Title of Invention

"A COMBINATION VACCINE FOR IMMUNIZING DOGS AGAINST CANINE PATHOGENS"

Abstract

The present invention relates to a vaccine for immunizing dogs against canine pathogens characterized in that said vaccine consists of: a) a Leptospira cell preparation of Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrhagiae, and Leptospira Pomona; wherein the amount of each Leptospira strain in the vaccine is in the range of about 100-3500 nephelometric units; b) an attenuated strain of canine distemper (CD) virus, c) an attenuated strain of canine adenovirus type 2 (CAV-2), d) an attenuated strain of canine parainfluenza (CPI) virus, e) an attenuated strain of canine parvovirus (CPV), and f) a carrier. wherein the amount of said attenuated strain of CD virus, said attenuated strain of CAV-2, said attenuated strain of CPI virus, and said attenuated strain of CPV, in said vaccine, are each in the range of l02 to l09 TCID50.

Full Text

The present invention relates to a combination vaccine for immunizing dogs against canine pathogens. FIELD OF THE INVENTION This invention relates to vaccines containing a Bordetella bronchiseptica p68 antigen and the use thereof for protecting dogs against infectious tracheobronchitis ("kennel cough") caused by Bordetella bronchiseptica. This invention also relates to combination vaccines containing a Bordetella bronchiseptica p68 antigen and one or more antigens of another canine pathogen such as canine distemper (CD) virus, canine adenovirus type 2 (CAV-2), canine parainfluenza (CPI) virus, canine coronavirus (CCV), canine parvovirus (CPV), Leptospira bratistava, Leptospira canicola, Leptospira grippotyphosa, Leptospira icteronaemorrbagiae or Leptospira pomona. Methods for protecting dogs against diseases caused by canine pathogens using combination vaccines are also provided. This invention relates to vaccines containing Leptospira bratistava and the use thereof for protecting dogs against Infections caused by Leptospira bratistava. TNs invention also relates to combination vaccines containing Leptospira bratistava and one or more antigens of another canine pathogen such as canine distemper (CD) virus, canine adenovirus type 2 (CAV-2). canine parainfluenza (CPI) virus, canine coronavirus (CCV), canine parvovirus (CPV), Leptospira canicofa, Leptospira gnppotyphosa, Leptospira icterohaemorrbagiae or Leptospira pomona This invention further relates to combination vaccines of said antigens without Leptospira bratistava Methods for protecting dogs against diseases caused by canine pathogens using the combination vaccines are also provided. BACKGROUND OF THE INVENTION The present commercially available canine Bordetella bronchiseptica vaccine product is composed of an inactivated, nonadjuvanted Bordatella bronchiseptica whole cell bacterin. Such whole cell bacterin can lead to cell protein rotated post-vaccination reactions. The p68 protein of B. bronchiseptica is antigenically similar to the Outer Membrane Protein (OMP) of B, pertussis and the OMP of ft parapertussis (Shahin et al., °Characterization of ihe Protective Capacity and Immunogenicity of the 69-kD Outer Membrane Protein of Bordetella pertussis°, J. Exp. Mad.. 171; 63-73,1990). A protective role of this OMP has been demonstrated for mice (Shahin et al., supra; Novotny et al., "Biologic and Protective Properties of the 69-kD Outer Membrane Protein of Bordetella pertussis: A Novel Formulation for a Acellufar Pertussis Vaccine", J. Infect. Pis, 164:114-22,1991), humans (He et al., "Protective Role of Immunoglobulin G Antibodies to Filamentous Hemagglutinin and Pertactin of Bordetella pertussis in Bordetetia parapertussis Infection*, Eur.J Clin Microbiol Infect Pis. 10:793-798, 1996) and swine (Kobisch et al., ° identification of a 68-Kilodaiton Outer Membrane Protein as the Major Protective Antigen of Bordetella bronchiseptica by Using Specific-Pathogen-Free Piglets". Infect. Immun. 58f2):352-357.1990). Prior to the present invention, there had been no showing that a Bordeteifa bronchiseptica p68 antigen can be a safe and effective vaccine in dogs. Therefore, there is a need to develop a BordeteHa bronchiseptica vaccine containing a p68 antigen that is suitable for canine use. It would be even more advantageous if such a Bordetetta bronchiseptica p68 vaccine is safe for administration to puppies and provides a long-term protection. CD is a universal, high-mortality viral disease with variable manifestations. Approximately 50% of nonvaccinated, nonimmune dogs infected with CD virus develop clinical signs, and approximately 90% of those dogs die. Infectious canine hepatitis or ICH, caused by canine adenovirus type 1 (CAV-1), is a universal, sometimes fatal, viral disease of dogs characterized by hepatic and generalized endotheliai lesions. CAV-2 causes respiratory disease, which, in severe cases, may include pneumonia and bronchopneumonia. CPI is a common viral upper respiratory disease. Uncomplicated CPI maybe mild or subdinicaJ, with signs becoming more severe if concurrent infection with other respiratory pathogens exists. CPV infection results in enteric disease characterized by sudden onset of vomiting and diarrhea, often hemorrhagic. Leukopenia commonly accompanies clinical signs. Susceptible dogs of any age can be affected, but mortality is greatest in puppies. In puppies 4-12 weeks of age CPV may occasionally cause myocarditis that can result in acute heart failure after a brief and inconspicuous illness. Following infection many dogs are refractory to the disease for a year or more. Similarly, seropositive bitches may transfer to their puppies CPV antibodies which can interfere with active immunization of the puppies through 1B weeks of age. CCV also causes enteric disease in suscepttole dogs of all ages worldwide. Highly contagious, the virus is transmitted primarily through direct contact with infectious feces, and may cause clinical enteritis within 1 -4 days after exposure. Severity of disease may be exacerbated by concurrent infection wfth other agents. Primary signs of CCV infection include anorexia, vomiting, and diarrhea. Frequency of vomiting usually diminishes within a day or 2 after onset of diarrhea, but diarrhea may linger through the course of Infection, and stools occasionally may contain streaks of blood. With CCV infection most dogs remain afebrite and leukopenia is not observed in uncomplcated cases. Leptospirosis occurs in dogs of all ages, with a wide range of clinical signs and chronic nephritis generally following acute infection. Some combination vaccines have been developed, including those sold under the Vanguard© tradename. However, prior to the present invention, there have been no effective combination vaccines that protect dogs against Bontetella bronchiseptica and one or more of other canine pathogens such as CD virus, CAV-2, CPI virus. CPV, CCV, and a Leptospira species such as L bratisfava, L canteola, L grippotypnosa, L icterohaemorrhagiae and L pomona. There also have been no effective combination vaccines comprising L Btatislava against these other canine pathogens but without Bordeteifa broncriiseptica. A problem in developing combination vaccines involves efficacy interference, namely a failure of one or more antigens In a combination composition to maintain or achieve efficacy because of the presence of the other antigens in the composition. This is believed to be a result of interference with an antigen in the composition administered to a host, e.g., a dog, in the immunotogical, antigenic, antibody or protective response such antigen induced in the host because of the other antigens present in the composition. However, for other hosts, such as cats, combination vaccines are known, it is believed that efficacy interference in dogs is due to some peculiarity of the canine biological system, or due to the reaction of the antigens with the canine biological system. There is a need, therefore, to develop a combination vaccine suitable for administration to dogs against BordeteHa bronchiseptica and one or more other canine pathogens, which does not exhibit efficacy interference in canines. There is also a need to develop such combination vaccines without Borcfetella bronchiseptica. It would be even more advantageous *rf such combination vaccines are safe for administration to puppies and provide long-term protection. SUMMARY OF THE INVENTION The present invention provides vaccines and methods for protecting dogs against diseases caused by canine pathogens. In one embodiment, the present invention provides p68 vaccines suitable for administration to dogs and capable of protecting dogs against disease caused by BordeteHa bronchiseptica. Such vaccines of the present invention include a BordeteUa bronchiseptica p68 antigen and a veterinary-acceptable carrier such as an adjuvant. In another embodiment, the present Invention provides methods of protecting dogs against disease caused by BordeteSa bronchiseptica by administering to a dog a vaccine which includes a BordeteHa bronchiseptica p68 antigen and a veterinary-acceptable carrier such as an adjuvant. In another embodiment, the present invention provides Leptospira bratislava vaccines suitable for administration to dogs and capable of protecting dogs against disease caused by Leptospira bratislava. Such vaccines of the present invention include a cell preparation of Leptospira bratislava and a veterinary-acceptable carrier such as an adjuvant In another embodiment, the present invention provides methods of protecting dogs against disease caused by Leptospira bratislava by administering to a dog a vaccine which includes a cell preparation of Leptospira bratislava and a veterinary-acceptable carrier such as an adjuvant In still another embodiment, the present invention provides combination vaccines suitable for administration to dogs. The combination vaccines of the present invention include a Bonteteila bronchiseptica p68 antigen in combination with at least one other antigen from other canine pathogens, capable of inducing a protective immune response in dogs against disease caused by such other pathogen(s). Such other pathogens can be selected from canine distemper (CD) virus, canine adenovirus type 2 (CAV-2), canine paralnfluenza (CPI) virus, canine parvovirus (CPV), canine coronavhus (CCV), canine hetpesvirus, rabies virus, Leptospira bratisiava, Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrhagiae, Leptospira pomona, Leptospira hardjobovis, Porphyromonas spp., Bactertodes spp., Leishmania spp., Borreiia spp., EMichia spp., Mycoplasma spp. and Microsporvm cants. A preferred combination of the present invention includes two or more antigens from canine pathogens, capable of inducing a protective immune response in dogs against disease caused by such pathogen(s). Such pathogens can be selected from canine distemper (CD) virus, canine adenovirus type 2 (CAV-2), canine parainfluenza (CPI) virus, canine parvovirus (CPV), canine coronavirus (CCV), canine herpesvirus, rabies virus, Leptospira bratisiava, Leptospira canicola, Leptospira grippotyphosa, Leptospira Icterohaamorrhagiag, Leptospira pomona, Leptospira hardjobovis, Porphyromonas spp., Bacteriodes spp., Leishmania spp., BorreJia spp., Ehrtichia spp., Mycoplasma spp. and Microsporum cants. A preferred combination vaccine of the present invention includes attenuated strains of canine distemper (CD) virus, canine adenovirus type 2 (CAV-2), canine parainfluenza (CPI) virus and canine parvovirus (CPV); an inactivated preparation of a strain of canine coronavirus (CCV); and a Bordetete bronchiseptica p68 antigen. A preferred combination vaccine of the present invention includes attenuated strains of canine distemper (CD) virus, canine adenovirus type 2 (CAV-2), canine parainfluenza (CPI) virus, and canine parvovirus (CPV); and an inactivated preparation of a strain of canine coronavirus (CCV). Another preferred combination vaccine of the present invention includes attenuated strains of canine distemper (CD) virus, canine adenovirus type 2 (CAV-2), canine parainfluenza (CPI) virus and canine parvovirus (CPV); an inactivated preparation of a strain of canine coronavirus (CCV); a Bordetstta bronchiseptica p68 protein, and an inactivated cell preparation of Five Leptospira serovars (Leptospira bratisiava, Leptospira canicola, Leptospira grippotyphosa, Leptospira fcterohaamorrhagiae and Leptospira pomona). Another preferred combination vaccine of the present invention includes attenuated strains of canine distemper (CD) virus, canine adenovirus type 2 (CAV-2), canine parainfluenza (CPI) virus, and canine parvovirus (CPV); and an inactivated preparation of a strain of canine coronavirus (CCV); and a celt preparation of five Leptospira serovars (Leptospira bratisiava, Leptospira canicola. Leptospira grippotyphosa, Leptospira icterohaemorrhagiae and Leptospira pomona). Another preferred combination vaccine of the present invention includes attenuated strains of canine distemper (CD) virus, canine adenovirus type 2 (CAV-2), canine parainfluenza (CPI) virus, and canine parvovirus (CPV); an inactivated preparation of a strain of canine coronavirus (CCV); and an inactivated cell preparation of four Leptospira serovars (Leptospira canicola, Leptospira grippotyphosa, Leptospira tcterohaemorrhagiae and Leptospira pomona). Still another preferred combination vaccine of the present Invention includes attenuated strains of CD virus, CAV-2, CPI virus, a CPV strain; and a Sorcfetefe bronchiseptica p68 antigen. Stil another preferred combination vaccine of the present invention includes attenuated strains of CD virus, CAV-2, CPI virus, a CPV strain. Another preferred combination vaccine of the present invention includes attenuated strains of CD virus, CAV-2, CPI virus, a CPV strain; a Bordeteila bmnchisepOca p68 antfgen; and an inactivated cell preparation of Leptospira canicola and Leptospira icterohaemorrhagiae. Another preferred combination vaccine of the present invention includes attenuated strains off CD virus, CAV-2, CPI virus, a CPV strain; and an inactivated ceil preparation of Leptospira canicofa and Leptospira icterohaernorrhagiae. Still another preferred combination vaccine of the present invention includes attenuated strains of CD virus, CAV-2, CPI virus, a CPV strain, a Bordetetia bronchiseptica p68 antigen and an inactivated cell preparation of five Leptospira serovars (Leptospira bfatislava, Leptospira canicofa, Leptospira grippotyphosa, Leptospira icterohaemorrhagiae and Leptospira pomona). Still another preferred combination vaccine of the present invention includes attenuated strains of CD virus, CAV-2, CPI virus, a CPV strain, and an inactivated cell preparation of five Leptospira serovars (Leptospira Bratislava, Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrhagiae and Leptospira pomona). Still another preferred combination vaccine of the present Invention includes attenuated strains of CD virus, CAV-2, CPI virus, a CPV strain, and an inactivated ceil preparation of four Leptospira serovars (Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrhagiae and Leptospira pomona). Another preferred combination vaccine includes a BonfeteHa bronchiseptica p66 antigen and an attenuated CPI virus. Still another preferred combination vaccine includes a Bordetefla bronchiseptica p68 antigen, an attenuated CPI virus and an inactivated call preparation of Leptospira canicola and Leptospira icterohaemorrhagiae. The present invention also provides methods of protecting dogs against disease • caused by a canine pathogen by administering to a dog a combination vaccine of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Summary of the geometric mean of 068 ELISA endpoint trters in unvaccinated and BordeteUa 068 (15 jig/dose) vaccinated dogs-aerosol challenge with Bordetefla bronchiseptica. Figure 2. Summary of Serum Amyloid A titers in dogs following aerosol challenge with BordeteHa bronchiseptica. Figure 3. Summary of the geometric mean of p68 ELISA endpoint liters in unvaccinated and Bordeteila p68 vaccinated dogs following vaccination and aerosol challenge with Bordeteila bronchiseptica. Figure 4. Summary of Serum Amyloid A triers in dogs following aerosol challenge with Bordeteila bronchiseptica. Figure 5. Western blot snowing reactivity of p68 monoclonal antibody Bord 2-7 to p68 whole cell lysate. DETAILED DESCRIPTION OF THE INVENTION In one embodiment, the present invention provides monovatent vaccines suitable for administration to dogs which are capable of protecting dogs against disease caused by BordeteHa bronchiseptica. The monovatent vaccines of the present invention include a recombinantly produced BordeteHa bronchiseptica p68 antigen and a veterinary-acceptable carrier such as an adjuvant. In another embodiment, the present invention provides methods of protecting dogs against disease caused by BordeteHa bronchiseptica by administering to a dog a monovatent vaccine which includes a recombinantly produced Bordeteila bronchiseptica p68 antigen and a veterinary-acceptable carrier such as an adjuvant. In still another embodiment, the present invention provides combination vaccines suitable for administration to dogs. The combination vaccines of the present Invention include a recombinantly produced BordeteHa bronchiseptica p68 antigen in combination with at least one other antigen capable of inducing a protective immune response in dogs against disease caused by such other antigen. Another embodiment of the present invention includes two or more antigens from canine pathogens, capable of inducing a protective immune response in dogs against disease caused by such pathogen(s). A preferred combination vaccine of the present invention Includes attenuated strains of canine distemper (CD) virus, canine adenovirus type 2 (CAV-2), canine parainfluenza (CPI) vims and canine parvovirus (CPV); an inactivated preparation of a strain of canine coronavirus (CCV); and a preparation of four Leptospira serovars (Leptospira carvcoia, Leptospira grippotyphosa, Leptospira icterohaemorrhagiae, and Leptospira pomona). Another preferred combination vaccine of the present invention includes attenuated strains of canine distemper (CD) virus, canine adenovirus type 2 (CAV-2), canine parainfluenza (CPI) virus and canine parvovirus (CPV); an inactivated preparation of a strain of canine coronavirus (CCV); and a preparation of five Leptospira serovars (Leptospira bratisteva, Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrhagiae and Leptospira pomona). Still another preferred combination vaccine of the present invention includes attenuated strains of CD virus, CAV-2, CPI vims, a CPV strain; and a preparation of four Leptospira serovars (Leptospira canicola, Leptospira grippotypftosa, Leptospira icterohaemorrhagiae and Leptospira pomona).. Another preferred combination vaccine of the present invention includes attenuated strains of CD virus, CAV-2, CPI virus, a CPV strain, a CCV strain; and a preparation of Leptospira canicola and Leptospira Icterohaemorrhagiae. Sb'll another preferred combination vaccine of the present invention includes attenuated strains of CD virus, CAV-2, CPI virus, a CPV strain and a preparation of five Leptospira seravars (Leptospira bratislava, Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrhagiae and Leptospira pomona). The present invention also provides methods of protecting dogs against disease caused by a canine pathogen by administering to a dog a combination vaccine of the present invention. For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the following subsections which describe or illustrate certain features, embodiments or applications of the invention. Definitions and Abbreviations The term "protecting a dog against a disease caused by a canine pathogen" as used herein means reducing or eliminating the risk of infection by the pathogen, ameliorating or alleviating the symptoms of an infection, or accelerating the recovery from an infection. Protection is achieved if there is a reduction in viral or bacterial toad, a reduction in viral or bacteria/ shedding, a decrease in incidence or duration of infections, reduced acute phase serum protein levels, reduced rectal temperatures, and/or increase in food uptake and/or growth, for example. The term °p68 antigen' refers to a protein with a molecular weight of 68 kDa as determined by SDS polyacrylamtde gel etectrophoresis, is recognized by the p68-specific monoclonal antibody Bord 2-7. (ATCC), and has an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence that is substantially identical to SEQ ID NO: 1. By "substantially identical' is meant a degree of sequence Identity of at least about 90%, preferably at least about 95%, or more preferably, at least about 98%. The term "monovalent vaccine" as used herein refers to a vaccine having one principal antigenic component For example, a p68 monovalent vaccine includes a Bordeteila bronchiseptica p68 antigen as the principal antigenic component of the vaccine and Is capable of protecting the animal to which the vaccine is administered against diseases caused by Bordetetla bmnchiseptica. Another example of a monovalent vaccine includes a cell preparation of Leptospira bratislava as the principal antigenic component of the vaccine and is capable of protecting the animal to which the vaccine is administered against diseases caused by Leptospira bratislava. The term "combination vaccine" is meant a bivalent or multivalent combination of antigens which are capable of inducing a protective immune response in dogs. The protective effects of a combination vaccine against a pathogen or pathogens are normally achieved by Inducing in the animal subject an immune response, either a cell-mediated or a humoral immune response or a combination of both. By "immunogenic" Is meant the capacity of a composition to provoke an immune response in dogs against a particular pathogen. The immune response can be a cellular immune response mediated primarily by cytotoxic T-cells and cytokine-producing T-cells, or a humoral immune response mediated primarily by helper T-cells, which in turn activates B-cells leading to antibody production. The term therapeutically effective amount' or "effective amount" refers to an amount of a monovatent or combination vaccine sufficient to elicit a protective immune response in the dog to which ft is administered. The immune response may comprise, without limitation, induction of cellular and/or humoral immunity. The amount of a vaccine that is therapeutically effective may vary depending on the particular antigen used in the vaccine, the age and condition of the dog, and/or the degree of infection, and can be determined by a veterinary physician. 068 Vaccines The present invention has demonstrated for the first time that a vaccine composition containing a Bordetetta bronchiseptica p68 antigen effectively protected dogs against disease caused by Bordatefla bronchiseptica. The vaccine composition of the present invention does not cause significant post-vaccination reactions, is safe for administration to puppies, and induces protective immunity in dogs that lasts for an extended period of time. Accordingly, one embodiment of the present invention is directed to a vaccine composition containing a Bordeteila bronchiseptica p68 antigen (or "a p68 vaccine"), that is suitable for administration to dogs and is capable of protecting dogs against disease caused by Bofdetella bronchiseptica, e.g., infectious tracheobnonchitis ("kennel cough*). For the purpose of the present invention, the term "p68 antigen" refers to a protein (see Rgure 5) with a molecular weight of 68 kDa as determined by SDS polyacrylamide gel electrophoresis, is recognized by the p68-specHic monoclonal antibody Bord 2-7 (ATCC#), and has an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence that is substantially identical to SEQ ID NO: 1. By "substantially identical" is meant a degree of sequence identity of at least about 90%, preferably at least about 95%, or more preferably, at least about 98%. An example of a p68 antigen having an amino add sequence substantially identical to SEQ ID NO: 1 is the p68 antigen described in WO 92/17587, which is set forth in SEQ ID NO: 3. The p68 specific monoclonal antibody of the present invention recognizes native p68 proteins, recombinant p68 proteins and p68 proteins on the surface of bacteria, for example. In accordance with the present invention, p68 antigens suitable for use in the present invention include both native p68 proteins (i.e., naturally occurring p68 proteins purified from Bordeteila bronchiseptica) and recombinantly produced p6B proteins. Purification of native p6B from Bordeteila bronchiseptica is described, e.g., in Montaraz et al., Infection and Immunity 47:744-751 (1985), and is also illustrated in the examples provided hereinbelow. Recombinant production of p68 can be achieved using any one of the molecular cloning and recombinant expression techniques known to those skilled in the art For example, a nucleic acid molecule encoding p68 can be introduced into an appropriate host cell, such as a bacterium, a yeast cell (e.g., a Pichia cell), an insect cell or a mammalian cefl (e.g., CHO cell). The pea-encoding nucleic acid molecule can be placed in an operable linkage to a promoter capable of effecting the expression of the p68 antigen in the host cell. p6B, which is expressed by the host cell, can be readily purified using routine protein purification techniques. In a preferred embodiment of the present invention, the nucteotide sequence as set forth in SEQ ID NO: 2 codng for the p68 antigen which has the amino acid sequence of SEQ ID NO: 1, is cloned in an expression vector and placed in an operable linkage to a temperature sensitive promoter. The expression vector is introduced into Escherichia and the p68 antigen is expressed upon heat induction. The cells are and the inclusion bodies where the 068 antigen accumulates are separated by centrifugation. The recombinant p68 in the inclusion bodies is solubilized using SOS or other solubilization agents known in the art such as urea, guanidine hydrochtoride, sodium cholate, taurocholate, and sodium deoxycholate. In accordance with the present invention, a purified native or recombinant p68 protein is combined with a veterinary-acceptable carrier to form a p68 vaccine composition. The term 'a veterinary-acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. Diluents can include water, saline, dextrose, ethanot, grycerol, and the like: Isotonic agents can include sodium chloride, dextrose, mannital, sorbftol, and lactose, among others. Stabilizers include albumin, among others. Adjuvants suitable for use in accordance with the present invention include, but are not limited to several adjuvant classes such as; mineral salts, e.g., Alum, aluminum hydroxide, aluminum phosphate and calcium phosphate; surface-active agents and micropartfcles, e.g., nonionfc block polymer surfactants (e.g., cholesterol), virosomes, saponins (e.g., Quil A, QS-21 and GPI-0100), proteosomes, immune stimulating complexes, cochleates, quarterinary amines (dimethyl diocatadecyl ammonium bromide (DDA)), avridine, vitamin A, vitamin E; bacterial products such as the RIBI adjuvant system (Ribi Inc.), cell wall skeleton of Mycobacterum phlei (Detox®), muramyl dipeptides (MDP) and tripeptides (MTP), monophosphoryl lipid A, Bacillus Calmete-Guerin, heat labile E. coii enterotoxins, cholera toxin, trehatose dimycolate, CpQ oligodeoxnudeotides; cytokines and hormones, e.g., , interieuklns (IL-1, IL-2, IL-6, IL-12, IL-15, IL-18), granulocyte-macrophage colony stimulating factor, dehydroepiandrosterone, 1,25-dfcydroxy vitamin D3; polyanions, e.g., dextran; polyacrylics (e.g., pofymethylmethacrylate, Carbopol 934P); carriers e.g., tetanus toxid, diptheria toxoid, cholera toxin B subnuit, mutant heat labile enterotoxin of enterotoxigenic E. coli (rmLT), heat shock proteins; oil-in-water emulsions e.g.,AMPHIGEN* (Hydronics, USA); and water-in-oO emulsions such as, e.g., Freund's complete and incomplete adjuvants. Preferred adjuvants for use in the vaccines of the present invention include Quil A and cholesterol. The p68 antigen and the veterinary-acceptable carrier can be combined in any convenient and practical manner to form a vaccine composition, e.g., by admixture, solution, suspension, emulsification, encapsulation, absorption and the like, and can be made in formulations such as tablets, capsules, powder, syrup, suspensions that are suitable for injections, implantations, inhalations, ingestions or the like. Preferably, the vaccine is formulated such that it can be administered to dogs by injection in a dose of about 0.1 to 5 ml, or preferably about 0.5 to 2.5 ml, or even more preferably, in a dose of about 1 ml. When appropriate, the pharmaceutical compositions of the present invention should be made sterile by well-known procedures. The amount of p68 in the vaccines should be immunizing-effective and is generally in the range of 0.5 -1000 per dose. Preferably, the amount of p68 is in the range of 1-260 pg per dose. More preferably, the amount of p68 is in the range of 10-100 ug per dose. Even more preferably, the amount of p68 is about 15 to 25 ug per dose. The amount of adjuvants suitable for use in the vaccines depends upon the nature of the adjuvant used. For example, when Quil A and cholesterol are used as adjuvant. Quil A is generally in an amount of about 1 -1000 ug per dose, preferably 30-100 ng per dose, and more preferably, about 50-75 ug per dose; and cholesterol is generally in an amount of about 1-1000 ug per dose, preferably about 30-100 ug per dose, and more preferably, about 50-75 ug per dose. In another embodiment, the present invention provides methods of protecting dogs against disease caused by Bordetella bronchiseptica by administering to a dog a p68 vaccine composition, as described hereinabove. In accordance with the present Invention, the p68 vaccine composition provides dogs with a long term immunity for at least about 4 months, preferably for at least about 6 months, or even more preferably, for about one year, In accordance with the present invention, a p68 vaccine can be administered to a dog by any known routes, including the oral, intranasal, mucosal. topical, transdermal, and parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular). Administration can also be achieved using needle-free delivery devices. Administration can be achieved using a combination of routes, e.g., first administration using a parental route and subsequent administration using a mucosal route. Preferred routes of administration include subcutaneous and intramuscular administrations. The p68 vaccine composition of the present invention can be administered to dogs of at least 6 weeks old, preferably at least 7 weeks old, and more preferably, at least 8 or 9 weeks old. Dogs can be vaccinated with one dose or-wfth more than one dose of a p6S vaccine. Preferably, two doses of a p68 vaccine are administered to dogs with an interval of about 2-4 weeks, preferably about 3 weeks, between the two administrations. H dogs are vaccinated before the age of 4 months, it is recommended that they be revaccinated with a single dose upon reaching 4 months of age, because maternal antibodies may interfere wfth development of an adequate immune response in puppies less than 4 months. Dogs can also be revaccinated annually with a single dose. Where B. bronchiseptica exposure is likely, such as breeding, boarding, and showing situations, an additional booster may be given within 1 year, or preferably 6 months, of the occurrence of these events. Combination Vaccines In another embodiment, the present invention provides combination vaccines and methods for protecting dogs against Bordetelfa bronchiseptica and/or one or more other canine pathogens by administering such combination vaccines. The combination vaccine compositions of the present invention do not exhibit efficacy interference and are safe for administration to puppies. The combination vaccines of the present invention include a BordeteHa bronchiseptica p68 antigen, which can be made as described hereinabove, in combination with at least one antigen from other canine pathogens capable of inducing a protective immune response in dogs against disease caused by such other pathogens. Such combination vaccines also include combinations of two or more such other canine pathogens without the p68 antigen. Such other pathogens include, but are not limited to, canine distemper (CD) virus, canine adenovirus type 2 (CAV-2), canine parainfluenza (CPI) virus, canine parvovirus (CPV), canine coronavirus (CCV), canine herpesvirus, and rabies virus. Antigens from these pathogens for use in the vaccine compositions of the present invention can be in the form of a modified live viral preparation or an inactivated viral preparation. Methods of attenuating virulent strains of these viruses and methods of making an inactivated viral preparation are known in the art and are deserted in, e.g., U.S. Patents 4,567,042 and 4,567,043. Other pathogens also include Leptospira bratislava, Leptospira canteota, Leptospira grfppotyphosa, Leptospira ictemhaemorrhagiae. Leptospira pomona, Leptospira hardjobovis, Porphymmonas sppn Bacteriodes spp., Leishmania spp,, Borrelfa spp., EhrBchia spp., Mycoplasma ssp. and Microsporum cants. Antigens from these pathogens for use in the vaccine compositions of the present invention can be in the form of an inactivated whole or partial cell preparation, using methods well-known in the art For example, methods of making an inactivated whole or partial Leptospira cell preparation are known in the art and are described in, e.g., Van, K-T, "Aspects of Immunity to Leptospira borgpetersenS ssrovar hardier, PhD Thesis, Appendix 1,1996. Faculty of Agriculture and Food Science, The Queen's University of Belfast Mackintosh et ai., The use of a hardjo-pornona vaccine to prevent leptosplruria in cattle exposed to natural challenge with Leptospia intemgans semvar hardj(f, Maw Zealand Vet. J. 28:174-177,1980;Bolin et. al., "Effect of vaccination with a pentavalent leptopsiral vaccine on Leptospira Interrogans serovarhardjo type hardjo-boivs infection of pregnant cattle*, Am. J. Vet. Res. 50:161 -165,1989. In accordance with the present invention, the combination vaccines generally include a veterinary-acceptable carrier. As described hereinabove, a veterinary-acceptable carrier includes any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, arrtibacteriaJ and arrtifungal agents, isotonic agents, adsorption delaying agents, and the like. Diluents can include water, saline, dextrose, ethanol, gfycerol, and the like, fsotonic agents can include sodium chloride, dextrose, mannitol, sorbftol, and lactose, among others. Stabilizers include albumin, among others. Adjuvants suitable for use in accordance with the present invention include, but are not limited to several adjuvant classes such as; mineral salts, e.g., Alum, aluminum hydroxide, aluminum phosphate and calcium phosphate; surface-active agents and microparticles, e.g., nonionic block polymer surfactants (e.g., cholesterol), virosomes, saponlns (e.g., Quil A, QS-21 and GPI-0100), proteosomes, immune stimulating complexes, cochleates, quarterinary amines (dimethyl dfbcatadecyi ammonium bromide (ODA)), avridine, vitamin A, vitamin E; bacten'al products such as the RIBJ adjuvant system (Rifai Inc.), cell wall skeleton of Mycobacterurn phlei (Detox®), muramyl cfipeptides (MDP) and tripeptkJes (MTP), monophosphoryl ipid A, Bacillus Calmete-Querin. heat labile E. coli enterotoxins, cholera toxin, trehalose dimycolate, CpG oligodeoxnucleotides; cytokines and hormones, e.g., interleukins (IL-1, IL-2, IL-6, IL-12, IL-15, IL-18), granulocyte-macrophage colony stimulating factor, dehydroepiandrosterone, 1 ,25-dihydroxy vitamin Dg; polyanions, e.g., dextran; polyacryllcs (e.g., pdymethvimethacrylate, Carbopol 934P); carriers e.g., tetanus toxld, diptheria toxoid, cholera toxin B subnuit, mutant heat labile enterotoxin of enterotoxkjenic E. coli (rrnLT), heat shock proteins; oiMn-water emulsions e.g.^MPHIGEN* (Hydronics, USA); and water-in-oil emulsions such as, e.g., Freund's complete and incomplete adjuvants. Preferred adjuvants for use in the combination vaccines in accordance with the present invention include 1) Quil A plus cholesterol; and 2) aluminum hydroxide. The amount of adjuvants suitable for use in the vaccines depends upon the nature of the adjuvant used. For example, when Quil A and cholesterol are used as adjuvant, Quil A I* generally in an . amount of about 1-1000 jig per dose, preferably 30-100 |ig per dose, and more preferably, about 50-75 ug per dose; and cholesterol is generally in an amount of about 1-1000 |tg per dose, preferably about 30-100 ug per dose, and more preferably, about 50-75 ug per dose. When aluminum hydroxide is used as adjuvant, it is generally in an amount of about 0.5-20%, preferably about 0.5-10%, and more preferably about 1-2%. The p6B antigen, one or more antigens from other pathogens, and/or the veterinary-acceptable carrier can be combined in any convenient and practical manner to form a combination vaccine composition, e.g., by admixture, solution, suspension, emuteiffcation, encapsulation, absorption and the like, and can be made in formulations such as tablets, capsules, powder, syrup, suspensions that are suitable for injections, implantations, inhalations, ingestfons or the like. Preferably, the vaccine is formulated such that ft can be administered to dogs by injection in a dose of about 0.1 to 5 ml, or preferably about 0.5 to 2.5 ml, or even more preferably, in a dose of about 1 ml. Combination vaccines may prepared by rehydrating a freeze-dried preparation of the attenuated viral strains (or a preparation made by other methods such as spray drying or desiccation) and viral preparation with a liquid preparation, which liquid preparation is composed of the Lepfosp/ra/ antigens, dissolved in sterile saline solution and adjuvanted with Quil A and cholesterol Such combination vaccine may also be prepared by rehydrating a freeze-dried preparation of the attenuated viral strains and Leptospira viral preparation (or a preparation made by other methods such as spray drying or desiccation) with a sterile solution, or rehydrating said freeze-dried preparation with CCV plus diluent. In accordance with the present invention, combination vaccines can be administered I to a dog of at least 6 weeks old, preferably at least 7 weeks old, and more preferably, at least 8 or 9 weeks old. The combination vaccines can be administered in 2 to 4 doses, preferably in 2 to 3 doses. The doses can be administered with 2 to 6 weeks between each dose, preferably with 2 to 4 weeks between each dose. The administration can be done by any known routes, including the oral, intranasal. mucosal topical, transdermal, and parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular). Administration can also be achieved using needle-free delivery devices. Administration can also be achieved using a combination of routes, e.g., first administration using a parental route and subsequent administration using a mucosal route. Preferred routes of administration include subcutaneous and intramuscular administrations. Preferred Combination Vaccines and Vaccination Methods A preferred combination vaccine of the present invention includes an attenuated strain of CD virus, an attenuated strain of CAV-2. an attenuated strain of CPI virus, an attenuated strain of CPV, an inactivated preparation of a strain of CCV, and a Bordetetia bronchiseptica p68antigen. An especially preferred combination vaccine includes the attenuated CD virus strain designated as the "Snyder Hill" strain (National Veterinary Service Laboratory, Ames, IA), the attenuated CAV-2 strain designated as the 'Manhattan" strain (National Veterinary Service Laboratory, Ames, IA), the attenuated CPI virus strain having the designation of "NL-CPI-5" (National Veterinary Service Laboratory, Ames, IA), the attenuated CPV strain having the designation of "NL-35-D" (National Veterinary Service Laboratory, Ames, !A), an inactivated preparation of the CCV strain having the designation of "NL-18" (National Veterinary Service Laboratory, Ames, IA), and the recombinant BorteteHa bronchiseptica p68 antigen having the sequence of SEQ ID NO: 1. Such combination vaccine, also referred to herein as "the D68/5CV combination vaccine", is preferably prepared by rehydrating a freeze-dried preparation of the attenuated viral strains and viral preparation with a liquid preparation, which liquid preparation is composed of the p68 antigen dissolved in sterile saline solution and adjuvanted with Quil A and cholesterol. This combination without the p68 antigen is referred to herein as the 5CV combination. Such combination vaccine is preferably prepared by rehydrating a freeze-dried preparation of the attenuated viral strains and viral preparation with a liquid preparation, which liquid preparation Is composed of sterile saline solution and adjuvanted with Quil A and cholesterol. Another especially preferred combination vaccine includes the antigenic components of the p68/5CV combination vaccine as well as inactivated whole cell preparations of five Leptospira species: Leptospira bratislava (e.g., a Leptospira bratislava strain which can be obtained from National Veterinary Service Laboratory, Ames, IA), Leptospira canicola {e.g., strain C-5, National Veterinary Service Laboratory, Ames, IA), Leptospira grippotyphosa (e.g., strain MAL1540, National Veterinary Service Laboratory, Ames, IA.), Leptospira icterohaemorrhagiaeie.g., strain NADL11403, National Veterinary Service Laboratory, Ames, IA) and Leptospira pomona (e.g., strain T262, National Veterinary Service Laboratory, Ames. IA). Such combination vaccine, also referred to herein as the p68/5CV-teptospira combination vaccine", is preferably prepared by rehydrating a f reeze-dried preparation of the attenuated viral strains (or a preparation made by other methods such as spray drying or desiccation) and viral preparation with a liquid preparation, which liquid preparation is composed of the p66 antigen and Leptasp/ra/antigens, dissolved in sterile saline solution and adjuvanted with Quil A and cholesterol. This combination without the p66 antigen is referred to herein as the 5CV-SLeptaspira combination. This combination without the p6B antigen and without Leptospira bratislava is referred to herein as the 5CV-4Leptosplra combination. The 5CV combination without the p68 antigen and with Leptospira canicola and Leptospira icterohaemorrhagiae is referred to herein as the 5CV-2Leptospira combination. Such combination vaccines are preferably prepared by rehydrating a freeze-dried preparation of the attenuated viral strains (or a preparation made by other methods such as spray drying or desiccation) and viral preparation with a liquid preparation, which liquid preparation is composed of the Leptospiral antigens, dissolved in sterile saline solution and adjuvanted with Qui A and cholesterol. Such combination vaccines are also preferably prepared by rehydrating a freeze-dried preparation of the attenuated viral strains and Leptospira viral preparation (or a' preparation made by other methods such as spray drying or desiccation) with a sterile solution, or rehydrating said freeze-dried preparation with CCV plus diluent. In accordance with the present invention, the p68/5CV, p68/5CV-Leptospira, 5CV, SCV-SLeptospira, 5CV-4Leptospira, and 5CV-2Leptospira combination vaccines can be administered to healthy dogs 4 weeks of age or older, preferably 6 weeks or older, and preferably In 3 doses, each administered about 3 weeks apart Dogs can be revaccinated annually with a single dose. Where B. bronchiseptica and/or canine virus exposure is likely, such as breeding, boarding, and showing situations, an additional booster may be given within 1 year, or preferably 6 months, of the occurrence of these events. .Still another preferred combination vaccine of the present invention includes an attenuated strain of CD virus, an attenuated strain of CAV-2, an attenuated strain of CPI virus, an attenuated strain of CPV, and a recombinant BordeteSa bronchiseptica p68 antigen. An especially preferred combination vaccine includes the attenuated CD virus strain designated as the "Synder Hill* strain (National Veterinary Service Laboratory, Ames, IA), the attenuated CAV-2 strain designated as the •Manhattan" strain (National Veterinary Service Laboratory, Ames, IA), the attenuated CPI virus strain having the designation of "NL-CPi-5" (National Veterinary Service Laboratory, Ames, IA), th& attenuated CPV strain designated as "NL-35-D" (National Veterinary Service Laboratory, Ames, IA), and the recombinant Bordetella bronchtsepticapQB antigen having the sequence of SEQ ID NO: 1. Such combination vaccine, also referred to herein as "the pSB/DAzPP combination vaccine", is preferably prepared by rehydrating a freeze-dried preparation of the attenuated viral strains (or a preparation made by other methods such as spray drying or desiccation) with a liquid preparation, which liquid preparation is composed of .the p68 antigen dissolved in sterile saline solution and adjuvarrted with Quil A and cholesterol. This combination vaccine without the p68 antigen is referred to as the DAaPP combination vaccine. Such combination vaccine fe preferably prepared by rehydrating a freeze-dried preparation of the attenuated viral strains (or a preparation made by other methods such as spray drying or desiccation) with a liquid preparation, which liquid preparation is composed of sterile saline solution and adjuvanted with Quil A and cholesterol. Another especially preferred combination vaccine includes the antigenic components of the p68/DAaPP combination vaccine as well as inactivated whole cell preparations of two Leptospira species: Leptospira canicola (e.g., strain C-51, National Veterinary Service Laboratory, Ames, IA), and Leptospira icterohaemorrhagiae (e.g., strain NADL11403, National Veterinary Service Laboratory, Ames, IA). Alternatively, a preferred combination, vaccine can include the antigenic components of the p68/DAgPP combination vaccine as well. as inactivated whole cell preparations of five Leptospira species: Leptospira bratisteva, Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrhagiae and Leptospira pomona. These combination vaccines, also referred to herein as the p68/DAaPP-Leptospira combination vaccines", are preferably prepared by rehydrating a freeze-dried preparation of the attenuated viral strains (or a preparation made by other methods such as spray drying or desiccation) and viral preparation with a liquid preparation, which liquid preparation is composed of the p68 antigen and Leptospira! antigens, dissolved in sterile saline solution and adjuvanted with Quil A and cholesterol. Alternatively, another preferred combination vaccine includes the antigenic components of the DAePP combination vaccine as well as inactivated whole cell preparations of five Leptospira species: Leptospira bratislava, Leptospira canicola, Leptospira grippotyphosa, Leptospira Icterohaemorrhagiae and Leptospira pomona. Still another preferred combination vaccine includes the antigenic components of the DAaPP combination vaccine as well as inactivated whole cell preparations of four Leptospira species: Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrhagiae and Leptospira pomona. These combination vaccines, also referred to herein as the DAaPP-Leptospira combination vaccines", are preferably prepared by rehydrating a freeze-dried preparation of the attenuated viral strains (or a preparation made by other methods such as spray drying or desiccation) and viral preparation with a liquid preparation, which Bquid preparation is composed of the Leptospiral antigens, dissolved in sterile saSne solution and adjuvanted with Quil A and cholesterol. In accordance with the present invention, the p68/DAaPP, pee/DAaPP-Leptospira, DA2PP, and DAaPP-Leptospira combination vaccines can be administered to healthy dogs 6 weeks or older, or preferably 8 weeks of age or older, and preferably in 2 doses, each administered about 3 weeks apart. A single dose may be sufficient if given to a dog at least 12 weeks of age. Dogs can be revaccinated annually with a single dose. Where B. bronchiseptica and/or canine vims exposure is likely, such as breeding, boarding, and showing situations, an additional booster may be given within 1 year, or preferably 6 months, of the occurrence of these events. Another preferred combination vaccine includes a p68 antigen, preferably the recomfainant p68 antigen having SEQ ID NO: 1, in combination with an attenuated strain of CPI. Still another preferred combination vaccine includes a p68 antigen, preferably the recombinant p68 antigen having SEQ ID NO: 1, an attenuated strain of CPI, and two at least two Leptospira species such as Leptospira canicota (e.g., strain C-51, National Veterinary Service Laboratory, Ames, IA), and Leptospira ktemha&norrhagiae (e.g., strain NADL11403, National Veterinary Service Laboratory, Ames, IA). The amount of the p68 antigen and the antigen(s) from one or more other pathogens in the combination vaccines of the present invention should be immunizing-effective. In general, the p68 antigen in a combination vaccine should be in an amount of at least about 0.5 ug per dose. The attenuated CD virus should be in an amount of at least about 102 to about 109 TCID50 per dose TCIDso (tissue culture infectious dose 50% cytopathfc effect) per dose, and preferably in the range of about 104 to about 10fl TCIDso per dose. The attenuated CAV-2 should be in an amount of at least about 10aTCIDso to about 109 TCID^per dose, preferably in the range of 10" to about 10*° TCIDa, per dose. The attenuated CPI virus should be in an amount of at least about 102 TCIDso to about 10* TCIDso per dose, and preferably in the range of 10* to about 108 TCID*) per dose. The attenuated CPV should be in an amount of at least about If/TCIDsoto about 10* TCIDso per dose, preferably, an amount in the range of 107 to about 109 TCIDso per dose. The amount of CCV in an inactivated viral preparation should be at least about 100 relative units per dose, and preferably in the range of 1000-4500 relative units per dose. Each Leptospira species in the vaccine should be in the range of about 100-3500 NU (nephetometric units) per vaccine dose, and preferably in the range of 200-2000 NU per dose. The combination vaccines are formulated such that the vaccines can be administered to dogs by injection in a dose of 0.1 ml to 5 ml, preferably from 0.5 ml to 2.5 ml, and more preferably, about 1 ml. The present invention is further adjust rated by the following non-limiting examples. EXAMPLE 1 VACCINE: The experimental vaccine antigen was a recombinant p68 outer membrane protein (SEQ ID NO: 1) of B. bronchiseptica produced by £. coti strain LVV68. The vaccfne contained varying levels of SOS (sodium dodecyl sulfale) solubilized p68, adjuvanted with 50 ug of QAC (Quil A/50ng cholesterol) in a 1 mL dose. CHALLENGE MATERIAL An aerosol of Bordetella broncfiiseptica, dog isolate #85B. passage #3, lot #051597, was used as the challenge material. The mean plate count was 1.59 X 10° CFU/ml. ANIMALS; Sixty male and female canine pups were randomly allocated to one of six treatment groups (10 pups per group). Pups were bled and trachea] swabs were taken 41 days prior to the first vaccination and again 28 days prior to first vaccination and any seroposftive or culture Dositive animals were removed from the study. Animals were randomly assigned to treatments and rooms according to a randomized jompletB block design. Post-vaccination observations were done without knowledge of vaccine assignment groups.. DESIGN (Table Removed) PROCEDURE: IVP Administration: Animals were vaccinated on Day 0 with either the placebo or the experimental vaccine. A second vaccination was administered on Day 21. The first vaccination was administered subcutaneously in the right neck and the second vaccination was administered subcutaneously in the left neck. Challenge Administration: All animals were challenged 28 days after the second vaccination with an aerosol of Bordetelta bfonchispetica. Animals were monitored for coughing for a period of 30 minutes, twice daily (once in the a.m. and once in the p.m.) on days two through fourteen fallowing challenge (Days 51 through 63). Observations and Samples Collection: All injection sites were palpated and measured three dimensionally for seven days following each vaccination (Days 0 through 7 and 21 through 28) and on the 14* day post each vaccination (Days 14 and 35). Rectal temperatures were recorded on the day of vaccination and for three days following each vaccination (Days 0 through 3 and 21 through 24). Blood was collected on the days of vaccination (Days 0 and 21) and on Days 42,50, and 63 and assayed by ELISA for specific antibodies against the p68 protein purified from B. bronchispetica. Blood was also colected on Days 42,49,50,52,54,56 and 58 and analyzed for Serum Amyloid A (SAA). All animals were trachea! swabbed for E bmnchispetica isolation and blood was collected for B, bronchispetica agglutination titers prior to vaccination (at the vendor, on Day -41 and Day -28) and on Day 49. Bondetetta P68 Dog and Mouse Antibody Trtration DAB ELISA, Purified native p68 was diluted to 600 ng/mL in 0.01 M Borate Buffer and was added to each well at 100 jiL/well. The plates were incubated overnight at 4°C. The plates were then washed once with excess PBS-Tween 20. 1 % nonfat dried milk in PBS was added to the plates at 200 uL/well. The plates were then incubated for 1 hour at 37°C. The plates were then washed once with excess PBS-Tween 20. Dog or mouse serum was added at a 1:50 dilution to the top row of the ELISA plates and two fold serially diluted serum was added all the way down the plate. The plates were incubated for 1 hour at 37°C. Subsequently, the plates were washed 3 times with excess PBS-Tween 20. To plates Incubated with dog serum above, peroxidase labeled goat anti-dog IgQ (H + L), diluted at a 1:2000 dilution, was added at 100 jxUWell. The plates were then incubated for 1 hour at 37°C. To plates incubated with mouse serum above, peroxidase labeled goat anti-mouse IgG (H + L)f diluted at a 1:4000 dilution, was added at 100 uUwell. The plates were / than incubated for 1 hour at 37°C. The plates were then washed 3 times with excess PBS-Tween 20. ABTS substrate was added at 100 Approximately 20 minutes later, the plates were read with a Molecular Devices or an equivalent plate reader at 405-490 nm. DATA ANALYSIS: Treatment differences in the number of dogs coughing were tested using Fisher's Exact Test The 5% level of significance was used. ELISA titers were log transformed prior to analysis using a general finear mixed model. The 95% level of confidence was used to assess treatment differences. Chalenge observations were monitored twice daily for 30 minutes each. RESULTS: . Trachea! Swab Outturn and Agglutination Triers Tracheal swab cultures and agglutination titers were evaluated to monitor the B. bronchiseptica status of animals enrolled in the study. A number of dogs demonstrated increased titers at various time points but no titer increased above 128 prior to challenge. Injection Site Observations Injection site reactions following the first vaccination are presented in Table 1. The largest injection site reactions were observed in T05 (64 jig) vaccinated animals, with tha largest mean injection site reaction measuring only 14.69 cm3 (two days post vaccination). T03 (4 ug), T04 (16 jig) and T06 (256 ug) vaccinated animals demonstrated varying injection site reactions up to 7 days post vaccination. T02 (1 ug) vaccinated animals only demonstrated reactions on Day 1 post vaccination. By the seventh day post vaccination, there was no statistically significant difference in injection site reactions among the treatment groups. By Day 14, aft injection site reactions had dissipated. Injection site reactions following the second vaccination are presented in Table 2. Following the second vaccination the largest mean injection site reactions were observed in T06 (256 jig), with the largest mean injection site reaction measuring 50.03 cm3 (one day post vaccination). Injection site reactions were demonstrated in T05 (64 ug) and T04 (16 ug) animals up to 7 days post second vaccination. Minimal injection site reactions were demonstrated in T03 (4 ug) and TQ2 (1 ug) animals up to 7 days post vaccination. Infection site reactions that were not statistically different from the placebo group were demonstrated in T02 (1 jig) and T03 (4 ug) post vaccination. Fourteen days post second vaccination no injection site reactions were observed. Frequency of injection site reactions following first vaccination is presented in Table 3. The highest overal LSM frequency, 76%, of injection sites exhibiting a reaction at any time post first vaccination resulted from vaccination with T06 (256 ug). The next most frequent were 72% of the injection sites showing a reaction following tha first vaccination with T05 (64 ug), 69% following the first vaccination with T04 (16 ug), and 63% following the first vaccination wfth T03 (4 ug). The lowest frequency, 38%, followed the first vaccination of T02 Frequency of injection site reactions following the second vaccination is presented in Table 4. The overall LSM frequency for each vaccine was consistent with that seen post first vaccination. Incidence and duration of injection site reactions following vaccination are summarized in Table 5. The incidence (or the number of dogs showing a reaction at any time) of a measurable injection site reaction was 100% for T03, T04, T05 and T06 (4 ng, 16 jig, 64 jig, and 256 fig, respectively) following the first and second vaccination. Animals that received T02 (1 fig) demonstrated the least incidence of injection site reactions post vaccination (57.1%). Duration of the reaction (expressed as a least squares means of days with a reaction shown in Table 5) was longer for T04, T05 and T06 (16 jig, 64 ug, and 256 fig, respectively) vaccinated animals following the first and second vaccinations (2.7 to 5.1 days post first vaccination and 6.0 to 6.7 days post second vaccination). T02 and T03 (1 ug and 4 ng, respectively) vaccinated animals demonstrated the fewest number of days with an injection site reaction following the first and second vaccinations (0.3 and 1.3 days post first vaccination and 1.9 and 4.5 days post second vaccination). Rectal Temperatures Mean rectal temperature measurements are summarized in Table 6. The LSM rectal temperature for T02 (1 ug) on Day 1 and 24, for T03 (4 fig) on Day 1,21, and 24, for T04 (16 jig) on Day 2 and 24, for T05 (64 ng) on Day 23, and for T06 (256 ug) on Day 0,1, and 24 were significantly different from the placebo. On Day 23 ail comparisons were not statistically significant (P>0.05) from the placebo. D6BELJSASenjloav Summary of p68 ELJSA data are presented in Table 7. The pre-vaccination geometric mean virus tilers of p68 ELISA specific antibodies in all groups were low (range 24.9 to 2B.9) and liters for the placebo remained low throughout the duration of the study. Twenty-one days following the first vaccination, 068 ELISA geometric mean liters had increased in the vaccinated treatment (range 55.2 to 4,411.7), however T02 (1 ug) titer was not statistically different from the Placebo (T01). Forty-two days after the second vaccination, geometric mean triers were further increased in all vaccinated groups (range 674.6 to 48,382.0) demonstrating good serological response to vaccination. Serum AmviaidA fSAA) Semiaav SAA tilers are summarized in Table 8. Prior to challenge, geometric mean SAA tilers were low in all the treatment groups (range 0.1 to 0.5). Post challenge, T01 GMT liters ranged from 1.5 to 146.0. where p68 treatment groups ranged from 0.3 to 23.1. All treatment groups were statistically different than the placebo on Days 50,52,54, and 56. No statistical differences were demonstrated among the p68 vaccines with the exception of T02 (1 ug) on Day 52 when it demonstrated a statistically different geometric mean from all other p€8 treatment groups. Challenge Resoose Challenge response data are presented in Table 9. The response was determined by monitoring following challenge and the observations were analyzed using two methods: least mean number of days with cough and two consecutive days of coughing (Instead of Disease). Analysis t the mean number of days coughing demonstrated no statistic By significant differs between the p68 treatment groups; but in the dogs vaccinated with placebo mean of 8.6 days whereas dogs administered p68 vaccines coughed significantly less, leans ranging between 2.2 to 4.7 days. When were evaluated using Incidence of Disease, all T01 (placebo) were observed for two consecutive days (100% Incidence of Disease}. T04 (16 ug) and T05 (64 fig) vaccieted dogs demonstrated an incidence of Disease of 55.6% and 66.7%, respectively.. Onl£8.6% of T02 (1 ug), 50% of T03 (4 ug), and 33.3% of T06 (256 ug) vaccinated dogs »re observed coughing for two consecutive days. DISCUSSION: In this stuff, the objective was to establish a relationship between antigen dose, immune response and protection in dogs. The p6B antigen doses examined were 1 fig, 4 jig, 16 jig, 64 ug, andE6 ug. Analysis 4 injection site reaction measurements demonstrated a negligible reaction in the p68 , with the exception of T06 (256 ug) on the first day post second vaccination. Reacfcns that were observed tended to be small, generally decreasing In size during the observfon periods. The size of these reactions was clinically insignificant and would most likely P unnoticed on unshaven dogs. Rectal temperatures post vaccination were unremarkable and were within normal limits for all dogs all groups. Serotogid response to vaccination was excellent in T03 through T06 groups. In these treatment, all demonstrated significantly higher p68 ELISA when compared to the placebo 21 through Day 63. T02 (1 ug) demonstrated significant p68 ELISA compared T01 (placebo) from Day 42 through Day 63. The highest were observed in T05 (* ug) and T06 (256 ug}. . of the SAA response in all p68 vaccinated dogs following challenge indicated a much mailer rise in the SAA post when compared to control dogs. No difference was dewnstrated between the p68 vaccine dose levels post-challenge with the exception of T02 |ug) on Day 52 that demonstrated a statistically different geometric mean from all other p68teatment groups. Post coughing observations were analyzed using {east squares means (LSM) of the days with cough or two consecutive days coughing (incidence of disease). Using LM of days coughing, a significant difference was demonstrated between placebo and all vaccinated groups although no difference was demonstrated between the different p68 vaccine dose levels. Using the Incidence of Disease T02 (1ug), T03 (4ug) and T06 (256ug) vaccinated dogs coughed significantly less than the placebo. CONCLUSIONS: The study was conducted to establish a relationship between antigen dose, immune response, and protection in dogs. The p68 antigen doses examined were 1 fig, 4 ug, 16 fig, 64ug,and256ug. All vaccines were safe as demonstrated by minimal injection site reactions, normal rectal temperatures and absence of adverse response to vaccination. The size of the injection site reactions and the duration of these reactions were less in the lower antigenic treatment groups. Serological response to vaccination as measured by ELISA titers was excellent with the higher antigenic dose groups demonstrating higher serologicaJ responses. When using LSM days coughing as a method of comparison, all treatment groups demonstrated a significant reduction in coughing when compared to placebo. No differences were noted between the treatment groups. When two consecutive days coughing (or Incidence of Disease) was used for comparison, TQ2 (1 jig), T03 (4 ug) and T06 (256 fig) vaccinated dogs coughed significantly less than the placebo. (Table Removed)EXAMPLE 2 Animals Forty-five male and female mixed breed dogs were purchased. A MLV parvovirus vaccine was administered to all puppies on the day the puppies arrived at the study site. No other vaccines, other than the experimental products, were administered to the puppies during the study. Dogs were approximately 9 weeks of age (± 1 week) on Day 0 (day of first vaccination). Dogs were kept in an isolation facility necessary to prevent exposure to Bordetelfa and other canine pathogens prior to challenge. After aerosol challenge with BordefeHa, isolation procedures were continued to prevent exposure to other canine pathogens. Vaccines Sterile saline was used as a placebo vaccine in treatment groups T01 and T02. Canine recombinant p68 Botdeletta Bronchiseptica Vaccine was used in treatment groups T03 and T04. The structural gene of the p68 antigen was cloned in Escherichia co//and expression of the gene was regulated by a temperature sensitive promoter. The cells were fysed and the inclusion bodies were separated by centrifugation. The recombinant p68 in the inclusion bodies was solubized by SOS treatment The recombinant p68 (15 ug per mL) was combined with 50 u,g of Quil A and 50 ug of cholesterol per mL in sterile saline as the diluent. Each one ml dose contained 0.28% of ethanol and 0.01% thimerosal. Challenge Inoculum Bordetetta brvnchiseptica Bihr Cat strain was prepared as the challenge inoculum using the method currently employed by Biologies Control Laboratories-Microbiology. Bordet-Genou agar plates were plated with a confluent growth of Bordetella brvnchiseptica - Blhr Cat strain and incubated for 48 hours at 37.5 +/- 2.5°C. Virulent phase I colonies were selected and streaked on Bordet-Qenou agar and incubated for 24 hours at 37.5 +/- 2.5°C. After incubation, Bordetella saline was used to wash colonies from the agar and the antigen was diluted to an optical density of 0.80 at 600 nm. A cell count was performed pre- and post-challenge for confirmation of the nephelometer reading. Challenge target concentration was approximately 1 X 10s CPU. The pre-challenge concentration was 2.37 x 10s CPU (100% Phase I) and post-challenge concentration count was 1.35 x 1 o9 CPU (100% Phase I). (Table Removed)Randomization/Blinding For the time period from vaccination to day of challenge, animals were assigned to treatments according to a generaized block design. Treatments were randomly assigned to rooms. On the day of challenge, animals were randomly assigned to challenge rooms by block. Qualified individuals, unaware of the assigned treatment groups, conducted microbiological and serological assays and assessments of injection sites, measurements of rectal temperatures, and observations of coughing. Data Analysis Post vaccination response variables consisted of injection she data, rectal temperatures and p68 ELISA tilers. Injection site data was summarized in the following ways: 1) number of animate having a measurable reaction by treatment and day of study, 2) number .of animal time points having a measurable reaction by treatment. 3) number of animals having a measurable reaction at any time point by treatment. Separately for first and second vaccinations, injection site volume (cubic cm), rectal temperatures and natural log transformed p68 ELISA titer data was analyzed using a general Inear mixed model. A priori linear contrasts of the treatment by observation time-point least squares mean were constructed to test treatment group deferences at each observation time-point and to compare time-points within each treatment The 5% level of significance was used for all comparisons. Post challenge response variables consisted of daily coughing observations. p68 ELISA tilers and serum amyloid A titers. Number of days coughing during the post challenge period was analyzed using a general linear mixed model. A priori contrasts of the treatment least squares mean was constructed to test treatment group differences. The 5% level of significance was used for all comparisons. Separately for first and second vaccinations, Fisher's Exact test was used to compare treatment groups for the incidence of two days of consecutive coughing. The 5% level of significance was used for all comparisons. For serum amyloid A (SAA) titer data post challenge, the natural log transformation was applied to titer values prior to analysis using a general linear mixed model. A linear contrasts of the treatment by observation time-point least squares mean was constructed to test treatment group differences at each observation time-point and to compare time-points within each treatment. The 5% level of significance was used for all comparisons. Study Procedure Detailed Animal Procedures Forty-five (45) seronegative and culture negative pups were randomly assigned to one of 4 treatment groups. Eight and seven dogs were allocated to the intramuscular (IM) or subcutaneous (SC) control groups respectively, for a total of 15 control dogs. Fifteen dogs were allocated to the p68 SC treatment group and 15 dogs were allocated to the p68 IM treatment group. Treatment Groups are detailed in the Study Design Section above. Day 0 was designated as the day of first vaccination. Vaccinations were administered on Day 0 and repeated 21 days later. For the first vaccination, the right side of the neck was used and for the second vaccination, the left side of the neck was used. Intramuscular injections were administered in the right and left semimembranosus muscle for the first and second vaccinations, respectively. All injection sites were measured three dimensionally for seven days following each vaccination with a follow-up measurement conducted 14 days following vaccination. Rectal temperatures were monitored on the day of vaccination (prior to vacdnation) and for three days following each .vaccination. On Day 35, all animals were trachea! swabbed for & brortchiseptica culture and blood was collected for agglutination Mere. Ad animals were negative by trachea! swab and serotogically negative to BordeteUa and deemed eigibie for challenge. On Day 45, twenty-four days after the second vaccination, an aerosol challenge of R bronchiseptica was administered to all dogs. Sedated dogs were challenged using a disposable nose cone, which was fitted snugly over the muzzle of the sedated dog. The nose cone was attached to a nebulizer which was attached to a vacuum pressure pump set at 5.5 to 6.0 psi. One mL of challenge material was placed in the nebulizer and the aerosolized challenge material was administered to each dog for 4 mutes. Personnel making observations were unaware of treatment group assignments. Animals were monitored for coughing for 14 days following challenge (Days 46-59). Observations were made in 2 (two), approximately 30-minute periods at approximately the same time each day, one conducted in the AM and one in the PM and results were recorded. Blood Collection Blood for agglutination liters was collected prior to first vaccination and prior to challenge. Blood for anti-p68 ELISA evaluation was collected prior to vaccination on Days 0 and 21 and on Days 35,45. and 59. Blood for Serum Amyloid A (SAA) assay was collected on the day of challenge (Day 45) and on Days 46,48,50, 52 and 54. Tests Performed on Samples Tracheal swabs were evaluated for the presence of ft bronchiseptica by culture. Each trachea! swab was streaked onto a Bordetela Selective Agar plate. Positive and negative controls were included. The plates were incubated at 37.5 ± 2.5° C for 48 ± 4 hours. The resulting colonies on each plate were compared to the positive control and any colony which appeared identical to the positive control was further tested to confirm the presence of B. bronchiseptica. Confirmational testing included the use of TSI, Citrate and Urea Agar and Nitrate Red media. Sera were evaluated for agglutination titers, p68 ELISA analysis or SAA analysis using the following methods: Agglutination titers - Sera were serially diluted in microtiter plates using Bordeteda saline. Positive and negative controls were included on each plate. B. bronchiseptica Strain . 87 (grown on Bordet Genou agar, harvested, inactivated and diluted to 20% T at 630nm) was used as the agglutinating antigen and was added to each well. Plates were shaken and incubated at 35 ± 2° C for 2 hours. Plates were read after a second incubation at room temperature for 22 hours. The endpoint titer was determined using the last well to show 50% agglutination. p68 ELISA titers - The recornbtnant p68 antigen was captured on a 96 well microtiter plate coated with a polyclonal antiserum specific to the Bordetefla p68 antigen. Serial two-fold dilutions of the canine serum were added to the plate and incubated. Positive and negative controls at a 1:1000 dilution were included on each plate. A peroxidase labeled affinity purified goat anti-dog IgG indicator conjugate was used to detect antibodies specific for the p68 antigen. A chromogenfc substrate ABTS was then added and the plate read when the positive control wells had an O.D. of 1.2 + 0.2. The titer of a given sample was calculated as the reciprocal of the last dilution with an optical density greater than the mean of the negative control serum dilution plus five standard deviations. SAA titers - The canine Serum Amyloid A titers were evaluated using a kit purchased from Accupfex Co., University of Nebraska Medical Center, Omaha, NE 68198. Briefly, canine SAA was captured on a microtiter plate coated with a monoclonal anti-canine SAA antibody. Diluted samples of the canine serum were added to the plate followed by a biotin labeled anti-canine antibody conjugate. Following incubation, a peroxidase conjugated streptavidin chromogenic substrate was added. The plate was read after 30 minutes. Results Rectal Temperatures Summary of rectal temperature measurements are presented in Tables 10 and 11. (Table Removed)No significant difference was noted between any groups on any day following the first vaccination. A significant difference was noted between saline vaccinated dogs and all p68 vaccinated dogs (p=0.0053 for J01T02 v T03T04) on Day 21. A significant difference (p=0.0124) between p68 SC and IM vaccinates was demonstrated on Day 24. Injection Site Reactions Injection site reactions are summarized in Tables 12 and 13. Due to technical oversight, no injection site observations were conducted at the 14-day observation following the second vaccination (Day 35). Measurable site reactions were observed in T03 (p68 SC) and were minimal in size. A small injection site reaction was noted in one dog in T04 (068 IM) on Day 3 but the minimal impact of the measurement is not reflected in the over O mean for the group. Table 12: Least squares mean (cubic cm) of injection site reactions in dogs following saline or p68 (15ug/dose) Bordetella vaccination (post first vaccination") (Table Removed)Significant differences in injection sites measurements are summarized in Table 14. A significant difference in injection site measurements between T02 (saJine SC) versus T03 (p68 ISug SC) was noted for six days following the first vaccination. By Day 7 and again on Day 14 (the two week re-evaluation observation), no significant differences were noted between any treatment group. A significant difference in injection site measurements between T02 (saline SC) versus T03 (p6815ug SC) was noted for seven days folowing the second vaccination. (Table Removed)068 ELISA liters p68 EUSA data are summarized in Table 15 and Figure 1. Due to the considerable titer response to p68 in the vaccinated dogs, various filiation minimums were used at different time-points in the study. Trtratfons for Days 0 and 21 were started at SO. For Days 35,45 and 59, filiations were begun at 200. Any value reported as "less than* was divided by 2 prior to analysis. The incremental rise observed In p68 EUSA values for control groups (T01 and TQ2) during the course of the study is due to these minimum titration values. Agglutination liters remained All p68 vaccinated animals demonstrated at least a four-fold Increase in ttters from the first day of vaccination to the day of challenge (Day 0 vs. Day 45) when compared to placebo vaccinated animals. (Table Removed)Significant differences for least squares mean of post vaccination and post challenge ELISA are in Table 16. No significant difference was noted between the p66 SC and p68 IM vaccinated animals after vaccination was complete. (Table Removed)Significant differences in SAA titers are summarized in Table 18. Saline controls demonstrated higher SAA titers than vaccinated dogs on Days 1,3 and 5 following challenge. Saline SC controls continued to demonstrate significantly higher SAA titers when compared to vaccinated SC dogs on Day 7 and 9 following challenge. Table 18. Significance values for a priori contrasts among least square mean of Serum Amyloid A titers Dostchallenge. (Table Removed) Coughing Observations Aerosol challenge for all treatment groups occurred 24 days following the second vaccination (Day 45). Coughing observations were examined using two methods - disease status based on two consecutive days coughing (presented in Table 19) and percentage of days coughing (presented in Tables 20 and 21). When dogs were evaluated using criteria of two consecutive days coughing, 80% of the p6B vaccinated dogs (SG and IM) coughed at least two consecutive days whereas the Saline SC and Saline IM vaccinated dogs coughed 100% and 87.5%, respectively. When dogs were evaluated using percentage of days observed coughing. p68 SC and IM vaccinated dogs coughed 38.72% and 41.05% of the days observed, respectively. Saine SC and Saline IM vaccinated dogs coughed 69.04% and 62.66%, respectively. Table 19. Summary of disease status in saline and p68 (15 ug/dose) Bordetella vaccinated dogs based on two consecutive days coughing following aerosol challenge with Bordetella bronchiseptfca(Table Removed) No significant difference was demonstrated between saline vaccinated and p68 vaccinated dogs when disease status was based on two consecutive days coughing. (Table Removed)A significant difference (p= 0.0112} was demonstrated between T02 (safine SC) and T03 (p6815 fig SC). No significant difference was demonstrated between T01 (saline IM) and T04(p6815uglM). (Table Removed)A significant difference (p=0.0022) was demonstrated between the saine controls and p68-vaccinated dogs. Discussion The study was designed to demonstrate the safety and efficacy of a p68 Bordeteila 15 tig/dose vaccine in dogs. Safety was examined using injection site and rectal temperature observations. Analysis of injection site reaction measurements demonstrated a negligible reaction in the IM vaccinated group and minimal reactions in SC vaccinated group. Reactions that were observed tended to be small, generally decreasing in size during the observation periods. The size of these reactions would most likely go unnoticed on unshaven dogs. Although a significant difference was observed between saline and vaccinated on Day 21 and between IM and SC vaccinates on Day 24, rectal temperatures were clinically unremarkable and were within normal limits for all dogs in all groups. Efficacy was examined using observations of measurement of p68 ELISA endpoint liters and coughing. Regardless of the route of administration, a good p68 antibody response was demonstrated in pea-vaccinated groups by Day 35. A good anamnestic response was observed in vaccinates post challenge. Although higher antibody responses have traditionally been obtained with the more vascular and less fatty IM route as compared to the SC route, the difference between p68 SC and IM vaccinates was not significant through the course of the study. Examination of the SAA response in vaccinated and unvaccinated p68 Bordeteila dogs following challenge indicated a much smaller rise in the SAA values in vaccinated animals groups especially on days 1,3 and 5 days post-challenge. Conclusions In this study, efficacy a 15 ug/dose p6S canine Bordetella vaccine was examined using a canine challenge model 24 days after vaccination. The vaccine was safe as demonstrated by normal rectal temperatures, minimal injection site reactions and efficacy was demonstrated in combined IM and SC groups. Comparison of SAA values demonstrated a significant difference between saline and p68 vaccinated dogs on Days 1,3 and 5 following aerosol challenge. EXAMPLES SIX MONTH DURATION OF IMMUNITY STUDY OF CANINE BORDETELLA p68 VACCINE Animals Ninety male and female mixed breed dogs were purchased and the majority of puppies were 9 weeks (±1 week) on the day of first vaccination. A MLV parvovirus vaccine was administered to dogs upon arrival at .the study site. To be eligible for the study, animals were determined to be negative to B. bronchiseptica by trachea! swab and agglutination tiler. No vaccines, other than the experimental products, were administered during the study. Dogs were kept in an isolation facility necessary to prevent exposure to B. brvftchiseptica and canine pathogens prior to challenge. After aerosol challenge with B. brvnchiseptica, isolation procedures were continued to prevent exposure to other canine pathogens. Vaccines Sterile safne was used as a placebo vaccine in treatment groups T01 and T02. Canine recombinant p68 Bordetella Bronchiseptica Vaccine was used in treatment groups T03 and T04. The structural gene of the p68 antigen was cloned in Escherichia cc/Fand expression of the gene was regulated by a temperature sensitive promoter. The cells were tysed and the inclusion bodies were separated by centrifugation. The recombinant p6B in the inclusion bodies was solubiiized by SOS treatment Separately, the 15 ug p68 and 60 ug p68 were combined with 50 tig of Quil A and 50 jig of cholesterol per ml in sterile Lepto saline as the diluent The combined components were mixed at 4°C for 24 hours and passed three times through a microf luidizer. Each one ml dose contained 2.7 ul of ethanol and 0.0001 % thimerosal. p€8 concentrations in the experimental vaccines were measured by p68 ELISA. All assays were done in replicates of five (5). All vaccines were used within 6 months of assembly. Challenge Inoculum Bordet-Genou agar plates were plated with Bortfeteffa bronchtseptica - Bihr Cat strain and incubated for 48 hours at 37.5 +/- 2.5°C. Virulent phase I colonies were selected and streaked on Bordet-Genou agar and incubated for 24 hours at 37.5 W- 2.5°C. After incubation, Bordetella saline was used to wash colonies from agar and the cells diluted to an optical density of 0,80 at 600 nm. A cell count was performed pre and post challenge for confirmation of the nephelometer reading. Challenge target concentration was approximately 1 X lO^FU. For Group I, the prechalienge concentration count was 1.94 X109 and the post challenge concentration count was 1.43 X109. For Group II, the prechalienge concentration count was 2.55 X109 and the post challenge concentration count was 2.13 X10°. Study Design Summary Table(Table Removed)The study was conducted in two phases or groups consisting of 48 dogs in Group I and 42 dogs in Group II. Vaccination #1 occurred on Day 0 for the each Group. Vaccination #2 occurred 20 days later. Events in Group 1 were offset .from the events in Group II by approximately 15 days. Dogs were aerosol challenged with & bronchissptica 181 days after the last vaccination. Animals were randomly assigned to treatments and rooms according to a complete randomized design. Fertile time period from vaccination #1 to day of challenge within each study group, animals were randomly assigned to treatments and rooms (3 to 5 dogs per room) using a randomization plan. On the day of challenge, the previously treated animals were randomized to challenge rooms within study group using a generalized block design. Qualified individuals, unaware of the assigned treatment groups, conducted microbiological and serological assays and assessments of coughing and injection sites. pata Analysis Post vaccination response variables consisted of injection site data, rectal temperatures and p68 ELISA titers. Injection site data was summarized as follows: 1) number of animals having a measurable reaction by treatment and day of study, 2) number of animal tone points having a measurable reaction by treatment, 3) number of animals having a measurable reaction at any time point by treatment, 4) duration of a measurable reaction for each animal. Separately for first and second vaccinations, injection site volume (cubic cm), rectal temperatures and natural log transformed p68 EUSA titer data was analyzed using a general linear mixed model. A prioriKnear contrasts of the treatment by observation time point feast squares mean was constructed to test treatment group differences at each observation time point and to compare time points within each treatment The specific comparisons of interest were T01 vs. T03, T01 vs. TOS, T03 vs. TOS, T02 vs. T04, T02 vs. T06, and T04 vs. T06. If the time point-by-treatment-by-study group interaction term was significant at P Post challenge response variables consisted of p68 ELISA liters, Serum Amyloid A liters and daily coughing observations. Post challenge p68 ELISA liters were analyzed as previously described. For Serum Amyloid A (SAA) titer. data post challenge, the natural tog transformation was applied to titer values prior to analysis using a general linear mixed modef. The analysis of coughing was amended to reflect USDA requirements. For each dog, the percentage of observation periods during which coughing was observed was calculated. Prior to analysis, the percentage was transformed using the arcsin square root transformation. A general inear mixed model was used for analysis of coughing. Least squares mean from this analysis were back-transformed to percentages and the percent reduction in coughing was calculated as: Percent Reduction = 100 X (control group mean,; treatment group mean) (control group mean) A priori linear contrasts of the treatment least squares mean was constructed to test treatment group differences. The specific comparisons of interest were T01 vs. T03, TOl vs. TOS, T03 vs. TOS, T02 vs. T04, T02 vs. T06, and T04 vs. T06. If the treatment-by-study group interaction term was significant at P Study Procedure Detailed Animal Procedures Prior to arrival on study premises and prior to the first vaccination, puppies were trachea! .swabbed for B. branchlseptica culture and blood was collected for agglutination liters. All animals were negative by trachea! swab and serologfcafly negative to Bortetella and deemed eligible for the study. Forty-eight puppies were randomly assigned to one of six treatment groups for Group I. The procedure was repeated using forty-two dogs for Group II. Animals were acclimated to the study site for at least five days. Groups and treatments are detailed in Section 7.4.A. Due to facflity constraints and to enhance trie accuracy of coughing observations following challenge, the vaccination and the respective challenge periods were staggered by 15 days to generate the two dog groups. Day 0 refers to the day of vaccination #1 for both Group I and II. Vaccination #2 occurred 20 days later. Treatments T01, T03, and TOS were administered via the subcutaneous route. Treatments T02, T04, and T06 were administered via the intramuscular route. Subcutaneous injections were admnistered in the dorsolateral aspect of the neck. For vaccination #1, the right side of the neck was used and for vaccination #2, the left side of the neck was used. Intramuscular injections were administered in the right and left semimembranosus muscle for vaccination #1 and vaccination #2, respectively. All injection sites were measured three dimensfonally for seven days following each vaccination with a follow-up measurement done 14 days following vaccination. Rectal temperatures were monitored on the day of vaccination (prior to vaccination) and for three days following each vaccination. Blood was collected prior to each vaccination (on Day -1 and Day 19) and on Day 50 for p68 ELISA titer determination. Each month, all dogs were trachea! swabbed for B. btvnchiseptica culture under sedation to confirm bronchiseptica negative status. Blood was also collected for agglutination and ELISA tilers. The procedure was repeated 7 days prior to challenge for each group. Evidence of a positive tracheal swab culture or a rising agglutination titer excluded the animal from the study. Challenge was administered to dogs 181 days after vaccination #2. Sedated dogs were challenged using a disposable nose cone, which was fitted snuggly over the muzzle of the sedated dog. The nose cone was attached to a nebulizer which was attached to a vacuum pressure pump set at 5.5 to 6.0 psi. One mi. of challenge material was placed in the nebulizer and the aerosolized challenge material was administered to each dog for 4 minutes. Post challenge coughing observations were amended prior to challenge to comply with USDA recommendations. After challenge, each group of dogs was observed between the third and tenth day following challenge, for a total of 8 days. Animals were observed twice daily for coughing for approximately 45 minutes at each observation period. The interval between observation periods was approximately 12 hours. Personnel unaware of the assigned treatment groups recorded coughing observations. Blood Collection Blood for agglutination liters was collected prior to first vaccination, monthly and prior to challenge for each group. Blood for anti-p6B ELISA evaluation was collected the day before vaccination #1 and #2, on Day 50 and at approximately 30 day intervals thereafter for each group. Blood was also collected the day of challenge and on the final day of post challenge observation. Blood for Serum Amyloid A (SAA) assay was collected on the day of challenge (prior to challenge) and on 1,3,5,7, and 9 days post challenge for each group. Tests Performed on Samples Trachea! swabs were evaluated for the presence of 3. bronchiseptica by culture. Each trachea! swab was streaked onto a Bordetella Selective Agar plate. Positive and negative controls are included. The plates were incubated at 37.5 ± 2.5°C for 48 ± 4 hours. The resulting colonies on each plate were compared to the positive control and any colony which appeared identical to the positive control was further tested to confirm the presence of a bronchiseptica. Conf irmational testing included the use of TSI, Citrate and Urea Agar and Nitrate Red media. Sera were evaluated for agglutination Wars, p68 ELISA analysis or SAA analysis using the following methods: Agglutination liters - Sera were serially diluted in microtiter plates using Bordetella saline. Positive and negative controls were included on each plate, ft bronchiseptica Strain 87 (grown on Bordet Genou agar, harvested, inactivated and diluted to 20%T at 630nm) was used as the agglutinating antigen and was added to each well. Plates were shaken and incubated at 35 ± 2°C for 2 hours. Plates were read after a second incubation at room temperature for 22 hours. The endpoint titer was determined using the last well to snow 50% agglutination. . i p68 ELISA tilers - The recombinant 068 antigen was captured on a 96 well microtiter plate coated with a porydonal antiserum specific to the Bordetella p68 antigen. Serial two fold dilutions of the canine serum were added to the plate and incubated. Positive and negative controls at a 1:1000 dilution were included on each plate. A peroxidase labeled affinity purified goat anti-dog IgG Indicator conjugate was used to detect antibodies specific for the rp68 antigen. A chromogenic substrate ABTS was then added and the plate read when the positive control weds had an O.D. of 15. ± 0.2. The tiler of a given sample was calculated as the reciprocal of the last dilution with an optical density greater than the mean'of the negative control serum dilution plus five standard deviations. SAA titers - The canine Serum Amyloid A was captured on a 96 well microtiter plate coated with a monoclonal anti-canine SAA antibody. Diluted samples of the canine serum were added to the plate and Incubated. A reference standard was added to obtain a standard curve from 0.31 ng/ml to 20 ng/ml. A Wotin labeled anti-canine antibody conjugate was added. Following the incubation of the biotin labeled anti-canine antibody, a peroxidase conjugated streptavidin was added. A chromogenic substrate TMB was added and the plate was read after 30 minutes. The concentration of Serum Amyloid A was determined by comparison the sample to the standard curve and multiplication by the appropriate dQution factor. Results Unless otherwise noted, results are the combined data from Group I and II. Trachea! Swab Culture and Agglutination Titers Positive trachea! swab cultures and/or rising agglutination titers were demonstrated in eleven dogs during the course of the study. These dogs and any dog housed with the positive dogs were removed from the study, resulting in a loss of 20 dogs. The number of dogs removed from each group was: T01-4 dogs, T02-2 dogs, T03-3 dogs, T04-1 dog, T05-5 dogs, T06-5 dogs. Injection Sfte Obssrvations Injection site reactions are summarized in Tables 22-25. Injection site information was. not collected for Dog 81595 on Day 21 for Group I due to technical oversight. The protocol was amended so that injection site reaction data was not collected for dogs in Group II on Day 22 therefore, summary of data from Day 22 contains only information from the eight dogs per treatment group in Group I. Injection site reactions were not observed for any dog receiving an IM treatment. Injection site measurements were minimal for both SC vaccinated treatment groups (T03 and T05). (Table Removed)Significant differences in injection sites measurements are summarized in Table 26. A significant difference in injection sites measurements between T01 (saline SC) versus T03 (60 ug SC) was noted for seven days after the first vaccination. A significant difference between T01 (saline SC) and T05 (15 ug SC) was noted for only the first four days after the first vaccination. A significant difference was found between T03 (60 ug SC) and T05 (15 ug SC) on Days 3 through 7. By Day 14 (the two week re-evaluation observation), no difference was noted between any of the groups. A significant difference (FMX0138) in injection sites measurements between T01 (saline SC) versus T03 (60 tig SC) and T05 (15 ug SC) was noted for seven days after the second vaccination. A significant difference was found between T03 (60 ug SC) and T05 (15 ug SC) on Days 23 through 27. By Day 34 (the two week re-evaluation observation), no difference was noted between any of the groups (Table Removed)A significant difference was noted in rectal temperatures between T02 (saline IM) and T04 (60 fig IM) on Day 1 after the first vaccination. No significant difference was found in rectal temperatures between any group on any day after the second vaccination. D68ELISA Titans Prechallenge p6S EUSA data are summarized in Table 29. Due to the response of Group I dogs in T06 on Day 19, an effect due to group was observed in the data analysis of p68 EUSA tilers. The effect was small and did not influence other analyzed timepoints. Therefore, data from Group I and tl are combined for reporting purposes. Due to the considerable titer response to p6S in the vaccinated dogs, various titration minimums were used at different timepoints in the study. Trtrations for Days -1 and 19 were started at 50. For days 50 through 195, trtrationa were begun at 200. Tftrations for samples collected on Day 201 and 211 were started at 1000. Any value reported as "less than" was divided by 2 prior to analysis. The incremental rise observed in p68 ELISA values for control groups (T01 and T02) during the course of the study is due to these minimum titration values. Agglutination titers, except as previously noted, remained All placebo vaccinated dogs had p68 liters (Table Removed)Significant Differences for least squares mean of post vaccination ELISA titers are listed in Table 30. No significant difference in p68 ELISA titers was observed between SC controls and SC vaccinates or IM controls and IM vaccinates prior to vaccination (Day -1}. p68 EL1SA tilers measured during the course of the study are summarized In Table 31 and illustrated in Figure 3. During the course of the study, every attempt was made to coordinate activities between Groups I and II. For pivotal data collection time points (i.e. events surrounding vaccination and challenge), this was achieved. In three instances during the interim of the study, blood and trachea! swab collection varied by 1 or 2 days between groups. In order to summarize and report p68 ELISA data for this interim period, data from these days were combined. Therefore, Day 79 contains combined data from Day 79 (Group I) and Day 81 (Group II), Day 111 corresponds to Day 110 (Group II) and Day 111 (Group I) and Day 169 corresponds to Day 169 (Group II) and Day 170 (Group I). Per the protocol, data analysis was not performed on p68 EUSA data beyond Day 50. (Table Removed)During the course of the study, every attempt was made to coordinate activities between Groups I and II. In three Instances, blood collection for the Groups varied by 1 or 2 days. Data from these days were combined for data summary. Analysis was not done on p68 ELISA liter values collected beyond Day 50. Day 79 corresponds to Day 79 and 81, Day 110 corresponds to Day 110 and 111. Day 169 corresponds to Day 169 and 170. Coughing Observations Aerosol challenge for both groups occurred 181 days following the second vaccination. To comply with USDA recommendations, coughing criteria was amended to approximately 45-minute observations, approximately twelve hours apart on the third through eighth day following challenge. Coughing observations are summarized in Tables 32 and 33. (Table Removed)Discussion The study was designed to demonstrate the safety and six-month efficacy of a recombinant p68 Bordetetfa vaccine in dogs. Safety of both the 15 fig/dose and the 60 jig/dose vaccine was demonstrated. The efficacy and 6 month duration of immunity of the 15 fig/dose was well supported in the study. Safety was examined using injection site and rectal temperature observations. Analysis of injection site reaction measurements demonstrated no reactions in the IM vaccinated groups and minimal reactions in both the 15 ^ig/dose and 60 (tg/dose SC vaccinated groups. Reactions that were observed tended to be smaller in the 1 Spxj/dose SC vaccinated dogs. Such reactions that were seen were transient, generally resolving in 14 days r less. The size of these reactions would most likely go unnoticed on dogs where injection sites were not shaven. Rectal temperatures post vaccination were unremarkable and were within normal limits for all dogs in all groups. Efficacy and duration of immunity were examined 181 days from vaccination using observations of coughing and measurement of p68 ELISA endpoint titers. The percentage of coughing in the saline controls (78.26%) indicated that the challenge administered to the study animate was acceptable. Percentage of time points coughing for the 15 tig/dose group (37.92%) demonstrated a 51.55% reduction in coughing when compared to the controls, satisfying the efficacy requirements mandated by the USDA. No protection was demonstrated in the 60 {igfctose group (89.58%) with dogs demonstrating minimal reduction in coughing when compared to the controls. Although not statistically compared, it can be seen from Tables 29 and 31 that SC vaccinates tended to have higher antibody responses when compared to IM vaccinates. For the purposes of the discussion, further comments regarding the different dose groups combine the results of the IM and SC routes of administration. Regardless of the route of administration, an excellent p68 antibody response was demonstrated in both vaccinated groups by Day 50 and was maintained by vaccinates throughout the course of the study. A good anamnestte response was observed in vaccinates post challenge. Comparison of the p68 ELISA ftera of the 15 fig/dose and 60 ug/dose during the course of the study indicated that the 60 ug/dose group showed a slightly higher liter response (Figure 3). This response, although excellent, cfd not correlate with protection following ' aerosol challenge. p68 EUSA titer responses were variable in dogs removed from the study with positive tracheal swabs or rising agglutination titers. Three of the six controls removed from the study with positive tracheal swab cultures and/or rising agglutination titers maintained p68 EUSA titers of Examination of the SAA response in vaccinated and unvaccinated p68 BordeteKa dogs following challenge indicated a much smaller rise in the SAA values in the 15 ug/dose groups especially on days 5 and 7 post-challenge. Conclusions In this study, efficacy a 15 ug/dose and 60 ug/dose of a p68 canine Bordetetta vaccine was examined using a canine challenge model 6 months after vaccination. Both vaccines were safe as demonstrated by normal rectal temperatures and minimal injection site reactions. Although both the 15ug/dose and the 60ug/dose vaccinated dogs showed good serotagical response to vaccination as measured by p68 EUSA titers, the response did not correlate with clinical protection in the 60 ug/dose vaccinated dogs. The 60ug/dose vaccinated dogs demonstrated no significant difference in coughing when compared to unvaccinaled controls. Good efficacy of the 1 Sjig/dose vaccine was demonstrated by a greater than 50% reduction In coughing when compared to controls. It is postulated that increased levels of SOS in the 60 u.g/dose vaccine may resuft in the demonstrated difference in protection. Comparison of SAA values demonstrated a difference between vaccinates and controls. EXAMPLE 4 Safety and Efficacy of VANGUARD® Plus S/CV-L VANGUARD® Plus S/CV-L is a freeze-dried preparation of attenuated strains of CD virus, CAV-2, CPI virus, CPV, and inactivated whole cultures of L canicola and L Kterohaemorrhagiae. plus a liquid preparation of inactivated CCV with an adjuvant. All viruses were propagated on established cell ines. The CPV fraction was attenuated by low passage on the canine cell Una which gave it the immunogenic properties capable of overriding maternal antibody interference at the levels indicated in Table 38. The liquid component was used to rehydrate the freeze-dried component, which had been packaged with inert gas in place of vacuum. Laboratory evaluation demonstrated that VANGUARD® Plus 5/CV-L immunized dogs against CO, ICH, CAV-2 and CPI respiratory disease, enteritis caused by CCV and CPV, and leptospirosis caused by L canicola and L icterohaemorrhagiae, and that no immunologtc interference existed among the vaccine fractions. Extensive field safety trials showed it to be safe and essentially reaction-free in dogs as young as 6 weeks of age under normal usage conditions. It was also demonstrated that CAV-2 vaccine cross-protects against ICH caused by CAV-1. Studies demonstrated that CAV-2 not only protects against ICH, but against CAV-2 respiratory disease as well. Canine adenovirus type 2 challenge virus was not recovered from CAV-2-vaccinated dogs in tests conducted. The CPV fraction in VANGUARD® Plus 5/CV-L was subjected to comprehensive safety and efficacy testing. It was shown to be safe and essentially reaction-free in laboratory tests and in clinical trials under field conditions. Product safety was further demonstrated by a backpassage study which included oral administration of multiple doses of the vaccine strain to susceptible dogs, all of whom remained normal. Research demonstrated that 3 doses of the vaccine with increased CPV virus titer can overcome serum neutralization (SIM) thers associated with maternal antibody. Serum neutralization titers as tow as 1:4 were shown by others to interfere with active immunization using conventional modified live vaccines. A clinical trial was conducted with fifty 6-week-old puppies [25 vaccinates (SN titer range -256) and 25 nonvaccinated controls (SN titer range 4-1024)] (Table 37). The group of vaccinates received 3 doses, with vaccinations administered 3 weeks apart beginning at 6 weeks of age. After 1 vaccination, 13/25 puppies exhibited a 4-foW or greater increase in CPV SN titer (seroconversfon) (Table 38). Twelve of these 13 puppies had maternal SN titers £1:16 at the time of the first vaccination with the remaining puppy having an SN titer of 1 £4. Another 9 puppies with initial SN titers between 1:16 and 1:256 seroconverted after the second vaccination. Their maternal anttoody SN titers had declined to £1:64 at the time of the second vaccination. Similarly, the last 3 vaccinates, with initial SN filers of 1:128, seroconverted after the third vaccination, after their maternal antibody CPVtiter dropped *1:64. Therefore, in this study, when 3 doses of vaccine were given beginning at 6 weeks of age, all 25 vaccinates, even those with the highest maternal antibody levels, became actively immunized (GM = 1:1176; range of SN liters 128-4096). All 50 dogs were challenged 3 weeks after the third vaccination with a heterologous CPV challenge virus. Fourteen of 25 nonvaca'nated control dogs died or showed illness severe enough to warrant euthanasia, wMe aH 26 vaccinates remained essentially healthy. The high-tiler, low-passage vaccine virus in VANGUARD® Plus oVCV-L was therefore highly immunogenfc and capable of stimulating active immunity in the presence of maternal antibodies. The efficacy of the CCV fraction of VANGUARD® Plus 5/CV-L was demonstrated in an extensive vaccination challenge study. Sixteen 7- to 8-week-old puppies were vaccinated with VANGUARD® Plus 5/CV-L (vaccinates) and 17 with Vanguard® Plus 5/L (controls). All puppies received three 1-mL closes at 3-week intervals. Three weeks following the third vaccination, puppies were chalenged with a virulent strain of CCV (CV-6). Clinical observations, temperatures, weights, and blood parameters were monitored for 21 days following infection. CCV vaccinates demonstrated a reduction in the occurrence of diarrhea and amount of vfrutent CCV shed when compared to controls. At 21 days postehallenge, fluorescent antibody staining for virulent CCV of smafl intestinal sections demonstrated a significant reduction (P) in detectable CCV antigen between CCV vaccinates and controls (Table 39). . t ' (Table Removed)Conclusions In this study, an adjuvantsd combination vaccine containing CD virus, CAV-2, CPI virus, CPV and inactivated whole cultures of L canicola and L icterohaemorrhagtae and CCV, was shown to be both safe and efficacious as a vaccine when used in puppies. The combination vaccine was also shown to overcome serum neutraJzation (SN) tilers associated with maternal antibody. EXAMPLES CANINE BOBDETELLA NATIVE 068IMMUNOGENICfTY STUDY Animals The study included two litters of SPF beagtes and two Titters of random source dogs. Dogs were assigned randomly to vaccinated or non-vaccinated groups. The study included a total of 10 vaccinated and 11 non-vaccinated dogs. Preparation of Experimental Vaccine S. bronchispetica (strain 11 OH) was harvested froma 48 hour Bordet-Gengou blood agar spread plates by washing the plate surface with 5 to 10 ml heat extraction buffer. Alternatively, ceUs grown in both culture (Charlotte Parker Defined Medium) were harvested by centrifugation discarding the supernatant fraction. Harvested cells were suspended in 25 mM Tris-HCL, pH 8.8 and incubated at 60°C for 1 hour. Cell debris was separated from heat extract by centrifugation at 20,000 x g at 4°C fro 30 minutes. Sodium azkte (0.01%) was added to the heat extracted supernatant fraction which was then further clarified by microporous filtration. Monoclonal antfcody affinity resin was prepared by conjugation of monoclonal antibody (designated Bord 2-7) to CNBr-activated Sepharose 4B using standard procedures. Approximately 30.35 mg of monoclonal antibody was conjugated to 1 gram of affinity resfn. Clarified heat extracted supernatant fraction (above) and Bord 2-7 affinity resin was combined at an approximate ratio of 1 liter extract to 20 ml resin. Binding of the native p68 to the resin was facilitated by incubating the mixture at ambient temperature, with gentle shaking, overnight, followed by resin settling and aspiration of the supernatant fraction. The resin was then packed into a 2.6 cm diameter column and the column washed sequentially with PBS, pH 7.5 and 10 mM phosphate buffer, pH 8.0 at a flow rate of 5 ml/mia When absorbance at 280 nm reached a baseline level, bound material was eluted using 100 mM trlethylamina and fractions under the single large peak of 260 nm absorbance were collected and tested for the presence of p68 by ELISA. Fractions containing p68 were pooled and dialyzed against PBS to remove triethylamine. An experimental vaccine serial formulated was formulated to contain approximately 100 micrograms of purified p68 and 1% aluminum hydroxide gel. Formalin (0.01%) was used as a preservative in a final vaccine dose volume of 1 mL Challenge Inoculum Challenge material was prepared essentially as described in examples above. Study Procedure Twenty-one (21) serbnegative and culture negative pups were randomly assigned to one of two treatment groups. Eleven dogs were assigned to the non-vaccmtated, control group and ten dogs to the vaccinated group. Day 0 was designated as the day of first vaccination. One ml of vaccine was administered subcutaneously on Day 0 and repeated 21 days later. Blood was collected for serdoglcal p68 ELJSA prior to first and second vaccination. On Day 35, fourteen days after the second vaccination, an aerosol challenge of a. bmnchiseptica was administered to all dogs as described above. Animals were monitored for coughing for 14 days fallowing challenge as described in previous examples. Results Summary of clinical observations and serologic responses to p68 are presented in Table 40. Table 40: Summary of ClinicaJ Observations and Serologic Responses (Table Removed)Discussion In this study, 10 of 10 control dogs coughed on at least two consecutive days. A dog is considered clinically diseased if it coughs for two consecutive days. By this criteria, 100% of the non-vaccinated control dogs were diseased. In the vaccinated group, one dog coughed on day 4 post-challenge and one dog coughed on days 4 and 6 post-challenge. Two dogs coughed on day 14. None of the vaccinated dogs coughed for two consecutive days. Therefore, 100% of the dogs in the native p68 vaccinated group were judged to remain normal following challenge. Conclusions The trial demonstrates the abiity of a native p68 vaccine to protect against B. brvnchiseptica disease. EXAMPLES Efficacy of rmifthralent canine vaccines against Leptospira bratislava challenge The purpose of this study was to demonstrate the efficacy of vaccines containing fractions to Leptospira serovars Bratislava, canicola, grippotyphosa, icterohaemorrhagiae and pomona against L bratisfava challenge in dogs. (MATERIALS Vaccines: Vaccines used were the following: 1. A lyophilized vaccine comprising canine distemper, adenovirus type 2, parainfluenza, and parvovirus antigens (VANGUARD ® PLUS 5) was used. The vaccine contained release antigen levels. Product code 13D122 2. A carine coronavirus vaccine in a liquid diuent formulation (FIRSTDOSE® CV) was used. The vaccine contained release antigen levels. Product code 14P5.20 3. A lyophilized vaccine comprising canine distemper, adenovirus type 2, parainf luenza, parvovirus, bratislava. canicola, grippotyphosa, Icterohaemorrhagiae and pomona antigens was used. The vaccine contained approximately 600 nephtos of each Leptospira serovar; and release antigen levels of the modified ive virus fractions (canine adenovirus, distemper virus, parainfluenza virus, parvovirus). Product code 4637.2A Study Animals: Beagle puppies of approximately 5-6 weeks of age of either sex were used. They were seronegative ( Challenge Organism: Each animal was administered one dose of approximately 2 mL (10'1 dilution of infected hamster liver tissue) of Leptospira bratislava as an intraperitoneal injection. (Table Removed)PROCEDURES Vaccination Phase: On Study Days 0 and 21,40 dogs in 4 vaccinate groups (10 animals/group) were given an injection of the control or test vaccines as. outlned below: T1:1 ml of control vaccine containing canine distemper, adenovirus type 2, parainfluenza, parvovirus vaccine reconstituted with canine coronavirus diluent and given by subcutaneous (SQ) injection. (Product Code 1301.22 reconstituted with Product Code 14P5.20) T£ 1 mL of control vaccine containing canine distemper, adenovirus type 2, parainfluenza, parvovirus vaccine reconstituted with canine coronavirus diluent and given by intramuscular (IM) injection. (Product Code 13D1.22 reconstituted with Product Code 14P5.20) T3; 1 ml of Leptospira vaccine containing canine distemper, adenovirus type 2, parainfluenza, parvovirus, bratislava. canfcola, grrppotyphosa, icterohaemorrhagiae and pomona vaccine reconstituted with canine coronavirus diluent and given by SQ injection. (Product Code 46J7.2A,- a combination of Product Codes 4337.2A and 14P5.20) T4:1 ml of Leptospira vaccine containing canine distemper, adenovirus type 2, parainfluenza, parvovirus, bratislava, cantcoia. grippotyphosa. tcterohaernormagiae and pomona vaccine reconstituted with canine coronavirus d'duent and given by IM Ejection. (Product Code 46J7.2A; a combination of Product Codes 4637.2A and 14P5.20) Post-vaccination observations for untoward systemic reactions were made at approximately 1 hour and 5 hours following vaccination. Leptospira ChaBenge: On Study Days 49, the test animals were chaBenged via an approximate 2 ml dose of L bratislava by intraperttoneai injection. Bfood Sample Collection: Serum samples were collected from available animals on Study Days 0,21,35,48,50,52,55,58,6t, 64,67 and 70. Similarly, plasma samples were collected on Study Days 48,50, 52,55, SB, 61,64, 67 and 70. Bacterial Seroiogy: Serum samples obtained on Study Days 0,21,35,48, 58 and 70 were assayed via a mfcroagglutination test for circulating antibodies to L. bratisiava. Spirochetemia: Plasma samples obtained on Study Days 48, 50,52,55,58,61,64, 67 and 70 were examined by dark-field microscopy for spirochetes and cultured for Leptospira re-isolation. Complete Blood Count / Serum Chemistry Panel: Plasma samples obtained on Study Days 48,50,52,55,58,61,64,67 and 70 were assayed for, but not limited to. platelet counts and sedimentation rate. Serum samples, obtained at those same intervals were assayed for, but not limited to, amytase, alanine aminotransferase (ALT), aspartate aminotransaminase (AST) and creatinine. A CBC and sedimentation rate was not completed for 2 animals (Nos. QPH3, R VG3) on Study Day 55, for one animal (No. RBH3) on Study Day 58, for 3 animals (Nos. OLH3, OUH3. PTG3) on Study Day 61, nor for one animal (No. OWG3) on Study Day 70 because the plasma sample collected clotted prior to testing. On Study Day 67, a sedimentation rate was not completed for 2 animals (Nos. OUH3, SAG3) because the plasma sample quantity was insufficient for testing. Those outcomes had no impact on these study parameters. Rectal Body Temperatures: Rectal body temperatures were recorded on Study Days 47-70. Post-challenge (Study Day 50), an elevated body temperature of Z 39.2 °C was considered indicative of leptospirosis. Urine Cultures: Urine samples obtained on Study Days 48,55 and 70 were cultured for Leptospira, and submitted for urinalysis. On Study Day 48, a urinalysis was not completed for one animal (No. CBC3) as the quantity of urine available at collection was insufficient for testing. On Study Day 55, a urinalysis was not completed for 2 animals (No. OPH3. RXG3) as the quantity of urine available at collection was insufficient for testing. On Study Day 70, a urinalysis was not completed for 10 animals (Nos. OYG3, PIG3, PUG3, QHG3, QQH3. QSG3, RDG3, RUG3, RVG3, RYG3) as the quantities of urine available at collection were insufficient for testing. Those outcomes had no impact on this study parameter. Necropsy and Leptospira Isolation: Animals euthanized during or at conclusion of the post-challenge period were necropsied Body fluids and tissues (i.e., fiver, kidney and urine) were collected and submitted to BCL for Leptospira re-isolation. On Study Day 55. bacteria] re-Isolation was not completed for 2 animals (No. OPK3, RXG3) as the quantity of urine available at collection was insufficient for testing. On Study Day 70, bacterial re-isolation was not completed for 4 animals (Nos. OYG3, PUG3, QSG3, RUG3) as the quantities of urine available at collection were insufficient for testing. Those outcomes had no impact on this study parameter. Health Observations: Animals were monitored daily for general health status. DATA SUMMARY AND ANALYSIS Data were analyzed with a mixed model or categorical (SAS/STAT Software Changes and Enhancements through Release 6.12, SAS Institute, Gary, North Carolina) procedure. A general linear repeated measures mixed model (fixed effect model terms are treatment, study day, and treatment by study day) was used to analyze temperature, serum antibody tilers, blood platelet count, sedimentation rate, amyiase, alanine aminotransferase (ALT), aspartate aminotransaminase (AST) and creatinine. Contrasts of interest were made 1 . •$'• ;..•' after detecting a significant (P $ 0.05) treatment'or treatment by day of study interaction effect. Trters were log-transformed as appropriate for analysis, and when transformed, the least-squares means were back-transformed to geometric means for presentation. Observations not analyzed (i.e., necropsy results and post-vaccination observations) were not entered into the database for summary. Frequency distributions of animals with platelet counts Animals were classified as normal or ill on each day post-chaflenge during the study. The presence of any of the following signs resulted in a classification of ill: conjunctivitis, depression, inappetence, muscle tremors, nasal discharge, pyrexia (5 39.2 °C) or watery A general linear mixed model (fixed effect model term is treatment) was used to analyze the number of days post-challenge that an animal was classified as HI. The binomial variable died or euthanized was analyzed with Fisher's Exact test Spirochetemia, and bacteria re-isolation In the urine, kidney and liver were analyzed using Fisher's Exact test to compare treatment groups. In the absence of a significant difference between the routes of administration and route by treatment interaction (PS0.05 two-sided), contrasts were used to compare the average of treatments T1 and T2 to the average of treatments T3 and T4 (PS0.05 one-skied). If the analysis was a repeated measures analysis, then the comparison was made at each time point data was collected. Otherwise, contrasts were used to compare T1 to T3 and T2 to T4 {PS0.05 one-sided). These comparisons were made at each time point if the analysis was a repeated measures. The efficacy of the Leptospira vaccine against L bratisfava was demonstrated by a lower incidence (P £ 0.05, one sided) of illness in the vaccinated animals. The following variables supported the efficacy of the Leptospira vaccine: (1) The mean platelet count was significantly (P $ 0.05, ona sfcted) higher for the vaccinates; (2) The m«an sedimentation rate was significantly (P RESULTS Clinical Signs Post-Challenge: The mean number of days that the test animals displayed clinical signs indicative of leptospirosis (e.g., conjunctivitis, depression, diarrhea, hematuria, icterus, inappetence, moribund, muscle tremors, pyrexia, vomiting) is presented in Table 1. Post-challenge {Study Days 50-70), the mean number of days the T1 and T2 controls were 31 was 4.3 and 3.3, respectively. Conversely, the means for the T3 and T4 vaccinates were 0.7 and 0.5, and those results were significantly improved (P -Conversely, the T3-T4 vaccinates remained otherwise healthy during that same interval, and that comparison (T1-T2 vs T3-T4) was significantly improved (P Spirochetemia Post-Challenge: The frequency of spirochetemia is presented in Table 3. The presence of spirochetes in the blood (detected via bacterial culture) is a clinical outcome demonstrating leptospirosis. One day post-challenge (Study Day 50), spirochetemia was established in 90% of the T1 controls, 60% of the T2 controls, 60% of the T3 vaccinates and 70% of the T4 vaccinates. Bacterial re-isolation was expected from the blood at 24 hours after intraperttoneal injections regardless of the status of vaccination. Thereafter, spirochetemia was established for the T1 controls as fallows: 100% on Day 3 post-challenge (Study Day 52), 56% on Day 6 (Study Day 55) and 33% of Day 9 (Study Day 58). Similarly, spirochetemia was established for the T2 controls as follows: 60% on Day 3 post-challenge, 50% on Day 6 and 29% of Day 9. Conversely, it was not established in the T3-T4 vaccinates during that same post-challenge period nor during the remainder of the post-challenge period (i.e., Study Days 50-70). Overall, the percentage of animals that were positive for spirochetemia including one day post-challenge (Study Days 50-70) was 60% -100% for the T1-T2 controls and 60%-70% for the T3-T4 vaccinates. In contrast, the percentage of animals that were positive excluding one day post-challenge (Study Days 52-70) was 60% -100% for the T1-T2 controls and 0% for the T3-T4 vaccinates. Leptospira Re-Isolation from Body Fluids and Tissues Post-Challenge: Leptospira re-isolatfon from Wood, urine, kidney and liver samples is presented in Table 4. A summary oi Leptospira re-isolation results from blood is provided in the preceding section. Beginning at 3 days post-challenge (i.e., excluding day 1 post-challenge), spirochetemia was established in 60-100% of the T1-T2 controls, and was not established in the T3-T4 vaccinates during that same period. Notably, that comparison (T1-T2 vs T3-T4) was significantly improved (P Kidney samples were collected at necropsy. Leptospira was re-isolated from 50% of the kidney samples obtained from the T1 and T2 controls. H was not re-isolated from samples derived from the T3-T4 vaccinates. That comparison (T1-T2 vs T3-T4) was significantly improved (P Liver samples were collected at necropsy. Leptospira was re-isolated from 10% and 20% of the liver samples obtained from the T1 and T2 controls, respectively. It was not re-Isolated from samples derived from the T3-T4 vaccinates. That comparison (T1-T2 vs T3-T4) was significantly improved (P Urine samples were collected at 2 intervals post-challenge and at necropsy. Leptospira was re-isolated from 2 T1 controls at 6 days post-challenge (Study Day 55). Leptospira was not re-isolated from any sample for the T2 controls nor T3-T4 vaccinates. Platelet Counts Post-Challenge: Mean platelet counts are presented in Table 5. A decrease in trie number of blood platelets (thrombocytopenia) is a clinical result indicative of leptospirbsis. Mean concentrations forme T1-T2 controls ranged between 61 and 937. During the same interval, the mean counts for the T3-T4 vaccinates ranged between 400 and 566. Notably, the platelet counts fortoe T3-T4 vaccinates were signifkantV improved (P . The frequency of animals with at least one platelet count Sedimentation Rates Post-Challenge: Mean sedimentation rates are presented in Table 6. An . increase in sedimentation rate u a clinical result indicative of leptospirosis. Mean rates for the T1-T2 controls ranged between 2.4 and 16.0. During the same interval, the mean rates for the T3-T4 vaccinates ranged between 1.5 and 6.3. Notably, the rates for the T3-T4 vaccinates were significantly lower (P ALT Concentrations Post-Challenge: Mean alanine aminotransferase (ALT) concentrations are presented in Table 7. An increase in ALT is a cSnical result indicative of bacterial infection (i.e., as liver function deteriorates, ALT levels rise). Mean concentrations for the T1-T2 controls ranged between 22 and 79. During the same interval, the mean concentrations for the T3-T4 vaccinates ranged between 21 and 61. Notably, the concentrations for the T3-T4 vaccinates were significantly tower (P Creatinine Concentrations Post-Challenge: Mean creatinine concentrations are presented in Table 8. An increase in creatinine concentrations is a dinfcal result indicative of bacterial infection (i.e., as kidney function deteriorates due to leptospirosis, creatinine levels rise). Mean concentrations for the T1 controls (Le.. SQ administration) ranged between 0.30 and .99. During the same interval, the mean concentrations for the T3 vaccinates (i.e., SQ administration) ranged between 0.26 and 0.40. Notably, the concentrations for the T3 vaccinates were significantly lower (P Amylase, AST and Urinafysis Post-Challenge: There were no significant differences in the mean concentrations for the T3-T4 vaccinates when compared to the T1-T2 controls. However, in general the post-challenge concentrations were more dramatically increased for the T1-T2 controls. By and large, post-challenge changes in AST (aspartata aminotransamlnase) and urinalysis results were not observed for the T1- T4 test animals. The results for these 3 parameters are not otherwise tabulated herein. Serum Antibody Triers: Mean serum L bratislava antibody tilers are presented in Table 9. During the vaccination phase of the investigation (Study Days 0-48), the mean titers for the T1-T2 controls were approximately 2 (i.e., seronegative). Correspondingly, the means for the T3-T4 vaccinates ranged between 2 (pre-vaccination) and 1181 (post-vaccination). Notably, the mean tilers for the T3-T4 vaccinates were significantly higher (P During the challenge phase of the investigation (Study Days 58 & 70), the mean titers for the T1-T2 controls ranged between 2135 and 41160. Correspondingly, the mean titers for the T3 and T4 vaccinates ranged between 727 and 10891, and were significantly lower (P Systemic Reactions Post-Vaccination: No post-vaccinal systemic reactions were observed in the T1-T4 test animals when evaluated at approximately 1 and 5 hours after the primary and booster vaccinations. That result is not tabulated herein. CONCLUSION The efficacy of a mirftivalent Leptospira vaccine given by SQ or IM injection against L bratislava was demonstrated post-challenge by, inter alia: (1) a significantly lower incidence of Leptospira-associated illness, (2) a significantly lower incidence of spirochetemia, (3) significantly higher platelet counts, and (4) significantiy bwer mean sedimentation rates, when compared to controls. (Table Removed) WE CLAIM: 1. A vaccine for immunizing dogs against canine pathogens characterized in that said vaccine consists of: a) a Leptospira cell preparation of Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrhagiae, and Leptospira Pomona; wherein the amount of each Leptospira strain in the vaccine is in the range of about 100-3500 nephelometric units; b) an attenuated strain of canine distemper (CD) virus, c) an attenuated strain of canine adenovirus type 2 (CAV-2), d) an attenuated strain of canine parainfluenza (CPI) virus, e) an attenuated strain of canine parvovirus (CPV), and f) a carrier. - wherein the amount of said attenuated strain of CD virus, said attenuated strain of CAV-2, said attenuated strain of CPI virus, and said attenuated strain of CPV, in said vaccine, are each in the range of l02 to l09 TCID50. 2. The vaccine as claimed in claim 1, optionally consisting of at least about 100 relative units of an inactivated whole or partial cell preparation of a strain of canine coronavirus (CCV). 3. The vaccine as claimed in either claim 1 or claim 2, wherein said carrier comprises saponin and a surfactant. 4. The vaccine as claimed in claim 3, wherein said saponin is Quil A and said surfactant is cholesterol. 5. The combination vaccine as claimed in either claim 1 or claim 2, wherein the carrier comprises aluminum hydroxide.

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