Methods and uses of fusobacterium necrophorum immunogenic compositions and vaccines

Abstract

Provided herein are methods and uses for treating and / or preventing ear tip necrosis (ETN) in a pig, comprising administering to the pig or use of an effective amount of a composition comprising an agent that targets Fusobacterium necrophorum (F. necrophorum) and a pharmaceutically acceptable carrier. Also provided herein are methods for screening for an agent that prevents and / or treats ETN. Further provided herein are methods for culturing F. necrophorum.

Description

METHODS AND USES OF FUSOBACTERIUM NECROPHORUM IMMUNOGENIC COMPOSITIONS AND VACCINESRELATED APPLICATION

[0001] This application claims benefit of US Provisional Application serial no. 63 / 627,509 filed on January 31 , 2024, incorporated herein by reference in its entirety.FIELD

[0002] The present disclosure relates to methods and uses of preventing and / or treating ear tip necrosis in a pig; and methods of screening for an agent that treats and / or prevents ear tip necrosis.INTRODUCTION

[0003] Porcine (or swine) ear-tip necrosis (ETN), porcine (swine) ear necrosis (PEN) or porcine (swine) ear necrosis syndrome (PENS) is an infectious disease that is characterized by progressive loss of ear tissue in nursery or grower pigs. Early lesions are localized on the tip of the ear, progressing to erosions that finally necrotize. Lesions can advance towards the base of the ear, resulting in complete loss of the external ear (pinna) in the most severe cases. The cause of ETN is unknown, thus it has been described as a "multifactorial disease". Many factors have been found in association with ETN, such as high stocking density, PCV-2, and mycotoxins (Park et al., 2013. Papatsiros, 2012).

[0004] ETN has been reported in all pork-producing countries across the globe. Surveillance performed by the Canada West Swine Health Intelligence Network (CWSHIN) found that 44% of practicing veterinarians in Western Canada (n=27) classify ETN as a common disease, and 11 % would describe it as very prevalent (CWSHIN, 1Q2020). In general, the disease is suggested to not severely affect animal performance. However, ETN is very likely a painful condition and an animal welfare concern with societal implications: affected pigs eventually become disfigured and mutilated, losing parts of one or both ears. Furthermore, severe cases can result in open wounds and may provide a port-of-entry for other pathogens that affect weight gain and animal performance (Park et al., 2013). It is common for producers to use antibiotics in an attempt to control ETN, resulting in increased production costs (secondary to ETN) due to medication, removal of animals from a raised-without-antibiotics flow, and the potential for secondary infections. Thus, the use of antibiotics in barns where ETN is prevalent also contributesto the development of antimicrobial-resistant bacteria. As the primary cause of ETN is unknown it is challenging to treat and / or prevent.

[0005] An initial study in pigs from Saskatchewan, Canada described that ETN is an infectious disease and that viruses are not detected in affected pigs (Costa et al., 2021).

[0006] A subsequent study in pigs in Ghent, Belgium detected pathogens, including bacteria and viruses) and mycotoxins in affected pigs (Malik et al., 2023).

[0007] As the etiology of ETN remains unclear, there remains a need for methods that control and / or prevent ETN in pigs.SUMMARY

[0008] The present disclosure relates to methods of treating and / or preventing ear tip necrosis (ETN) in a pig, comprising administering to the pig an effective amount of a composition comprising an agent that targets Fusobacterium necrophorum (F. necrophorurri) and a pharmaceutically acceptable carrier. Also disclosed is use of a composition comprising an agent that targets F. necrophorum and a pharmaceutically acceptable carrier for treating and / or preventing ear tip necrosis (ETN) in a pig. Further disclosed is use of a composition comprising an agent that targets F. necrophorum and a pharmaceutically acceptable carrier in the manufacture of a medicament for treating and / or preventing ear tip necrosis (ETN) in a pig. Even further disclosed is a composition comprising an agent that targets F. necrophorum and a pharmaceutically acceptable carrier for use in treating and / or preventing ear tip necrosis (ETN) in a pig.

[0009] In some embodiments, the methods and uses are for preventing ETN.

[0010] In some embodiments, the methods and uses are for treating ETN.

[0011] In some embodiments, the agent is an attenuated or an inactive F. necrophorum, optionally Fusobacterium necrophorum subspecies necrophorum biotype A.

[0012] In some embodiments, the agent is a F. necrophorum bacterin.

[0013] In some embodiments, the attenuated or the inactive F. necrophorum or theF. necrophorum bacterin is prepared by culturing F. necrophorum in brain heart infusion (BHI) media supplemented with about 5 pg / mL to about 25 pg / mL hemin for about 20hours to about 48 hours. In some embodiments, the BHI media supplemented with hemin is anaerobic.

[0014] In some embodiments, the agent is Fusogard™.

[0015] In some embodiments, the pharmaceutically acceptable carrier is a polymeric adjuvant, optionally the polymeric adjuvant is Montanide™ Gel.

[0016] In some embodiments, the pharmaceutically acceptable carrier is oil-in water, water-in oil-in-water, or aluminum hydroxide.

[0017] In some embodiments, the composition comprises a ratio of agent:pharmaceutically acceptable carrier of 15:85 v / v.

[0018] In some embodiments, the composition is administered in or for use as a single dose.

[0019] In some embodiments, the composition is administered in or for use as two doses, optionally wherein the two doses are 21 days apart.

[0020] In some embodiments, the composition is administered or for use intradermally, subcutaneously, or intramuscularly.

[0021] In some embodiments, the composition is administered or for use at a volume of 0.5 mL to 2 mL, 0.5 mL to 1 mL, about 1 mL, or about 0.5 mL.

[0022] In some embodiments, the composition is administered or for use at 104cfu / mL to 1011cfu / mL, optionally the composition is administered or for use at about 108cfu / mL.

[0023] In some embodiments, the methods and uses further comprise coadministering one or more further agents for treating ETN.

[0024] In some embodiments, the one or more further agents comprise an antibiotic, optionally the antibiotic is tetracycline, penicillin, and / or ampicillin.

[0025] In some embodiments, the methods and uses further comprise, prior to administration or use, obtaining the agent from an infected pig of the same herd.

[0026] Also disclosed is a method for screening for an agent that prevents ear tip necrosis (ETN), the method comprising: a) administering, at least once, a test compound, to a pig;b) inoculating the pig with Fusobacterium necrophorum (F. necrophorumy and c) scoring at least one clinical sign of ETN to evaluate if the test compound is effective for preventing ETN in the pig; wherein the test compound is identified as an agent that prevents ETN if the pig has a reduction in at least one clinical sign of ETN compared to a control.

[0027] In some embodiments, the at least one clinical sign of ETN comprises: change in weight, abnormal skin colour, impaired responsiveness, edema, hyperemia, lesions, scabbing, necrosis, or tissue loss.

[0028] In some embodiments, administering, at least once, comprises administering the test compound in two doses.

[0029] In some embodiments, the two doses are 21 days apart.

[0030] In some embodiments, step b) inoculating the pig with F. necrophorum is 14 days after step a) administering, at least once, the test compound to the pig, optionally inoculating the pig with F. necrophorum is 14 days after administering a second dose of the test compound.

[0031] In some embodiments, step c) scoring at least one clinical sign of ETN is at least 1 to at least 20 days after the step of inoculating the pig with F. necrophorum.

[0032] Also disclosed is a method for screening for an agent that treats ear tip necrosis (ETN), the method comprising: a) inoculating a pig with Fusobacterium necrophorum (F. necrophorumy b) administering, at least once, a test compound to the pig; and c) scoring at least one clinical sign of ETN to evaluate if the test compound is effective for treating ETN in the pig; wherein the test compound is identified as an agent that treats ETN if the pig has a reduction in at least one clinical sign of ETN compared to a control.

[0033] In some embodiments, the at least one clinical sign of ETN comprises: change in weight, abnormal skin colour, impaired responsiveness, edema, hyperemia, lesions, scabbing, necrosis, or tissue loss.

[0034] In some embodiments, administering, at least once, comprises administering the test compound in two doses.

[0035] In some embodiments, the two doses are 21 days apart.

[0036] In some embodiments, step a) inoculating the pig with F. necrophorum and step b) administering, at least once, a test compound to the pig, are at least one day apart.

[0037] In some embodiments, step c) scoring at least one clinical sign of ETN is at least 1 to at least 20 days after the step of administering, at least once, a test compound to the pig.

[0038] In some embodiments, inoculating the pig comprises an intradermal injection of F. necrophorum into the ear of the pig, optionally at a dose of 108cfu / mL. In some embodiments, inoculating the pig comprises an intradermal injection of F. necrophorum into the ear of the pig, optionally at a dose of 106cfu / mL.

[0039] In some embodiments, the F. necrophorum for inoculating the pig is prepared by culturing the F. necrophorum in BHI media supplemented with about 5 pg / mL to about 25 pg / mL hemin for about 20 hours to about 48 hours. In some embodiments, the BHI media supplemented with hemin is anaerobic.

[0040] Also disclosed is an in vitro method for screening an agent that prevents ear tip necrosis (ETN), the method comprising: a) contacting an epithelial layer of cells with a test compound; b) culturing the epithelial layer of cells with Fusobacterium necrophorum (F. necrophorumy and c) measuring tissue viability and / or stress response in the epithelial layer of cells to evaluate if the test compound is effective for preventing ETN; wherein the test compound is identified as an agent that prevents ETN if the epithelial layer of cells has improved tissue viability and / or stress response compared to a control epithelial layer of cells without the test compound.

[0041] Further disclosed is an in vitro method for screening an agent that treats ear tip necrosis (ETN), the method comprising: a) culturing an epithelial layer of cells with Fusobacterium necrophorum (F. necrophorumy b) contacting the epithelial layer of cells with a test compound; and c) measuring tissue viability and / or stress response in the epithelial layer of cells to evaluate if the test compound is effective for treating ETN;wherein the test compound is identified as an agent that treats ETN if the epithelial layer of cells has improved tissue viability and / or stress response compared to a control cell without the test compound.

[0042] In some embodiments, the epithelial layer of cells is a primary skin culture or an explant culture.

[0043] In some embodiments, measuring cell viability and / or stress response comprises histology, electron microscopy, fluorescence microscopy, enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), or high-throughput sequencing.

[0044] In some embodiments, the F. necrophorum for culturing with the epithelial layer of cells is prepared by culturing the F. necrophorum in brain heart infusion (BHI) media supplemented with about 5 pg / mL to about 25 pg / mL hemin for about 20 hours to about 48 hours. In some embodiments, the BHI media supplemented with hemin is anaerobic.

[0045] A further aspect of the disclosure is a method of culturing Fusobacterium necrophorum (F. necrophorum), comprising growing the F. necrophorum in brain heart infusion (BHI) media supplemented with about 5 pg / mL to about 25 pg / mL hemin for about 20 hours to about 48 hours.

[0046] In some embodiments, the method further comprises agitating the F. necrophorum.

[0047] In some embodiments, growing the F. necrophorum comprises culturing the F. necrophorum for about 24 hours.

[0048] In some embodiments, the concentration of hemin is about 5 pg / mL.

[0049] In some embodiments, the method further comprises measuring the optical density of the F. necrophorum, optionally stopping growth of the F. necrophorum when the OD at 600nm is about 0.6 to about 1.1.

[0050] In some embodiments, the method further comprises killing or inactivating the F. necrophorum.

[0051] In some embodiments, the killed or inactivated F. necrophorum is formulated for administration as a bacterin.

[0052] In some embodiments, the F. necrophorum is formulated for inoculation of a pig.

[0053] In some embodiments, inoculation of the pig comprises an intradermal injection of F. necrophorum into the ear of the pig, optionally at a dose of about 104cfu / mL to about 1011cfu / mL, further optionally at a dose of about 106cfu / mL.

[0054] Another aspect of the disclosure is a method of preparing a Fusobacterium necrophorum (F. necrophorum) bacterin, the method comprising: a) growing the F. necrophorum in brain heart infusion (BHI) media supplemented with about 5 pg / mL to about 25 pg / mL hemin for about 20 hours to about 48 hours; b) optionally, measuring the optical density of the F. necrophorum; c) inactivating or killing the F. necrophorum; and d) optionally, mixing the inactivated or killed F. necrophorum with an adjuvant.

[0055] In some embodiments, the concentration of hemin is about 5 pg / mL.

[0056] In some embodiments, growing the F. necrophorum comprises culturing for about 24 hours.

[0057] In some embodiments, growing the F. necrophorum is stopped when the optical density at 600 nm is about 0.6 to about 1.1.

[0058] In some embodiments, the method further comprises formulating the inactivated or killed F. necrophorum for administration to a pig.

[0059] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the disclosure, are given by way of illustration only and the scope of the claims should not be limited by these embodiments but should be given the broadest interpretation consistent with the description as a whole.DRAWINGS

[0060] For a better understanding of the embodiments described herein and to show more clearly how they may be carried into effect, reference will now be made, byway of example only, to the accompanying drawings which show at least one exemplary embodiment, and in which:

[0061] Figure 1 shows, in an embodiment of the disclosure, lesion progression following challenge in vaccinated (bacterin or Fusogard™) and control animals. Tissue loss is observed in the ear tip of controls pigs, but not in the ears of pigs receiving either vaccine.

[0062] Figure 2 shows, in an embodiment of the disclosure, lesion progression following challenge in bacterin vaccinated and control animals at post-inoculation day 0, day 4, day 5, day 7, day 10, day 14 and day 17.

[0063] Figure 3 shows the scoring schema for assessing ear tip necrosis in an embodiment of the disclosure.

[0064] Figure 4 shows, in an embodiment of the disclosure, growth of Fusobacterium necrophorum cultured in BHI, BHI supplemented with hemin (5 pg / mL) or BHI supplemented with 1 % FBS. Growth was measured by optical density at 4 hours, 25 hours and 36 hours.

[0065] Figure 5, shows, in an embodiment of the disclosure, growth of Fusobacterium necrophorum cultured in BHI supplemented with hemin with or without agitation. Growth was measured by optical density at 24 hours and 36 hours.DESCRIPTION OF VARIOUS EMBODIMENTS

[0066] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

[0067] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearlyindicated to the contrary. In particular, any feature described herein may be combined with any other feature or features described herein.I. Definitions

[0068] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0069] As used herein, the terms “ear tip necrosis”, “ETN”, “porcine ear necrosis”, “PEN”, “porcine ear necrosis syndrome”, “PENS”, “necrotic ear syndrome in swine”, “necrotic auricular dermatitis”, “ulcerative spirochetosis of the ear”, “streptococcal auricular dermatitis”, “ear biting” or “ear necrosis” refer to a condition in pigs characterized by progressive loss of ear tissue in nursery and early-grower pigs. Early lesions are localized on the tip of the ear, progressing to erosions that finally necrotize. Lesions can advance towards the base of the ear, resulting in complete loss of the external ear (pinna) in the most severe cases.

[0070] As used herein, the term “clinical signs of ETN” refers to the physical presentation of ETN and possibly secondary infection, including without limitation, lesions and scabbing of the ear, tissue loss, including loss of the external ear (pinna), edema, hyperemia, change in weight (e.g., weight loss, or impaired weight gain), abnormal skin colour, and impaired responsiveness to stimuli (i.e. alertness). Clinical signs of ETN may be scored, for example, using a scoring scheme of 0-4, wherein: score 0) no visual abnormalities; score 1) edema and hyperemia visible, initial lesions are closed and located at the tip of the ear or corresponding with the most distal end of the marginal ear vein; score 2) the initial swelling erodes through the skin surface to form a scab; score 3) advanced ear necrosis involving the tip and margins of both ears; score 4) healed lesions with loss of ear tissue.

[0071] As used herein, the term “bacterin” refers to a suspension of killed or attenuated bacteria. As contemplated herein the bacterin may be administered or for useas a vaccine or immunogenic composition. A bacterin may be prepared by methods known to a person skilled in the art. For example, use of formaldehyde, heat, sonication, freeze-thaw cycles, ultraviolet radiation, antibiotics, sodium hydroxide, ethanol, or any method of shearing the cell wall using methods known to the skilled person.

[0072] The term “attenuated” as used herein refers to a pathogen, for example a bacterium, that has been manipulated or weakened to reduce its virulence.

[0073] The term “Fusobacterium necrophorum" or “F. necrophorum" as used herein, refers to an anaerobic, gram-negative bacteria. F. necrophorum refers to any strain of F. necrophorum, and suitably includes Fusobacterium necrophorum subspecies necrophorum biotype A.

[0074] The term “Fusogard™” as used herein refers to the commercial vaccine produced by Elanco containing a Fusobacterium necrophorum bacterin for the treatment of footrot in cattle. Fusogard™ is described in US Patent No. 6,632,439 incorporated herein by reference.

[0075] The term "pharmaceutically acceptable carrier" refers to any carrier, agent, excipient, or vehicle that is not biologically or otherwise undesirable. Except where the carrier, agent, excipient or vehicle is incompatible with the active ingredient, its use in the therapeutic formulations is contemplated. The use of such pharmaceutically acceptable carrier is well known in the art.

[0076] The term “effective amount” as used herein refers to any amount of a compound or a composition that is sufficient to generate a desired response, including but not limited to preventing or reducing the chance of disease onset, slowing disease progression, and alleviating symptoms. As used herein, the term can for example refer to an amount that is sufficient to treat or prevent ETN in a pig.

[0077] The term "treating" or “treatment” as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. "Treating" and "Treatment" can also mean reducingthe clinical signs of ETN. For example, a subject with ETN can be treated with a composition described herein to mitigate lesion progression. "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. For example, a pig with ETN can be treated with a composition described herein to prevent a secondary infection, or to reduce mortality rate associated with the secondary infection.

[0078] The term “prevention” or “prophylaxis”, as used herein refers to a reduction in the risk or probability of a subject becoming afflicted with a disease, disorder or condition or manifesting a symptom associated with a disease, disorder or condition. The disease, disorder or condition can for example be ETN or an infection of F. necrophorum.

[0079] The term “control” as used herein refers to a control pig with known ETN status, or a control value or dataset that can be used to prognose or classify the pig e.g., decrease in clinical signs of ETN, and / or absence of necrosis or reference decrease in clinical signs of ETN and / or reference absence of necrosis obtained from a test pig or pigs associated with a known outcome. In one embodiment, the dataset may be obtained from a pig or a group of pigs known to have ear tip necrosis and have clinical signs of ETN and / or the presence of necrosis. The clinical signs of ETN, and / or presence of necrosis in the dataset can be used to create a “control value” that is used in testing a test agent in a pig. A control value may be obtained from historical data including, scoring clinical signs of ETN and / or presence of necrosis for a pig or pool of pigs with a known ETN status.

[0080] The term “test compound” as used herein refers to any compound being assessed for efficacy in treating and / or preventing ETN in a pig. A compound may include, without limitation, bacteria, viruses, cells, cell and tissue extracts, nucleic acids, antisense oligonucleotide molecules (antisense nucleic acid molecules), DNA, RNA, shRNA, siRNA, proteins, protein mimetics, peptides, peptidomimetics, antibodies (and fragments thereof), aptamers, small molecules, large molecules, chemicals, analogs that mimic the binding site of an enzyme, receptor, or other protein, e.g., that is involved in signal transduction, gene editing agents, therapeutic agents, pharmaceutical compositions, drugs, other substances directed at the target expression or activity and combinations of these.

[0081] In understanding the scope of the present disclosure, the term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps,but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

[0082] The term “consisting” and its derivatives, as used herein, are intended to be closed ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0083] All numerical values herein are modified by “about” or “approximately” the indicated value, and take into account experimental error and variations that would be expected by a person having ordinary skill in the art.

[0084] The terms "about", “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies or unless the context suggests otherwise to a person skilled in the art.

[0085] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, a composition containing “a compound” includes a mixture of two or more compounds. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0086] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0087] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0088] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Eachaspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary.

[0089] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, examples of methods and materials are now described.II. Methods and Uses

[0090] It is demonstrated herein that agents that target F. necrophorum can prevent and / or treat ear tip necrosis in a pig.

[0091] Accordingly, in one aspect, there is provided a method of treating and / or preventing ear tip necrosis (ETN) in a pig, comprising administering to the pig an effective amount of a composition comprising an agent that targets Fusobacterium necrophorum (F. necrophorum) and a pharmaceutically acceptable carrier. Also provided herein is a use of a composition comprising an agent that targets F. necrophorum and a pharmaceutically acceptable carrier for treating and / or preventing ear tip necrosis (ETN) in a pig. Further provided is a use of a composition comprising an agent that targets F. necrophorum and a pharmaceutically acceptable carrier in the manufacture of a medicament for treating and / or preventing ear tip necrosis (ETN) in a pig. Even further provided is a composition comprising an agent that targets F. necrophorum and a pharmaceutically acceptable carrier for use in treating and / or preventing ear tip necrosis (ETN) in a pig.

[0092] In an embodiment, the method or use is for preventing ETN.

[0093] In an embodiment, the method or use is for treating ETN. For example, treatment may include mitigating lesions (e.g., reduction of lesion area, mitigation of lesion progression) as compared to a control that has not received the agent.

[0094] The agent can act directly or indirectly to target F. necrophorum. For example, the agent may elicit an immune response that then targets F. necrophorum. Accordingly, in an embodiment, the agent is an attenuated or an inactive F. necrophorum. In an embodiment, the agent is a F. necrophorum bacterin, optionally Fusobacterium necrophorum subspecies necrophorum biotype A. In an embodiment, the Fusobacterium necrophorum subspecies necrophorum biotype A is a clinical sample, obtained from any livestock, including a pig with ETN. In an embodiment, the agent is Fusobacterium necrophorum subspecies necrophorum ATCC 25286, VPI 2891 [2358, JCM 3718], In anembodiment, the agent is Fusobacterium necrophorum subspecies necrophorum strain VPI 6054-A. In an embodiment, the agent is Fusobacterium necrophorum subspecies necrophorum strain NCTC 10576. In an embodiment, the agent is Fusobacterium necrophorum subspecies, necrophorum strain NCTC 10577. In an embodiment, the agent is Fusobacterium necrophorum subspecies necrophorum strain A200. In an embodiment, the agent is Fusobacterium necrophorum subspecies necrophorum strain DSM 20698. In an embodiment, the agent is Fusobacterium necrophorum subspecies necrophorum strain NCTC 13730.

[0095] Methods of obtaining a clinical sample from a pig with ETN are known to those skilled in the art. For example, a scraping from the ear of a pig with ETN can be collected, as described in Costa et al., 2021 , incorporated herein by reference, and cultured to generate a F. necrophorum isolate, for example as described in Example 1. Methods of culturing an F. necrophorum isolate may be prepared by conventional methods known to a person skilled in the art.

[0096] In one embodiment, the attenuated or the inactive F. necrophorum is prepared by culturing the F. necrophorum in brain heart infusion (BHI) media supplemented with about 5 pg / mL to about 25 pg / mL hemin for about 20 hours to about 48 hours. In one embodiment, the F. necrophorum bacterin is prepared by culturing the F. necrophorum in BHI media supplemented with about 5 pg / mL to about 25 pg / mLhemin for about 20 hours to about 48 hours. In some embodiments, the BHI media supplemented with hemin is anaerobic.

[0097] In some embodiments, the concentration of hemin is about 5 pg / mL.

[0098] In some embodiments, preparing the attenuated or the inactive F. necrophorum comprises culturing the F. necrophorum for about 24 hours. In some embodiments, preparing the F. necrophorum bacterin comprises culturing the F. necrophorum for about 24 hours.

[0099] In one embodiment, the agent is Fusogard™.

[0100] Fusogard™ described in US patent number 6,632,439, incorporated herein by reference, is a vaccine comprising a killed biotype A (FNN) Fusobacterium necrophorum isolate taken from a bovine species member. The F. necrophorum isolate is identified as strain number 021496, ATCC Accession No. PTA-917. Fusogard™ further comprises a suitable adjuvant. The Fusobacterium necrophorum is killed withformaldehyde. Fusogard™ has been previously described for treating diseases such as footrot and liver abscesses in a ruminant.

[0101] The composition disclosed herein can be used as a vaccine or as an immunogenic composition, which is typically administered to or used in healthy pigs to, for example, prevent and / or reduce the likelihood of disease onset caused by an infection and / or to reduce severity of the disease in case of infection. The composition can also be used as a treatment. For example, the composition can be administered to or used in pigs having or suspected of having ETN.

[0102] The composition described herein can be prepared by any known methods for the preparation of pharmaceutically acceptable compositions such that an effective amount can be administered to or used in a pig such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable carrier to for example, elicit a prophylactic or therapeutic immune response.

[0103] Pharmaceutical compositions include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially compatible with the tissues or the blood of an intended recipient. Other components that may be present in such compositions include water, surfactants (such as Tween), alcohols, polyols, glycerin and vegetable oils, for example. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions. The composition may be supplied, for example but not by way of limitation, as a lyophilized powder which is reconstituted with sterile water or saline prior to administration to the patient. In an embodiment, the composition disclosed herein further comprises other agents suitable for preventing and / or treating ETN. Suitable agents include, without limitation, tetracycline, penicillin, and ampicillin.

[0104] In an embodiment, the pharmaceutically acceptable carrier is a polymeric adjuvant, optionally the polymeric adjuvant is Montanide™ Gel.

[0105] In an embodiment, the pharmaceutically acceptable carrier is oil-in water, water-in oil-in-water, or aluminum hydroxide.

[0106] In an embodiment, the composition comprises at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the agent.

[0107] In an embodiment, the composition comprises a ratio of agent:pharmaceutically acceptable carrier of 15:85 v / v.

[0108] The composition for use with the methods and uses described herein can be formulated for any suitable route of administration and using any suitable dosage form, for example, by parenteral, intravenous, subcutaneous, intramuscular, intraperitoneal, inhalation or spray (e.g. via aerosol), mucosal administration, rectal administration, vaginal administration, skin patch or skin application or oral administration, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. For example, the compositions can be formulated for mucosal administration (such as intranasal, oral, ophthalmic, rectal, vaginal, sublingual) or for injection (such as subcutaneous, intramuscular, intravenous).

[0109] Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003 - 20thedition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

[0110] In an embodiment, the composition is administered or formulated for use intradermally, subcutaneously or intramuscularly.

[0111] In an embodiment, the composition is administered or formulated for use intradermally.

[0112] In another embodiment, the composition is administered or formulated for use subcutaneously.

[0113] In a further embodiment, the composition is administered or for use intramuscularly.

[0114] The effective amount to be administered or used can be determined by conventional methods known to a person skilled in the art. An effective amount can, for example, be determined by starting with a low dose and incrementally increased until a desirable outcome is reached.

[0115] For example, the composition can be administered or used at a volume of 0.5-2mL intradermally, subcutaneously, or intramuscularly.

[0116] In an embodiment, the composition is administered or used at a volume of 0.5 mL. In an embodiment, the composition is administered or used at a volume of 0.6 mL. In an embodiment, the composition is administered or used at a volume of 0.7 mL. In an embodiment, the composition is administered or used at a volume of 0.8 mL. In an embodiment, the composition is administered or used at a volume of 0.9 mL. In an embodiment, the composition is administered or used at a volume of 1.0 mL. In an embodiment, the composition is administered or used at a volume of 1.1 mL. In an embodiment, the composition is administered or used at a volume of 1.2 mL. In an embodiment, the composition is administered or used at a volume of 1.3 mL. In an embodiment, the composition is administered or used at a volume of 1.4 mL. In an embodiment, the composition is administered or used at a volume of 1.5 mL. In an embodiment, the composition is administered or used at a volume of 1.6 mL. In an embodiment, the composition is administered or used at a volume of 1.7 mL. In an embodiment, the composition is administered or used at a volume of 1.8 mL. In an embodiment, the composition is administered or used at a volume of 1.9 mL. In an embodiment, the composition is administered or used at a volume of 2.0 mL.

[0117] In an embodiment, the composition is administered or used at 0.5 mL subcutaneously. In an embodiment, the composition is administered or used at 0.5 mL intramuscularly. In an embodiment, the composition is administered or used at 1 mL subcutaneously. In an embodiment, the composition is administered or used at 1 mL intramuscularly.

[0118] In an embodiment, the composition is administered or used at 104- 1011cfu / mL. In an embodiment, the composition is administered or used at 105- 1011cfu / mL. In an embodiment, the composition is administered or used at 106- 1011cfu / mL. In an embodiment, the composition is administered or used at 107- 1011cfu / mL. In an embodiment, the composition is administered or used at 107- 1010cfu / mL. In an embodiment, the composition is administered or used at 108- 1011cfu / mL. In an embodiment, the composition is administered or used at 108- 1010cfu / mL.

[0119] In an embodiment, the composition is administered or used at about 104cfu / mL. In an embodiment, the composition is administered or used at about 105cfu / mL. In an embodiment, the composition is administered or used at about 106cfu / mL. In an embodiment, the composition is administered or used at about 107cfu / mL. In an embodiment, the composition is administered or used at about 108cfu / mL. In anembodiment, the composition is administered or used at about 109cfu / mL. In an embodiment, the composition is administered or used at about 1010cfu / mL. In an embodiment, the composition is administered or used at about 1011cfu / mL.

[0120] In a particular embodiment, the composition is administered or used at about 108cfu / mL.

[0121] The composition disclosed herein may be administered or used according to any suitable schedule, for example as a single dose, or multiple doses (e.g. two doses), separated by a suitable period of time, or any other suitable number of doses and / or period of time, depending for example on the formulation, and / or route of administration.

[0122] In an embodiment, the composition is administered or used in a single dose. In an embodiment, the composition is administered or used in a 2-dose regimen, with at least 21 days between the 2 doses.

[0123] In an embodiment, the composition is administered or used in 2 doses with 21 days apart.

[0124] The methods and uses disclosed herein can further comprise administering or using one or more further agents suitable for treating ETN. Suitable agents for treating ETN include without limitation antibiotics, optionally, tetracycline, penicillin, and ampicillin.

[0125] The one or more agents can be co-administered or used with the composition disclosed herein. For co-administration or use, the one or more agents and the composition disclosed herein can be formulated separately or combined in a single formulation if suitable as determined by a skilled person. Accordingly, the present description provides a single unit dosage form comprising the composition described herein, a therapeutic agent, and a pharmaceutically acceptable carrier. The term “coadministration” shall mean that at least two compounds or compositions are administered to or used in the animal such that effective amounts or concentrations of each of the two or more compounds may be found in the pig at a given point in time. Although compounds according to the present disclosure may be co-administered to or used in a pig at the same time, the term embraces both administration or use of two or more agents at the same time or at different times, provided that effective concentrations of all coadministered compounds or compositions are found in the pig at a given time. The exact details of the administration will depend on the pharmacokinetics of the two substances in the presence of each other, and can include administering or using the two substanceswithin a few hours of each other, or even administering or using one substance within 24 hours of administration or use of the other, if the pharmacokinetics are suitable. Design of suitable dosing regimens is routine for one skilled in the I art. In particular embodiments, two substances will be administered or used substantially simultaneously, i.e., within minutes of each other, or in a single composition that contains both substances. In other embodiments, the combination of agents is administered or used in a noncontemporaneous fashion.

[0126] In an embodiment, the method further comprises, prior to administration, obtaining the agent from an infected pig of the same herd.

[0127] Obtaining the agent from an infected pig of the same herd to which the agent will be delivered is commonly known as an autogenous vaccine or an autogenous immunogenic composition. Methods of making an autogenous vaccine or autogenous immunogenic composition are known in the art. Briefly, autogenous vaccines and autogenous immunogenic compositions are inactivated (killed) immunological medicinal products aimed at inducing immunity or eliciting an immune response against a single specific pathogenic organism derived from animals from the same herd where the agent will be used.

[0128] In a further aspect, there is provided a method for screening an agent that prevents ear tip necrosis (ETN), the method comprising: a) administering, at least once, a test compound to a pig; b) inoculating the pig with Fusobacterium necrophorum (F. necrophorum and c) scoring at least one clinical sign of ETN to evaluate if the test compound is effective for preventing ETN in the pig; wherein the test compound is identified as an agent that prevents ETN if the pig has a reduction in at least one clinical sign of ETN compared to a control.

[0129] In an embodiment, the at least one clinical sign of ETN comprises: change in weight, abnormal skin colour, impaired responsiveness to stimuli, edema, hyperemia, lesions, scabbing, necrosis, or tissue loss.

[0130] In an embodiment, the administering, at least once, comprises administering the test compound in two doses. In an embodiment, the two doses are 21 days apart.

[0131] In an embodiment, the step of inoculating the pig with F. necrophorum is 7 days to 50 days, 10 days to 40 days, 10 days to 30 days, 10 days to 20 days, after administering, at least once, the test compound to a pig. In an embodiment, the step of inoculating the pig with F. necrophorum is at least 7 days, at least 10 days, at least 20 days, at least 30 days, or at least 50 days after administering, at least once, the test compound to a pig. In an embodiment, the step of inoculating the pig with F. necrophorum is 35 days after a first administration of the test compound. In an embodiment, the step of inoculating the pig with F. necrophorum is 43 days after the first administration of the test compound. In embodiment, the step of inoculating the pig with F. necrophorum is 5 to 14 days after administering, at least once, the test compound to a pig, optionally, 5 to 14 days after administering a second dose of the test compound to the pig.

[0132] In an embodiment, the step of scoring at least one clinical sign of ETN is performed at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days after the step of inoculating the pig with F. necrophorum.

[0133] In an embodiment, scoring at least one clinical sign of ETN is done daily for at least one day, at least one week, at least two weeks, or at least three weeks after the step of inoculating the pig with F. necrophorum.

[0134] In a further aspect, there is provided a method for screening an agent that treats ear tip necrosis (ETN), the method comprising: a) inoculating a pig with Fusobacterium necrophorum (F. necrophorum b) administering, at least once, a test compound to a pig; and c) scoring at least one clinical sign of ETN to evaluate if the test compound is effective for treating ETN in the pig; wherein the test compound is identified as an agent that treats ETN if the pig has a reduction in at least one clinical sign of ETN compared to a control.

[0135] In an embodiment, the at least one clinical sign of ETN comprises: change in weight, abnormal skin colour, impaired responsiveness to stimuli, edema, hyperemia, lesions, scabbing, necrosis, or tissue loss.

[0136] In an embodiment, the administering, at least once, comprises administering the test compound in two doses. In an embodiment, the two doses are 21 days apart.

[0137] In an embodiment, the step of administering, at least once, the test compound is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days after the step of inoculating the pig with F. necrophorum.

[0138] In an embodiment, the step of scoring at least one clinical sign of ETN is performed at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days after the step of administering at least once, a test compound to the pig.

[0139] In an embodiment, scoring at least one clinical sign of ETN is done daily for at least one day, at least one week, at least two weeks, or at least three weeks after the step of administering at least once, a test compound to a pig.

[0140] In an embodiment, inoculating the pig comprises an intradermal injection of F. necrophorum into the ear of the pig, optionally at a dose of 108cfu / mL. In an embodiment, inoculating the pig comprises an intradermal injection of F. necrophorum into the ear of the pig, optionally at a dose of 106cfu / mL.

[0141] In a further aspect, there is provided an in vitro method for screening an agent that prevents ear tip necrosis (ETN), the method comprising: a) contacting an epithelial layer of cells with a test compound; b) culturing the epithelial layer of cells with Fusobacterium necrophorum (F. necrophorum and c) measuring tissue viability and / or stress response in the epithelial layer of cells to evaluate if the test compound is effective for preventing ETN; wherein the test compound is identified as an agent that prevents ETN if the epithelial layer of cells has improved tissue viability and / or stress response compared to a control epithelial layer of cells without the test compound.

[0142] In a further aspect, there is provided an in vitro method for screening an agent that treats ear tip necrosis (ETN), the method comprising:a) culturing an epithelial layer of cells with Fusobacterium necrophorum (F. necrophorumy b) contacting the epithelial layer of cells with a test compound; and c) measuring tissue viability and / or stress response in the epithelial layer of cells to evaluate if the test compound is effective for treating ETN; wherein the test compound is identified as an agent that treats ETN if the epithelial layer of cells has improved tissue viability and / or stress response compared to a control cell without the test compound.

[0143] In an embodiment, the epithelial layer of cells is a primary skin culture, generated from the pinnae skin of a pig. In an embodiment, the epithelial layer of cells is an explant culture. Methods of generating the epithelial layer of cells are known and may be prepared by conventional methods known to a person skilled in the art. Without wishing to be bound by theory, the epithelial layer of cells may be prepared as described in Example 3.

[0144] Methods of measuring the tissue viability and / or stress response in the epithelial layer of cells are known to those skilled in the art. For example, histology, electron microscopy, fluorescent microscopy, or any other method to visualize or quantify cell viability. Stress response may be measured, for example, by quantification of stress response factors produced by the epithelial layer of cells, such as cytokines and chemokines. Stress response factors may be measured by any suitable method known to the skilled person, including, for example, enzyme linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR).

[0145] In some embodiments, the F. necrophorum as used in the methods and uses herein, is prepared by culturing the F. necrophorum in BHI media supplemented with about 5 pg / mL to about 25 pg / mL hemin for about 20 hours to about 48 hours. In some embodiments, the BHI media supplemented with hemin is anaerobic.

[0146] In some embodiments, the BHI media is supplemented with about 5 pg / mL hemin.

[0147] In some embodiments, the F. necrophorum is grown in the BHI media supplemented with hemin for about 24 hours.

[0148] In another aspect of the disclosure is a method of culturing Fusobacterium necrophorum (F. necrophorum comprising growing the F. necrophorum in brain heartinfusion (BHI) media supplemented with about 5 pg / mL to about 25 pg / mL hemin for about 20 hours to about 48 hours.

[0149] In some embodiments, the BHI media supplemented with hemin is anaerobic.

[0150] In some embodiments, the method further comprises agitating the F. necrophorum. In one embodiment, agitating the F. necrophorum comprises shaking at 200 rpm.

[0151] In some embodiments, growing the F. necrophorum comprises culturing the F. necrophorum in BHI media supplemented with hemin for about 24 hours.

[0152] In some embodiments, the concentration of hemin is about 5 pg / mL.

[0153] In some embodiments, the method further comprises measuring the optical density of the F. necrophorum. In one embodiment, measuring the optical density is at 600 nm (OD600). In some embodiments, growing the F. necrophorum is stopped when the OD600 is about 0.6 to about 1.1.

[0154] In some embodiments, the method further comprises killing or inactivating the F. necrophorum. Methods of killing or inactivating F. necrophorum are known to the skilled person. For example, F. necrophorum is killed by exposure to formaldehyde at4°C for 24 hours as shown in Example 1 .

[0155] In some embodiments, the killed or inactivated F. necrophorum is formulated for administration as a bacterin. Methods of formulating a bacterin are known to the skilled person. For example, the killed or inactivated F. necrophorum is mixed with an adjuvant, such as Montanide™ gel.

[0156] In some embodiments, the method further comprises formulating the F. necrophorum for inoculation of a pig. In some embodiments, inoculation of the pig comprises an intradermal injection of F. necrophorum into the ear of the pig, optionally at a dose of about 104cfu / mL to about 1011cfu / mL. In some embodiments, inoculation of the pig comprises a dose of about 106cfu / mL.

[0157] A further aspect of the disclosure is a method of preparing a Fusobacterium necrophorum (F. necrophorum) bacterin, the method comprising:a) growing the F. necrophorum in brain heart infusion (BHI) media supplemented with about 5 pg / mL to about 25 pg / mL hemin for about 20 hours to about 48 hours; b) optionally, measuring the optical density of the F. necrophorum; c) inactivating or killing the F. necrophorum; and d) optionally, mixing the inactivated or killed F. necrophorum with an adjuvant

[0158] In some embodiments, the BHI media supplemented with hemin is anaerobic.

[0159] In some embodiments, the concentration of hemin is about 5 pg / mL.

[0160] In some embodiments, growing the F. necrophorum comprises culturing the F. necrophorum for about 24 hours.

[0161] In some embodiments, growing the F. necrophorum is stopped when the optical density at 600 nm is about 0.6 to about 1.1.

[0162] In some embodiments, the method further comprises formulating the inactivated or killed F. necrophorum for administration to a pig.

[0163] In some embodiments, the adjuvant comprises Montanide™ gel.

[0164] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.EXAMPLESExample 1 : Preparation of a Fusobacterium necrophorum bacterin

[0165] A bacterin formulation of Fusobacterium necrophorum (F. necrophorum) was prepared by growing F. necrophorum (biotype A) in BHI media for 96h at 37°C anaerobically to an optical density at 600nm of 1.1 (approximately 108cfu / mL). Next, bacterial culture was killed by exposure to formaldehyde at 4°C for 24 hours, washed in PBS 4 times and resuspended in PBS. This was then mixed with an adjuvant (Montanide(Seppic) Gel 02) at 15 antigen:85 adjuvant ratio and thoroughly mixed until an homogenous solution was formed. The vaccine was then stored at 4°C until use.Example 2: F. necrophorum bacterin protects against ETN

[0166] Healthy, 4-week old crossbred males (mean body weight 5.6 kg), free of ETN-like clinical signs (n = 18) were transported from the source farm and acclimatized in a BSL-2 facility for 7 days. Pigs were scored twice daily for any clinical abnormalities. This included body condition (change in weight), skin color, responsiveness (impaired responsiveness / alertness), and ear lesions. Ear lesions were scored as shown in Figure 3: 1 ) edema and hyperemia visible, initial lesions are closed and located at the tip of the ear or corresponding with the most distal end of the marginal ear vein; 2) the initial swelling erodes through the skin surface to form a scab; 3) advanced bilateral ear necrosis involving the tip and margins of both ears; 4) healed lesions with loss of ear helix. A score of 0 (not shown) represents a pig without ETN.

[0167] Two vaccine preparations were evaluated: 1- Fusogard™ (commercially distributed by Elanco, labelled for use in cattle for the prevention of liver abscesses) and 2- a bacterin formulation using F. necrophorum prepared as described in Example 1.

[0168] At 0 days post-first-dose (dpfd), pigs received 0.5 mL Fusogard™ subcutaneously (n=8) or 1 mL bacterin (n=7) intramuscularly. A second dose was applied on 21 dpfd. On 43 dpfd, the left ear of each pig was inoculated intradermally with 1 mL of a pure culture of F. necrophorum at 108CFU / mL. Presence or absence of necrosis (characterized as tissue loss at the day of necropsy) was compared between groups using Fisher’s exact test. Vaccine effectiveness was calculated as VE = incidence in unvaccinated-incidence in vaccinated incidence in unvaccinated

[0169] Mild, transient fever (<12h duration) was observed in 7 / 8 pigs following second Fusogard™ dose, and in 4 / 7 bacterin pigs, no other clinical signs were detected. All control / unvaccinated pigs developed necrosis and had tissue loss. Six out of 8 pigs vaccinated with Fusogard™ developed necrosis and tissue loss (P=0.46). Four out of 7 pigs vaccinated with the F. necrophorum bacterin had necrosis and tissue loss (P=0.056). In summary, Fusogard™ vaccine effectiveness was 25%, while the bacterin had an effectiveness of 43%. Figure 1 shows clinical progression of ETN was lessened in pigs vaccinated with Fusogard™ and the F. necrophorum bacterin.

[0170] By 7 days post-inoculation on the left ears (56 dpfd), all pigs had no lesions in any of the ears (T able 1 ).Table 1 : Ear necrosis scores following challenge with F. necrophorumDays post-inoculation 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16Example 3: Skin culture

[0171] Pinnae skin with no signs of dermatologic disease were sampled, from donor pigs, by an incision on the apex of pinnae, separating the cartilage from the dermis. Prior to sampling, the target site was cleaned with pressurized water to remove any slurry attached, dried with paper towels, rinsed with 70% ethanol and left to dry. Full thickness skin biopsies were surgically excised and transported for processing in refrigerated Phosphate-buffered saline (PBS, pH 7.4) within 90 minutes of euthanasia. Forceps and scissors were used to remove the hypodermis, resulting in explants with approximately 0.5 cm of thickness. Explants were cut into 1 cm x 1 cm sections. Petri dishes received 8 mL of culture media (KBM Gold Bullet Kit, Lonza, Breda, Netherlands) supplemented with 1.5 mMol of CaCI (Sigma, Zwijndrecht, Netherlands). Cell strainers with 75 pl pore-size(Thermo Fisher Scientific, Bremen, Germany) were used as an attachment matrix for the explants, that were then cultured at 37° Celsius on 5% CO2 gas mix for 48 hours.

[0172] The resulting explants may be used as an epithelial cell layer to screen for agents that prevent and / or treat ETN.

[0173] Exposure of a test compound and F. necrophorum to the keratinized surface of the skin will follow to evaluated potential skin damage. Explant samples are evaluated by histology, fluorescent microscopy, electron microscopy, gene expression by PCR or high-throughput sequencing, or cytokine levels by ELISA.Example 4: BHI media supplemented with hemin improved growth of F. necrophorum

[0174] Methods: F. necrophorum was cultured in BHI media supplemented with hemin (5 / g / mL), BHI media supplemented with 1 % FBS or BHI alone. All conditions were assessed in triplicate.

[0175] Results: BHI and BHI + 1 % FBS groups did not grow above an OD600 of 0. At 24 hours, the BHI + Hemin group had a mean±standard deviation of 0.594±0.214. By 36 hours, this group reached 1.096±0.258 (FIG. 1).

[0176] Supplementing BHI media with hemin accelerated the growth rate of F. necrophorum, reaching GD600 by 24 hours, and increased the inoculation success rate. (FIG. 1), compared to growth in BHI media, which achieved an GD600 by 96 hours (see Example 1 ). Further, supplementing BHI media with hemin facilitated real-time optical density measurements, which would otherwise not be possible with other supplements such as defibrinated blood.Example 5: Agitation of BHI media supplemented with hemin improved growth of F. necrophorum

[0177] Methods: F. necrophorum was cultured in BHI with hemin 5 / g / mL, either agitated (n=3) at 200rpm or static (n=3).

[0178] Results: At 24 hours, BHI supplemented with hemin (1X) without agitation had a mean±standard deviation of 0.0.083±0.031 , growing to 0.762±0.086 at 36 hours. BHI media supplemented with hemin (1X) with agitation grew to GD600 of 0.429±0.128 and 1.207±0.147 at 24 and 36 hours, respectively (FIG. 5).

[0179] Accordingly, agitation of BHI media supplemented with hemin demonstrated further growth acceleration than static BHI media supplemented with hemin (e.g. example 4).Example 6: Pigs inoculated with F. necrophorum grown in BHI media supplemented with hemin developed ear tip necrosis

[0180] Methods: Pigs (n=6) were sedated prior to challenge with dexmedetomidine (20 mg / kg), alfaxalone (4mg / kg). Following confirmation of sedation, the right ear of each pig was injected using a 9-pin, 32G, adjustable length needle (Panacea medical, South Korea) set at 2 mm, with 1 mL (106CFU) of a log-phase F. necrophorum culture in BHI supplemented with hemin. Animals were monitored for 14 days following inoculation.

[0181] Results: Within 40 minutes, all 6 pigs developed purple discoloration on the inoculated ear. By 24 hours post challenge, inflammation was observed. All inoculated pigs developed necrosis between 3-5 days post-challenge.

[0182] While the present disclosure has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples but should be given the broadest interpretation consistent with the description as a whole.

[0183] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Specifically, the sequences associated with each accession numbers provided herein including for example accession numbers and / or biomarker sequences (e.g. protein and / or nucleic acid) provided in the Tables or elsewhere, are incorporated by reference in its entirely.ReferencesCosta et al., “Partial reproduction of ear-tip necrosis suggests an infectious, initially bacterial aetiology”, Veterinary microbiology 262 (2021) 109246.Malik M., “Porcine ear necrosis: characterization of lesions and associated pathogens”, Veterinary Research (2023) 54:85.Papatsiros V. Ear necrosis syndrome in weaning pigs associated with PCV2 infection: A case report. Vet Res Forum. 2012 Summer;3(3):217-20.Park J, Friendship RM, Poljak Z, DeLay J, Slavic D, Dewey CE. An investigation of ear necrosis in pigs. Can Vet J. 2013 May;54(5):491-5.

Claims

CLAIMS:1 . Use of a composition comprising an agent that targets Fusobacterium necrophorum (F. necrophorum) and a pharmaceutically acceptable carrier for treating and / or preventing ear tip necrosis (ETN) in a pig.

2. Use of a composition comprising an agent that targets F. necrophorum and a pharmaceutically acceptable carrier in the manufacture of a medicament for treating and / or preventing ear tip necrosis (ETN) in a pig.

3. The use of claim 1 or 2, for preventing ETN.

4. The use of claim 1 or 2, for treating ETN.

5. The use of any one of claims 1 to 4, wherein the agent is an attenuated or an inactiveF. necrophorum, optionally Fusobacterium necrophorum subspecies necrophorum biotype A.

6. The use of any one of claims 1 to 5, wherein the agent is a F. necrophorum bacterin.

7. The use of claim 6, wherein the F. necrophorum bacterin is prepared by culturing F. necrophorum in brain heart infusion (BHI) media supplemented with about 5 pg / mL to about 25 pg / mL hemin for about 20 hours to about 48 hours.

8. The use of any one of claims 1 to 6, wherein the agent is Fusogard™.

9. The use of any one of claims 1 to 8, wherein the pharmaceutically acceptable carrier is a polymeric adjuvant, optionally the polymeric adjuvant is Montanide™ Gel.

10. The use of any one of claims 1 to 8, wherein the pharmaceutically acceptable carrier is oil-in water, water-in oil-in-water, or aluminum hydroxide.

11. The use of any one of claims 1 to 10, wherein the composition comprises a ratio of agent: pharmaceutically acceptable carrier of 15:85 v / v.

12. The use of any one of claims 1 to 11 , wherein the composition is for use in a single dose.

13. The use of any one of claims 1 to 11 , wherein the composition is for use in two doses, optionally wherein the two doses are 21 days apart.

14. The use of any one of claims 1 to 13, wherein the composition is for use intradermally, subcutaneously or intramuscularly.

15. The use of any one of claims 1 to 14, wherein the composition is for use at a volume of 0.5 mL to 2 mL, 0.5 mL to 1 mL, about 1 mL, or about 0.5 mL.

16. The use of any one of claims 1 to 15, wherein the composition is for use at 104cfu / mL to 1011cfu / mL, optionally the composition is for use at about 108cfu / mL.

17. The use of any one of claims 1 to 16, further comprising use of one or more further agents for treating ETN.

18. The use of claim 17, wherein the one or more further agents comprise an antibiotic, optionally the antibiotic is tetracycline, penicillin, and / or ampicillin.

19. The use of any one of claims 1 to 18, wherein the agent was previously obtained from an infected pig of the same herd.

20. A composition comprising an agent that targets F. necrophorum and a pharmaceutically acceptable carrier for use in treating and / or preventing ear tip necrosis (ETN) in a pig.21 . The composition for use of claim 20, for preventing ETN.

22. The composition for use of claim 20, for treating ETN.

23. The composition for use of any one of claims 20-22, wherein the agent is an attenuated or an inactive F. necrophorum, optionally Fusobacterium necrophorum subspecies necrophorum biotype A.

24. The composition for use of any one of claims 20-23, wherein the agent is a F. necrophorum bacterin.

25. The composition for use of claim 24, wherein F. necrophorum bacterin is prepared by culturing F. necrophorum in brain heart infusion (BHI) media supplemented with about 5 pg / mL to about 25 pg / mL hemin for about 20 hours to about 48 hours.

26. The composition for use of any one of claims 20 to 25, wherein the agent is Fusogard™.

27. The composition for use of any one of claims 20 to 26, wherein the pharmaceutically acceptable carrier is a polymeric adjuvant, optionally the polymeric adjuvant is Montanide™ Gel.

28. The composition for use of any one of claims 20 to 26, wherein the pharmaceutically acceptable carrier is oil-in water, water-in oil-in-water, or aluminum hydroxide.

29. The composition for use of any one of claims 20 to 28, wherein the composition comprises a ratio of agent:pharmaceutically acceptable carrier of 15:85 v / v.

30. The composition for use of any one of claims 20 to 29, wherein the composition is for use in a single dose.31 . The composition for use of any one of claims 20 to 29, wherein the composition is for use in two doses, optionally wherein the two doses are 21 days apart.

32. The composition for use of any one of claims 20 to 31 , wherein the composition is for use intradermally, subcutaneously or intramuscularly.

33. The composition for use of any one of claims 20 to 32, wherein the composition is for use at a volume of 0.5 mL to 2 mL, 0.5 mL to 1 mL, about 1 mL, or about 0.5 mL.

34. The composition for use of any one of claims 20 to 33, wherein the composition is for use at 104cfu / mL to 1011cfu / mL, optionally the composition is for use at about 108cfu / mL.

35. The composition for use of any one of claims 20 to 34, further comprising use of one or more further agents for treating ETN.

36. The composition for use of claim 35, wherein the one or more further agents comprise an antibiotic, optionally the antibiotic is tetracycline, penicillin, and / or ampicillin.

37. The composition for use of any one of claims 20 to 36, wherein the agent was previously obtained from an infected pig of the same herd.

38. A method for screening for an agent that prevents ear tip necrosis (ETN), the method comprising: a) administering, at least once, a test compound, to a pig; b) inoculating the pig with Fusobacterium necrophorum (F. necrophorum)', and c) scoring at least one clinical sign of ETN to evaluate if the test compound is effective for preventing ETN in the pig; wherein the test compound is identified as an agent that prevents ETN if the pig has a reduction in at least one clinical sign of ETN compared to a control.

39. The method of claim 38, wherein the at least one clinical sign of ETN comprises: change in weight, abnormal skin colour, impaired responsiveness, edema, hyperemia, lesions, scabbing, necrosis, or tissue loss.

40. The method of claim 38 or 39, wherein administering, at least once, comprises administering the test compound in two doses.41 . The method of claim 40, wherein the two doses are 21 days apart.

42. The method of any one of claims 38 to 41 , wherein step b) inoculating the pig with F. necrophorum is 14 days after step a) administering, at least once, the test compound to the pig, optionally inoculating the pig with F. necrophorum is 14 days after administering a second dose of the test compound.

43. The method of any one of claims 38 to 42, wherein step c) scoring at least one clinical sign of ETN is at least 1 day to at least 20 days after the step of inoculating the pig with F. necrophorum.

44. A method for screening for an agent that treats ear tip necrosis (ETN), the method comprising: a) inoculating a pig with Fusobacterium necrophorum (F. necrophorum)', b) administering, at least once, a test compound to the pig; and c) scoring at least one clinical sign of ETN to evaluate if the test compound is effective for treating ETN in the pig; wherein the test compound is identified as an agent that treats ETN if the pig has a reduction in at least one clinical sign of ETN compared to a control.

45. The method of claim 44, wherein the at least one clinical sign of ETN comprises: change in weight, abnormal skin colour, impaired responsiveness, edema, hyperemia, lesions, scabbing, necrosis, or tissue loss.

46. The method of claim 44 or 45, wherein administering, at least once, comprises administering the test compound in two doses.

47. The method of claim 46, wherein the two doses are 21 days apart.

48. The method of any one of claims 44 to 47, wherein step a) inoculating the pig with F. necrophorum and step b) administering, at least once, the test compound to the pig, are at least one day apart.

49. The method of any one of claims 44 to 48, wherein step c) scoring at least one clinical sign of ETN is at least 1 day to at least 20 days after the step of administering, at least once, the test compound to the pig.

50. The method of any one of claims 44 to 49, wherein inoculating the pig comprises an intradermal injection of F. necrophorum into the ear of the pig, optionally at a dose of 108cfu / mL.51 . An in vitro method for screening an agent that prevents ear tip necrosis (ETN), the method comprising: a) contacting an epithelial layer of cells with a test compound;b) culturing the epithelial layer of cells with Fusobacterium necrophorum (F. necrophorum)-, and c) measuring tissue viability and / or stress response in the epithelial layer of cells to evaluate if the test compound is effective for preventing ETN; wherein the test compound is identified as an agent that prevents ETN if the epithelial layer of cells has improved tissue viability and / or stress response compared to a control epithelial layer of cells without the test compound.

52. An in vitro method for screening an agent that treats ear tip necrosis (ETN), the method comprising: a) culturing an epithelial layer of cells with Fusobacterium necrophorum (F. necrophorum)-, b) contacting the epithelial layer of cells with a test compound; and c) measuring tissue viability and / or stress response in the epithelial layer of cells to evaluate if the test compound is effective for treating ETN; wherein the test compound is identified as an agent that treats ETN if the epithelial layer of cells has improved tissue viability and / or stress response compared to a control cell without the test compound.

53. The method of claim 51 or 52, wherein the epithelial layer of cells is a primary skin culture or an explant culture.

54. The method of any one of claims 51 to 53, wherein measuring cell viability and / or stress response comprises histology, electron microscopy, fluorescence microscopy, enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), or high- throughput sequencing.

55. The method of any one of claims 38 to 54, wherein the F. necrophorum is prepared by culturing F. necrophorum in brain heart infusion (BHI) media supplemented with about 5 pg / mL to about 25 pg / mL hemin for about 20 hours to about 48 hours.

56. A method of culturing Fusobacterium necrophorum (F. necrophorum), comprising growing the F. necrophorum in brain heart infusion (BHI) media supplemented with about 5 pg / mL to about 25 pg / mL hemin for about 20 hours to about 48 hours.

57. The method of claim 56, further comprising agitating the F. necrophorum.

58. The method of claim 56 or 57, wherein growing the F. necrophorum comprises culturing the F. necrophorum for about 24 hours.

59. The method of any one of claims 56-58, wherein the concentration of hemin is about 5 pg / mL.

60. The method of any one of claims 56-59, further comprising measuring the optical density (OD) of the F. necrophorum, optionally stopping growth of the F. necrophorum when the OD at 600nm is about 0.6 to about 1.1.61 . The method of any one of claims 56-60, further comprising killing or inactivating the F. necrophorum.

62. The method of claim 61 , wherein the killed or inactivated F. necrophorum is formulated for administration as a bacterin.

63. The method of any one of claims 56-60, wherein the F. necrophorum is formulated for inoculation of a pig.

64. The method of claim 63, wherein inoculation of the pig comprises an intradermal injection of F. necrophorum into the ear of the pig, optionally at a dose of about 104cfu / mL to about 1011cfu / mL, further optionally at a dose of 106cfu / mL.

65. A method of preparing a Fusobacterium necrophorum (F. necrophorum) bacterin, the method comprising: a) growing the F. necrophorum in brain heart infusion (BHI) media supplemented with about 5 pg / mL to about 25 pg / mL hemin for about 20 hours to about 48 hours; b) optionally, measuring the optical density of the F. necrophorum;c) inactivating or killing the F. necrophorum; and d) optionally mixing the F. necrophorum with an adjuvant.

66. The method of claim 65, wherein the concentration of hemin is about 5 pg / mL.

67. The method of claim 65 or 66, wherein growing the F. necrophorum comprises culturing for about 24 hours.

68. The method of any one of claims 65-67, wherein growing the F. necrophorum is stopped when the optical density at 600nm is about 0.6 to about 1.1.

69. The method of any one of claims 65-68, further comprising formulating the inactivated or killed F. necrophorum for administration to a pig.