Note: Descriptions are shown in the official language in which they were submitted.
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Sea Lice Treatment
The present invention relates to the treatment and/or prevention of a sea lice
infestation on fish, and compositions for that use. 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
oncorhynch0 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 (Salm 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
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- 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.
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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 etal., 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, chain deacetylases, chitosanases and lytic polysaccharide
monooxygenases
(LPM0s), 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 13-1,4 linked N-acetylglucosamine (GIcNAc) units. The
deacetylated form of
chitin, called chitosan, is a water-soluble heteropolymer of 13-1,4-linked
GIcNAc and D-
glucosamine (GIcN). 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 etal., 2010,
Marine Drugs, 8(5), p1482-1517). Other interesting applications are the
suggested use of
chitinases as a fungicide (Bliffeld of al., 1999, Theoret. Appl. Genet., 98(6-
7), p1079-1086;
Lorito etal., 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).
Whilst changes in protease activity in the skin mucus of salmon in response to
sea
lice infections have been observed (Firth etal., 2000, J. Parasit., 86(6),
p1199-1205; Ross et
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at, 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 method for treating
or
preventing sea lice infestation on a fish, said method comprising
administering a composition
containing one or more chitinolytic enzymes to said fish.
Alternatively viewed, the present invention also 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 invention provides 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.
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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
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(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 Salm, Oncorhynchus, Hucho, Salvinus or
Lenok.
Particularly preferred species which may be treated using the methods and
compositions of
the present invention thus include, but are not limited to, SaImo salar
(Atlantic salmon),
SaImo 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 p-1,4 linked N-acetylglucosamine (GIcNAc) units.
Chitin forms
structures with the related polymer chitosan, being the deacetylated form of
chitin, and
comprising alternating 13-1,4-linked GIcNAc and D-glucosamine (GIcN) 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
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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, pmol/min).
Enzymes having chitinolytic activity (i.e. chitinolytic enzymes) are
classified into
different families in the carbohydrate-active enzymes database (CaZY,
http://www.cazy.orgi)
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.
Chitinolytic enzyme CaZy family
Chitinases GH18 and GH19
Chitobiases GH20
Chitin deacetylases CE4
Chitosanases GH5, GH7, GH8, GH46, GH75 and
GH80
LPM0s AA9 (formerly GH61), AA10 (formerly
CBM33) and AA11
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
(LPM0s), 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 GIcNAc, 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
GIcNAc
monomers.
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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)-6-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 LPM0s may be used to
work
synergistically with chitinases. The LPM0s 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 LPM0s 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 eta!, 2009, Biomacromol., 10(4),
p892-899; Hobel
etal., 2005, Extremophiles, 9(1), p53-64). Some chitosanases may also work on
highly
acetylated chitosans (chitins) (Heggset etal., 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
CA 02920768 2016-02-12
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separately, sequentially or simultaneously. Where different enzymes are used,
the enzymes
may exhibit a symbiotic effect wherein the degradation of chitinichitosan 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
'10 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
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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), A0A059111TO (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. 083009 (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 LPM0s (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 chitin-
degrading 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 chitin-
degrading 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
CA 02920768 2016-02-12
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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(1,4-1342-acetamido-2-deoxy-D-
glucosidel)
glycanohydrolase from Serratia marcescens (C7809) or Streptomyces griseus
(C6137);
Chitosan N-acetylglucosaminohydrolase from Streptomyces griseus (C9830); N-
acetyl-f3-
glucosaminidase 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
CA 02920768 2016-02-12
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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 in-
between, 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 4-20 C). 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
CA 02920768 2016-02-12
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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,
CA 02920768 2016-02-12
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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.
CA 02920768 2016-02-12
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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 55 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 m3, or may be larger, for instance
10,000 m3 -
30,000 m3. 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 m3 of
water, preferably 1000-2000 m3 of water. More preferably a wellboat may
contain
approximately 1500 m3 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 m3, e.g. between 10-30,000 m3 (preferably 20,000-30,000 m3)
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
CA 02920768 2016-02-12
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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 NaCI 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
CA 02920768 2016-02-12
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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 (SaImo
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) witch 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.
CA 02920768 2016-02-12
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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 eta!,, 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 7Ø The
protein standard
used is High Range Rainbow Molecular Weight Markers (Amersham, GE Healthcare
Life
Sciences, UK).
CA 02920768 2016-02-12
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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 GIcNAc, whereas in the control reaction (Tris-HCl added) no GIcNAc was
released,
when measured by HPLC. The amount of GIcNAc 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 7.3 C to 12.0 C, and is specified
for each
experiment.
Preparation of colloidal chitin
To prepare colloidal chitin, a-chitin from the Arctic cold water shrimp
Panda/us
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 HCI (37% v/v) while
stirring. When
the chitin was evenly distributed, the HCI-suspension was slowly heated to 37
C, with
moderate stirring. The suspension then changed into a less viscous and more
transparent
- 20 -
solution. This solution was filtered through glass-wool, and slowly
transferred to ice cold
dH20, 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 -otor, type TTH-
750 (Thermo
Scientific, MA, USA), 10 minutes at 4000 rpm and 4 C). The pellet was washed
several times
with dl-I20, and finally resuspended in 500 mL dH20 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/I.
Preparation of a natural chitinolytic enzyme mix
Basal salt and buffer (BSB) media with colloidal chitin (Bromke & Venuti,
1999, Can.
J,, Microbial., 45(1), p88-91) was prepared by mixing 0.3759 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 dH20 to 900 mL. pH was then adjusted to pH 6.5, using NaOH, and the volume
adjusted
to 1 L using dH20. 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, 10 000 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 pm (Pall,
MI, USA) to remove any remaining bacteria and obtain a sterile enzyme
solution.
The chitinolytic enzymes present in the supernatant were concentrated
approximately
TM
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 Bic labs, 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 pm PES
membrane-
filter (VWR International, PA, USA) to make it sterile, and analysed using
sodium dodecyl
Date Recue/Date Received 2022-06-06
- 21 -
sulphate polyacrylamide gel electrophoresis (SOS-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 As, Norway. The enzymes were produced and purified according to standard
routines
described previously (Brurberg etal., 1994, FEMS Microbiol. Left., 124(3),
p399-404;
Brurberg et al., 1996, Microbiology-UK, 142, p1581-1589; Synstad at al., 2008,
Biosci.
Biotech. Biochem., 72(3), p715-723; Vaaje-Kolstad etal., 2005, J. Biol. Chem.,
280(12),
p11313-11319). The proteins were shipped on ice as solutions in 50 mM Tris-
HCI, 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 dH20. The
solution was
transferred to Snake Skin Dialysis tubing with a 10K MVVCO cut-off, and
dialyzed against
sterile sea water, pH 7.0, adjusted with HCI (see Figure 3). The chitosanase-
solution was
then filtered through a 0.2 urn 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 I ¨ Killing of sea lice with recombinant chitinolvtic 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 chitinotytic enzymes alone. The sea lice were
collected the same
day as the experiment was conducted as described above. The sea temperature
that day
was 12.0 C. They were transferred to the reaction beakers with a sterile
inoculating loop,
Materials and methods:
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CA 02920768 2016-02-12
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Three different treatments were compared in three 100 ml beakers, containing
23 sea
lice and 60 ml sterile sea-water (Marine SeaSalt, Tetra, MeIle, Germany) each.
The sea
water naturally had a pH of approximately 8Ø 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 set-
ups, 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
CA 02920768 2016-02-12
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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 8Ø 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 Chimax0, 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 7.3 C. 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,
Melte, Germany)
each. The pH of the sea water was adjusted to pH 7.0 using HCI. 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
CA 02920768 2016-02-12
- 24 -
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 etal., 2004, Appl. Environ.
Microbiol., 70(8),
p4522-4531). At pH 7.0, Chimax-0 only retains 30 % of its maximum activity
(Choi etal.,
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 chitinolvtic
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 10 C.
- 25 -
Materials and methods
Two single sea lice were first washed three times with 0.75 mL dH20, 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 100p150 mM
TrisHCI pH 8.0,
at 50 C. The second sea louse was incubated with 100 pinatural Serratla
marcescens
chitinolytic enzyme mix (34 mg/ml) at 50 C. Preparation of the natural
chitinolytic enzyme
-rm
mix is described above. As a blank, an Eppendorf tube containing 100p150 mM
TrisHCI 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, GIcNAc). 10 pl of sample was added 10 pl 50 mM H2SO4 to
stop the
reaction, and then filtered using a 0.45 pm Duraporex membrane Multiscreen 96
well plate
(Millipore). 8 pl 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
auto-
injector (Dionex Corp.) and an Ultimate 3000 column compartment (Dionex
Corp.), The liquid
phase consisted of 5mM H2504, 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 GIcNAc was detected
in
the control sample containing no chitinolytic enzymes, even after 24 hours
incubation. In
contrast, G1cNAc was detected in the sample containing the natural Serratia
marrescens
chitinolytic enzyme cocktail. Furthermore, an increase in the amount GicNAc
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 GIcNAc was detected in the blank (results not
shown).
Example 4 ¨ Protocol for the treatment of sea lice Jr 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
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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.
