Electron beam (EBEAM)-killed multi-bacterial vaccines

Abstract

The present disclosure pertains to an avian vaccine that includes a plurality of electron beam (eBeam) -treated bacteria. The present disclosure also pertains to methods of immunizing an avian subject by administering an avian vaccine of the present disclosure to the avian subject. The present disclosure also pertains to methods of making an avian vaccine of the present disclosure by exposing a plurality of bacteria to eBeam treatment.

Description

TITLEELECTRON BEAM (EBEAM)-KILLED MULTI-BACTERIAL VACCINESSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under 58-6022-2-002 awarded by the United States Department of Agriculture. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 661,335, filed on June 18, 2024. The entirety of the aforementioned application is incorporated herein by reference.BACKGROUND

[0003] A need exists for improved compositions and methods for treating and preventing avian infections. Numerous embodiments of the present disclosure aim to address the aforementioned need.SUMMARY

[0004] In some embodiments, the present disclosure pertains to an avian vaccine. In some embodiments, the avian vaccine includes a plurality of electron beam (eBeam)-treated bacteria. Additional embodiments of the present disclosure pertain to methods of immunizing an avian subject by administering an avian vaccine of the present disclosure to the avian subject. Further embodiments of the present disclosure pertain to methods of making an avian vaccine of the present disclosure by exposing a plurality of bacteria to eBeam treatment.

[0005] In some embodiments, the eBeam-treated bacteria include at least two different strains of Staphylococcus species. In some embodiments, the Staphylococcus species include, without limitation Staphylococcus aureus, Staphylococcus agnetis, Staphylococcus cohnii, Staphylococcus lenlus, or combinations thereof. In some embodiments, the Staphylococcus species include Staphylococcus aureus and Staphylococcus agnetis. In some embodiments, the Staphylococcus species include Staphylococcus aureus, Staphylococcus agnetis, Staphylococcus cohnii, and Staphylococcus lentus.

[0006] The immunization methods of the present disclosure may have various applications. For instance, in some embodiments, the methods of the present disclosure may be utilized to treat or prevent a disease in an avian subject. In some embodiments, the disease includes, without limitation, a bacterial infection, a staphylococcus infection, bacterial chondronecrosis with osteomyelitis (BCO), or combinations thereof. In some embodiments, the disease to be treated or prevented includes BCO.DRAWINGS

[0007] FIG. 1 provides a timeline of a vaccination study set forth in Example 1.1.

[0008] FIG. 2 provides a variation of percentage cumulative lameness in treatment groups from d30 to d56 for the vaccination study in Example 1.1.

[0009] FIG. 3 provides a percentage lameness reduction of treatment groups at d56.

[0010] FIG. 4 provides the percentage of femoral and tibial bacterial chondronecrosis with osteomyelitis (BCO) lesions over treatment groups at d56.

[0011] FIG. 5 provides a timeline of a vaccination study set forth in Example 1.2.

[0012] FIGS. 6A-6C provide fluorescence microscopy images (lOOx magnification) following Baclight viability staining of fresh, untreated cells of 5. aureus (FIG. 6A), eBeam-killed 5. aureus cells at 8 kGy dose (FIG. 6B), and Formalin killed cells of .S', aureus visualized by overlaying 530 nm (green) and 630 nm (red) emission wavelength channels (FIG. 6C). Undamaged cells (with intact membranes) appear green while dead cells (with compromised cell membranes) appear red / orange. Results show almost intact membrane integrity in untreated cells in 8kGy eBeam-treated cells, while that of formalin-treated cells is highly compromised.

[0013] FIG. 7 shows the variation of percentage cumulative lameness in treatment groups from d30 to d56.

[0014] FIG. 8 shows the percentage lameness reduction of treatment groups at d56. The results show a lameness reduction of 50.94% for the eBeam treatment group.

[0015] FIG. 9 provides the percentages of femoral necroses over treatment groups at d56.DETAILED DESCRIPTION

[0016] It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that include more than one unit unless specifically stated otherwise.

[0017] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0018] Avian infections present an agricultural and economic threat. For instance, broiler chicken lameness caused by bacterial chondronecrosis with osteomyelitis (BCO) was first reported in 1972 and is presently amongst the top-most economic and animal welfare issues faced by the poultry industry. BCO lameness results in hundreds of millions of dollars in lost revenue annually in the United States and worldwide due to bird condemnation at the marketing age. BCO is caused by multiple opportunistic bacterial pathogens in the respiratory and gastrointestinal (GI) tracts.

[0019] Moreover, inhaled bacterial pathogens from the aerosol of chicken houses may leak from the respiratory system to the blood and eventually colonize the growth plates of long bones, leading to lameness. Similarly, bacterial species ingested with the diet can leak from the GI tract to the bloodstream via compromised intestinal tight junctions and colonize the growth plates of long bones.

[0020] Necrosis of cartilage and bone tissue by such colonized bacteria (typically in the femoral and tibial heads) ultimately causes the birds to be lame. Subclinical BCO lameness infections are widespread and can adversely impact animal well-being and meat quality, which is, in turn, detrimental to the sustainability and financial profitability of the broiler industry.

[0021] For instance, the regular lameness rate in broiler chickens varies from 3% up to 15%, resulting in mortality rates ranging from 5% to 10%. In the early 2010s, 12.5 billion broiler chickens were estimated to experience leg-related disorders worldwide. Considering the broiler production value of $50.4 billion of the United States in 2022, the current estimate of the regular economic loss in chicken meat production due to lameness in the US ranges from $900 million to $1.8 billion.

[0022] As such, a need exists for improved compositions and methods for treating and preventing avian infections. For instance, the aforementioned impact on the poultry industry, added to the absence of efficacious mitigatory measures, necessitates the need for the development of a successful commercial vaccine for the control of BCO lameness worldwide. Numerous embodiments of the present disclosure aim to address the aforementioned need.

[0023] In some embodiments, the present disclosure pertains to an avian vaccine. In some embodiments, the avian vaccine includes a plurality of eBeam-treated bacteria. Additional embodiments of the present disclosure pertain to methods of immunizing an avian subject by administering an avian vaccine of the present disclosure to the avian subject. Further embodiments of the present disclosure pertain to methods of making an avian vaccine of the present disclosure by exposing a plurality of bacteria to eBeam treatment. As set forth in more detail herein, the avian vaccines and methods of the present disclosure can have numerous embodiments.

[0024] eBeam Treatment

[0025] The avian vaccines of the present disclosure can include various eBeam-treated bacteria. Additionally, the avian vaccine formation methods of the present disclosure may expose bacteria to various types of eBeam treatments.

[0026] In some embodiments, bacteria are eBeam treated under conditions effective to kill or attenuate the bacteria while retaining the ability of the bacteria to elicit a protective immune response in an avian subject against the bacteria. In some embodiments, an effective eBeam treatment for killing the cells may include killing about 99.99% or more of the viable cells, without destroying the cell surface properties or lysing the cells but still retaining the ability of the cells to elicit an antibody response in the avian subject.

[0027] The eBeam-treated bacteria of the present disclosure may be in various forms. For instance, in some embodiments, the eBeam-treated bacteria are in an attenuated form. In some embodiments, the eBeam-treated bacteria are incapable of effective multiplication. In some embodiments, the eBeam-treated bacteria include intact cell wall structures. In some embodiments, the eBeam-treated bacteria include immunogenic cell walls.

[0028] Bacteria may be eBeam treated at various doses. For instance, in some embodiments, the bacteria are eBeam treated at a dose between 2 kGy and 15 kGy. In some embodiments, the bacteria are eBeam treated at a dose between 2 kGy and 10 kGy. In some embodiments, the bacteria are eBeam treated at a dose of 10 kGy. In some embodiments, the bacteria are eBeam treated at a dose of 8 kGy.

[0029] Bacteria may be eBeam treated by various devices. For instance, in some embodiments, the devices include conventional high-energy linear accelerators that are suitable for generating electron beams. In some embodiments, the high-energy linear accelerators are suitable for generating electron beams at energies above approximately 10.0 MeV. In some embodiments, suitable electron beam accelerators include, without limitation, electrostatic direct-current (DC), electrodynamic DC, linear accelerators (LINACS), magnetic-induction LINACs, continuous- wave (CW) accelerators, or combinations thereof.

[0030] Bacteria may be eBeam treated under various conditions. For instance, in some embodiments, bacteria may first be suspended in a liquid, such as a culture medium, a diluent and / or a physiologically buffered saline. In some embodiments, the suspension may be prepared or adjusted to provide an approximate concentration between lxl06and IxlO7colony forming units (CFU) / ml.

[0031] eBeam-treated bacteria

[0032] The avian vaccines of the present disclosure may include various types of eBeam-treated bacteria. Additionally, the avian vaccine formation methods of the present disclosure may be utilized to form various types of eBeam-treated bacteria.

[0033] In some embodiments, the eBeam-treated bacteria include a plurality of different strains and / or species of bacteria. In some embodiments, the eBeam-treated bacteria include a plurality of different strains of bacteria. In some embodiments, the eBeam-treated bacteria include a plurality of different species of bacteria.

[0034] In some embodiments, the eBeam-treated bacteria include, without limitation, Staphylococcus species, Enterococcus species, Escherichia species, or combinations thereof. In some embodiments, the eBeam-treated bacteria include one or more Staphylococcus species. In some embodiments, the eBeam-treated bacteria include at least two different strains of Staphylococcus species. In some embodiments, the Staphylococcus species include, without limitation Staphylococcus aureus, Staphylococcus agnetis, Staphylococcus cohnii, Staphylococcus lentus, or combinations thereof. In some embodiments, the Staphylococcus species include Staphylococcus aureus and Staphylococcus agnetis. In some embodiments, the Staphylococcus species include Staphylococcus aureus, Staphylococcus agnetis, Staphylococcus cohnii, and Staphylococcus lentus.

[0035] In some embodiments, the eBeam-treated bacteria include one or more Enterococcus species. In some embodiments, one or more Enterococcus species include Enterococcus cecorum.

[0036] In some embodiments, the eBeam-treated bacteria include one or more Escherichia species. In some embodiments, one or more Escherichia species include Escherichia coli.

[0037] In some embodiments, the avian vaccine formation methods of the present disclosure also include a step of growing the bacteria prior to eBeam treatment.

[0038] Vaccine compositions

[0039] In addition to eBeam-treated bacteria, the avian vaccines of the present disclosure may have various compositions. For instance, in some embodiments, the avian vaccines of the present disclosure may also include a pharmaceutically acceptable carrier. In some embodiments, the avian vaccines of the present disclosure may further include an adjuvant. In some embodiments, the adjuvant includes, without limitation, Freund's incomplete adjuvant, Freund's complete adjuvant, alum, microparticles, nanoparticles, beads, oil, or combinations thereof.

[0040] The avian vaccines of the present disclosure may have various uses. For instance, in some embodiments, the avian vaccines of the present disclosure may be suitable for use in immunizing an avian subject. In some embodiments, the avian vaccines of the present disclosure may be suitable for use in immunizing an avian subject in accordance with the immunization methods set forth herein.

[0041] Methods of immunizing an avian subject

[0042] Additional embodiments of the present disclosure pertain to methods of immunizing an avian subject. In some embodiments, the immunization methods include administering a vaccine of the present disclosure to the avian subject. In some embodiments, immunity may be considered as having been induced in a population of treated avian subjects when the level of protection for the population is evidenced by one or more of the following: reducing bacterial shedding, reducing the average bacterial concentration in the different target organs, reducing the duration of bacterial colonization, or lowering the percentage of animals colonized with bacteria when compared to an unvaccinated group.

[0043] Avian subjects

[0044] The immunization methods of the present disclosure may be utilized to immunize various avian subjects. For instance, in some embodiments, the avian subjects include, without limitation, birds, ducks, geese, chickens, broiler chickens, or combinations thereof. In some embodiments, the avian subject is a chicken.

[0045] The immunization methods of the present disclosure may be utilized to immunize avian subjects at various life stages. For instance, in some embodiments, the avian subject is at a stage that includes an egg stage, a hatching stage, a nestling stage, a fledgling stage, a juvenile stage, a sub-adult stage, an adult stage, or combinations thereof.

[0046] In some embodiments, vaccine administration includes administering the vaccines of the present disclosure to an avian subject at an egg stage. In some of such embodiments, the vaccines of the present disclosure may be administered through in-ovo delivery.

[0047] In some embodiments, vaccine administration includes administering the vaccines of the present disclosure to an avian subject after the egg stage. For instance, in some embodiments, vaccine administration includes administering the vaccines of the present disclosure to an avian subject at a hatching stage, a nestling stage, a fledgling stage, a juvenile stage, a sub-adult stage, an adult stage, or combinations thereof. In some of such embodiments, the vaccines of the present disclosure may be administered through intramuscular administration. In some of such embodiments, the vaccines of the present disclosure may be administered intravenously. In some of such embodiments, the vaccines of the present disclosure may be administered through oral administration.

[0048] In some embodiments, the vaccines of the present disclosure may be administered in a single dose or a plurality of doses. In some embodiments, the vaccines of the present disclosure may be administered in multiple doses, the timing of which may be readily determined by the skilled artisan.

[0049] Treatment or prevention of diseases

[0050] The immunization methods of the present disclosure may have various applications. For instance, in some embodiments, the methods of the present disclosure may be utilized to treat or prevent a disease in an avian subject. In some embodiments, the methods of the present disclosure may be utilized to prevent a disease in an avian subject. In some embodiments, the disease includes, without limitation, a bacterial infection, a staphylococcus infection, bacterial chondronecrosis with osteomyelitis (BCO), or combinations thereof.

[0051] In some embodiments, the disease to be treated or prevented includes BCO. For instance, in some embodiments, the methods of the present disclosure may be utilized to prevent BCO lameness.

[0052] Additional embodiments

[0053] Reference will now be made to more specific embodiments of the present disclosure and experimental results supporting such embodiments. However, Applicant notes that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0054] Example 1. Electron beam (eBeam) for attenuation in vaccine development

[0055] In this Example, Applicants developed and tested an eBeam-killed multispecies bacterial vaccine in vitro. In addition, Applicants conducted two in vivo field studies to test the efficacy of the vaccine. Based on both studies, eBeam-killed vaccines developed against Staphylococcus species reduce lameness in broiler chickens by 50%. Currently, there are no commercial vaccines available, and the 50% reduction of bacterial chondronecrosis with osteomyelitis (BCO)-induced lameness would be an optimal benefit for the poultry industry.

[0056] Bacterial chondronecrosis with osteomyelitis (BCO) lameness in broilers is caused by several bacterial pathogens, including Enterococcus cecorum, Streptococcus spp., Staphylococcus spp, and E. coli. BCO is often caused by several species of Staphylococcus and vaccines arc known to enhance immunity, thereby reducing colonization, and preventing diseases. It is known that eBeam-killed- Salmonella, and eBeam-killed-Clostridium perfringens prevented colonization in chicken.

[0057] eBeam technology combines the safety of inactivated or killed vaccines while retaining the immunogenicity of live attenuated vaccines. eBeam technology halts bacterial multiplication by irreversibly breaking multiple directly opposed double- stranded DNA break and preventing DNA replication. Nevertheless, the cell membranes of eBeam-inactivated cells are structurally intact without alteration of antigenic epitopes. Therefore, eBeam-killed bacterial cells have no risks of reversible virulence yet are highly immunogenic owing to their conserved epitopes.

[0058] Applicants conducted two vaccine field testing studies to test the efficacy of the vaccine. As illustrated in FIGS. 1 and 5, each vaccine study was conducted over 56 days. Broiler chicken embryos were vaccinated in ovo on dl8 of embryogenesis. Secondly, birds were challenged with live bacteria and were observed for any signs of lameness from day (d) 22 to d56. Birds expressing clinical signs of lameness (resistance to stand up / walk) were humanely euthanized and investigated for the severity of BCO lesions in the tibial and femoral heads of leg bones. Birds were placed on fresh pine shavings and provided with age and species-appropriate temperatures and environmental conditions, ad libitum access to water, and age & speciesspecific feed throughout the study period. Photoperiod was set to 23h light and one hour dark to accelerate the expression of lameness within the study period by increasing their activity.

[0059] Based on both studies, eBeam-killed vaccines developed against Staphylococcus species reduce lameness in broiler chickens. Currently, there are no commercial vaccines available, and the 50% reduction of BCO-induced lameness would be an excellent benefit for the poultry industry.

[0060] eBeam technology combines the safety of inactivated or killed vaccines while retaining the immunogenicity of live attenuated vaccines. eBeam technology halts bacterial multiplication by irreversibly breaking multiple directly opposed double-stranded DNA and preventing DNA replication. Nevertheless, the cell membranes of eBeam-inactivated cells are structurally intact without alteration of antigenic epitopes.

[0061] In the first study, Applicants used two species of Staphylococcus (Staphylococcus aureus and Staphylococcus agnetis) in the vaccine preparation. In the second study, Applicants used five different strains of Staphylococcus. In both studies, Applicants vaccinated dl8 broiler chicken embryos (in ovo vaccination). The vaccinated and non- vaccinated birds were housed on floor pens on the hatch day in the first study and challenged the birds with S. agnetis (~lxl04CFU / mL) on d20 and d21 through the water. Applicants collected blood samples before and after the challenge to determine the immune parameters. The birds were scored for lameness regularly every day after 22 days post-hatch. All birds were killed on d56.

[0062] Example 1,1. First Study

[0063] For the development of eBeam vaccines in the first study, a high energy electron beam (10 MeV, 18 kW) was applied overnight to grown cultures of Staphylococcus agnetis and Staphylococcus aureus (grown separately and mixed in equal amounts) in Tryptic Soy Broth (TSB) at a concentration of -IxlO8CFU / mL -exposed to a dose of 10 kGy. For the development of formalin-killed vaccines, similar bacterial cultures were resuspended in TSB after treatment with 0.6% (v / v) formaldehyde for 48h at room temperature. This was followed by purification.

[0064] For negative controls, in ovo vaccination occurred with a blank compound without a bacterial challenge. The study was initiated with two pens / treatment with 30 birds / pen. The birds were culled down to 25 / pen on dl4 of the trial. For the bacterial challenge, on d20 and d21 of the trial, the eBeam and formalin treatment group birds were challenged with -IxlO4CFU / mL of Staphylococcus agnetis.

[0065] Compared to the negative control group, a significant reduction of 35.29% in daily cumulative lameness was observed in the eBeam vaccinated group (FIGS. 2-4). In contrast, the Formalin-killed vaccinated group showed no significant reduction. Data were analyzed using Microsoft Excel 365 (Microsoft, Redmond, WA, USA) and a generalized linear model (GLM) module in R x64 4.2.1 was used to evaluate the significant difference in lameness (P-values) between treatments, at a significance level of P < 0.05.

[0066] Example 1,2, Second Study

[0067] In the second study, the mode of challenge was similar to the natural route by rearing positive control birds on wire-floored pens, which will act as the source of infection for lameness (FIG. 5). This is a proven model (aerosol transmission model) of challenge.

[0068] For the development of eBeam vaccines in the second study, a high energy electron beam (10 MeV, 18 kW) was applied to an overnight grown culture of the following strains of Staphylococcus (grown separately and mixed in equal amounts) in Tryptic Soy Broth (TSB) at a concentration of -IxlO8CFU / mL (exposed to a dose of 8 kGy): Staphylococcus agnetis; Staphylococcus cohnii (2 distinct strains); Staphylococcus lentus; and Staphylococcus aureus.

[0069] For the development of formalin-killed vaccines, similar bacterial cultures were resuspended in TSB after treatment with 0.6% (v / v) Formaldehyde for 48h at room temperature. This was followed by purification.

[0070] For the positive controls, in ovo vaccination occurred with fresh TSB (sham vaccine) and raised on wire flooring (explained further in Example 1.3). For the negative controls, in ovo vaccination occurred with a sham, and raised on litter floor as the eBeam and formalin-killed vaccine groups.

[0071] The study was initiated with four pens / treatment with 60 birds / pen. The birds were culled down to 50 / pen on dl4 of the trial. The positive control (wire-flooring) group had only 2 pens. The vaccines were modified to include more strains of Staphylococcus, hypothesizing that this would provide more optimal protection. The dose of eBeam exposure was reduced to 8 kGy upon further observation that membrane intactness and cell viability are better preserved under this dose while preventing the multiplication of and the ability to culture the eBeam exposed bacteria (FIG. 6).

[0072] The results indicate that, compared to the negative control group, a significant reduction of 50.94% in daily cumulative lameness was observed in the eBeam vaccinated group (FIGS. 7- 9). Meanwhile, the Formalin-killed vaccinated group showed no significant reduction. As expected, including multiple strains in the vaccine and optimizing of the treatment dose improved the effect of lameness reduction. Data were analyzed using Microsoft Excel 365 (Microsoft, Redmond, WA, USA) and a generalized linear model (GLM) module in R x64 4.2.1 was used to evaluate the significant difference in lameness (P-values) between treatments, at a significance level of P < 0.05.

[0073] Example 1,3. Wire-floored Pens

[0074] The second study utilized wire-floored pens (FIG. 5). The wire-floored pens were the first two pens at the front of the house by the cooling pads-they act as the source of infection / positive control. Based on the findings of Applicants’ research, the wire-floored pens act as the source of infection for lameness by expediting the leg-bone necrosis owing to continued mechanical stress on the bones by the wire floor, which immunosuppresses the birds, allowing opportunistic bacteria to settle and colonize in the bone fractures / crevices caused by the mechanical stress.

[0075] Applicants used an adaptation of this model with the two front-most pens being wire- floored and the rest of the pens being litter-floored, with four tunnel fans at the opposite (rear) end of the house, whose action disseminates the infectious bacteria aerosolized by the wire- floored birds to the other pens via air, as the tunnel fans pull the air out. This model (aerosol transmission model) successfully induces >70% lameness, as the birds are challenged by the natural opportunistic microbiota of their system.

[0076] Example 1.4. Conclusion

[0077] As the above evidence proves, eBeam treatment is preferable for bacterial attenuation, and the vaccine developed against different species of Staphylococcus reduced BCO lameness in broiler chickens. The vaccines presented in this Example provide numerous advantages. To begin with, eBeam-killed vaccines are inexpensive to produce because they rely on the massive production of vaccines using eBeam technology. In addition, eBeam vaccines are safe because the pathogens in the vaccine are inactivated. Moreover, eBeam vaccines arc effective because they stimulate a robust immune response. The in-ova delivery of the vaccine is similar to the commercial application of vaccines against other poultry diseases, which makes it convenient, easy to deliver, and more acceptable to the poultry industry.

[0078] Moreover, the eBeam vaccines presented in this Example reduce lameness by 50%, improve animal health, and reduce the financial losses resulting from the condemnation of lame birds. BCO lameness results in hundreds of millions of dollars in lost revenue. The vaccine helps build an immune response that reduces Staphylococcus infection in broiler chickens. The alternative to vaccines are treatments with antimicrobials, which can negatively impact the meat quality and increase antimicrobial resistance.

[0079] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.

Claims

CLAIMS:

1. An avian vaccine, wherein the avian vaccine comprises a plurality of electron beam (eBeam)- treated bacteria, wherein the plurality of eBeam-treated bacteria comprise a plurality of different strains or species of bacteria.

2. The avian vaccine of claim 1, wherein the bacteria are eBeam treated under conditions effective to kill or attenuate the bacteria while retaining the ability of the bacteria to elicit a protective immune response in an avian subject against the bacteria.

3. The avian vaccine of claim 1, wherein the plurality of eBeam-treated bacteria are selected from the group consisting of Staphylococcus species, Enterococcus species, Escherichia species, or combinations thereof.

4. The avian vaccine of claim 1, wherein the plurality of eBeam-treated bacteria comprise at least two different strains of Staphylococcus species.

5. The avian vaccine of claim 4, wherein the Staphylococcus species are selected from the group consisting of Staphylococcus aureus, Staphylococcus agnetis, Staphylococcus cohnii, Staphylococcus lentus, or combinations thereof.

6. The avian vaccine of claim 4, wherein the Staphylococcus species comprise Staphylococcus aureus and Staphylococcus agnetis.

7. The avian vaccine of claim 4, wherein the Staphylococcus species comprise Staphylococcus aureus, Staphylococcus agnetis, Staphylococcus cohnii, and Staphylococcus lentus.

8. The avian vaccine of claim 1, wherein the plurality of eBeam-treated bacteria comprise one or more Enterococcus species, wherein the one or more Enterococcus species comprise Enterococcus cecorum.

9. The avian vaccine of claim 1, wherein the plurality of eBeam-treated bacteria comprise one or more Escherichia species, wherein the one or more Escherichia species comprise Escherichia coli.

10. The avian vaccine of claim 1, wherein the avian vaccine is suitable for use in immunizing an avian subject.

11. A method of immunizing an avian subject against a disease, said method comprising: administering an avian vaccine to the avian subject, wherein the avian vaccine comprises a plurality of electron beam (eBeam)-treated bacteria, and wherein the plurality of eBeam-treated bacteria comprise a plurality of different strains or species of bacteria.

12. The method of claim 11, wherein the avian subject is selected from the group consisting of birds, ducks, geese, chickens, broiler chickens, or combinations thereof.

13. The method of claim 11, wherein the avian subject is a chicken.

14. The method of claim 11, wherein the administering comprises administering the vaccine to the avian subject at an egg stage, wherein the administering comprises in-ovo delivery of the vaccine.

15. The method of claim 11, wherein the administering comprises administering the vaccine to the avian subject at a hatching stage, a nestling stage, a fledgling stage, a juvenile stage, a subadult stage, an adult stage, or combinations thereof.

16. The method of claim 15, wherein the administering comprises intramuscular administration.

17. The method of claim 11, wherein the disease is selected from the group consisting of a bacterial infection, a Staphylococcus infection, bacterial chondronecrosis with osteomyelitis (BCO), or combinations thereof.

18. The method of claim 11, wherein the disease comprises bacterial chondronecrosis with osteomyelitis (BCO).

19. The method of claim 18, wherein the method is utilized to prevent BCO lameness.

20. The method of claim 11, wherein the plurality of eBeam-treated bacteria are selected from the group consisting of Staphylococcus species, Enterococcus species, Escherichia species, or combinations thereof.

21. The method of claim 11, wherein the plurality of eBeam-treated bacteria comprise at least two different strains of Staphylococcus species.

22. The method of claim 21, wherein the Staphylococcus species are selected from the group consisting of Staphylococcus aureus, Staphylococcus agnetis, Staphylococcus cohnii, Staphylococcus lentus, or combinations thereof.

23. The method of claim 21, wherein the Staphylococcus species comprise Staphylococcus aureus and Staphylococcus agnetis.

24. The method of claim 21, wherein the Staphylococcus species comprise Staphylococcus aureus, Staphylococcus agnetis, Staphylococcus cohnii, and Staphylococcus lentus.

25. A method of making an avian vaccine, said method comprising: exposing a plurality of bacteria to an electron beam (eBeam), wherein the plurality of eBeam-treated bacteria comprise a plurality of different strains or species of bacteria.

26. The method of claim 25, wherein the bacteria are eBeam treated under conditions effective to kill or attenuate the bacteria while retaining the ability of the bacteria to elicit a protective immune response in an avian subject against the bacteria.

27. The method of claim 25, wherein the bacteria are eBeam treated at a dose between 2 kGy and 10 kGy.

28. The method of claim 25, wherein the plurality of bacteria are selected from the group consisting of Staphylococcus species, Enterococcus species, Escherichia species, or combinations thereof.

29. The method of claim 28, wherein the plurality of bacteria comprise one or more Staphylococcus species.

30. The method of claim 29, wherein the one or more Staphylococcus species are selected from the group consisting of Staphylococcus aureus, Staphylococcus agnetis, Staphylococcus cohnii, Staphylococcus lentus, or combinations thereof.

31. The method of claim 25, wherein the plurality of bacteria comprise one or more Enterococcus species, wherein the one or more Enterococcus species comprise Enterococcus cecorum.

33. The method of claim 25, wherein the plurality of bacteria comprise one or more Escherichia species, wherein the one or more Escherichia species comprise Escherichia coli.

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