CA1324075C - Bordetella bronchiseptica vaccine - Google Patents

Abstract

Title Bordetella Bronchiseptica Vaccine Abstract of the Disclosure A vaccine for protecting canines against infection by Bordetella bronchiseptica is prepared by inactivating whole cells with glutaraldehyde.

Description

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TITLE
~; Bordetella Bronchiseptica Vaccine ~ . .
FIELD OF THE INVENTION
~ This invention relates to inactivation of -~ 15 pathogenic bacteria and use of such inactivated bacteria in a vaccine.

BACXGROUND INFORMATION
The members of the genus Bordetella, which ~;~ - 20 presently includes B. pertussis, B. p~raPertussis and B.
~` bronchisePtica, are small, gram-negative coccobacilli, 0.2 to 0.3 ~m by 0.5 to 1.0 ~m. They appear singly, in ` pairs and in small clusters. Their morphology and physiology are more fully described in Zinsser MicrobiolosY~ 17th edit., edit. by Joklik et al., 1980, Appleton-Century-Crafts, New York, pages 614-624.
Bordetella species are serious pathogens of the respiratory tract in a wide variety of mammals, including man. B. bronchisePtica is a primary causati~e agent of -` 30 atrophic rhinitis and pneumonia in pigs and of infectious tracheobronchitis (~Kennel Cough~) in dogs. B. ~ertussis and, to a lesser extent, B. parapertussis and B.
bronchiseDtica cause whooping cough ln man.
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1 Peppler et al., Infect. Immun. 44(3):681 (1984), describe phenotypic variation of Phase I, II and III B.
; bronchiseptica strains. Similar phenotypic variation occurs in B. Pertussis and B. ParaPertuSSiS.
Musser et al., J. Bact. 166:230 (1986), discuss the interrelatedness of Bordetella species.
, Goodnow et al., Am. J. Vet. Res. 44:207 (1983), report presence of a heat-labile toxin in B. bronchisePtica ,. .
s extracts and suggest a role for such toxin in pathogenesis.
.; 10 Robinson et al., Infect. Immun. 40:523 (1983), report that a protein produced by B. pertussis known variously as lymphocytosis-producing factor hemagglutinin ~: (LPF), histamine-sensitizing factor, islet-activating .~ protein, pertussis to~in and pertussigen is important for protection in an intracerebral (i.c.) mouse protection ,, :
: test. Affinity purified LPF was treated with glutaralde-hyde to detosify the protein.
Sekiya et al., Infect. Immun. 41:598 ~1983), report vaccination of mice with formalin-inactivated B. bronchisePtica.
Relyveld et al., C.R. Acad. Sc. Paris, t277, Serie D, 613-616 (1973), and Relyveld et al., Toxicon 1976, Suppl. 1 (Toxins: Animal, Plant and Microbiol.), ~; pages 1045-1065, disclose preparation of a 8. pertussis s 25 vaccine by glutaraldehyde inactivation.
~,~ To date, there has not been a satisfactory ~ vaccine for protecting canine animals against infection by -~ 8Ordetella bronchisePtica. The formalinized or otherwise inactivated whole cell vaccines which have been employed cause undesirable side effects, predominantly including transient lethargy, anorexia and vomiting.

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SUMMARY OF THE INVENTION
It has now been found that a safe and effective 5 vaccine for protecting canines from disease caused by Bordetella bronchisePtica can be prepared by inactivating whole cells by treatment with glutaraldehyde. Thus, in ~' one aspect, this invention is a vaccine for protecting a canine animal against disease caused by Bordetella 10 bronchiseptica comprising a vaccinal amount of whole Bordetella bronchisePtica organisms which have been inactivated by treatment with glutaraldehyde.
In a second aspect, the invention is such vaccine which further comprises a vaccinal amount of one or more 15 other antigenic components for protecting a canine animal ' against disease caused by one or more other pathogenic ` microorganisms, viruses or cells.
In a further aspect, the invention is a method for protecting a canine animal from disease which r 20 comprises internally administering the vaccine of the invention.

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DETAILED DESCRIPTION OF THE INVENTION
The vaccine of the invention comprises a vaccinal, i.e., effective and non-toxic, amount of w~ole 80rdetella bronchisePtica organisms which have been inactivated by treatment with glutaraldehyde. Typically, the vaccine is prepared by addition of glutaraldehyde to a culture of a non-attenuated strain of B. bronchiseptica.
80rdetella organisms for use in preparing a vaccine o~ this invention are available from a vaeiety of sources including various public and private depositories and clinical infections. Bordetella colonies on Bordet-Gengou agar are known to exist in distinct morphological types. These have heen classed into Phase I, II and III. See, Peppler et al., Infect. Immun. 44 (3):681 ~1984). In this invention, non-attenuated Phase I
Bordetella are preferably employed.

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.,, 1 For initial growth and propagation, the Bordetella organisms can be cultured on a variety of ~ nutrient media. Bordet-Gengou medium (BG) and synthetic T~ minimal medium, as described by Parker, Adv. ApPl. Micro.
20:27-41(1976), ("Charlotte Parker~s minimal medium, i.e., HCPM"), are prefered. Both can be prepared using ~ commercially available media. BG, for example, can be -~ prepared by adding 20% defibrinated sheep blood to basal medium such as is supplied by B~, Division of Becton Dickinson, Cockeysville, Maryland. Bordetella organisms are cultured at 30 to 45C, preferably 35-40C and most preferably at 36-38~, at approximately neutral pH, e.g., ~- 6.5-7.5, preferably 6.7-7.1. For best growth, liquid cultures are agitated and aerated throughout fermentation. To prepire production cultures, seed ~` cultures are serially propagated in accordance with '~ standard techniques. ProductiDn cultures are seeded with at least a 1%, preferably 5 to 10~, inoculum of actively ~. growing cells. Fermentation, prior to inactivation, is j 20 typically continued until nutrients are substantially depleted, i.e., depleted or nearly depleted, e.g., 18 to ~.: 36 hours. The organisms used in bacterin preparation are ;~ passaged for propagation and preferably remain substantially non-attenuated throughout serial passage and production culture.
To prepare the bacterin used in vaccine formu-lation, glutaraldehyde is added to an active culture having achieved a cell density of at least 3,000 nephelo-metric units, preferably 4500 to 10,000 nephelometric $
:` t`` 30 units. Nephelometric units are determined using a Nepho-^~ Colorimeter which has been calibrated with a standard of g: certified accuracy, for example, a Coleman Certified Nephelo Standard N ~ 78, and a dilution blank solution, for example, physiological saline. The method used to ~ 35 calibrate the Nephelo-Colorimeter is commonly referred to ;. ,~
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Samples of unknown nephelometric values are diluted stoichiometrically in dilution blank solution so that when measured in the calibrated instrument, the value of the diluted sample is betweeen zero and 78 nephelometric units. This value is then multiplied by the dilution factor to determine the nephelometric value of the undiluted sample.
Glutaraldehyde is added to a final concentration of 0.15 to 0.25 weight percent (wt. %), preferably 0.20, Swt percent glutaraldehyde per 5000 nephelometric units.
The culture is incubated in a presence of glutaraldehyde until complete inactivation has occurred, typically at least 12 hours. An equimolar amount of L-lysine can then be added to complex residual free glutaraldehyde.
20Finally, a preservative such as thimerosal or other known ~ disinfecting agent suitable for internal administration s`~ can be added to the bacterin. TypicaLly, the bacterin ~s will then be diluted to prepare a vaccine formulation ~ containing an effective amount per unit dose. The diluent ;~ 25can be any pharmaceutically acceptable solvent, e.g., ~, physiological saline.
An effective amount of the bacterin is determined by standard techniques as that amount which does not cause :~ unacceptable adverse reactions but which is effective in 30inducing an immune response in a canine animal such that.
` ~ the host is substantially protected against development Oe clinical symptoms associated with infection by Bordetella b onch.septica, i.e., Kennel Cough. ~n effective amount for protectinq canine animals is typically in the range of 35250 to 750, preferably about 500, nephelometric units per dose (based on nephelometric determination at time oE
~s~- harvest, prior to inactivation). Such dose typically is .. ..

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--~ 1 324075 administered to a canine animal in a dose volume of 0.5 to ;` 3 ml, preferably about 1 ml in a two dose course, the second dose being 1 to 5, preferably 2-4, weeks after the - 5 first. The vaccine is administered internally, e.g., parenterally, preferably by subcutaneous or intramuscular injection such as in the neck region of a dog or other domestic animal. The vaccine is preferably administered to puppies 6 to 12, preferably about 9, weeks old.
10 Intramuscular may be the preferred route of administration in puppies. Annual revaccination with a single dose is recommended for all dogs.
~- Vaccination with the vaccine of this invention - has been shown to be effective in preventing respiratory i 15 disease symptoms associated with a. bronchiseptica infection in dogs and, surprisingly, to be free of the undesirable side efects typically associated with known whole cell vaccines. For example, thimerosal-inactivated bacterins and attenuated live vaccines for ~-~ 20 protecting canine animals against Kennel Cough have been ~ associated with transient lethargy, anorexia and vomiting -~i 1-6 hours following vaccination in up to 1/3 of dogs inoculated. Such effects are typically not observed with the vaccine of the invention. In an extensive field trial 25 in which over 9,000 doses of the B. bronchisePtica vaccine of the invention were administered to dogs, there were only 1 in 400 reports of such side effects (0.23%).
A typical preparation of a vaccine for protecting canines against the clinical symptoms of infection by * 30 B. bronchiseptica is as follows.
~ A master seed stock of non-attenuated B.
-~ bronchiseptica is maintained as a 1yophl1ized culture at ~; 2-7C. For preparation Oe workinq seed, a san?le is restored with sterile distil1ed water and spread onto a 35 Bordet-Genqou plate. Followinq incubation at 27 + 1C for ;~ 24 to 48 hours, typical smooth colonies (Phase I) are selected ~or culture in liquid medium.

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~ _ 7 _ t 324075 1 Seed culture and production cultures are examined microscopically for motility and by gram stain for morphology. Cultures are also confirmed for (+) citrate utilization, t+) nitrate reduction, (-) H2S production and (+) urea utilization.
Following serial passage for propagation, a seed culture is inoculated into minimal medium in a fermentor and incubated at 37 + 1C to a cell density of at least 4500 nephelometric units (typically 18-36 hours, often 10 times 20-30 hours) with continuous agitation and aeration.
At harvest, the pH of the culture is adjusted to approximately 6.8 with concentrated HCl and a nephelometric i value is determined. Glutaraldehyde is added to yield a ,~ 15 final concentration of 0.10 to 0.30 weight percent of `~ glutaraldehyde per 5,000 neohelometric units of cells.
The treated culture material is stirred in the fermentor at 15 to 45C, preferably 35 to 40C, for about an hour.
~s The material is then transferred aseptically into a glass 20 storage vessel wherein incubation at 15 to 30C, ~referably 20 to 24C, with gentle stirring is continued ~.
for approximately 24 hours after ~hich an equimolar amount of sterile lysine is added. The temperature is then - lowered to 2 to 7C until assembly into dosage unit forms.
To assemble dosage unit forms, the bacterin is diluted with saline to a final cell density of 250 to 750, preferably 500, nephelometric units per 0.5 to 3, preferably 1 ml and packaged in 1.0 ml units in sterile vials. Thus, based on the calculation at time of harvest, 30 each unit dose contains 250 to 750, preferably about 500, nephelometric u~its. If desired, the bacterin can be lyophili3ed for subsequent reconstitution with saline or other parenterally acceptabl~ diluent or excipient in accordance with standard veterinary pharmaceutical 35 techniques.

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Included within this invention is a combination vaccine for protecting animals against infection by ; Bordetella and by one or more other pathogenic microorganisms, cells or viruses. Such other vaccinal agent contains additional antigenic components such as a killed or modified whole cell vaccine or a subunit vaccine which, in any event, is combined with the Bordetella bacterin such that all antigenic components are provided in vaccinal amounts. For example, the B. bronchisePtica r; 10 bacterin can be combined with, for esample, modified live Canine Distemper Virus (ML-CDV), modified live canine Adenovirus Type 2 (ML-CAV2), modified live Canine Parainfluenza Virus (ML-CPl) and a LeptosPira, e.g., canicola and ictohemmoraqhiae bacterin. Techniques for preparing such combination vaccines are well known. For example, Jaeger et al., Vet. Bull. 44:2163 (abstract) 1974, report a trivalent vaccine comprising inactivated ~` rabies virus, ML-CDV and inactivated canine viral hepatitis virus (CVH) and a tetravalent vaccine comprising ; 20 inactivated rabies virus, ML-CDV, inactivated CVH and killed leptospira; Ackermann et al., Vet. Bull 46:1894 , (abstract) 1976, report a tetravalent vaccine comprising inactivated rabies virus, ML-CDV, inactivated CvH and : ~.
killed leptospira; Bijlenga, U.S. 4,351,827, disclose a bivalent vaccine comprising ML rabies virus and ML-CDV.
To prepare a combination vaccine of the invention, a vaccinal amount of the Bordetella bacterin of the ~ invention, e.g., 250 to 750 nephelometric units per dose as described above, is contained with one or more of such other vaccine components.
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:, , ExAMPLES
The Examples which follow are illustrative and not limiting of the vaccine and method of the invention.

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MATERIALS AND METHODS
Cultures. B. bronchisePtica strain 87 was s~; received from Dr. L.E. Carmichael, Cornell University, Ithaca, New York. The organism was originally isolated by Dr. D.A. Bemis from a dog at the James A. Baker Institute ;~ for Animal Health. B. bronchise~tica strain BC was ~-~ isolated from a cat by Mr. Jerry G. Bihr of Norden ~; Laboratories, Lincoln, Nebraska.
~ Media. Trypticase Soy Agar (DIFCO Laboratories, -~` Detroit, Michigan) supplemented with 100,000 units/litar of U.S.P. Potassium Penicillin-G and lS0 mg/liter of i;~. "Furaltadone" (Norwich Pharmacal Co.) was used as a ;- i- 20 selective medium for B. bronchisePtica. Bordet-Gengou medium was prepared from basal medium (8BL, Cockeysville, . Maryland) supplemented with 20% defibrinated sheep's m blood. The medium used for vaccine production was the synthetic minimal medium described by Parker, Adv. APPl.
Micro. 20:27(1976).
The medium is preferrred by (i) dissolving, in order, L-proline (2.40 9), L-glutamic acid (0.67 9), NaCl 9), RH2PO4 (0.S0 9), KC1 (0.20 g), Mg ~; C12-6H2O (0.10 g), CaC12-2H2O (0.0265 9) and Trizma base (6.075 9) (Sigma Chemicals, St. Louis, ~-~; Missouri) in 900 ml of water, (ii) adjusting the pH to about pH 7.2 with HCl, (iii) adding water to a total of 970 ml, (iv) sterilizing the medium by autoclaving and (v) adding to the medium filter-sterilized aliquots (10 ml) of . , ~ 3S A:L-cystine (.04 g), B:FeSO4-7H2O (0.01 9) and : C:Ascorbic acid (0.02 g), nicotinamide (0.004 9) and ~. , '~
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,, 1 324 075- lo sodium acetate (0.2 9), thereby bringing the total volume from 970 ml to 1 L.
. t Agqlutination Test Preparation of Antiqen: B. bronchisePtica strain 87 was grown in 750 ml of synthetic medium at 37C with * vigorous shaking. Following incubation for about 22 hr, ~`- 0.8 ml of formalin was added. After standing for 24 hours ~, 10 at 37C the cells were harvested by centrifugation at ~- 10,000 x g for 30 min at 5C. The cells were resuspended to approximately 200 ml in saline containing 0.037 percent i:
formaldehyde. The concentrate was stored under refrigeration. For use, it was diluted 1/20 in Dulbecco's saline solution prepared by dissolving NaCl (6.4 g), CaC12 (0.01 g), and MgC12 6H2O (0.01 g) in water to a total volume of 100 ml (q.s.).
Test Procedure: The test was performed in conical bottomed 96 well plastic trays. ~ilutions of serum were made in Dulbecco's saline supplemented with 0.01% bovine serl~ albumin in a volume of 0.05 ml. Sera to : ~,.
be tested were added in a volume of 0.05 ml to the first well and dilutions are made manually. To each well, 0.5 -~^ ml of antigen was added. The eirst well was then considered to be a 1:4 dilution of serum. Standard ~; controls with known positive and negative sera were included within each series. The plates were shaken x mechanically for 2 minutes, incubated at 37 for 2 hr and -~ then overnight at 4C. The test was read usinq a stand t with a magnifying mirror. A reference to this procedure , may be found in a publication by D.L. Harris and W.P.
~- Switzer entitled, "Immunization of Pigs Against Bordetella ;~; bronchiseptica Infection by Parenteral Vaccination,N
published in the Am. J. Veterin. Res. 33:197S-1984.
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,~ Aerosol Exposure of Dogs ;, Dogs were sedated prior to treatment by inocula-tion of 44 mg of xylazine (nRompum," Chemago Division of 5 Bay Chemical Corp., Kansas City, Missouri) per kilogram of body weight. The challenge dose was administered with a model 40 DeVilbiss glass nebulizer (DeVilbiss Company, Medical Products Division, Somerset, Pennsylvania) which ~' was inserted through a hole in the bottom of a styrofoam 10 cup fitted over the doq's muzzle. The culture concentrate was administered from four to eight minutes using 0.7 Rg/cm2 (10 lbs/sq. inch) of air pressure. In a sin~le ~, experiment, the same time period of administration was :
; used for each dog.
;; The culture for challenge was prepared by J'.,'~' reconstituting a vial of lyophilized challenge culture (B.
bronchiseptica Strain BC) into a flask of Trypticase Soy Broth or Charlotte Parker's Medium. After incubation at 37C for approximately 24 hour~, a drop of broth culture 20 was transferred to plates of Bordet-Gengou Medium. After r~: an additional 24 hours incubation, the growth was removed and resuspended in saline. It has been found that cultures standardized by nephelometry to 1 X 107 orqanisms/ml consistently produced coughing in susceptible 25 dogs-. .
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Selection of Doqs All dogs used for these studies were screened for ~ antibodies against B. bronchiseptica and cultured to 6~ ~ 30 verify they were not colonized, and therefore susceptible to infection. Antibodies were detected by the aqqlutination test. Dogs having serum titers of 1:16 or hiqher were excluded from the test. Cultures were t collected from deep tracheal swabs and plated on selective 35 mediu~. If a dog was infected, a pure culture of B.
:~ bronchiseptica was usually obtained. Isolates which were ' ,: :
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,,, 1 not Bordetella were tentatively identiied by a gram stain. Dogs were segregated by litters in isolation ' rooms. Additional swabs were taken prior to challenge and two weeks following challenge. All cultures taken prior '~ 5 to challenge had to be negative for B. bronchisePtica .~`r. before approving the dog for experimental use.

Collection of Tracheal Swabs For the routine examination of dogs, it was found that deep tracheal swabs were preferable to nasal swabs.
~ If infected, tracheal swabs were usually pure cultures of -~ B. bronchisePtica. Dogs were anesthetized with ~Suritol~
(Park Davis, Detroit, Michigan) using about 0.1 ml per pound of body weight. While the mouth was held open, a sterile swab was inserted deep into the trachea. The swab . was transported in a screw cap tube with 0.1 ml saline.
For culturing, the swab was streaked onto the Selective . Medium. Cultures were incubated at 37C and examined at 48 hours. Growth on selective medium was subcultured to 20 differential media for biochemical identification of B.
bronchisePtica.
7~:~ Seed. A working seed has been prepared from the master seed by five passages on Bordet-Gengou blood plates, overnight growth in synthetic medium, and 25 lyophilized. The working seed was checked and found satisfactory.

Bacterin PreParation .~
,~ Growth of Orqanisms. Each experimental lot of ;~ 30 bacterin was derived from a freshly reconstituted vial of ~` working seed. A drop of reconstituted seed was placed :~ onto Bordet-Gengou blood agar and the plate was struck with a sterile loop to obtain isolated colonies. After 48 hours incubation at 37C, 10 to 20 typical small, smooth . ~
colonies were picked, transferred to 5 to 10 ml of sterile synthetic medium which was immediately transferred to 50 :

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t 324075 13 -'~. 1 ml of synthetic medium in a 300 ml flask. This starter culture, and the subsequent step-up cultures, were ' incubated for 18 to 24 hours at 37C with vigorous shaking. Step-up growth to 10 liter production fermentors ~, occurred in 500 ml of synthetic medium in a 2 liter flask. Once the 10 liter fermentor was seeded, the culture was incubated at 37C for 8 to 24 hours; that is, ~ until the organism no longer demanded oxygen. Dissolved `~ 10 oxygen content was monitored and controlled by aeration with sterile air. The culture was stirred continuously during the growth period. Sterile antifoam solution was used to control foam.
Inactivation. At the time of inactivation, the 15 culture density was determined by nephelometry. The pH of ;` the culture was adjusted to approximately 6.8 and . f sufficient glutaraldehyde was added to give a ratio of glutaraldehyde to cell density of 0.20 weight percent ~lutaraldehyde to 5000 nephelometric units of cells. To 20 ensure free aldehyde groups no longer remained, an equimolecular amount of sterile lysine ~as subsequently ~i added.
~, Assemblv. Four small lots of bacterin were prepared and were assembled according to the proportions ~ 25 shown in Table 1 such that one dose contained 500 ;~ nephelometric units of inactivated B. bronchiseptica per ml. Sterile 10~ thimerosal was added to a concentration not exceeding 0.01% (W~V).

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., 1 -Tahle 1. Assembly of Experimental Lots of B. bronchiseptica Bacterin Experimental Lot ComPonent Ferm. Run No. 10 11 12 13 NEPHELOMETRIC
~'~ VALUE 6000 5000 5000 7200 Vol. of Culture (ml) 20.8 25 25 28 ' 10 , Vol. of Sterile , Saline (ml)229.2 225 225 372 Vol of ? Sterile 10%
Th imerosal i 15 (ml) 0.025 0.025 0.025 0.4 Vaccination and Challenge. Litters of mongrel pups ranging in age from 8 to 20 weeks were purchased locally and brought into our isolation units where they ~` 20 were held as litters throu~hout the entire test period.
Prior to use, all pups were screened for antibod ies against B. bronchiseptica and checked twice by tracheal culture for absence of this or~anism. Pups were kept by '5' litters and, in so far as practical, controls and 25 vaccinates for each serial were selected sequentially.
Two exper iments were conducted. The first experiment tested experimental Lots 10, 11, and 12; the second tested experimental lots 11, 12,and 13. Pups were ~ vaccinated intramuscularly two times at a two week `55 30 inte rval with 1.0 ml dose. Nonvaccinated control dogs received a sham vaccination of saline. Twelve to 28 days eollowinq the second vaccination the pups were challenged by aerosol. The pups on the first test were vaccinated in two group.s; the first qroup, Days 0 and 14, and the second, Days 14 and 28. All pups were challeng~-l on Day 42. The pups on the second test were vaccinated on Days 0 and 14 and challenged on Day 30.
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~ 1 Other Tests. The esperimental lots were tested `.~ for purity, safety, and viricidal activity in accordance with 9 CFR 113.26, 113.38, and 113.35, respectively.

RESULTS
' PuritY, SafetY, and Viricidal ActivitY. Purity, safety, and viricidal activity tests were performed by the quality assurance division of Norden Laboratories. The results were satisfactory for all tests performed.
, 10 Untoward Reactions. All dogs were observed 'L continuously for four hours immediately following vaccination for indications of lethargy, malaise, and seizures of vomiting, and daily thereafter. All dogs ~ remained normal throughout the observation period.
.~ 15 SeroloqY. Serum samples taken prior to vaccination, two weeks later at the second vaccination, prior to challenge, and two weeks post challenge were evaluated for antibody levels by agglutination. Serum from vaccinated dogs showed increased antibody levels prior to challenge, whereas, serum from sham (saline) vaccinated controls dogs did not. Titers from all dogs increased following challenge. The results are summarized in Tables 2 and 3.

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Table 3. Results of Serum Agglutination Tests Reciprocal Titers E~perimental Dog Pre- Pre Pre 2 Weeks Lot No.Vac2nd Vac.Challenae Post Challence - 11 A26 4 8 1024 >8192 0 A30 4 16 1024 >8192 ` A35 ~4 8 2048 8192 A39 4 64 4092 >8192 , A43 4 32 2048 >8192 ~ A47 4 16 2048 4096 -~ A50 4 16 2048 4096 12 A27 <4 8 1024 >8192 A31 4 8 512 >8192 i~ A36 4 16 2048 8192 A44 4 16 4096 >8192 ;~ A51 4 32 4096 >8192 13 A28 4 32 2048 >8192 A41 4 64 2048 >8192 ~, 25 A52 4 8 128 4096 ~ Sham (Saline) A25 4 4 4 32 '~ Vaccinated A29 4 4 4 128 Controls for A33 4 4 4 256 Lots 11, 12, A34 <4 <4 <4 512 13 A38 4 4 4 lZ8 A42 <4 4 <4 128 A46 4 4 4 2p48 . .

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,, ~ - 18 ~ 1 324075 - 1 Chalienqe. Following challenge, pups were observed twice each day for 14 consecutive days for clinical signs of disease. Failure to protect was defined as coughing or other indication of clinical disease on two consecutive days. Artificial stimulation to produce the coughing reflex was not used. The results are presented in Tables 4 and 5 and summarized in Table 6.
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Clinical Remained 5ExPerimentLot No.Siqns (couqhinq) Normal s~ 10 Control 5 :.~ 2 11 1 7 .'.~. 12 2 6 ~, 15 Control 8 0 -',~, .
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- 22 _ 1 324075 s 1 Throat swabs collected from selected pups (vaccinates and controls) two weeks post challenge uniformly showed colonization with B. bronchisePtica.
- Analysis of results by the Chi-Square Test shows .,. .
that all four esperimental lots are satisfactory at P<5%.
Lot 10 was satisfactory in experiment 1. Lots 11 and 12 were inconclusive because of the number of dogs in the . test. In experiment 2, lots 11, 12, and 13 were satisfactory. The combined results from both experiments ' 10 also showed lots 11 and 12 to be satisfactory.
The canine potency test used to evaluate this product is reliable because it closely approximates the disease naturally observed in the host animal and depends --.;
.~ upon a natural route of infection for esposure. The difficulty with it is in obtaining dogs without prior ., esposure. Our criteria for dogs were strict. Dogs must have been serologically negative and free from B.
~ bronchisePtica as determined by two tracheal swabs taken -, two weeks apart so that dogs are in isolation for at least twb weeks before the second specimen is collected.
One problem with the Bordetella bacterins, heretGfore, has been that they produced an unacceptable tosic reaction in too great a percentage of pups shortly -after administration. Within the next few hours after leaving the veterinary clinic, a vaccinated dog's owner . might observe lethargy, malaise, or seizures of vomiting.
Despite the fact that the product provided protection, veterinarians were reluctant to use this product.
An adjuvant was not used in preparing these ., ` 30 bacterins because good potency was obtained without one.
However; aluminum hydroxide as well as other parenterally-~ tolerated adjuvants can be employed in the vaccine of the ;~ invention.

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., MATERIALS AND METHODS
Canine Parainfluenza Vaccine. Canine parain-fluenza virus strain NL-CPI-5, a strain licensed by the United States Department of Agriculture, was cultured and harvested in Norden Laboratories production facility. A
small esperimental serial was prepared by serially passaging the virus in a canine kidney cell line. The ` 10 virus was harvested, combined with a stabilizer (40~ by . volume) and lyophilized in one ml dosage units. Each ~; dosage unit contained 107-3 TCID50 of modified live s virus.
Bordetella Bronchiseptica Bacterin. A Pilot r, 15 Serial of a Bordetella Bronchiseptica Bacterin was ~ prepared substantially as described in Example 1, above.
-~ Selection of Doqs. All dogs used for this study ~ were screened for antibodies against B. bronchisePtica and ;;~ canine parainfluenza virus. Further, a throat swab was taken from each dog substantially as described above to verify that they were not colonized with B. bronchiseptica.
Dogs colonized with B. bronchisePtica or having serum titers of 1:16 or higher were excluded from the test.
Dogs having titers to canine parainfluenza of 1:4 or greater were likewise excluded.
Vaccination. The Bordetella bronchisePtica bacterin was used to aseptically rehydrate the Canine Parainfluenza Vaccine. Vaccinations were administered intramuscularly as a 1.0 ml dose on 2 occasions with a 14 day interval. Dogs were closely observed for 3 to 4 hours following each administration of vaccine for development of untoward reactions.
Seroloqical Evaluation. Sera were collected prior to the initial, and two weeks following the final, vaccinations. Antibody levels to B. bronchisePtica were determined by an agglutination test. Antibody levels to '~
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RESULTS
Untoward Reactions. All dogs were observed ; continuously for 3 to 4 hours immediately following , vaccination for indications of lethargy, malaise, and -` seizures of vomiting, and daily thereafter. All dogs remained normal throughout the observation period.
SeroloqY. Results of serological evaluation of -~ pre- and post-vaccination sera are shown in Table 1-2.
Prior to vaccination, the sera of all dogs were essentially negative for antibodies to both components. The serum antibody levels for all dogs increased following the vaccination regimen and were consistent with titers of dogs which have been shown to be protected from challenge . with virulent strains of either pathogen.
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1 Results of serological evaluation of the Pilot Serial of 80rdetella Bronchiseptica 8acterin are presented in Table 2-2. we evaluated an increase in serum antibody level, as measured by agglutination, to reflect exposure ; 5 to 8. bronchise~tica surface antigens, either through vaccination or by natural infection. Although the agglutination titer may not be a direct measurement of antibody levels to protective antigens, it is the strongest indicator of a post vaccination response at our disposal and, historically, has been consistent with protection in challenge of immunity studies. Some dogs demonstrate indications of disease regardless of their serum agglutination titer. Conversely, other data show protection in dogs having a titer of as low as 4 at the time of challenge. We evaluated the agglutination titer for B. bronchisePtica herein to be satisfactory when ` compared to antibody levels observed in dogs, at the time : of challenge, which were protected.
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NO. PRE POST
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~ 10 A59 NEG 4096 5" A60 NEG 1024 ~ A64 NEG 4096 .; A65 NEG 2048 .~ 15 A66 NEG 4096 ~,. Geometric Mean NEG 2299 ~: NON-VACCINATED 205 NEG NEG
: CONTROLS 206 NEG NEG
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1 The purpose of the combination vaccine described herein is to elicit a protective level of immunity to virulent strains of canine parainfluenza virus and B.
bronchiseptica. The data presented herein demonstrate that neither fraction interfered with the immunological response of the other. Further, the levels of serum antibody to both canine parainfluenza virus and 3.
- bronchisèPtica were consistent with the levels in animals found to be protected from clinical disease following 10 rigorous challenge.
In a similar study, a combination bacterin containing the gluturaldehyde-inactivated B. bronchisePtica fraction of the invention and inactivated LePtosPira canicola and Leptospira icterhemmorhaaiae was used to 15 restore dessicated ML-CDV, ML-CAV2, ML-CPI and ML-CPV as ; contained in VANGUARD0 Canine Distemper-Adenovirus Type . ~i ~; 2 - Parainfluenza-Parvovirus Vaccine ~Norden Laboratories, Lincoln, Nebraska, U.S.A.). This seven component combination vaccine was administered in l ml doses, . ~ 20 subcutaneously or intramuscularly to dogs. All dogs ; developed an immune response indicative of protection to all fractions following a single vaccination except that a ;~ booster dose, e.g., S to 17 days later, was required to : ~.
stimulate the desired response to the B. bronchisePtica $ 25 fraction.
The above description and examples fully disclose - this ~n~ention and the preferred embodiments thereof. The ;~ invention, however, is not limited to the precise constructions herein described but, rather, encompasses all modifications and improvements coming within the scope of the claims which follow.
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Claims (21)

1. A vaccine for protecting a canine animal against disease caused by Bordetella bronchiseptica which comprises a vaccinal amount of whole Bordetella bronchiseptica cells which have been inactivated by treatment with glutaraldehyde.

2. The vaccine of claim 1 which comprises a glutaraldehyde inactivated culture of B. bronchiseptica grown in liquid medium at 35 to 40°C with aeration for 18-36 hrs until nutrients are substantially depleted.

3. The vaccine of claim 1 which comprises 250 to 750 nephelometric units, prior to inactivation, within each vaccine dose.

4. The vaccine of claims 1, 2 or 3 which further comprises L-lysine in an amount required to complex residual free glutaraldehyde groups.

5. The vaccine of claims 1, 2 or 3 which comprises 500 nephelometric units, prior to inactivation, per ml.

6. The vaccine of claim 5 which further comprises an amount of L-lysine equimolar to the amount of glutaraldehyde and .001 to .01% thimerosal.

7. The vaccine of claim 1 which further comprises a vaccinal amount of one or more additional antigenic components for protecting a canine animal against disease caused by one or more other pathogenic microorganisms, cells or viruses.

8. The vaccine of claim 7 in which the additional antigenic components are one or more of inactivated Leptospira canicola, inactivated Leptospira icterhemmorhagiae, modified canine distemper virus, modified canine adenovirus type 2, modified canine parainfluenza virus and modified canine parvovirus.

9. The use of a vaccine comprising a vaccinal amount of whole Bordetella bronchiseptica cells which have been inactivated by treatment with glutaraldehyde to protect a canine animal against disease caused by Bordetella bronchiseptica, wherein said vaccine is internally administered to the animal.

10. The use of claim 9 in which the vaccine comprises a glutaraldehyde inactivated culture of B.
bronchiseptica grown in liquid medium at 35 to 40°C for 18-36 hrs with aeration until nutrients are substantially depleted.

11. The use of claim 9 in which the vaccine comprises 250 to 750 nephelometric units, prior to inactivation, within each vaccine dose.

12. The use of claims 9, 10 or 11 in which the vaccine further comprises L-lysine in an amount required to complex residual free aldehyde groups.

13. The use of claims 9, 10 or 11 which the vaccine comprises 500 nephelometric units, prior to inactivation, per ml.

14. The use of claim 13 in which the vaccine further comprises an amount of L-lysine equimolar to the amount of glutaraldehyde and .001 to .01% thimerosal.

15. The use of claim 9 in which the vaccine further comprises a vaccinal amount of one or more additional antigenic components for protecting a canine animal against disease caused by one or more other pathogenic microorganisms, cells or viruses.

16. The use of claim 15 in which the additional antigenic components are one or more of inactivated Leptospira canicola inactivated, Leptospira icterhemmorhagiae, modified canine distemper virus, modified canine adenovirus type 2, modified canine parainfluenza virus and modified canine parvovirus.

17. A process for preparing a vaccine for protecting a canine animal against disease caused by Bordetella bronchiseptica which comprises inactivating whole Bordetella bronchiseptica cells with glutaraldehyde and adding L-lysine in an amount sufficient to complex residual free glutaraldehyde groups.

18. The process of claim 17 which comprises culturing B. bronchiseptica in a liquid medium at 35 to 40° C with aeration for 18-36 hours until nutrients are substantially depleted prior to inactivating the cells,

19. The process of claim 18 in which the B
bronchiseptica is cultured until it comprises 250 to 750 nephelometric units per vaccine dose prior to inactivating the cells.

20. The process of claim 18 in which the B
bronchiseptica is cultured until it comprises 500 nephelometric units per vaccine dose prior to inactivating the cells.

21. The process of claim 17, 18, 19 or 20 in which L-lysine is added in an amount equimolar to the amount of glutaraldehyde and which further comprises adding thimerosal to a final concentration of .001 to .01%.