AU2018200954B2 - Live Vaccine Strains - Google Patents

Abstract

The present specification relates generally to the field of vaccination of farm animals. Provided herein is a live vaccine strain and methods for immunising farm animals against an intestinal spirochaete. The subject specification discloses an isolated live vaccine strain of Brachyspira hyodysenteriae and methods of immunising an animal against infection by B. hyodysenteriae or a related microorganism.

Description

Live Vaccine Strains

FIELD

[0001] The present specification relates generally to the field of vaccination of farm animals. Provided herein is a live vaccine strain and methods for immunising farm animals against an intestinal spirochaete. The subject specification discloses an isolated live vaccine strain of Brachyspira hyodysenteriae and methods of immunising an animal against infection by B. hyodysenteriae or a related microorganism.

BACKGROUND

[0002] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

[0003] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgement or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0004] Swine dysentery is a bacterial disease in pigs which has symptoms of colitis and bloody mucoid diarrhoea. Swine dysentery is endemic in most regions of the world. It is widespread among pig herds in Australia and has recently re-emerged in North and South America. The disease leads to heavy economic losses in pig farms due to mortality, morbidity, depression of growth and feed conversion efficiency.

[0005] The intestinal spirochaete, Brachyspira hyodysenteriae, is typically the causative agent of swine dysentery (SD). Other related causative agents include Brachyspira suanatina and Brachyspira hampsonii. The diagnosis of swine dysentery is typically based on post-mortem examinations, laboratory tests on faecal smears, isolation and identification of bacteria by serological and biochemical tests and DNA analysis.

[0006] Antimicrobials are typically used to treat swine dysentery. For example, the pigs may be treated with carbadox, lincomycin or tiamulin, However, the widespread use of antimicrobials leading to increased resistance is becoming a major concern. Furthermore, multi-drug resistant strains of Brachyspira hyodysenteriae have also been reported in Europe and Australia. A longer term solution is needed.

[0007] Accordingly, there is a need to provide alternative methods to treat or prevent B. hyodysenteriae infection in pigs.

[0008] There is also a need to develop a safe and effective vaccine strains to prevent B. hyodysenteriae infection in pigs.

SUMMARY

[0009] The present specification discloses a live vaccine strain and methods for preventing infection by B. hyodysenteriae or a microorganism related to B. hyodysenteriae in farm animals. The ability to prevent or control infection by this microorganism is important in the pig industry where diseases such as swine dysentery are common. The present invention provides an isolated live vaccine strain of B. hyodysenteriae, wherein: (a) the live vaccine strain lacks one or more functional virulence factor(s); and (b) the live vaccine strain lacks one or more functional hemolysis factor(s) or has low hemolysis activity as compared to a positive control B. hyodysenteriae strain. In an embodiment, the vaccine is used to protect a farm animal from an intestinal spirochaete. In an embodiment, the farm animal is a pig.

[00010] There is also provided a method of preparing a live vaccine strain of B. hyodysenteriae, comprising the steps of (in any order): (a) obtaining a virulent strain of B. hyodysenteriae', (b) deleting, inactivating or modifying one or more functional virulence factor(s) and one or more functional hemolysis factor(s) in said strain of B. hyodysenteriae', and (c) isolating a live B. hyodysenteriae strain containing said modifications.

[00011] There is also provided a method of identifying a candidate live vaccine strain of B. hyodysenteriae, comprising the steps of (in any order): (a) obtaining a stain of B. hyodysenteriae', (b) determining the presence or absence of one or more functional virulence factor(s) and one or more hemolysis factor(s) in said strain, wherein the absence of one or more functional virulence factor(s) and one or more hemolysis factor indicates that the B. hyodysenteriae strain is suitable as a vaccine.

[00012] There is also provided a method of identifying a candidate live vaccine strain of B. hyodysenteriae, comprising the steps of (in any order): (a) obtaining a strain of B. hyodysenteriae', (b) determining the presence or absence of one or more functional virulence factor(s) in said strain; (c) comparing the hemolytic activity of said strain to a positive control B. hyodysenteriae strain, wherein low hemolytic activity as compared to the positive control B. hyodysenteriae strain and the absence of one or more functional virulence factor(s) indicates that the B. hyodysenteriae strain is suitable as a vaccine.

[00013] Provided herein is a vaccine composition, comprising in a pharmaceutically acceptable vehicle, at least one vaccine strain of B. hyodysenteriae as defined herein or at least one vaccine strain of B. hyodysenteriae obtained by the method as defined herein.

[00014] Provided herein is a method of preventing a diarrhoeal disease in an animal comprising administering to said animal an effective amount of an isolated vaccine strain of B. hyodysenteriae as defined herein, an isolated vaccine strain of B. hyodysenteriae obtained according to the methods as defined herein, or a vaccine composition as defined herein.

[00015] Provided herein is a method of immunising an animal against infection by B. hyodysenteriae or a related microorganism, comprising administering to said animal an effective amount of an isolated vaccine strain of B. hyodysenteriae as defined herein, an isolated vaccine strain of B. hyodysenteriae obtained according to the methods as defined herein, or a vaccine composition as defined herein.

[00016] Provided herein is an isolated vaccine strain of B. hyodysenteriae as defined herein, an isolated vaccine strain of B. hyodysenteriae obtained according to the methods as defined herein, or a vaccine composition as defined herein for use in preventing a diarrhoeal disease in an animal.

[00017] Provided herein is an isolated vaccine strain of B. hyodysenteriae as defined herein, an isolated vaccine strain of B. hyodysenteriae obtained according to the methods as defined herein, or a vaccine composition as defined herein for use in immunising an animal against infection by B. hyodysenteriae or a related microorganism.

[00018] Provided herein is the use of an effective amount of an isolated vaccine strain of B. hyodysenteriae as defined herein, an isolated vaccine strain of B. hyodysenteriae obtained according to the methods as defined herein, or a vaccine composition as defined herein in the manufacture of a medicament for preventing a diarrhoeal disease in an animal.

[00019] Provided herein is the use of an effective amount of an isolated vaccine strain of B. hyodysenteriae as defined herein, an isolated vaccine strain of B. hyodysenteriae obtained according to the methods as defined herein, or a vaccine composition as defined herein in the manufacture of a medicament for immunising an animal against infection by B. hyodysenteriae or a related microorganism.

BRIEF DESCRIPTION OF THE FIGURES

[00020] Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

[00021] Figure 1 shows a Multi-locus Sequence Typing (MSLT) dendogram showing the 30 sequence types (STs) of the 96 B. hyodysenteriae isolates from 2014/16. The dendrogram is based on consensus sequences constructed from combined individual distance matrices of nucleotide sequences from seven genes adh, alp, est, gdh, glpK,pgm and thi. STs shared with historic isolates found in the PubMLST database are indicated in bold (these were all of Australian origin). The STs of the two weakly hemolytic B. hyodysenteriae isolates are indicated with an asterisk. The length of the scale bar represents 10-nucleotide substitution in 100 base pairs of the sequenced gene fragment.

[00022] Figure 2 shows a Minimum Spanning Tree (MST) showing the relationships of 96 B. hyodysenteriae isolates from 2014/16 and the three categories of herd-health status that were originally reported. In the MST, each labelled node represents a different ST and the colour represents the original reported herd health status (negative, positive or of uncertain health status). The STs of the weakly hemolytic isolates identified in this study are indicated with black arrows.

[00023] Figure 3 shows the in vitro hemolytic activity of the weakly hemolytic B. hyodysenteriae strains MUVI and MUV2.

DETAILED DESCRIPTION

[00024] As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a single method, as well as two or more methods; reference to "an agent" includes a single agent, as well as two or more agents; reference to "the disclosure" includes a single and multiple aspects taught by the disclosure; and so forth. Aspects taught and enabled herein are encompassed by the term "invention". Any variants and derivatives contemplated herein are encompassed by "forms" of the invention.

[00025] The present specification discloses a live vaccine strain for preventing B. hyodysenteriae infection in pigs. In particular, there is provided an isolated live vaccine strain of B. hyodysenteriae, wherein (a) the live vaccine strain lacks one or more functional virulence factor and (b) the live vaccine strain lacks one or more functional hemolysis factor or has low hemolysis activity as compared to a positive control B. hyodysenteriae strain.

[00026] The term “isolated” as used herein means altered "by the hand of man" from its natural state; i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.

[00027] The term “vaccine” includes an agent which may be used to stimulate the immune system of an animal. In this way, immune protection may be provided against an antigen not recognized as a self-antigen by the immune system.

[00028] The vaccine strain of the present invention is a live strain of B. hyodysenteriae. The term "live" is used herein to indicate that the B. hyodysenteriae bacteria in the vaccine strain is able to grow and reproduce as opposed to a dead cell vaccine. The term "strain", as used herein, describes variants of a bacterial species that can be distinguished by one or more characteristics, such as ribosomal RNA sequence variation, DNA polymorphisms, serological typing, or toxin production, from other strains within that species. B. hyodysenteriae strains may be distinguished by their virulence status, i.e. strains are classified as virulent or avirulent. Examples of virulent B. hyodysenteriae strains include WAI, B204, Vic2, BW1, NSW5, Q17, NSW15, while examples of avirulent strains include B78T, SA2206, VS1, B234, R301, B6933, FM 88.90 and Al.

[00029] In one example, the B. hyodysenteriae strain is an attenuated strain. The term “attenuated” refers to a virulent strain of B. hyodysenteriae that has been modified so that it is no longer capable of causing disease (i.e., the modified strain is avirulent). The attenuated (or modified) B. hyodysenteriae strain would therefore be different from a naturally-occurring B. hyodysenteriae strain.

[00030] The “live vaccine strain” may be used in a porcine animal. The porcine animal may be a domestic pig. Alternatively, the porcine animal may be, for example, a wild boar, a Bornean bearded pig, a Palawan Bearded Pig, Vietnamese Warty Pig, Visayan Warty Pig, Celebes Warty Pig, Flores Warty Pig, Mindoro Warty Pig, Philippine Warty Pig or Javan Warty Pig. The porcine animal may also be a hog.

[00031] Alternatively, the “live vaccine strain” may be used in other mammalian and avian species, including humans, companion animals such as dogs and cats, domestic animals such as chicken and geese, horses, cattle and sheep, or zoo mammals such as non-human primates, felids, canids and bovids.

[00032] The phrase “lacks one or more functional virulence factor(s)” means that when one or more functional virulence factor(s) is absent or modified, then overall virulence activity of the bacterial strain is lost or sufficiently impaired. The lack of one or more functional virulence factor(s) may result in about 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction of the overall virulence activity of the bacterial strain. In one example, the lack of one or more functional virulence factor results in about 50% or more reduction of the overall virulence activity of the bacteria. This may be expressed as a range of from 40% to 100% reduction including 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

[00033] Similarly, the phrase “lacks one or more functional hemolysis factor(s)” means that when one or more functional hemolysis factor(s) is absent or modified, then overall hemolytic activity of the bacterial strain is lost or sufficiently impaired. The lack of one or more functional hemolysis factor may result in about 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction of the overall hemolytic activity of the bacterial strain. In one example, the lack of one or more functional hemolysis factor results in about 50% or more reduction of the overall hemolytic activity of the bacteria. Again, this may be represented as a range of from 40% to 100% as defined above.

[00034] The one or more functional virulence factor(s) or hemolysis factor(s) may naturally be missing or lacking. Alternatively, the one or more functional virulence or hemolysis factor may be deleted, inactivated or modified.

[00035] The term “virulence factor” may refer to a factor(s) that enables a bacterium to cause disease (i.e. determine its pathogenicity). The “virulence factor” may be a gene or a protein. A bacterium that lacks one or more “virulence factor” may have a lower likelihood of causing disease. For example, a B. hyodysenteriae bacterium that has low virulence may be able to colonize the pig gut without inducing much lesions. The “virulence factor” may be a hemolysin, phospholipase, lipooligosaccharide or a factor associated with chemotaxis, motility, accessory factor for substrate utilisation, iron binding, aerotolerance, and cell surface lipoprotein.

[00036] The one or more functional “virulence factor(s)” may be Fe-S oxidoreductase containing radical SAM domain (one of SEQ ID NOs: 1-3), glycosyl transferase, group 1-like protein (SEQ ID NO:4), NAD dependent epimerase (SEQ ID NO:5) or dTDP-4-dehydrorhamnose 3, 5-epimerase (rfbC) (SEQ ID NO:6).

[00037] The one or more functional virulence factor(s) may be encoded by one or more polynucleotide sequences that is at least 90% identical to one or more of the nucleic acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[00038] In one example, the live vaccine strain lacks all six functional virulence factors encoded by the nucleic acid sequences of SEQ ID NOs: 1-6.

[00039] In one example, the virulence factor is not a hemolysis factor (i.e. a non-hemolytic virulence factor).

[00040] The terms "nucleic acid" or “polynucleotide" as used herein refer to deoxyribonucleic acid and ribonucleic acid in all their forms, i.e., single and double-stranded DNA, cDNA, mRNA, and the like.

[00041] The term "encode" includes nucleotides and/or amino acids that correspond to other nucleotides or amino acids in the transcriptional and/or translational sense.

[00042] The term “percentage identity” or “% identity” between two nucleic acid sequences means the percentage of identical nucleotide residues in corresponding positions in the two optimally aligned sequences. Methods for aligning sequences are well known in the art. For example, various bioinformatics or computer programs and alignment algorithms such as ClusterW and Sequencher may be used to determine the “% identity” between two nucleic acid sequences.

[00043] The term “hemolysis factor” may refer to a factor or factors that allows a bacteria to rupture (or lyse) red blood cells. The “hemolysis factor” may be a gene or a protein. The “hemolysis factor” may be acyl carrier protein (SEQ ID NO:7), tlyA (SEQ ID NO:8), tlyB (SEQ ID NO:9), tlyC (SEQ ID NO:10), hemolysin III (SEQ ID NO:11), hemolysin activation protein (SEQ ID NO: 12), hemolysin channel protein (SEQ ID NO: 13) or hemolysin (SEQ ID NO: 14). The acyl carrier protein (SEQ ID NO: 7) is an acyl carrier protein that possess hemolytic activity.

[00044] The one or more functional hemolysis factor(s) may be encoded by one or more polynucleotide sequences that is at least 90% identical to one or more of the nucleic acid sequences selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQIDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.

[00045] In one example, the isolated live vaccine strain of B. hyodysenteriae has been modified such that it lacks one or more functional hemolysis factor.

[00046] The presence or absence of one or more functional virulence or hemolysis factor(s) may be determined by the analysis of transcription and/or translation of the polynucleotide or gene encoding a virulence or hemolysis factor, including analyzing the RNA and protein expression levels, as well as the presence of the DNA sequence within the chromosome or cytoplasm. The polynucleotide or gene encoding a virulence or hemolysis factor may also be sequenced to determine if there are any mutations that inactivate the virulence or hemolysis factor which may cause the virulence or hemolysis factor to be non-functional.

[00047] A B. hyodysenteriae strain may lack one or more functional virulence or hemolysis factor(s) due to naturally-occurring mutations in polynucleotides or genes encoding virulence or hemolytic factors. The mutations may occur in the coding regions, regulatory or promoter regions of the virulence factor or hemolysis factor. This may result in non-expression or reduced expression of the virulence or hemolysis factor at the RNA or protein level. The naturally-occurring B. hyodysenteriae strain, once isolated by standard techniques, can, if required, be subjected to further mutagenesis or recombinant DNA techniques to construct a double or multiple mutant strains. Further, the B. hyodysenteriae strain may contain deletions of whole genes encoding virulence or hemolysis factors.

[00048] Techniques for identifying bacteria that have one or more mutations in genes encoding virulence or hemolysis factors are known by one skilled in the art. These techniques include Northern and Western blotting, PCR, ELISAs and cytotoxicity assays.

[00049] The B. hyodysenteriae strain may be modified by any of a number of methods known in the art such as multiple serial passages, temperature sensitive attenuation, mutation, or the like, such that the resultant strain is attenuated and not capable of causing disease in an animal.

[00050] A number of techniques are well known in the art for reducing or abolishing polynucleotide expression. For example, a mutation may be introduced at a predetermined site, such as the promoter region or within the coding sequence to produce a nonsense mutation, using recombinant DNA-technology. Recombinant DNA techniques comprise cloning the gene of interest, modification of the gene sequence by site-directed mutagenesis, restriction enzyme digestion followed by re-ligation and subsequent replacement of the wild type gene with the mutant gene.

[00051] A mutation may also be introduced at a predetermined site in chromosomal or extrachromosomal DNA (eg. a plasmid) via an insertion, a deletion, or a substitution of one nucleotide by another, such as a point mutation, which leads to a mutated gene that has reduced or no expression. The mutation should produce a B. hyodysenteriae strain that has a reduced capacity to cause diarrhoeal diseases, such as swine dysentery. The mutation may, for example, be a deletion mutation, where disruption of the gene is caused by the excision of nucleic acids. Such a mutation can, for example, be made by the deletion of a contiguous span of base pairs. Even very small deletions such as stretches of 10 base pairs can cause the gene to encode no protein or a non-functional protein. Even the deletion of one single base pair may lead to no protein or a non-functional protein, since as a result of such a mutation, the other base pairs are no longer in the correct reading frame or transcription has been inhibited or diminished. In an embodiment, a longer stretch is removed e.g. 100 base pairs. In an embodiment, the whole gene is deleted.

[00052] Well-defined and deliberately made mutations involving the deletion of fragments or the whole gene, or combinations thereof, have the advantage, in comparison to classically induced mutations, that they will not revert to wild-type. Thus, a mutation in a gene encoding a virulence or hemolysis factor may comprise a deletion or an insertion to disrupt the polynucleotide sequence encoding the virulence factor so that no corresponding protein is produced or the protein is non-functional.

[00053] Genes encoding the virulence or hemolysis factors of the present invention may be plasmid-bome. In one example, the modification to a virulent B. hyodysenteriae strain comprises curing the strain of one or more plasmids. The term "plasmid" as used herein refers to cytoplasmic DNA that replicates independently of the bacterial chromosome. A variety of methods involving chemical and physical agents for eliminating or "curing" plasmids from a bacterial strain are known in the art. The curing of a bacterial strain of a plasmid does not involve the physical removal of the plasmid directly, but instead concerns interfering with the replication and/or partitioning of the plasmid so as to increase the rate at which plasmid-free partitions occur.

[00054] Techniques for identifying cured derivatives are known by one skilled in the art. These include Northern and Western blotting, ELISAs and cytotoxicity assays. In one example, the single colonies are screened for the loss of a plasmid by PCR.

[00055] It would be apparent to one of skill in the art that these same techniques could be applied to identify naturally occurring avirulent strains of B. hyodysenteriae that lack one or more plasmids containing virulence genes .

[00056] The live vaccine strain may have low hemolysis activity as compared to a positive control B. hyodysenteriae strain. The term “hemolysis activity” may refer to the ability for a bacterium to rupture (or lyse) red blood cells. The “hemolysis activity” of a bacterium may be compared to a positive control B. hyodysenteriae strain.

[00057] The “hemolysis activity” of a bacterial strain may be measured by methods that are well known in the art. For example, the “hemolysis activity” may be measured by quantifying the release of hemoglobin from lysed red blood cells by absorbance. In one example, hemolytic activity is determined by the measuring the decrease in absorbance at 450nm following lysis of the red blood cells as shown in the examples. The “hemolysis activity” of a bacterial strain may also be estimated by culturing the bacterial strain on blood agar plates and observing visually its ability to induce hemolysis around bacterial growth on the plates.

[00058] The live vaccine strain may have 60%, 50%, 40%, 30%, 20%, 10% or 0% hemolysis activity as compared to the hemolysis activity of the positive control B. hyodysenteriae strain.

In one example, the live vaccine strain has 50% hemolysis activity as compared to the hemolysis activity of the positive control B. hyodysenteriae strain. This may be expressed as a range of from 60% to 0% which includes 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,7, 6,5,4,3,2, 1 or 0%.

[00059] The live vaccine strain may be of different MLST sequence types. In one example, the live vaccine strain is MUV1 (V17/004760) or MUV2 (V17/004761). MUV1 was deposited at the National Measurement Institute with an accession number of VI7/004760 on 8 March 2017. MUV1 was deposited at the National Measurement Institute with an accession number of VI7/004761 on 8 March 2017.

[00060] A positive control B. hyodysenteriae strain may be WAI (ATCC49526).

[00061] In one example, there is provided a method of preparing a live vaccine strain of B. hyodysenteriae, comprising the steps of: (a) obtaining a virulent strain of B. hyodysenteriae', (b) deleting, inactivating or modifying one or more functional virulence factor(s) and one or more functional hemolysis factor(s) in said strain of B. hyodysenteriae', and (c) isolating a live B. hyodysenteriae strain containing said modifications.

[00062] In one example, there is provided a method of identifying a candidate live vaccine strain of B. hyodysenteriae, comprising the steps of: (a) obtaining a stain of B. hyodysenteriae', (b) determining the presence or absence of one or more functional virulence factor(s) and one or more hemolysis factor(s) in said strain, wherein the absence of one or more functional virulence factor and one or more hemolysis factor indicates that the B. hyodysenteriae strain is suitable as a vaccine.

[00063] In one example, there is provided a method of identifying a candidate live vaccine strain of B. hyodysenteriae, comprising the steps of: (a) obtaining a strain of B. hyodysenteriae', (b) determining the presence or absence of one or more functional virulence factor(s) in said strain; (c) comparing the hemolytic activity of said strain to a positive control B. hyodysenteriae strain, wherein low hemolytic activity as compared to the positive control B. hyodysenteriae strain and the absence of one or more functional virulence factor(s) indicates that the B. hyodysenteriae strain is suitable as a vaccine. These steps may be performed in any order without departing from the present invention.

[00064] Vaccine compositions are also provided herein. In one example, there is provided a vaccine composition comprising in a pharmaceutically acceptable vehicle at least one vaccine strain of B. hyodysenteriae as defined herein or at least one vaccine strain of B. hyodysenteriae obtained by the method as defined herein.

[00065] The term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to the subject. Useful carriers are well known in the art, and include, e.g., water, buffered w water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. The term "veterinarily acceptable" may also be used with the same meaning.

[00066] The vaccine composition may comprise or be an adjuvant. The adjuvant may be a substance that increases the immunological response of the subject (e.g. pig) to the vaccine. Suitable adjuvants include, but are not limited to, aluminum hydroxide (alum), immunostimulating complexes (ISCOMS), non-ionic block polymers or copolymers, cytokines (like IL-1, IL-2, IL-7, IFN-a, IFN-β, IFN-γ, etc.), saponins, monophosphoryl lipid A (MLA), muramyl dipeptides (MDP) and the like. Other suitable adjuvants include, for example, aluminium potassium sulfate, heat-labile or heat-stable enterotoxin isolated from Escherichia coli, cholera toxin or the B subunit thereof, diphtheria toxin, tetanus toxin, pertussis toxin, Freund's incomplete or complete adjuvant, etc. Toxin-based adjuvants, such as diphtheria toxin, tetanus toxin and pertussis toxin may be inactivated prior to use, for example, by treatment with formaldehyde.

[00067] In one example, the vaccine strain of B. hyodysenteriae is in a freeze-dried or lyophilized formulation. The vaccine strain may also be reconstituted for use as a vaccine.

[00068] In one example, there is provided a method of preventing a diarrhoeal disease in an animal comprising administering to said animal an effective amount of an isolated vaccine strain of B. hyodysenteriae as defined herein, an isolated vaccine strain of B. hyodysenteriae obtained according to the method as defined herein, or a vaccine composition as defined herein.

[00069] In one example, there is provided a method of immunising an animal against infection by B. hyodysenteriae or a related microorganism, comprising administering to said animal an effective amount of an isolated vaccine strain of B. hyodysenteriae as defined herein, an isolated vaccine strain of B. hyodysenteriae obtained according to the method as defined herein, or a vaccine composition as defined herein.

[00070] The isolated vaccine strain of B. hyodysenteriae that is administered to the animal may be MUV1 (VI7/004760) or MUV2 (VI7/004761).

[00071] In one example, about lxlO8, 2xl08, 3xl08, 4xl08, 5xl08, 6xl08, 7xl08, 8xl08, 9xl08, lxlO9, 2xl09, 3xl09, 4xl09, 5xl09, 6xl09, 7xl09, 8xl09, 9xl09, lxlO10, 2xlO10, 3xl010, 4xlO10, 5xlO10, 6xlO10, 7xlO10, 8xl010, 9xlO10, lxlO11, 2xlOn, 3xl0n, 4xlOn, 5χ10π, 6χ10π, 7χ10π, 8χ10π or 9χ10π of live vaccine Β. hyodysenteriae cells or a value inbetween these amounts are administered to the animal. In one example, about lxl09 live vaccine B. hyodysenteriae cells are administered to the animal. These amounts may also be expressed as a range of from 1x10 to 9x10 or any value inbetween.

[00072] In one example, the B. hyodysenteriae cells are administered to the animal once a day consecutively for a period of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days. In one example, the B. hyodysenteriae cells are administered to the animal once a day consecutively for a period of 4 days. The period may also be expressed as a range of from 2 to 30 days or a time period inbetween.

[00073] The term “preventing” as used herein may refer to the delaying or impeding of the onset of a disease (e.g. a diarrhoeal disease), condition or disorder. It may also refer to the reduction of the frequency or severity of symptoms associated with the disease, condition or disorder. In other words, the vaccine strain may be used as a prophylactic.

[00074] The term “diarrhoeal disease” as used herein may be dysentery or Swine Dysentery.

[00075] The term “immunising” may refer to inducing a protective immune response in an animal whereby the animal produces its own immune response against B. hyodysenteriae. The induction of the “protective immune response” may increase the resistance of the animal to infection by and/or disease caused by future challenge with B. hyodysenteriae. The immune response may be a production of antibodies against B. hyodysenteriae. The immune response may also be a mucosal immune response.

[00076] The immunisation may protect an animal against a microorganism related to B. hyodysenteriae. A "related microorganism" includes an immunological cross reactive relative. Hence, immunisation may protect against an immunological cross reactive relative of B. hyodysenteriae that has infected an animal. This is because the immune response may also recognize the “immunological cross reactive relative” and therefore increase the resistance of the mammal to infection by and/or disease cased by future challenge of the “immunological cross reactive relative”. A “related microorganism” may include other pathogenic Brachyspira species such as Brachyspira hampsonii, Brachyspira suanatina, Brachyspira intermedia, Brachyspira pilosicoli or Brachyspira murdochi.

[00077] The term “administering” as used herein may refer to contacting, applying, delivering or providing an isolated vaccine strain of B. hyodysenteriae or a vaccine composition to an animal by any appropriate means. The vaccine strains or compositions may be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts, taking into consideration such factors as the age, sex, weight, species and condition of the recipient animal, and the route of administration. The route of administration can be percutaneous, via mucosal administration (e.g., oral, nasal, anal, vaginal) or via a parenteral route (intradermal, intramuscular, subcutaneous, intravenous, or intraperitoneal). Vaccine strains or compositions can be administered alone, or can be co-administered or sequentially administered with other treatments or therapies. Forms of administration may include suspensions, syrups or elixirs, and preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration) such as sterile suspensions or emulsions.

[00078] For oral administration, the formulation of the vaccine preparation may be presented as capsules, tablets, powders, granules, or as a suspension. The preparation may have conventional additives, such as lactose, mannitol, com starch, or potato starch. The preparation also may be presented with binders, such as crystalline cellulose, cellulose derivatives, acacia, com starch, or gelatins. Additionally, the preparation may be presented with disintegrators, such as corn starch, potato starch, or sodium carboxymethylcellulose. The preparation may be further presented with dibasic calcium phosphate anhydrous or sodium starch glycolate. The preparation may be presented with lubricants, such as talc or magnesium stearate.

[00079] In one example, the live vaccine strain is administered orally to the animal.

[00080] For intravenous, cutaneous, or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity, and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants, and/or other additives can be included, as required.

[00081] For intranasal administration (e.g., nasal sprays) and/or pulmonary administration (administration by inhalation), formulations of the vaccine preparation, including aerosol formulations, may be prepared in accordance with procedures well known to persons of skill in the art. Aerosol formulations may comprise either solid particles or solutions (aqueous or non-aqueous). Nebulizers (e.g., jet nebulizers, ultrasonic nebulizers, etc.) and atomizers may be used to produce aerosols from solutions (e.g., using a solvent such as ethanol); metered-dose inhalers and dry-powder inhalers may be used to generate small-particle aerosols. The desired aerosol particle size can be obtained by employing any one of a number of methods known in the art, including, without limitation, jet-milling, spray drying, and critical -point condensation.

[00082] Supplementary active ingredients can also be incorporated into the vaccine preparations. The latter is particularly contemplated as far as the present invention extends to multi-component vaccines. Accordingly, in another embodiment, the vaccine preparations of the present invention may comprise in addition to B. hyodysenteriae, one or more other active compounds such as antigens and or immune stimulating compounds.

[00083] Optionally, one or more compounds having adjuvant activity may be added to the vaccine preparation.

[00084] In one example, there is provided an isolated vaccine strain of B. hyodysenteriae as defined herein, an isolated vaccine strain of B. hyodysenteriae obtained according to the method as defined herein, or a vaccine composition as defined herein for use in preventing a diarrhoeal disease in an animal.

[00085] In one example, there is provided an isolated vaccine strain of B. hyodysenteriae as defined herein, an isolated vaccine strain of B. hyodysenteriae obtained according to the method as defined herein, or a vaccine composition as defined herein for use in immunising an animal against infection by B. hyodysenteriae or a related microorganism.

[00086] In one example, there is provided a use of an effective amount of an isolated vaccine strain of B. hyodysenteriae as defined herein, an isolated vaccine strain of B. hyodysenteriae obtained according to the method as defined herein, or a vaccine composition as defined herein in the manufacture of a medicament for preventing a diarrhoeal disease in an animal.

[00087] In one example, there is provided the use of an effective amount of an isolated vaccine strain of B. hyodysenteriae as defined herein, an isolated vaccine strain of B. hyodysenteriae obtained according to the method as defined herein, or a vaccine composition as defined herein in the manufacture of a medicament for immunising an animal against infection by B. hyodysenteriae or a related microorganism.

EXAMPLES

[00088] Aspects disclosed herein are further described by the following non-limiting Examples. EXAMPLE 1

Surveillance for B. hyodysenteriae in Australian herds and characterisation of isolates

The Sample collection [00089] The project involved samples. Samples were requested from herds with a history of SD, whether or not they showed disease at the time of sampling, from herds with mild enteritis of uncertain aetiology, and from herds that did not have disease but had given positive reactions (“false positives”) in a serological ELISA for SD in a previous study.

Bacteriological culture [00090] All faecal or colonic samples that were received were swabbed onto Trypticase Soy agar (TSA) plates containing 5% (v/v) defibrinated ovine blood, and cultured for 5 to 7 days at 37°C in a culture jar with an anaerobic atmosphere generated by an AnaeroGen (Trade Mark) 2.5L Sachet (Oxoid). Zones of hemolysis around the inoculated area indicated growth, and surface growth was suspended in phosphate buffered saline (PBS) and viewed with a phase-contrast microscope to confirm that spirochaetes were present. The samples that contained spirochaetes were retained for testing by Polymerase Chain Reaction (PCR).

Diagnostic PCR

[00091] The identification of the Brachyspira species cultured from the field samples was determined using published PCR tests for B. hyodysenteriae (La, T., Phillips, N.D. and Hampson, D.J. (2003), Journal of Clinical Microbiology 41:3372-3375), Brachyspira intermedia (Phillips, N.D., La, T. and Hampson, D.J. (2006), Veterinary Microbiology 116:239-245) and Brachyspira pilosicoli (La et al., 2003, supra). Newly developed PCR tests for B. hampsonii and B. suanatina using unique primers targeting the hemolysin A gene (tlyA) and DNA-dependent RNA polymerase gene (rpoC), respectively, were used. A genusspecific PCR for Brachyspira spp. (Phillips et al., 2006, supra) was also used.

[00092] For the field samples, PCR assays were applied to growth harvested from the primary isolation plates and resuspended in sterile water. The PCR assays were performed in 25 μ! reactions consisting of lx PCR buffer, 1.5 mM MgCfi, 0.5 U Taq DNA polymerase, 0.2 mM of each dNTP and 0.5 μΜ of forward and reverse primers. Cycling conditions involved an initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 15 s and primer extension at 72°C for 1 min. The products were separated by gel electrophoresis and visualized under UV light after staining with ethidium bromide.

Antimicrobial Susceptibility Testing [00093] Isolates were subcultured to purity. The spirochaetes to be tested for antimicrobial susceptibility were cultured on a fresh TSA plate at 37°C for 5 days, as described above. The cells were harvested from the agar plate by resuspending the surface growth with 2 ml sterile PBS. These cells were transferred into a micro fuge tube and counted using a hemocytometer. Spirochaete cells were collected by centrifuging the bacterial suspension at 5,000 g, and the pellet was then resuspended with sterile PBS to a density of 106 cells per ml.

[00094] Antimicrobial susceptibility of the field isolates was assessed using the agardilution method. The test plates consisted of TSA containing 5 % (v/v) defibrinated ovine blood and an appropriate antibiotic concentration. Control plates did not include antibiotics. The plates were incubated for 5 days at 37 °C in anaerobic jars and were observed for hemolysis. The isolates were tested for susceptibility to varying concentrations of tiamulin (0.25, 0.5, 1, 4 and 8 pg/ml), tylosin (1, 5, 25, 50 and 100 pg/ml) and lincomycin (2, 4, 16, 36, and 72 pg/ml).

[00095] A total of 105 cells were drop-inoculated onto the control and sensitivity plates. Each isolate was tested in duplicate and B. hyodysenteriae control strain WAI was included in each batch of tests. Growth of the strains on the control and sensitivity plates was checked visually after 5 days incubation. Zones of hemolysis present around the growth on the control plates were determined, and isolates were recorded as being susceptible to the antimicrobial concentration in the test plates if no such zones were observed. Any surface growth was scraped off the plate and examined under a phase contrast microscope to confirm its purity and determine the endpoint.

[00096] The first sensitive colony zone and the last resistant colonies were checked for spirochaete growth by phase-contrast microscopy. The minimum inhibiting concentration (MIC) of the antibiotic was reported as the lowest concentration of antimicrobial that inhibited growth. MIC breakpoints used to assist interpretation of the results are presented in Table 1.

Table 1. M1C breakpoints (gg/ml) for in-vitro antimicrobial susceptibility tests performed on Brachyspira isolates recovered from field samples.

Antimicrobial Sensitive Intermediate Resistant____________Reference__________

Tiamulin <0.25 >0.25 <2 >2 Pringle et al, 2012

Tylosin <1 > 1 < 4 >4 Ronne and Szancer, 1990

Lincomycin <4 >4 <36 >36 Ronne and Szancer, 1990

Multi-locus Sequence Typing (MLST) [00097] Ninety-six B. hyodysenteriae field isolates were typed by MLST. The isolates were subcultured to purity prior to use. All isolates were cultured on a fresh TSA plate at 37°C for 5 days as described above. The cells were harvested from the agar plate by resuspending the surface growth with 2 ml sterile PBS. The cells were transferred into a microfuge tube and counted using a hemocytometer. The cells were collected by centrifuging at 5,000 g for extraction of high molecular weight DNA using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s instructions. Cell pellets were resuspended in 180 pl of lysis buffer containing 20 μΐ of proteinase K (10 mg/ml) and incubated at 55°C for 30 min. After all the cells had been lysed, 180 μΐ of AL Buffer was added and the sample incubated at 70°C for 10 min. Two hundred μΐ of absolute ethanol was immediately added to the sample and this was transferred to a DNeasy column. Column wash buffers AW1 and AW2 were added sequentially to the columns and centrifuged at 6,000 x g. The flow-through was discarded, and the DNA was eluted with elution buffer and stored at -20°C.

[00098] MLST was conducted as previously described (La, T., Phillips, N.D., Harland, B.L., Wanchanthuek, P., Bellgard, M.I. and Hampson, D.J. (2009), Veterinary Microbiology 138:330-338). PCR assays were performed in 50 μΐ reactions consisting of lx PCR buffer, 1.5 mM MgSCfi, 0.5 U HotStar HiFidelity DNA polymerase (Qiagen), 0.2 mM of each dNTP and 0.5 μΜ of forward and reverse primers. Cycling conditions involved an initial enzyme activation step at 95°C for 15 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 15 s and primer extension at 72°C for 1 min. The products were separated by gel electrophoresis and visualized after staining with ethidium bromide. PCR products used for sequencing were purified with the UltraClean (Trade Mark) PCR Clean-up Kit (Mo Bio Laboratories) according to the manufacturer’s instructions. The purified PCR products were used for sequencing. Sequencing reactions were performed in 10 μΐ volumes using the BigDye (Registered Trade Mark) Terminator v3.1 Cycle Sequencing Kit (Life Technologies) according to the manufacturer’s instructions. An annealing temperature of 50°C was used. The sequencing products were analyzed using the Applied Biosystems 3730XL DNA analyzer.

[00099] The raw sequences were edited and analyzed using Geneious version 9.1.2 (Biomatters Ltd). For each locus the consensus sequences were aligned with the original B. hyodysenteriae strain WAI sequence downloaded from the PubMLST database (www.pubmlst.org) and the aligned loci sequences were trimmed for subsequent MLST analysis as previously reported (La et al., 2009, supra). Allele designations for each locus were obtained by a query search from the PubMLST website. New sequence types (STs) were assigned by the PubMLST curator for the Brachyspira MLST schemes (Dr Tom La).

[000100] Dendograms and minimum spanning trees (MST) were constructed from the data matrix of allelic mismatches using the UPGMA (unweighted-pair group method with allelic arithmetic means) method with 1000 bootstrap replicates using the PHYLOViZ software (Francisco, A.P., Vaz, C., Monteiro, P.T., Melo-Cristino, J., Ramirez, M. and Carriqo, J.A. (2012), BMC Bioinformatics 13:87). Color coding of the MSTs was added to highlight the region where the isolates originated and source/supplier of the pigs. The sequence types (STs) of the isolates were compared with those of historic isolates of B. hyodysenteriae recovered in Australia since the 1980s, deposited in PubMLST

Plasmid Virulence Gene Testing of B. hyodysenteriae isolates [000101] High molecular weight DNA extracted from the 96 B. hyodysenteriae field isolates for MLST analysis was used as template for the PCR of virulence-associated plasmid genes. PCR tests targeting six plasmid-encoded genes recently described as possible virulence genes in B. hyodysenteriae (La, T., Phillips, N.D., Thomson, J.R. and Hampson, D.J. (2014), Veterinary Research 45:131) were used for the prediction of virulence in B. hyodysenteriae isolates. For each plasmid-encoded gene, three primer pairs were designed for PCR amplification. PCR assays were performed in 25 μΐ reactions, as described above with the exception that the annealing temperature used for each primer was set at 5°C less than the optimal annealing temperature to allow for a moderate stringency reaction. Amplification products were electrophoresed through an agarose gel, stained with ethidium bromide and viewed over ultraviolet light. Isolates were predicted to have reduced virulence potential when one or more of the six virulence-associated genes gave a negative result. ELISA for swine dysentery

Serum samples [000102] Serum samples were collected from slaughter age pigs and were provided by the consulting veterinarians. These included samples from herds that did not have the disease but were previously tested positive for Swine Dysentery with the ELISA assay. The samples also came from herds with clinical SD, and herds with no reported clinical disease. ELISA method [000103] The prototype commercial ELISA from Prionics is no longer available. The test was set up using the original reagents and conditions. Wells in a 96-well microtitre plate (MicroIon, Greiner Bio-One) were coated with recombinant Hl 14 protein at 5 pg/mL in 100μ1 100 mM carbonate buffer (pH 9.6) at 4°C overnight. The wells were blocked with 3% (v/v) skim-milk powder in phosphate buffered saline (PBS, pH 7.2) for lh before being washed three times with PBS containing 0.05% (v/v) Tween 20 (PBST). Serum samples were diluted 600-fold with PBST before 100 μΐ of the diluted serum was added to each well in the ELISA plate. The wells were incubated with the diluted serum samples for 2h at room temperature (RT) and washed three times with PBST. Each well was incubated with 100 μΐ mouse antiswine IgGl (1:1,000 dilution, Serotec) for lh before being washed three times with PBST. Goat anti-mouse IgG conjugated with HRP (1:50,000 dilution, Serotec) was added to the wells and incubated at RT for lh followed by three washes with PBST. 100 pL of 3,3',5,5'-tetramethylbenzidine liquid substrate (Sigma) were added to each well, and the plate was incubated in the dark at RT. The color reaction was stopped after 20min by adding 100 μΐ 0.5 M sulphuric acid. The optical density (OD) was measured at 450 nm on a microplate reader (BioRad Model 3550-UV).

[000104] For cross-plate standardization, a positive control was added in triplicate to each plate. The positive control consisted of serum collected from a pig that had recovered from experimental infection with B. hyodysenteriae. Normalized optical densities were calculated according to the following formula: (OD value of test sample - OD value of blank control)/average of (OD value of control sample - OD value of blank control). Samples with normalized OD values greater than 0.52 were considered positive according to the ELISA test.

Sampling of potential avian reservoirs of B. hyodysenteriae

Samples collected [000105] Faecal samples from aquatic waterfowl were obtained from researchers conducting surveillance projects for Avian Influenza Virus in South Australia (n = 179), Tasmania (n = 26) and Western Australia (n = 100). Bird species sampled included the Red Necked Stint (Calidris ruficollis), Ruddy Turnstone (Arenaria interpres). Hooded Plover (Thinornis rubricollis), Sanderling (Calidris alba), Silver Gull (Chroicocephalus novaehollandiae), Masked Lapwing (Vanellus miles), Pacific Black Duck (Anas superciliosa) and Magpie Goose (Anseranas semipalmata).

[000106] Faecal samples also were collected from Australian White Ibis (Threskiornis molucca) found around water bodies located at a farm in Western Australia. Colon samples collected from herds on this farm had been tested positive for B. hyodysenteriae around the time of sampling.

Bacteriological culture and PCR

[000107] Bacteriological culture and PCR identification of Brachspira species were performed on the avian samples as described above.

Outcomes -Detection of B. hyodysenteriae in Australian herds and characterization of isolates B. hyodysenteriae isolates [000108] Overall a total of 929 samples, comprising 472 faecal samples and 457 colon samples were provided for analysis. These samples originated from 75 farms across five Australian States.

[000109] Brachyspira hyodysenteriae was detected in 127 (13.7%) of the 929 samples originating from 24 of the 75 herds examined (32%). A number of the more recently sampled herds had previously been tested and were sent as follow-up samples for confirmation and/or antimicrobial susceptibility testing. Five herds were sampled because they had been recorded as “false positives” in a previous study using a prototype SD serological ELISA test kit (Hampson, D.J., La, T., Phillips, N.D., Holyoake, P.K. (2016),Veterinary Microbiology 191:15-9). They were considered false positive in the ELISA because no clinical disease was recorded. Two of the five herds had B. hyodysenteriae isolated and the samples from the other three herds were negative. Sera had also been collected at the time of sampling for comparative analysis using the SD ELISA test.

[000110] In addition, the potential pathogen Brachyspira pilosicoli was identified in 78 (8.4%) of the 927 samples, B. intermedia (of uncertain clinical significance) in 59 (6.4%) and non-pathogenic B. innocens/B. murdochii in 21 (2.3%) of the samples. B. hampsonii and B. suanatina were not found in any of the samples tested.

[000111] A total of 78 B. hyodysenteriae isolates were successfully recovered in pure culture, of which 65 isolates were further analyzed. Since many of the current samples came from farms that had been previously tested, the isolates recovered were analyzed further. As a result, a total of 96 B. hyodysenteriae isolates originating from 30 herds were further investigated (Table 2). Of the 30 herds identified as being colonized by B. hyodysenteriae, 13 herds were originally thought to be free of SD, 11 herds were reported to have SD based on clinical signs or past history, and 6 herds were of uncertain health status.

[000112] Two B. hyodysenteriae isolates, one each from herd 28 (ST151) and herd 86 (ST161) respectively, were weakly beta-hemolytic, and both herds were considered to be free ofSD.

[000113] Table 2. Comparison of sequence type (ST), plasmid gene profile and antimicrobial susceptibility profiles of 96 B. hyodysenteriae isolates recovered between 2014 and 2016 present in 30 herds in different Australian States that either were thought to be free of swine dysentery (SD), to have the disease, or to be of uncertain health status.

Herd 2____________1____________140___________4__________________R (>72)___________________R(>100)_________________1 (<0,5)__________

Herd 2____________3____________143___________4__________________R (>72)___________________R(>100)_________________1 (<0,5)__________

Herd 3 1 145 1 S (<2) R(>5<25) 1 (<0.5)

Herd 3 1 145 1 I (>4<16) R(>50<100) S (<0.25)

Herd 3 1 146 1 NTf NT NT

Herd 3____________1____________154___________1__________________I (>4<16)________________R(>50<100)_______________S (<0.25)_________

Herd 21___________2____________49___________1__________________R (>72)__________________R (>5<25)_________________1 (<0.5)__________

Herd 37___________1____________152___________1__________________R (>72)___________________R(>100)_________________1 (<0,5)__________

Herd 46___________2___________150__________7_________________R (>72)__________________R(>100)_________________R (>8)_________

Herd 49___________1___________158__________2________________R (>36>72)________________R(>100)________________R (>4<8)________

Herd 71 4 166 3 R(>72) R(>100) R(>8)

Herd 71___________1___________166__________4_________________R (>72)__________________R(>100)_________________R (>8)_________

Herd 73 1 155 1 I (>4<16) R(>100) 1 (<0.5)

Herd 73 1 156 1 1 (>16<36) R(>100) 1 (<0.5)

Herd 73___________8____________144___________1__________________R (>72)___________________R(>100)________________S (<0.25)_________

Herd 75___________1____________149___________1__________________R (>72)___________________R(>100)_________________1 (0,5)__________

Herd 78___________1____________163___________4_________________I (>4<16)_________________R(>5<25)________________S (0.25)_________

Herd 85___________1___________143__________4_________________R (>72)__________________R(>100)________________S (0,25)________

Herds of uncertain disease status (6 herds, 8 STs, 22 isolates)___________________________________________________________________________________

Herd 8____________4____________165___________1__________________R (>72)___________________R(>100)_________________1 (0.5)__________

Herd 12___________1____________164___________3__________________R (>72)___________________R (>100)_________________1 (0,5)__________

Herd 13 1 141 1 NT NT NT

Herd 13__________1___________142__________1__________________NT_____________________NT____________________NT__________

Herd 47___________5___________150__________7_________________R (>72)_________________R (>5<25)________________R(>8)_________

Herd 95___________7____________31___________5___________________S (<2)___________________R(>100)________________S (0.25)_________

Herd 98 1 144 1 I (>4<16) R(>100) 1 (0.5)

Herd 98___________2___________140__________4_________________R (>72)__________________R(>100)________________1 (0.5)_________ dST, sequence type in MLST; S, sensitive; I, intermediate; R, resistant.

Tsolates marked with an asterisk are weakly hemolytic dSTs 140, 144 and 150 were recovered from pigs from all three categories of health status, and STs 49 and 143 were found in two categories. The other STs were only found in one health status category. cPt, plasmid type: see Table 4 for definitions. fNT, Not tested.

MLST

[000114] The 96 B. hyodysenteriae isolates recovered belonged to 30 sequence types (STs) (Table 2). The 39 isolates from 13 herds that were originally considered not to have SD were divided into 12 STs. Out of these, ST50 was found in two herds and ST150 in five of the 13 herds. One of the latter five was a breeding herd, and the other four herds with this ST are likely to have received pigs from it. This ST was also found in one herd with SD and one of the herds of uncertain health status, making it the most common ST found. The 35 isolates from the 11 herds that were originally believed to have SD belonged to 17 STs, whilst the 22 isolates from the six herds of uncertain SD-status belonged to 8 STs. STs 140, 144 and 150 were found in pigs from all three categories of health status, whilst STs 49 and 143 each were found in two health categories. The other STs each were only found in herds of a single health category. STs 31, 49 and 50 previously have been described in Australian herds sampled in earlier decades.

[000115] Multiple isolates (2-10) were available for analysis from 20 of the herds. Ten of these were herds reported as not having SD, and in four of them more than one ST was identified; five of the herds were reported to have SD, and multiple STs were found in three of these; and five of the herds were of unknown health status, of which two had multiple STs, with four STs found in herd 2 and three in herd 3.

[000116] A MLST dendrogram demonstrating the relationships of the various STs for isolates for the period 2014/16 is shown as Figure 1. Four clonal complexes were identified by the eBURST program and are marked on the tree. Most isolates were relatively closely related, although STI61 and to a lesser extent ST 154 were outliers. The former was weakly hemolytic, although the second weakly hemolytic isolate in ST151 was in the main body of the tree and was not closely related to ST161.

[000117] A minimum spanning tree (MST) showing the relationship of the 2014/16 isolates of B. hyodysenteriae belonging to 30 different STs and the reported disease status of their herds of origin is shown as Figure 2. The STs containing isolates from the three disease status categories were dispersed across the tree, with no clear and consistent clustering of STs related to the health status. Four STs (49, 140, 144 and 150) included multiple isolates from herds with different reported health categories.

Antimicrobial Susceptibility Testing [000118] The antimicrobial susceptibility status to lincomycin, tylosin and tiamulin for isolates in STs of B. hyodysenteriae is shown in Table 2, and a summary comparing the results with previously reported results for Australian isolates (Hampson, D.J. (2008), Report prepared for the Co-operative Research Centre for High Integrity Australian Pork, Roseworthy, South Australia) is presented in Table 3. Most or all isolates from all three periods were resistant to tylosin. Approximately 61% of isolates from 2014/16 were resistant to lincomycin, and this figure was considerably higher as compared to the results from both earlier periods. Tiamulin resistance in 2014/16 was more common (15.2%) than amongst the isolates from 2006-2007, and there was a high percentage (58.7%) of isolates of intermediate susceptibility in 2014/16. The distribution of susceptibility for tiamulin for the isolates from 2002-2006 was more similar to the data for the 2014/16 isolates than for the 2006-2007 isolates.

[000119] Amongst the isolates from 2014/16, the prevalence of resistance to each of the antimicrobials tested was similar regardless of whether the isolates came from herds with disease or from apparently healthy herds.

[000120] Isolates belonging to 4 STs (STs 150, 158, 159 and 166) were not susceptible to all three of the antimicrobials that were tested (ie were multi-drug resistant isolates), and were identified in five herds from the same state, with ST150 being present in two herds. Two of these herds supplied breeding pigs to other herds and were thought to be free of infection. Other isolates belonging to ST 150 in five other herds had different patterns of susceptibility to the three antimicrobials.

Plasmid Virulence Gene Testing [000121] The distribution of members of the block of six plasmid genes in STs of B. hyodysenteriae isolates from 2014/16 is shown in Tables 2 and 4 for each of the three health statuses that were originally reported. The distribution of STs with different numbers of the block of six plasmid genes was not obviously different across the three reported health statuses. Between half and three quarters of the STs in the three categories had plasmid profile 1, lacking all six genes, whilst few STs had isolates with all six of the block of plasmid genes. No significant differences were found between the distribution of isolates with plasmid types 1 and 7 in the three reported health statuses using Fisher’s exact test. Three isolates, one each from herd 73 (ST155), herd 28 (ST151), and herd 86 (ST161) respectively lacked the entire plasmid. The latter two isolates also were weakly hemolytic. Herds 5, 63, 2, 71 and 98 contained isolates with different STs that had different plasmid types (e.g. types 1 and 7 both were found in isolates from herds 5 and 63).

Table 3. Classification of the B. hyodysenteriae isolates collected in 2014/2016 as being susceptible, intermediate or resistant to the three antimicrobials, and comparison with reported results for Australian isolates from previous periods.

Period No. (%) No. (%) No. (%)

Antimicrobial (no. of isolates)____________________________susceptible_______intermediate_________resistant_____ 2OI4-2OI6(n-46)11 Lincomycin 9(19.6%) 9(19.6%) 28 (60.9%)

Tylosin - 4(8.9%) 42(91.3%) _________________________Tiamulin_________12(26,1%)________27 (58,7%)_________7 (15,2%) 2006-2007 (n-60)b Lincomycin 19(31.6%) 31(51.6%) 10(16.6%)

Tylosin - - 60 (100.0%) __________________________Tiamulinc__________57 (95%)__________2 (3,3%)___________1 (1,6%) 2002-2006 (n-89)b Lincomycin 26 (29.2%) 57 (64%) 6 (6.7%)

Tylosind - 2(2.7%) 73 (97.3%) ________________________Tiamulinc_________16 (18%)__________62 (70%)_________11 (12,4%) “Multiple isolates from the same herd with the same overall profile only counted once bResults from Hampson, 2008, supra “Results recalibrated according to the criteria of Pringle et al, 2012. dFourteen isolates not tested

Table 4. Number and percentage of the 30 STs of B. hyodysenteriae isolates from the 2014/16 period possessing different combinations of the six plasmid-bome virulence-associated genes (plasmid types)

Number and percentage of STsa with different plasmid types_______ h Amongst 13 herds Amongst 11 herds Amongst 6 herds of

Plasmid type” thought to be reported to have SD uncertain health ___________________________uninfected___________________________________status________________ 1 (N, N, N, N, N, N) 9 (75%) 11 (64.7%) 4 (50%) 2 (N, N, N, N, N, P) - 1 (5.9%) - 3 (N, N, P, N, P, P) - - 1 (12.5%) 4 (N, N, P, P, P, P) 1(8.3%) 4(23.5%) 1(12.5%) 5 (N, P, P, N, P, P) - - 1 (12.5%) 6 (P, P, P, N, P, P) - - - 7 (P, P, P, P, P, P)_________2 (16,7%)______________1 (5,9%)________________1 (12,5%)_____________

Total number of STs______12___________________17___________________8___________________ “ST, sequence type in MLST (total of 30 STs identified). Three STs (140, 144 and 150) were common to all three of the health status categories, and two ST (49 and 143) each were common to two categories. '’Plasmid types defined by the absence (N) or presence (P) of the six plasmid genes in numerical order (orfll, orfl2, orfl3, orfl4, orfl5, orfl6).

Potential reservoirs of B. hyodysenteriae [000122] A total of 316 faecal samples from aquatic waterfowl were received and tested for Brachyspira. These samples were collected from the Ruddy Turnstones (n = 145), Red Necked Stints (n = 47), Silver Gulls (n = 5), Sanderlings (n = 4), Masked Lapwings (n = 3), Hooded Plovers (n = 1), Pacific Black Duck (n = 50), Magpie Geese (n = 50) and Australian White Ibis (n = 11). B. pilosicoli was isolated from two Pacific Black Duck samples and one Australian White Ibis sample. B. innocen/B. murdochii was recovered from two Magpie Geese samples and two Australian White Ibis samples. No other Brachyspira was detected. ELISA test for swine dysentery [000123] A total of 473 serum samples from 16 herds were received (Table 5). These included 8 herds with clinical SD, six herds where no clinical disease had been reported and two of uncertain health status. Seven of the 16 herds previously had been tested in the commercial ELISA test and five of these herds had given positive reactions (“false positives”: marked with an asterisk in Table 5). Of the 5 “false-positive” herds, two herds were subsequently identified as being infected with B. hyodysenteriae by culture (herds 49 and 5). The remaining three “false-positive” herds were negative for B. hyodysenteriae by culture of the faecal samples that were submitted. All five “false positive” herds were again positive in this study.

[000124] Overall, 14 of the 16 herds gave serological reactions that indicated that the herds were infected, using the selected cut-off values. All but one of the eight herds that were reported to be infected were positive in the ELISA. On the other hand, five of the six herds reported as “negative” were serologically positive, including four that previously had been considered “false positive” in the commercial ELISA. One of these herds had a B. hyodysenteriae isolate recovered. Both of the herds of unknown health status were ELISA and culture positive.

Table 5. Comparison of reported health status of 16 herds with current ELISA test results and bacteriological culture of B. hyodysenteriae. ______Herd___________Reported health status__________ELISA results________Culture results

Herd 2 Positive 24/31 positive Positive

Herd 3 Positive 6/31 positive Positive

Herd 49* Positive 18/25 positive Positive

Herd 75 Positive 8/24 positive Positive

Herd 73 Positive 14/15 positive Positive

Herd 99 Positive 13/31 positive Positive

Herd 100 Positive 13/15 positive Positive

Herd 101_____________________Positive__________________30/30 negative_______________nt_________

Herd 5* Negative 21/25 positive Positive

Herd 70 Negative 60/60 negative Negative

Herd 102 Negative 1/20 positive nt

Herd 103* Negative 16/35 positive Negative

Herd 104* Negative 26/40 positive Negative

Herd 105*___________________Negative__________________10/21 positive_____________Negative______

Herd 95 Unknown 18/30 positive Positive

Herd 98____________________Unknown________________24/40 positive____________Positive______ nt, not tested * “false-positive” herds when previously tested with the Prionics prototype commercial ELISA, EXAMPLE 2

Identification of weakly hemolytic B. hyodysenteriae strains

Sample collection [000125] Colon samples were obtained from herds with no history of SD but were showing mild enteritis of uncertain aetiology. Colon samples were also obtained from herds that had did not have disease but had given positive reactions (“false positives”) in a prototype serological ELISA for SD.

Bacteriological culture [000126] All colonic samples were swabbed onto Trypticase Soy agar (TSA) plates containing 5% (v/v) defibrinated ovine blood, and cultured for 5 to 7 days at 37°C in a culture jar with an anaerobic atmosphere generated by an AnaeroGen (Trade Mark) 2.5L Sachet (Oxoid). Zones of hemolysis around the inoculated area indicated growth, and confirmation was obtained by resuspending surface growth in phosphate buffered saline (PBS) and viewing with a phasecontrast microscope. Samples which contained motile spirochaetes were retained for testing by Polymerase Chain Reaction (PCR). Samples which had spirochaetes present and which produced weakly hemolytic growth were noted.

Diagnostic PCR

[000127] The identification of the Brachyspira species cultured from the field samples was determined using published PCR tests for B. hyodysenteriae, Brachyspira intermedia, Brachyspira pilosicoli, Brachyspira hampsonii and “B. suanatina”.

[000128] PCR assays were applied to growth harvested from the primary isolation plates and resuspended in sterile water. The PCR assays were performed in 25 μΐ reactions consisting of lx PCR buffer, 1.5 mM MgCfi, 0.5 U Taq DNA polymerase, 0.2 mM of each dNTP and 0.5 μΜ of forward and reverse primers. Cycling conditions involved an initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 15 s and primer extension at 72°C for 1 min. The products were separated by gel electrophoresis and visualised over UV light after staining with ethidium bromide.

Confirmation of weak hemolysis in B. hyodysenteriae isolates

Growth of spirochaetes [000129] Field isolates which were positive in the PCR for B. hyodysenteriae and also had weakly hemolytic grown on agar media were cultured on a fresh TSA plate at 37°C for 5 days, as previously described. A 5mm x 5mm section of the agar plate containing spirochaete growth was excised using a sterile scalpel blade and transferred to a 10ml tube of Kunkle's pre-reduced anaerobic broth containing 2% (v/v) foetal bovine serum and a 1% (v/v) ethanolic cholesterol solution (Kunkle, R.A., Harris, D.L., Kinyon, J.M. (1986), Journal of Clinical Microbiology 24:669-671). The cells were incubated at 37°C and grown to exponential log phase. The cells were counted using a hemocytometer before the supernatant was collected by centrifuging at 5,000 g and filtered using a 0.2 micron syringe filter.

In vitro hemolysis assay [000130] A 10% (v/v) suspension of red blood cells was made by adding 1ml of whole sheep blood to 9ml of sterile physiological saline. The red blood cells were collected by centrifugation and the cell pellet washed three times with 10ml of sterile physiological saline. The washed red blood cells were resuspended with 10ml sterile physiological saline and 50μ1 of the resuspended red blood cells was added to the well of a 96-well flat bottom microtitre plate. Fifty microliters of the B. hyodysenteriae supernatant was added to the red blood cells in the microtitre plate and incubated under anaerobic condition for 2 h at 37°C. After incubation, the absorption at 450 nm was determined using an ELISA plate reader for all samples. Hemolytic activity was represented by a decrease in absorbance following lysis of the red blood cells.

[000131] Sterile water was included as the control for complete hemolysis and sterile physiological saline was used as the control for no hemolysis. The strongly hemolytic B. hyodysenteriae strain WAI was also included as a test positive control. All samples were tested in triplicate.

Genome sequencing and in silico analysis of B. hyodysenteriae isolates

Growth of spirochaetes [000132] The B. hyodysenteriae isolates were cultured on a fresh TSA plate at 37°C for 5 days, as previously described. The cells were harvested from the agar plate by resuspending the surface growth with 2 ml sterile PBS. The cells were then transferred into a microfuge tube and then counted using a hemocytometer. The cells were collected by centrifuging at 5,000 g and the cell pellet was resuspended with sterile PBS to a density of 106 cells per ml. DNA extraction [000133] High molecular weight DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s instructions. Ten ml of a 10 cells per ml culture of Brachyspira was harvested by centrifugation at 5,000 g. The cell pellet was resuspended in 180 μΐ of lysis buffer containing 20 μΐ of proteinase K (10 mg/ml) and incubated at 55°C for 30 min. After all the cells had been lysed, 180 μΐ of AL Buffer was added and the sample incubated at 70°C for 10 min. Two hundred μΐ of absolute ethanol was immediately added to the sample and this was transferred to a DNeasy column. Column wash buffers AW1 and AW2 were added sequentially to the columns and centrifuged at 6,000 x g. The flow-through was discarded, and the DNA was eluted with elution buffer and stored at -20°C.

Whole genomic sequencing [000134] Genome sequences of the B. hyodysenteriae strains were generated on an Illumina MiSeq using v3 chemistry and 300 base pair (bp) paired-end reads using dual indexed Nextera XT libraries. The mean insert size was around 250-300 bp and the sequencing was performed at 70x depth of coverage. De novo assembly was performed using Geneious R9 (Biomatters Ltd, Auckland, New Zealand).

In silico multi-locus sequence typing (MLST) [000135] MLST was conducted as previously described (La et al, 2009, supra), except that for each MLST locus, the first allele sequence was used to identify the allele sequences for each sequenced B. hyodysenteriae strain using the BlastN function of the Geneious R9 software.

Allele designations for each locus were then obtained by a query search from the PubMLST website and the sequence type (ST) designations were identified.

In silico identification of the B. hyodysenteriae plasmid genes [000136] High molecular weight DNA used for the MLST analysis was also used for the analysis of plasmid genes. The 32 B. hyodysenteriae strain WAI plasmid gene sequences were obtained from GenBank (accession number CP001360). The B. hyodysenteriae genome sequences were individually searched using the BlastN function of Geneious R9 software for the presence of the 32 plasmid genes.

Outcomes

Identification and isolation of weakly hemolytic B. hyodysenteriae strains [000137] Two B. hyodysenteriae isolates (designated “MUV1” and “MUV2”) were recovered from farms with no clinical swine dysentery and showed weakly hemolytic growth on TSA plates containing blood.

In vitro hemolysis assay [000138] The in vitro hemolysis of the B. hyodysenteriae strains are shown in Figure 6. The strength of hemolysis for the two weakly hemolytic strains MUV1 (2.66 ± 0.167) and MUV2 (2.694 ± 0.173) was approximately two times less than the strongly hemolytic strain WAI (1.398 ± 0.134). In comparison, the weakly hemolytic Brachyspira innocens strain B256T (2.652 ± 0.186) and Brachyspira pilosicoli strain P43/6/78T (2.705 ± 0.187) showed similar hemolytic activity as strains MUV1 and MUV2.

Multi-locus Sequence Typing [000139] The sequence types (ST) for B. hyodysenteriae strains MUV1 and MUV2 are listed in Table 6. The STs identified for the strains were unique and different from those previously identified for Australian and international isolates.

Table 6. MLST profiles for the weakly hemolytic B. hyodysenteriae isolates MLST Profile

Strain ad alp „ ,, . SI est gdh glpr^ pgm thi MUV1 2Ϊ8 15 30 23 28 34 2032_ MUV2 161 16 30 24 29 35 1933

Identification of plasmid genes [000140] It was found that strains MUV1 and MUV2 did not possess any of the 32 B. hyodysenteriae plasmid genes. EXAMPLE 3

Experimental challenge of pigs with atypical B. hyodysenteriae isolates

Pigs and housing [000141] The study was conducted with the approval of the Murdoch University Animal Ethics Committee. The pigs were housed in the Animal Isolation House at Murdoch University. [000142] Two pig trials were conducted for this study. Each trial utilized 30 castrated male pigs (Large White x Landrace x Duroc) of approximately 10 kg body weight that were purchased from a commercial piggery that is free of swine dysentery as determined by regular laboratory and clinical testing. The pigs were weighed and ear-tagged. Faecal samples were taken and cultured to exclude the possible presence of B. hyodysenteriae and other Brachyspira species as described above. The pigs were randomly assigned to three housing groups, comprising 10 pigs per group. Each group was housed in a single pen in a different room of an isolation animal house. Strict biosecurity protocols, including the use of different sets of protective clothing in the different rooms, were maintained to prevent transmission of infection between the rooms. The pigs were fed ad libidum on a commercial grower diet that did not contain antimicrobials.

Experimental groups [000143] Two separate trials were performed for this study. For each trial, three experimental groups were used, with each group comprising 10 pigs.

For the first pig trial, one group of pigs (group 1A) were challenged with a strain of B. hyodysenteriae that was weakly hemolytic on selective agar plates and lacked the virulence-associated plasmid. The second group of pigs (group IB) was challenged with a strain of B. hyodysenteriae that was strongly hemolytic on selective agar plates and predicted to be “avirulent” due to the absence of six virulence-associated plasmid genes. The third group of pigs (group 1C) was challenged with a virulent strain of B. hyodysenteriae that had been shown to be cause swine dysentery in previous experimental challenge experiments (La, T., Phillips, N.D., Reichel, M.P. and Hampson, D.J. (2004), Veterinary Microbiology 102:97-107). Both strains used in groups 1A and IB were recently isolated from herds where clinical SD had not been observed.

[000144] For the second pig trial, one group of pigs (group 2A) was challenged with the “avirulenf ’ strain of B. hyodysenteriae used in group 1A of the first trial. The second group of pigs (group 2B) was challenged with the strain of B. hyodysenteriae that was strongly hemolytic on selective agar plates and predicted to be “avirulent” due to the absence of the whole virulence-associated plasmid. This strain was recently recovered from a herd that did not have any history of swine dysentery. The third group of pigs (group 2C) was challenged with a strongly hemolytic strain of B. hyodysenteriae that had recently been recovered from a herd with an outbreak of swine dysentery.

Experimental infection [000145] Following arrival at the Animal House and allocation into groups, the pigs were grown until they reached an average weight of approximately 18-20 kg. The pigs were challenged with an appropriate amount of B. hyodysenteriae. This was done by feeding each animal with 10 blood agar plates containing mid log-phase growth of spirochaete cells that were mixed into a handful of feed pellets. Each pig was challenged individually using a piglet feeding/drinking dish. The pigs were monitored until all pigs had completely consumed the inoculum. This procedure was repeated daily on the following four days.

Health monitoring [000146] The pigs were observed daily for signs of ill-health. Starting three days after the end of the experimental infection, rectal swabs were taken from each pig twice weekly and cultured for B. hyodysenteriae as described above. When diarrhoea containing fresh blood and mucus was observed the pigs were recorded as having swine dysentery, and they were removed for post-mortem examination. For the first trial, the remaining pigs that did not develop clinical signs were killed four weeks after the start of the experimental infection. In the second trial, the remaining pigs that did not develop clinical disease were killed seven weeks after the start of the experimental infection.

Post-mortem examination [000147] The pigs were stunned using a captive bolt pistol and then exsanguinated by severing the carotid artery. The carcase was opened and the intestinal tract removed. The large intestine was opened along its length and intestinal contents were collected from the caecum and proximal colon for spirochaete culture. Observations of gross pathological changes and their distribution in the large intestine were recorded.

Analysis [000148] Fisher’s exact test was used to make comparisons between the three pig groups in: i) number of animals showing signs of disease; ii) number of animals that have excreted spirochaetes post-infection, iii) number of animals that were culture positive at post-mortem, and iv) number of animals that have gross large intestinal lesions at post-mortem.

Outcomes

Pig Trial 1 [000149] Two B. hyodysenteriae field isolates identified in project 2A-111, predicted to be “avirulenf ’ based on the absence of plasmid-bome virulence-associated genes, were each used to experimentally infect groups of ten pigs. One strain (“a”, group 1A) lacked the entire plasmid and was also weakly hemolytic on blood agar plates. The other strain (“b”, group IB) lacked six putative virulence-associated genes and was strongly hemolytic. A positive control group (group 1C) was challenged with B. hyodysenteriae strain “c”, which was strongly hemolytic and possessed the plasmid. The B. hyodysenteriae strain “c” was originally isolated from a pig with SD.

[000150] A summary of the colonisation of pigs after inoculation as evidenced by faecal shedding is shown in Table 7, and observations of lesions in the colon and caecum of the pigs at post-mortem is shown in Table 8. Following inoculation, the pigs in all three groups became colonized with the respective B. hyodysenteriae strains used in their challenge. Colonization was most frequent and heaviest for the pigs in group IB, followed by 1C and then 1A. The maximum colonization rate for group 1A was 60% (6 out of 10 pigs) at 17 days postinoculation; all the pigs from group IB were colonized by day 14 post-infection and all pigs continued to be colonized for the duration of the trial; for group 1C 80% (8 out of 10 pigs) were culture positive at 21 days post-inoculation. Only two pigs showed clinical signs of disease during the three-week post-inoculation period (one developed diarrhoea and one developed typical SD at the end of the experimental period). Both animals were challenged with strain “b”, the strain lacking the block of six plasmid genes (group IB).

Table 7. Detection of B. hyodysenteriae in the faeces of pigs in the pig trial 1. Group 1A was challenged with a weakly hemolytic B. hyodysenteriae strain that lacked the entire plasmid (strain “a”); Group IB was challenged with a strain that lacked the six putative virulence-associated plasmid genes (strain “b); Group 1C was challenged with a field strain isolated from a pig with SD (strain “c”).

Pig Pig Culture Result (days following first day of challenge)3 group number 0_______7_______10______14______17______21______24_____PMb 1A 1 0 0 0 0 1 1 00 200000000 300000000 4 0 0 0 0 1 1 11 5 0 0 0 1 1 1 11 6 0 0 0 0 1 1 11 700001222 800000000 9 0 0 0 1 1 2 21 __10 00000000 IB 11 00254333 12 00355555 13 00255555 14 00255333 15 00555555 16 00045555 17 00255555 18 00045555 19 00255444 ______________20 0 0________4________4________5________5________5________5 1C 21 00000000 22 00132333 23 00022222 24 00013333 25 00003333 26 00002333 27 00000000 28 00000220 29 0 0 0 2 1 1 1 1 ________________30 0 0__________1__________3_________2__________1__________1__________1 “ Culture score; 1 to 5, weak to strong growth on selective agar plates b Post-mortem at 29 days after first day of challenge [000151] At post-mortem, six of the pigs from positive control group 1C showed mild to moderate lesions in the colon and one had moderate to severe lesions consistent with SD (Table 8). Of the pigs from group IB, three had mild to moderate lesions, two had moderate lesions, two had moderate to severe lesions and three had severe lesions in the colon. In the caecum, two pigs had mild lesions and two had mild to moderate lesions. Three of the pigs from group 1A had mild changes in the colon but no changes in the caecum. The other pigs in these two groups had colons and caecums with normal gross appearance. The colons of all pigs contained the respective challenge strains, although the colonisation scores in pigs from group 1A were lower than in the other two groups.

Table 8. Clinical signs and gross lesions observed at post-mortem examination in the caecum and large intestine of pigs in the first pig trial.

Pig Pig Clinical Lesions observed at post-mortem Culture3 group number_____signs________Caecum______________Colon___________CaecumColon ΙΑ 1 Normal Normal Normal 01 2 Normal Normal Normal 01 3 Normal Normal Normal 01 4 Mild patchy lesion

Normal Normal 02 upper 1/3 5 Normal Normal Normal 11 6 xt 1 X, i Mild patchy lesion

Normal Normal ,,-, 02 upper 1/3 7 X, , X, 1 Mild patchy lesion

Normal Normal ,,-, 02 upper 1/2 8 Normal Normal Normal 01 9 Normal Normal Normal 01 __________10______Normal_______Normal______________Normal_____________0_________1_____ IB 11 Normal Normal ModMHC 25 12 rx mild/mod_

Dysentery ..... Severe MHC 55 typhititis 13 Normal Normal ModMHC 05 14 Normal Normal Mild colitis entire length 04 15 rx- 1. mild/mod c cc

Diarrhoea ..... Severe MHC 55 typhititis 16 Normal Severe MHC 25 reddening

Normal patchy Mod/Severe MHC 25 reddening 18 Normal Normal Mild colitis 05 19 Normal Normal Mild colitis 04 __________20______Normal_______Normal_________Mod/Severe MHC_________1_________5____ 1C 21 Normal Normal Normal 01 22 Normal Normal Moderate - severe colitis 15 23 xr , xr , mild localized lesions in „„

Normal Normal , 03 upper 1/3 24 x r , x r , mild localized lesions in „_

Normal Normal ,03 upper 1/2 25 xr , xr , mild localized lesions in „„

Normal Normal ,03 upper 1/2 26 xr , xr , mild localized lesions in „

Normal Normal , 04 upper 1/3 27 Normal Normal Normal 03 __________28______Normal_______Normal______________Normal_____________0_________1_____ 29 , , mild localized lesions m „,

Normal Normal , ,, 03 upper 1/3 30 , , mild localized lesions in „,

Normal Normal , ,_ 03 __________________________________________________________upper 1/2_______________________________ “ Culture score; 1 to 5, weak to strong growth on selective agar plates; 0, no growth MHC, mucohemorrhagic colitis [000152] Interpretation of relative virulence was made difficult by the mild nature of the disease in the positive control group. The strain used had been passaged multiple times, and it is possible that this attenuated its virulence. A different more recent strain was selected as the positive control strain for pig trial 2.

Pig Trial 2 [000153] In the second pig trail, 30 pigs were divided into three groups of ten pigs and challenged with different strains of B. hyodysenteriae identified and isolated during project 2A-111. One group of pigs (group 2A) was challenged with the weakly hemolytic B. hyodysenteriae strain “a” lacking the plasmid (from group 1A of the first trial). Pigs in group 2B were challenged with a strain of B. hyodysenteriae (“d”) that lacked the entire plasmid and was strongly hemolytic on blood agar plates. Positive control group 2C was challenged with a recent isolate of B. hyodysenteriae (“e”) recovered from a pig reported to have severe clinical signs of SD, which was strongly hemolytic and possessed the plasmid.

[000154] Table 9 shows colonisation of the pigs after inoculation as measured by faecal shedding, and Table 10 shows the observation of lesions in the colon and caecum of the pigs at post-mortem. At 17 days post-infection, all pigs in the positive control group (2C) were colonized and these pigs continued to be colonized for the duration of the experiment. In the group challenged with the hemolytic strain lacking the plasmid (group 2B), all ten pigs were colonized 21 days post-infection. In three pigs, shedding of B. hyodysenteriae in the faeces could not be detected after 28, 38 and 42 days post-infection. The remaining pigs continued to be colonized for the duration of the experiment. In the group of pigs challenged with the weakly hemolytic strain (group 2A), all pigs were colonized 24 days post-infection. One pig stopped shedding B. hyodysenteriae after 28 days post-infection and three pigs stopped shedding after 31 days post-infection. The remaining pigs were colonized for the duration of the experiment, although at lower numbers than the other groups. Five pigs from the positive control group (2C) and three pigs from group 2B showed signs of disease (diarrhoea) during the seven-week post-inoculation period.

[000155] At post-mortem, the colons of all pigs in all groups were colonized with the respective challenge strains. All pigs in the positive control group 2C were also shedding B. hyodysenteriae in the faeces compared to seven pigs from group 2B and five pigs from group 2A. Nine pigs in the control group and eight pigs from group 2B were colonized in the caecum. Only one pig in the group challenged with the weakly hemolytic strain “a” was colonized in the caecum. Five of the control pigs (group 2C) had mild to moderate lesions in the colon while two pigs from group 2B had mild to moderate lesions in the colon and two pigs from group 2A has mild lesions in the colon. The lesions in the colon seen in all groups were localized to the upper one third (ascending colon). In the caecum, eight pigs from the control group (2C) had lesions with one pig having severe lesions in the caecum. This was compared to six pigs from group 2B showing lesions in the caecum and two pigs from group 1A having mild lesions. The other pigs in these groups had colons and ceca with normal gross appearance.

[000156] Table 9. Shedding of B. hyodysenteriae in the faeces of pigs in the second pig trial detected by culture on selective media. Group 2A was challenged with a B. hyodysenteriae strain (“a”) that lacked six putative virulence-associated genes. Group 2B was challenged with a strongly hemolytic strain that lacked the entire plasmid (strain “d”). Group 2C was challenged with a current field strain isolated from a pig with SD (strain “e”).

Pig Pig Culture Result (days following first day of challenge)3

Group number 0 3_______7 10 14 17 21 24 28 31 35 38 42 45 PMb 2A 1 000011122221111 2000012111121222 3 0000001 12100000 4 000000022100000 5000011222110111 6000011122110111 7000000111111100 8 000012221 100000 9000111553333221 _____________10 0 0 0 0 0 0 1_________1_________1 0 0 0 0 0 0 2B 1100001 1 1 12222222 12 000013233544455 13 000002544444444 14 000001121111000 15 000000221222222 16 000001221000000 17 000001223222222 18 0001 12233333333 19 00000013331 1 100 _________________20 0 0 0 0___________1___________1___________1___________1___________1___________1___________1___________1___________1___________1___________1 2C 21 000144444444444 22 000001111111111 23 000011211111111 24 000001 134444455 25 000001111111111 26 000011111122222 _27 000033544444444 28 0001 12444444555 29 000001 1 12233344 _30 000444444444444 a Culture score; 1 to 5, weak to strong growth on selective agar plates; 0, no growth b Post-mortem at 49 days after first day of challenge

Table 10. Observation of clinical signs and gross lesions at post-mortem examination in the caecum and colon of pigs in the second pig trial.

Pig Pig Clinical Lesions observed at post-mortem Culture3 group number signs________Caecum______________Colon__________CaecumColon 2 A 1 Normal Normal Normal 01 2 Normal Mild patchy Mild patchy lesion upper reddening1/3 3 Normal Normal Normal 01 4 Normal Normal Normal 01 5 Normal Normal Normal 01 6 Normal Normal Normal 11 7 Normal Normal Normal 01 8 Normal Normal Normal 01 9 Normal Mild patchy Mild patchy lesion upper reddening1/3 __________10_____Normal_______Normal______________Normal_____________01 2B 11 Normal Mild Normal 23 12 τχ. , .., mild localized lesions in

Diarrhoea Moderate . ,, 55 upper 1/3 13 τχ. , .., mild localized lesions in ,

Diarrhoea Moderate , ,, 35 upper 1/3 14 Normal Normal Normal 02 15 Normal Mild Normal 34 16 Normal Normal Normal 12 17 Normal Moderate Normal 42 18 Diarrhoea Moderate Normal 35 19 Normal Normal Normal 03 __________20_____Normal_______Normal______________Normal_____________12 2C 21 τχ. , .., mild localized lesions in Λr

Diarrhoea Moderate . ,, 45 upper 1/3 22 Normal Normal Normal 13 23 Normal Normal Normal 03 24 .,, mild localized lesions in

Diarrhoea Moderate . ,, 55 upper 1/3 25 Normal Mild Normal 33 26 Normal Mild Normal 34 27 τχ. , .., mild localized lesions in

Diarrhoea Moderate . ,, 55 upper 1/3 28 , _ Patchy moderate lesions

Diarrhoea Severe .,, 55 upper 1/3 29 Normal Moderate Normal 55 30 τχ. , .., mild localized lesions in

Diarrhoea Moderate . ,, 55 ____________________________________________________________upper 1/3_____________________________ a Culture score; 1 to 5, weak to strong growth on selective agar plates; 0, no growth MHC, mucohemorrhagic colitis

Application of Research B. hyodysenteriae in Australian herds [000157] The study confirmed that B. hyodysenteriae is widely distributed amongst Australian pig herds, and that it occurs in herds that do not have clinical signs and/or which were considered to be uninfected. The occurrence of the pathogen in healthy herds poses a significant risk of inadvertent transmission of the spirochaete to other herds, particularly when breeding herds are colonized.

[000158] Strain typing by MLST was useful for identifying strains that may have been transmitted by movement of pigs. Comparison of isolates either recovered from herds that were considered to be infected or which had thought themselves to be free of infection did not identify any consistent differences between isolates or their plasmid gene profile from the two categories. Other potential differences between strains could be analyzed by whole genomic sequencing and analysis, but this was outside the scope of the current project. It remains possible that other factors such as diet or intestinal microbiota could influence disease expression, and this deserves further investigation.

[000159] Analysis of antimicrobial susceptibility profiles demonstrated that many Australian strains of B. hyodysenteriae are resistant to one or more key antimicrobials, and a number of different strains are multidrug resistant. This is of considerable concern and requires continuing monitoring and development of new means of control such as through vaccination or by quarantine and/or eradication.

Potential avian reservoirs of B. hyodysenteriae in Australia [000160] Examination of a limited number of faecal samples from different aquatic bird species failed to detect B. hyodysenteriae or other novel agents that potentially could cause SD. This did not exclude the possibility that they occur, but does indicate that, if present, they are rare. The potential pathogen B. pilosicoli was detected in samples from two Pacific Black Duck and one Australian White Ibis. This emphasizes the importance of preventing pigs from becoming exposed to aquatic birds. This is particularly difficult to achieve in outdoors piggeries. ELISA test for Swine Dysentery [000161] The ELISA test correctly identified seven of the eight herds that were considered to be infected by the referring veterinarians. Only 30 samples were tested from the eighth herd, and this was less than the recommended number to test. In comparison, five of the six herds reported not to be infected also gave positive serological results. In one of these herds B. hyodysenteriae was isolated from colonic samples, indicating that it actually was infected (previously it had been considered to be a “false positive”). Three other herds had given “false positives” in the earlier study using the Prionics test kit, and these again were positive. It remains unclear whether these really are false positives or not. No colonic or faecal samples were available from the final “false positive” herd to try to determine its true colonization status. The final two herds were of unknown status and were serologically positive, and these subsequently had B. hyodysenteriae isolated, demonstrating that they were infected.

[000162] Overall the ELISA performed well in detecting infected herds, but questions about whether it may generate false positive reactions remain. Additional faecal and colonic samples are being collected from herds that have given “false positive” serological reactions and will be tested to determine whether strains of B. hyodysenteriae are present.

Experimental challenge of pigs with “avirulent” B. hyodysenteriae [000163] Interpretation of the first experimental infection trial was complicated by the fact that of the pigs inoculated with the positive control strain BW1 (strain “c”) only one developed moderate/severe colitis, whilst the others mostly only developed mild colitis. Unexpectedly, more severe lesions were seen in the pigs infected with strain SAI9 (strain “b”) (lacking the six plasmid virulence-associated genes), and this suggests that the plasmid genes are not essential for disease. Nevertheless, they may still play an indirect role in the pathogenesis, for example by facilitating colonization and hence reducing the infectious dose required to cause disease in the field. It is clear, however, that the plasmid genes are not the only determinant of pathogenic potential. Strain MUV1 (strain “a”, group 1A) caused minimum changes, but it was also weakly hemolytic, and it is possible that the combination of lack of plasmid genes and weak hemolysis both contribute to the relative lack of virulence of this strain. The hemolytic activity of B. hyodysenteriae is thought to contribute to colonic lesion production.

[000164] In the second trial strain MUV1 was again tested to determine whether the results could be replicated, and new positive control strain, NSW54 (strain “e”) was used to try to induce more disease in the control group. The other isolate, Vic210 (strain “d”) lacked the 36 Kb plasmid. Unfortunately although five of the positive control group developed diarrhoea, and all were heavily colonized, caecal and colonic lesions were mainly only moderate or mild. Three pigs inoculated with Vic20 developed diarrhoea, and although all pigs showed heavy colonization, only two had mild lesions in the colon. Again the group inoculated with MUV1 remained healthy and only two had mild lesions, despite all the pigs being relatively lightly colonized.

[000165] In both trials, the MUV1 strain which lacked the plasmid genes and was weakly hemolytic colonized the pigs, but to a lower level than with the other strains, and without causing disease. This supports the suggestion that the plasmid genes may facilitate colonization, whilst the hemolytic activity is likely to be necessary for severe lesion production. A further study was carried out to confirm these results and to assess whether MUV1 is suitable to be used as a live vaccine. EXAMPLE 4

Vaccination and experimental challenge of pigs

Pigs and housing [000166] The study was conducted with the approval of the Murdoch University Animal Ethics Committee. The pigs were housed in the Animal Isolation House at Murdoch University. [000167] Thirty-six castrated male pigs (Large White x Landrace x Duroc) of approximately 10 kg body weight were purchased from a commercial piggery that is free of SD as determined by clinical examination and regular laboratory testing. The pigs were weighed and ear-tagged. Faecal samples were taken and cultured to exclude the possible presence of B. hyodysenteriae and other Brachyspira species, as described below. The pigs were randomly assigned to three housing groups, comprising 12 pigs per group. Each group was housed in a single pen in a different room of an isolation animal house. Strict biosecurity protocols, including the use of different sets of protective clothing in the different rooms, were maintained to prevent possible transmission of infection between the rooms. The pigs were fed ad libidum on a commercial weaner diet that did not contain antimicrobials, and then transferred to an antimicrobial-free commercial grower diet prior to vaccination.

Experimental groups [000168] Two groups of pigs (group A and group B) were challenged with a vaccine strain of B. hyodysenteriae that was weakly hemolytic on selective agar plates and lacked the virulence-associated plasmid (designated “MUV1”). The third group of pigs (group C) were left unchallenged. Four weeks after challenge with MUV1, pigs in groups B and C were challenged with an equal mixture of five virulent strain of B. hyodysenteriae that had been shown to be cause SD in previous experimental challenge experiments (strains WAI, BW1, Vic2, NSW5 and Q10: La et al., 2004, supra). Forty-three days after the day of challenge with the virulent strain mixture (“cocktail”) all remaining pigs were slaughtered. At post-mortem, samples were collected from the caecum and colon for spirochaete isolation and evaluation of any pathological changes.

Experimental infection [000169] Following arrival at the Animal House and allocation into groups, the pigs were grown until they reached an average weight of approximately 10-12 kg. Pigs in groups A and B were challenged with vaccine strain MUV1 by feeding each animal 10 blood agar plates containing a dense surface growth of mid log-phase spirochaete cells mixed into a handful of

O feed pellets. Each plate contained a surface growth of ~10 spirochaete cells. Each pig was challenged individually using a piglet feeding/drinking dish. The pigs were monitored until they all had completely consumed the inoculum. This procedure was repeated daily on the following four days. Thirty days after the vaccination protocol started, the pigs in groups B and C were challenged in the same manner using the cocktail of virulent strains, with each strain grown on two plates per pig/day. The challenge was repeated on the four following days.

Health monitoring [000170] All pigs were monitored daily for signs of disease, and faeces was collected twice weekly for spirochaete culture. When diarrhoea containing fresh blood and mucus was observed, the pigs were recorded as having SD, and they were removed for post-mortem examination. The remaining pigs that did not develop clinical signs were killed 43 days after the commencement of experimental infection with the cocktail of virulent strains.

Bacteriological culture [000171] All faecal samples were swabbed onto Trypticase Soy agar (TSA) plates containing 5% (v/v) defibrinated ovine blood, and cultured for 5 to 7 days at 37°C in a jar with an anaerobic atmosphere generated by an AnaeroGen (Trade Mark) 2.5L Sachet (Oxoid). Zones of hemolysis around the inoculated area indicated growth, and surface growth was suspended in phosphate buffered saline (PBS) and viewed with a phase-contrast microscope to confirm that spirochaetes were present. Any samples that contained spirochaetes were tested for B. hyodysenteriae using a Polymerase Chain Reaction test (La et al, 2003, supra). The extent of growth was scored on a scale from one to five, with one representing light growth in the inoculum and five indicating heavy growth through to the last streak on the isolation plate.

Post-mortem examination [000172] The pigs were stunned using a captive bolt pistol and then exsanguinated by severing the carotid artery. The carcase was opened and the intestinal tract removed. The large intestine was opened along its length and intestinal contents were collected from the caecum and proximal colon for spirochaete culture. Observations of gross pathological changes and their distribution in the large intestine were recorded.

Analysis [000173] Fisher’s exact test was used to make comparisons between the three pig groups in: i) numbers of animals showing or not showing signs of disease; ii) numbers that excreted spirochaetes post-infection, iii) numbers that were or were not culture positive at post-mortem, and iv) numbers that had or did not have gross large intestinal lesions at post-mortem.

Outcomes [000174] A summary of the colonisation of pigs after inoculation, as evidenced by faecal shedding, is shown in Table 11. Observations of lesions in the colon and caecum of the pigs at post-mortem is shown in Table 12.

[000175] All vaccinated pigs in Group A were colonized by MUV1 as shown by faecal excretion of the spirochaete (Table 11). In each case, growth on the plates was scored as one, indicating light growth. Excretion varied in duration, with one animal only being culture positive at one sampling time (pig 26) and another being culture positive over a period of 40 days (pig 6).

[000176] All but one (pig 5) of the 12 pigs in group B shed MUV1 prior to subsequent challenge with the virulent strain cocktail. Five of these pigs (9, 19, 23, 24, 25 and 32) shed low numbers of spirochetes for periods of up to 3 weeks following challenge with MUV1. Subsequently, 31-35 days after challenge with the cocktail, two other vaccinated pigs (12 and 14) excreted high levels of spirochaetes, and one developed dysentery. Seven of the pigs in group B had moderate to high numbers of spirochaetes in their faeces at post-mortem. One pig in this group was removed before the end of the experiment as it had developed dysentery.

[000177] In contrast to group B, 11 of the 12 pigs in group C that were just challenged with the cocktail of virulent strains showed high rates of excretion with large numbers of spirochaetes over the experimental period. The earliest time point that excretion was recorded was 10 days after challenge. Seven pigs developed dysentery and were removed, and overall, 10 of the 12 pigs had heavy growth of the spirochaete in their faeces at post-mortem. This rate was significantly greater than seen in the vaccinated pigs of group B (P = 0.0006 in Fisher’s exact test).

[000178] None of the pigs from group A that were vaccinated with MUV1 developed diarrhoea or dysentery and none showed lesions in the caecum or colon at post-mortem (Table 12). Four pigs were culture positive in the colon at this time, with slight to moderate growth recorded.

[000179] In group B one of the pigs that had received MUV1 and then been challenged with the virulent strains developed diarrhoea and one had dysentery. The pig with dysentery had severe mucohemorrhagic lesions in the caecum and colon at post-mortem, and the pig with diarrhoea had moderate lesions. Two other pigs had mild localized lesions in the upper colon. Seven pigs had heavy spirochaetal growth in the colons, but no pigs had growth in the caecum.

[000180] In group C two pigs developed diarrhoea and seven had dysentery following challenge with the cocktail of virulent strains. Rates of clinical disease were significantly higher in pigs from group C (9 out of 12) than in pigs from group B (2 out of 12) (P = 0.012). Severe mucohemorrhagic colitis was recorded in 8 of the pigs from group C, with moderate lesions found in two other pigs. Two pigs had no lesions. Comparison of rates of moderate and severe lesions in groups B (2 out of 12) and C (10 out of 12) again revealed a significant difference between them (P = 0.003). Eleven of the 12 pigs in group C had heavy spirochaetal growth in the colon at post-mortem, and nine had moderate to heavy growth in the caecum.

Summary [000181] In summary, all but one of 24 pigs in groups A and B receiving MUV1 shed low numbers of the strain in their faeces in the first 30 days following exposure. Four of 12 pigs in group A were still culture positive in the colon at post-mortem ten weeks later, and none of the animals from group A developed clinical signs or had lesions in the large intestine at the time of slaughter. This demonstrates that MUV1 can colonise pigs for up to 10 weeks without causing disease. Consequently it appears that MUV1 is safe.

[000182] Significantly fewer pigs in group B that were exposed to MUV1 and then challenged with virulent strains were colonised with virulent strains (P = 0.0006 in Fisher’s exact test), developed swine dysentery or diarrhoea (P = 0.012) or had colonic lesions (P = 0.003) compared to the unvaccinated pigs in group C that were challenged with virulent strains in the same manner. These results clearly show that the use of MUV 1 as a live vaccine provides a significant level of protection against colonisation and disease with virulent strains of B. hyodysenteriae.

[000183] This study has provided evidence that MUV1 has good potential as a live vaccine strain to control swine dysentery caused by B. hyodysenteriae. The study has confirmed that the B. hyodysenteriae vaccine strain MUV1 colonizes pigs following oral exposure, and does not induce disease. Colonization of the colon lasts for at least up to seven weeks in a proportion of pigs. Exposure to and colonization with MUV1 provides a highly significant level of protection against colonization and development of disease following subsequent challenge with five different virulent strains of B. hyodysenteriae.

Table 11. Shedding of B. hyodysenteriae in the faeces of pigs as detected by culture on selective media. Group A was experimentally infected (vaccinated) with a weakly hemolytic B. hyodysenteriae strain MUV1 that lacked the plasmid containing the six putative virulence-associated genes. Group B was infected with MU VI and subsequently challenged with a mixture of five virulent field isolate of B. hyodysenteriae. Group C was only challenged with the virulent Isolates.

Pig Pig Culture Result (days after first day of vaccination)3 Culture Result (days after first day of challenge)11

Group number 8 11 15 18 22 25 29 7 10 14 17 21 24 28 31 35 38 FM A 1 000110000000000000 2 000011111100000000 3 001111100000000000 4 00001 1000000000000 6 001111111111000000 7 000111100000000000 10 000001110000000000 15 000010100000000000 16 000000000000000000 18 0001 1 1000000000000 22 00001 1000000000000 _______________26________0_______0 0 0________1________0 0_______0 0 0_______0_______0 0_______0 0_______0 0_______0 B 5 000000000000000000 9 000111110000000003 12 000001 1000000055 dead 5 14 000010000000000453 19 001111111100000005 23 000111111111000002 24 000001011101000003 25 000101111100000005 27 00001 1000000000000 29 001111100000000000 32 000010111100000000 ________________33_________0________0________1________1________1________0 0________0 0 0________0________0 0________0 0________0 0________0 C 8 0000000000005555 dead 5 11 000000000000005555 13 000000000000055555 17 0000000005555 dead dead dead dead 5 20 0000000035555 dead dead dead dead 5 ______________21________0_______0 0 0_______0 0 0 0 0 4_______5_______5_______5 dead dead dead dead 5 28 0000000000005555 dead 5 30 00000000555 dead dead dead dead dead dead 5 31 0000000000005555 dead 5 34 000000000000005555 35 000000000000000550 _______________36________0_______0 0_______0_______0_______0 0_______0 0 0_______0_______0 0_______0 0_______0 0________1 b Culture score; 1 to 5, weak to strong growth on selective agar plates; 0, no growth c Post-mortem at 43 days after first day of challenge with virulent isolate (Isolate B) dead; indicates pigs developed clinical signs and were removed for post-mortem

Table 12. Clinical signs and gross lesions observed in the caecum and large intestine of pigs at postmortem examination.

Pig Pig Clinical ______Lesions observed at post-mortem____________Culture3_____ group number_____signs______Caecum_____________Colon____________CaecumColon A 1 Normal Normal Normal 00 2 Normal Normal Normal 00 3 Normal Normal Normal 00 4 Normal Normal Normal 00 6 Normal Normal Normal 00 7 Normal Normal Normal 00 10 Normal Normal Normal 00 15 Normal Normal Normal 03 16 Normal Normal Normal 01 18 Normal Normal Normal 01 22 Normal Normal Normal 03 26 Normal Normal Normal 00 B 5 Normal Normal Normal 00 9 Normal Normal Normal 05 12 Moderate

Dysentery SMHC entire length 05 typhititis 14 Diarrhoea Normal Moderate lesions upper 1/3 05 19 Normal Normal Normal 05 23 Normal Normal Mild localized lesions upper 1/3 05 24 Normal Normal Mild localized lesions upper 1/3 05 25 Normal Normal Normal 05 27 Normal Normal Normal 00 29 Normal Normal Normal 00 32 Normal Normal Normal 00 ___________33_______Normal______Normal______________Normal________________00 C 8 Patchy

Dysentery SMHC+F entire length 45 reddening 11 Diarrhoea Normal Moderate lesions upper 1/3 05 13 Diarrhoea Normal Moderate lesions upper 1/3 25 17 Dysentery Severe SMHC+F entire length 55 20 Dysentery Severe SMHC+F entire length 55 21 Dysentery Severe SMHC+F entire length 55 28 Dysentery Normal SMHC+F entire length 35 30 Dysentery Severe SMHC+F entire length 55 31 Dysentery Normal SMHC+F entire length 55 34 Dysentery Patchy SMHC entire length 55 reddening 35 Normal Normal Normal 00 36 Normal Normal Normal 05 d Culture score; 1 to 5, weak to strong growth on selective agar plates; 0, no growth SMHC, severe mucohemorrhagic colitis SMHC+F, severe mucohemorrhagic colitis with fibrin

Reference

Francisco, A.P., Vaz, C., Monteiro, P.T., Melo-Cristino, J., Ramirez, M. and Carriqo, J.A. (2012), BMC Bioinformatics 13:87.

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Claims (18)

WHAT IS CLAIMED IS:

1. An isolated live vaccine strain of Brachyspira hyodysenteriae, wherein (a) the live vaccine strain lacks six functional virulence factors encoded by the nucleic acid sequences of SEQ ID NOs 1-6; and (b) the live vaccine strain lacks one or more functional hemolysis factor(s) or has low hemolysis activity as compared to a positive control B. hyodysenteriae strain, .

2. The isolated live vaccine strain of Claim 1, wherein the one or more functional hemolysis factor(s) is encoded by one or more polynucleotide sequence(s) that is at least 90% identical to one or more of the nucleic acid sequences selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 13 and SEQ ID NO: 14.

3. The isolated live vaccine strain of Claim 1 or 2, wherein the six functional virulence factors or one or more functional hemolysis factor(s) is deleted, inactivated or modified.

4. The isolated live vaccine strain of any one of Claims 1-3, wherein the positive control B. hyodysenteriae strain is WAI (ATCC49526).

5. The isolated live vaccine strain of any one of Claims 1-4, wherein the live vaccine strain is MUV1 (V17/004760) or MUV2 (V17/004761).

6. A method of preparing a live vaccine strain of B. hyodysenteriae, comprising the steps of: (a) obtaining a virulent strain of B. hyodysenteriae', (b) deleting, inactivating or modifying six functional virulence factors encoded by the nucleic acid sequences of SEQ ID NOs 1-6 and one or more functional hemolysis factor(s) in said strain of B. hyodysenteriae', and (c) isolating a live B. hyodysenteriae strain containing said modifications.

7. A method of identifying a candidate live vaccine strain of B. hyodysenteriae, comprising the steps of: (a) obtaining a stain of B. hyodysenteriae', and (b) determining the presence or absence of six functional virulence factors encoded by the nucleic acid sequences of SEQ ID NOs 1-6 and one or more functional hemolysis factor(s) in said strain, wherein the absence of the six functional virulence factors and one or more functional hemolysis factor(s) indicate that the B. hyodysenteriae strain is suitable as a vaccine.

8. A method of identifying a candidate live vaccine strain of B. hyodysenteriae, comprising the steps of: (a) obtaining a strain of B. hyodysenteriae', (b) determining the presence or absence of six virulence factors encoded by the nucleic acid sequences of SEQ ID NOs 1-6 in said strain; and (c) comparing the hemolytic activity of said strain to a positive control B. hyodysenteriae strain, wherein low hemolytic activity as compared to the positive control B. hyodysenteriae strain and the absence of the six functional virulence factors indicate that the B. hyodysenteriae strain is suitable as a vaccine.

9. The method of Claim 6 or 7, wherein the at least one or more functional hemolysis factor(s) is encoded by one or more polynucleotide sequences that is at least 90% identical to one or more of the nucleic acid sequences selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 SEQ ID NO: 13, and SEQ ID NO: 14.

10. The method of Claim 8, wherein the positive control B. hyodysenteriae strain is WAI (ATCC 49526).

11. A vaccine composition comprising in a pharmaceutically acceptable vehicle at least one vaccine strain of B. hyodysenteriae according to any one of Claims 1 to 5 or at least one vaccine strain of B. hyodysenteriae obtained by the method of any one of Claims 6-10.

12. A vaccine composition according to Claim 11, wherein the vaccine composition comprises an adjuvant.

13. A method of preventing a diarrhoeal disease in an animal comprising administering to said animal an effective amount of an isolated vaccine strain of B. hyodysenteriae according to any one of Claims 1 to 5, an isolated vaccine strain of B. hyodysenteriae obtained according to the method of any one of Claims 6-10, or a vaccine composition according to Claim 11 or 12.

14. A method of immunising an animal against infection by B. hyodysenteriae or a related microorganism, comprising administering to said animal an effective amount of an isolated vaccine strain of B. hyodysenteriae according to any one of Claims 1 to 5, an isolated vaccine strain of B. hyodysenteriae obtained according to the method of any one of Claims 6-10, or a vaccine composition according to Claim 11 or 12.

15. An isolated vaccine strain of B. hyodysenteriae according to any one of Claims 1 to 5, an isolated vaccine strain of B. hyodysenteriae obtained according to the method of any one of Claims 6-10, or a vaccine composition according to Claim 11 or 12 for use in preventing a diarrhoeal disease in an animal.

16. An isolated vaccine strain of B. hyodysenteriae according to any one of Claims 1 to 5, an isolated vaccine strain of B. hyodysenteriae obtained according to the method of any one of Claims 6-10, or a vaccine composition according to Claim 11 or 12 for use in immunising an animal against infection by B. hyodysenteriae or a related microorganism.

17. Use of an effective amount of an isolated vaccine strain of B. hyodysenteriae according to any one of Claims 1 to 5, an isolated vaccine strain of B. hyodysenteriae obtained according to the method of any one of Claims 6-10, or a vaccine composition according to Claim 11 or 12 in the manufacture of a medicament for preventing a diarrhoeal disease in an animal.

18. Use of an effective amount of an isolated vaccine strain of B. hyodysenteriae according to any one of Claims 1 to 5, an isolated vaccine strain of B. hyodysenteriae obtained according to the method of any one of Claims 6-10, or a vaccine composition according to Claim 11 or 12 in the manufacture of a medicament for immunising an animal against infection by B. hyodysenteriae or a related microorganism.