Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
. ..Vbr~ 92106599 PCT/GB91/01828
VACCINE COMPOSITIONS FOR FISH
IO
This invention relates to compositions useful for
oral administration of sensitive materials such as
vaccines to aduatic animals, especially fish.
The intensive rearing of fish such as salmon, which
today is practised vn a wide scale, suffers from the
disadvantage that the entire stock of fish in a facility
may become infected with disease. The commercial
consequences of a serious disease outbreak can be
enormous. Several well-recognised fish diseases are
causing severe problems in the fish farming industry.
Examples are bacterial-kidney disease, caused by
~ten~bacterium salmo inarum; enteric redmouth, caused by
Yersinia ruc a '; and Vibriosis, caused by various strains
of Vibrio, notably V anauillarum and V salmonicida.
Currently, the most significant disease, at least as far
3o as the farming in T3orthern Europe of Atlantic salmon
Salma salary is concerned, is furunculosis; this is
caused by the bacterium Aeromonas salmo~i,cida.
3S
PC'I'/GB91/01~2~ ,.,~
i~t~ 92/06599
The desirability of immunising farmed fish against
such diseases has long been recognised. However,
technical progress in this area has been slow. This
applies both to the effectiveness of the commercially
available vaccines (a wholly effective vaccine for salmon
against furunculosis has yet to be developed), and also to
the means by which such vaccines are administered to the
fish. Traditionally, administration of vaccines to fish
has either been by immersion (which j.s wasteful as well as
of limited efficacy), or by injection. Although injection
to
of individual fish provides a sure way of delivering a
vaccine, it suffers from the disadvantages that it is very
labour-intensive, and moreover the handling and injection
cause considerable stress to the fish, and can precipitate
disease problems or at least cause temporarily retarded
1~
growth. Usually fish are anaesthetised prior to
injection. .
There is a clear commercial need for a vaccine
delivery system for fish which is less labour-intensive,
and which causes little or no stress to the fish.
For some while it has been recognised that an oral
delivery system, in which a vaccine is administered to the
fish either as part of the regular diet or in a
composition administered together with the regular diet,
would be beneficial. Reported experiments involving
attempted oral administration of fish vaccines have
yielded inconsistent results, and no effective oral
3o vaccine has yet become available commercially. Particular
problems recognised in the oral route are: possible loss
of or damage to essential vaccine components during
manufacture of the composition; possible loss of
water-soluble vaccine components in the aqueous
environment ~n which the fish live; and possible
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degradation of the vaccine within the intestine of the
fish before the vaccine has induced a protective response.
By the present invention we provide a particulate
composition for oral administration to aquatic creatures,
especially fish, comprising a water-in-coil emulsion
containing a sensitive agent such as a vaccine or the
like, the emulsion being carried on a solid edible carrier
material, preferably particles of a feed-stuff appropriate
to for the aquatic creatures.
The skilled reader will readily appreciate that the
invention can be adapted far use with aquatic creatures
other than true fish, for example crustacea such as
prawns, shrimps, lobsters and crabs, and molluscs such as
oysters. For convenience,. the invention will be described
in relation to fish, and the term "fish°' should be
understood as encompassing other aquatic life farms that
may benefit from the oral administration of vaccines and
2o the like. Bxamples of true fish that are reared on a
substantial scale in captivity in various parts of the
world are: Atlantic salmon, Pacific salmon, rainbow
trout, brown trout, catfish, halibut, turbot, carp and
tilapia.
Generally, the sensitive agent will be water-soluble
rather than oil-soluble, and hence dispersed in the
aqueous phase of the emulsion. The emulsion should
contain sufficient of the sensitive agent to provide the
3o desired effect, eg. an effective immune response, when the
composition is administered to the fish.
Preferably the emulsion is applied to particulate
fish feed~that is nutritionally deficient in oil.
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Preferably the emulsion comprises a water: oil mixture
containing not more than about 70% by weight water. More
preferably, the water content is not greater than about
65% by weight. Nevertheless, the emulsion must contain
sufficient water to act as a carrier for the water-soluble
agent, and a particularly preferred water: oil weight ratio
range is 6:4 to 4:6.
Preferably the emulsion comprises at least about 1%
to by weight of the final composition. More preferably, the
emulsion comprises at least about 2%, and ideally at least
about 3%, by weight of the composition. Generally, the
amount of emulsion is not greater than about 10%~by
weight.
The emulsion should have a viscous consistency, ie.
creamy and flowable, to enable it to be applied uniformly
to the particulate carrier.
2o The carrier should be relatively dry and non-oily so
that the applied emulsion can be absorbed, at least
partially, by the particles. Preferably the particulate
carrier is nn-oiled feed in granular or pelletted form.
The composition of the feed is not critical to the
invention, as long as the formulation is appropriate for
the fish to which the composition is to be fed, and at the
time of adding the emulsion the particles do not contain
oil ar other fluid ingredients in such amounts that the
emulsion cannot be absorbed by the particles. After the
3o aPPlication of the emulsion, the particles should still be
sufficiently dry and free-flowing to be handled in the
same manner as conventional fish feed. Typical fish feeds
are high in protein, and are conventionally based an fish
meal with added components such as cereals, and ail as an
energy source. Fish meal naturally contains a certain
?p~yf~'~~
W~0 92/06599 PCT/GB91/01828
amount of ail, but usually this needs to be boosted for
nutritional reasons. The additional oil is usually added
to the granules or pellets after these have been formed;
the oil-deficient material is herein termed "un-oiled'°.
The size of fish feed pellets varies widely, depending on
the type and age of the fish; for salmon the pellets
typically have a diameter from about Z to about 6mm, with
pellets of about 3mm being appropriate at the smolt stage.
Fish feeds can also contain minor components such as
vitamins, minerals, preservatives and pigments.
to
Alternatively, non-feed carriers can be used, but these
should provide final compositions that are perceived by
the fish as normal feed, otherwise the fish may ignore ar
reject the composition. Fish tend to be very critical in
Z5 accepting feed particles, and many factors such as size,
shape, colour and density, which all affect the
"behaviour" of the particles in water, can be very
influential. A composition of the invention should
therefore be made to have physical properties as similar
2o as passible to those of the normal feed to which the fish
are accustomed.
The emulsion should be sufficiently stable to protect
the aqueous phase for a time sufficient to enable the
25 composition to be produced and administered to the fish.
In general, the emulsion should not °'crack'°, ie. separate
into distinct aqueous and oil phases, in less than one
week. Preferably, the emulsion is stable for at least one
month.
The oil is preferably a neutral oil, because the
presence of free fatty acids can sometimes interfere with
the formation of an adequately stable emulsion.
Preferably, the level of free fatty acids should b~ not
greater than about 5a by weight of the oil, and ideally
W~ 92/06599 PCTlGB91/01~2~ '. ~ '
not greater than about 3~. Preferred oils are whole fish
body oil and neutral marine oil. If desired, the emulsion '
can incorporate an antioxidant such as butylated
hydroxytoluene or ethoxyquin. '
The emulsifier should be food grade, and is
preferably a lecithin. An ideal emulsifier is soya
lysolecithin (a modified phospholipid) and examples are
available commercially from Unimills BV under the trade
name "Bolek". Generally, the emulsifier will comprise
from about 0.1 to about 5% by weight of the total
emulsion.
The emulsion can be prepared by blending the oil and
aqueous phases, generally at ambient temperature, using a
homogeniser. In-line homogenising equipment is preferred.
Preferably the components can be recycled two: or more
times through the homogeniser if desired.
The invention also provides a process for the
preparation of an orally administrable composition for
fish, wherein a stable water-in-oil emulsion containing a
sensitive agent such as a vaccine, is applied to a
particulate feedstuff, preferably by pan-coating or the
like.
In a preferred embodiment of the invention,
additional oil, eg. from about 1 to about 3% by weicJht, is
applied to the particulate composition after the
aPPlication thereto of the emulsion. This can also be
achieved by pan-coating.
An important aspect of the invention is the use of an
oral delivery system to reduce the total effective
administered dose level of a vaccine that would otherwise
CA 02071974 2001-11-28
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need to be administered by a non-oral route (generally by
injection or immersion) in a substantially higher total
dose. We have found that administration of a vaccine in a
composition according to the invention surprisingly
enables an effective protective response in fish to be
achieved at a much lower dose level. In some
circumstances, a 10-fold reduction in the effective dose
level can be achieved.
l0 For example, we have conducted trials with a
commercially-available furunculosis vaccine ("Furogen")~
which is recommended for single administration by
parenteral injection at a dose level of 0.1 ml per fish.
We have administered this commercially-available vaccine
orally to salmon using a composition of the invention. We
have found that a total dose level of only 0.001 ml
"Furogen" per fish, administered orally over a ten-day
period, provided protection at least as effective as the
single recommended 0.1 ml dose administered by injection.
2o In contrast to the injection route, oral administration of
the vaccine in accordance with the invention did not cause
any stress to the fish, and could be conducted as part of
their normal feeding regime.
Although the theory behind the effectiveness of the
invention is not yet fully understood, we believe that the
oral administration of a furunculosis vaccine in
accordance with the invention promotes a cellular response
in the fish, which leads to enhanced protection. This
3o protection seems unrelated to the quantity of antibodies
within the circulation system of the fish. Administration
of the same vaccine by injection may lead to enhanced
circulating antibody levels, but apparently its impact at
the cellular level has not been studied.
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T/ 4~.~ t n ~n
!V0 92/06599 ~ ~ ° ~ '' 3 ~ fCT/GB91/01828
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We believe that the oil in a composition of the
invention protects the antigenic components of the vaccine
from degradation in the upper acidic regions of the gut of
the fish, while allowing the antigenic components to be
released in the lower alkaline regions where the oil is
digested enzymatically.
An oral composition of the invention, containing a
vaccine, can be used in place of conventional inject:able
and/or immersion vaccines. Alternatively, a combination
1o
of routes of administration can be employed, for example
by using the oral route to provide a basic level of
protection which can be boosted at an appropriate occasion
(eg. when fish are moved or counted, or at a time of
greater perceived infection risk) by a supplementary
injection or immersion.
Although it is envisaged that a composition of the
invention will generally be administered to farmed fish,
at can also be distributed "in the wild" to reduce the
incidence of fish disease in the natural environment, so
benefiting the indigenous fish population and enhancing
global fish resources.
z5 The oral composition of the invention can be prepared
"on the spat" for immediate administration to fish.
Alternatively, a composition of the invention can be
prepared and packaged in any manner conventionally uses
for commercially-available fish feeds, for example in
3o grease-proof sacks, and supplied as a commercial product.
The shelf-life of the composition can be enhanced, if
necessary, by the incorporation of preservatives, eg. in '
the applied emulsion.
CA 02071974 2001-11-28
_ g _
The invention can be used to administer any fish
vaccine that can induce an immune response via the gut.
For example, the vaccine can be a simple bacterin
composition, ie. a killed whole culture of an infective
organism, or an extract of a killed culture. The vaccine
s
can comprise toxoided components, ie, toxic factors
associated with the infective organism which have been
treated, eg. by chemical means such as formaldehyde or
glutaraldehyde treatment, to reduce their toxic effect
without seriously impairing their antigenic properties.
Where appropriate, the vaccine can comprise live
organisms,.preferably attenuated, eg. by controlled
culture or by genetic manipulation. The active components
of the vaccine can include, or indeed can consist entirely
is of, factors originally identified in the disease-causing
organism but subsequently produced for the purpose of the
vaccine by expression in genetically-modified organisms
such as E.coli.
By way of example only, a composition in accordance
with the invention can be made as follows.
Example 1
25 An approximately 1:1 mixture of whole fish body oil
and diluted aqueous "Furogen" injectable vaccine (an
aqueous composition containing a bacterin derived from
killed Aeromonas organisms) was blended by multiple passes
through an ultrasonic homogeniser, to form a stable creamy
;o water-in-oil emulsion, with the aid of 3~ (total weight)
of "Bolek~K" lecithin emulsifier. "Bolek K" may no longer
be commercially available, but other lecithin emulsifiers
from the "Bolek" range, such as "Bolek M", can be regarded
as identical for the practical purposes of this invention.
3s "Furogen" is available commercially from Aquahealth
i
fVO 92/06599 ~ ~ ~ ~ ~ ~ PC'f/G~91/01828 ~.'
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Limited, Canada and supplied in two formulations: for
parenteral injection and for immersion.
The emulsion was soaked into un-oil.ed conventianal
extruded 3mm diameter fish feed pellets (based on fish
meal and cereal), by pan-coating, using 5% emulsion by
weight of the feed. An additional 2.5% fish oil by weight
was added subsequently to the feed by the same method.
Fish normally consume 1-3% body weight/day, and the
quantity of °'Furogen" was adjusted, by dilution with
crater, to provide a total of about 0.001 ml of the
commercially-supplied injectable composition over a 10-day
trickle feed period.
The resulting composition was physically
indistinguishable from conventional oiled fish feed
pellets, and could be handled and fed to fish.in any
conventional manner, eg. by hand or by mechanical feeders.
sample 2
Trials in Atlantic salmon Salmo salary were
conducted as follows, using oral compositions made as in
2S Example 1, with a control group of fish receiving the
commercially-available injectable composition.
Groins and treatment
3o All fish were 1990 smolts from a commercial salmon
hatchery.
a) 1000 fish; composition of Example 1 containincf
"Furogen" immersion vaccine formulation @ 0.1
ml/fish*.
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b) 1000 fish; composition of Example 1 containing
"Furogen" injectable vaccine formulation @ 0.1
ml/f ish*.
c) 1000 fish; composition of Example Z containing
"Furogen" immersion vaccine formulation @ 0.01
ml/fish*.
d) 1000 fish; composition of Example 1 containing
''Furogen" immersion vaccine formulation @ 0.001
lfl
ml/fish*.
Control: 750 fish receiving a single dose of °'Fur~gen°' by
injection @ 0.1 ml/fish.
*Total received on average per fish over a lo-day feeding
period.
Method
To identify the groups, all fish were marked using a
panjetter and Alcian blue dye ~4%) before the beginning of
the experiment.
Fish were fed their respective vaccines over a 10-day
period in separate tanks, with a common water supply.
The fish were sample weighed and cultured far
A.salmonicida. Low-level infection was already present,
3o representing a typical on-farm situation. Further
challenge was natural, from the local water supply.
The results are shown in Table 1, and indicate the
percentage cumulative mortality of the trial groups over a
35 one-month period commencing from the start of vaccination.
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These results demonstrate that the oral route is an
effective delivery system, and produces results at least
as good as the injection raute. Indeed, the results
indicate that the oral route can enable lo~rer administered
vaccine levels to be used and at the same time induce
enhanced protection.
Table 1
Composition Dose (mljfish) Mortality ~~~)
Control 0.1 15.0
a~ 0.1.. 12.6
b) 0.1 7.0
c, 0.01 12.8
d~ 0.001 1.4
y
25
35
