Treatment of spotty liver disease

Abstract

The disclosure relates to the identification and characterisation of a cytotoxin from Campylobacter hepaticus and use thereof for treating Spotty Liver Disease (SLD) in avian animals.

Description

[0001] "Treatment of Spotty Liver Disease"

[0002] Incorporation by reference

[0003] All documents cited or referenced herein, and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference in their entirety.

[0004] This specification claims priority to Australian provisional patent application no. 2024903957 filed 29 November 2024, the entire contents of which are herein incorporated by reference.

[0005] The entire content of the electronic submission of the sequence listing is incorporated by reference in its entirety for all purposes.

[0006] Field

[0007] The disclosure relates to the identification and characterisation of a cytotoxin from Campylobacter hepaticus and use thereof for treating Spotty Liver Disease (SLD) in avian animals (e.g. poultry).

[0008] Background

[0009] With the constant increase of the world’s population, there is a growing need to provide more high-quality affordable protein to feed the population. The rapidly expanding poultry industry produces meat and eggs to address this ever-growing need. The size of the Australian layer industry increased nearly 2-fold between 2005 and 2021 (13 to 22 million hens), and Australian egg farmers produce 18.3 million eggs every day; 6.7 billion eggs each year. Similarly, the global production of eggs is also rapidly increasing. The world egg production increased 39%, between 2011 and 2021. Importantly, the increase in egg production has also been associated with a change in primary production systems, with cage free eggs now accounting for almost two-thirds of all eggs produced worldwide.

[0010] The move from cages to free range and barn systems has coincided with increasing incidences of Spotty Liver Disease (SLD). SLD is characterised by liver damage including multifocal, 1 -2 mm grey-white lesions and is now considered one of the most significant bacterial diseases of layer flocks. It can cause reductions in egg production of between 10 and 35% and mortalities of up to 15% in affected flocks (Sevoian M et al., (1958) Avian Diseases 2:348). SLD is a global concern and, given the costs of this disease have been estimated to range between $5,000 to $43,000 per 10,000 birds (Courtice JM et al., (2020) AVPA Scientific Meeting), the cost of SLD can be extrapolated up to 95 million dollars for the Australian poultry industry each year and at least 16 billion dollars for the 33 billion chickens used for egg production globally. With the phase-out date of caged eggs within the next 10 years, a solution for the poultry industry regarding SLD is urgently needed.

[0011] The causative agent of SLD remained elusive until 2015 when it was demonstrated that a novel Campylobacter could be isolated from infected flocks in England and parenteral administration of this organism could induce micro-foci of pathology similar to that observed in the field. Subsequently, the causative agent was characterized as Campylobacter hepaticus in Australia (Van TTH et al., (2016) Ins J Syst Evol Microbiol 66:4518) and Koch’s postulates were finally satisfied with the demonstration that this agent could induce what is now recognised as SLD.

[0012] Currently, the only efficacious treatment for C. hepaticus infection is antibiotic therapy, something that the global food production market is seeking to reduce to help prevent the development of antimicrobial resistance in avians. Therefore, the animal production industries are looking for alternative disease treatments so that antibiotic use can be reduced.

[0013] SLD causes significant problems for the poultry industry. Simple vaccines (killed whole bacterin) are not effective in preventing SLD and despite many years of research into vaccines against other Campylobacters, progress has been slow. Currently, there is no effective vaccine or other prevention approach for SLD or any other Campylobacter in poultry.

[0014] There is a need in the art for a vaccine which is able to vaccinate poultry against SLD and therefore is able to reduce the economic burden of this disease.

[0015] Summary of the disclosure

[0016] The present disclosure is based on the finding that Campylobacter hepaticus produces a cytotoxin that can damage host chicken liver cells. C. hepaticus is the cause of Spotty Liver Disease (SLD. When expressed recombinantly, the cytotoxin exhibited significant cytotoxic effects on leghorn male hepatoma (LMH) cells, a proxy for liver cells. However, this cytotoxicity could be neutralised when the cytotoxic protein was fused at its N-terminus to a small ubiquitin modifying (SUMO) protein.

[0017] In one aspect, there is provided a protein or antigenic portion thereof from Campylobacter hepaticus for use or when used to treat or prevent Spotty Liver Disease (SLD) in an avian animal. In one example, the protein in its native form is cytotoxic.

[0018] In one example, the protein in its native form is cytotoxic to avian liver cells, more particularly to chicken liver cells. In one example, the chicken liver cells are a hepatic carcinoma cell line. In one example, the hepatic carcinoma cell line is the chicken liver cell line LMH. In one example, the protein comprises or consists of the sequence of SEQ ID NO:1 or a sequence at least 90% identical thereto, wherein SEQ ID NO:1 has the sequence set forth in: ADGATLFKKCAICHGAKAEKVYLNKVPALNTLSAEEITQALKEYKAGTRNKFGQGGIMKIQLTSL SEADLEAIAQYIQTLK.

[0019] In another example, the cytotoxic protein comprises or consists of a sequence having at least 91 %, 92%, 95%, 97% or 99% identity to SEQ ID NO:1 across its full length. In a further example, the cytotoxic protein comprises or consists of the sequence of SEQ ID NO:1 .

[0020] In one example, the protein is modified relative to its native form by substitution of the amino acid residue at position 14 histidine (H) to a leucine (L) to attenuate the cytotoxic activity of the native protein.

[0021] In one example, the protein comprises or consists of the sequence set forth in: ADGATLFKKCAICLGAKAEKVYLNKVPALNTLSAEEITQALKEYKAGTRNKFGQGGIMKIQLTSL SEADLEAIAQYIQTLK (SEQ ID NO:2).

[0022] In some examples, the cytotoxic protein described herein further comprises a second protein which assists in maintaining solubility of the cytotoxic protein. Preferably, the second protein is a protein which does not interfere with the ability of the cytotoxic protein antigen to elicit an immune response in the avian animal.

[0023] The skilled person will appreciate that in some examples, the second protein may be any protein which assists in the isolation and purification of the cytotoxic protein. Suitable examples include a small ubiquitin modifying (SUMO) protein, maltose binding protein (MBP), glutathione S-transferase (GST) or histidine tag (e.g. His6). In one example, the SUMO protein comprises the sequence set forth in SEQ ID NO:3:

[0024] DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFL YDGIRIQADQAPEDLDMEDNDIIEAHREQIGG (SEQ ID NO:3).

[0025] In some examples, cytotoxic protein described herein is produced in recombinant form. In some examples, the protein is produced as a SUMO fusion protein.

[0026] In a further example, the protein comprises the sequence of SEQ ID NO:4 or a sequence having at least 90% identity thereto, wherein SEQ ID NO:4 comprises or consists of the sequence set forth in: MNWSHPQFEKSSGSSGGHHHHHHGGSGGSGSDSEVNQEAKPEVKPEVKPETHINLKVSDG SSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQI GGADGATLFKKCAICHGAKAEKVYLNKVPALNTLSAEEITQALKEYKAGTRNKFGQGGIMKI

[0027] QLTSLSEADLEAIAQYIQTLK (SEQ ID NO:4) wherein the N-terminal sequence MNWSHPQFEKSSGSSGG is a leader sequence, HHHHHH is a histidine tag, the sequence GGSGGSGS is a linker sequence, the SUMO sequence is underlined and the cytotoxic protein is in bold. In another aspect, the disclosure provides a vector comprising the nucleic acid sequence encoding the sequence comprising or consisting of the sequence of SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:4.

[0028] The cytotoxin protein and second protein (e.g. SUMO protein) may be fused, linked or conjugated together using standard methods known in the art. In some examples, the second protein is fused to the N-terminus of the cytotoxic protein. In some examples, the second protein is fused to the C-terminus of the cytotoxic protein.

[0029] In some examples, the cytotoxin protein is synthetically produced. In some examples, the cytotoxin protein is recombinantly produced.

[0030] In some examples, the cytotoxic protein comprises an antigenic portion of SEQ ID NO:1 . The antigenic portion may be any fragment that consists of 15, 20, 25, 30, 40, 50, or 75 or greater contiguous amino acids of the amino acid sequence of SEQ ID NO:1 . The antigenic portion may correspond to a fragment having at least 90,% 92%, 95%, 97%, 99% or 100% identity. In some examples, the antigenic portion is able to elicit an immune response but is not cytotoxic to chicken liver cells.

[0031] In some examples, the cytotoxic protein is a fusion protein comprising the cytotoxic protein and an antigen derived from Campylobacter. In one example, the antigen is a cell surface antigen derived from C. hepaticus or C. jejuni. In another example, the antigenic protein is selected from the group consisting of flagellin, Campylobacter ad hesins, C. jejuni PEB1 protein, C. jejuni PEB3, protein membrane -associated protein A, Omp18, CjA, Pal, Peb1A, Cj0404, RpIL, Tuf and Cbf2 / Peb4A. In one example, the antigen derived from Campylobacter is CjaA.

[0032] In one example, the fusion protein comprises or consists of the sequence set forth in SEQ ID NO:5 or a sequence at least 90% identical thereto.

[0033] MKK / I / I / LALAGLYLAFSASAADQNATGGDQNATGGDQNATGGDQNATDSKTLNSLDKIKQNGV VRIGVFGDKPPFGYVDEKGNNQGYDIALAKRIAKELFGDENKVQFVLVEAANRVEFLKSNKVDI ILANFTQTPQRAEQVDFCLPYMKVALGVAVPKDSNITSVEDLKDADGATLFKKCAICLGAKAE KVYLNKVPALNTLSAEEITQALKEYKAGTRNKFGQGGIMKIQLTSLSEADLEAIAQYIQTLKKT LLLNKGTTADAYFTQNYPNIKTLKYDQNTETFAALMDKRGDALSHDNTLLFAWVKDHPDFKMG IKELGNKDVIAPAVKKGDKELKEFIDNLIIKLGQEQFFHKAYDEILKAHFGDDVKADDVVIEGGKI RSGGLKDQNATGGDQNATGGDQNATGGDQNATHHHHHH* (SEQ ID NO:5) wherein the sequence in bold corresponds to the cytotoxic protein. HHHHHH is a histidine tag. The italicised region is the leader sequence, the underlined sequence represents CjaA and the remainder of the sequence (ADQNATGGDQNATGGDQNATGGDQNATD and

[0034] GGLKDQNATGGDQNATGGDQNATGGDQNAT) are targets of protein glycosylation.

[0035] In another aspect, the disclosure provides a vector comprising the nucleic acid sequence encoding the sequence comprising or consisting of the sequence of SEQ ID NO:5. In another aspect, there is provided an immunogenic composition comprising the cytotoxic protein or fusion protein comprising the cytotoxic protein described herein, for example the cytotoxic protein according to SEQ ID NO:1 , SEQ ID NO:2 or SEQ ID NO:5.

[0036] In another aspect, there is provided an immunogenic composition comprising the cytotoxic protein or fusion protein comprising the cytotoxic protein described herein for use or when used to treat or prevent Spotty Liver Disease (SLD) in an avian animal. In certain examples, the SLD is caused by C. hepaticus and / or C. bills. Preferably, the composition comprises one or more of a physiologically acceptable diluent, excipient, adjuvant or carrier. In some examples, the avian animal is a poultry animal.

[0037] In some examples, the immunogenic composition further comprises an antigenic protein which is a Campylobacter cell surface antigen derived from C. hepaticus or C. jejuni. In another example, the antigenic protein is selected from the group consisting of flagellin, Campylobacter adhesins, C. jejuni PEB1 protein, C. jejuni PEB3, protein membrane -associated protein A, Omp18, CjA, Pal, Peb1A, Cj0404, RpIL, Tuf and Cbf2 / Peb4A. In some examples, the antigenic protein is provided concurrently with the cytotoxic protein composition.

[0038] In some examples, the immunogenic composition is a vaccine.

[0039] In some examples, the compositions described herein may further comprise an antigenic protein derived from Campylobacter, Salmonella or Influenza.

[0040] In another aspect, there is provided a polynucleotide sequence encoding the mature cytotoxic protein of SEQ ID NO:1 or SEQ ID NO:2 or encoding an antigenic portion thereof. In a particular example, the polynucleotide comprises or consists of the sequence set forth in: TTTTAAGGTT TGAATATATT GAGCAATAGC TTCTAAATCA GCTTCACTTA AAGAAGTCAA TTGTATTTTC ATAATACCGC CTTGACCAAA TTTATTTCTT GTACCTGCTT TATATTCTTT AAGCGCTTGA GTGATTTCTT CTGCACTTAA AGTATTAAGA GCTGGAACTT TATTTAAATA AACTTTTTCA GCTTTTGCAC CATGACATAT AGCGCATTTT TTAAATAAAG TAGCACCGTC TGC (SEQ ID NO:6).

[0041] In another aspect, there is provided a vector comprising a polynucleotide sequence encoding the sequence of the cytotoxic protein or fusion protein comprising the cytotoxic protein described herein. The vector may be any vector that can conveniently subjected to recombinant DNA procedures and can bring about expression of the nucleic acid molecule described herein. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. In some examples, the vector is suitable for use in avians and poultry. In one example, the vector is a fowl poxvirus (FPV), fowl adenovirus (FAdV), Newcastle disease virus (NDV), Marek’s disease virus (MDV) and herpesvirus of turkey (HVT). In some examples, the vector is selected from Trovac®-NDV, Trovac® Al H5, Vectormune® FP LT, Newxxitek™ HVT + ND, VAXXITEK® HVT + IBD + ILT, and Innovax® ND-IBD. In one example, the vector comprises a polynucleotide sequence of SEQ ID NO:6 or encodes a sequence selected from of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. The vector preferably comprises further regulatory elements for driving expression of the polynucleotide sequence. In one example, the vector comprises a promoter for driving expression of the polynucleotide.

[0042] In another aspect, there is provided a carrier cell comprising the vector described herein. In one example, the carrier cell is a bacteria, such as Salmonella spp., Lactobacillus spp, Escherichia spp, Lactococcus spp. etc. In a further example the carrier is a live attenuated bacteria. In one example, the carrier cell is transformed with the vector. In some examples, the vector is for use or when used to treat or prevent SLD caused by C. hepaticus.

[0043] In another aspect, there is provided a vaccine comprising the cytotoxic protein or fusion protein comprising the cytotoxic protein described herein. In one example, the vaccine is administered together with an adjuvant.

[0044] In another aspect, there is provided use of the cytotoxic protein, fusion protein comprising the cytotoxic protein, composition, vector or vaccine described herein in the manufacture of a vaccine for treating or preventing SLD caused by C. hepaticus. In certain examples, the cytotoxic protein, fusion protein comprising the cytotoxic protein, composition or vector described herein further comprise one or more of a physiologically acceptable diluent, excipient, adjuvant or carrier.

[0045] In another aspect, there is provided the cytotoxic protein, composition, vector or vaccine described herein for use in treating or preventing SLD caused by C. hepaticus.

[0046] In some examples, the compositions and vaccines described herein may be combined with another avian vaccine, preferably a Campylobacter \ / acc'\re or a Salmonella vaccine. In other example, the compositions and vaccines described herein may be combined with an influenza vaccine. The vaccines may be formulated for sequential or combined administration.

[0047] In further examples, the cytotoxic protein, fusion protein comprising the cytotoxic protein, composition, vector or vaccine described herein may be combined with an antibiotic and / or immunomodulatory agent.

[0048] In another aspect, there is provided a method for inducing an immune response to SLD in an avian animal, the method comprising administering to the animal the cytotoxic protein, fusion protein comprising the cytotoxic protein, composition, vector or vaccine described herein.

[0049] In another aspect, there is provided a method of vaccinating an avian animal against SLD, the method comprising administering to the animal the cytotoxic protein, fusion protein comprising the cytotoxic protein, composition, vector or vaccine described herein. Preferably, the cytotoxic protein, fusion protein, composition or vector is provided in a therapeutically effective amount. In another aspect, there is provided a method for treating or preventing SLD in an avian animal, the method comprising administering to the animal the cytotoxic protein or antigenic portion thereof, composition, vector or vaccine as described herein.

[0050] In certain examples according to the methods herein, the cytotoxic protein or antigenic portion thereof, fusion protein comprising the cytotoxic protein, composition, vector or vaccine is administered in a therapeutically effective amount. In certain examples, the methods reduce the number of liver spots compared to the number of liver spots present in unvaccinated avians. In certain examples, the methods described herein maintain or reduce avian mortality compared to avians infected with Campylobacter, particularly Campylobacter hepaticus. In certain examples, the methods described herein maintain normal egg production in avians compared to avians infected with Campylobacter, particularly Campylobacter hepaticus.

[0051] The proteins, compositions, vectors or vaccines described herein may be formulated for administration by any suitable means. Administration routes may include orally, as injectables, parentally, by inhalation spray, intranasally, rectally, mucosally, topically, or for administration by oral gavage or ad libitum feeding, for example, in drinking water, either as liquid solutions or suspension, in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. In some examples, the vaccine is sprayed onto shells prior to hatching or provided in the feed.

[0052] In some examples, parenteral administration includes subcutaneous, intravenous, intraarterial, intramuscular, intrasternal, intratendinous, intravitreal, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneally. In some examples, the proteins, compositions, vectors and vaccines are administered as a first dose followed by a booster dose. In some examples, the proteins, compositions, vectors or vaccines may be in a solid form suitable for solution in, or suspension in, liquid prior to injection. The proteins, compositions, vectors or vaccines may also, for example, be emulsified, or be encapsulated in liposomes or microparticles. The proteins, compositions, vectors or vaccines can also be formulated for addition to livestock feed, feed additives or water, and for administration to an avian animal.

[0053] In one example, the avian animal is a poultry animal. The avian may be selected from the group consisting of a chicken, a turkey, duck, geese, quail, and pheasant. In one example, the avian is a chicken. In one example, the avian is a broiler chicken. In one example, the avian chicken is a layer chicken.

[0054] Description of the Figures

[0055] Figure 1 shows LDH cytotoxicity assay of C. hepaticus HV10Ttoxin on LMH cells. Briefly, 1 x 105cells / well were treated with mature / native (SUMO tagged cleaved) and fusion (SUMO tagged) from 5-100 pg for 24 hrs and assayed according to the manufacturer’s specifications. For C. jejuni and C. hepaticus HV10Tsamples, supernatant was concentrated 200x times using a Vivispin 3 kDa centrifuge unit and diluted to 10% in complete Waymouth media. For the heat inactivated control, 30pg of mature toxin was heated to 100°C for 10 min before treatment. For the buffer control, LMH cells were treated with the toxin purification buffer (20 mM Tris-HCI, 150 mM NaCI, pH 7.4). Finally, mature toxin (30 pg) was treated with 50 pl of proteinase K (PK) for 2 hours at 37°C. The assay was completed in triplicates (n=3). Unpaired t-test using Welch’s correction was used at P=0.05 was used to measure statistical significance against the heat inactivated mature toxin control. For treatments which were not normally distributed, a Mann Whitney’s t-test was implemented. The mature proteins (with the SUMO tag removed) showed a significant increase in cytotoxic effects on LMH cells compared to the control group (heat- inactivated mature toxin).

[0056] Figure 2 shows LDH cytotoxic assay of LMH cells treated with mature C. hepaticus HV10Ttoxin pre-incubated with rabbit-derived polyclonal antibodies (pAb) to measure neutralization of cytotoxic effect of toxin. Mature toxin and pAb were pre-incubated for 4 h at 4°C in native purification buffer (20 mM Tris-HCI pH 7.5, 150 mM NaCI) at the following ratios: 10 pg mature toxin, 10 pg pAb, 10 pg mature toxin:5 pg pAb and 10 pg mature toxin:2.5 pg pAb. Mixtures were diluted in complete Waymouth medium (Waymouth media, 10% Fetal Bovine Serum and 1 % Penicillin-Streptomycin) and applied to LMH cells (1 x 105cells / well) for 24 hours. LDH release was measured according to the kit manufacturer’s instructions. LMH cells were treated with 5 and 10 pg of pAb to ensure there was no cytotoxic effect of the pAb. For the positive control, LMH cells were treated with 10 pg of mature toxin to obtain the highest cytotoxicity level. SUMO tagged / fusion toxin (2.5 pg) and toxin purification buffer (20 mM Tris-HCI pH 7.5, 150 mM NaCI) are negative controls. Results are based on three independent LDH assays, completed in triplicate (n=3). Unpaired t-test using Welch’s correction was used at P=0.05 was used to measure statistical significance against the heat inactivated mature toxin control. For treatments which were not normally distributed, a Mann Whitney’s t-test was implemented. The cytotoxic activity was neutralised by the polyclonal antibody raised against the recombinant cytotoxic protein when the 5 ug or more mature of the mature toxin was used in the assay.

[0057] Figure 3 shows protection against spotty liver disease in layer chickens. Data is from 2 independent trials. Positive controls n=24 in both trials and all other groups n=10-12. Figures show lesion scores recorded macroscopically at necropsy using the following scale; 0=no lesions, 1 =1-5 lesions, 2=6-20 lesions, 3=21 -100 lesions, 4=101 -1000 lesions and 5=1000+ lesions. Black bar represents the average for each group. Groups were subject to one-way ANOVA with Tukey’s multiple comparison test with * p<0.05, ** p>0.01 and *** p>0.001 . Group names denotes which antigen was used to vaccinate the animals priorto experimental challenge.

[0058] The no challenge control group received adjuvant only mock vaccinations.

[0059] Figure 4 shows ELISA results using pooled serum (n= 1 1-12) from vaccinated birds. Wells of a 96-well plate were coated with 25 ng of protein: purified 209 (CjaA+Toxin), 209* (CjaA only) and toxin only. Serum came from groups vaccinated with 209 (CjaA+Toxin), 209* (CjaA only), toxin only and adjuvant only and were collected immediately prior to pathogen challenge. Results are marked with a ‘V’ showing from which vaccinated group the sera was derived and a ‘P’ showing what protein coated the corresponding well. Assays were performed at triplicate and TMB stopped with 0.5 M sulphuric acid was used to generate colorimetric results which were read at 450 nm. Groups with different letters are significantly different to one another (p<0.05).

[0060] Sequences

[0061] SEQ ID NO:1 amino acid sequence of the cytotoxin protein

[0062] SEQ ID NO:2 amino acid sequence of the attenuated cytotoxic protein

[0063] SEQ ID NO:3 amino acid sequence of the small ubiquitin modifying (SUMO) protein.

[0064] SEQ ID NO:4 amino acid sequence of the cytotoxin-SUMO fusion protein.

[0065] SEQ ID NO:5 amino acid sequence of the fusion protein

[0066] SEQ ID NO:6 polynucleotide sequence of the mature cytotoxic protein.

[0067] Detailed Description

[0068] General

[0069] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs (e.g., molecular biology, vaccinology, microbiology, RNA or DNA detection, pharmacology, protein chemistry, and biochemistry). Any materials and methods similar or equivalent to those described herein can be used to practice or test the present disclosure. Practitioners are particularly directed to Ausubel et al., Current Protocols in Molecular Biology, Supplement 47, John Wiley & Sons, New York, 1999; CoIowick and Kaplan, eds., Methods In Enzymology, Academic Press, Inc.; Weir and Blackwell, eds., Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications, 1986; Remington's Pharmaceutical Sciences (18th ed., Mack Easton, Pa. (1990)), for definitions and terms of the art and other methods known to the person skilled in the art.

[0070] Throughout this specification, unless specifically stated otherwise, orthe context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, group of steps, or group of compositions of matter.

[0071] Those skilled in the art will appreciate that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0072] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally- equivalent products, compositions and methods are clearly within the scope of the disclosure.

[0073] Any example disclosed herein shall be taken to apply mutatis mutandis to any other example unless specifically stated otherwise.

[0074] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0075] A list or features including the phrase “and / or” between the second last and last feature means that any one or more of the listed features may be present in any combination.

[0076] Reference to the singular forms “a”, “an” and “the” is also understood to imply the inclusion of plural forms unless the context dictates otherwise.

[0077] As used in this disclosure, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or”. That is, unless specified otherwise, or clear from context, “X employs A or B” is intended to mean any of the natural inclusive permutations. That is, if X employs A; X employs B; or X employs both A and B, then “X employs A or B” is satisfied under any of the foregoing instances. Further, at least one of A and B and / or the like generally means A or B or both A and B.

[0078] The term “sequence identity” as used herein means the percentage of identical nucleotide or amino acid residues at corresponding portions in two or more sequences when sequences are aligned to maximise sequence matching, i.e. taking into account gaps and insertions. Identity can be readily calculated using known methods, including, but not limited to those described in Computational Molecular Biology, Lesk AM ed. Oxford University Press New York, 1988; Computer Analysis of Sequence data, Part I Griffin AM and Griffin HG eds., Humana Press, New Jersey, 1994; Sequence analysis in molecular biology, von Heinje G, Academic Press, New Jersey, 1994). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determined identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequence include, but are not limited to, the GCG program package, BLASTP, BLASTN and FASTA. The well-known Smith Waterman algorithm may also be used to determine identity.

[0079] All documents cited or referenced herein, and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference in their entirety.

[0080] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally- equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

[0081] Selected Definitions

[0082] The term “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0083] The term “mature cytotoxic protein” as used herein refers to a derivative of the initial translation product that has the secretion signal removed.

[0084] The term “pharmaceutically acceptable carrier” is understood to be a compound that does not adversely affect the health of the animal to be vaccinated at least not to the extent that the adverse effect is worse than the effects seen when the animal is not vaccinated. A pharmaceutically acceptable carrier can be e.g. sterile water or a sterile physiological salt solution. In a more complex form, the carrier can e.g. be a buffer. The term “fragment” or “antigenic fragment” as used herein refers to a peptide that has an amino-terminal and / or carboxy-terminal and / or internal deletion, but where the remaining amino acid sequence is identical to the corresponding positions of the sequence of the full length protein (e.g. a protein according to SEQ ID NO:1). It will be appreciated that for inducing an immune response and in general for vaccination purposes, a protein need not be full length nor have all of its wild type function, and fragments of the protein are equally useful. The person skilled in the art will appreciate that changes can be made to a protein e.g. by amino acid substitutions, deletions, additions etc. using routine molecular biological procedures. Generally, conservative amino acid substitutions may be applied without loss of function or immunogenicity of the polypeptide. This can easily be checked according to routine procedures known to persons skilled in the art.

[0085] The term “immunogenic composition” as used herein refers to the ability of a substance (e.g. cytotoxic protein described herein) to produce an immune response in an avian animal. The term “isolated” as referred to herein refers to a polynucleotide which has been removed from its natural surrounding nucleic acid sequences. Such isolated nucleic acid sequences may comprise additional sequences useful for promoting expression and / or purification of the encoded protein, including, but not limited to poly A sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, secretory signals, nuclear localisation signals and plasma membrane localisation signals as appropriate for a given use.

[0086] It will be understood that reference to “protein” includes isolated proteins. Protein refers to a protein of recombinant or synthetic origin or some combination thereof, which by virtue of its origin or source of derivation the protein is not associated with proteins found in nature, is free of other proteins from the same source, is expressed by a cell from a different species or does not occur in nature.

[0087] The term “live, attenuated” refers to a vaccine in which the bacterial carrier host has reduced virulence but remains replication competent. Ways of creating attenuated bacteria are known to persons skilled in the art. One example includes repeated passage in tissue culture. Other methods include growing the bacteria in sub-optimal culture methods such as altering the temperature requirements or culture medium.

[0088] As used herein the term “treating”, “treat” or “treatment” include administering a therapeutically effective amount of the immunogenic composition or vaccine according to the present disclosure sufficient to reduce or eliminate at least one symptom of SLD. In one example, the treatment involves administering a therapeutically effective amount of the immunogenic composition or vaccine to treat or prevent SLD. In one example, treatment also refers to prophylactic treatment.

[0089] As used herein, the terms “preventing”, “prevent” or “prevention” include administering a therapeutically effective amount of the immunogenic composition of the present disclosure sufficient to stop or hinder the development of at least one symptom of SLD.

[0090] The term “therapeutically effective amount” refers to an amount of therapeutic agent that when administered alone or in combination with another therapeutic agent to a cell, tissue or subject is effective to prevent or ameliorate the disease condition or the progression of the disease.

[0091] Spotty Liver Disease

[0092] Spotty Liver Disease (SLD) presents as multiple grey / white lesions in the liver and reduction in egg output. It is prevalent within the layer industry, particularly the free-range sector of the industry. The clinical signs include a brief period of depression in laying birds. Often birds are found dead without any prior clinical signs of disease. The causative agents of SLD include Camplyobacter hepaticus (Van TTH et al, (2016) Int. J. Syst. Evol. Microbiol., 66:4518) and Campylobacter bills (Van TTH et al., (2023) Veterinary Microbiology vol 276:109603).

[0093] The present disclosure is based on the finding that a cytotoxin produced by Campylobacter hepaticus can damage host chicken liver cells. C. hepaticus is the major cause of Spotty Liver Disease (SLD) in chickens, however the mechanism by which this happens was previously unknown. Previous attempts to produce vaccines using C. hepaticus have proven unsuccessful because killed bacterin has been of low or variable effectiveness.

[0094] The cytotoxin is unlike any other bacterial toxin previously identified and its function and activity were unexpected. This is because the protein was shown to have high homology to cytochrome C proteins and further because toxins like the present toxin described herein have not previously been identified in any bacterial species.

[0095] SLD results in significant loss to the poultry industry, as it can cause flock mortalities of up to 15% and can reduce egg production by 35%. The costs of this disease have been estimated to range between AUD$5,000 to AUD$43,000 per 10,000 birds. The cost of SLD can be extrapolated up to 95 million dollars for the Australian poultry industry each year and at least 16 billion dollars for the 33 billion chickens used for egg production globally.

[0096] SLD is also an emerging disease around the globe and so there is a market for any anti- SLD treatments in all countries that have modern egg production facilities. Antibiotics are currently used to treat the disease, but such use is undesirable due to the risk of developing and transferring resistance traits. Therefore, the animal production industries are looking for alternative disease treatments so that antibiotic use can be reduced.

[0097] Currently, there is no effective vaccine or other prevention approach for SLD or any other Campylobacter in avian animals, particularly poultry. Given the decrease in egg production and increase in mortality rates as a consequence of infection with C. hepaticus, there is a need in the art for vaccine approaches that maintain commercial egg production.

[0098] Immunogenic compositions

[0099] The present disclosure provides compositions comprising the cytotoxic protein from C. hepaticus described herein, including fusion proteins comprising same and / or antigenic fragments thereof. Preferably, the compositions are provided as a vaccine. The composition may further comprise one or more of a physiologically acceptable diluent, excipient, adjuvant or carrier. The compositions are useful for stimulating an immune response in an avian animal at risk of C. hepaticus infection or colonisation. More particularly, the compositions are useful to treating or preventing Spotty Liver Disease (SLD) in an avian animal.

[0100] Antigenic fragments of the cytotoxic protein may consist of 15, 20, 25, 30, 40, 50, or 75 or greater contiguous amino acids of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In various further examples, at least one polynucleotide encodes a protein comprising or consisting of SEQ ID NO:1 or SEQ ID NO:2.

[0101] The immunogenic compositions of the disclosure may comprise proteins modified in any suitable way. In one embodiment, the protein component(s) of the composition are treated to extend in vivo half-life by, for example, such as by PEGylation, HESylation, PASylation, or glycosylation. The proteins may also be glycosylated as deemed appropriate, using standard techniques in the art. In another embodiment, those protein components in the immunogenic compositions that possess N-glycosylation sequences (NXS or NXT) may be glycosylated, to help further stimulate the immune response.

[0102] In some examples, the proteins are attenuated by means known in the art. Such attenuation methods may include the introduction of specific mutations e.g. alanine or leucine as described herein. Attenuation may also be achieved by the use of chemical compounds including, for example carvacrol, thymol, resveratrol, N-acetylcysteine and corticosteroids.

[0103] The immunogenic compositions may be formulated by any means known in the art. The composition is typically formulated as a pharmaceutical composition.

[0104] The immunogenic compositions described herein may also contain an adjuvant. Adjuvants in general comprise substances that boost the immune response of the host in a nonspecific manner. A number of different adjuvants are known in the art. Examples of adjuvants are Freund’s Complete and Incomplete adjuvant, vitamin E, non-ionic block polymers and polyamines such as dextransulphate, carbopol and pyran. Also very suitable are surface active substances such as Span, Tween, hexadecylamine, lysolecitin, methoxyhexadecylglycerol and saponins (i.a. Quil A(R). Furthermore, peptides such as muramyldipeptides, dimethylglycine, tuftsin, are often used. Next to these adjuvants, Immune-stimulating Complexes (ISCOMS), mineral oil e.g. Bayol® or Markol®, vegetable oils or emulsions thereof and Diluvac® Forte can advantageously be used. In particular examples, the adjuvant is Montanide™, including Montanide ISA 78 VG, Montanide™ ISA, Montanide™ IMS, Montanide™ Gel and Montanide™ GR which are commonly used in poultry. Montanide™ ISA is an oil-based adjuvant composed of a mineral oil and a surfactant from the mannide monooleate family.

[0105] The compositions described herein may also comprise a vehicle. A vehicle is a compound to which the polypeptide adheres, without being covalently bound to it. Often used vehicle compounds are e.g. aluminium hydroxide, — phosphate, sulphate or — oxide, silica, Kaolin, and Bentonite. A special form of such a vehicle, in which the antigen is partially embedded in the vehicle, is the so-called ISCOM (EP 109.942, EP 180.564, EP 242.380).

[0106] Suitable excipients include water, saline, dextrose, glycerol, ethanol and the like and combinations thereof. Often, the vaccine is mixed with stabilisers, e.g. to protect degradation-prone polypeptides from being degraded, to enhance the shelf-life of the vaccine, or to improve freeze drying efficiency. Useful stabilisers are i.a. SPGA (Bovarnik et al; J. Bacteriology 59: 509 (1950)), skimmed milk, gelatin, bovine serum albumin, carbohydrates e.g. sorbitol mannitol, trehalose, starch, sucrose, dextran or glucose, proteins such as albumin or casein or degradation products thereof, and buffers, such as alkali metal phosphates.

[0107] The compositions according to the present disclosure may also comprise preservatives such as sodium azide, thimerosal, gentamicin, neomycin, and polymyxin.

[0108] Freeze-drying is an efficient method for conservation. Freeze-dried material can be stored stable for many years. Storage temperatures for freeze-dried material may well be above zero degrees, without being detrimental to the material. Freeze-drying can be done according to all well-known standard freeze-drying procedures. However, the immunogenic compositions of the invention may be stored in any suitable manner. For example, the immunogenic compositions could be lyophilized or otherwise stabilized and stored in food or water for delivery.

[0109] In some examples, the composition may be combined with an antibiotic or an immunomodulatory agent, for example an isoquinoline alkaloid. Examples of suitable alkaloids include sanguinarine and chelerythrine (see for example, Quinteros JA et al., (2021) Poultry Science 100(11):101423).

[0110] Producing modified forms of cytotoxin

[0111] In some embodiments, a mutant form of the cytotoxin protein may be utilised to enhance the immune response while reducing potential toxicity. One example is the use of fusion proteins where the antigen is combined with another protein to improve its immunogenicity and stability. This approach can help target the immune response more effectively and minimise adverse effects. In other examples, single amino acids can be changed to ablate the toxic activity of the protein while maintaining its immunogenicity.

[0112] Methods of post translational modification of proteins are known in the art and are described for example in Zhong Q et al., (2023) MedComm 4:e261 . Such modifications include SUMOylation as described herein.

[0113] Vaccine production methods

[0114] The C. hepaticus cytotoxin and fusion proteins comprising same as described herein may be produced by recombinant or synthetic routes as known in the art. In an example, the cytotoxin is produced from a deoxyribonucleic acid encoding the cytotoxin protein as described herein (e.g. according to SEQ ID NO:1 or SEQ ID NO:2). In an example, the cytotoxin is produced from a vector comprising a deoxyribonucleic acid encoding the cytotoxin protein as described herein. The expression vectors may encode the whole mature cytotoxic protein or and antigenic portion thereof. As referred to herein an antigenic portion is any fragment of 10 or more contiguous amino acids in the recited amino acid sequence. In various further examples, the antigenic portion is any fragment is 15, 20, 25, 30, 40, 50, 75 or greater contiguous amino acids of the recited amino acid sequence.

[0115] The isolated polynucleotides encoding the cytotoxic protein may be single or double stranded DNA, RNA, or cDNA. The isolated polynucleotide may be any nucleic acid encoding the cytotoxic protein antigen, antigenic fragment thereof, or fusion protein described herein. In one example, the isolated polynucleotide comprises or consists of the sequence of SEQ ID NO:6 or portion thereof. It will be apparent to the skilled person, based on the teachings herein what polynucleotide sequences will encode the cytotoxic polypeptide or fragment thereof described herein.

[0116] Any expression vector suitable for an intended use can be used in the compositions of the present disclosure. Such expression vectors can be of any type known in the art, including, but not limited to plasmid, bacterial and viral-based expression vectors. The construction of expression vectors for use in transfecting prokaryotic cells is also well known in the art, and thus can be accomplished via standard techniques. (See, for example, Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA. In a preferred embodiment, the expression vector comprises a plasmid. However, the disclosure is intended to include other expression vectors that serve equivalent functions, such as viral vectors. Specifics of the expression vector will depend on the ultimate desired use. Designing appropriate expression vectors for an intended use is well within the level of those of skill in the art based on the teachings herein.

[0117] Any suitable promoter may be used that can direct expression (i.e.: is “operatively linked”) of the encoded proteins. The term “promoter” includes any nucleic acid sequence sufficient to direct expression of the encoded protein(s), including inducible promoters, repressible promoters and constitutive promoters. If inducible, there are sequences present which mediate regulation of protein expression so that the polynucleotide is transcribed only when an inducer molecule is present. Such cis-active sequences for regulated expression of an associated polynucleotide in response to environmental signals are well known to the art. The expression vector may comprise any other control sequences as may be suitable for an intended use. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered “operably linked” to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, enhancers, termination signals, and ribosome binding sites.

[0118] The vector is preferably one which expresses the polynucleotide to produce a protein, preferably a protein comprising the sequence of SEQ ID NO:1 or SEQ ID NO:2 or an immunogenic fragment thereof. The expressed protein encoded by the expression vector may comprise the sequence of SEQ ID NO:1 or a sequence having at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity over the full length of the recited amino acid sequence or antigenic fragment thereof.

[0119] The immunogenic compositions described herein may be used or inducing an immune response by administration as naked protein (e.g. sub-unit antigen) or as naked DNA using standard methods. Alternatively, the expression vectors may comprise viral expression vectors, including, but not limited to recombinant adeno-associated virus (AAV) gene delivery vector. In this embodiment, the expression vector is bounded on the 5' and 3' end by functional AAV inverted terminal repeat (ITR) sequences. By “functional AAV ITR sequences” is meant that the ITR sequences function as intended for the rescue, replication and packaging of the AAV virion. Recombinant AAV (rAAV) virions encapsidating the expression vectors of the invention may be produced using standard methodology. In one embodiment, an AAV expression vector according to the invention is introduced into a producer cell, followed by introduction of an AAV helper construct, where the helper construct includes AAV coding regions capable of being expressed in the producer cell and which complement AAV helper functions absent in the AAV vector. This is followed by introduction of helper virus and / or additional vectors into the producer cell, wherein the helper virus and / or additional vectors provide accessory functions capable of supporting efficient rAAV virus production. The producer cells are then cultured to produce rAAV. These steps are carried out using standard methodology. Replication-defective AAV virions encapsulating the recombinant AAV vectors of the instant invention are made by standard techniques known in the art using AAV packaging cells and packaging technology. Examples of these methods may be found, for example, in U.S. Pat. Nos. 5,436,146; 5,753,500, 6,040,183, 6,093,570 and 6,548,286, expressly incorporated by reference herein in their entirety. Further compositions and methods for packaging are described in Wang et al. (US 2002 / 0168342), also incorporated by reference herein in its entirety

[0120] In an aspect, the disclosure provides a carrier cell comprising the deoxyribonucleic acid as described herein, e.g. SEQ ID NO:6 or the vector described herein.

[0121] In another embodiment, the one or more expression vectors are present in a carrier cell, including but not limited to an avirulent, Campylobacter and non-Campylobacter bacterial carrier cell. Live bacterial vaccine “vectors” (i.e.: bacterial cells comprising immunogenic compositions) have been used successfully to elicit effective immune responses in order to prevent infection. Recombinant attenuated bacterial cell delivered vaccines have been adapted to stably express protective antigens at high levels. They are capable of stimulating strong primary humoral, mucosal and lasting memory immune responses without significant tissue damage or other performance reducing effects. In various non-limiting embodiments, the bacterial carrier cell is an avirulent bacterial cell selected from the group consisting of live or attenuated E. coli, L. monocytogenes, Salmonella spp., V. cholerae, Shigella spp., M. bovis BCG, Y. enterocolitica, B. anthracis, S. gordonii, Lactobacillus spp., Lactococcus spp., Enterococcus spp., Streptococcus spp., and Staphylococcus spp.

[0122] As used herein, “attenuated” means that the bacteria is reduced in causing disease symptoms in a host it is delivered to compared to a non-attenuated bacterial vector. Suitable attenuated bacteria can be any species or strain that is or can be sufficiently attenuated to allow for its non-pathological administration to avians in live and / or dead form. A particularly suitable example is the BIOPROPERTIES (BPL) Salmonella typhimurium (STM) vaccine (Vaxsafe® ST vaccine).

[0123] In other embodiments, an attenuated Salmonella species is used. In exemplary embodiments, Salmonella that can be used include, but are not limited to Salmonella enterica strains selected from the group consisting of S. Typhimurium, S. Enteriditis, S. Sofia, S. Heidelberg, S. Gallinarum, S. Hadar, S. Agona, S. Kentucky, S. Typhi, S. Paratyphi and S. Infantis. S. Typhimurium is especially useful for vaccination purposes because the genome sequence is fully characterized and many animal studies confirm its safe medical use. Recombinant attenuated Salmonella vaccines (RASVs) have been constructed to deliver antigens from other pathogens to induce immunity to those pathogens in vaccinated hosts; see, for example, Curtiss et al., Crit Rev Immunol. 2010; 30(3):255-70; 2010; Qiu et al., J. Virological Methods 188:108; Strugnell et al., Infect. Immunol. 1992, 60:3994; Layton et al., Clinical and Vaccine Immunology march 201 1 , 449-454; Al-Ojali et al., Microbial Pathogenesis 52:326 (2012); and (Wyszynska et al., 2004) Wyszyriska et al. (2004). In one embodiment, the RASV comprises attenuating mutations in the pmi (mannose-6-phosphate isomerase), fur (ferric uptake regulator) and crp (cAMP regulatory protein) genes (see, for example, (Li et al., PNAS 106:592-597 2009, Curtiss et al., 2009) and U.S. Pat. No. 8,133,493. In another embodiment, the RASV comprises the x9992 vector disclosed in U.S. Pat. No. 8,133,493. In another embodiment, the RASV is one that is commercially available, such as Megan®Vac1 (Lohman Animal Health, US).

[0124] Attenuated bacterial cells can be transfected with the one or more expression vectors using standard techniques in the art. In an aspect, the disclosure provides a method of producing the cytotoxin protein as described herein comprising culturing the carrier cell as described herein in culture medium to produce the protein. In an example, the method further comprising isolating the cytotoxin antigen from the cell and / or cell culture medium.

[0125] In some embodiments, the cytotoxin protein is fused to second protein that facilitates solubilisation of the protein. In one example, the cytotoxin vaccine antigen is fused to SUMO. In a further example, the cytotoxin vaccine antigen is fused to CjA or other membrane antigen from Campylobacter.

[0126] It will be understood that the compositions and vaccines of the present disclosure are not limited to those described above. The skilled person will appreciate that the methods of the disclosure can be carried out using any suitable vaccine approach available to those skilled in the art. Examples of suitable vaccines within the scope of the present disclosure include bacterial vectored, sub-unit vaccines, protein-polysaccharide conjugates, or viral vectored vaccines.

[0127] Subjects and modes of administration

[0128] The term “avian” as used herein is intended to include males and females of avian species, but is primarily intended to encompass poultry which are commercially raised for eggs, meat or as pets. Accordingly, the term “avian” is particularly intended to chickens, turkeys, ducks, geese, quail, pheasant, parakeets, parrots, and the like. Chickens and turkeys are the preferred avian subjects, with chickens being most preferred. The avian subject may be a hatched bird, including newly-hatched (i.e., about the first three days after hatch), adolescent, and adult birds.

[0129] The compositions described herein may be formulated for administration by any suitable route, including orally, as injectables, parentally, by inhalation spray, intranasally, rectally, mucosally, topically, or for administration by oral gavage or ad libitum feeding, for example, in drinking water, either as liquid solutions or suspension, in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes, subcutaneous, intravenous, intra-arterial, intramuscular, intrasternal, intratendinous, intraspinal, intravitreous intracranial, intrathoracic, infusion techniques or intraperitoneally. Solid forms suitable for solution in, or suspension in, liquid prior to injection or other administration may also be prepared. The compositions may also, for example, be emulsified, or the encapsulated in liposomes or microparticles. The immunogenic compositions may also be present in and / or expressed by transgenic plants.

[0130] The compositions may be formulated for mucosal administration, such as by admixing of the composition with drinking water or food, or admixing for use as a spray, to mist over the animals to uptake the immunogenic composition during grooming behaviour. However, any suitable method of administration can be used for any subject. Thus, in various examples, the compositions are formulated for intraocular, intranasal, or transdermal administration. In some examples, the vaccine is sprayed onto shells.

[0131] In an example, the administration is performed about 14 days after hatching. Thus, in one example a first administration is carried out by about 14 days after hatching and an additional booster administration is given at any suitable time, such as by about 21 days after hatching.

[0132] In another example, the administration is performed when the animal (e.g. avian) is about 12 weeks of age. In an example, the administration is performed when the animal is about 16 weeks of age. In an example, the administration is performed when the animal is about 20 weeks of age, or later.

[0133] In an example, the administration is performed on the animal two or more times.

[0134] In an example, the administration is performed on the animal three or more times. In an example, the first administration is performed when the animal is about 12 weeks of age, the second administration is performed when the animal is about 16 weeks of age, and the third administration is performed when the animal is about 20 weeks of age.

[0135] In an example, the administration is performed during moulting of the animal. In an example, the administration is performed on the animal two or more times. In an example, the administration is performed on the animal two or more times, wherein the second administration is performed about 4 weeks after the first administration.

[0136] The compositions described herein may be administered in an effective amount.

[0137] In an example, the effective amount of the composition or vaccine is between about 0.1 ml to about 1 ml. In an example, the effective amount of the vaccine is about 0.10 ml. In an example, the effective amount of the vaccine is about 0.25 ml. In an example, the effective amount of the vaccine is about 0.50 ml. In an example, the effective amount of the vaccine is about 0.75 ml. In an example, the effective amount of the vaccine is about 1 ml.

[0138] In an example, the cytotoxic protein or fusion protein is administered in an amount of between about 0.1 ug / kg-100 mg / kg body weight. In other examples, the immunogenic composition or vaccine may comprise 0.5 ug / kg to 50 mg / kg; 1 ug / kg to 25 mg / kg, or 5 ug / kg to 10 mg / kg body weight.

[0139] In certain examples, the vaccine comprises a live attenuated C. hepaticus mutant bacterium. In one example, the effective amount of the vaccine comprises bacterium at a dose of between about 1 x 106and 1 x 1010total cell count (TCC). In an example, the effective amount of the vaccine comprises bacterium at a dose of about 1 x 106TCC. In an example, the effective amount of the vaccine comprises bacterium at a dose of about 1 x 107TCC. In an example, the effective amount of the vaccine comprises bacterium at a dose of about 1 x 108TCC. In an example, the effective amount of the vaccine comprises bacterium at a dose of about 1 x 109 TCC. In an example, the effective amount of the vaccine comprises bacterium at a dose of about 1 x 1010TCC.

[0140] Methods of Treatment

[0141] The present disclosure provides methods for stimulating an immune response against C. hepaticus comprising administering to an avian animal an effective amount of the cytotoxic protein, fusion protein, composition, vector or vaccine according to any aspect or embodiment to treat or prevent Spotty Liver Disease (SLD) in the animal.

[0142] As referred to herein, methods for stimulating an immune response result in one or more effects (e.g. maturation, proliferation, direct-or cross-presentation of antigen, gene expression profile) on cells of either the innate or adaptive immune system. For example, the immune response may involve, effect, or be detected in innate immune cells such as, for example, dendritic cells, monocytes, macrophages, natural killer cells, and / or granulocytes (e.g., neutrophils, basophils or eosinophils). The immune response may also involve, effect, or be detected in adaptive immune cells including, for example, lymphocytes (e.g., T cells and / or B cells). The immune response may be observed by detecting such involvement or effects including, for example, the presence, absence, or altered (e.g., increased or decreased) expression or activity of one or more immunomodulators. The immune response may stimulate a de novo or previously undetected antibody response, or enhance or suppress an existing response against the immunogen by, for example, causing an increased antibody response (e.g., amount of antibody, increased affinity / avidity) or an increased cellular response (e.g., increased number of activated T cells, and / or increased affinity / avidity of T cell receptors. In certain embodiments, the immune response may be protective, meaning that the immune response may be capable of preventing initiation or continued infection of or growth within a host of C. hepaticus.

[0143] In some instances, elimination of an agent from the host may mean that the method is therapeutic, in that the method is used to treat an animal already infected with C. hepaticus. When the method is therapeutic, the method may comprise treating a C. hepaticus infection, wherein “treating” means accomplishing one or more of the following: (a) reducing the severity of the infection; (b) limiting or preventing development of symptoms characteristic of the infection; (c) inhibiting worsening of symptoms characteristic of the infection; (d) limiting or preventing recurrence of the disorder(s) in subjects that have previously had the infection; and (e) limiting or preventing recurrence of symptoms in subjects that were previously symptomatic for the infection.

[0144] In particular examples, the animal is able to stimulate an immune response against the cytotoxin antigen of C. hepaticus which is evidenced by prevention of, or a reduction in the number of liver spots observed in the liver of the avian animal. The liver spots can be seen by eye. Scoring systems such as those described in Van TTH et al., (2022) Front Vet Sci 9:1039774 can be used to record the number of lesions found on the surface of the liver. The score is based on a scale from 0 to 5, wherein 0= no visible lesions, 1 =1 -5 lesions, 2=6-20 lesions, 3=21-100 lesions, 4= 101-1000 spots and 5= more than 1 ,000 lesions.

[0145] In some examples, the immunogenic composition or vaccine described herein is able to reduce the number of liver lesions compared to control, unvaccinated animals by at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100%. Vaccine effectiveness may also be indicated by changes in the expression of host gene markers that have been found to be correlated with SLD severity and progression.

[0146] In some examples, the immunogenic composition or vaccine described herein reduces mortality, and / or egg production loss of vaccinated animals by at least 40%, 50%. 60%, 70%, 80%, or 90% of greater compared to unvaccinated animals.

[0147] In another aspect, the present disclosure also provides methods for treating or preventing SLD in an avian animal, comprising administering to the animal an immunogenic composition or vaccine described herein. Preferably the composition or vaccine is administered to the animal in a therapeutically effective amount.

[0148] In one embodiment, the immunogenic composition orvaccine is able to protect the animal against challenge with C. hepaticus.

[0149] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. The present disclosure includes the following non-limiting examples.

[0150] EXAMPLES

[0151] Methods

[0152] Identification and purification of cytotoxic protein

[0153] C. hepaticus supernatant was filter sterilised using 0.45 and 0.22 pM filter unit and initially purified via cation exchange using HiTrap SP HP columns (Cytiva, USA). Various buffers at different pH’s ranging from 1 .4 to 8.8 were used to screen fractions of interests. For all buffers, a 15 column volumeswash step was applied and fractions were collected using a gradient elution (0.2-1 .0 M NaCI). The proposed toxin eluted in 50 mM phosphate, pH 7.0. Fractions were pooled and buffered exchanged in 20 mM Tris pH 7.5, 150 mM NaCI, 1 mM dithiothreitol (DTT) using Amicon centrifugal filter unit 30 MWCO.

[0154] Eluted fractions from cation exchange which demonstrated cytotoxic activity on LMH cells were further purified using Size Exclusion Chromatography (SEC) using a Superdex 200 Increase 10 / 300 GL column (Cytiva, USA) at a flow rate of 0.75 ml / min with 20 mM Tris pH 7.5, 150 mM NaCI, 1 mM dithiothreitol (DTT). All purification steps were performed using the KTA pure™ purification system (Cytiva, USA).

[0155] The identification of the toxin candidate was performed using nanoLC ESI MS / MS using the QExactive Plus 2 (Thermo Scientific) mass spectrometer. An Acclaim PepMap RSLC analytical column (75 pm x 50 cm, nanoViper, C18, 2 pm, 100A; Thermo Scientific) was used as the analytical column. The data was analysed against the C. hepaticus HV10 reference genome using the Byronic (ProteinMetrics) search engine. Carbamidomethylation was used as the fixed modification. The protein FDR cut-off was 1 %,

[0156] LMH cytotoxic assay

[0157] The toxic activity of C. hepaticus HV10Tmature / native (SUMO tagged cleaved) and fusion (SUMO tagged) proteins on LMH cells were measured via lactase dehydrogenase released from dead cells using a colorimetric LDH assay (CyQUANT LDH Cytotoxicity Assay, Invitrogen, USA). The cells were grown in Waymouth tissue culture media (ThermoFisher Scientific, Gibco, USA), supplemented with 10% Fetal Bovine Serum (FBS) (Corning, USA) and 1 % Penicillin-Streptomycin-Glutamine (ThermoFisher Scientific, Gibco, USA) and grown at 37°C in 5% O2. LMH cells were seeded 1 x105per well in 96-well plates for approximately 24 hours and were then treated with mature and fusion proteins from 5-100 pg for 24 hrs and assayed according to the manufacturer’s specifications. Colorimetric changes were measured using a POLARStar Omega spectrophotometer (BMG LabTech, USA) at 490 nm. Controls include: C. jejuni and C. hepaticus HV10Tsupernatant- concentrated 200X times using a Vivispin 3kDa MWCO centrifuge filter unit and diluted to 10% in complete Waymouth media; heat inactivated control- 30 pg of mature toxin was heated to 100°C for 10 min before treatment; buffer control- the toxin purification buffer (20 mM Tris-HCI, 150 mM NaCI, pH 7.4); Proteinase K (PK) digestion of the toxin- mature toxin (30 pg) was treated with 50 pl of PK for 2 hours at 37°C. The assay was completed in triplicate (n=3).

[0158] For assessing the neutralisation of the cytotoxin on LMH cells by a polyclonal antibody raised against the cytotoxic protein, LMH cells treated with mature C. hepaticus HV10Ttoxin preincubated with rabbit-derived polyclonal antibodies (pAb, Genscript). Mature toxin (SEQ ID NO:1) and pAb were pre-incubated for 4 h at 4°C in native purification buffer (20 mM Tris-HCI pH 7.5, 150 mM NaCI) at the following ratios: 10 pg mature toxin, 10 pg pAb, 10 pg mature toxin:5 pg pAb and 10 pg mature toxin:2.5 pg pAb. Mixtures were diluted in complete Waymouth medium (Waymouth media, 10% Fetal Bovine Serum and 1 % Penicillin-Streptomycin) and applied to LMH cells (1 x 105cells / well) for 24 hours. LDH release was measured according to the kit manufacturer’s instructions (CyQUANT™ LDH Cytotoxicity Assay). LMH cells were treated with 5 and 10 pg of pAb to ensure there was no cytotoxic effect of the pAb. For the positive control, LMH cells were treated with 10 pg of mature toxin to obtain the highest cytotoxicity level. SUMO tagged / fusion toxin (2.5 pg) and toxin purification buffer (20 mM Tris-HCI pH 7.5, 150 mM NaCI) are negative controls. Results are based on three independent LDH assays, completed in triplicate (n=3).

[0159] Vaccine vector

[0160] The fusion of CjaA and the cytotoxic protein was generated via a 54 bp deletion of the ChuA fragment of the CjaA_ChuA (#9) construct (Mcdonald JB et al. Front. Vet. Sci. 12:1518231). This deleted fragment was replaced with the 283 bp fragment of the cytotoxic protein via Gibson assembly using the primer pair 5’- aatatattcaaaccttaaaaAAAACCTTGCTTTTAAACAAAGGTACAACAG-3’ and 5’- aataaagtagcaccgtctgcATCTTTTAAATCTTCTACGCTAGTTATATTACTATCCTTTGGT-3’ to generate the vector amplification product using purified vector DNA as a template and primer pair 5’-GCGTAGAAGATTTAAAAGATgcagacggtgctactttatttaaaaaatgc-3’ and 5’- TTGTTTAAAAGCAAGGTTTTttttaaggtttgaatatattgagcaatagcttctaaatcagc-3’ with C. hepaticus HV10Tgenomic DNA being used as the template for the amplification of the cytotoxic protein encoding gene. The construct was constructed on a pUC57 backbone. Inverse PCR was subsequently employed to introduce the single amino acid change at position 14 (H to L) using primer pair 5’-Atatgtcttggtgcaaaagctg-3’ and 5’-agcgcattttttaaataaagtagc-3’

[0161] Vaccination of animals

[0162] Protein for vaccination was first filter sterilised using 0.45 and 0.22 pM filter unit. Protein was then heat inactivated at 90 degrees for 5 minutes immediately prior to vaccine formulation. Each vaccine dose consisted of a mix of 70% (v / v) Montanide ISA 78 VG adjuvant (Seppic) and 30% (v / v) of purified protein (50 pg) in sterile PBS in a volume of 0.5 ml which was thoroughly homogenised via vigorous vortexing and passing through a 20-21 gauge needle multiple times. Commercial layer chickens were first vaccinated at approximately 17 weeks of age with booster vaccinations occurring at 2-3 week intervals twice. The vaccination comprised injection intramuscularly into the breast muscle. As controls some animals received adjuvant only with no protein and others were left untreated. Birds were challenged with the C. hepaticus pathogen 2 weeks after the third vaccination. Challenge was via oral gavage with approximately 1 x 109CFU of C. hepaticus 44L strain in a 1 ml volume. These conditions were used for all vaccine trials.

[0163] Disease score

[0164] Disease score was assessed by counting the number of disease spots (lesions) observed in the liver of each animal macroscopically. This process was performed at the time of necrosis. The carcass was opened and the liver examined in situ. Disease score was categorised as follows: 0 = no disease; 1 = 1 -5 spots; 2 = 6-20 spots; 3 = 21 -100 spots; 4 =101 to 1000 spots and 5 = 1000+ spots.

[0165] ELISA

[0166] ELISA was performed using serum collected from a second vaccine trial conducted in 2025 (Figure 3, right panel). The serum were collected by wing bleed from trial chickens immediately prior to pathogen challenge and two weeks post the third vaccination. Wells of a NUNC MaxiSorp 96-well plate were coated with 25 ng of protein and then blocked using a 5% skim milk powder solution in PBST in a volume of 100 p overnight at 4 degrees. Protein used was purified 209 (CjaA+Toxin, SEQ ID NO: 5), 209* (CjaA only) and toxin only. Primary anti-sera used was the pooled sera of birds (n= 10-12) derived from blood collected immediately prior to pathogen challenge. The groups used were birds vaccinated with 209 (CjaA+Toxin, SEQ ID NO:5), 209* (CjaA only), toxin only or adjuvant only. Primary sera was added to wells at a 1 :500 dilution in PBST in a volume of 50 pl for 2 hours at room temperature with gentle shaking before being washed 3 times with PBST. Secondary antibody was goat-anti chicken IgG HRP conjugate and was applied at 1 :5000 dilution in PBST in a volume of 50 pl for 1 hour at room temperature with gentle shaking before being washed 3 times with PBST. 50 pl TMB was added to wells to develop signal in the dark for 15 minutes before the reaction was stopped with 50 pl of 0.2 M sulphuric acid. Colorimetric changes were measured using a POLARStar Omega spectrophotometer (BMG LabTech, USA) at 450 nm. Background correction was applied using protein coated wells that received secondary but no primary antibodies. Statistical analysis

[0167] Disease trial data was analysed via one-way ANOVA with Tukey’s multiple comparison test. The positive control group had an n=24 and the other groups had n=10-12. ELISA data was analysed via ANOVA with Tukey’s multiple comparison test. All ELISA data was performed in triplicate.

[0168] For the LMH cytotoxic assay, unpaired t-test using Welch’s correction was used at P=0.05 was used to measure statistical significance against the heat inactivated mature toxin control. For treatments which were not normally distributed, a Mann Whitney’s t-test was implemented.

[0169] Example 1 Identification of a novel cytotoxin from Campylobacter hepaticus, the cause of Spotty Liver Disease in chickens

[0170] The inventors undertook an investigation of C. hepaticus proteins that play a role in Spotty Liver Disease (SLD) pathogenesis. The inventors found that C. hepaticus culture supernatant was cytotoxic as were purified fractions from the supernatant on immortalised chicken liver cells (LHM cells). The culture supernatant was subjected to cation exchange chromatography and the fractions obtained from cation exchange chromatography further purified using size-exclusion chromatography. The purified fractions with the most cytotoxic activity were subjected to SDS- PAGE protein gel electrophoresis. Fourteen protein bands were obtained and identified using mass spectrometry, based on the analysis of peptides generated through proteolytic digestion and comparison to the predicted coding sequences (genes) identified from our whole-genome sequencing of HV10T. Based on abundance, percent coverage and predicted function, a candidate cytotoxin protein was identified. This candidate protein was not related to any known classical bacterial toxin. Analysis of the candidate protein showed that it has high homology to cytochrome C proteins. Some cytochrome C proteins have a role in cell apoptosis and so it was hypothesised that it could be driving cytotoxicity via an apoptotic mechanism.

[0171] A recombinant version of the putative cytotoxic protein was produced as a small ubiquitin modifying (SUMO) fusion protein according to manufacturer’s instructions and as described in Panavas T et al., (2009) In: Ulrich, H.D (eds) SUMO Protocols. Methods in Molecular Biology vol 497 Humana Press, Totowa, NJ). The SUMO tag was used as a mechanism to keep the toxin in the soluble fraction as it had been predicted that the toxin would likely be insoluble by itself in the Escherichia coli production system. The SUMO-cytotoxin fusion protein was designed so that the protein could be proteolytically cleaved to release the mature native toxin sequence. The protein sequence was subcloned into the E.coli expression vector pSUMO-M (Addgene plasmid #177848; Zhang et al., (2015) Nature 10;525(7568):269) via the Stu\ and Hind\\\ restriction sites. The details of the subcloned sequence as translated are provided below: MNWSHPQFEKSSGSSGGHHHHHHGGSGGSGSDSEVNQEAKPEVKPEVKPETHINLKVSDG SSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHRE QIGGADGA TLFKKCAICHGAKAEKVYLNKVPALNTLSAEEITQALKEYKAGTRNKFGQGGIMKI QLTSLSEADLEAIAQYIQTLK (SEQ ID NO:4).

[0172] Wherein MNWSHPQFEKSSGSSGG is the leader sequence, the underlined H corresponds to a His6 tag and GGSGGSGS corresponds to a linker sequence (GGS)2GS. The bold and underlined sequence corresponds to the sequence of the SUMO sequence, the sequence of the putative cytotoxic protein is italicized.

[0173] Example 2 Characterisation of the cytotoxin protein

[0174] A BLAST search was conducted on the putative cytotoxic protein. As shown in Table 1 below, the sequence showed less than 86% sequence identity to the C-type cytochrome gene of other Campylobacter species and 90 % identity to that of C. bills, another species causing SLD.

[0175] The expressed putative C. hepaticus cytotoxic protein sequence was found to correspond to SEQ ID NO:1 which sequence is set forth below.

[0176] ADGATLFKKCAICHGAKAEKVYLNKVPALNTLSAEEITQALKEYKAGTRNKFGQGGIMKIQLTSL SEADLEAIAQYIQTLK (SEQ ID NO:1).

[0177] For animal trials this sequence was rationally attenuated whereby amino acid residue position 14 H (Histidine) was replaced with L (Leucine) as set forth below. ADGATLFKKCAICLGAKAEKVYLNKVPALNTLSAEEITQALKEYKAGTRNKFGQGGIMKIQLTSL SEADLEAIAQYIQTLK (SEQ ID NO:2).

[0178] Bioinformatic analysis of the toxin identified it as a CBB3 type cytochrome. This family of cytochromes have a heme binding domain as their active site with the conserved motif CXXCH where X is any amino acid and the H binds the heme molecule. The H at position 14 of the sequence of SEQ ID NO:1 is replaced with L. This single amino acid change was made to disrupt the critical function of the binding site thus attenuating cytotoxic activity. An H to L substitution was selected as it is a conservative amino acid change and thus least likely to adversely affect overall protein structure. Table 1 Campylobacter c-type cytochrome showing alignment with the putative cytotoxic protein

[0179] The activity of the putative cytotoxic protein was investigated in vitro on immortalised chicken liver cells (LMH cells). As demonstrated in Figure 1 , the recombinant putative cytotoxic protein exhibited the same cytotoxic effects as the C. hepaticus supernatant on LMH cells. The mature proteins (with the SUMO tag removed) demonstrated cytotoxic effects on LMH cells, while the SUMO-cytotoxin fusion version did not. Further work showed that the cytotoxic activity was neutralised by a polyclonal antibody raised against the recombinant cytotoxic protein, demonstrating the cytotoxic activity was caused by the cytotoxin (Figure 2).

[0180] The inventors also produced another fusion version of the toxin in which is joined to the antigen, C. jejuni, CjaA derived from Campylobacter. This fusion protein has some potential advantages over the SUMO-toxic fusion protein when used as a vaccine antigen as it contains another potentially relevant antigen, rather than the immunologically irrelevant SUMO protein. The protein sequence of the construct is presented below, with the cytotoxin sequence highlighted in bold within the CjaA antigen.

[0181] MKKIWLALAGLVLAFSASAADQNATGGDQNATGGDQNATGGDQNATDSKTLNSLDKIKQNGV VRIGVFGDKPPFGYVDEKGNNQGYDIALAKRIAKELFGDENKVQFVLVEAANRVEFLKSNKVDI ILANFTQTPQRAEQVDFCLPYMKVALGVAVPKDSNITSVEDLKDADGATLFKKCAICLGAKAE KVYLNKVPALNTLSAEEITQALKEYKAGTRNKFGQGGIMKIQLTSLSEADLEAIAQYIQTLKKT LLLNKGTTADAYFTQNYPNIKTLKYDQNTETFAALMDKRGDALSHDNTLLFAWVKDHPDFKMG IKELGNKDVIAPAVKKGDKELKEFIDNLIIKLGQEQFFHKAYDEILKAHFGDDVKADDVVIEGGKI RSGGLKDQNATGGDQNATGGDQNATGGDQNATHHHHHHfSEQ ID NO:5).

[0182] The underlined sequence corresponds to the CjaA antigen.

[0183] Example 3 Vaccine preparation and testing

[0184] There has been no approach to date using antigen specific to C. hepaticus for a SLD vaccine. An antigen that is specific to SLD disease is the key to a successful vaccine against C. hepaticus.

[0185] Work by the present inventors has demonstrated that conventional killed vaccines are ineffective in the treatment of SLD, accordingly a sub-unit form or fusion form of the antigen is expected to be the most effective approach for vaccination.

[0186] For vaccination, layer chickens (12 birds per group) were divided into groups with one group vaccinated with PBS (negative control group), or test vaccine (fusion versions of the cytotoxin or mature toxin that underwent heat inactivation at 95°C for 5 minutes), which has shown no toxic effect on chicken cells. Animals were challenged with wild type C. hepaticus 44L strain. The birds were sacrificed 6 days post-challenge. The ovaries, spleen, liver, bile and blood were collected. Anti-C. hepaticus antibody from the serum samples were measured by ELISA as described in Muralidharan et al., (2022) Front Vet Sci 9:1082358. Additionally, disease output was measured by physical examination for liver spots using a known scoring system as described in Van TTH et al., (2022) Front Vet Sci 9:1039774. The score is based on a scale from 0 to 5, wherein 0= no visible lesions, 1 =1 -5 lesions, 2=6-20 lesions, 3=21 -100 lesions, 4= 101-1000 spots and 5= more than 1 ,000 lesions.

[0187] RNA from the tissues will be extracted and sequenced. Gene expression level and its fold changes will be calculated using CLC genomics workbench to identify genes with an absolute fold change greater than 2 and a false discovery rate less than 0.05. C. hepaticus levels will be quantified from bile samples using qPCR as described in in Van TTH et al., (2017) Vet Microbiol 207:226-230,

[0188] Example 4 Animal trials

[0189] Evidence of disease protection using liver lesion scores

[0190] Approximately 16 week old commercial layer chickens, in groups of 12 birds, were vaccinated at 2-3 week intervals a total of 3 times prior to challenge with the C. hepaticus pathogen 2 weeks after the third vaccination. Challenge was via oral gavage with approximately 1 x 109CFU in a 1 ml volume. Significantly lower liver lesion scores in the group vaccinated with the recombinant protein containing a fusion of CjaA and Toxin (SEQ ID NO:5), compared to the positive control group, demonstrated that the recombinant protein provided protection against liver damage caused by C. hepaticus. Birds vaccinated with CjaA alone did not provide protection, providing evidence that the cytotoxic protein is responsible for protection. These results are shown in Figure 3A and B. The analysis was performed using one way ANOVA with Tukey’s post hoc test (comparing all means against each other). As shown in Figure 3, the disease significantly reduced when birds vaccinated with a fusion of CjaA and Toxin compared to the control groups.

[0191] ELISA analysis

[0192] The results of the ELISA are shown in Figure 4. Each result was labelled with a “V” and a “P”. The “V” values correspond to the antigen that the animals were vaccinated with and “P” corresponds to the protein coated in the ELISA plate. Protein used was purified 209 (CjaA+Toxin (SEQ ID NO:5)), 209* (CjaA only) and toxin only (SEQ ID NO:1). Primary anti-sera used was the pooled sera of birds (n= 10-12) derived from blood collected immediately prior to pathogen challenge. The groups used were birds vaccinated with 209 (CjaA+Toxin), 209* (CjaA only), toxin only or adjuvant only. All data were performed in triplicate and the background corrected against protein coated wells with secondary antibody but without primary serum.. V negative corresponds to pooled serum from the adjuvant only (70% (v / v) Montanide ISA 78 VG adjuvant (Seppic) to 30% (v / v) sterile PBS) negative control group.

[0193] Serum from birds vaccinated with the recombinant protein containing a fusion of CjaA and Toxin showed a significantly higher response against the toxin than serum from birds vaccinated with adjuvant only or with CjaA only (vaccination with CjaA alone did not provide protection), indicating a toxin-specific reaction responsible for protection.

[0194] Example 5 Vaccine administration reduced egg production loss in commercial layer chickens

[0195] In this prophetic example, approximately 16 week old commercial layer chickens, in groups of 100-200 birds, will be vaccinated with CjA+Toxin (SEQ ID NO:5) at 2-3 week intervals a total of 3 times. The birds will be challenged 2 weeks after the third vaccination. The birds will be assessed from day 1 to 4 weeks following the challenge for changes in egg production compared to unvaccinated chickens or chickens vaccinated with CjA alone.

[0196] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:1 . A protein or antigenic portion thereof from Campylobacter hepaticus and / or C. bills for use to treat or prevent Spotty Liver Disease (SLD) in an avian animal, wherein the protein is cytotoxic to avian cells.

2. The protein according to claim 1 , comprising the sequence of SEQ ID NO:1 or a sequence at least 90% identical thereto, wherein SEQ ID NO:1 has the sequence set forth in: ADGATLFKKCAICHGAKAEKVYLNKVPALNTLSAEEITQALKEYKAGTRNKFGQGGIMKIQLTSL SEADLEAIAQYIQTLK.

3. The protein according to claim 2, wherein the protein comprises the sequence set forth in SEQ ID NO:2.

4. The protein according to claim 3, comprising the sequence of SEQ ID NO:4 or SEQ ID NO:5.

5. The protein or antigenic portion thereof, wherein the antigenic portion is a fragment consisting of 15, 20, 25, 30, 40, 50, or 75 or greater contiguous amino acids of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

6. A vector comprising a nucleic acid sequence encoding the sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:5.

7. An immunogenic composition comprising the protein according to any one of claims 1 to 4 or vector according to claim 6.

8. An immunogenic composition comprising the protein of SEQ ID NO:1 or SEQ ID NO:2 or antigenic portion thereof for use or when used to treat or prevent Spotty Liver Disease (SLD) in an avian animal, together with a pharmaceutically acceptable diluent, excipient, adjuvant or carrier.

9. The composition according to claim 5 which is a vaccine.

10. The composition according to claim 9 further comprising an adjuvant.11 . The composition according to any one of claims 8 to 10, further comprising an antigenic protein derived from Campylobacter, Salmonella or Influenza.

12. A polynucleotide sequence encoding the mature cytotoxic protein of SEQ ID NO:1 or SEQ ID NO:2 or antigenic portion thereof.

13. The polynucleotide according to claim 12 comprising the sequence of SEQ ID NO:6.

14. A vector comprising a polynucleotide sequence comprising the sequence of SEQ ID NO:6.

15. A carrier cell comprising the vector according to claim 6 or 14.

16. A vaccine comprising the protein according to any one of claims 1 to 4.

17. Use of the protein comprising the sequence of SEQ ID NO:1 or SEQ ID NO:2 or antigenic portion thereof, or composition according to claim 8 or vector according to 14 in the manufacture of a vaccine for treating or preventing SLD caused by C. hepaticus.

18. The immunogenic composition according to any one of claims 8 to 11 further comprising an antibiotic and / or immunomodulatory agent.

19. A method for inducing an immune response to SLD in an avian animal, the method comprising administering to the animal the protein comprising the sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:5 or antigenic portion thereof, or composition according to any one of claims 8 to 11 , vector according to claim 14, or vaccine according to claim 16.

20. A method for inducing an immune response to SLD in an avian animal, the method comprising administering to the animal the protein comprising the sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:5 or antigenic portion thereof, or composition according to any one of claims 8 to 11 , vector according to claim 14, or vaccine according to claim 16.21 . A method of vaccinating an avian animal against SLD, the method comprising administering to the animal the protein comprising the sequence of SEQ ID NO:1 , SEQ ID NO:2,SEQ ID NO:4 or SEQ ID NO:5 or antigenic portion thereof, or composition according to any one of claims 8 to 11 , vector according to claim 14, or vaccine according to claim 16.

22. A method for treating or preventing SLD in an avian animal, the method comprising administering to the animal the protein comprising the sequence of SEQ ID NO:1 , SEQ ID NO:2,SEQ ID NO:4 or SEQ ID NO:5 or antigenic portion thereof, or composition according to any one of claims 8 to 11 , vector according to claim 14, or vaccine according to claim 16.

23. The vaccine according to claim 16 which is formulated for administration in the drinking water, intranasally, sprayed on shells prior to hatch, provided in the feed, or injected.

24. The method according to any one of claims 19 to 23 wherein the avian is selected from a chicken, a turkey, duck, geese, quail, and pheasant.

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