Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~53310
_1_ A~03
ANTIGENIC COMPOSITIONS
This invention relates to antigenic
compositions, vaccines thereo*, their
preparation and use for the prevention and
treatment of foot-rot, especially in sheep.
5. Ovine foot-rot is a widely-occurring,
contagious disease, affecting the epidermal
tissues of the foot and caused by the synergic
action of two Gram-negative anaerobic bacteria,
Bacteroides nodosus (Fusiformis nodosus) and
10. Fusobacterium necrophorum (Fusiformis
necrophorum). The disease occurs only when
both these organisms are present.
F. necroPhorum is fol~nd normally in the
alimentary tract, and is excreted in the
15. faece~, so the organism i9 usually available
in the immediate environment of the sheep's
feet to participate in the infection. B. nodosus,
on the other hand, is unable to survive under
natural conditions for more than a few days
20. outside the lesions of foot-rot. The infected
foot is its only natural habitat and accordingly
B. nodosus is described frequently as the
specific causal agent of the disease. The
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elimination of B. nodosus from a flock of sheep,
which could be accomplished by curing all cases of
foot-rot present or by the specific destruction of
the organism, would eradicate the disease since
F. necrophorum cannot cause foot-rot in the absence
of B. nodosus.
In past years, foot-rot has been controlled
by isolation of the affected animals, followed by
treatment comprising extensive paring of the
affected areas of the feet and external application
of disinfectants or external or parenteral admini-
stration of suitable antibiotics. More recently, the
successful large-scale culture of B. nodosus has
permitted the vaccination of sheep using emulsion or
aluminium adjuvenated vaccines, see for example UK
Patent Specification No 1 375 544. In many instances
of the use of such vaccines, incomplete control of the
disease has been achieved, and intense efforts have been
made to discover the cause of failure and to improve the
efficacy of existing vaccines.
It has now been found that an antigenic preparation
of the present invention produces a greater antibody
titre and a better control of the disease than B. nodosus
vaccines used hitherto. Accordingly, the present
invention provides an antigenic composition comprising
a two-phase water-in-oil emulsion containing antigenic
JDM/LM/23 June 1980
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material derived from or consisting of Bacteroides
nodosus organisms and an aluminium adjuvant in the
aqueous phase of the emulsion.
The antigenic material of the composition may
comprise an immunogenic extract of the organisms but
preferably the material comprises the whole killed
organisms in the form of an anaculture or harvested
cells. Any one immunogenic strain or variant OT any
combination of any number of strain(s) and/or
variant(s), representative of any one or more B. nodosus
serotypets), may be used. Satisfactory protection has
especially been obtained by using organisms grown from
fimbriate colonies in which the organisms are highly
piliated (see for example J A Short et al, J. appl. Bact.
1976, 40, 301-315).~_. nodosus organisms grown on solid
media are highly piliate'd, a~d in the performance of the
present invention, growth conditions in liquid culture
should be chosen so as to ensure the maximal degree of
piliation in order to enhance the efficacy of the
resultant vaccines. By highly piliated B. nodosus
organisms, as described herein, are meant organisms
having at least 20 pili per organism, and preferably
more than 100 per organism. For maximal efficacy, the
antigenic compositions and vaccines derived therefrom
should consist wholly of highly piliated variants of
B. nodosus, but as a practical matter adequate
protection is obtained where 90~ of the organisms are
JDM/~M/23 June 1980
.
~ ;33~0
highly piliated variants. The highly piliated
variant of strain CSIR0 No 198 (Wellcome
Laboratories Culture No 6476) ~deposited with the
American Type Culture Co-llection on 19 July 1979 under
No 31545) is especially valuable. Preferably9 protection
may be afforded against homologous challenge (i.e.
challenge with one or more serotype ~s~ homologous with
the B. nodosus serotype (s) of the composition) since
protection against heterologous challenge is weaker.
Thus, challenge with organisms representative of m~re
than one B. nodosus serotype will preferably be met with
a composition comprising more than one strain,each
strain being representative of each serotype comprising
the challenge organisms.
The B. nodosus organisms for use in compositions
-
of the present invention may be grown or cultured by
any method known in the art, for example by the method
described in UK Patent Specification No 1 375 544; or
by the methods described in the paper of Short et al
(ibid) which produces highly piliated organisms.
The aluminium adjuvant is one which in its own right
enhances the antibody response to the antigenic material,
and may be chosen from those well Xnown in the art such
as potash alum, aluminium hydroxide and aluminium
phosphate. Particularly preferred are thixotropic
~-type aluminium hydroxide gels
~'
~;3310
A603
--5--
which carry a positive charge in the absence
of electrolytes. An example of such a gel is
'Alhydrogel' (Trade Name3. Preferably the
compositions should contain between 0.25 and
5 . "4, o % vv/v of the aluminium adjuvant."
The emulsion has a single oil continuous
phase and a single aqueous dispersed phase
(that is to say, it is a ~w'o-'ph'a'~e wa`të.r-i''n-oi'l
emulsion), and may be prepared by the use of
10. one or more emulsifiers and an oil. Naturally,
the emulsifier or emulsifiers for inclusion
in the compositions must be non-toxic and
compatible with the antigenic components of thè
vaccine. They are preferably of the non-ionic
15. type so that there is less tendency for them
to be precipitated by components of the
antigen preparation than when a cationic or
anionic emulsifier is used. There is also less
danger of denaturation of the protein of the
20. antigen if a non-ionic emulsifier is used~ The
combination, quantities and proportions of
emulsifier chosen should also be those which,
when included in the vaccine,produce the maximal
degree of enhancement of antigenicity and
25. stability of the emulsion, and preferably
should con~ist of a lipophilic and a hydrophilic
JDM/LM/2 July 1979
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A603
--6--
emulsifier.
The lipophilic emulsifiers are preferably
non-ionic surface active agents having a low
hydrophile-lipophile balance of between 8 and 20.
5. ~William C Griffin, 'Calculation of HLB values
of non-ionic surfactants', Journal of the Society
of Cosmetic Chemists (1954) volume 5, pages
249-256; Osipow, 'Surface Chemistry ACS
Monograph 153', pages 295-314, Reinhold, 1962.
10. This is the outer limit of the range of HLB
values of emulsifiers most suitable for
incorporating in the oil phase and an optimal
limit of HLB values is 2 to 6. Among suitable
lipophilic emulsifiers are di- and tri- esters
15. of polyhydric alcohols and fatty acids, oxidised
~ fatty oils, and partial esters of common fatty
acids, e.~g. palmitic, lauric, stearic and oleic
acids with hexitol anhydrides derived from
sorbitol or mannitol. Examples of specific
20. emulsifiers are mannitan and sorbitan fatty
acid esters such as mannide monooleate (Arlacel
A, HLB 4.3), sorbitan monooleate (Arlacel 80,
HLB 4.3), sorbitan monopalmitate (Arlacel 40,
HLB 6.7), sorbitan monostearate (Arlacel 60,
25. HLB 4.7), and sorbitan sesquioleate (Arlacel C,
HLB 3.7)-
JDM/LM/2 July 1979
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~.~L5i3310
A603
-7-
The hydrophilic emulsifier for use in the
compositions should preferably have an HLB value
of between about 9 and20 and optimally between
11 and 18. Suitable classes are polyglycol fatty
5. acid esters~ polyoxyethylene modified fatty acid
esters, polyoxyethylene-polyol fatty acid esters,
polyoxypropylene fatty alcohol ethers, and
--- polypropylene glycol fatty acid esters. A
particularly useful class are polyoxyalkylene
10. derivatives of polyhydric alcohol fatty acid
esters such as polyoxyethylene derivatives of
partial esters of lauric, palmitic, stearic and
oleic acids and hexitol anhydrides. Examples of
emulsifiers are polyoxyethylene (20) sorbitan
15. ~ monolaurate (Tween 20, HLB 16.7), polyoxyethylene
,. , ~
(20) sorbitan monopalmitate (Tween 40, HLB 15.6),
' polyoxyethylene ~20) sorbitan monostearate (Tween
- 60, HLB 14.9), polyoxyethylene (20) sorbitan
monooleate (Tween 80, HLB 15)S polyoxyethylene
20. (20) sorbitan trioleate (Tween 85, HLB 11.0) and
polyoxyethylene (8) laurate (G 2127, HLB 12.8).
Any non-toxic oil of vegetable or mineral
origin compatible with the antigenic material
may be used in the compositions of this invention.
25. Mineral oils of a pharmaceutical grade are
especially preferred as they in general produce
JDM/LM/2 July 1979
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~ 1 ~3310
-~ -8- A603
a substantially more stable emulsion and less
tissue irritation. Light liquid paraffin oils
~` (Bayol F and Drakeol No 6R) have produced
satisfactory emulsions and compositions.
The liquid of the aqueous phase may consist
of the culture medium or, where the cells are
extracted or harvested, aqueous saline or water.
The compositions of the invention may be made
by any of the techniques known in the art for the
preparation of emulsions of biological materials
and may in fact be prepared by VigoTous agitation
of an aqueous antigen/aluminium adjuvant mixture
with the oil and emulsifier~s). The aqueous antigen/
aluminium adjuvant mixture is prepared by inactivating
the B. nodosus organisms, using for example formalin ,
concentrating the culture or anaculture to about 30~
pre~erably
of the original culture volume, but~no less than 10~,
by any of the techniques known in the art and adding
the aluminium adjuvant to the aqueous antigen. The
aluminium adjuvant may be added after concentration
or the concentration process may itself use an
aluminium adjuvant. An example of the former technique
is isoelectric point precipitation and an example
of the latter technique is co-precipitation with a
flocculating agent.DIsoelectric point precipitation
is especially applicable to antigenic material comprising
highly piliated B. nodosus organisms. In this
technique, the pH of the culture or anaculture
JDM/LM/23 June 1980
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is lowered, by the addition of acid, to approximately
the isoelectric point of the cellsj although a lower
pH may be used. Suitable acids may be chosen from
mineral acids such as hydrochloric, phosphoric and
especially sulphuric acids and strong organic acids
such as acetic and lactic acids. The pH is suitably
less than 6.0, preferably less than 5.6 and more
preferably in the range of from 4.8 to 5.2. Below
pH 4.8, precipitation should be complete but, depending
upon the particular antigenic material used, damage to
the antigen may occur. After lowering of the pH, the
temperature of the culture or anaculture should be
lowered to between 4 and 12C.k o-precipitation using
a flocculating agent may be a,chieved by adding a
5 sterile solution of the flocculating agent to the
for example
anaculture and the pH adjusted~with alkali to give
optimal precipitation. After settling in the cold
~say 2 days or more) the clear supernatant liquor is
removed to waste, thereby effecting a concentration of
the cells and proteinaceous matter (including pili) in
the anaculture. Although some of the flocculating agent
may be discarded with the supernatant liquor, it is
preferable to restrict the quantity used for
precipitation since the larger the amount used the more
appears in the precipitate (cell concentrate) and the
more irritant the ensuing vaccine. Suitable flocculating
agents include the sulphates, chlorides and insoluble
hydroxides of polyvalent cations, and polyanionic
JDM/LM/23 June 1980
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10- A603
polymers such as sodium carboxymethyl cellulose and
hydroxypropylmethyl cellulose. Especially suitable for
the present invention is potash alum since this may also
function as the aluminium adjuvant. It has been found
that 1 volume of 10% w/v potash alum added to 7
volumes of anaculture ~1.25% w/v alum overall) and
with the pH adjusted to 5.4, a precipitate of
suitable properties and clear supernatant liquor
develops. The higher the pH te.g. 5.8, 6.4) the
longer the time required for complete clearing of the
supernatant. The lower the concentration of alum the
longer the clearing time required. The bulk of
precipitate also is dependent on the amount of
alum added to the system; thus the addition of 1
volume of 10%iw/v alum to 11 volumes of anaculture
tO.83~ alum w/;v overall) results in a barely acceptable
precipitate with an inconveniently long settling time,
whereas the addition of 1 volume of alum added to 4
volumes of anaculture t2.0% alum w/v) results in a
very bulky precipitate which does not pack sufficiently
well without centrifugation to be of use in the
harvesting of cells. In similar manner the purified
~a~
aluminium hydroxide substance 'Alhydrogel' tTrade N~ffle)
when added to anacultures of B. nodosus, remains in the
dispersed phase for a long while, so that harvesting of
absorbed cells/protein needs to be accomplished by
centrifugation ~Another technique for concentrating
the culture or anaculture besides centrifugation is
ultra-filtration, and either of these latter two
JDM/LM/23 June 1980
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techniques may be used in conjunction with
isoelectric point precipitation or co-precipitation.
~In` the case where more than one serotype of B. nodosus
is comprised in the antigenic material, an aqueous
antigen/aluminium adjuvant mixture is prepared as
described above for each strain or variant,then about
equal amounts of each mixture are blended prior to
emulsification. Preferably, the emulsion is prepared
by mixing the oil with the lipophilic emulsifier,
the aqueous antigen/aluminium component with the
hydrophilic emulsifier, and combining the oil and
aqueous phases. It is preferable to add the aqueous
phase slowly to the oil phase with agitation in order
to minimise any tendency to foTm an oil-in-water
emulsion. Finer dispersions of the aqueous phase can
be obtained by homogenising or milling in a colloidal
mill, and this may be continued until no further
decrease in particle size occurs.
JDM/LM/23 June l980
,
.
~L53310
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The optimal proportion of oil to water
phase in a vaccine will depend on a variety of
factors such as the concentration of antigenic material
5. in the aqueous phase, the desired viscositv of
the vaccine., and the antigenic~ty of the antigenic material.
The aqueous phase preferably does not exceed
600/o and does not fall below 10% of the total
volume of the emulsion. A greater proportion
10. of water is undesirable because of the increase
in viscosity and instability of the emulsion,
and a proportion of less than 10% makes it
difficult to incorporate sufficient antigen
without having an unacceptably large dose
15. volume. It has been found that satisfactory
~ , .
vaccines are obtained when the aqu~ous phase
comprises, from 20 to 50/0 by volume of the whole
- - - vaccine. If a large proportion of water is
required, for example above 40%~ then precautions
20. should be taken to avoid formation of an oil-
in-water emulsion and instability of the emulsion.
It has been found that such emulsions containing
polyoxyethylene (20) sorbitan trioleate may be
stabilised by the addition of Falba - a stabiliser
25. containing beeswax paraffin oils of various
viscositi~s and oxycholesterins extracted from
lanolin.
JDM/LM/2 July 1979
~;33~0
-13- ~603
The amount of emulsifier needed in the
respective phases will depend on a variety of
factors and particularly on the HLB values of
the emulsifiers and the choice of oil, and it
5. is estimated th~t from 2.5 to 15% by volume of
a lipophilic emulsifier and from 0.5 to 10.0%
by volume of a hydrophilic emulsifier in the
respective phases will provide vaccines of the
properties described herein. A considerable
10. enhancement of the antigenicity of the antigens
has been obtained using from 5 to 12% by volume
of the oil phase and from 0. 2 to 7~ by volume
of theaqueous phase, as the respective emulsi~iers.
The compositions of the present invention
15~ may be formulated into a vaccine suitable for
administration by incorporating the composition
in an appropriate concentration into a container
as a sterile preparation.
It is necessary to sterilise the composition
20. after emul ification or to sterilise the individual
components beforehand and emulsify under sterile
conditiong, ~ompositions of the present invention
may be sterilised by the use of heat and filtration
to sterilise the components, or by the use of
25. gamma-radiation without any loss of potency or stability.
JDM/LM/2 July 1979
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The ~inal vaccine
is introduced into sterile containers and sealed.
The vaccine desirably contains a bacteriostat of
the kind generally employed in killed bacterial
vaccines, and 0. 01% (w/v) thiomersal(sodium
ethyLmercurithiosalicylate) may be incorporated
for this purpose, in the aqu~ous phase of the
composit~on.
Conveniently the final vaccine preparation
contains, in total, from-2.5 x 107 to l x 1011, and
desirably between i x 10 and 1.25 x 109,
killed organisms per millilitre of the preparation.
The vaccines of the present invention are
desirably administered to animals by subcutaneous,
intramuscular or intraperitoneal injection. It
is advantageous to administer the vaccine at a
site where any ~ocal reaction upon administration
is of little concern to the farmer, for example,
~0 on the anterior dorsal aspect of the neck or on
the lower mandible. The most preferred dosage range
is between 1 x 108 and 1 x 10 killed organisms
per dose, in particular between 1 and 10 and 1 x 10
per dose, wherein the dose is the total quantity of
antigenic material administered at one time to an
animal such as a sheep in a suitable volume
JDM/YA/2g July 1979
~, ~53310
~603
-15-
of liquid, for example 1 to 2 ml. The dose of
vaccine may be administered to an animal as one
unit or as a multiplicity of sub-doses, and the
latter may be administered over a period of time,
5. or by simultaneous injections of the sub-doses
at different sites. A suitable immuni-sation
schedule may comprise a single inj ection of the
~ose, or two injections, each comprising a sub-
dose, given simultaneously at. different sites
10. on the ~nimal. The optimal dosage schedule will
of course vary according to the strains of
organism chosen for the basis of the vaccine,
: the total number of organisms employed, the
nature of the adjuvant and t:he route of
15. administration. It has been found especially
suitable to administer a vaccine comprising
, 3 x 10~ organisms/ml in a single 1 ml or 2 ml
do~e by the subcutaneous route followed by a
second injection of the same volume and
20. strength and by the same route after a~ interval
of at least 6 weeks, preferably of 12 weeks.
Boo~ter doses should be given just before the
a~ticipated seasonal spread of foot-rot~
In addition to containing antigenic
25. material derived from B. nodosus, a vaccine
of the present invention may also contain
JDM/LM/2 July 1979
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.
331~
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antigenic material derived from other bacteria
that are causative organisms of diseases in,
for example, sheep, in particular F. necorphorum,
or clostridia. A particularly preferred combination
is a multiple component vaccine comprising a
preparation of one or more clostridial antigens
described in UK patent specification No 1 143 545,
in association with a B. nodosus antigenic
composition produced as hereinbefore described.
According to the present invention there is therefore
. , _ .. . . .. . .. . . .
provided: ~
(a) A~ antigenic composition comprising a two-phase
water-in-oil emulsion containing antigenic material
derived from or consisting of Bacteroides nodosus
organisms and an aluminium adjuvant in the aqueous
phase of the emulsion.
(b) A method of preparing an antigenic composition
as defined in paragraph (a) above comprising~the
emulsification of an aqueous medium containing antigenic
. . . . . , . . . . . . . , . . . , . . ~ . _ . . . . . .
material derived from or consisting of B nodosus
organisms and an aluminium adjuvant with an oil.
(c) A vaccine comprising a sterile preparation of a
composition as defined in paragraph (a) above.
(d) A method for the prophylaxis or treatment of a
foot-rot infection of sheep comprising the administration
to the sheep of an effective, non-toxic dose of a
vaccine as defined in paragraph (c) above.
JDM/Y~ 25 July 1979
`~ ~1533~0
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The following are Examples of this invention
but should not be construed as comprising any
limitation thereof.
5. Reference Example 1 - Growth of organisms
B. nodosus (Strain CSIR0 198) organisms
obtained from seed culture, were inoculated in
the medium (described by Thorley,C.M.(1976)
' J. Appl, Bact. 40, 301-309 at page 302)
10. contained in screw-capped bottles, and the air
displaced by a mixture of 10% carbon dioxide
and 90% nitrogen. The cultures wer-~ maintained
at 37 C for between 20 and 40 hours, and the
organisms killed by addition of formalin (0. 5%
- 15- ' w/w). Examination of the organisms under the
electron microscope showed that at least 90/0
were highly piliated (>100 pili per organism).
, ' Example 2,- AntiFenic com~osition
A formolised anaculture (3,200 ml) of
20. highl~piliated B. nodosus organism~ obtained
in the manner described in Example 1 was treated
with an aqueous solution '( 10% w/v) of potash
alum (320 ml) and the pH of the mixture
adjusted to 6.2 by the addition of aqueous
25. sodium hydroxide (400/o w/v). After maintaining
the mixture at 4C for 24 hours, supernatant
JDM/LM/2 July 1979
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liquid above the precipitated organisms was
discarded until the volume was reduced to
1,200 ml.
Tween 80 (30 ml; 400/o v/v aqueous solution)
5. and thiomersal (1.2 ml; 10% aqueous solution)
were added, and the mixture emulsified with a
mixture (2.8 1) of Arlacel A (10% v/v) and
Marcol (90% v/v).
Examples 3-6 - Anti~enic Compositions
10. Highly piliated B. nodosus organisms were
collected by isoelectric point precipitation by
treating the anacultures with sufficient dilute
aqueous sulphuric acid to lower the pH to 5Ø
After standing for three days at 16-20C, the
15. organisms were concentrated 9-10 times by
discarding 133.7 litres of supernatant. The
or~anisms were used in the preparation of vaccine
formulations of Examples 3-6 as shown in Table 1.
In each instance, the concentrated
. 20. anaculture was diluted (Co.lumn 3) with aqueôus
sodium chloride solution, and thiomersal added
to the aqueous mixture (Column 7) to produce a
concentration of 0. 01% w/v prior to emulsification
with the oil (Column 8). In Exampl~ 4 to 6, the
25. pH was adjusted after the addition of the
hydrophilic emulsifier.
JDM/LM/2 July 1979
-19~ 53310 A603
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-20- A603
Example 7 - Antigenic Composition
Following the procedure described in
Example 2, a further composition was prepared
using highly piliated B. nodosus organi~ms
5. (obtained in the manner described in Example
1), as shown in Table 1.
Exa le 8 - Vaccines
mp .
The compositions described in Examples
3-7 may be filled into l, 2 and 10 ml sterile
10. vials, sealed and sterilised by gamma
irradiation.
Example 9 - EfficacY
In this experiment, four groups of five
sheep were vaccinated subcutaneously on the
15. lower mandible with 2 ml~vaccine prepared as
described in Example 2 (Vaccine A) and with
3 other vaccine preparations ( Vaccines B, C
and D), Six weeks later, a second dose of
vaccine similar in all aspects to the first
20. was given to all sheep, and five sheep from
each of the vaccine groups were challenged by
having a live culture of the vaccine strain
applied directly to each (predisposed) foot.
Thereafter, each group of five sheep was
25. challenged at monthly intervals, 50 that the
last challenge oc~urred twelve weeks post
JDM/LM/2 July 1979
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second vaccination. Feet were assessed for
clinical foot-rot three weeks after each
challenge. A score of one or more feet affected
with u~der-r~mning foot-rot or severe inter-
5. digital dermatitis in 2 or more feet on two or
more of the five animals was deemed to fail the
vacci~e on grounds of insufficient protection,
and further testing of that vaccine was omitted.
The four experimental vaccines (vaccines
10, A, B, C, and D} were tested simultaneously in
comparison with a non-vaccinate control group
of sheep to check the virulence of the challenge
, culture., Five lots of 4 x 5 sheep were used. Blood
, samp}es were collected at 0 and six weeks and at
15. the time of foot assessment. Agglutination titres
were determined and geometric mean titres found.
- The results of the trial are given in Table
2.
It can be seen that piliate alum/oil vaccine
20. A of the invention was the only one to confer
protection on sheep at the fourth challenge. Two
weeks subsequent to foot assessment the foot-
lesions in the one sheep to become infected in
this vaccine group had resolved whereas those in
25. the five control sheep were as severe as ever.
Furthermore, a distinct difference was found
JDM/L~/2 Jul~ 1979
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between serum agglutination titres o~ sheep
immunised with Vaccine A and sheep receiving
the other vaccines.
~hallenge 2 was invalid on account of
5. three control sheep failing to become infected.
Challenge 4 was very severe, the control sheep
being badly infected at a preliminary ten day-
post-challenge examination.
lt is believed that this experiment has
10. demonstrated for the first time the possibility
of protecting sheep by vaccination against
foot-rot for a period of twelve weeks following
the last dose of vaccine.
Vaccines B, C and D were identical to
15. vaccine A except in that:-
Vaccine B: the organisms were non-virulent
and were genetically non-piliated, that is to
say~ none were found to have more than 20 pili
per bacterium.
20. Vaccine C: the aluminium adjuvant was
omitted.
Vaccine D: the aluminium adjuvant was
omitted and the organisms were non-piliate as
in Vaccine B.
JDM/LM/2 July 1979
~.~53310
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_
o o o o
~ ~ C~
h u~ u~ ~
~t ~ ~ 5
~ a~
R 0
_l cl
P~o ~;
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,~ , ~ ~". .
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h ~ cr~ oo ~ a)
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~ ~53310
-24- A603
Example 10 - Efficac~ of vaccines
In this experiment, vaccines prepared from
the compositions described in Examples 3 to 7
were compared with vaccines adjuvenated by alum
5. alone (Vaccines F and G). Sheep free of foot-rot
were treated either with a single dose of a
Vaccine of Examples 3 to 7 or with two doses
of Vaccines F or G spread 6 weeks apart. At
intervals post-vaccination, groups of the
10. vaccinated sheep were compared with non-vaccinate
controls for resistance to infection against
artificial challenge with a living culture of
B. nodosus as described in Example 9, The
! . . results are given in Table 3.
JDM/LM/2 July 1979
~11533J,0
-25- A603
TABLE
. ~
Vaccine Dose of Challenge post-vaccinationtsheep
(ml) .affected) .
. 8 weeks12 weeks 15 weeks
_ _ . _
Ex 3 1 1/5 o/4 _
.- . _
Ex 4 1 0/5 ¦ /5 _
. ,, . I . .
Ex 5 2 _ _ /5
_ . . ,
Ex 6 1 /5 _ /5
, _
. Ex 7 1 /5 1/5 _
.
Ex 7 2 . 0/4 /5 _
_ ,
F 2x2 4/5
G 2x2 4/5 _
. _ _ . .
Controls 4/5 5/5 4/5
JDM/LM/2 July 1979
