Note: Descriptions are shown in the official language in which they were submitted.
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:~j BACKGROUND AND PRIOR ART ~
:, ,`, ,
Bordetella b nchiseptica has been established as the
;~ principal cause`of the widespread disease in swine commonly referred
~to as "turbinate atrophy", because, fol1owing the primary infection,
the nasal turbinate bones frequently undergo serious deterioration.
. .~ ,: . .
See Canad. J. Comp. Med., 31, 53-57 (1967), and literature references
s cited therein. Bordetella bronchiseptica bacteria can persist in the ; ~i
nasal cavitiesr leading to lnfection of the susceptible offspring of
;~ breeding sows.
~, Baby pigs become infected with B. bronchiseptica early in
., .
O life (under 4 weeks of age) which makes the problem of effective ~ ;
. . .
.,, :
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58
protection particularly difficult, since the immunity system of
very young p:igs does not respond s~tisfactorily to vaccines. ~'
For effec-tive antibody production, vaccines are usually admin
istered to pigs at an age of about 6 to 8 weeks. By that age,
however, if the pigs have become infected with B. bronchiseptica
damage to the turbinate bones may well occur, even though the
pigs become immunized to the infection.
The first treatment proven to be of value in reducing
the incidence of infection of young pigs was the administration
~-~ 10 of a sulfonamide therapeutic agent, such as sulEamethazine or
`` sulfaethoxypyridazine, in the rations fed to the breeding herds.
-' This method was reported in the literature and pat~nted by
Dr, William P. Switzer: Vet.Med , 58, 571-574 (1963); and
United States Patent 3,336,190. However, the effectiveness of
sulfonamide therapy in eliminating Borae'tella b
~"A' infection has been limited by development of sulfonamide-
~, resistant strains of the organism. For example, a study in 1967'
: ! ' ' . ~
found that isolates of B. bronchiseptica recoverea rom 80% of
the herds inspected were resistant to sulfonamide. Am. J. Vet. ;'
~' 2Q Res., 3~ 1621-1624 (Sep-t., 1969). This data emphasized the
recognized need for additional drugs, or, in particular, effec-t-
; i~e immunizin~ agents. Prior to the present invention, however,
' no other preventative treatment suitable for use in the commer-
cial raising of swine has been developed. ' '~
It was found that the introduction of live cells of a
low-virulence strain of B, bronchiseptica into the nasal cavities ;~
; of non-immune swine would cause a relatively mild infection, and
., . : : .
'' that thereafter the swine would be immune to urther infect1on~
including infection by more virulent strains of the organismO
, 30 See J_ Vet. Res,, 30, 1161-1166 (July, 1969J. One such low
.
virulent strain, which was not publicly available, was identified ~ '-
by the private code designa-tion of "Strain D-l". This is the
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same strain used for prepariny the killed whole cell parenteral
vaccine of the presen-t invention. However, in prior work,
live cells oE the Strain D -l were found -to persist in the nasal
cavities of recovered swine. Therefore/ Strain D-l was ruled
out as a live whole cell intranasal vaccine. More~ver, no
other strain of B. bronchiseptica was known which would produce
immunity, and thereafter be self-clearing from the nasal
passages of the immunized swine.
; Attempts were also made to develop a vaccine from ` '~
killed cells of B. bronchiseptica which would induce immunity `~
` by parenteral administration. One of the first such experi- ' ~'
.. ..
mental vaccines was prepared from a virulent s-train of
~ B. bronchiseptica (identified as "Strain B")~ But this whole
'~ cell-vaccine failed to induce resistance to nasal infection as
reported by Harris, D.L., and Switzer, W.P.: A~. J Vet. Res., ~ ;
30, 1161-1166 (July, 1969). Because of -the failure of such `~
whole-cell parenteral vaccines, Harris and Switzer postulated ~ '
:, .
that "exposure of the pig to the internal antigens of
';~ B. bronchiseptica'may be necessary for the production of resist-
'`~ 20 ance against nasal infection." Am. J. Vet. Res., 33, 1975,
''~, at 1981-1982 (Qct., 1972) This theory proved incorrect~ '
Vaccines prepared from disrupted cells (sonicated) of ~ '
~' B. bronchiseptica, Strain D-l, failed to prevent B. bronchiseptica
~; infection, but induced an accelerated clearance by ~0 days post
challenge (A.J, Vet.~ Res., ~33, 1975, at 1979 and 19~1), It was
' concluded that the type of resistance lnduced by a parenteral
~ vaccine containing the liberated antigens of killed cells was `
'~ different than the immunity of swine which had recovered from ~; ~
'`I
intranasal infection with live B, bronchiseptica. The goal of
. 2'~ 30 effective immunization against the infection remained to be
'0 achieved, and no answer to this baffling problem was apparent.
''i The state of the art was summarized by D.O. Farrington
., ~
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~5~658
and W.P. Switzer, the inventors named in this application, in
a paper entitled "Resistance to Bordetella Rhlnitis",
Proceedings, The George A. Young Conference on Advances in
Swine Repopulation and the Thirteenth Annual Nebraska SPF
Conference, Lincoln, Nebraska, July 23-24, 1973, pages 44-52
as there stated (p. 46) "parenteral immunization of swine
with B. bronchiseptica bacterins is known to accelerate nasal
. _
clearance of B. bronchiseptica infections. In addition, develop~
ment of the gross lesions associated with atrophic rhinitis
appears to be significantly inhibited in suitably immunized
swine." Trials with experimental B. bronchiseptica bacterins
are summari~ed in Table 1. It was concluded that while
"parenteral bacterins have shown some degree of accelerated
clearance of B. bronchiseptica nasal infections'~, that "many
unanswered questions remain before the development of a prac-
tical immunizing agent against Bordetella rhinitis becomes a
j ~ reality."
DESCRIPTION OF THE INVENTION -
As indicated above, Bordetella bronchiseptica infection
,.,~. , . :
is the primary cause of atrophic rhinitis. Frequently, in the
~' aftermath of the infection, the turbinate bones undergo serious `
.... . .
.
~ atrophy. In the colonization of the nasal passages by the
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Bordetella organisms, they penetrate the mucous layer and anchor
themselves in colonies to the ep~thelial cells. During the
~s infec-tion, toxic substances are produced, which reach the under-
~ lying tissue, and! in particular/ the bone~forming cells of the
s nasal turbinate. Such toxic substances are believed to produce
the lesions causing turbinate atrophy. The present invention
provides a means for effectively immunizing against the toxic
substances which cause turbinate a~rophy without preventing
the primary B. bronchiseptica infection. More specifically, in
the work leading to the present invention, it was discovered
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that a killed whole cell vaccine could be prepared ~rom a
strain of B. bronchisep-tica identi~ied herein by ATCC No.
31124; which, when administered in a critical dosage, was
capable of effectively protecting swine against development
of the turbinate atrophy. In addition, although the vaccin- "
, ~ ~
ated swine are not immune to infection, an accelerated
clearance of the B. bronchise tica infection is usually obtained.
~ However, the principal new result of the present invention is
`~ that it immuni~es against turbinate lesions accompanying
- 1~ B b onchiseptica lnfection. This is important since such
_ ~
lesions and the resulting nasal bone deterioration are a major
.. ' ` ,,:: .
cause of economic loss to swine raisers. Because af the preva-
lence of Bordetella bronchiseptica organisms in breeding herds,
i and the great practical difficulty of maintaining breeding
1 herds free of B. bronchiseptica it is extremely di~ficult to
il avoid infection of young pigs at a susceptible age.
;l Further important details with respect to the prepar-
ation of vaccines in dosage form in accordance with the present
~;' invention, and the method of using such vaccines, are disclosed
below. Killed whole cell parenteral bacterins prepared from
Strain ATCC No. 31124 have been found to be effective in pre~
; venting turbinate atrophy and in reducing the duration of
B. bronchiseptica infection. Furthermore, it has been found
:., . , . . .~
that baby pigs at ages under 4 weeks respond satisfactorily to
the bacterins of this invention. The present invention there-
~ fore provides a means for providing baby pigs wlth antibody -
i; protection against turbinate atrophy before they have had an
opportunity to become infected with resulting nasal bone lesions.
Prior to the filing of the present application, there
~ ' ! ''
was placed on deposit with the American Type Culture Collection, -~
~, Rockville, Maryland, viable samples of the strain of
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~l B. b , which can be used as seed cultures for
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producing the vaccin~ o~ th~ pr~s~nt invention. The deposited
identical samples, earlier identified by the private code
designation "Strain ~-1", have been assigned ACTT No. 31124,
which will therefore be used as the identifying reference in
this spécification and appended claims. The available taxo-
nomic description of B. bronchiseptica Strain ATCC No. 31124
~ is ~
; Taxonomic D~scription ~-
ATCC No. 311~4 was recovered from the nasal and tracheal
exudate of a young mongrel pup that had clinical signs of canine
distemper. This strain was subsequently determined to be
avirulent for swine, and to have the follo~ing identifying
1. Small gram-negative rod.
2. Aerobic.
, 3. Colony approximately 1 mm in diameter after
`, incubation on 5% horse blood agar for 48 hours
. , .
at 37C. `
4. Colony circular, low convex, entire, opaque,
` 1! 20 smooth, homogeneous with undulate margin on 5%
i horse blood agar.
;~ S. Alkalinization of lactose broth in 18~24 hours
without gas formation.
6. Alkalinization of dextrose broth in 18-24 hours
without gas formation. `
7. Urease positive within 2~12 hours. ;
8. Citrate positive within 12-24 hours.
,, ~ .
9. Nitrate positive.
10. Alkalinization of litmus milk within 48 hours.
;~ 30 11. Motile by means of peritrichous flagella. ;~
12. Grows on MacConkeys agar in the presence of bile ;~
; . .~
~ salts. ~
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13. Sensitive -to sul~amethazine at 5 mg/ml level on
disc assay.
14. Resistant to 0.02 mg/ml furaltadone (NF-260J. ;;;
15. Hemagglutination positive (2.8% suspension of
sheep red blood cells1. ;~
16. Hemolytic on 5% horse blood agar after incubation
at 37C~ for 2~ hours. ~ ~
17. Non spore forming. ~;
18. Catalase positive.
19. Oxidase positive.
20. Indole negative.
21. Hydrogen sulfide negative.
, 22. Gelatin liquefaction negative.
- In preparing the vaccine of the present invention,
viable cells of B. bronchiseptica strain ATCC No. 31124, which
3 may have been subjected to freeze-drying or preservation, are
introduced into a suitable culture medium, which is then
incubated at a temperature favoxing the growth of the organism.
In ge~eral, published procedures for culturing B. bronchisep-tica
. ,, . ~
organisms can be employed. See, for example, Am. J. Vet. Res.,
~,~ 30, 1161, 1162 ~1969); and Am. J. Vet. Res~, 33, 1975, at 1976
''!1 . (1972). More specifically, tryptose phosphate broth (TPB) may
be used for propagation of the organism. One suitable source
of such a TPB medium is Difco Laboratories, Inc~, Detroit,
Michigan. Other usable culture mediums include: Bordet-Gengou
Agar ~Difco3 9 Brain-Heart Infusion Broth (Difco), tryptone soya
.: . :
i broth (Oxoid Limited, London, England). Propagation temperatures
:! of 36 to 38C. are favorable. More detailed propagation
::, . .
directlons are set in the following examples.
After propagation, the culture of ATCC No. 31124 is ;
killed without disruption of the cell walls. Suitable killing ~
agents include a final concentration of 0.2~ formaldehyde or ~ -
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1 lo, ono thimerosal (sodium ethylmercurithio.salicylate). A
preferred p~ocedure is -to add formalin, a 37% aqueous solution
of formaldehyde. Final formaldehyde concentrations o~ 0.1 to
0.2% are sufficient for complete inactivation where the cells
are held for 24 hours a-t room temperature (20-25C.). After
killing, the intact cells can be stored under refrigera-tion
(viz., at 4C.). Preferred concentrations of the bacteria
range from about 109 to 101l cells per milliliter, such as
substantially 101 cells per millilitex. Such killed cell ~-
concentrates are well suited for preparing vaccines in the
dosage form for practicing the present invention.
As is known to the art of preparing parenteral vaccines, -
a suitable adjuvant can be mixed with the killed cells in pre-
paring the vaccine for administration. An effective amount of
. ~ . , .
an adjuvant mixed with the killed cells can inGrease antibody
production in the vaccinated animals. For meat producin~
animals, such as swine, it is desirable that the adjuvant be
absorbable, whether injected subcutaneously or intramuscularly~
It is therefore desirable, at least for use in khe United States,
to employ an adjuvant which is approved by the U.S. Department
of Agriculture, Veterinary Biologics Division, for administration
.
to meat-producing animals. Aluminum hydroxide ~alumina cream)
has value as an adjuvant in the vaccines of the present inventon,
and is approved for use in mea-t-producing animals. The use of
this adjuvant is illustrated in the examples~ Another usable
adjuvant is one prepared as described in U.S. Patent 3~149rO36.
.:., . ~
A commercial form of this adjuvant is sold by Merck & Co., Inc.,
Rahway, New Jersey, as Merck Adjuvant 65~. The preparation an~
use of this adjuvant is also described in the examples.
. ~ - ~, .
The vaccine of this invention in parenterally inject~
able dose form consists essentially of the adjuvant in admixture
with from about 109 to 101l Xilled whole cells of said strain
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ATCC No. 3112~. The preerxed vacGine dose contains from 5
to 15 x 10 9 cells of said strain. Such vaccine doses are
particularly suitable for immunizing swine against development
of turbinate atrophy. The total dose volume of the vaccine
will include the cell volume plus the volume of the injectable
absorbable adjuvant. The dose volume will usually be more than
0 5 milliliters and less than 3.0 milliliters, but should be of
sufficient si~e to include an effective amount of the adjuvant.
Dose volumes of from 0.8 to 2.5 milliliters are particularly
suitable for intramuscular or subcutaneous administration In
one preferred preparation, from 10 to 15 x 109 cells are present, '~
and the total dose volume is from 1.8 to 2.2 milliliters. ~;
The vaccine of this invention can also be used for
protecting the offspring of a pregnant sow against the deYe
ment of turbinate atrophy. For example, at least one dose of
the ~accine containing from 109 to 1011 killed whole cells of
Strain ATCC No. 31124 is parenterally administered to the ~'
: ~:
~' pregnant sow not less than 14 days prior to farrowing. In a
', preferred procedure, from two to three doses of the vaccine are
administered to the'pregnant sow at intervals of not less than
~' 5 days between doses. For example, two injections of the' ~-
; vaccine may be given, the first one'approximately 28 days before
'~' farrowing, and the second one approximately 14 days before "
farrowing.
~' Procedures for practicing the present invention in
~ .
`';'!its various embodiments are illustrated by the following examplesO
'~ Example I
Bacterins for use in practicing the present invention '~
I can be produced by the following proceduxe:
;l1 30 1, Lyophilized D~l Strain (ATC~ No. 31124) is removed
'''~' , from storage at -20C and incubated overnight at
-'` 37C. in TPB (Tryptose phosphate broth~
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2, Transfer TPB culture into desired quantity of TPB
in a suitable container.
3. Aerobically incubate for 24 to 48 hours.
4. Purit~ check and determination of colony ~orming ~ ''units on 5~ horse blood agar. -~
. .
5~ Add formaldehyde, 37% solution (J.T. Baker,
Phillipsburg, N.J.~ to a ~inal formalin concen- ~'
tration of 1:1000. Mix well.
6. Incubate overnight at room temperature. Mix well~ ;Inactivation and sterility check on 5~ horse b1ood
agar.
' 7. Store at 4C.
r Example II ~ ;
AdJuvants suitable for use in practicing the present
1 invention can be prepared as follows: ' ~ ,
'J` Aluminum Hydroxide Adjuvant
:,
, 1, A suitable amount,of aluminum hydroxide compressed
~, , gel, eg. 384g., containing about 905% Al2ol3 is ,~
'~ , mixed with deionized water in variable amounts to
~ . ~
make a final 2G% aluminum hydroxide solution.
~ 2. The final solution will provide approximately 2%
) ' A1209.
3. Autoclave sample. ,,;
~ 4. Store at room temperature.
',, Peanut Oil Adjuvant
'~, , 1. The adjuvant is composed of 85.0% peanut oil, 1~.6
mannide monooleate ~Arlacel A~ and 4.4% aluminum
',, monostearate. , , '~
~' 2. A suitable quantity of each ingredient is mixed
.. " . . .
in the above proportions in a suitable container on ,
, a heater-stirrer.
,,, 3. The temperature is raised at the rate of 4C per
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minu-te ~o 120C.
4. The mixture is constan-t]y s-tirred with a glass
rod during the heating process.
5. The homogeneous material is dispensed into amber
; bottles and autoclaved.
6. Store at room temperature. ;
This adjuvant is referred to as ~djuvant 65, and i5 `
obtainable from Merck & Co., Inc.l Rahway, N.~. tSee U.S. Patent ~
No. 3,149,036.~ ~ ;
J 10 Example lII ! ~ ~ :
Suitable procedures for preparing vaccines using the
adjuvan$s of Example II are set out below.
Pre aration of Vaccine with Aluminum Hydroxide Adjuvant
P . , ` ~: ~
1. Equal volumes of the aluminum hydroxide adjuvant
:; - :
(20% solution) and inactivated TPB D-l strain
i broth culture are thoroughly mixed in a suitable
container to form the complete vaccine~
2, The vaccine will contain approximately 1% Al203. ,~
3. The final concentration of the vaccine contains
approximately 101 cells per ml~ ;
4. The vaccine is stored at 4C. until use in 100 ml.
; glass, rubber stoppered, sealed, vials.
- 5. For administration to baby pi~s, two ml. of the
:. . , . :
~ vaccine would be administered subcutaneously.
!Pre aration of Vaccine with Peanut Oil Adiuvant
P
~ 1 :
1 1.~ Equal volumes of the peanut oil adjuvant and in-
activated TPB D-l strain broth culture are
'l thoroughly emulsified to form the complete vaccine.
~1 :'' :'
2. The final~concentration of the vaccine contains ~ ;~
~`~ 30 approximately 101 cells per ml.
--~ 3. The vaccine is stored at 4C. until use in 100 ml.
~ glass, rubber stoppered, sealed vials.
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4. For administration to baby pigs, two ml. of the ;~
vaccine would be administered subcutaneously.
Example IV
A preferred vaccination procedure for ba~y pigs is as
~ollows: .
. .
1. A two ml. dose of either of the vaccine products
of Example III is administered to each pig at 1
week and at 4 weeks of age.
2. The vaccine is injected subcutaneously.
Example V ~;
A preferred vaccination procedure for sows prior to
:
farrowing is as follows;
A two ml. dose of either of the vaccine products
of Example III is administered to each dam at 4
weeks and at 2 weeks prior to farrowing.
;: .
il 2. The vaacine is injected subcutaneously.
3. Females with high levels of humoral antibody
against B. bronchiseptica can be expected to ~ -
transfer high levels of anti-Bordetella bronchi-
septica antibody via the colostrum to the ;~
.. ~
'`~ - - susceptible young pigs. ~
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