NO344699B1 - Composition for use in treating or preventing sea lice infestation on fish and a method of killing or damaging sea lice - Google Patents

Description

The present invention relates to compositions for that use in the treatment and/or prevention of a sea lice infestation on fish. Methods of killing or damaging sea lice are also encompassed. More specifically, the present invention provides methods for the treatment and/or prevention of a sea lice infestation on a fish using a composition comprising one or more chitinolytic enzymes.

Parasitic infestation of fish stocks represents a major source of economic loss for fish farmers, and severely impacts the welfare of the farmed animals. Sea lice, including Lepeophteirus salmonis (Lepeophteirus salmonis salmonis and Lepeophteirus salmonis oncorhynchi) and Caligus elongates, are parasitic crustaceans which are found in marine waters, and are known to be a threat to both farmed and wild fish stocks, in particular Atlantic Salmon (Salmo salar) and trout (Oncorhynchus mykiss). Sea lice live on the surface of fishes’ skin, and consume the skin, mucus and blood of their hosts, which may result in the formation of severe wounds. These wounds may in turn be the entry point for further sources of infection, for instance fungal, bacterial or viral infections. Lice infestations in farmed fish stocks thus result in economic losses from increased costs associated with handling and treating infestations, slower growth rates, and lower slaughter weight and classification of slaughtered fish, as well as reduced fish welfare.

L. salmonis mate on their host, and females carry fertilised eggs in a pair of egg strings containing from 100 to 1000 eggs. When the eggs hatch, the larvae undergo several life stages on their way to adulthood. The first three stages of life (Nauplius I and II, and Copepoditt) are free-living and planktonic, and larvae are capable of dispersing over a large area. In the life stages that follow (Chalimus I-IV), the lice are attached to the host’s skin by a special frontal filament, and during the pre-adult and adult life stages the lice move freely over the skin of the fish to feed (mobile life stages). In particular, the head and back regions of the fish are typically prone to infestation by foraging lice.

At present several different treatments exist to treat sea lice infestations in fish.

Countries such as Norway have also implemented a joint strategy to reduce sea lice infestations (Heuch et al., 2005, Aquaculture, 246(1-4), p79-92), which includes:

- the placement of sea farms at appropriate geographical locations,

- arranging sea farms into zones with synchronised production and farrowing,

- use of biological treatments (such as wrasse – Labridae) as cleaner fish to remove sea lice from the skin of farmed fish,

- monitoring and reporting of sea lice populations,

- monitoring and reporting sea lice sensitivity towards chemotherapeutic agents, and - strategic and optimised use of chemotherapeutic agents.

Biological treatments such as cleaner wrasse are an effective method for eliminating sea lice, and four species of wrasse are currently used in the production of salmon and rainbow trout in Norway: goldsinny wrasse (Ctenrolabrus rupestris), corkwing wrasse (Symphodus melops), rock cook (Centrolabrus exoletus) and juvenile ballan wrasse (Labrus bergylta).

However, there are limitations associated with the use of cleaner wrasse which diminish the effectiveness of their use. It is only possible to use cleaner wrasse during summer months (when sea lice populations are at their lowest). The cages and nets used in salmon must also be kept free from marine growth as cleaner wrasse will preferentially use this as their food supply. Finally, wrasse must be captured alive and transported to fish farms, as commercial attempts to farm wrasse have so far proved unsuccessful.

Chemotherapeutic treatments are important control strategies which have been adopted by the salmon industry. Currently used chemotherapeutic agents include pyretroids (such as Deltametrine and Cypermetrin), organophosphases (such as Azamethiphos), oxidative agents (such as hydrogen peroxide), benzamides which inhibit chitin synthase (such as Diflubenzuron and Teflubenzuron) and avermectins (such as Emamectin benzoate). However, the emergence of resistance towards several existing chemotherapeutic agents has reduced the efficacy of these treatments (Igboeli et al.2012, Aquaculture, 344, p40-47), and resistant lice can quickly recolonise an area that has been treated with a chemotherapeutic agent due to their short generation time and efficiency of dispersal. It is thus anticipated that the problem of resistance to chemotherapeutic agents will only grow with time.

It is also possible that such treatments may represent a risk to human health, and may accumulate in food chains and cause widespread damage to the marine environment. As such, there are problems with all of the current sea lice treatments, and safe and effective treatments for the elimination of sea lice in farmed fish stocks are required.

Several new approaches for preventing sea lice infestation have been proposed, including land-based fish farms, vaccination against sea lice, and breeding of lice-resistance fish. However, vaccination and breeding programmes are yet to be shown to be effective, and land-based fish farms produce high levels of waste and are expensive. Alternative treatments, including laser treatment, flushing sea lice off fish and warm water treatment are yet to be adopted by industry. The development of appropriate, commercially viable, effective, non-toxic, environmentally friendly treatments for the treatment of sea lice in fish are therefore needed.

Sea lice have been found to comprise genes encoding chitinase enzymes in their genomes, and sea lice with disrupted chitinase genes have been found to be deformed and have a highly reduced ability to infect salmon (Eichner et al., 2015, Experimental Parasitology, in press). It is surprising therefore that it has now been found that chitinolytic enzymes are effective in treating sea lice infestations.

The chitinolytic enzymes act on chitin and chitosan and include chitinases, chitobiases, chitin deacetylases, chitosanases and lytic polysaccharide monooxygenases (LPMOs), which are described in more detail hereinafter. The chitinolytic enzymes are capable of breaking down chitin which is an abundant, linear water-insoluble oligosaccharide, consisting of β-1,4 linked N-acetylglucosamine (GlcNAc) units. The deacetylated form of chitin, called chitosan, is a water-soluble heteropolymer of β-1,4-linked GlcNAc and D-glucosamine (GlcN). Chitosans differ in terms of the degree of acetylation, the distribution of acetyl-groups along the chain, and the length of their chain (Aiba, 1991, Int. J. Biol.

Macromol., 13(1), p40-44; Kubota & Eguchi, 1997, Polymer J., 29(2), p123-127; Rinaudo & Domard, 1989, Solution properties of chitosan, New York: Elsevier Applied Science).

However, chitin and chitosans are highly related, and it is difficult to divide them strictly into groups as the borderline is arbitrary; chitinous material found in nature varies in the degree of acetylation. Chitinous material tends to comprise chitin/chitosan polymer molecules as part of co-polymeric structures, i.e. structures where they associate in situ with several other (macro-) molecules e.g. proteins, carotenoids, glucans (Tharanathan & Kittur, 2003, Critical Rev. Food Sci. Nutr., 43(1), 61-87). In nature chitinous materials are important structural components in fungal cell-walls and in arthropods (Adams, 2004, Microbiology-Sgm, 150, p2029-2035; Gooday, 1990, Advances in Microbiol. Ecology, 11, p387-430; Raabe et al, 2007, Acta Biomaterialia, 3(6), p882-895; Rinaudo, 2006, Prog. Polym. Sci., 31(7), p603-632).

Even though several areas of applications have been suggested for chitinolytic enzymes, their use is still limited. The enzymatic conversion of chitin into well-defined bioactive chito-oligosaccharides is one of the main focus areas in the field (Aam et al., 2010, Marine Drugs, 8(5), p1482-1517). Other interesting applications are the suggested use of chitinases as a fungicide (Bliffeld et al., 1999, Theoret. Appl. Genet., 98(6-7), p1079-1086; Lorito et al., 1994, Microbiology-UK, 40, p623-629) or as an insect controlling compound (Cohen, 1993, Arch. Insect Biochem. Physiol., 22(1-2), p245-261; Doucet & Retnakaran, 2012, Adv. Insect Physiol., vol 43, p437-511; Kramer & Muthukrishnan, 1997, Insect Biochem. Mol. Biol., 27(11), p887-900). EP 1356733 A2 discloses pesticide and antiparasitic compositions containing chitinase. The compositions are intended to be used against animal and plant parasites. US 5935572 A discloses compositions containing a protease and a glycosidase, such as chitinase, to break down and destroy head lice and head lice eggs. TU et al; in J. of Vertebrate Path., 2010, vol.104, no.2, p.75-82 discloses chitinases from S.

marcescens identified on the basis that such strains contain a miticidal activity against the bee mite V. destructor. S. marcescens is formerly known as an effective chitin degrader. The results show that both S. marcescens culture and the isolated kinases have miticidal effect, probably by breaking down the mite’s scale layer.

Whilst changes in protease activity in the skin mucus of salmon in response to sea lice infections have been observed (Firth et al., 2000, J. Parasit., 86(6), p1199-1205; Ross et al., 2000, Diseases Aquat. Org., 41(1), p43-51), the use of enzymes which act on chitin and/or chitosan to treat sea lice has not been suggested. Surprisingly, and as discussed in more detail below, such enzymes have been found to be of particular utility in the treatment of sea lice. Contrary to expectation these enzymes may be used effectively in environments compatible with the treatment of cold-water fish and are not immediately degraded or washed away from the target sea lice even when used in an aqueous environment. However, unlike other chemotherapeutic chitinase inhibitors, these enzymes do not accumulate in the marine environment. These enzymes thus offer non-toxic, biological tools for controlling sea lice in a way that avoids environmental damage but effectively removes sea lice to enhance commercial efficacy of fish farming and improve animal welfare.

Thus, in a first aspect, the present invention provides a composition containing one or more chitinolytic enzymes for use in treating or preventing a sea lice infestation on a fish. When more than one chitinolytic enzyme is to be used, the present invention provides a product containing at least two chitinolytic enzymes as a combined preparation for simultaneous, separate or sequential use in treating or preventing a sea lice infestation on a fish.

Additionally, the present description disclose the use of one or more chitinolytic enzymes in the preparation of a medicament for the treatment or prevention of sea lice infestation on a fish.

The ‘treatment’ of a sea lice infestation on a fish refers to reducing, alleviating or eliminating one or more symptoms of the sea lice infestation, relative to a fish without a sea lice infestation. Preferably the symptom to be treated is the presence of live sea lice on the fish and the treatment serves to eliminate and/or kill sea lice on the fish. The treatment may be absolute, in terms of eliminating and/or killing all sea lice or may be partial, e.g. achieving a reduction in the number of live sea lice on a fish. Preferably live sea lice are reduced by at least 25, 50 or 75% relative to the number of live sea lice before the treatment.

The ‘prevention’ of a sea lice infestation on a fish refers to reducing the number of live sea lice on a fish relative to the number that would be present without the preventative treatment. This prevention may be facilitated by, for example, reducing or eliminating the ability of a sea louse to bind to a fish, and/or reducing or eliminating the ability of a sea louse to propagate on a fish (i.e. the extent of infestation by a sea louse is reduced). Preferably the preventative treatment reduces the presence of live sea lice by at least 25, 50 or 75% relative to the number of live sea lice on a fish to which the treatment has not been applied.

The present invention thus acts by killing sea lice, and/or inhibiting or retarding growth and/or development of a sea louse, and/or reducing reproductive ability of a sea louse, and/or weakening of a sea louse. The methods and uses described herein may be achieved by killing or damaging the sea louse. ‘Damaging’ a sea louse refers to decreasing the viability of the sea louse, e.g. by retarding or inhibiting the growth and/or shortening its life cycle and/or development of the sea louse, and/or reducing the ability of the sea louse to reproduce, and/or weakening the sea louse (i.e. decreased mobility and/or motility, binding to a fish, and/or feeding).

The term ‘sea lice’ refers to any species of crustacean within the order Siphonostomatoida, family Caligidae. Such lice are capable of infesting a fish (i.e. attaching to a fish and parasitically feeding off the skin, scales, mucus and blood of a fish). Preferred sea lice are of the genera Lepeophtheirus or Caligus. Particularly preferred species include Lepeophteirus salmonis (Lepeophteirus salmonis salmonis and Lepeophteirus salmonis oncorhynchi) and Caligus elongates. Although the present invention may be used in treating or preventing infestation on a fish by sea lice of any stage of development, it may have particular utility in treating or preventing infestation of a fish by adult sea lice, i.e. sea lice which have reached maturity. This may include female sea lice and mobile (i.e. planktonic) sea lice. Thus, in preferred aspects the methods and uses are not used to treat or prevent infestation by non-adult sea lice.

Sea lice ‘on’ a fish may be present on the skin, scales, eyes, nasal passages, gills, mouth, or fins of a fish, or any surface of fish which may be accessible to water and/or an externally administered therapeutic composition (referred to herein as a composition, medicament or product). An infestation denotes the presence of at least one (preferably at least 1, 2, 5, 10 or more) sea louse on a fish host. It will be appreciated, however, that an infestation may also denote the presence of less than 1 sea louse per fish when averaged for a population of fish (i.e. less than one sea louse per fish, e.g.0.75, 0.5.0.25 or 0.1 sea lice per fish).

As referred to herein, the step of ‘administering’ the composition to the fish may be performed by any convenient means as described in more detail hereinafter. Conveniently, to allow application to large numbers of fish the composition (or chitinolytic enzymes) are added to water in which the fish are present. For the purposes of the administration the fish are preferably collected in a small volume tank or vessel to reduce the amount of enzyme required. Thus the fish may be held in a vessel or container used to contain fish, such as a pen, net, cage, hatching pond, or holding tank a vessel or a vessel or container used to transport fish, such as a fish carrier boat (wellboat) or a pipe, or any other transporting tank (e.g. a bucket or similar container) and the enzymes may be added to the water (or aqueous environment as described hereinafter) within the vessel or container.

Alternatively, the enzymes may be administered directly to the fish, e.g. by dipping or bathing fish in a solution containing the enzyme(s), or topically applying a composition to the fish e.g. by smearing, brushing, rubbing or spraying a suitable preparation of the enzymes on the fish.

It is contemplated that the methods and uses of the present invention may be used to treat or prevent sea lice infestation in any species of fish which is susceptible to infection or infestation by sea lice. In a preferred embodiment, the present invention relates to the treatment of sea lice in fish of the family Salmonidiae, such as species of salmon or trout, which may preferably be from the genera Salmo, Oncorhynchus, Hucho, Salvinus or Lenok. Particularly preferred species which may be treated using the methods and compositions of the present invention thus include, Salmo salar (Atlantic salmon), Salmo trutta, (brown trout), Oncorhynchus clarkia (cutthroat trout), Oncorhynchus gorbuscha (Humpback salmon), Oncorhynchus keta (dog salmon/keta salmon), Oncorhynchus kisutch (coho salmon), Oncorhynchus masou (masu salmon/cherry salmon), Oncorhynchus mykiss (Rainbow trout), Oncorhynchus nerka (Sockeye salmon), Oncorhynchus tshawytscha, and Oncorhynchus tshawytscha (Chinook salmon) and Salvelinus (charr) species.

The methods of the present invention utilise a composition comprising one or more enzymes having chitinolytic activity (chitinolytic enzymes). A ‘chitinolytic enzyme’ refers to an enzyme which is capable of hydrolysing, degrading or weakening a structure comprising chitin, chitinous material (such as chitosan) or chitooligomers (also known as ‘chitinolytic activity’). Hydrolysis may involve, for example, the lysis of bonds connecting the monomers, or deacetylation of chitin. Degradation or weakening refers to alterations to the substrate which ultimately yield to or assist lysis of the chitin, chitinous material or chitooligomers.

Chitin is a biological polymer which comprises the linear water-insoluble oligosaccharide consisting of β-1,4 linked N-acetylglucosamine (GlcNAc) units. Chitin forms structures with the related polymer chitosan, being the deacetylated form of chitin, and comprising alternating β-1,4-linked GlcNAc and D-glucosamine (GlcN) repeats. The degree of acetylation of chitin/chitosan varies, and thus for the purposes of the present invention the term ‘chitin’ may be used interchangeably with ‘chitinous material’, and refers to biological material which comprises chitin and/or chitosan monomers, e.g. in a copolymer.

Chitooligomers refers to smaller polymers which may be derived from chitin and encompasses di and tri-saccharides.

Thus, ‘chitinolytic activity’ of an enzyme should not be considered to be limiting to only enzymes capable of hydrolysing, degrading or weakening chitin within chitinous material, and this term should equally be considered to encompass enzymes which are capable of hydrolysing, degrading or weakening chitosan and/or chitooligomers.

As referred to herein, the activity of the enzyme denotes the ability of the enzyme to convert its substrate in a unit time (under standard conditions of the substrate, e.g. chitin, chitosan or chitooligomers, temperature, pH etc.). For convenience this is expressed in enzyme units (U, µmol/min).

Enzymes having chitinolytic activity (i.e. chitinolytic enzymes) are classified into different families in the carbohydrate-active enzymes database (CaZY, http://www.cazy.org/) based on their amino acid sequence. An overview of chitinolytic enzymes and their CaZY classification is presented in Table 1.

Table 1. Overview of the chitinolytic enzymes and their classification into families according to the CaZY database.

Abbreviations used: GH = Glycoside hydrolase family (EC 3.2.1), CE = carbohydrate esterase family, AA = auxiliary activity family.

Chitinolytic enzymes may fall within the families of enzymes including chitinases, chitobiases, chitin deacetylases, chitosanases and lytic polysaccharide monoxygenases (LPMOs), and enzymes selected from any of these families may be used in the methods and uses of the present invention. Thus, one or more chitinolytic enzymes may be selected from the group consisting of chitinases, chitobiases, chitin deacetylases, chitosanases and lytic polysaccharide monooxygenases.

Chitinases hydrolyse chitin into oligosaccharides of GlcNAc, also called chitooligomers, and are found in glycoside hydrolase (GH) families 18 and 19 (EC 3.2.1.14). GH18 chitinases act by a substrate-assisted retaining double displacement mechanism, whereas GH19 enzymes use an inverting direct displacement mechanism. GH20, also referred to as chitobiases (EC 3.2.1.52), further degrades chitooligomers into GlcNAc monomers.

Enzymes that can reduce the degree of acetylation of chitin and convert chitin into chitosan by deacetylation are called chitin deacetylases (EC 3.5.1.41), and are found in carbohydrate esterase (CE) family 4. Hydrolysis of the (1-4)-β-glycosidic bond in chitosan is carried out by chitosanases (EC 3.2.1.132), which have been detected in families GH5, GH7, GH8, GH46, GH75 and GH80. Finally, enzymes in auxiliary activity (AA) families AA9 (formerly GH61), AA10 (formerly CBM33) and AA11, are lytic polysaccharide monooxygenases, several of which act on chitin. These LPMOs may be used to work synergistically with chitinases. The LPMOs include chitin-binding proteins (CBPs) which contain carbohydrate-binding modules of family 33 (CBM33) which are thought to facilitate chitinase accessibility to chitinous matrices by introducing breaks in the chitin chains.

However, some CBM33 domains of CBPs are able to enzymatically cleave chitin and hence they are classified as LPMOs in family AA10.

The different chitinolytic enzymes act on most forms of chitin/chitosan. For example, chitinases hydrolyse highly deacetylated chitins (chitosans) as well as chitin (Aiba, 1991, Int. J Biol. Macromol., 13(1), p40-44; Heggset et al, 2009, Biomacromol., 10(4), p892-899; Hobel et al., 2005, Extremophiles, 9(1), p53-64). Some chitosanases may also work on highly acetylated chitosans (chitins) (Heggset et al., 2010, Biomacromol., 11(9), p2487-2497). Chitinolytic enzymes as described herein, particularly from any of the above families, encompass functionally equivalent molecules, i.e. comprising one or more amino acid substitutions, insertions or deletions relative to a ‘wild-type’ chitinolytic enzyme and may also be used in the methods and uses of the present invention. Thus, for example, such variant molecules may have more than 70, 80, 90, 95, 96, 97, 98 or 99% amino acid sequence identity to wild type sequences (as assessed over the full length of the sequence), particularly those described herein. Functionally equivalent molecules are those which have the same or substantially the same enzymatic function (activity) as the wild type molecule to which they are compared (e.g. when used under the same conditions on a particular substrate, produce at least 90% of the amount of the same product or degrade at least 90% of the same substrate as the wild-type enzyme or have at least 90% of the wild-type enzyme’s activity). Preferably, variant molecules will catalyse the same reaction (i.e. the same substrates are used and the same products are formed) as the wild-type sequences. However, it is also contemplated that variant molecules may show alterations in specificity and/or product production.

The methods of the present invention utilise a composition comprising at least one enzyme having chitinolytic activity. The composition may comprise a single chitinolytic enzyme, or alternatively may comprise more than one (i.e. two, three, four, five or more) different enzymes. When multiple enzymes are to be used, they may be administered separately, sequentially or simultaneously. Where different enzymes are used, the enzymes may exhibit a symbiotic effect wherein the degradation of chitin/chitosan is enhanced relative to the use of a single enzyme.

‘Different’ enzymes refers to two or more chitinolytic enzymes having different (i.e. non-identical) amino acid sequences. This may refer to two or more enzymes capable of catalysing the same chemical reaction (having the same enzymatic activity) but having different amino acid sequences. The different enzymes may therefore be from the same CaZY family (or EC designation), as defined above. It is anticipated that the different enzymes may be derived from different organisms or may be alternative variant forms of the same chitinolytic enzyme (i.e. one or more of the enzymes may be a mutant enzyme). In one embodiment, the different enzymes having the same enzymatic activity may have optimal enzymatic activity at different temperatures or pH values. The term ‘different’ may also refer to two or more enzymes capable of catalysing different chemical reactions, (having different enzymatic activities). The different enzymes may thus be from different CaZY families (or EC designations).

Thus in one embodiment, the composition may comprising two or more different enzymes having the same enzymatic activity, or from the same family of chitinolytic enzymes (i.e. having the same CaZY designations (or EC designation)). In an alternative embodiment, the composition may comprise two or more different enzymes having different enzymatic activities, i.e. selected from different families of chitinolytic enzymes (i.e. having different CaZY designations (or EC designations)). In a further embodiment the composition may comprise enzymes having different enzymatic activities, (i.e. selected from two or more different families of chitinolytic enzymes), and may further comprise more than one (i.e. two, three, four, five of more) different enzymes from within each enzyme family, or having the same enzymatic activity.

As discussed above, any enzyme having chitinolytic activity may be used in the methods of the present invention. Chitinolytic enzymes may thus be derived from any source, i.e. a chitinolytic enzyme from any organism may be used. In nature, chitinolytic enzymes are secreted by chitin-degrading organisms, including bacteria (such as Serratia marcescens), fungi (such as Trichoderma) and actinomycetes (such as Streptomyces), and can be obtained from chitin-degrading organisms through culture using chitin as the sole carbon source. These organisms typically produce and secrete a series of chitinolytic enzymes belonging to different families of chitinolytic enzymes, and thus an enzyme mixture can be obtained using this method (e.g. as described in the Examples herein). Preferably, a chitinolytic enzyme may be derived from a chitin-degrading organism (i.e. an organism that naturally degrades chitin), including bacteria, fungi and actinomycetes, however, a chitinolytic enzyme may alternatively be obtained from other sources such as insects or animals.

In a preferred embodiment, the chitinase enzyme is a GH18 family enzyme and preferably may be selected from the list consisting of ChiA, ChiB and ChiC (or a combination of two or all three of these enzymes may be used). In a particularly preferred embodiment, the enzyme may be derived from Serratia marcescens. The sequences of ChiA, ChiB and ChiC, from Serratia marcescens may be found in Uniprot Database Accession No. B3VK60 (version 1 sequence submitted 2 September 2008), A0A059UJTO (version 1 sequence submitted 3 September 2014), and Q068W1 (version 1 sequence submitted 31 October 2006), respectively and sequences related to these sequences by the sequence identity levels indicated hereinbefore are preferred.

In a further preferred aspect, the composition comprises an LPMO, particularly a chitinolytic enzyme from the AA10 CaZy classification. In a particularly preferred embodiment, the LPMO is a CBP, e.g. CBP21. In a particularly preferred embodiment, the enzyme may be derived from Serratia marcescens. The sequence of CBP21 from Serratia marcescens may be found in Uniprot Database Accession No. O83009 (version 1 sequence submitted 1 November 1998) and sequences related to this sequence by the sequence identity levels indicated hereinbefore are preferred.

Preferred combinations of chitinolytic enzymes that may be used in accordance with the invention include one or more chitinases (preferably from the GH18 family) with one or more LPMOs (preferably from the AA10 family), e.g. one or more chitinases selected from ChiA, ChiB and ChiC in combination with a CBP such as CBP21.

A chitinolytic enzyme may be produced by expression of a gene encoding a chitinolytic enzyme in a host cell. In a first embodiment, a chitinolytic enzyme may be expressed as a native (i.e. homologous) chitinolytic enzyme in a chitin-degrading organism. In a preferred embodiment, a mixture of chitinolytic enzymes may be obtained from a chitindegrading organism from the expression of more than one (i.e. two, three, four, five or more) native chitinolytic enzymes. In a preferred embodiment of the present invention, the chitindegrading organism may be grown (cultured) using chitin as the sole carbon source.

In an alternative embodiment, a gene encoding an enzyme having chitinolytic activity may be inserted into a non-native expression vector or construct and inserted into a host cell which does not naturally comprise said gene (heterologous expression). In other words, a host cell may be modified to recombinantly express a chitinolytic enzyme. This approach also allows for the possibility of engineering or modifying the enzymes in order to alter their properties, if desired, e.g. temperature-optimum, stability, pH-optimum etc. This method may also be used to engineer and screen mutants to identify those with appropriate enzyme characteristics. In a further embodiment, more than one (i.e. two, three, four, five or more) genes encoding chitinolytic enzymes may be inserted into the same expression vector or construct prior to insertion of the vector or construct into the host cell or may be inserted into separate constructs and inserted into the host cell independently.

In methods in which the chitinolytic enzyme(s) is to be purified from the source material or from cells modified to express the enzyme(s), following expression of a chitinolytic enzyme (or chitinolytic enzymes), it is preferred to recover the enzyme from the culture medium. The chitinolytic enzyme may be secreted by the host cell, and thus may be present in the soluble (liquid) phase of the culture medium. In such an embodiment, cells may be removed from the culture medium (e.g. by centrifugation or filtration), and the resulting supernatant may be used in the methods of the present invention. Alternatively, the chitinolytic enzyme may be retained within the host cell, and thus may not be present in the soluble phase of the culture medium. It may thus be desirable to separate the host cells (e.g. by centrifugation or filtration) and discard the culture medium, before lysing the cells to produce a cell lysate comprising a chitinolytic enzyme(s), which may be used in the methods or uses of the present invention. Optionally, the enzyme may be further purified from the culture supernatant or cell lysate before being used in the methods or uses of the present invention using any protein purification methods known in the art. In a preferred embodiment, the proteins may be purified using chitin-coated beads or a purification column comprising immobilised chitin. Preferably the enzyme(s) as used herein has at least 50, 60, 70, 80 or 90% purity.

The chitinolytic enzymes may also be modified to comprise one or more tags which may be used to assist the purification of the enzymes, such as a 6xHis tag or FLAG tag. Other tags may, however, also be used.

Chitinolytic enzymes may also be obtained from commercial sources, such as from Sigma-Aldrich (e.g. Chitodextrinase, Poly(1,4-β-[2-acetamido-2-deoxy-D-glucoside]) glycanohydrolase from Serratia marcescens (C7809) or Streptomyces griseus (C6137); Chitosan N-acetylglucosaminohydrolase from Streptomyces griseus (C9830); N-acetyl-βglucosaminidase and chitodextrinase from Trichoderma viride (C8241); or Glucanex (Lysing enzymes) from Trichoderma harzianum (L1412).

It is desirable that the chitinolytic enzyme(s) which is used in methods and uses of the invention has chitinolytic activity at conditions under which the method or use of the present invention is performed. Thus, an appropriate enzyme or enzymes having activity under these conditions should be selected, or as mentioned above one or more appropriate enzymes may be engineered or modified to comprise one or more amino acid insertions, deletions or substitutions relative to its native sequence, in order to alter the temperature, pH and/or salinity at which the enzyme has maximal (optimal) chitinolytic activity. Where necessary, the method may be modified to accommodate the enzyme which is used and, for example, the pH and/or temperature may be modified to closer to the enzyme(s) preferred conditions such that at least 20, 30, 40 or 50% of the enzyme(s)’s optimum activity is retained. Furthermore, administration methods may be used to optimize the enzyme(s)’s activity, e.g. direct application to the fish rather than into water which may not have the optimum temperature or pH. Nevertheless, as noted in the Examples, chitinolytic enzymes used outside their optimal pH or temperature are also effective in methods or uses of the invention.

Preferably, an enzyme will have activity at a temperature of between 0-25°C, more preferably at a temperature of between 4-20°C, and more preferably at a temperature of between 10-19, 12-17 or 13-15, or in the alternative 7-15°C, 8-14°C, 9-13°C, or 10-12°C. The enzyme will preferably have chitinolytic activity at a temperature of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C or at any temperature inbetween, or at higher temperatures, such as 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35°C.

Preferably, the enzyme will retain activity when water is heated, for instance when water is heated to treat a sea lice infestation. Thus, in a one embodiment, the enzyme may be obtained from an extremophile organism, preferably a thermophilic organism. In an alternative embodiment, however, the enzyme has optimal activity at a lower temperature (e.g. at 4-20ºC). Such enzymes may be wild-type or may be derived by modifying a wild-type enzyme to have enhanced activity at a low temperature. Preferably said enzyme has optimum activity (or at least 20, 30, 40 or 50% of optimum activity) within the above indicated range or temperature indicated above.

It is also desirable that the enzyme will have activity at a pH range of between pH 4-11, more preferably at a pH range of between pH 6-10, or pH 6.5-9.5, pH 7-9, or pH 7.5-8.5. Preferably said enzyme has optimum activity (or at least 20, 30, 40 or 50% of optimum activity) within the above indicated pH range indicated above. Preferably the enzyme retains its activity and remains stable at a pH range outside its optimal pH range.

Conveniently the enzyme(s) is selected to have activity in either sea water (salt water, or saline conditions) and/or in brackish water.

The composition of the present invention may optionally comprise further proteinaceous and/or non-proteinaceous components, in addition to the one or more chitinolytic enzymes. In a preferred embodiment, the composition may comprise further enzymes, e.g. with hydrolytic properties, such as one or more proteases.

Nevertheless, the chitinolytic enzymes form the major active component in the composition or for use in the methods or uses of the invention. Thus, in a preferred embodiment, the composition comprises at least 20% (w/w) chitinolytic enzymes as a percentage of the total proteinaceous material (preferably of the total enzymatic proteinaceous material) in the composition which is used. Thus the composition may comprise at least 25, 30, 35, 40, 45, 50, 60, 70, 80 or 90% (w/w) chitinolytic enzymes as a percentage of proteinaceous material (preferably of the total enzymatic proteinaceous material), or the proteinaceous material (or preferably the enzymatic proteinaceous material) in said composition may be solely comprised of chitinolytic enzymes (i.e.100% (w/w) chitinolytic enzymes as a percentage of total proteinaceous, preferably enzymatic, material). However, the composition may alternatively comprise lower proportions of chitinolytic enzymes, for instance at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.12.5 or 15% (w/w) chitinolytic enzymes as a percentage of the composition’s total proteinaceous (preferably enzymatic) material. In a particularly preferred embodiment, the chitinolytic enzymes are the principal enzymatic component of the composition, i.e. comprise >50% of the enzymatic components of the composition.

In addition to the proteinaceous material the composition may comprise any pharmaceutically acceptable diluent, carrier or excipient. "Pharmaceutically acceptable" as referred to herein refers to ingredients that are compatible with other ingredients of the compositions as well as physiologically acceptable to the recipient. The nature of the composition and carriers or excipient materials, dosages etc. may be selected in routine manner according to choice and the desired route of administration, pH, temperature etc.

Thus, the active ingredient (the chitinolytic enzyme(s)) may be incorporated, optionally together with other active substances as a combined preparation, with one or more conventional carriers, diluents and/or excipients, to produce conventional galenic preparations such as powders, sachets, suspensions, emulsions, solutions, aerosols (as a solid or in a liquid medium), ointments, and the like. The compositions may be stabilized by use of freeze-drying, undercooling or Permazyme.

Suitable excipients, carriers or diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, calcium carbonate, calcium lactose, corn starch, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, water, water/ethanol, water/glycol, water/polyethylene, glycol, propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof. Agents for obtaining sustained release formulations, such as carboxypolymethylene, carboxymethyl cellulose, cellulose acetate phthalate, or polyvinylacetate may also be used. The compositions may additionally include lubricating agents, wetting agents, emulsifying agents, viscosity increasing agents, granulating agents, disintegrating agents, binding agents, osmotic active agents, suspending agents, preserving agents, adsorption enhancers (e.g. lice penetrating agents), organic solvent, antioxidant, stabilizing agents, emollients, silicone, alpha-hydroxy acid, demulcent, anti-foaming agent, moisturizing agent, vitamin, ionic or non-ionic thickeners, surfactants, filler, ionic or non-ionic thickener, sequestrant, polymer, propellant, alkalinizing or acidifying agent, opacifier, colouring agents and fatty compounds and the like.

The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the fish by employing techniques well known in the art.

The use of solutions, suspensions, gels and emulsions (or powders which may be made into such forms) are preferred, e.g. the active ingredient may be carried in water, a water-based liquid, an oil, a gel, an emulsion, an oil-in water or water-in-oil emulsion, a dispersion or a mixture thereof.

Topical compositions and administration are use which include gels, creams, ointments, sprays, lotions, salves, sticks, soaps, powders, films, aerosols, drops, foams, solutions, emulsions, suspensions, dispersions e.g. non-ionic vesicle dispersions, milks and any other conventional pharmaceutical forms in the art.

Ointments, gels and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will, in general, also contain one or more emulsifying, dispersing, suspending, thickening or colouring agents. Powders may be formed with the aid of any suitable powder base. Drops and solutions may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing, solubilising or suspending agents. Aerosol sprays are conveniently delivered from pressurised packs, with the use of a suitable propellant.

In addition to the pharmaceutically acceptable carrier or excipient, the composition may comprise at least 0.0005% chitinolytic enzymes (w/w) as a percentage of its total composition. Thus, the composition (at the point of administration) may comprise at least 0.0005, 0.001 or 0.005 to 25%, e.g.0.005 to 1% or 0.001 to 10%, such as 0.005 to 0.5% chitinolytic enzyme(s) (w/w of the final preparation for administration, particularly for topical administration). Said concentrations are determined by reference to the amount of the chitinolytic enzymes themselves and thus appropriate allowances should be made to take into account the purity of the enzymes. Effective single doses may lie in the range of from 1-100mg/day, preferably 2-10mg/day, per fish, administered as a single dose. Conveniently the composition may be provided in more concentrated form (e.g. for dilution 100- or 1000-fold) for administration to the aqueous environment containing the fish during the treatment. Thus, the composition may contain as little as 0.0005% w/w chitinolytic enzyme(s) when used directly, or may contain more e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 20, 25, 30, 35, 40, 45 or 50% chitinolytic enzyme(s) as a percentage of its total composition (w/w) when applied indirectly. Higher concentrations may be used for more severe infestations. Thus, for example, the above doses may be considered appropriate for moderately infested fish (e.g. with ≤5 lice per fish) and may be elevated proportionally for fish with higher levels of infestation.

Dosages to be used in methods and uses of the invention may be determined in routine manner and may depend upon the nature of the enzyme(s) (or components of the composition), mode of administration, extent of infestation, age and size of the fish, and size of the container in which the fish are present (when indirect administration is used) etc.

As discussed hereinbefore, administration may be by direct (topical) or indirect (additional to aqueous environment) methods.

Thus, in a preferred embodiment of the present invention, the fish to be treated will be present in an aqueous environment. An ‘aqueous environment’ refers to any body of water or water within a container in which a fish may be found, and includes a natural body of water, such as an ocean, sea, lake, pond, river or stream, or a vessel, or water within a container used to contain fish, such as a pen, net, sea cage, hatching pond, or holding tank. In a preferred embodiment, a fish may be in a sea cage or net. A sea cage or net may typically have a volume of 1,000 - 5,000 m<3>, or may be larger, for instance 10,000 m<3>-30,000 m<3>. A sea net may hold up to 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or 100,000 fish, depending on its size. In one embodiment, the cage/net may be lifted up and a waterproof sail/canvas placed underneath it to produce an aquarium cage. It is also anticipated that the fish may be present within a vessel or container used to transport fish, such as a fish carrier boat (wellboat) or a pipe, or any other transporting tank (e.g. a bucket or similar container). A wellboat may contain 500-3000 m<3>of water, preferably 1000-2000 m<3>of water. More preferably a wellboat may contain approximately 1500 m<3>of water. A wellboat may contain up to 100,000 fish, for instance up to 75,000, 50,000, 40,000, 30,000, 20,000 or 10,000 fish.

Preferably 50,000 – 100,000 fish are treated in the method of the invention.

Preferably when treatment is conducted in the vessel or container, the volume of water is less than 30,000 m<3>, e.g. between 10-30,000 m<3>(preferably 20,000-30,000 m<3>) and may contain from 10-100,000 fish (preferably 50,000-100,000) fish.

The composition is added to the aqueous environment to provide a suitable concentration (as described hereinbefore) in the water surrounding the fish. The enzymes may be evenly distributed in the water (e.g. when vessels or containers are used) or may be unevenly distributed with local concentrations in the location of the fish, with concentrations in these areas at the levels indicated hereinbefore. Thus, concentrated forms of the compositions (e.g. as powders or solutions) may be added, for example, at feeding areas when the fish have access to a large aqueous environment. The composition (or chitinolytic enzymes) may be added to the water directly, or in the form of a slow-release tablet, pill or pellet, to allow the continuous release of the enzymes. The enzymes may also be provided in fish feed.

In the alternative, the enzymes are administered directly to the fish. In this case fish may be removed from a larger aqueous environment and temporarily immersed in dips or baths containing the enzyme(s). Alternatively, topical mixtures may be applied directly to the fish, e.g. a solution, gel, emulsion etc., may be directly applied to the surface of the fish infested with sea lice, or at risk of being infested with sea lice, e.g. by use of loaded brushes or other applicators such as aerosols. The fish may also pass through an oil film containing the enzymes.

The composition or enzyme may also be administered in combination with other sea lice treatment techniques. In particular, the composition or enzymes may be administered in combination with a thermal de-lousing treatment in which a fish is passed into warm water in order to remove lice (i.e. the composition may be added to the heated water). In such an embodiment, the fish may be subjected to an increased temperature for a period of time, such as 10, 20, 30, 40, 50 or 60 seconds, during which time the fish may also come into contact with the composition or enzymes of the invention.

The methods may be performed in either sea water (salt water, or saline conditions) or in brackish or fresh water. As referred to herein, sea water has a salinity of from 530-650mM. Saline may have a NaCl concentration of from 100-200mM, preferably 150mM. The method may also be performed in brackish water, i.e. where a body of fresh water meets a body of sea water.

Preferably the methods of the present invention are performed under conditions which are similar to the natural environment in which the fish may be found in order to reduce the distress or discomfort caused to the fish. In a particularly preferred embodiment, the present invention is performed in situ, i.e. in the fishes’ natural environment. It is thus desirable that the composition comprising one or more chitinolytic enzymes has activity under the conditions in which the fish which are to be treated may be found.

Preferably, the method may be performed at the temperature of sea water in the location of the fish which are to be treated. Thus the method may be performed at a temperature of between 0-25°C, more preferably at a temperature of between 4-20°C, and more preferably at a temperature of between 7-15°C, or 8-14°C, 9-13°C, or 10-12°C. The method may be performed at a temperature of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C or at any temperature in-between, or at higher temperatures, such as 30°C.

It is also desirable that the method is performed at a suitable pH. Ideally, the method may be performed at the pH of sea water in the location of the fish which are to be treated. Thus in a preferred embodiment the method may be performed at a pH range of between pH 5-11, more preferably at a pH range of between pH 6-10, or pH 6.5-9.5, pH 7-9, or pH 7.5-8.5. The compositions as used herein, which may form a further aspect of the invention, preferably have the pH indicated above.

The methods of the present invention may find particular utility in treating sea lice in farmed fish, i.e. fish which are kept in fish farms. In particular, the present invention relates to the treatment of sea lice in farmed Salmonidiae species, such as Atlantic salmon (Salmo salar) and trout (Oncorhynchus mykiss). To avoid environmental effects, the activity of the enzymes used during treatment may be controlled by addition of chitin to the aqueous environment after the treatment (and forms a preferred aspect of the invention).

The composition may be administered to a fish in one or more doses (i.e. two, three, four, five or more doses) which may each, or collectively, provide a dose of enzyme as described hereinbefore. All of the composition which forms a single dose may be administered at the same time or over a period of time. Thus, for example the composition of one dose may be applied to the aqueous environment or the fish directly, at a single time point. In the alternative, the full dose may be provided over a course of minutes, hours or days. The latter is particularly appropriate when the enzyme(s) may be diluted with the passage of time (e.g. in large aqueous environments). The fish may thus be exposed to said composition for a period of 1, 2, 3, 4, 5, 10, 15, 30 or 60 seconds (e.g. less than a minute), or 2, 3, 4, 5, 10, 15, 30 or 60 minutes, or 2, 3, 4, 5, 6, 9, 12, 15, 18 or 24 hours, or 2, 3, 4, 5, 6 or 7 days for each dose. In total, the treatment (including treatments for prevention) which may include multiple doses may be performed over the above periods for a single dose, or longer, e.g. for 2, 3, 4 or 5 weeks. Conveniently, a period of time may be left between successive doses, e.g.5, 15, 30 or 60 minutes, or 2, 5, 12, 18 or 24 hours, or 2, 4, 7, 14, 30, 60 days. Preferably, a single dose is administered to the fish for a period of up to 24 hours, e.g. from 5 minutes to 24 hours. It will be appreciated that depending on the administration method, the fish may remain exposed to the enzymes beyond the dosing time (albeit at lower concentrations) due to remaining enzyme in the aqueous environment or in the topical compositions applied to the fish. The above dosing times refer to times at which the dosages are in the therapeutic/prevention range as described hereinbefore.

In a further aspect, the present invention provides a method of killing or damaging a sea louse, said method comprising contacting said sea louse with a composition of the invention. This method may be performed under the conditions defined above.

As referred to herein, ‘contacting’ refers to allowing the enzyme(s) to come into spatial proximity to the sea louse in an appropriate medium. As defined hereinbefore in relation to administration methods, this may be by application of the composition to an aqueous medium in which the sea louse is present or may be by direct application to the sea louse. ‘Damaging’ has the meaning as given hereinbefore.

Such a method may thus be performed in the presence or absence of a fish, i.e. the sea louse need not be present on a fish. Conveniently the method may be applied to a vessel or container as defined herein that has been used, is being used, or will be used to contain or transport fish.

The methods and compositions of the present invention may be better understood with reference to the following Examples and Figures, in which:

Figure 1 shows an SDS-PAGE gel of the natural chitinolytic enzyme mix from Serratia marcescens after purification on chitin beads. ChiA, ChiB, ChiC and CBP21 were identified by comparing with literature data for Serratia chitinases and are all highlighted by arrows. The protein standard used is High Range Rainbow Molecular Weight Markers (Amersham, GE Healthcare Life Sciences, UK).

Figure 2 shows an SDS-PAGE gel of recombinantly produced chitinolytic enzymes from Serratia marcescens; ChiA, ChiB, ChiC and CBP21. ChiC is prone to proteolysis between the catalytic domain and C-terminally located additional domains involved in substrate binding, and thus lane 4 has an upper (full length) and lower band (catalytic domain only – Gal et al., 1998, FEMS Microbiol. Lett., 160(1), p151-158). The protein standard used is High Range Rainbow Molecular Weight Markers (Amersham, GE Healthcare Life Sciences, UK).

Figure 3 shows an SDS-PAGE gel of Chimax-O, a chitosanase belonging to the GH8 CaZY family (Amicogen Inc., Korea). Lane 1 shows Chimax-O dissolved in water before dialysis, lane 2 shows Chimax-O after dialysis against sterile sea water, pH 7.0. The protein standard used is High Range Rainbow Molecular Weight Markers (Amersham, GE Healthcare Life Sciences, UK).

Figure 4 shows a scatter plot showing a comparison of the decrease in viability after treatment of sea lice with 1) only sea water (diamonds); 2) boiled recombinant enzyme mix (squares); 3) recombinant enzyme mix (triangles); recombinant enzyme mix contains ChiA, ChiB, ChiC and CBP21.

Figure 5 shows a scatter plot showing a comparison of the decrease in viability after treatment of sea lice with 1) only sea water (diamonds); 2) boiled Chimax-O (squares); and Chimax-O (triangles). A – sea lice were identified as dead when they did not move. B – sea lice were identified as dead when they changed colour from brown/grey to red.

Figure 6 shows that sea lice treated with Serratia marcescens chitinolytic enzyme cocktail release GlcNAc, whereas in the control reaction (Tris-HCl added) no GlcNAc was released, when measured by HPLC. The amount of GlcNAc released by the sample containing the chitinolytic enzymes increased from 7 hours to 24 hours.

Examples

General methods

Preparation and storage of sea lice

L. salmonis, or sea lice, were collected at sea farms in the Rogaland and Hordaland region on the west coast of Norway the same day as the experiments were conducted. The sea lice for our experiments were always collected from a farm that was not undergoing any current treatment. Sea lice are found on the surface of the fish, referred to here as host fish. Host fish were anesthetized according to normal routines in the industry, and sea lice were then carefully removed from the fish skin. The majority of the sea lice were in their mobile life stage, but attached sea lice were also observed. The sea lice were then transferred to a container filled with water from the specific sea farm, and taken to the laboratory immediately. The sea temperature varied from 7.3⁰C to 12.0⁰C, and is specified for each experiment.

Preparation of colloidal chitin

To prepare colloidal chitin, α-chitin from the Arctic cold water shrimp Pandalus borealis (Chitinor AS, Senjahopen, Norway) was first passed through a 30-mesh sieve.30 g was then slowly added to 800 mL ice cold, concentrated HCl (37% v/v) while stirring. When the chitin was evenly distributed, the HCl-suspension was slowly heated to 37°C, with moderate stirring. The suspension then changed into a less viscous and more transparent solution. This solution was filtered through glass-wool, and slowly transferred to ice cold dH2O, which leads to precipitation of chitin. This suspension was stirred for 30 minutes, and then kept at 4°C overnight. The following day the supernatant was decanted, the remaining mixture was then centrifuged (using 750 mL bottles in a Heraeus Multifuge 3SR+ centrifuge, Thermo Scientific, MA, USA) and a Sorvall Heraeus swing-out rotor, type TTH-750 (Thermo Scientific, MA, USA), 10 minutes at 4000 rpm and 4°C). The pellet was washed several times with dH2O, and finally resuspended in 500 mL dH2O and autoclaved. The colloidal chitin was incubated at 4°C in the dark. The dry matter concentration in this colloidal chitin suspension was approximately 37 g/l.

Preparation of a natural chitinolytic enzyme mix

Basal salt and buffer (BSB) media with colloidal chitin (Bromke & Venuti, 1999, Can. J., Microbiol., 45(1), p88-91) was prepared by mixing 0.375 g Na2CO3(Sigma-Aldrich, MO, USA), 0.375 g KH2PO4(Sigma-Aldrich, MO, USA), 0.325 g (NH4)2SO4(Sigma-Aldrich, MO, USA), 0.250 g NaCl (Sigma-Aldrich, MO, USA), 0.125 g MgSO4(Sigma-Aldrich, MO, USA) and 33.35 g HEPES buffer (Sigma-Aldrich, MO, USA), 62.5 mL colloidal chitin (37 mg/ml) and dH2O to 900 mL. pH was then adjusted to pH 6.5, using NaOH, and the volume adjusted to 1 L using dH2O. The BSB medium with colloidal chitin was then autoclaved at 121°C for 15 minutes. Serratia marcescens BJL200 was grown in BSB media with colloidal chitin at 225 rpm shaking at 30°C for approximately 48 hours. Cells were harvested by centrifugation (250 mL bottles, 10000 rpm, 10 minutes at 4°C in a Heraeus Multifuge 3SR+ centrifuge with a F14-6x250LE FiberLite rotor; Thermo Scientific, MA, USA). The supernatant was centrifuged once more, and subsequently filtered through a VacuCap 90 PF Filter Unit, 0.8/0.2 µm (Pall, MI, USA) to remove any remaining bacteria and obtain a sterile enzyme solution.

The chitinolytic enzymes present in the supernatant were concentrated approximately 100 times using a Vivaflow-200 system with a 10,000 Da cut-off PES-membrane (Sartorius, VWR International, PA, USA). Purification of the enzymes was carried out using chitin beads (New England Biolabs, MA, USA).10 mL chitin beads were packed in a glass-column with an inner diameter of 1.0 cm and washed with 0.14 M HEPES-buffer pH 6.5.4 ml with an approximate protein concentration 3 mg/ml was then carefully applied, before the column was washed with several column volumes of HEPES-buffer. The proteins were eluted with 20 mM acetic acid and kept on ice. The pH of the eluted protein solution was immediately adjusted to a pH between 6.0 and 8.0 using NaOH. The proteins were dialyzed against BisTris buffer, pH 6.0 using Snake Skin Dialysis tubing with a 10K MWCO cut-off (Thermo Scientific, MA, USA). Finally, the proteins were filtered through a 0.2 µm PES membranefilter (VWR International, PA, USA) to make it sterile, and analysed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to confirm their size and purity (see Figure 1). Protein concentrations were determined using the BioRad Protein Assay (Bio-Rad, Hercules, CA, USA) with BSA as a standard (Bradford, 1976, Anal. Biochem., 72(1-2), p248-254).

Preparation of recombinant chitinolytic enzymes

ChiA, ChiB, ChiC and CBP21 from Serratia marcescens were all purchased from Professor Vincent Eijsink’s laboratory at the Norwegian University of Life Sciences (NMBU) in Ås, Norway. The enzymes were produced and purified according to standard routines described previously (Brurberg et al., 1994, FEMS Microbiol. Lett., 124(3), p399-404;

Brurberg et al., 1996, Microbiology-UK, 142, p1581-1589; Synstad et al., 2008, Biosci.

Biotech. Biochem., 72(3), p715-723; Vaaje-Kolstad et al., 2005, J. Biol. Chem., 280(12), p11313-11319). The proteins were shipped on ice as solutions in 50 mM Tris-HCl, pH 8.0, and stored at 4°C after arrival. The proteins were dialyzed against sterile sea-water using Snake Skin Dialysis tubing with a 10K MWCO cut-off, and analysed using SDS-PAGE to confirm their size and purity (see Figure 2). Protein concentrations were determined using the BioRad Protein Assay with BSA as a standard (Bradford, 1976, supra).

Chimax-O, a chitosanase belonging to family GH8 and originating from a Bacillus sp. strain, was purchased from Amicogen Inc, Korea www.amicogen.en.ec21.com/Enzymes--2606798_2606800.html.1 g of the product was dissolved in 4 mL of dH2O. The solution was transferred to Snake Skin Dialysis tubing with a 10K MWCO cut-off, and dialyzed against sterile sea water, pH 7.0, adjusted with HCl (see Figure 3). The chitosanase-solution was then filtered through a 0.2 µm PES membrane-filter (VWR International, PA, USA) to make it sterile. The protein concentration was determined using BioRad Protein Assay with BSA as a standard (Bradford, 1976, supra).

Example 1 – Killing of sea lice with recombinant chitinolytic enzyme mix

In this experiment a recombinant chitinolytic enzyme mix, containing enzymes from the gram-negative soil bacterium Serratia marcescens obtained as described above, was applied to kill or weaken sea lice. Purified enzymes were used to ensure that the observed effects were caused by the chitinolytic enzymes alone. The sea lice were collected the same day as the experiment was conducted as described above. The sea temperature that day was 12.0⁰C. They were transferred to the reaction beakers with a sterile inoculating loop.

Materials and methods:

Three different treatments were compared in three 100 ml beakers, containing 23 sea lice and 60 ml sterile sea-water (Marine SeaSalt, Tetra, Melle, Germany) each. The sea water naturally had a pH of approximately 8.0. The three different treatments were as follows:

Beaker 1: Only sterile sea water was added (control)

Beaker 2: Boiled recombinant enzyme mix was added (control)

Beaker 3: Recombinant enzyme mix was added

The recombinant enzyme mix consisted of purified Serratia marcescens ChiA, ChiB, ChiC, and CBP21. Preparation of the recombinant enzymes is described above. Purified ChiA, ChiB, ChiC and CBP21, all in sterile sea water, were mixed into a stock solution. The stock solution was added to beaker 2 and 3, and masses of the enzymes added were 2.4 mg, 1.8 mg, 0.57 mg and 1.4 mg of CBP21, ChiA, ChiB and ChiC, respectively. Beaker 1 contained sea water alone. The three beakers were then incubated at 15°C. After two hours fresh air was added into each beaker using a Maxima R air pump and air stone (JBL ProSilent Aeras Micro S2), to improve the conditions for the sea lice. Dead sea lice were identified, counted and removed from the beakers every 24 hour. The criteria for the sea lice being verified as dead were extremely strict; sea lice were only counted as dead when they had changed colour from brown/grey into red. Notably, they stopped moving before the colour change.

Results

Results from this experiment are shown in Figure 4. The data clearly show that sea lice die faster in the presence of the recombinant enzyme mix (Figure 4, series no.3 -triangles). The sea lice clearly lived for longer in the two control samples (Figure 4, series no.

1 and no.2 – diamonds and squares respectively), with no added enzyme and addition of a boiled recombinant enzyme mix, respectively. Figure 4 also shows that, generally, it is difficult to keep the sea lice alive for a longer period under laboratory conditions. In all setups, eventually, the sea lice die.

Discussion

The results show that after several days, the sea lice die even when no or inactivated (boiled) enzyme has been added. Since the sea lice feed on their host, they will die naturally after a period of time when removed from the fish surface. However, we can see a difference during the first few days where sea lice die faster with chitinolytic enzymes present. We also observed less movement in the beaker with enzyme present.

The terms chosen for this experiment were carefully considered according to several factors: 1) Pumping fresh air into the beakers was performed to improve the conditions for the sea-lice, giving them the best possible opportunity to survive for a longer period of time away from their host.2) The pH was chosen according to the natural salmon breeding environment. Sea water usually has a pH of approximately 8.0. The enzymes used have different pH-optima, which are all below 8.0 (Synstad et al., 2004, 2008). The pH used in this experiment reflects the optimum for salmon-breeding.3) The temperature in our experiment was set at 15 °C. When temperatures are above 20°C, there are usually no problems concerning sea lice in the sea. We therefore chose a temperature well below 20°C, but still relatively high to ensure enzyme activity.

Example 2 – Killing of sea lice with ChimaxO, a chitinolytic enzyme mix

In this experiment Chimax-O, a commercially available chitosanase, was applied to kill or weaken sea lice. The experiment was conducted to analyse whether chitosanases may have the same effect on sea lice as chitinases (previously shown in Example 1). The sea lice were collected the same day as the experiment was conducted as described above. Sea temperature this day was 7.3⁰C. They were transferred to the Erlenmeyer flasks using a sterile inoculating loop.

Materials and methods

Three different treatments were compared in three 100 ml Erlenmeyer flasks, containing 25 sea lice and 100 ml sterile sea-water (Marine SeaSalt, Tetra, Melle, Germany) each. The pH of the sea water was adjusted to pH 7.0 using HCl. The three different treatments were as follows:

Flask 1: Only sterile sea water was added (control)

Flask 2: Boiled Chimax-O was added (control)

Flask 3: Chimax-O was added

Preparation of the Chimax-O is described above. The Chimax-O solution was added to beaker 2 and 3, boiled and un-boiled, respectively.7 mg of Chimax-O was added to each beaker. Beaker 1 contained sea water alone. The three beakers were then incubated at 15°C. After two hours, fresh air was continually pumped into to each flask using a Maxima R air pump and air stone (JBL ProSilent Aeras Micro S2), to improve the conditions for the sea lice. Dead sea lice were identified, counted and removed every 24 hour. Sea lice were qualified as dead using two alternative criteria, meaning that two datasets were generated. In the first method, the sea lice were verified as dead when they changed colour from grey to red (they stopped moving earlier), identical to the criteria used in Example 1 (= colour criteria). In the second method, sea lice were presumed dead when they stopped moving, even when pushed with a sterile inoculating loop (= movement criteria).

Results

The results from this experiment are presented in Figure 5a (counting lice using the movement criteria) and Figure 5b (counting lice using the colour criteria). The results very clearly show that sea lice die faster in the presence of Chimax-O (series no.3 - triangles) compared to the controls (series no.1 and 2). Comparison of Figures 5a and 5b shows the same trend, albeit with a small time delay between the observed effects in Figure 5b and Figure 5a.

Discussion

The results are presented in two different scatter plots, in Figure 5b the sea lice were detected as dead when they changed colour from brown/grey into red, whereas in Figure 5a the sea lice where detected as dead when they stopped moving. Both detection methods showed the same overall results, which demonstrate that adding Chimax-O kills or weaken the sea lice. On the basis of the similarity between Figures 5a and 5b we suggest that the sea lice are already dead when they are no longer moving, and that the colour change is just a subsequent step in the degradation-process. Use of the movement criteria gives an even better separation of the treated versus control samples, as shown in Figure 5a.

The conditions chosen for this experiment were carefully considered, taking into account several factors: The optimum activity of the enzyme is found in the pH-range between pH 4.0 and pH 6.0 for Chimax-O (Choi et al., 2004, Appl. Environ. Microbiol., 70(8), p4522-4531). At pH 7.0, Chimax-O only retains 30 % of its maximum activity (Choi et al., 2004, supra). The pH of the sea water was therefore adjusted to pH 7.0, to ensure the presence of sufficient enzyme activity.

Example 3 - Detection of the degradation of lice chitin, using a natural cocktail of chitinolytic enzymes

In this experiment, single sea lice were degraded using chitinolytic enzymes to confirm that the sea lice contain chitinous material. Sea lice were collected as previously described. Sea temperature this day was 10⁰C.

Materials and methods

Two single sea lice were first washed three times with 0.75 mL dH2O, and then dried at 50°C in an oven for approximately 72 hours. The sea lice were then transferred to one eppendorf tube each. The first sea louse was then incubated in 100µl 50 mM TrisHCl pH 8.0, at 50°C. The second sea louse was incubated with 100 µl natural Serratia marcescens chitinolytic enzyme mix (34 mg/ml) at 50°C. Preparation of the natural chitinolytic enzyme mix is described above. As a blank, an Eppendorf tube containing 100µl 50 mM TrisHCl pH 8.0 was also incubated at 50°C. After 7 and 24 hours, samples were taken from all Eppendorf tubes for analysis of breakdown products of chitin (since we were using a complete enzyme cocktail, the (almost) only product expected and detected was monomeric N-acetylglucosamine, GlcNAc).10 µl of sample was added 10 µl 50 mM H2SO4to stop the reaction, and then filtered using a 0.45 µm Duraporex membrane Multiscreen 96 well plate (Millipore).8 µl portions of the inactivated and filtered samples were analysed by HPLC using Dionex Ultimate 3000 HPLC System (Dionex Corporation, Sunnivale, CA, USA), equipped with a Rezex RFQ-Fast H+ (8%, 100x7.8 mm) from Phenomenex, an Ultimate 3000 autoinjector (Dionex Corp.) and an Ultimate 3000 column compartment (Dionex Corp.). The liquid phase consisted of 5mM H2SO4, the flow rate was 1.0 ml/min, and the temperature of the column compartment was 85°C. Eluted oligosaccharides were detected at 195 nm.

Quantification of peaks was performed by comparing with standard samples, using Chromeleon, version 6.80 (Dionex Corp.).

Results and discussion

Results from the experiment are presented in Figure 6. No GlcNAc was detected in the control sample containing no chitinolytic enzymes, even after 24 hours incubation. In contrast, GlcNAc was detected in the sample containing the natural Serratia marcescens chitinolytic enzyme cocktail. Furthermore, an increase in the amount GlcNAc was observed after 24 hours, confirming that the release is a result of enzyme activity. These results confirm that sea lice contain chitin, as sea lice were the only substrate for the chitinolytic enzymes in this reaction. No GlcNAc was detected in the blank (results not shown).

Example 4 – Protocol for the treatment of sea lice in fish

Farmed fish (typically salmon) are treated using the composition of the present invention in a wellboat or a sealed cage in order to treat or prevent a sea lice infestation. The fish are collected in the vessel in their normal aqueous environment and held in a sealed container (i.e. a body of water not continuously mixing with sea water). Ideally the water volume is reduced by appropriate means. The composition (or enzymes) of the invention are added at a dose as indicated previously. The composition or enzymes is added directly to the water in the vessel. The water in which the fish are kept is continually oxygenated, and treatment takes place under normal conditions for the fish, i.e. the same pH and temperature as the sea water in order to reduce the distress caused to the fish. Fish are safely and humanely kept under such conditions for up to 24 hours.

The treatment may be conducted in conjunction with other sea lice treatment methods, for instance a thermal treatment method in which fish are passed into warm water for a period of approximately 20-30 seconds. The composition or enzymes of the present invention may be added to the warmed water, i.e. so that the fish are passed into warm water containing one or more chitinolytic enzymes.

Claims (25)

Claims

1. A composition containing one or more chitinolytic enzymes for use in treating or preventing a sea lice infestation on a fish.

2. The composition for use as claimed in claim 1 wherein said one or more chitinolytic enzymes are selected from the group consisting of chitinases, chitobiases, chitin deacetylases, chitosanases and lytic polysaccharide monooxygenases.

3. The composition for use as claimed in claim 1 or 2 wherein said composition comprises two or more different chitinolytic enzymes.

4. The composition for use as claimed in claim 3 wherein said two or more different chitinolytic enzymes have different enzymatic activities.

5. The composition for use as claimed in any one of claims 1 to 4 wherein said composition comprises one or more of ChiA, ChiB and ChiC.

6. The composition for use as claimed in any one of claims 1 to 5 wherein said composition comprises CBP21.

7. The composition for use as claimed in any one of claims 1 to 6 wherein said chitinolytic enzymes comprise at least 20% (w/w) of the enzymatic proteinaceous material in said composition.

8. The composition for use as claimed in any one of claims 1 to 7 wherein said composition is for use in said treatment or prevention which is to be performed at a temperature of between 4°C and 20°C.

9. The composition for use as claimed in any one of claims 1 to 8 wherein said composition is for use in said treatment or prevention which is to be performed at a pH of between 7.0 and 9.0.

10. The composition for use as claimed in any one of claims 1 to 9 wherein said sea lice are Lepeophteirus salmonis or Caligus elongates.

11. The composition for use as claimed in any one of claims 1 to 10 wherein said sea lice are adult.

12. The composition for use as claimed in any one of claims 1 to 11 wherein said fish is from the family Salmonidiae.

13. The composition for use as claimed in claim 12 wherein said fish is a salmon or trout.

14. The composition for use as claimed in claim 13, wherein said fish is selected from the group consisting of Salmo salar, Salmo trutta, Oncorhynchus clarkii, Oncorhynchus gorbuscha, Oncorhynchus keta, Oncorhynchus kisutch, Oncorhynchus masou, Oncorhynchus mykiss, Oncorhynchus nerka, Oncorhynchus tshawytscha and Salvelinus species.

15. The composition for use as claimed in any one of claims 1 to 14 wherein said fish are farmed fish.

16. The composition for use as claimed in any one of claims 1 to 15 wherein said fish is present in an aqueous environment and said composition is to be administered to said environment.

17. The composition for use as claimed in any one of claims 1 to 16 wherein said composition is to be administered to said fish in one or more doses.

18. The composition for use as claimed in claim 17 wherein all of the composition to be used in said one dose are to be administered at the same time or over a period of time.

19. The composition for use as claimed in claim 17 or 18 wherein said fish is exposed to said composition for up to 24 hours for each dose.

20. A method of killing or damaging a sea louse, said method comprising contacting said sea louse with a composition containing one or more chitinolytic enzymes, wherein said composition is as defined in any one of claims 1 to 7.

21. The method as claimed in claim 20 wherein said method is performed under the conditions defined in claim 8 or 9 and/or said sea louse is as defined in claim 10 or 11.

22. The method as claimed in claim 20 or 21, wherein said sea louse is present in an aqueous environment and said composition is administered to said environment.

23. The method as claimed in claim 22, wherein said aqueous environment is the water in a vessel or container used to transport or hold fish.

24. The method as claimed in any one of claims 20 to 23 wherein said composition is administered to said sea louse in one or more doses, wherein all of the composition used in said one dose may be administered at the same time or over a period of time.

25. The method as claimed in claim 24 wherein said sea louse is exposed to said composition for up to 24 hours for each dose.