which the animal is forced to swim, and into which bubbles may be conveniently injected. The site of injection may be chosen e.g. to optimise bubble placement in preferred areas of parasite infestation (see above), or in areas of optimal bubble entrainment.

The ultrasound zone (or region) of the apparatus or step in the method or system of the invention may include ultrasound transducers that generate sound waves or an acoustic field that may be widely generated in the bulk of the liquid or targeted towards the animal. Targeting towards the animal may be obtained, for example, by using the same or a similar constricted volume as that used for targeted bubble placement. In some embodiments, the location of ultrasound wave generation may be chosen e.g. to coincide with the optimal bubble placement areas as described above. In some embodiments, the location and timing of ultrasound wave generation may be adapted such that the induced collapse of bubbles generates a radial flow that may shear the parasite from the surface of the animal. A convenient mechanism for guiding the animal into an effector or treatment zone (e.g. comprising bubble placement and/or ultrasound zones) is by use of a tunnel within the bulk liquid, into which a fish can swim. In some embodiments at least a portion of the tunnel may have a diameter only slightly larger than a diameter of the fish so as to control its trajectory. The apparatus of the invention may include a funnelled region (e.g. preceding a tunnel region) within the bulk liquid to direct animals towards the effector or treatment zone.

In both the bubble placement and ultrasound zones, the apparatus may include sensors to detect the presence of an animal and trigger the appropriate action, i.e. bubble or ultrasound generation. In some embodiments, this may be in the form of one or more magic eye photodiodes. The sensors further communicate with a processor for calculating or estimating animal speed and/or velocity to improve the targeting or timing of bubble and/or ultrasound generation.

In some embodiments, the animals may be encouraged to travel through the bubble placement and/or ultrasound zone(s) by means of a flow in the liquid. For example, in the case of salmon, a flow may be created to encourage ‘upstream’ movement through a treatment zone. Appropriate means of directing animals to particular zones will depend on the animal and may include the use of light, chemicals, baits, current, behavioural training (e.g. positive or negative reinforcement) or any other means that can attract an animal towards a zone of the apparatus or detract them from another zone.

In particularly advantageous embodiments, the apparatus may include two tanks separated by a constricted region, e.g. a tunnel or tube. A flow may be generated to encourage movement of animals from the first tank, through the tunnel / tube in which a treatment or process of the invention may be applied, to the second tank. Animals in the second tank would therefore have reduced parasite infestation or may be free of parasite infestation.

In some embodiments, the process may be conducted in a single tank, where bubbles are placed on the animal or parasites either by injection or by bringing at least a portion of the animals in contact with the air (e.g. by lifting the floor of a cage); and an acoustic field or targeted ultrasounds are generated in the tank, either simultaneously or after bubble placement has occurred.

In preferred embodiments, standing wave fields may be developed within the treatment zone for refined accuracy of bubble targeting and reduced effective pressure (and therefore animal exposure), as well as running cost optimisation.

The process described above may be performed once or repeated multiple times, as required to obtain a population of animals that is substantially free of parasites or where the parasite infestation is satisfactorily reduced. In some embodiments, a satisfactory result may be obtained when at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the animal population is substantially free of parasites. In some embodiments, a satisfactory result may be obtained when the parasite population on one or more animals has been reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the number of parasites detected on the animal before and after application of the method of the invention, as estimated by any appropriate statistical measure of the location or the distribution of numbers of parasites per animal (e.g. median or average number of parasites per fish in a representative sample).

In cases where multiple tanks or zones are used, repeating the process may involve an inversion or a change of direction of movement of the animals between zones in the apparatus. In preferred embodiments where animals are guided through an effector zone between two tanks, this may be achieved by inverting the direction of movement of the animals between the two tanks. In particular, where the treatment zone is a constricted area with a flow to encourage upstream movement through the treatment zone, the process may be repeated by inverting the direction of the flow in the treatment zone. Each pass through an effector or treatment zone may be considered a ‘cycle’ of the method of the invention. A course of more than one cycle of application or treatment is therefore encompassed in the methods of the invention. In embodiments that comprise a plurality of cycles / passes, second and subsequent cycles may be performed only once the parasites removed from an animal in a previous cycle of the process have been removed from the medium. For example, this may be performed by filtering the liquid. Advantageously, recovering the parasites removed by the process may be used to monitor the parasite infestation and the efficiency of the process. In combination with knowledge of the expected number of parasites per fish, data on the number of parasites recovered (such as e.g. by weighing or counting recovered parasites) may be used to estimate the number of fish successfully cleaned.

In some embodiments, the liquid containing dead, disabled, attenuated or live parasites dislodged / removed from the animals may be treated to ensure all free parasites are killed or otherwise prevented from becoming a risk of re-infestation. Any appropriate mechanism, such as chemical or radiation treatment of the affected liquid may be used.

The invention will now be further illustrated by way of the following non-limiting examples.

Examples

Example 1 - Fish Louse Study

Cuticle strength in adult lice can be studied to determine appropriate ultrasound intensity, frequency and pressure for use in the apparatus of the invention.

Samples of lice are cultivated on agar or equivalent substrates including upon host animals, and Young's modulus, E, is determined using scanning probe microscopy (SPM) or alternative metrology. This involves the capture of force distance curves that are obtained by pressing on the cuticle to well defined depths using a standardised and [stiffness-] calibrated cantilever. Once E has been determined, the critical tension, Tc, for cuticle rupture will be measured experimentally also using SPM.

Example 2 - Fish Treatment

An effector or treatment chamber or zone (2) of the invention is illustrated in Figure 4. The effector zone (2) includes a tunnel or tube (4) which is filled with water and is arranged to connect between first and second water tanks (not shown). A fish (6) is induced to swim into tunnel (4), for example, by generating a favourable fluid flow condition to encourage a unidirectional swim vector. When the fish (6) is a salmon, the salmon may preferentially swim against the direction of fluid flow. In the illustrated embodiment, the fish swims through the tunnel (4) in the direction of the arrows.

At least a pair of photodiodes (8a, 8b) are arranged in the wall of the tunnel (4) spaced from one another along the axis of travel for the fish, so that the photodiode (8a) is activated by movement of the fish (6) before the photodiode (8b). The photodiodes (8a, 8b) detect the fish (6) as it passes an invisible plane or line through the tunnel (4) indicated by each of the dashed lines (10a, 10b), and send a signal to a controller (12; not shown) indicating that a fish (6) has been detected passing either of the detection points (10a, 10b). In this way, the approach of the fish towards the bubble generation zone (14), in which bubbles are indicated by the dotted line (14a), can be predicted or timed.

Bubbles (14a) may be generated continuously or specifically in response to the detection of an approaching fish (6), and are delivered into the tunnel (4) at or towards the bottom surface of the tunnel (4a). In this way, the bubbles (14a) spontaneously travel upwards around and past the fish (6) so as to contact the fish (6) and collect on the fish’s outer surface. The apparatus may include a mechanism, such as outlets with one-way gas-release valves (not shown) for enabling bubbles (14a), but not fluid or water (16), from leaving the tunnel (4) through an upper surface (4b) thereof. This will prevent build-up of gas in the tunnel.

Bubbles (14a) may be introduced into the tunnel (4) at a point source so as to produce a narrow targeted stream, or at a number of points so as to produce a ‘curtain’ effect to ensure that all relevant surfaces of the fish come into contact with bubbles (14a). Since a fish (6) may be discouraged from swimming along the tunnel (4) in the direction of the arrows by a continuous curtain of bubbles (14a), in some embodiments it can be desirable that the bubbles (14a) are introduced into the tunnel (4) only as the fish approaches the bubble generation zone (14). In other embodiments the source of bubbles is targeted towards specific points on the fish’s (6) outer surface.

Simultaneously with or coincidently with at least a portion of the bubble flow the photodiode (8a, 8b) detection system can be used to time and trigger activation of ultrasound waves, which may collapse bubbles entrained on the surface of the fish (6). In some embodiments the ultrasound waves are generated at one or more specific locations along or around the tunnel (6). In some embodiments the ultrasound generators are arranged at a number of locations within an ultrasound generation zone (16).

In the embodiment depicted (see Figure 4b) ultrasound generators (16a, 16b) are located at both the left (TL) and right (TR) sides of the tunnel (4). As depicted the ultrasound generators (16a, 16b) may include focussed transducers which focus the ultrasound energy at the bubbles and/or the surface of the fish. Alternatively the ultrasound energy may be introduced so as to broadly cover the cross-section of the tunnel (4). It will be appreciated that there may be one or more ultrasound generating transducers (16a, 16a) in the zone (16), and they may be arranged at any convenient location around and along the tunnel (4), according to preferences. It will be appreciated that the ultrasound generating zone (16) is desirably located upstream of the bubble generating zone (14) in relation to the swimming direction of the fish.

In other embodiments there may be more than one bubble generation zone (14). For example, there may be a bubble generation zone at the entrance to the tunnel (4) and another bubble generation zone (14) immediately adjacent the ultrasound generation zone (16) to increase the number of bubbles (14a) or the efficiency of bubble entrainment on the surface of the fish (6). Similarly, the apparatus may comprise both a broad, non-targeted bubble generation device and also a targeted bubble generation device proximate to the ultrasound generation zone (16). This may help to increase the number of bubbles (14a) that entrain onto desired target areas of the fish or parasites. For example, bubbles (14a) may be fed into a ‘pre-treatment pond’ in the absence of ultrasound waves (not shown) where attachment to lice might be encouraged in free swimming fish (6).

The ultrasound generators (16a, 16b) may generate continuous ultrasounds, when activated, or may generate ultrasound pulses of a desired frequency and duration. By triggering of the ultrasound generators (16a, 16b) on approach of a fish (6) towards the effector zone the running cost and power demand of the apparatus may be reduced, and transducer longevity may be enhanced whilst ensuring that ultrasound is only applied once a fish is in the correct region / sub-zone of the effector zone. In combination with the use of relatively low pressure ultrasounds, this may also minimise collateral damage to the fish population.

Causing the fish (6) to pass through a curtain of bubbles (14a) or to swim freely in a bubble pre-treatment pond can be particularly beneficial for optimising process effectiveness / efficiency where the specific locations of parasite infestation on the fish’s (6) skin is not known, because bubbles are more likely to become entrained directly on the parasites. It can be advantageous, for similar reasons, to expose a fish to ultrasounds across substantially the whole of its surface area. Targeting bubbles (14a) and/or ultrasounds to specific locations on the fish’s surface (according to expected or known parasite infestation locations can further improve the efficiency of the process. With this in mind, bubble generator and ultrasound generator locations may be adjustable within the apparatus of the invention.

The effector / treatment zone (2), especially the tube (4), may include a parasite (louse) harvesting collection area where removed parasites collect and can be subsequently removed.

Example 3 - Bubble placement

Subjecting bubbles placed on parasites in control environments to ultrasounds of various pressures, and generated from various directions, may help determine the optimal configuration of the system in order to remove parasites from the surface of an animal.

Figure 5 is a schematic representation of a suitable bubble placement on parasites according to an embodiment of the invention. Bubbles (14a) may be created on the surface of a parasite (18) resting on a skin equivalent surface (not shown). Bubbles may then be subjected to a range of ultrasound pressures generated from multiple directions in order to ascertain the direction and pressure that allows the generation of a radial flow on collapse of the bubble, such that the parasite is detached from the substrate. Imaging means (not shown) may be used to study bubble placement on the parasites (18) and collapse characteristics.

The optimal direction and timing of ultrasound generation may depend on the behaviour of the parasite. Salmon lice are primarily positioned on the surface of the fish with the leading edge of the louse directed against the predominant flow direction (20) in such a way as to create a downwards force. Therefore, bubble collapse induced to the rear of the leading edge at an acute angle Θ (22) may be most likely to create radial flows that may prise the louse from the skin. Accordingly, targeting the ultrasound to preferentially induce the collapse of optimally placed bubbles may increase the efficiency of the process.

Optimal characteristics of ultrasound generation in order to dislodge the parasites may then be used to define the placement and timing of ultrasound generation in relation to an animal swimming in a treatment zone, such that bubble collapse at the optimal angle relative to the surface of the animal is preferentially induced.

Additionally, measurement of jet velocities in such a system, using high speed imaging, may be used to infer water hammer pressure experienced by the system at the skin interface. This may allow definition of the pressure threshold for shear of the parasite and the upper threshold for acceptable damage or stress to the animal.

Example 4 - Bubble collapse for parasite lysis

Figure 6a shows the damage to a single cell arising from a microjet impact; and Figure 6b shows the topographical cross-section corresponding to the white arrow in Figure 6a (see Prentice eta!., Nature Physics, 2005, 1(107):107-110).

Ultrasound frequency and pressure as well as bubble size may be selectively chosen using experiments where the damage to single cells is assessed. In this example, a human breast cancer cell line (MCF-7) cultured in Dulbeccos modified Eagle’s medium was exposed to bubbles of 4-5 pm diameter and 20 ps bursts of 1 MHz ultrasound at peak negative pressure of 1.39 MPa (±14%).

Example 5 - Bubble collapse for dislodging

Figure 7 shows a top down view of a bubble attached to a biofilm before (left) and after (right) ultrasound activation. A quiescent microbubble was put in contact with and below an E. coli PHL628 biofilm grown for 24 hours on a horizontal coverslip (Figure 7a). 50 pulses (50 ps) of 1 MHz, 0.6 MPa (peak negative pressure) ultrasounds were applied and the same region was monitored after 9 minutes (Figure 7b). A biofilm clearance area (shaded) of circa 30 pm in diameter was observed at the location where the bubble initially rested. Similarly introduced surface shear flows may remove sea lice without harming the host salmon.

Example 6 - Bubble collapse for molecular delivery

Figure 8 shows the formation of large clearance zones in a cell monolayer and molecular (small molecule) uptake into cells upon bubble collapse.

Cell monolayers grown on a polymeric slip to confluence were subjected to insonation. Figure 8a is an optical microscopy image showing a large clearance zone (CZ) on a cell monolayer that had been previously fully confluent prior to insonation. Figure 8b is a fluorescence microscopy image of the same area as Figure 8a, showing that molecular uptake of calcein has occurred, predominantly in those cells on the periphery of the clearance zones.

The clearance zone size is a function of acoustic pressure applied to the bubble and bubble displacement from the substrate film.

Claims

Claims

1. A method of reducing parasite infestation on an aquatic animal which includes the steps of: a) providing bubbles formed in a liquid on or near the surface of the animal or parasite; and b) exposing the local environment surrounding the animal or parasite to sound waves, wherein the sound waves induce resonance and asymmetric collapse of the bubbles.
2. A non-therapeutic method for improving the condition, appearance, meat quality or growth rate of an aquatic animal, the method comprising the steps of: a) providing bubbles in a liquid medium on or near the surface of the animal; and b) exposing the bubbles to sound waves so as to induce resonance and asymmetric collapse of the bubbles.
3. The method of claim 1 or claim 2, wherein the intensity of the sound waves is selected so that the collapsing of the bubbles generates a jet that has sufficient energy to kill, dislodge or stun a parasite.
4. The method of claim 1 or claim 2, wherein the intensity of the sound waves is selected so that the collapsing of the bubbles creates a laminar radial flow on the surface of an aquatic animal that is sufficient to shear off a parasite, or so that the acoustically active bubble or bubble ensemble generates microstreaming locally in order to shear off parasites.
5. The method of any preceding claim, wherein the intensity of the sound waves and duration of exposure are selected to: a) kill parasites by formation of an energetic liquid jet upon asymmetric collapse of the bubbles by using a high intensity short pulse train; b) remove parasites from the surface of the animal by creating a jet impact with radial flow upon asymmetric collapse of the bubbles using an intermediate intensity short pulse train; c) remove parasites from the surface of the animal by creating a microstreaming shear flow using low to intermediate intensity ultrasound and intermediate to long exposure periods; or d) any combination of a), b), and/or c).
6. The method of any preceding claim, wherein the frequency of the sound waves is determined using the formula:

where ro is the average or median radius of the bubbles, γ is the polytropic coefficient, po is the ambient pressure and p is the density of the liquid.
7. The method of any preceding claim, wherein the sound waves frequency is between 20 kHz and 2 MHz.
8. The method of any preceding claim, wherein the bubbles are created by injection of gas in the liquid, or wherein the bubbles are pre-formed with a specific diameter or range of diameters and released into the liquid.
9. The method of claim 8, wherein the bubbles are injected or released in the vicinity of an aquatic animal.
10. The method of claim 9, wherein the bubble injection or release is targeted towards preferential locations of parasite infestation on the animal.
11. The method of any of claims 1 to 7, wherein the bubbles are provided or placed by bringing at least a portion of the animal into contact with air and entraining bubbles at the surface of the animal or parasites upon re-immersion in the liquid, or wherein acoustically active bubbles are created in a macroscopic water jet used to shower the animal when partially in contact with the air.
12. The method of claim 11, wherein bringing at least a portion of the animal into contact with the air is achieved by lifting or forcing the animal temporarily out of the liquid.
13. The method of any preceding claim, wherein the aquatic animal is a fish.
14. The method of claim 13, wherein the fish is a salmon.
15. The method of any preceding claim, wherein the parasite is non-pathogenic.
16. The method of any preceding claim, wherein the parasite comprises one or more Lepeophtheirus or Caligus species.
17. The method of claim 16, wherein the parasite comprises one or more of Lepeophtheirus salmonis, Caligus clemensi and Caligus rogercresseyi.
18. The method of any preceding claim, wherein the bubbles are stabilised within the liquid.
19. The method of claim 18, wherein stabilising the bubbles includes creating a film at the gas-liquid interface, or by including a surfactant in the liquid.
20. The method of claim 19, wherein the film comprises a surfactant.
21. The method of any of claims 18 to 20, wherein the film comprises a lipid compound.
22. The method of any preceding claim, wherein the sound waves have intensity and duration that are selected to avoid significant damage to the aquatic animal.
23. The method of any of claims 19 to 21, wherein the film comprises molecules for delivery to the animal or parasite, and the ultrasound exposure parameters are adapted to allow molecular delivery to the animal or cells of the animal.
24. The method of claim 23, wherein the film comprises one or more of: a vaccine, therapeutic agent, or antibiotic.
25. An apparatus for reducing parasite infestation of an aquatic animal, for improving the condition, appearance, meat quality or growth rate of an aquatic animal, wherein the apparatus comprises: a) means for providing bubbles in a liquid on or near the surface of the animal or parasite; b) means for exposing the bubbles to sound waves, wherein the sound waves induce resonance and asymmetric collapse of the bubbles.
26. The apparatus of claim 25, wherein the means for providing bubbles comprises a mechanism for bringing at least a portion of the animal into contact with air such that bubbles are entrained on the surface of the animal or parasites upon re-immersion of the animal.
27. The apparatus of claim 26, wherein the mechanism for bringing at least a portion of the animal into contact with air comprise means for lifting or forcing the aquatic animals out of the liquid.
28. The apparatus of claim 25, wherein the means for providing bubbles comprises a device for injection of bubbles in the liquid, or for release of pre-formed bubbles, preferably in the vicinity of the aquatic animals.
29. The apparatus of any of claims 25 to 28, wherein the means for providing bubbles comprises a device for injecting and/or releasing bubbles of controlled size, for example, of a predetermined range of diameters.
30. The apparatus of claim 28 or claim 29, wherein the device for injecting bubbles or releasing bubbles is adapted to target bubbles to preferential locations of parasite infestation on the animal.
31. The apparatus of any of claims 25 to 30, wherein the means for exposing the bubbles to sound waves is adapted to target sound waves to the animal.
32. The apparatus of claim 29, wherein the means for exposing the bubbles to sound waves is adapted to target sound waves to preferential locations of parasite infestation on the animal.
33. The apparatus of any of claims 25 to 27, which comprises a device for creating a macroscopic water jet containing acoustically active bubbles.
34. The apparatus of claim 33, wherein the device for creating a macroscopic water jet containing acoustically active bubbles is arranged such that when the animal is brought partially into contact with air the macroscopic water jet containing acoustically active bubbles showers the animal while it is partially in contact with the air.
35. The apparatus of any of claims 25 to 34, which comprises a treatment zone in which sound waves are delivered.
36. The apparatus of claim 35, wherein the treatment zone comprises a channel through which an aquatic creature can move.
37. The apparatus of claim 35 or claim 36, wherein the means for providing bubbles comprises a device arranged to inject bubbles into the treatment zone.
38. The apparatus of claim 35 or claim 36, wherein the means for providing bubbles is arranged to provide bubbles on the surface of the animal or parasites prior to entry of the animal into the treatment zone.
39. The apparatus of any of claims 35 to 38, further comprising means for detecting the presence of an animal in the treatment zone.
40. The apparatus of claim 39, wherein the means for exposing the bubbles to sound waves is arranged to generate sound waves in the treatment zone upon detection of an animal.
41. The apparatus of claim 39 or claim 40, which is adapted to inject bubbles into the treatment zone upon detection of an animal.
42. The apparatus of claim 41, wherein the means for providing bubbles comprises a device for targeted injection of bubbles towards the animal, preferably towards locations of parasite infestation on the animal.
43. The apparatus of any of claims 25 to 42, further comprising a liquid medium and wherein the liquid is adapted to stabilise the bubbles.
44. The apparatus of claim 43, wherein the liquid comprises a film at the gas-liquid interface in order to stabilise the bubbles.
45. The apparatus of claim 44, wherein the film comprises a surfactant.
46. The apparatus of claim 44 or claim 45, wherein the film comprises a lipid compound and optionally wherein the film comprises molecules for delivery to the animal or parasite, and wherein the apparatus is adapted to deliver sound waves at a frequency, intensity and/or duration to allow molecular delivery in addition to parasite removal.
47. The apparatus of any of claims 25 to 46, wherein the means for providing sound waves includes a device for generating sound waves at a desired frequency and wherein the sound waves frequency is selected using the formula:

where ro is the average or median radius of the bubbles, γ is the polytropic coefficient, po is the ambient pressure and p is the density of the liquid.
48. The apparatus of any of claims 25 to 47, wherein the apparatus is adapted to deliver sound waves at a frequency of between 20 kHz and 2 MHz.
49. The apparatus of any of claims 25 to 48, wherein the means of providing sound waves is adapted to deliver sound waves at a pressure that is sufficient to cause collapsing of bubbles and generation of a jet that can kill or stun parasites, and/or a radial flow on the surface of the animal that can dislodge parasites.
50. The apparatus of any of claims 25 to 49, which is adapted to deliver sound waves at a pressure and duration that are low enough that the collapsing of bubbles does not significantly damage the surface of the aquatic animal.
51. A method for the treatment of a parasitic infestation of an aquatic animal, the method comprising: a) providing bubbles in a liquid on or near the surface of the animal or parasite; and b) exposing the local environment surrounding the animal or parasite to sound waves, wherein the sound waves induce resonance and asymmetric collapse of the bubbles.
52. The method of claim 51, wherein the sound waves have an intensity and the intensity of the sound waves is selected so that the collapsing of the bubbles generates a jet that has sufficient energy to kill or stun a parasite.
53. The method of claim 51, wherein the sound waves have an intensity and the intensity of the sound waves is selected so that the collapsing of the bubbles creates a laminar radial flow on the surface of an aquatic animal that is sufficient to shear off a parasite.
54. The method of any of claims 51 to 53, wherein the frequency of the sound waves is determined using the formula:

where ro is the average or median radius of the bubbles, γ is the polytropic coefficient, po is the ambient pressure and p is the density of the liquid.
55. The method of any of claims 51 to 54, wherein the sound wave frequency is between 20 kHz and 100 kHz.
56. The method of any of claims 51 to 55, wherein the bubbles are created by injection of gas in the liquid or by release of pre-formed bubbles.
57. The method of claim 56, wherein the bubbles are injected or released in the vicinity of an aquatic animal.
58. The method of claim 57, wherein the bubble injection is targeted towards preferential locations of parasite infestation on the animal.
59. The method of any of claims 51 to 55, wherein the bubbles are placed by bringing at least a portion of the animal into contact with the air so as to entrain bubbles at the surface of the animal or parasites upon re-immersion.
60. The method of claim 59, wherein bringing at least a portion of the animal into contact with the air is achieved by lifting or forcing the aquatic animal out of the liquid.
61. The method of any of claims 51 to 60, wherein the aquatic animal is a fish.
62. The method of claim 61, wherein the fish is a salmon.
63. The method of claim 62, wherein the parasites comprise one or more Lepeophtheirus or Caligus species.
64. The method of claim 63, wherein the parasites comprise one or more of Lepeophtheirus salmonis, Caligus clemensi and Caligus rogercresseyi.
65. The method of any of claims 51 to 64, wherein the bubbles are stabilised within the liquid.
66. The method of claim 65, wherein stabilising the bubbles includes creating a film at the gas-liquid interface or by inclusion of surfactants in the liquid.
67. The method of claim 66, wherein the film comprises a surfactant.
68. The method of claim 66 of claim 67, wherein the film comprises a lipid compound.
69. The method of any of claims 66 to 68, wherein the film comprises molecules for delivery to the animal or parasite, and the ultrasound exposure parameters are adapted to allow molecular delivery in addition to parasite removal.
70. The method of any of claims 51 to 69, wherein the sound waves have an intensity and duration that are selected to avoid significant damage to the aquatic animal.
71. A method for the delivery of molecules to an aquatic animal, the method comprising: a) providing bubbles in a liquid on or near the surface of the animal or a parasite on the surface of the animal; b) stabilising the bubbles by providing a film at the gas-liquid interface or by inclusion of surfactants in the bulk of the liquid, wherein the film or liquid comprises molecules for delivery to the animal or parasite; and c) exposing the local environment surrounding the animal or parasite to sound waves, wherein the sound waves induce resonance and asymmetric collapse of the bubbles; and the ultrasound exposure parameters are adapted to allow molecular delivery to the aquatic animal or to cells of the aquatic animal.
72. The method of claim 71, wherein the film comprises a surfactant and/or a lipid compound.
73. The method of claim 71 or claim 72, wherein the sound waves have an intensity and the intensity of the sound waves is selected so that the collapsing of the bubbles creates a laminar radial flow on the surface of an aquatic animal.
74. The method of any of claims 71 to 73, wherein the frequency of the sound waves is determined using the formula:

where ro is the average or median radius of the bubbles, γ is the polytropic coefficient, po is the ambient pressure and p is the density of the liquid.
75. The method of any of claims 71 to 74, wherein the sound waves frequency is between 20 kHz and 100 kHz.
76. The method of any of claims 71 to 75, wherein the bubbles are created by injection of gas in the liquid or by localised release of pre-formed bubbles.
77. The method of claim 76, wherein the bubbles are injected or released in the vicinity of an aquatic animal.
78. The method of any of claims 71 to 77, wherein the bubbles are placed by bringing at least a portion of the animal into contact with the air and so as to entraining bubbles at the surface of the animal or parasites upon re-immersion.
79. The method of claim 78, wherein bringing at least a portion of the animal into contact with the air is achieved by lifting or forcing the aquatic animals out of the liquid.
80. The method of any of claims 71 to 79, wherein the aquatic animal is a fish.
81. The method of claim 80, wherein the fish is a salmon.
82. The method of any of claims 71 to 81, wherein the molecule comprises a vaccine, therapeutic agent and/or an antibiotic.
83. The method of any of claims 1 to 24 or 51 to 82, wherein the population of bubbles is substantially within a predefined size or diameter range.
84. A composition comprising water for use in a method for the treatment of a parasitic infestation of an aquatic animal, the method comprising the steps of: a) providing bubbles in a liquid on or near the surface of the animal or parasite: and b) exposing the local environment surrounding the animal or parasite to sound waves, wherein the sound waves induce resonance and asymmetric collapse of the bubbles.
85. The composition for use of claim 84, which further comprises a gas.
86. The composition for use of claim 84 or claim 85, wherein the gas comprises air or oxygen.
87. The composition for use of any of claims 84 to 86, wherein the sound waves have an intensity and the intensity of the sound waves is selected so that the collapsing of the bubbles generates a jet that has sufficient energy to kill or stun a parasite.
88. The composition for use of any of claims 84 to 87, wherein the sound waves have an intensity and the intensity of the sound waves is selected so that the collapsing of the bubbles creates a laminar radial flow on the surface of an aquatic animal that is sufficient to shear off a parasite.
89. The composition for use of any of claims 84 to 88, wherein the frequency of the sound waves is determined using the formula:

where ro is the average or median radius of the bubbles, y is the polytropic coefficient, po is the ambient pressure and p is the density of the liquid.
90. The composition for use of any of claims 84 to 89, wherein the sound wave frequency is between 20 kHz and 100 kHz.
91. The composition for use of any of claims 84 to 90, wherein the bubbles are created by injection of gas in the liquid or by release of pre-formed bubbles.
92. The composition for use of claim 91, wherein the bubbles are injected or released in the vicinity of an aquatic animal.
93. The composition for use of claim 92, wherein bubble injection is targeted towards preferential locations of parasite infestation on the animal.
94. The composition for use of any of claims 84 to 90, wherein bubbles are placed by bringing at least a portion of the animal into contact with air so as to entrain bubbles at the surface of the animal or parasites upon re-immersion.
95. The composition for use of claim 94, wherein bringing at least a portion of the animal into contact with air is achieved by lifting or forcing the aquatic animal out of the liquid.
96. The composition for use of any of claims 84 to 95, wherein the aquatic animal is a fish.
97. The composition for use of claim 96, wherein the fish is a salmon.
98. The composition for use of claim 96 or claim 97, wherein the parasites comprise one or more Lepeophtheirus or Caligus species.
99. The composition for use of claim of any of claims 96 to 98, wherein the parasites comprise one or more of Lepeophtheirus salmonis, Caligus clemensi and Caligus rogercresseyi.
100. The composition for use of any of claims 84 to 99, wherein the bubbles are stabilised within the liquid.
101. The composition for use of claim 100, wherein stabilising the bubbles includes creating a film at the gas-liquid interface or by inclusion of surfactants in the liquid.
102. The composition for use of claim 101, wherein the film comprises a surfactant.
103. The composition for use of claim 101 of claim 102, wherein the film comprises a lipid compound.
104. The composition for use of any of claims 101 to 103, wherein the film comprises molecules for delivery to the animal or parasite, and the ultrasound exposure parameters are adapted to allow molecular delivery in addition to parasite removal.
105. The composition for use of any of claims 84 to 104, wherein the sound waves have an intensity and duration that are selected to avoid significant damage to the aquatic animal.
106. The composition for use of any of claims 84 to 105, wherein the method comprises providing a population of bubbles having substantially a pre-defined size or diameter range.