Inactivated salmonella vaccine formulations

Abstract

The present disclosure provides formulations, kits, and vaccines directed to immunization of animals against Salmonella. Methods of usng the formulations, kits, and vaccines for protection of avians against one or more species of Salmonella are also provided. For instance, the formulations, kits, and vaccines can induce an immune response in an animal against a Salmonella infection in which the Salmonella infection is caused by more than one Salmonella group bacterium (e.g., a Group B bacterium, a Group C bacterium, and a Group D bacterium).

Description

[0001] 78045-429191

[0002] INACTIVATED SALMONELLA VACCINE FORMULATIONS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Serial No. 63 / 684,648, filed on August 19, 2024. the entire disclosure of which is incorporated herein by reference.

[0005] TECHNICAL FIELD

[0006] The disclosure relates to formulations, kits, and vaccines directed to immunization of animals against Salmonella. In one aspect, the present disclosure provides compositions and methods directed to protection of avians against one or more species of Salmonella.

[0007] BACKGROUND AND SUMMARY OF THE INVENTION

[0008] Salmonella is a genus of Gram-negative bacteria belonging to the family Enterobacteriaceae . Generally, Salmonella infections can be the result of a food borne illness and can potentially be caused by a Group B bacterium, a Group C bacterium, and / or a Group D bacterium. In particular, Salmonella lyphimurium is known to cause infection and Salmonella infantis is an emerging bacterium that poses an unmet medical need.

[0009] Salmonella infantis is capable of internal colonialization of avians. For instance, Salmonella infantis colonization of chicken intestines and internal organs (e.g., ceca, liver and spleen) are problematic. This bacterial colonization can result in contamination of food products from the avians, such as meat and eggs derived from broiler and egg laying chickens. Products contaminated with Salmonella are the leading cause of human food-bome gastrointestinal disease. However, there is no current product that controls transmission of Salmonella infantis in avians such as chickens. Thus, there exists a need for preventing bacterial colonization of Salmonella in avians in order to improve food safety for humans consuming products derived from avians.

[0010] Accordingly, the present disclosure provides formulations, kits, and vaccines directed to immunization of animals against Salmonella. The exemplary' embodiments of the present disclosure include several desirable features. For instance, the formulations, kits, and vaccines can induce an immune response in an animal against a Salmonella infection in which the Salmonella infection is caused by more than one Salmonella group bacterium (e.g., a Group 78045-429191

[0011] B bacterium, a Group C bacterium, and a Group D bacterium). In particular, the formulations, kits, and vaccines can provide induction of an immune response against a Salmonella typhimurium infection, a Salmonella infantis infection, or both.

[0012] Various aspects of the disclosure are described more fully below. However, different aspects of the disclosure may be implemented in many ways and should not be construed as limited to the aspects set forth herein. The following detailed description is, therefore, not to be taken in a limiting sense.

[0013] BRIEF DESCRIPTIONS OF THE DRAWING

[0014] The detailed description particularly refers to the accompanying figure in which: FIGURE 1 shows an exemplary process for making formulations according to the present disclosure.

[0015] DETAILED DESCRIPTION

[0016] Various embodiments are described herein as follows. In an illustrative aspect, a composition comprising i) an first antigen and ii) a second antigen is provided, wherein the first antigen is an inactivated Salmonella typhimurium antigen and wherein the second antigen is an inactivated Salmonella infantis antigen. As used herein, “inactivated” refers to a pathogen that is killed, or otherwise incapable of replication, infection and / or pathogenicity in a host, while retaining immunogenicity. Nonlimiting examples of inactivating treatments include heating, irradiation, freeze-thawing, genetic modification, and / or chemical treatments.

[0017] In an embodiment, the inactivated Salmonella infantis antigen is European- derived. As used herein, “European-derived” refers to an antigen, pathogen, or part of a pathogen that originates from a European country. It is known in the art that different geographic areas of the world can possess differences in virulence and / or immunogenicity.

[0018] In an embodiment, the composition induces an immune response in an animal against a Salmonella infection in the animal, wherein the Salmonella infection is caused by at least tw o of a Salmonella Group B bacterium, a Salmonella Group C bacterium, and a Salmonella Group D bacterium. In an embodiment, the composition induces an immune response in an animal against a Salmonella typhimurium infection in the animal and a Salmonella infantis infection in the animal. In an embodiment, the Salmonella infantis infection is European-derived. 78045-429191

[0019] In an illustrative aspect, a vaccine comprising i) an inactivated Salmonella typhimurium antigen, ii) an inactivated Salmonella infantis antigen, and an adjuvant is provided. In one illustrative aspect, the term “antigen” can refer, for example, to a whole Salmonella bacterial cell or a fragment or portion thereof.

[0020] In an embodiment, the inactivated Salmonella infantis antigen is European- derived. In an embodiment, the adjuvant is water-in-oil. In an embodiment, the vaccine further comprises a water-in-oil (W / O) component. In an embodiment, the adjuvant is water-in-oil-in- water. In an embodiment, the vaccine further comprising a water-in-oil-in-water (W / O / W) component. In an embodiment, the vaccine is configured as a multi-dose vial.

[0021] In an embodiment, the vaccine induces an immune response in an animal against a Salmonella infection in the animal, wherein the Salmonella infection is caused by at least two of a Salmonella Group B bacterium, a Salmonella Group C bacterium, and a Salmonella Group D bacterium. In an embodiment, the vaccine induces an immune response in an animal against a Salmonella typhimurium infection in the animal and a Salmonella infantis infection in the animal. In an embodiment, the Salmonella infantis infection is European-derived.

[0022] In an embodiment, the inactivated Salmonella typhimurium antigen is at a dose of between about 1 x 106and 1 x 1010In an embodiment, the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 106. In an embodiment, the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 107. In an embodiment, the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 108. In an embodiment, the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 109. In an embodiment, the inactivated Salmonella typhimurium antigen is at a dose of about 1 x IO10.

[0023] In an embodiment, the inactivated Salmonella typhimurium antigen is at a dose of between about 1 x 106and 1 x IO10. In an embodiment, the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 106. In an embodiment, the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 107. In an embodiment, the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 108. In an embodiment, the inactivated Salmonella typhimurium antigen is at a dose of about 1 x IO9In an embodiment, the inactivated Salmonella typhimurium antigen is at a dose of about 1 x IO10.

[0024] In an illustrative aspect, a kit comprising a vaccine as described herein and one or more vials is provided, wherein the vaccine is contained in the one or more vials.

[0025] In an embodiment, the kit further comprises a syringe. In an embodiment, the kit further comprises materials to substantially maintain the kit at a temperature between 2 °C and 78045-429191

[0026] 8 °C. In an embodiment, the temperature is maintained for a period of time between 1 day and 7 days.

[0027] In an embodiment, the kit is capable of maintaining stability at a temperature between 2 °C and 8 °C for a period of time of 6 months. In an embodiment, the kit is capable of maintaining stability at a temperature between 2 °C and 8 °C for a period of time of 12 months. In an embodiment, the kit is capable of maintaining stability at a temperature between 2 °C and 8 °C for a period of time of 18 months. In an embodiment, the kit is capable of maintaining stability at a temperature between 2 °C and 8 °C for a period of time of 24 months. As used herein, ‘'maintaining stability’’ can refer to stability of the first antigen, stability of the second antigen, or both. For instance, maintaining stability7can refer to the ability of the kit to provide induction of an immune response in an animal to the first antigen, the second antigen, or both.

[0028] In an embodiment, the inactivated Salmonella typhimurium antigen and the inactivated Salmonella infantis antigen are contained in the same vial. In an embodiment, the kit comprises at least two vials, and wherein a first vial contains the inactivated Salmonella typhimurium antigen and a second vial contains the inactivated Salmonella infantis antigen.

[0029] In an embodiment, the kit comprises a vial comprising a diluent. In an embodiment, the kit comprises a vial comprising a resuspension reagent. In an embodiment, the inactivated Salmonella typhimurium antigen is lyophilized, In an embodiment, the inactivated Salmonella infantis antigen is lyophilized.

[0030] In an embodiment, the kit further comprises a second veterinary composition. In an embodiment, the second veterinary composition is a second vaccine. In an embodiment, the second vaccine comprises a Salmonella enteritidis bacterium. In an embodiment, the second vaccine comprises a. Salmonella infantis bacterium.

[0031] In an illustrative aspect, a method of administering a vaccine to an animal is provided. The method comprises a step of administering to the animal an effective amount of a vaccine as described herein.

[0032] In an embodiment, the method provides at least 85% efficacy against a Salmonella infection. In an embodiment, the Salmonella infection is caused by a Salmonella Group B bacterium, a Salmonella Group C bacterium, a Salmonella Group D bacterium, or any combination thereof. In an embodiment, the method provides at least 90% efficacy. In an embodiment, the method provides at least 95% efficacy.

[0033] In an embodiment, the method provides efficacy in one or more organs of the animal. In an embodiment, the method provides efficacy in intestine of the animal. 78045-429191

[0034] In an embodiment, the animal is an avian. In an embodiment, the avian is selected from the group consisting of a chicken, a turkey, and a duck. In an embodiment, the avian is a chicken. In an embodiment, the chicken is a broiler chicken. In an embodiment, the chicken is a layer chicken.

[0035] In an embodiment, the administration is a subcutaneous injection. In an embodiment, the administration is an intramuscular injection.

[0036] In an embodiment, the administration is performed when the animal is about 12 weeks of age. In an embodiment, the administration is performed when the animal is about 16 weeks of age. In an embodiment, the administration is performed when the animal is about 20 weeks of age.

[0037] In an embodiment, the administration is performed on the animal two or more times. In an embodiment, the first administration is performed when the animal is about 12 weeks of age, and the second administration is performed when the animal is about 16 weeks of age.

[0038] In an embodiment, the administration is performed on the animal three or more times. In an embodiment, the first administration is performed when the animal is about 12 weeks of age, the second administration is performed when the animal is about 16 weeks of age, and the third administration is performed when the animal is about 20 weeks of age.

[0039] In an embodiment, the administration is performed during molting of the animal. In an embodiment, the administration is performed on the animal two or more times. In an embodiment, the administration is performed on the animal two or more times, wherein the second administration is performed about 4 weeks after the first administration.

[0040] In an embodiment, the effective amount of the vaccine is between about 0. 1 ml to about 1 ml. In an embodiment, the effective amount of the vaccine is about 0.10 ml. In an embodiment, the effective amount of the vaccine is about 0.25 ml. In an embodiment, the effective amount of the vaccine is about 0.50 ml. In an embodiment, the effective amount of the vaccine is about 0.75 ml. In an embodiment, the effective amount of the vaccine is about 1 ml.

[0041] In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella typhimurlum antigen at a dose of between about 1 x 106and 1 x 1010. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella typhimurlum antigen at a dose of about 1 x 106. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella typhimurlum antigen at a dose of about 1 x 107. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella 78045-429191 typhimurium antigen at a dose of about 1 x 108. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 109. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x IO10. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of between about 1 x 106and 1 x IO10. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 106. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 107. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 108. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 109. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x IO10.

[0042] In an embodiment, the method further comprises administration of a second veterinary composition. In an embodiment, the second veterinary composition is a second vaccine. In an embodiment, the second vaccine comprises a Salmonella Enteritidis bacterin.

[0043] In an illustrative aspect, a method of inducing an immune response in an animal is provided. The method comprises a step of administering to the animal an effective amount of a vaccine as described herein.

[0044] In an embodiment, the immune response is against one or more of a Salmonella Group B bacterium, a Salmonella Group C bacterium, a Salmonella Group D bacterium, or any combination thereof. In an embodiment, the immune response is against a Salmonella Group B bacterium. In an embodiment, the immune response is against a Salmonella Group C bacterium. In an embodiment, the immune response is against a Salmonella Group D bacterium.

[0045] In an embodiment, the immune response is against an inactivated Salmonella typhimurium antigen. In an embodiment, the immune response is against an inactivated Salmonella infantis antigen. In an embodiment, the inactivated Salmonella infantis antigen is European-derived. In an embodiment, the immune response comprises an immune response against an inactivated Salmonella typhimurium antigen and an immune response against an inactivated Salmonella infantis antigen. In an embodiment, the inactivated Salmonella infantis antigen is European-derived.

[0046] In an embodiment, the animal is an avian. In an embodiment, the avian is selected from the group consisting of a chicken, a turkey, and a duck. In an embodiment, the 78045-429191 avian is a chicken. In an embodiment, the chicken is a broiler chicken. In an embodiment, the chicken is a layer chicken.

[0047] In an embodiment, the administration is a subcutaneous inj ection. In an embodiment, the administration is an intramuscular injection.

[0048] In an embodiment, the administration is performed when the animal is about 12 weeks of age. In an embodiment, the administration is performed when the animal is about 16 weeks of age. In an embodiment, the administration is performed on the animal two or more times. In an embodiment, the first administration is performed when the animal is about 12 weeks of age and the second administration is performed when the animal is about 16 weeks of age.

[0049] In an embodiment, the administration is performed during molting of the animal. In an embodiment, the administration is performed on the animal two or more times. In an embodiment, the administration is performed on the animal two or more times, wherein the second administration is performed about 4 weeks after the first administration.

[0050] In an embodiment, the effective amount of the vaccine is between about 0. 1 ml to about 1 ml. In an embodiment, the effective amount of the vaccine is about 0.10 ml. In an embodiment, the effective amount of the vaccine is about 0.25 ml. In an embodiment, the effective amount of the vaccine is about 0.50 ml. In an embodiment, the effective amount of the vaccine is about 0.75 ml. In an embodiment, the effective amount of the vaccine is about 1 ml.

[0051] In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of between about 1 x 106and 1 x 1010. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 106. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 107. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 108. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about I x lO9In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 1010. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of between about 1 x 106and 1 x 1010. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 106. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x IO7 78045-429191

[0052] In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 108. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 109. In an embodiment, the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x IO10.

[0053] In an embodiment, the method further comprises administration of a second veterinary composition. In an embodiment, the second veterinary' composition is a second vaccine.

[0054] The following numbered embodiments are contemplated and are non-limiting:

[0055] 1. A composition comprising i) an first antigen and ii) a second antigen, wherein the first antigen is an inactivated Salmonella typhimurium antigen and wherein the second antigen is an inactivated Salmonella infantis antigen.

[0056] 2. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella infantis antigen is European-derived.

[0057] 3. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the composition induces an immune response in an animal against a Salmonella infection in the animal, and wherein the Salmonella infection is caused by at least two of a Salmonella Group B bacterium, a Salmonella Group C bacterium, and a Salmonella Group D bacterium.

[0058] 4. The composition of clause 1, any other suitable clause, or any combination of suitable clauses, wherein the composition induces an immune response in an animal against a Salmonella typhimurium infection in the animal and a Salmonella infantis infection in the animal.

[0059] 5. The composition of clause 4, any other suitable clause, or any combination of suitable clauses, wherein the Salmonella infantis infection is European-derived.

[0060] 6. A vaccine comprising i) an inactivated Salmonella typhimurium antigen, ii) an inactivated Salmonella infantis antigen, and iii) an adjuvant.

[0061] 7. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella infantis antigen is European-derived.

[0062] 8. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the adjuvant is water-in-oil.

[0063] 9. The vaccine of clause 6, further comprising a water-in-oil (W / O) component.

[0064] 10. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the adjuvant is water-in-oil-in-water. 78045-429191

[0065] 11. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, further comprising a water-in-oil-in-water (W / O / W) component.

[0066] 12. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the vaccine is configured as a multi-dose vial.

[0067] 13. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the vaccine induces an immune response in an animal against a Salmonella infection in the animal, and wherein the Salmonella infection is caused by at least two of a Salmonella Group B bacterium, a Salmonella Group C bacterium, and a Salmonella Group D bacterium.

[0068] 14. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the vaccine induces an immune response in an animal against a Salmonella typhimurium infection in the animal and a Salmonella infantis infection in the animal.

[0069] 15. The vaccine of clause 14, any other suitable clause, or any combination of suitable clauses, wherein the Salmonella infantis infection is European-derived.

[0070] 16. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella typhimurium antigen is at a dose of between about 1 x 106and 1 x IO10.

[0071] 17. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 106.

[0072] 18. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x IO7

[0073] 19. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 108.

[0074] 20. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 109.

[0075] 21. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x IO10.

[0076] 22. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella typhimurium antigen is at a dose of between about 1 x 106and 1 x IO10.

[0077] 23. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 106.

[0078] 24. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x IO7 78045-429191

[0079] 25. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 108.

[0080] 26. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 109.

[0081] 27. The vaccine of clause 6, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 1010

[0082] 28. A kit comprising the vaccine of any one of clauses 6 to 27 and one or more vials, any other suitable clause, or any combination of suitable clauses, wherein the vaccine is contained in the one or more vials.

[0083] 29. The kit of clause 28, any other suitable clause, or any combination of suitable clauses, wherein the kit further comprises a syringe.

[0084] 30. The kit of clause 28, any other suitable clause, or any combination of suitable clauses, wherein the kit further comprises materials to substantially maintain stability of the kit at a temperature between 2 °C and 8 °C.

[0085] 31. The kit of clause 30, any other suitable clause, or any combination of suitable clauses, wherein the temperature is maintained for a period of time between 1 day and 7 days.

[0086] 32. The kit of clause 28, any other suitable clause, or any combination of suitable clauses, wherein the kit is capable of maintaining stability at a temperature between 2 °C and 8 °C for a period of time of 6 months.

[0087] 33. The kit of clause 28, any other suitable clause, or any combination of suitable clauses, wherein the kit is capable of maintaining stability at a temperature between 2 °C and 8 °C for a period of time of 12 months.

[0088] 34. The kit of clause 28, any other suitable clause, or any combination of suitable clauses, wherein the kit is capable of maintaining stability at a temperature between 2 °C and 8 °C for a period of time of 18 months.

[0089] 35. The kit of clause 28, any other suitable clause, or any combination of suitable clauses, wherein the kit is capable of maintaining stability at a temperature between 2 °C and 8 °C for a period of time of 24 months.

[0090] 36. The kit of clause 28, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella typhimurium antigen and the inactivated Salmonella infantis antigen are contained in the same vial.

[0091] 37. The kit of clause 28, any other suitable clause, or any combination of suitable clauses, wherein the kit comprises at least two vials, and wherein a first vial contains the inactivated 78045-429191

[0092] Salmonella typhimurium antigen and a second vial contains the inactivated Salmonella infantis antigen.

[0093] 38. The kit of clause 28, any other suitable clause, or any combination of suitable clauses, wherein the kit comprises a vial comprising a diluent.

[0094] 39. The kit of clause 28, any other suitable clause, or any combination of suitable clauses, wherein the kit comprises a vial comprising a resuspension reagent.

[0095] 40. The kit of clause 28, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella typhimurium antigen is lyophilized,

[0096] 41. The kit of clause 28, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella infantis antigen is lyophilized.

[0097] 42. The kit of clause 28, any other suitable clause, or any combination of suitable clauses, wherein the kit further comprises a second veterinary composition.

[0098] 43. The kit of clause 42, any other suitable clause, or any combination of suitable clauses, wherein the second veterinary composition is a second vaccine.

[0099] 44. The kit of clause 43, any other suitable clause, or any combination of suitable clauses, wherein the second vaccine comprises a Salmonella enter itidis bacterium.

[0100] 45. The kit of clause 43, any other suitable clause, or any combination of suitable clauses, wherein the second vaccine comprises a Salmonella infantis bacterium.

[0101] 46. A method of administering a vaccine to an animal, said method comprising a step of administering to the animal an effective amount of the vaccine of any one of clauses 6 to 27.

[0102] 47. The method of clause 46. any other suitable clause, or any combination of suitable clauses, wherein the method provides at least 85% efficacy against a Salmonella infection.

[0103] 48. The method of clause 47, any other suitable clause, or any combination of suitable clauses, wherein the Salmonella infection is caused by a Salmonella Group B bacterium, a Salmonella Group C bacterium, a Salmonella Group D bacterium, or any combination thereof.

[0104] 49. The method of clause 47. any other suitable clause, or any combination of suitable clauses, wherein the method provides at least 90% efficacy.

[0105] 50. The method of clause 47, any other suitable clause, or any combination of suitable clauses, wherein the method provides at least 95% efficacy.

[0106] 51. The method of clause 47. any other suitable clause, or any combination of suitable clauses, wherein the method provides efficacy in one or more organs of the animal.

[0107] 52. The method of clause 47, any other suitable clause, or any combination of suitable clauses, wherein the method provides efficacy in intestine of the animal. 78045-429191

[0108] 53. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the animal is an avian.

[0109] 54. The method of clause 53. any other suitable clause, or any combination of suitable clauses, wherein the avian is selected from the group consisting of a chicken, a turkey, and a duck.

[0110] 55. The method of clause 53, any other suitable clause, or any combination of suitable clauses, wherein the avian is a chicken.

[0111] 56. The method of clause 55, any other suitable clause, or any combination of suitable clauses, wherein the chicken is a broiler chicken.

[0112] 57. The method of clause 55, any other suitable clause, or any combination of suitable clauses, wherein the chicken is a layer chicken.

[0113] 58. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the administration is a subcutaneous injection.

[0114] 59. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the administration is an intramuscular injection.

[0115] 60. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the administration is performed when the animal is about 12 weeks of age.

[0116] 61. The method of clause 46. any other suitable clause, or any combination of suitable clauses, wherein the administration is performed when the animal is about 16 weeks of age.

[0117] 62. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the administration is performed when the animal is about 20 weeks of age.

[0118] 63. The method of clause 46. any other suitable clause, or any combination of suitable clauses, wherein the administration is performed on the animal two or more times.

[0119] 64. The method of clause 63, any other suitable clause, or any combination of suitable clauses, wherein the first administration is performed when the animal is about 12 weeks of age, and the second administration is performed when the animal is about 16 weeks of age.

[0120] 65. The method of clause 46. any other suitable clause, or any combination of suitable clauses, wherein the administration is performed on the animal three or more times.

[0121] 66. The method of clause 64, any other suitable clause, or any combination of suitable clauses, wherein the first administration is performed when the animal is about 12 weeks of age, the second administration is performed when the animal is about 16 weeks of age, and the third administration is performed when the animal is about 20 weeks of age.

[0122] 67. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the administration is performed during molting of the animal. 78045-429191

[0123] 68. The method of clause 67, any other suitable clause, or any combination of suitable clauses, wherein the administration is performed on the animal two or more times.

[0124] 69. The method of clause 46. any other suitable clause, or any combination of suitable clauses, wherein the administration is performed on the animal two or more times, and wherein the second administration is performed about 4 weeks after the first administration.

[0125] 70. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine is between about 0. 1 ml to about 1 ml.

[0126] 71. The method of clause 46. any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine is about 0. 10 ml.

[0127] 72. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine is about 0.25 ml.

[0128] 73. The method of clause 46. any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine is about 0.50 ml.

[0129] 74. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine is about 0.75 ml.

[0130] 75. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine is about 1 ml.

[0131] 76. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of between about 1 x 106and 1 x 1010.

[0132] 77. The method of clause 46. any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 106.

[0133] 78. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 107.

[0134] 79. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 108.

[0135] 80. The method of clause 46. any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 109. 78045-429191

[0136] 81. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x IO10.

[0137] 82. The method of clause 46. any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of between about 1 x 106and 1 x IO10.

[0138] 83. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 106.

[0139] 84. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x IO7

[0140] 85. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 108.

[0141] 86. The method of clause 34, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 109.

[0142] 87. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x IO10

[0143] 88. The method of clause 46, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises administration of a second veterinary composition.

[0144] 89. The method of clause 88, any other suitable clause, or any combination of suitable clauses, wherein the second veterinary composition is a second vaccine.

[0145] 90. The method of clause 89. any other suitable clause, or any combination of suitable clauses, wherein the second vaccine comprises a Salmonella Enteritidis bacterin.

[0146] 91. A method of inducing an immune response in a non-human animal, said method comprising a step of administering to the animal an effective amount of a vaccine of any one of clauses 6 to 27.

[0147] 92. The method of clause 91 , any other suitable clause, or any combination of suitable clauses, wherein the immune response is against one or more of a Salmonella Group B bacterium, a Salmonella Group C bacterium, a Salmonella Group D bacterium, or any combination thereof. 78045-429191

[0148] 93. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the immune response is against a Salmonella Group B bacterium.

[0149] 94. The method of clause 91. any other suitable clause, or any combination of suitable clauses, wherein the immune response is against a Salmonella Group C bacterium.

[0150] 95. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the immune response is against a Salmonella Group D bacterium.

[0151] 96. The method of clause 91 , any other suitable clause, or any combination of suitable clauses, wherein the immune response is against an inactivated Salmonella typhimurium antigen.

[0152] 97. The method of clause 91 , any other suitable clause, or any combination of suitable clauses, wherein the immune response is against an inactivated Salmonella infantis antigen.

[0153] 98. The method of clause 97, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella infantis antigen is European-derived.

[0154] 99. The method of clause 91 , any other suitable clause, or any combination of suitable clauses, wherein the immune response comprises an immune response against an inactivated Salmonella typhimurium antigen and an immune response against an inactivated Salmonella infantis antigen.

[0155] 100. The method of clause 99, any other suitable clause, or any combination of suitable clauses, wherein the inactivated Salmonella infantis antigen is European-derived.

[0156] 101. The method of clause 91 , any other suitable clause, or any combination of suitable clauses. wherein the animal is an avian.

[0157] 102. The method of clause 101, any other suitable clause, or any combination of suitable clauses, wherein the avian is selected from the group consisting of a chicken, a turkey, and a duck.

[0158] 103. The method of clause 101, any other suitable clause, or any combination of suitable clauses, wherein the avian is a chicken.

[0159] 104. The method of clause 103, any other suitable clause, or any combination of suitable clauses, wherein the chicken is a broiler chicken.

[0160] 105. The method of clause 103, any other suitable clause, or any combination of suitable clauses, wherein the chicken is a layer chicken.

[0161] 106. The method of clause 91 , any other suitable clause, or any combination of suitable clauses, wherein the administration is a subcutaneous injection.

[0162] 107. The method of clause 91 , any other suitable clause, or any combination of suitable clauses, wherein the administration is an intramuscular injection.

[0163] 108. The method of clause 91 , any other suitable clause, or any combination of suitable clauses, wherein the administration is performed when the animal is about 12 weeks of age. 78045-429191

[0164] 109. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the administration is performed when the animal is about 16 weeks of age.

[0165] 110. The method of clause 91. any other suitable clause, or any combination of suitable clauses, wherein the administration is performed on the animal two or more times.

[0166] 111. The method of clause 110, any other suitable clause, or any combination of suitable clauses, wherein the first administration is performed when the animal is about 12 weeks of age and the second administration is performed when the animal is about 16 weeks of age.

[0167] 112. The method of clause 91. any other suitable clause, or any combination of suitable clauses, wherein the administration is performed during molting of the animal.

[0168] 113. The method of clause 112, any other suitable clause, or any combination of suitable clauses, wherein the administration is performed on the animal two or more times.

[0169] 114. The method of clause 112, any other suitable clause, or any combination of suitable clauses, wherein the administration is performed on the animal two or more times, any other suitable clause, or any combination of suitable clauses, wherein the second administration is performed about 4 weeks after the first administration.

[0170] 115. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine is between about 0. 1 ml to about 1 ml.

[0171] 116. The method of clause 91 , any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine is about 0. 10 ml.

[0172] 117. The method of clause 91 , any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine is about 0.25 ml.

[0173] 118. The method of clause 91 , any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine is about 0.50 ml.

[0174] 119. The method of clause 91 , any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine is about 0.75 ml.

[0175] 120. The method of clause 91. any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine is about 1 ml.

[0176] 121. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of between about 1 x 106and 1 x 1010.

[0177] 122. The method of clause 91 , any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 106. 78045-429191

[0178] 123. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 107.

[0179] 124. The method of clause 91. any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 108.

[0180] 125. The method of clause 91 , any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 109.

[0181] 126. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x IO10.

[0182] 127. The method of clause 91 , any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of between about 1 x 106and 1 x IO10.

[0183] 128. The method of clause 91 , any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 106.

[0184] 129. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x IO7

[0185] 130. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 108.

[0186] 131. The method of clause 91. any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 109.

[0187] 132. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x IO10

[0188] 133. The method of clause 91, any other suitable clause, or any combination of suitable clauses, wherein the method further comprises administration of a second veterinary composition.

[0189] 134. The method of clause 133, any other suitable clause, or any combination of suitable clauses, wherein the second veterinary’ composition is a second vaccine. 78045-429191

[0190] EXAMPLE 1

[0191] Exemplary Process for Making Formulations

[0192] Exemplary formulations according to the present disclosure can be made according to the following exemplary process. For instance, the forumulation can be a water- in-oil emulsion.

[0193] For the instant example, a 70% / 30% water-in-oil emulsion forumulation was prepared. Phases can be prepared using an aqueous phase (eg, contin ing antigen, Tween, formalin, and saline) that is separate from an oil phase (eg, containing Span 80 and mineral oil). The antigen content of the forumulation was obtained using a formalin-inactivated Salmonella infantis strain and a formalin-inactivated Salmonella typhimurium strain, which were added to the formulation at a targeted total cell count (TCC) per dose. For instance, exemplary formulations can be prepared using at 5.0X108TCC / 0.25 mL dose of each of the Salmonella infantis and Salmonella typhimurium antigens.

[0194] After the aqueous phase and oil phase have been prepared, the aqueous phase can be combined with the oil phase, followed by mixing to prepare the blend. Thereafter, the blend can be passed through a High Sheer Mixer (HSM) head and passaged until the resultant emulsion formulation is complete.

[0195] Figure 1 shows the exemplary process for the instant example.

[0196] EXAMPLE 2

[0197] Efficacy Evaluation - Salmonella Infantis Challenge

[0198] In the instant example, the efficacy of three exemplary formulations was evaluated. In particular, the efficacy evaluation was performed using a challenge against Salmonella Infantis (SI) in chickens. Three different dose levels of the exemplary formulations were utilized to immunize 12-week-old chickens against challenge at 20 weeks of age for the reduction of Salmonella infantis colonization in the intestines (duodenumjejunum and ileum) and organs (liver and spleen).

[0199] For the instant example, efficacy was assessed by evaluating the presence of Salmonella infantis colonization in the intestines and the organs. Necropsy and samples for Salmonella re-isolation were performed in 21 -week-old chickens. 78045-429191

[0200] Using a total of 84 chickens, 4 treatment groups with 20 chickens per treatment were formed. There were 21 chickens placed (20 per treatment and 1 extra) in each of Treatment Groups 1. 2, 3 and 4. Birds were randomized to four treatment groups (1. 2, 3, and 4) and one extra bird per group. The chickens were of similar size and were identified by unique identification numbers Each Treatment Group was housed in a pen and the extra chickens were removed and not utilized for the instant example.

[0201] The various Treatment Groups are presented in Table 1, along with the exemplary formulation used for each Group. Table 1.

[0202] Chickens in each of Treatment Groups 1, 2, 3, and 4 were administered the exemplary formulation or placebo via Subcutaneous (SC) injection under the skin on the dorsal side of the neck using a 20-gauge needle attached to a 1 mL syringe. Admnistrations w ere performed according to the following schedule as shown in Table 2.

[0203] Table 2. 78045-429191

[0204] A pathogenic nalidixic acid resistant SI challenge strain was prepared as follows. Briefly, the day prior to challenge, a frozen vial of SI challenge strain was plated for isolation on Tryptic Soy Agar (TSA) and incubated overnight at 37°±2°C. The next day, single colonies from the SI plates were inoculated at a rate of 2 colonies per 10 mL into Brain Heart Infusion (BHI) broth and incubated at 37°±2°C with agitation until the proper Optical Density (OD) of 1.276 (3.25 hours of incubation) was reached (determined by lab growth curve studies). Dilution of the challenge pool was performed (61.5 mL inoculum into 38.5 mL room temperature PBS) to achieve the optimal SI challenge titer of 8.0 x 108cfu / 1.0 mL dose. The challenge pool was assigned a lot number and placed on ice for transportation to the animal facility.

[0205] Titration of the prepared challenge pool was performed 5X with the appropriate 10-fold dilutions to determine the actual dose administered. Briefly, five replicate 10-fold serial dilution sets were prepared in PBS. Each of the appropriate dilutions were plated on TSA at 100 pL per plate and one plate per dilution. The inoculum was aseptically spread across agar plate surface and incubated at 37°C ± 2°C for approximately 24 hours. Dilutions with counts equal to or below 300 were counted and recorded to document the actual titer administered. Titaration results are presented in Table 3 below'.

[0206] Table 3.

[0207] At 20 w eeks of age, chickens in Treatment Groups 1, 2, 3, and 4 were challenged by the oral gavage route using a gavage needle attached to a 6 mL syringe to administer 1.0 mL / chicken of the challenge matenal. Chickens were held and observed for a brief time to ensure inoculum is not regurgitated.

[0208] Sample collection bags w ere pre-filled with Brilliant Green Tetrathionate Broth (BGTB) and labeled with chicken ID number, study number, pool identification (organ or intestine), and date collected. On necropsy day, the remaining chickens from Treatment Groups 78045-429191

[0209] 1, 2, 3 and 4 were euthanized, necropsied, and samples removed for Salmonella re-isolation testing. Chickens were humanely euthanized by CO2 gas asphyxiation and transported in plastic containment bags to the necropsy facility. The chickens were removed from plastic bags and were dipped in a soapy water solution to eliminate dander and fecal contamination. A new set of scissors and forceps were utilized per chicken for tissue sample collections. Final necropsy samples (organs and intestines) were aseptically collected and placed into bags of BGTB. A plastic ruler and a quarter were available during necropsy to use as guides for consistently collecting these samples.

[0210] For the organ pool collection, approximately a quarter sized section of the liver and approximately half of the spleen were collected and placed into a labeled sterile Whirl- Pak® bag containing 10.0 mL BGTB for each chicken necropsied. Each re-isolation bag was kept at room temperature until delivered to lab personnel for processing.

[0211] For the intestine pool collection, approximately a 30-40 mm section of the duodenum, (bottom of the duodenal loop below the pancreas), jejunum (region of the yolk sac diverticulum), and the ileum (anterior to the ileocecal junction) were collected from each chicken. The intestinal contents were expressed out of each section. The samples (3 expressed intestine sections) were pooled into a labeled sterile Whirl-Pak® bag containing 10.0 mL Brilliant Green Tetrathionate Broth (BGTB). Each re-isolation bag was kept at room temperature until delivered to lab personnel for processing.

[0212] The necropsy results obtained according to the instant example are presented in Table 4 below.

[0213] Table 4. Necropsy Salmonella inf antis Re-isolation Results

[0214] *Statistically significantly different from the Placebo Treatment Group 4. 78045-429191

[0215] The results demonstrate that a water-in-oil inactivated Salmonella typhimurium- Salmonella infantis formulation, when injected subcutaneously at 0.25 mL / dose at all three inputs tested (1.0 x 108tcc / dose. 5.0 x 108tcc / dose and 1.0 x 109tcc / dose) at 12 and 16 weeks of age is efficacious against a subsequent Salmonella infantis challenge.

[0216] EXAMPLE 3

[0217] Efficacy Evaluation - Salmonella Typhimurium Challenge

[0218] In the instant example, the efficacy of four exemplary formulations was evaluated. In particular, the efficacy evaluation was performed using a challenge against Salmonella typhimurium (ST) in chickens. Three different dose levels of the exemplary7formulations were utilized to immunize 12-week-old chickens against challenge at 20 weeks of age for the reduction of Salmonella typhimurium colonization in the intestines (duodenum, jejunum and ileum) and organs (liver and spleen).

[0219] For the instant example, efficacy was assessed by evaluating the presence of Salmonella typhimurium colonization in the intestines and the organs. Necropsy and samples for Salmonella re-isolation were performed in 21 -week-old chickens.

[0220] Using a total of 104 chickens, 5 treatment groups with 20 chickens per treatment were formed. There were 21 chickens placed (20 per treatment and 1 extra) in each of Treatment Groups 1, 2, 3 and 4 and 20 chickens were placed for Treatment Group 5. Birds were randomized to five treatment groups (1, 2, 3, 4, and 5). The chickens were of similar size and were identified by unique identification numbers Each Treatment Group was housed in a pen and the extra chickens were removed and not utilized for the instant example.

[0221] The various Treatment Groups are presented in Table 5, along with the exemplary formulation used for each Group.

[0222] Table 5. 78045-429191

[0223] Chickens in each of Treatment Groups 1, 2, 3, 4, and 5 were administered the exemplary formulation or placebo via Subcutaneous (SC) injection under the skin on the dorsal side of the neck using a 20-gauge needle attached to a 1 mL syringe. Admnistrations were performed according to the following schedule as shown in Table 6.

[0224] Table 6.

[0225] A pathogenic nalidixic acid resistant ST challenge strain was prepared as follows. Briefly, the day prior to challenge, a frozen vial of ST challenge strain was plated for isolation on Tryptic Soy Agar (TSA) and incubated overnight at 37°±2°C. The next day, single colonies from the ST plates were inoculated at a rate of 2 colonies per 10 mL into Brain Heart Infusion (BHI) broth and incubated at 37°±2°C with agitation until the proper Optical Density (OD) of 1.067 was reached (determined by lab grow th curve studies). Dilution of the challenge pool was performed (3.0 mL of the stock into 27.0 mL room temperature PBS then 15 mL of that dilution was put into 135.0 mL room temperature PBS) to achieve the optimal ST challenge 78045-429191 titer of 5.0 x 106cfu / 1.0 mL dose. Challenge pool was assigned a lot number and placed on ice for transportation to the animal facility'.

[0226] Titration of the prepared challenge pool was performed 5X with the appropriate 10-fold dilutions to determine the actual dose administered. Briefly, five replicate 10-fold serial dilution sets were prepared in PBS. Each of the appropriate dilutions were plated on TSA at 100 pL per plate and one plate per dilution. The inoculum was aseptically spread across agar plate surface and incubated at 37°C ± 2°C for approximately 24 hours. Dilutions with counts equal to or below 300 were counted and recorded to document the actual titer administered. Titaration results are presented in Table 7 below.

[0227] Table 7.

[0228] At 20 weeks of age, chickens in Treatment Groups 1, 2, 3, 4, and 5 were challenged by the oral gavage route using a gavage needle attached to a 6 mL syringe to administer 1.0 mL / chicken of the challenge material. Chickens were held and observed for a brief time to ensure inoculum is not regurgitated.

[0229] Sample collection bags were pre-filled with Brilliant Green Tetrathionate Broth (BGTB) and labeled with chicken identification number, study number, pool identification (organ or intestine), and date collected. On necropsy day, the remaining chickens from Treatment Groups 1. 2, 3, 4, and 5 were euthanized, necropsied, and samples removed for Salmonella re-isolation testing. Chickens were humanely euthanized by CO2 gas asphyxiation and transported in plastic containment bags to the necropsy facility. The chickens were removed from plastic bags and were dipped in a soapy water solution to eliminate dander and fecal contamination. A new set of scissors and forceps were utilized per chicken for tissue sample collections. Final necropsy samples (organs and intestines) were aseptically collected and placed into bags of BGTB. A plastic ruler and a quarter were available during necropsy to use as guides for consistently collecting these samples.

[0230] For the organ pool collection, approximately a quarter sized section of the liver and approximately half of the spleen were collected and placed into a labeled sterile Whirl- Pak® bag containing 10.0 mL BGTB for each chicken necropsied. Each re-isolation bag was kept at room temperature until delivered to lab personnel for processing. 78045-429191

[0231] For the intestine pool collection, approximately a 30-40 mm section of the duodenum, (bottom of the duodenal loop below the pancreas), jejunum (region of the yolk sac diverticulum), and the ileum (anterior to the ileocecal junction) were collected from each chicken. The intestinal contents were expressed out of each section. The samples (3 expressed intestine sections) were pooled into a labeled sterile Whirl-Pak® bag containing 10.0 mL Brilliant Green Tetrathionate Broth (BGTB). Each re-isolation bag was kept at room temperature until delivered to lab personnel for processing.

[0232] The necropsy results obtained according to the instant example are presented in Table 8 below.

[0233] Table 8. Necropsy Salmonella typhimurium Re-isolation Results

[0234] *Statistically significantly different from the Placebo Treatment Group 4.

[0235] The results demonstrate that a water-in-oil inactivated Salmonella typhimurium- Salmonella infantis formulation, when injected subcutaneously at 0.25 mL / dose at all three inputs tested (1.0 x 108tcc / dose. 5.0 x 108tcc / dose and 1.0 x 109tcc / dose) at 12 and 16 weeks of age is efficacious against a subsequent Salmonella typhimurium challenge.

Claims

78045-429191WHAT IS CLAIMED IS:

1. A method of administering a vaccine to an avian, said method comprising a step of administering to the avian an effective amount of a vaccine comprising i) an inactivated Salmonella typhimurium antigen, ii) an inactivated Salmonella infantis antigen, and iii) an adjuvant.

2. The method of claim 1, wherein the method provides at least 85% efficacy against a Salmonella infection.

3. The method of claim 2. wherein the Salmonella infection is caused by a Salmonella Group B bacterium, a Salmonella Group C bacterium, a Salmonella Group D bacterium, or any combination thereof.

4. The method of claim 2, wherein the method provides at least 90% efficacy.

5. The method of claim 2. wherein the method provides at least 95% efficacy.

6. The method of claim 2, wherein the method provides efficacy in one or more organs of the avian.

7. The method of claim 2. wherein the method provides efficacy in intestine of the avian.

8. The method of claim 1 , wherein the avian is selected from the group consisting of a chicken, a turkey, and a duck.

9. The method of claim 1, wherein the avian is a chicken.

10. The method of claim 9. wherein the chicken is a broiler chicken.

11. The method of claim 9, wherein the chicken is a layer chicken.

12. The method of claim 1, wherein the administration is a subcutaneous injection.

13. The method of claim 1. wherein the administration is an intramuscular injection.

14. The method of claim 1, wherein the administration is performed when the avian is about 12 weeks of age.

15. The method of claim 1. wherein the administration is performed when the avian is about 16 weeks of age.

16. The method of claim 1, wherein the administration is performed when the avian is about 20 weeks of age.78045-42919117. The method of claim 1, wherein the administration is performed on the avian two or more times.

18. The method of claim 17, wherein the first administration is performed when the avian is about 12 weeks of age, and the second administration is performed when the avian is about 16 weeks of age.

19. The method of claim 1, wherein the administration is performed on the avian three or more times.

20. The method of claim 19, wherein the first administration is performed when the avian is about 12 weeks of age, the second administration is performed when the avian is about 16 weeks of age, and the third administration is performed when the avian is about 20 weeks of age.

21. The method of claim 1. wherein the administration is performed during molting of the avian.

22. The method of claim 1, wherein the administration is performed on the avian two or more times, wherein the second administration is performed about 4 w eeks after the first administration.

23. The method of claim 1. wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of between about 1 x 106and 1 x 1010.

24. The method of claim 1, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 106.

25. The method of claim 1, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 10726. The method of claim 1, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 108.

27. The method of claim 1. wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x IO928. The method of claim 1, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 1010.

29. The method of claim 1. wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of between about 1 x 106and 1 x 1010.

30. The method of claim 1, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 106.78045-42919131. The method of claim 1, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x IO732. The method of claim 1. wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 108.

33. The method of claim 1, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 109.

34. The method of claim 1. wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x IO10.

35. A method of inducing an immune response in an avian, said method comprising a step of administering to the avian an effective amount of a vaccine comprising i) an inactivated Salmonella typhimurium antigen, ii) an inactivated Salmonella infantis antigen, and iii) an adjuvant.

36. The method of claim 35, wherein the immune response is against one or more of a Salmonella Group B bacterium, a Salmonella Group C bacterium, a Salmonella Group D bacterium, or any combination thereof.

37. The method of claim 35, wherein the immune response is against a Salmonella Group B bacterium.

38. The method of claim 35, wherein the immune response is against a Salmonella Group C bacterium.

39. The method of claim 35, wherein the immune response is against a Salmonella Group D bacterium.

40. The method of claim 35, wherein the immune response is against an inactivated Salmonella typhimurium antigen.

41. The method of claim 35, wherein the immune response is against an inactivated Salmonella infantis antigen.

42. The method of claim 41, wherein the inactivated Salmonella infantis antigen is European-derived.

43. The method of claim 35, wherein the immune response comprises an immune response against an inactivated Salmonella typhimurium antigen and an immune response against an inactivated Salmonella infantis antigen.

44. The method of claim 43, wherein the inactivated Salmonella infantis antigen is European-derived.

45. The method of claim 35, wherein the avian is selected from the group consisting of a chicken, a turkey, and a duck.78045-42919146. The method of claim 35, wherein the avian is a chicken.

47. The method of claim 46, wherein the chicken is a broiler chicken.

48. The method of claim 46, wherein the chicken is a layer chicken.

49. The method of claim 35, wherein the administration is performed when the avian is about 12 weeks of age.

50. The method of 35, wherein the administration is performed when the avian is about 16 weeks of age.

51. The method of claim 35, wherein the administration is performed on the avian two or more times.

52. The method of claim 51, wherein the first administration is performed when the avian is about 12 weeks of age and the second administration is performed when the avian is about 16 weeks of age.

53. The method of claim 51 , wherein the administration is performed on the avian two or more times, wherein the second administration is performed about 4 weeks after the first administration.

54. The method of claim 35, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of between about 1 x 106and 1 x I O10.

55. The method of claim 35, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 106.

56. The method of claim 35, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x IO757. The method of claim 35, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 108.

58. The method of claim 35, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x 109.

59. The method of claim 35, wherein the effective amount of the vaccine comprises an inactivated Salmonella typhimurium antigen at a dose of about 1 x IO10.

60. The method of claim 35, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of between about 1 x 106and 1 x 1010.

61. The method of claim 35, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 106.78045-42919162. The method of claim 35, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x IO763. The method of claim 35, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 108.

64. The method of claim 35, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x 109.

65. The method of claim 35, wherein the effective amount of the vaccine comprises an inactivated Salmonella infantis antigen at a dose of about 1 x IO10.

66. A composition comprising i) an first antigen and ii) a second antigen, wherein the first antigen is an inactivated Salmonella typhimurium antigen and wherein the second antigen is an inactivated Salmonella infantis antigen.

67. The composition of claim 66, wherein the inactivated Salmonella infantis antigen is European-derived.

68. The composition of claim 66, wherein the composition induces an immune response in an avian against a Salmonella infection in the avian, wherein the Salmonella infection is caused by at least two of a Salmonella Group B bacterium, a Salmonella Group C bacterium, and a Salmonella Group D bacterium.

69. The composition of claim 66, wherein the composition induces an immune response in an avian against a Salmonella typhimurium infection in the avian and a Salmonella infantis infection in the avian.

70. The composition of claim 69, wherein the Salmonella infantis infection is European-derived.

71. A vaccine comprising i) an inactivated Salmonella typhimurium antigen, ii) an inactivated Salmonella infantis antigen, and iii) an adjuvant.

72. The vaccine of claim 71, wherein the inactivated Salmonella infantis antigen is European-derived.

73. The vaccine of claim 71, wherein the inactivated Salmonella typhimurium antigen is at a dose of between about 1 x 106and 1 x 1010.

74. The vaccine of claim 71, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 106.

75. The vaccine of claim 71, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 107.

76. The vaccine of claim 71, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 108.78045-42919177. The vaccine of claim 71, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x IO978. The vaccine of claim 71, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x IO10.

79. The vaccine of claim 71, wherein the inactivated Salmonella typhimurium antigen is at a dose of between about 1 x 106and 1 x IO10.

80. The vaccine of claim 71, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 106.

81. The vaccine of claim 71, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 107.

82. The vaccine of claim 71, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 108.

83. The vaccine of claim 71, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x 109.

84. The vaccine of claim 71, wherein the inactivated Salmonella typhimurium antigen is at a dose of about 1 x IO10.

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