AU780980B2 - Novel therapeutic compositions for treating infection by lawsonia SPP - Google Patents

Description

WO 02/038594 PCT/AU01/01462 -1- Novel therapeutic compositions for treating infection by Lawsonia spp.

FIELD OF THE INVENTION The present invention relates generally to therapeutic compositions for the treatment and/or prophylaxis of intestinal disease conditions in animals and birds caused or exacerbated by Lawsonia intracellularis or similar or otherwise related microorganism. In particular, the present invention provides a novel gene derived from L. intracellularis which encodes an immunogenic polypeptide. The polypeptide described herein, selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, or a homologue, analogue or derivative of any one or more of said polypeptides, is particularly useful as an antigen in vaccine preparation for conferring humoral immunity against L. intracellularis and related pathogens in animal hosts. The present invention is also directed to methods for the treatment and/or prophylaxis of such intestinal disease conditions and to diagnostic agents and procedures for detecting L. intracellularis or similar or otherwise related microorganisms.

GENERAL

Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.

Reference hereinafter to "Lawsonia intracellularis" or its abbreviation "L.

intracellularis" includes all microorganisms similar to or otherwise related to this microorganism, as described by Stills (1991) or Jones et al.(1997) or Lawson et al.

(1993) or McOrist et al. (1995).

WO 02/038594 PCT/AU01/01462 -2- References herein to "AGAL" shall be taken to mean a reference to the Australian Government Analytical Laboratories located at 1 Suakin Street, Pymble, New South Wales 2073, Australia. All biological deposits referred to herein in respect of the plasmids assigned AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); and NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN) have been made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

As used herein, the word "flhB", or the term "flhB gene", shall be taken to refer to a gene encoding the antigenic-flhB polypeptide of the present invention, which gene comprises the nucleotide sequence set forth in SEQ ID NO: 1 or the nucleotide sequence of the L. intracellularis gene contained in the plasmid pGTE#2 which has been deposited under AGAL Accession No. NM00/16477. The word "flhB"or the term "flhB gene" shall further be taken to include a degenerate or complementary nucleotide sequence to SEQ ID NO: 1 or the nucleotide sequence of the L.

intracellularis gene contained in the plasmidpGTE#2 which has been deposited under AGAL Accession No. NM00/16477. It shall also be understood that the term "flhB polypeptide" refers to a polypeptide of the invention which comprises the amino acid sequence set forth in SEQ ID NO: 2 or a polypeptide encoded by the L. intracellularis gene contained in the plasmid pGTE#2 which has been deposited under AGAL Accession No. NM00/16477. The term "flhB polypeptide" shall further be taken to include a polypeptide which is functionally-related to or immunologically crossreactive with the polypeptide of SEQ ID NO: 2 or a polypeptide encoded by the L.

intracellularis gene contained in the plasmid pGTE#2 which has been deposited -under AGAL Accession No. NM00/16477.

As used herein, the word "fiR", or the term "fiR gene", shall be taken to refer to a gene encoding the antigenic fliR polypeptide of the present invention, which gene comprises the nucleotide sequence set forth in SEQ ID NO: 3 or the nucleotide WO 02/038594 PCT/AU01/01462 -3sequence of the L. intracellularis gene contained in the plasmid pGTE#3 which has been deposited under AGAL Accession No.NM00/16478. The word fliR"or the term "fliR gene" shall further be taken to include a degenerate or complementary nucleotide sequence to SEQ ID NO: 3, or the nucleotide sequence of the L.

intracellularis gene contained in the plasmid pGTE#3 which has been deposited under AGAL Accession No.NM00/16478. It shall also be understood that the term "fliR polypeptide" refers to a polypeptide of the invention which comprises the amino acid sequence set forth in SEQ ID NO: 4 or a polypeptide encoded by the L.

intracellularis gene contained in the plasmid pGTE#3 which has been deposited under AGAL Accession No.NM00/16478. The term "fliR polypeptide" shall further be taken to include a polypeptide which is functionally-related to or immunologically cross-reactive with the polypeptide of SEQ ID NO: 4 or a polypeptide encoded by the L. intracellularis gene contained in the plasmid pGTE#3 which has been deposited under AGAL Accession No.NM00/16478.

As used herein, the word "ntrC", or the term "ntrC gene", shall be taken to refer to a gene encoding the antigenic ntrC polypeptide of the present invention, which gene comprises the nucleotide sequence set forth in SEQ ID NO: 5 or the nucleotide sequence of the L. intracellularis gene contained in the plasmid pGTE#6 which has been deposited under AGAL Accession No.NM00/16481. The word '"trC"or the term "ntrC gene" shall further be taken to include a degenerate or complementary nucleotide sequence to SEQ ID NO: 5, or the nucleotide sequence of the L.

intracellularis gene contained in the plasmid pGTE#6 which has been deposited under AGAL Accession No.NM00/16481. It shall also be understood that the term "ntrC polypeptide" refers to a polypeptide of the invention which comprises the amino acid sequence set forth in SEQ ID NO: 6 or a polypeptide encoded by the L.

intracellularis gene contained in the plasmid pGTE#6 which has been deposited under AGAL Accession No.NM00/16481. The term "ntrC polypeptide" shall further be taken to include a polypeptide which is functionally-related to or immunologically cross-reactive with the polypeptide of SEQ ID NO: 6 or a polypeptide encoded by the L. intracellularis gene contained in the plasmid pGTE#6 which has been deposited under AGAL Accession No.NM00/16481.

WO 02/038594 PCT/AU01/01462 -4- As used herein, the word "ginH", or the term "gInH gene", shall be taken to refer to a gene encoding the antigenic glnH polypeptide of the present invention, which gene comprises the nucleotide sequence set forth in SEQ ID NO: 7 or the nucleotide sequence of the L. intracellularis gene contained in the plasmid pGTE#1 which has been deposited under AGAL Accession No.NM00/16476. The word "gnH"or the term "ginH gene" shall further be taken to include a degenerate or complementary nucleotide sequence to SEQ ID NO: 7, or the nucleotide sequence of the L.

intracellularis gene contained in the plasmid pGTE#1 which has been deposited under AGAL Accession No.NM00/16476. It shall also be understood that the term "glnH polypeptide" refers to a polypeptide of the invention which comprises the amino acid sequence set forth in SEQ ID NO: 8 or a polypeptide encoded by the L.

intracellularis gene contained in the plasmid pGTE#1 which has been deposited under AGAL Accession No.NM00/16476. The term "gInH polypeptide" shall further be taken to include a polypeptide which is functionally-related to or immunologically cross-reactive with the polypeptide of SEQ ID NO: 8 or a polypeptide encoded by the L. intracellularis gene contained in the plasmid pGTE#1 which has been deposited under AGAL Accession No.NM00/16476.

As used herein, the word "motA", or the term "motA gene", shall be taken to refer to a gene encoding the antigenic motA polypeptide of the present invention, which gene comprises the nucleotide sequence set forth in SEQ ID NO: 9, or to the nucleotide sequence of the L. intracellularis gene contained in the plasmid pGTE#4 which has been deposited under AGAL Accession No.NM00/16479 and which has homology to SEQ ID NO: 9. The word tnotA "or the term "motA gene" shall further be taken to include a degenerate or complementary nucleotide sequence to SEQ ID NO: 9, or the nucleotide sequence of the L. intracellularis gene contained in the plasmid pGTE#4 which has been deposited under AGAL Accession No.NM00/16479 and which has homology to SEQ ID NO: 9. It shall also be understood that the term "motA polypeptide" refers to a polypeptide of the invention which comprises the amino acid sequence set forth in SEQ ID NO: 10 or a polypeptide encoded by the L.

intracellularis gene contained in the plasmid pGTE#4 which has been deposited WO 02/038594 PCT/AU01/01462 under AGAL Accession No.NM00/16479 and which has homology to SEQ ID NO: 9.

The term "motA polypeptide" shall further be taken to include a polypeptide which is functionally-related to or immunologically cross-reactive with the polypeptide of SEQ ID NO: 10 or a polypeptide encoded by the L. intracellularis gene contained in the plasmid pGTE#4 which has been deposited under AGAL Accession No.NM00/16479 and having homology to SEQ ID NO: 9.

As used herein, the word "motB", or the term "motB gene", shall be taken to refer to a gene encoding the antigenic motB polypeptide of the present invention, which gene comprises the nucleotide sequence set forth in SEQ ID NO: 11 or the nucleotide sequence of the L. intracellularis gene contained in the plasmid pGTE#4 which has been deposited under AGAL Accession No.NM00/16479 and having homology to SEQ ID NO: 11. The word "motB"or the term "motB gene" shall further be-taken to include a degenerate or complementary nucleotide sequence to SEQ ID NO: 11, or the nucleotide sequence of the L. intracellularis gene contained in the plasmid pGTE#4 which has been deposited under AGAL Accession No.NMOO/16479 and having homology to SEQ ID NO: 11. It shall also be understood that the term "motB polypeptide" refers to a polypeptide of the invention which comprises the amino acid sequence set forth in SEQ ID NO: 12 or a polypeptide encoded by the L.

intracellularis gene contained in the plasmid pGTE#4 which has been deposited under AGAL Accession No.NM00/16479 and having homology to SEQ ID NO: 11.

The term "motB polypeptide" shall further be taken to include a polypeptide which is functionally-related to or immunologically cross-reactive with the polypeptide of SEQ ID NO: 12 or a polypeptide encoded by the L. intracellularis gene contained in the plasmid pGTE#4 which has been deposited under AGAL Accession No.NM00/16479 and having homology to SEQ ID NO: 11.

As used herein, the word "tlyC", or the term "tlyC gene", shall be taken to refer to a gene encoding the antigenic tlyC polypeptide of the present invention, which gene comprises the nucleotide sequence set forth in SEQ ID NO: 13 or the nucleotide sequence of the L. intracellularis gene contained in the plasmid pGTE#5 which has been deposited under AGAL Accession No.NM00/16480. The word 'tlyC"or the term WO 02/038594 PCT/AU01/01462 -6- "tlyC gene" shall further be taken to include a degenerate or complementary nucleotide sequence to SEQ ID NO: 13, or the nucleotide sequence of the L.

intracellularis gene contained in the plasmid pGTE#5 which has been deposited under AGAL Accession No.NM00/16480. It shall also be understood that the term "tlyC polypeptide" refers to a polypeptide of the invention which comprises the amino acid sequence set forth in SEQ ID NO: 14 or a polypeptide encoded by the L.

intracellularis gene contained in the plasmid pGTE#5 which has been deposited under AGAL Accession No.NM00/16480. The term "tlyC polypeptide" shall further be taken to include a polypeptide which is functionally-related to or immunologically cross-reactive with the polypeptide of SEQ ID NO: 14 or a polypeptide encoded by the L. intracellularis gene contained in the plasmid pGTE#5 which has been deposited under AGAL Accession No.NM00/16480.

As used herein, the word "ytfM", or the term "ytfM gene", shall be taken to refer to a gene encoding the antigenic ytfM polypeptide of the present invention, which gene comprises the nucleotide sequence set forth in SEQ ID NO: 15 or the nucleotide sequence of the L. intracellularis gene contained in the plasmid pGTE#7 which has been deposited under AGAL Accession No.NM00/16482. The word ytfM"or the term "ytfM gene" shall further be taken to include a degenerate or complementary nucleotide sequence to SEQ ID NO: 15, or the nucleotide sequence of the L.

intracellularis gene contained in the plasmid pGTE#7 which has been deposited under AGAL Accession No.NM00/16482. It shall also be understood that the term "ytfM polypeptide" refers to a polypeptide of the invention which comprises the amino acid sequence set forth in SEQ ID NO: 16 or a polypeptide encoded by the L.

intracellularis gene contained in the plasmid pGTE#7 which has been deposited under AGAL Accession No.NM00/16482. The term "ytfM polypeptide" shall further be taken to include a polypeptide which is functionally-related to or immunologically cross-reactive with the polypeptide of SEQ ID NO: 16 or a polypeptide encoded by the L. intracellularis gene contained in the plasmid pGTE#7 which has been deposited under AGAL Accession No.NM00/16482.

As used herein, the word "ytfN", or the term "ytfN gene", shall be taken to refer to a WO 02/038594 PCT/AU01/01462 -7gene encoding the antigenic ytfN polypeptide of the present invention, which gene comprises the nucleotide sequence set forth in SEQ ID NO: 17 or the nucleotide sequence of the L. intracellularis gene contained in the plasmid pGTE#8 which has been deposited under AGAL Accession No. NM01/23286. The word oytfN"or the term "ytfN gene" shall further be taken to include a degenerate or complementary nucleotide sequence to SEQ ID NO: 17 or the nucleotide sequence of the L.

intracellularis gene contained in the plasmid pGTE#8 which has been deposited under AGAL Accession No. NM01/23286. It shall also be understood that the term "ytfN polypeptide" refers to a polypeptide of the invention which comprises the amino acid sequence set forth in SEQ ID NO: 18 or a polypeptide encoded by the L.

intracellularis gene contained in the plasmid pGTE#8 which has been deposited under AGAL Accession No. NM01/23286. The term "ytfN polypeptide" shall further be taken-to include-a-polypeptide which is functionally-related to or immunologically cross-reactive with the polypeptide of SEQ ID NO: 18 or a polypeptide encoded by the L. intracellularis gene contained in the plasmid pGTE#8 which has been deposited under AGAL Accession No. NM01/23286.

As used herein the words "from" or and the term "derived from" shall be taken to indicate that a specified product, in particular a macromolecule such as a polypeptide, protein, gene or nucleic acid molecule, antibody molecule, Ig fraction, or other macromolecule, or a biological sample comprising said macromolecule, may be obtained from a particular source, organism, tissue, organ or cell, albeit not necessarily directly from that source, organism, tissue, organ or cell.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described.

WO 02/038594 PCT/AU01/01462 -8- It is to be understood that the invention includes all such variations and modifications.

The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps, features, compositions and compounds.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only.

Functionally equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

BACKGROUND OF THE INVENTION The meat-producing sector of the agricultural industry is dependent upon the health of its livestock and there is a need to maintain disease-free livestock for human consumption. The industry is subject to rapid economic downturn in response to disease conditions adversely affecting livestock and the quality of meat products derived therefrom, including those diseases which may potentially be transmitted to humans. It is important, therefore, to have well defined treatments and prophylactic and diagnostic procedures available to deal with infections or potential infections in livestock animals and humans.

Meat products derived from porcine and avian species are significant commercial products in the agriculture industry. In particular, pigs form a major component of the meat industry. However, pigs are sensitive to a wide spectrum of intestinal diseases collectively referred to as porcine proliferative enteropathy (PPE). These diseases have previously been known as intestinal adenomatosis complex (Barker and van Drumel, 1985), porcine intestinal adenomatosis (PIA), necrotic enteritis (Rowland and Lawson, 1976), proliferative haemorrhagic enteropathy (Love and Love, 1977), regional ileitis (Jonsson and Martinsson, 1976), haemorrhagic bowel syndrome (O'Neil, 1970), porcine proliferative enteritis and Campylobacter spp induced enteritis (Straw, 1990).

WO 02/038594 PCT/AU01/01462 -9- There are two main forms of PPE: a non-haemorrhagic form represented by intestinal adenomatosis which frequently causes growth retardation and mild diarrhoea; and a haemorrhagic form, which is often fatal, represented by proliferative haemorrhagic enteropathy (PHE), where the distal small intestine lumen becomes engorged with blood. PPE has been reported in a number of animal species including pigs (McOrist et al, 1993), hamsters (Stills, 1991), ferrets (Fox et al, 1989), guinea pigs (Elwell et al, 1981), rabbits (Schodeb and Fox, 1990) as well as avian species (Mason et al, 1998).

PPE is a significant cost component associated with the pig industry, especially in terms of stock losses, medication costs, reduced growth rates of pigs and increased feed costs. PPE also contributes to downstream indirect costs in, for example, additional labour costs and environmental costs in dealing with-antibiotic-residue contamination, and in control measures to prevent the organism from- being passed on or carried to other animals or humans.

L. intracellularis is a causative agent of PPE (McOrist et al, 1995). L. intracellularis is an intracellular, possibly obligate intracellular, bacterium. It can only be cultured in vitro with tissue culture cells (Jones et al., 1997; Lawson et al., 1993; McOrist et al, 1995; International Patent Application No. PCT/US96/09576). L. intracellularis is located in the cytoplasm of the villus cells and intestinal crypt cells of infected animals. Pigs suffering from PPE are characterised by irregularities in the villus cells and intestinal crypt structure with epithelial cell dysplasia, wherein crypt abscesses form as the villi and intestinal crypts become branched and fill with inflammatory cells.

Current control strategies for PPE rely on the use of antibacterials. However, such a strategy is considered to only be short to medium term, especially since govemmental regulatory pressures tend to discourage animal husbandry practices which involve the use of prophylactic antibiotics. There is a need, therefore, to develop effective, safe and low cost alternatives to the use of antibiotics and, in particular, to develop vaccine preparations capable of conferring protective immunity against L. intracellularis infection in livestock animals.

WO 02/038594 PCT/AU01/01462 The most effective vaccine preparations are generally comprised of a highly antigenic component, such as a polypeptide or other macromolecule which is derived from the pathogenic organism against which the vaccine is directed, wherein said antigenic component produces little or no contraindications when administered to a susceptible host animal, and produces little or no antigenic cross-reactivity with desirable organisms, such as non-pathogenic organisms that are a part of the normal flora of the intestinal tract or other tissues of said host animal. In summary, an effective vaccine preparation must be immunogenic, specific and safe.

Accordingly, there is a need to identify highly immunogenic antigens produced by the bacterium L. intracellularis.

Intemational Patent Application No. PCT/AU96/00767 describes several L.

intracellularis partial genetic sequences, and partial polypeptides encoded thereby.

However, there is a need to further identify polypeptide immunogens produced by the bacterium L. intracellularis and immunogenic peptides derived therefrom, including those immunogens which are genus- or species-specific, for use in improved vaccine compositions. The presently-described invention provides such immunogens.

SUMMARY OF THE INVENTION One aspect of the present invention is directed to an isolated or recombinant immunogenic polypeptide which comprises, mimics or cross-reacts with a B-cell or T-cell epitope of a polypeptide derived from Lawsonia spp, in particular a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, or a homologue, analogue or derivative of any one or more of said polypeptides.

Preferably, the isolated or recombinant immunogenic polypeptide is selected from the group consisting of the following: a polypeptide which comprises an amino acid sequence which has at WO 02/038594 PCT/AU01/01462 11 least about 60% sequence identity overall to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; (ii) a polypeptide which comprises an amino acid sequence which has at least about 60% sequence identity overall to an amino acid sequence encoded by L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 gInH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); (iii) a polypeptide which comprises at least about 5 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; (iv) a polypeptide which comprises at least about 5 contiguous amino acids of an amino acid sequence encoded by L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM0/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); and a homologue, analogue or derivative of any one of to (iv) which mimics a B-cell or T-cell epitope of Lawsonia spp.

In an altemative preferred embodiment, the isolated or recombinant immunogenic polypeptide is selected from the group consisting of the following: a polypeptide which comprises an amino acid sequence encoded by a nucleotide sequence having at least about 60% sequence identity overall to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; (ii) a polypeptide which comprises an amino acid sequence encoded by a nucleotide sequence having at least about 60% sequence identity overall to WO 02/038594 WO 02/38594PCT/AU01/01462 12the nucleotide sequence of L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM0O/16476 (plasmid pGTE#1 gInH); NMOO/1 6477 (plasmid pGTE#2 fihB); NMOO/1 6478 (plasmid pGTE#3 MiR); NMOO/16479 (plasmid pGTE#4 motAB); NMO/16480 (plasmid pGTE#5 UlyC); NMOO/16481 (plasmid pGTE#6 ntrC); NMOO/16482 (plasmid pGTE#7 ytfM); and NI1/23286 (plasmid pGTE#8 ytfN); (iii) a polypeptide encoded by at least about 15 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5,7, 9,11, 13, 15, and 17; (iv) a polypeptide encoded by at least about 15 contiguous nucleotides of a nucleotide sequence of L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NMOO/16476 (plasmid pGTE#1 gInH); NMOO/1 6477 (plasmid pGTE#2 flhB); NMOO/1 64-78 (plasmid pGTE#3 fMR); NM0O/1 6479 (plasmid pGTE#4 motAIB); NMOO/1 6480 (plasmid pGTE#5 tlyC); NMOO/1 6481 (plasmid pGTE#6 ntrC); NMOO/1 6482 (plasmid pGTE#7 ytfM); and NMOI1/23286 (plasmid pGTE#8 ytfN); and a homologue, analogue or derivative of any one of to (iv) which mimics a B-cell or T-cell epitope of Lawsonia spp.

In a particularly preferred embodiment, the polypeptide of the present invention comprises; or consists of an amino acid sequence selected from the group consisting of: an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; and (ii) an amino acid sequence encoded by L. intracellularis DNA contained within a deposited plasmid selected from the group consisting of AGAL Accession Nos: NMOO/1 6476 (plasmid pGTE#1 glnH); NMOO/1 6477 (plasmid pGTE#2 flhB); NMOO/1 6478 (plasmid pGTE#3 fliR); NMOO/1 6479 (plasmid pGTE#4 motAIB); NMOO/16480 (plasmid pGTE#5 UyC); NMOO/16481 (plasmid pGTE#6 ntrC); NMOO/16482 (plasmid pGTE#7 ytfM); and NI1/23286 (plasmid pGTE#8 ytfN).

WO 02/038594 PCT/AU01/01462 -13- A further aspect of the present invention provides a vaccine composition for the prophylaxis or treatment of infection in an animal, such as a pig or bird, by L.

intracellularis or a similar or otherwise related microorganism, said vaccine composition comprising an immunologically effective amount of an immunogenic component which comprises an isolated or recombinant polypeptide selected from the group consisting of flhB, fliR, ntrC, ginH, motA, motB, tlyC, ytfM, and ytfN polypeptides as described herein and one or more carriers, diluents and/or adjuvants suitable for veterinary or pharmaceutical use.

A further aspect of the invention extends to an immunologically interactive molecule, such as an antibody or antibody fragment, which is capable of binding to an immunogenic polypeptide of the invention selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides.

A further aspect of the invention provides a method of diagnosing infection of an animal by L. intracellularis or a related microorganism, said method comprising the steps of contacting a biological sample derived from said animal with an immunologically interactive molecule of the present invention for a time and under conditions sufficient for a complex, such as an antigen:antibody complex, to form, and then detecting said complex formation.

A further aspect of the present invention contemplates a method of determining whether or not an animal has suffered from a past infection, or is currently infected, by L. intracellularis or a related microorganism, said method comprising contacting a tissue or fluid sample, such as blood or serum derived from said animal, with an immunogenic polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, or a peptide derived therefrom, for a time and under conditions sufficient for a complex, such as an antigen:antibody complex, to form, and then detecting said complex formation.

A further aspect of the present invention provides an isolated nucleic acid molecule which comprises a sequence of nucleotides that encodes, or is complementary to a WO 02/038594 PCT/AU01/01462 -14nucleic acid molecule that encodes, a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, including any and all genes selected from the group consisting of flhB, fliR, ntrC, gInH, motA, motB, tlyC, ytfM, and ytfN genes as defined hereinabove.

In a preferred embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide that is immunologically cross-reactive with L.

intracellularis or other causative agent of PPE, wherein said nucleotide sequence is selected from the group consisting of: a nucleotide sequence having at least about 60% sequence identity overall to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; (ii) a nucleotide sequence-having at least about 60% sequence identity overall to L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); (iii) a nucleotide sequence which comprises at least about 15 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 1,3, 5, 7, 9, 11, 13, 15, and 17; (iv) a nucleotide sequence which comprises at least about 15 contiguous nucleotides of L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 gInH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM0/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); a nucleotide sequence which hybridizes under at least low stringency, more preferably moderate stringency, and most preferably high stringency conditions to a nucleotide sequence selected from the group consisting of WO 02/038594 PCT/AU01/01462 SEQ ID NOs: 1, 3, 5, 7, 9,11, 13, 15, and 17 or a complementary nucleotide sequence thereto; (vi) a nucleotide sequence which hybridizes under at least low stringency, more preferably moderate stringency, and most preferably high stringency conditions to L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motAIB); NM00/16480 (plasmid tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); and (vii) a homologue, analogue or derivative of any one of to (vi) which encodes a polypeptide which mimics a B-cell or T-cell epitope of Lawsonia spp.

In a particularly preferred embodiment, the isolated nucleic acid molecule of the present invention comprises or consists of a nucleotide sequence selected from the group consisting of: a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1,3, 5, 7, 9, 11, 13, 15, and 17; or a degenerate variant thereof; (ii) a nucleotide sequence of the L. intracellularis DNA contained within a deposited plasmid selected from the group consisting of AGAL Accession Nos: NMOO/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); and (iii) a nucleotide sequence that encodes the same polypeptide as or (ii), wherein said polypeptide is selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN; (iv) a nucleotide sequence that is complementary to or (ii) or (iii); and a nucleotide sequence that hybridises under high stringency conditions to the complement of a sequence selected from the group consisting of: SEQ WO 02/038594 PCT/AU01/01462 -16- ID NOs: 1, 3, 5, 7, 9, 11, 13, 15 and 17, wherein said nucleotide sequence is the complement of a sequence that encodes a polypeptide that is immunologically cross-reactive to a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN.

A still further aspect of the invention provides a diagnostic method of detecting L.

intracellularis or related microorganism in a biological sample derived from an animal subject, said method comprising the steps of hybridising one or more polynucleotide or oligonucleotide probes or primers derived from a gene selected from the group consisting of flhB, fiR, ntrC, glnH, motA, motB, tlyC, yffM, and ytfN genes, or a homologue, analogue or derivative thereof, to said sample, and then detecting said hybridisation using a detection means. The detection means according to this aspect of the invention is any nucleic acid-based hybridisation or amplification reaction.

A further aspect of the invention provides an isolated probe or primer derived from a gene selected from the group consisting of flhB, fiR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN genes. In a particularly preferred embodiment, the probe or primer of the invention is useful for isolating the ytfM and/or ytfN genes described herein. More preferably, the probe or primer of the invention comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 19 to SEQ ID NO: 68 or a complementary nucleotide sequence thereto.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a copy of a photographic representation showing expression of recombinant YtfN protein. The 5' portion of the gene up to the Bglll site was cloned into pET-30a. A plasmid with the fragment inserted in the proper orientation was transformed into E. coli BL21 (DE3) cells, and a single clone was propagated.

Induction was at OD 6 2 5 =2.9 with 0.1 mM IPTG. Lane 1, whole cell lysate (WCL) from uninduced cells; lanes 2 and 3, WCL at 2.25 and 3 hrs post-induction, respectively.

Arrow indicates the position of recombinant YtfN protein.

DETAILED DESCRIPTION OF THE INVENTION WO 02/038594 PCT/AU01/01462 -17- In work leading up to the present invention, the inventors sought to identify immunogenic proteins of L. intracellularis for use in vaccines for the prophylaxis and treatment of PPE in animals, including pigs and birds.

Accordingly, one aspect of the present invention is directed to an isolated or recombinant immunogenic polypeptide which comprises, mimics or cross-reacts with a B-cell or T-cell epitope of a polypeptide derived from Lawsonia spp, selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN, or a homologue, analogue or derivative of any one or more of said polypeptides.

Epitopes of Lawsonia spp. may be B cell epitopes or T-cell epitopes. It is well-known that antibody-binding sites (B-cell epitopes) involve linear as well as conformational epitopes (van Regenmortel, 1992). B-cell epitopes-are predominantly-conformational.

In contrast, T-cells recognize predominantly linear epitope sequences in combination with MHC class II molecules.

A precise identification and careful selection of epitopes of Lawsonia spp. facilitates the development of diagnostic reagents and vaccine compositions for the effective treatment or prophylaxis of Lawsonia infections. Epitope identification and characterization determination of the molecular weight, amino acid sequence, and structure of epitopes of Lawsonia spp.) may be performed using art-recognised techniques. For the detection of conformational epitopes, degrading and denaturing of the epitope molecule must be avoided in order to conserve the three-dimensional structure, because the antigen-antibody reaction will be diminished if the secondary structure of the epitope is altered significantly. In practice, the characterisation and isolation of linear non-conformational epitopes is easier, because any immunoreactive regions are contained within a single polypeptide or peptide fragment which is capable of being purified under a range of conditions.

Both non-conformational and conformational epitopes may be identified by virtue of their ability to bind detectable amounts of antibodies (such as IgM or IgG) from sera of animals immunised against or infected with Lawsonia spp. and, in particular L.

WO 02/038594 PCT/AU01/01462 -18intracellularis, or an isolated polypeptide derived therefrom or, alternatively, by virtue of their ability to bind detectable amounts of antibodies in a purified Ig fraction derived from such sera. The antibodies may be derived from or contained within pools of polyclonal sera, or may be monoclonal antibodies. Antibody fragments or recombinant antibodies, such as those expressed on the surface of a bacteriophage or virus particle, such as in a phage display library, may also be employed.

The determination of T-cell epitopes is performed by analysing the ability of the epitope peptides to induce the proliferation of peripheral blood lymphocytes or T-cell clones. The identification of T-cell epitopes is accomplished using a variety of methods as known in the art, including the use of whole and fragmented native or recombinant antigenic protein, as well as the more commonly employed "overlapping peptide" method. In the-lattermethod, overlapping peptides which span the entire sequence of a polypeptide derived from Lawsonia spp. are synthesized and tested for their capacity to stimulate T-cell cytotoxic or proliferative responses in vitro.

Structure determination of both conformational non-linear and non-conformational linear epitopes may be performed by nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallographic analysis. The determination of epitopes using X-ray techniques requires the protein-antibody complex to be crystallized, whereas NMR allows analysis of the complex in a liquid state. NMR measures the amount of amino acids as well as the neighbourhood of protons of different amino acid residues, wherein the alternating effect of two protons along the carbon backbone is characteristic of a particular epitope.

A successful method to recognize non-conformational linear epitopes is the immunoblot and in particular, the Westem blot. Peptides may be generated from a complete Lawsonia spp. polypeptide by digestion with site-specific proteases, such as trypsin or chymotrypsin, and the peptides generated thereby can be separated using standard electrophoretic or chromatographic procedures. For example, after electrophoresis according to molecular weight using SDS/PAGE (SDS/polyacrylamide gel electrophoresis) and/or according to isoelectric point using WO 02/038594 PCT/AU01/01462 -19- IEF (isoelectric focussing) or altematively, by two-dimensional electrophoresis, the peptides can be transferred to immobilizing nylon or nitrocellulose membranes and incubated with sera raised against the intact polypeptides. Peptides that comprise immunogenic regions B-cell or T-cell epitopes) are bound by the antibodies in the sera and the bound antibodies may be detected using secondary antibodies, such as anti-lgG antibodies, that have been labelled radioactively or enzymatically.

The epitopes may then be characterised by purification based upon their size, charge or ability to bind specifically to antibodies against the intact polypeptide, using one or more techniques, such as size-exclusion chromatography, ion-exchange chromatography, affinity chromatography or ELISA among others. After purification of the epitope, only one band or spot should be detectable with gel electrophoresis.

The N-terminal or total sequencing of the polypeptide or peptide fragment offers the possibility to compare the amino acid sequence with known proteins in databases.

Several computer-driven algorithms have now been devised to search for T-cell.

epitopes in proteins (Margalit et al, 1987; Vajda and C. DeLisi, 1990; Altuvia et al., 1994; Parker et al.1994; DeGroot et al.,1995; Gabriel et al, 1995; Meister et al., 1995). These algorithms search the amino acid sequence of a given protein for characteristics believed to be common to immunogenic peptides, locating regions that are likely to induce a cellular immune response in vitro. Computer-driven algorithms can identify regions of a Lawsonia spp. polypeptide that contain epitopes and are less variable among different isolates. Alternatively, computer-driven algorithms can rapidly identify regions of each isolate's more variable proteins that should be included in a multivalent vaccine.

The AMPHI algorithm (Margalit et al.,1987), which is based on the periodicity of T cell epitopes, has been widely used for the prediction of T-cell antigenic sites from sequence information alone. Essentially, AMPHI describes a common structural pattern of MHC binding motifs,.since MHC binding motifs patterns of amino acids that appear to be common to most of the peptides that bind to a specific MHC molecule) appear to exhibit the same periodicity as an alpha helix. Identification of T-cell epitopes by locating MHC binding motifs in an amino acid sequence provides WO 02/038594 PCT/AU01/01462 an effective means of identifying immunogenic epitopes in diagnostic assays.

The EpiMer algorithm (Meister et al., 1995; Gabriel et al., 1995; DeGroot et al., 1995) locates clustered MHC binding motifs in amino acid sequences of proteins, based upon the correlation between MHC binding motif-dense regions and peptides that may have the capacity to bind to a variety of MHC molecules (promiscuous or multi-determinant binders) and to stimulate an immune response in these various MHC contexts as well (promiscuous or multi-determinant epitopes). The EpiMer algorithm uses a library of MHC binding motifs for multiple class I and class II HLA alleles to predict antigenic sites within a protein that have the potential to induce an immune response in subjects with a variety of genetic backgrounds. EpiMer locates matches to each MHC-binding motif within the primary sequence of a given protein antigen. The relative density of these motif matches is determined along the length of the antigen, resulting in the generation of a motif-density histogram. Finally, the algorithm identifies protein regions in this histogram with a motif match density above an algorithm-defined cutoff density value, and produces a list of subsequences representing these clustered, or motif-rich regions. The regions selected by EpiMer may be more likely to act as multi-determinant binding peptides than randomly chosen peptides from the same antigen, due to their concentration of MHC-binding motif matches. The selection of regions that are MHC binding motif-dense increases the likelihood that the predicted polypeptide or peptide fragment.contains a "valid" motif, and furthermore, that the reiteration of identical motifs may contribute to binding.

Additional MHC binding motif-based algorithms have been described by Parker et a/.(1994) and Altuvia et a/.(1994). In these algorithms, binding to a given MHC molecule is predicted by a linear function of the residues at each position, based on empirically defined parameters, and in the case of the Altuvia et a1.(1994) algorithm, known crystallographic structures may also be taken into consideration.

Recombinant methods offer the opportunity to obtain well characterized epitopes of high purity for the production of diagnostic reagents and epitope-specific vaccine WO 02/038594 PCT/AU01/01462 -21formulations (Mohapatra et al., 1995). Based upon the amino acid sequence of a linear epitope and identification of the corresponding nucleotide sequence encoding same, polymerase chain reaction (PCR) may be performed to amplify the epitopeencoding region from cDNA. After cloning and expression in a suitable vector/host system, a large amount of epitopes of high purity can be extracted. Accordingly, the present invention clearly extends to both isolated non-recombinant polypeptides and recombinant polypeptides in an impure or isolated form.

The term "polypeptide" as used herein shall be taken to refer to any polymer consisting of amino acids linked by covalent bonds and includes within its scope the full-length amino acids disclosed herein, and any parts or fragments thereof such as, for example, peptides consisting of about 5-50 amino acid residues in length, preferably about 5-30 amino acid residues in length, more preferably about 5-20 amino acid residues in length, and even more preferably about 5-10 amino acid residues in length. Also included within the scope of the definition of a "polypeptide" are amino acid sequence variants, containing one or more preferably conservative amino acid substitutions, deletions, or insertions, which do not alter at least one essential property of said polypeptide such as, for example, its immunogenicity, use as a diagnostic reagent, or effectiveness as a vaccine against Lawsonia spp, amongst others. Accordingly, a polypeptide may be isolated from a source in nature, or chemically synthesized. Furthermore, a polypeptide may be derived from a fulllength protein by chemical or enzymatic cleavage, using reagents such as CNBr, trypsin, or chymotrypsin, amongst others.

Conservative amino acid substitutions are well-known in the art. For example, one or more amino acid residues of a native flagellar hook protein of the present invention can be substituted conservatively with an amino acid residue of similar charge, size or polarity, with the resulting polypeptide retaining an ability to function in a vaccine or as a diagnostic reagent as described herein. Rules for making such substitutions include those described by Dayhof (1978). More specifically, conservative amino acid substitutions are those that generally take place within a family of amino acids that are related in their side chains. Genetically-encoded amino acids are generally WO 02/038594 PCT/AU01/01462 -22divided into four groups: acidic=aspartate, glutamate; basic=lysine, arginine, and histidine; non-polar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and uncharged polar= glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Phenylalanine, tyrosine and tryptophan are also jointly classified as aromatic amino acids. One or more replacements within any particular group such as, for example, the substitution of leucine for isoleucine or valine or alternatively, the substitution of aspartate for glutamate or threonine for serine, or of any other amino acid residue with a structurally-related amino acid residue, will generally have an insignificant effect on the function of the resulting polypeptide.

The present invention is not limited by the source of the subject immunogen and clearly extends to isolated and recombinant polypeptides which are derived from a natural or a non-natural occurring source.

The term "recombinant polypeptide" as used herein shall be taken to refer to a polypeptide which is produced in vitro or in a host cell by the expression of a genetic sequence encoding said polypeptide, which genetic sequence is under the control of a suitable promoter, wherein a genetic manipulation has been performed in order to achieve said expression. Accordingly, the term "recombinant polypeptide" clearly encompasses polypeptides produced by the expression of genetic sequences contained in viral vectors, cosmids or plasmids that have been introduced into prokaryotic or eukaryotic cells, tissues or organs. Genetic manipulations which may be used in this context will be known to those skilled in the art and include, but are not limited to, nucleic acid isolation, restriction endonuclease digestion, exonuclease digestion, end-filling using the Klenow fragment of E. coil DNA polymerase I or T4 DNA polymerase enzymes, blunt-ending of DNA molecules using T4 DNA polymerase or Exolll enzymes, site-directed mutagenesis, ligation, and amplification reactions. As will be known to those skilled in the art, additional techniques such as nucleic acid hybridisations and nucleotide sequence analysis may also be utilised in the preparation of recombinant polypeptides, in confirming the identity of a nucleic acid molecule encoding a desired recombinant polypeptide and a genetic construct WO 02/038594 PCT/AU01/01462 -23comprising the nucleic acid molecule.

Wherein the polypeptide of the present invention is a recombinant polypeptide, it may be produced in and, if desirable, isolated from a recombinant viral vector expression system or host cell. As will be known to those skilled in the relevant art, a cell for production of a recombinant polypeptide is selected on the basis of several parameters including the genetic constructs used to express the polypeptide under consideration, as well as the stability and activity of said polypeptide. It will also be known to those skilled in the art that the stability or activity of a recombinant polypeptide may be determined, at least in part, by post-translational modifications to the polypeptide such as, for example, glycosylation, acylation or alkylation reactions, amongst others, which may vary between cell lines used to produce the recombinant polypeptide.

Accordingly, in a more particularly preferred embodiment, the present invention extends to a recombinant polypeptide or a derivative, homologue or analogue thereof as present in a virus particle, or as produced in prokaryotic or eukaryotic host cell, or in a virus or cell culture thereof.

The present invention also extends to a recombinant polypeptide according to any of the foregoing embodiments which is produced in a bacterial cell belonging to the genus Lawsonia, in particular a cell of L. intracellularis, or a culture thereof.

The term "isolated polypeptide" refers to a polypeptide of the present invention which has been purified to some extent, preferably to at least about 20% by weight of protein, preferably to at least about 50% by weight of protein, more preferably to at least about 60% by weight of protein, still more preferably to at least about 70% by weight of.protein and even more preferably to at least about 80% by weight of protein or greater, from its natural source or, in the case of non-naturally-occurring polypeptides, from the culture medium or cellular environment in which it was produced. Such isolation may be performed to improve the immunogenicity of the polypeptide of the present invention, or to improve the specificity of the immune WO 02/038594 PCT/AU01/01462 -24response against that polypeptide, or to remove toxic or undesirable contaminants therefrom. The necessary or required degree of purity of an isolated polypeptide will vary depending upon the purpose for which the polypeptide is intended, and for many applications it will be sufficient for the polypeptide preparation to contain no contaminants which would reduce the immunogenicity of the polypeptide when administered to a host animal, in particular a porcine or avian animal being immunized against PPE or, altematively, which would inhibit immuno-specific binding in an immunoassay for the diagnosis of PPE or a causative agent thereof.

The purity of an isolated polypeptide of the present invention may be determined by any means known to those skilled in the art, including the degree of homogeneity of a protein preparation as assessed by SDS/polyacrylamide gel electrophoresis, 2dimensional electrophoresis, or amino acid composition analysis-or sequence analysis.

Preferably, the polypeptide of the present invention will be substantially homogeneous or substantially free of nonspecific proteins, as assessed by SDS/polyacrylamide gel electrophoresis, 2-dimensional electrophoresis, or amino acid composition analysis or sequence analysis.

The polypeptide of the present invention can be purified for use as a component of a vaccine composition by any one or a combination of methods known to those of ordinary skill in the art, including, for example, reverse phase chromatography, HPLC, ion-exchange chromatography, and affinity chromatography, among others.

In a preferred embodiment, the isolated or recombinant polypeptide of the invention functions is secretable into the periplasmic space of a cell, preferably into the periplasm of a prokaryotic cell, such as, for example, Escherichia coli. or L.

intracellularis, or, alternatively, is immunologically cross-reactive with a L.

intracellularis polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN.

WO 02/038594 PCT/AU01/01462 In a particularly preferred embodiment, the isolated or recombinant polypeptide of the invention is derived from Lawsonia spp. or other pathogenic agent associated with the onset and/or development of PPE and more preferably, the subject polypeptide is derived from L. intracellularis.

A B-cell or T-cell epitope of a polypeptide selected from the group consisting of flhB, fliR, ntrC, gInH, motA, motB, tlyC, ytfM, and ytfN polypeptides, or a homologue, analogue or derivative of any one or more of said polypeptides, may comprise one or more of the following: the primary, amino acid sequence of any one of said polypeptides, as determined by an art-accepted methodology to comprise a continuous non-conformational epitope; (ii) the secondary structure which any one of said polypeptides adopt, as determined by an art-accepted methodology to comprise a continuous conformational epitope; (iii) the tertiary structure which any one of said polypeptides adopt in contact with another region of the same polypeptide molecule, as determined by an art-accepted methodology to comprise a discontinuous conformational epitope; or (iv) the quatemary structure which any one of said polypeptides adopt in contact with a region of another polypeptide molecule, as determined by an art-accepted methodology to comprise a discontinuous conformational epitope.

Accordingly, immunogenic polypeptides or derivatives, homologues or analogues thereof comprising the same, or substantially the same primary amino acid sequence -are hereinafter defined as "immunogens which comprise a B-cell or T-cell epitope" or similar term.

Immunogenic polypeptides or derivatives, homologues, or analogues thereof comprising different primary amino acid sequences may comprise immunologically WO 02/038594 PCT/AU01/01462 -26identical immunogens, because they possess conformational B-cell or T-cell epitopes that are recognised by the immune system of a host species to be identical. Such immunogenic polypeptides or derivatives, homologues or analogues thereof are hereinafter defined as "immunogens which mimic or cross-react with a B-cell or T-cell epitope", or similar term.

Accordingly, the present invention extends to an immunogen which comprises, mimics, or cross-reacts with a B-cell or T-cell epitope of an isolated or recombinant polypeptide according to any one of the foregoing embodiments or a derivative, homologue or analogue thereof. In a particularly preferred embodiment, the present invention provides an immunogen which comprises, mimics, or cross-reacts with a B-cell or T-cell epitope of an isolated or recombinant polypeptide which in its native form is obtainable from a species of Lawsonia such as, but not limited to L.

intracellularis and which polypeptide preferably has the same biological function as a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN, as hereinbefore defined.

Preferably, such immunogenic polypeptides will not comprise a primary amino acid sequence which is highly-conserved between L. intracellularis and another nonpathogenic microorganism which is normally resident in the gut or other organ of an animal, in particular a porcine or avian animal. The significance of this exclusion to those embodiments of the invention wherein specificity is essential to performance (eg vaccine and diagnostic applications) will be apparent to those skilled in the art.

To improve the immunogenicity of a subject polypeptide of the present invention one or more amino acids not corresponding to the original protein sequence can be added to the amino or carboxyl terminus of the polypeptide. Such extra amino acids are useful for coupling the polypeptide to another peptide or polypeptide, to a large carrier protein or to a solid support. Amino acids that are useful for these purposes include but are not limited to tyrosine, lysine, glutamic acid, aspartic acid, cysteine and derivatives thereof. Additional protein modification techniques can be used such as, NH 2 -acetylation or COOH-terminal amidation, to provide additional means WO 02/038594 PCT/AU01/01462 -27for coupling the polypeptide to another polypeptide or peptide molecule, or to a solid support. Procedures for coupling polypeptides to each other, or to carrier proteins or solid supports, are well known in the art. Polypeptides containing the abovementioned extra amino acid residues at either the carboxyl- or amino-termini and either uncoupled or coupled to a carrer or solid support, are consequently within the scope of the present invention.

Furthermore, the polypeptide can be immobilised to a polymeric carrier or support material.

In an altemative embodiment, the immunogenicity of a polypeptide of the present invention may be improved using molecular biology techniques to produce a fusion protein containing one or more polypeptides of the present invention fused to a carrier molecules such as a highly immunogenic protein. For example, a fusion protein containing a polypeptide of the present invention fused to the highly immunogenic B subunit of cholera toxin can be used to increase the immune response to the polypeptide. The present invention also contemplates fusion proteins comprising a cytokine, such as an interleukin, fused to the subject polypeptide of the present invention, and genes encoding same.

Preferably, the polypeptide of the present invention, or a derivative, homologue or analogue thereof, when administered to a mammal, induces an immune response in said mammal. More preferably, the polypeptide of the present invention, when administered to a mammal, in particular a porcine animal a pig) induces a protective immune response against Lawsonia spp., and preferably against L.

intracellularis, therein. As used herein, the phrase "induction of a protective immune response", and the like, refers to the ability of the administered polypeptide of the present invention to prevent or detectably slow the onset, development, or progression of symptoms associated with Lawsonia infection, and preferably, to prevent or detectably slow the onset, development, or progression of symptoms associated with PPE in pigs.

WO 02/038594 PCT/AU01/01462 -28- Preferably, the isolated or recombinant immunogenic polypeptide is selected from the group consisting of the following: a polypeptide which comprises an amino acid sequence which has at least about 60% sequence identity overall to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; (ii) a polypeptide which comprises an amino acid sequence which has at least about 60% sequence identity overall to an amino acid sequence encoded by L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 gInH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); (iii) a polypeptide which comprises at least about 5 contiguous amino acids, of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; (iv) a polypeptide which comprises at least about 5 contiguous amino acids of an amino acid sequence encoded by L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motAIB); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); a homologue, analogue or derivative of any one of to (iv) which mimics a B-cell or T-cell epitope of Lawsonia spp.

-In an alternative-preferred embodiment, the isolated or recombinant immunogenic polypeptide is selected from the group consisting of the following: a polypeptide which comprises an amino acid sequence encoded by a nucleotide sequence having at least about 60% sequence identity overall to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, WO 02/038594 PCT/AU01/01462 -29- 3, 5, 7, 9, 11, 13, 15, and 17; (ii) a polypeptide which comprises an amino acid sequence encoded by a nucleotide sequence having at least about 60% sequence identity overall to the nucleotide sequence of L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motAIB); NM00/16480 (plasmid pGTE#5 tlyC);'NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); (iii) a polypeptide encoded by at least about 15 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; (iv) a polypeptide-encoded by at least about 15 contiguous nucleotides of a nucleotide sequence of L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); a homologue, analogue or derivative of any one of to (iv) which mimics a B-cell or T-cell epitope of Lawsonia spp.

Preferably, the immunogenic polypeptide encompassed by the present invention has at least about 70% identity, more preferably at least about 80% identity, even more preferably at least about 90% identity, and still even more preferably at least about identity to the amino acid sequence of a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, as hereinbefore defined.

In determining whether or not two amino acid sequences fall within these percentage limits, those skilled in the art will be aware that it is necessary to conduct a side-byside comparison or multiple alignment of sequences. In such comparisons or WO 02/038594 PCT/AU01/01462 alignments, differences will arise in the positioning of non-identical residues, depending upon the algorithm used to perform the alignment. In the present context, reference to a percentage sequence identity or similarity between two or more amino acid sequences shall be taken to refer to the number of identical and similar residues respectively, between said sequences as determined using any standard algorithm known to those skilled in the art. For example, amino acid sequence identities or similarities may be calculated using the GAP programme of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America (Devereaux et al, 1984). The GAP programme utilizes the algorithm of Needleman and Wunsch (1970) to maximise the number of identical/similar residues and to minimise the number and/or length of sequence gaps in the alignment. Altematively or in addition, where more than two amino acid sequences are being compared, the ClustalW programme of Thompson et a (1994) can be used.

Preferably, the isolated or recombinant immunogenic polypeptide of the invention comprises at least about 10 contiguous amino acids of a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, as hereinbefore defined. More preferably, the isolated or recombinant immunogenic polypeptide of the invention comprises at least about 20 contiguous amino acid residues of a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, as hereinbefore defined.

Even more preferably, the isolated or recombinant immunogenic polypeptide of the invention comprises at least about 30 contiguous amino acid residues of a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, as hereinbefore defined, and still even more preferably, at least about 40 contiguous amino acid residues of said flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN polypeptides.

The present invention further encompasses homologues, analogues and derivatives of a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, as hereinbefore defined.

WO 02/038594 PCT/AU01/01462 -31- "Homologues" of a polypeptide are those immunogenic polypeptides that are derived from a full-length L. intracellularis polypeptides described herein, or have sequence similarity to a full-length L. intracellularis polypeptide, notwithstanding one or more amino acid substitutions, deletions and/or additions relative to the full-length L.

intracellularis polypeptide. A homologue may also retain the biological activity or catalytic activity of the full-length polypeptide. In such homologues, one or more amino acids can be replaced by other amino acids having similar properties such as, for example, hydrophobicity, hydrophilicity, hydrophobic moment, antigenicity, propensity to form or break a-helical structures of -sheet structures, and so on.

Substitutional variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1-10 amino acid residues. and deletions will range from about 1-20 residues.

Preferably, amino acid substitutions will comprise conservative amino acid substitutions, such as those described supra.

Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein. Insertions can comprise amino-terminal and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than amino or carboxyl terminal fusions, of the order of about 1 to 4 residues.

Deletional variants are characterised by the removal of one or more amino acids from the sequence.

Amino acid variants of the polypeptide of the present invention may readily be made using polypeptide synthetic techniques well known in the art, such as solid phase synthesis and the like, or by recombinant DNA manipulations. The manipulation of DNA sequences to produce variant proteins which manifest as substitutional, WO 02/038594 PCT/AU01/01462 -32insertional or deletional variants are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA having known sequence are well known to those skilled in the art, such as by M13 mutagenesis or other site-directed mutagenesis protocol.

"Analogues" are defined as those immunogenic polypeptides that are derived from a full-length L. intracellularis polypeptides described herein, or have sequence similarity to a full-length L. intracellularis polypeptide, notwithstanding one or more non-naturally occurring or modified amino acid residues relative to the naturallyoccurring full-length L. intracellularis polypeptide. The term "analogue" shall also be taken to include an amino acid sequence which is not similar to an amino acid sequence of a full-length L. intracellularis polypeptide set forth herein, however mimics or cross-reacts with a B-cell or T-cell-epitope-of Lawsonia spp. and preferably, mimics or cross-reacts with a B-cell or T-cell epitope of L. intracellularis, such as, for example, a polypeptide which is derived from a computational prediction or empirical data revealing the secondary, tertiary or quaternary structure of the full-length polypeptide or an epitope thereof.

For example, mimotopes (polypeptide analogues that cross-react with a B-cell or Tcell epitope of the Lawsonia polypeptide of the invention but, however, comprise a different amino acid sequence to said epitope) may be identified by screening random amino acid sequences in polypeptide libraries with antibodies that bind to a desired T-cell or B-cell epitope. As with techniques for the identification of B-cell or T-cell epitopes as described supra, the antibodies used to identify such mimotopes may be polyclonal or monoclonal or recombinant antibodies, in crude or purified form.

Mimotopes of a T-cell epitope may then be assayed further for their ability to stimulate T-cell cytotoxic or proliferative responses in vitro. Mimotopes are particularly useful as analogues of non-linear conformational) epitopes of the polypeptide of the present invention, because conformational epitopes are generally formed from non-contiguous regions in a polypeptide, and the mimotopes provide immunogenic equivalents thereof in the form of a single polypeptide molecule.

WO 02/038594 PCT/AU01/01462 -33- Additionally, the use of polypeptide analogues can result in polypeptides with increased immunogenic and/or antigenic activity, that are less sensitive to enzymatic degradation, and which are more selective. A suitable proline analogue is 2aminocyclopentane carboxylic acid (PACSc) which has been shown to increase the immunogenic activity of a native polypeptide more than 20 times (Mierke et al, 1990; Portoghese et al, 1990; Goodman et al, 1987).

"Derivatives" of a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, as hereinbefore defined, are those peptides or polypeptides which comprise at least about five contiguous amino acid residues of any one or more of said flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN polypeptides.

A "derivative" may further comprise additional naturally-occurring, altered glycosylated, acylated or non-naturally occurring amino acid residues compared to the amino acid sequence of a flhB, or fliR, or ntrC, or glnH, or motA, or motB, or tlyC, or ytfM, or ytfN polypeptide, as hereinbefore defined. Altematively or in addition, a derivative may comprise one or more non-amino acid substituents such as, for example, a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence of a flhB, or fliR, or ntrC, or glnH, or motA, or motB, or tlyC, or ytfM, or ytfN polypeptide, such as, for example, a reporter molecule which is bound thereto to facilitate its detection.

Other examples of recombinant or synthetic mutants and derivatives of a polypeptide immunogen of the present invention include those incorporating single or multiple substitutions in the amino acid sequence of a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides.

Recombinant or synthetic mutants and derivatives produced by making deletions from the amino acid sequence of a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, are also included within the scope of preferred derivatives. Additionally, recombinant or synthetic mutants and derivatives produced by making additions to a polypeptide WO 02/038594 PCT/AU01/01462 34 selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, such as, for example, using carbohydrates, lipids and/or proteins or polypeptides, are also encompassed by the present invention.

Naturally-occurring or altered glycosylated or acylated forms of the flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN polypeptides are particularly contemplated by the present invention.

Additionally, homopolymers or heteropolymers comprising one or more copies of the reference polypeptides, or one or more derivatives, homologues or-analogues thereof, are clearly within the scope of the present invention.

Preferably, homologues, analogues and derivatives of the flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN polypeptides of the invention are "immunogenic", defined hereinafter as the ability of said polypeptide, or a derivative, homologue or analogue thereof, to elicit B cell and/or T cell responses in the host, in response to immunization.

Preferred homologues, analogues and derivatives of the flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN polypeptides of the invention include any amino acid variant that functions as B cell or T cell epitope of any one of said polypeptides, wherein said variant is capable of mediating an immune response, such as, for example, a mimotope of the immunogenic polypeptide which has been produced by synthetic means, such as by Fmoc chemistry. The only requirement of such variant molecules is that they cross-react immunologically with a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN, as hereinbefore defined, or an epitope of said polypeptide.

As will be apparent to those skilled in the art, such homologues, analogues and derivatives of the polypeptides of the invention molecules will be useful to prepare antibodies that cross-react with antibodies against said polypeptide and/or to elicit a protective immune response of similar specificity to that elicited by said polypeptide.

WO 02/038594 PCT/AU01/01462 Such molecules will also be useful in diagnostic and other applications that are immunological in nature such as, for example, diagnostics which utilise one or more immunoassay formats (eg. ELISA, RIA and the like).

Accordingly, the immunogen of the present invention or a derivative, homologue or analogue thereof is useful in vaccine compositions that protect an individual against infection by L. intracellularis and/or as an antigen to elicit polyclonal or monoclonal antibody production and/or in the detection of antibodies against L. intracellularis in infected animals, particularly in porcine and avian animals.

The polypeptides of the present invention may comprise leader sequences to facilitate their secretion into the periplasmic space, either as part of the native protein, or alternatively, added by recombinant engineering means. Suchmay haveimproved immunogenicity compared to non-secreted or non-secretable polypeptides of L.

intracellularis, or non-secreted or non-secretable polypeptides of other causative agents of PPE. The particular advantages of such peptides will be immediately apparent to those skilled in the production of vaccine compositions, where the inherent immunogenicity of the immunogen is an important consideration for a protective immune response to be conferred.

Moreover, unique regions of the L. intracellularis polypeptides exemplified herein are promising antigenic peptides for the formulation of Lawsonia-specific vaccines and diagnostics for the specific detection of Lawsonia spp. in biological samples.

A second aspect of the present invention provides a vaccine composition for the prophylaxis or treatment of infection in a mammal or bird by L. intracellularis or similar or otherwise related microorganism, said vaccine composition comprising: an immunogenic component which comprises an isolated or recombinant polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides or an immunogenic homologue, analogue or derivative of any one of said polypeptides which is immunologically cross-reactive with L. intracellularis; and WO 02/038594 PCT/AU01/01462 -36- (ii) one or more carriers, diluents and/or adjuvants suitable for veterinary or pharmaceutical use.

As used herein, the term "immunogenic component" refers to a polypeptide encoded by DNA from, or derived from, L. intracellularis or a related microorganism thereto which is capable of inducing a protective immune response in an animal, in particular a porcine or avian animal, whether or not said polypeptide is in an isolated or recombinant form. Accordingly, the vaccine composition clearly encompasses those vaccine compositions which comprise attenuated, killed or non-pathogenic isolates or forms of L. intracellularis or related microorganisms thereto which comprise or express said polypeptide.

By "protective immune response" is-meant that the immunogenic component elicits an immune response irrthe animal-to which the vaccine composition is administered at the humoral and/or cellular level which is sufficient to prevent infection by L.

intracellularis or a related microorganism thereto and/or which is sufficient to detectably reduce one or more symptoms or conditions, or to detectably slow the onset of one or more symptoms or conditions, associated with infection by L.

intracellularis or a related microorganism thereto in an animal host, as compared to a control infected animal. The term "effective amount" of an immunogenic component present in the vaccine composition refers to that amount of said immunogenic component that is capable of inducing a protective immune response after a single complete dose has been administered, or after several divided doses have been administered.

Preferably, the polypeptide component of the subject vaccine composition comprises an amino acid sequence which is both immunogenic and specific, by virtue of its immunological cross-reactivity with the causative agent of PPE, L intracellularis. In this regard, it will be apparent from the preceding description that such polypeptide components may comprise the amino acid sequence of a polypeptide of L.

intracellularis as exemplified herein, or alternatively, an immunologically crossreactive homologue, analogue or derivative of said amino acid sequence, such as, WO 02/038594 PCT/AU01/01462 -37for example, a mimotope of said sequence.

The immunogenic polypeptide or immunogenic homologue, analogue or derivative may be a naturally-occurring polypeptide in isolated or recombinant form according to any of the embodiments described supra or exemplified herein. Preferably, the immunogenic polypeptide or immunogenic homologue, analogue or derivative is derived from Lawsonia spp., in particular L. intracellularis or a microorganism that is related thereto.

Preferably, the immunogenic component has undergone at least one purification step or at least partial concentration from a cell culture comprising L. intracellularis or a related microorganism thereto, or from a lysed preparation of L. intracellularis cells or related microorganism, or from another culture in which the immunogenic component is recombinantly expressed. The purity of such a component which has the requisite immunogenic properties is preferably at least about 20% by weight of protein in a particular preparation, more preferably at least about 50%, even more preferably at least about 60%, still more preferably at least about 70% and even more preferably at least about 80% or greater.

The immunogenic component of the vaccine of the present invention can comprise a single polypeptide, or a range or combination of different polypeptides covering different or similar epitopes. In addition or, altematively, a single polypeptide can be provided with multiple epitopes. The latter type of vaccine is referred to as a polyvalent vaccine. A multiple epitope includes two or more epitopes located within a polypeptide molecule.

The formulation of vaccines is generally known in the art and reference can conveniently be made to Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pennsylvania, USA.

A particularly useful form of the vaccine is a recombinant vaccine produced, for example, in a vaccine vector, such as but not limited to a mammalian cell transfected WO 02/038594 PCT/AU01/01462 -38with a vaccinia virus vector, an insect cell transfected with a baculovirus vector, or a bacterial cell transfected with a plasmid or cosmid, the only requirement being that the vector expresses the immunogenic component.

The present invention clearly extends to recombinant vaccine compositions in which the immunogenic component at least is contained within killed vaccine vectors prepared, for example, by heat, formalin or other chemical treatment, electric shock or high or low pressure forces. According to this embodiment, the immunogenic component of the vaccine is generally synthesized in a live vaccine vector which is killed prior to administration to an animal.

Furthermore, the vaccine vector expressing the immunogenic component may be non-pathogenic or attenuated. Within the scope of this embodiment are cells'that have been transfected with non-pathogenic or attenuated viruses encoding the immunogenic component of the vaccine and non-pathogenic or attenuated cells that directly express the immunogenic component.

Attenuated or non-pathogenic host cells include those cells which are not harmful to an animal to which the subject vaccine is administered. As will be known to those skilled in the art, "live vaccines" can comprise an attenuated virus vector encoding the immunogenic component or a host cell comprising same, which is capable of replicating in an animal to which it is administered, and using host cell machinery to express the immunogenic component albeit producing no adverse side-effects therein. Such vaccine vectors may colonise the gut or other organ of the vaccinated animal. Such live vaccine vectors are efficacious by virtue of their ability to continually express the immunogenic component in the host animal for a time and at a level sufficient to confer protective immunity against a pathogen which expresses animmunogenic equivalent of said immunogenic component. The present invention clearly encompasses the use of such attenuated or non-pathogenic vectors and live vaccine preparations.

The vaccine vector may be a virus, bacterial cell or a eukaryotic cell such as an WO 02/038594 PCT/AU01/01462 -39insect, avian, porcine or other mammalian cell or a yeast cell or a cell line such as COS, VERO, HeLa, mouse C127, Chinese hamster ovary (CHO), WI-38, baby hamster kidney (BHK) or MDCK cell lines. Suitable prokaryotic cells include Mycobacterium spp., Corynebacterium spp., Salmonella spp., Escherichia coli, Bacillus spp. and Pseudomonas spp, amongst others. Bacterial strains which are suitable for the present purpose are well-known in the relevant art (Ausubel et al, 1987; Sambrook et al, 1989).

Such cells and cell lines are capable of expression of a genetic sequence encoding a polypeptide of the present invention from L. intracellularis, or a homologue, analogue or derivative thereof, in a manner effective to induce a protective immune response in the animal. For example, a non-pathogenic bacterium can be prepared containing an expression vector which comprises-a nucleotidesequence encoding a polypeptide selected from the group consisting-offlhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, or a homologue, analogue, or derivative thereof, wherein said nucleotide sequence is placed operably under the control of a constitutive or inducible promoter sequence. The bacterium is then permitted to colonise suitable locations in a pig's gut, where it replicates and expresses the said polypeptide in amount sufficient to induce a protective immune response against L.

intracellularis.

In a further altemative embodiment, the vaccine can be a DNA or RNA vaccine comprising a DNA or RNA molecule encoding a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides or homologues, analogues or derivatives thereof, wherein said vaccine is injected into muscular tissue or other suitable tissue in a pig under conditions sufficient to permit transient expression of said DNA or RNA to produce an effective amount of said polypeptide to induce a protective immune response. In a preferred embodiment, the DNA vaccine is in the form of a plasmid, in which the DNA is operably connected with a promoter region capable of expressing the nucleotide sequence encoding the immunogen in cells of the immunized animal.

WO 02/038594 PCT/AU01/01462 In the production of a recombinant vaccine, except for a DNA vaccine described herein, it is therefore necessary to express the immunogenic component in a suitable vector system. For the present purpose, the immunogenic component can be expressed by: placing an isolated nucleic acid molecule in an expressible format, said nucleic acid molecule comprising the coding region of a gene selected from the group consisting of flhB, fliR, ntrC, gInH, motA, motB, tlyC, ytfM, and ytfN genes, or a protein-encoding homologue, analogue or derivative thereof; (ii) introducing the isolated nucleic acid molecule of in an expressible format into a suitable vaccine vector, and (iii) incubating or growing the vaccine vector for a time and under conditions sufficient for expression of the immunogenic component encoded by said nucleic-acid molecule to occur.

It will be apparent from the preceding discussion that the protein-encoding region of a flhB, fliR, ntrC, gInH, motA, motB, tlyC, ytfM, or ytfN gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, and 17, or alternatively or in addition, a protein-encoding nucleotide sequence of L.

intracellularis DNA contained within a deposited plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN).

Preferred homologues of the protein-encoding region of a flhB, fiR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN gene include those nucleotide sequences selected from the group consisting of: a protein-encoding nucleotide sequence having at least about sequence identity overall to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17 or a degenerate variant thereof; WO 02/038594 PCT/AU01/01462 -41- (ii) a protein-encoding nucleotide sequence having at least about sequence identity overall to the protein-encoding sequence of L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motAIB); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); (iii) a protein-encoding nucleotide sequence which comprises at least about 15 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; (iv) a protein-encoding nucleotide sequence which comprises at least about contiguous nucleotides of the protein-encoding sequence of L.

intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NMOO/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); a protein-encoding nucleotide sequence which hybridizes under at least low stringency conditions to the complement of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 17; and (vi) a protein-encoding nucleotide sequence which hybridizes under at least low stringency conditions to the non-coding strand of L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2- flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motAIB); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN).

WO 02/038594 PCT/AU01/01462 -42- The present invention clearly extends to analogues or derivatives of any one of to (vi) which encode a polypeptide which mimics a B-cell or T-cell epitope of Lawsonia spp.

For the present purpose, a preferred homologue of the protein-encoding region of a flhB, fliR, ntrC, gInH, motA, motB, tlyC, ytfM, or ytfN gene will have at least about nucleotide sequence identity to the coding region of said gene, still more preferably at least about 90% identity, and yet still more preferably at least about identity.

In determining whether or not two nucleotide sequences fall within these percentage limits, those skilled in the art will be aware that it is necessary to conduct a side-byside comparison or multiple alignment of sequences. In such comparisons or alignments, differences may arise in the positioning of non-identical residues, depending upon the algorithm used to perform the alignment. In the present context, reference to a percentage identity between two or more nucleotide sequences shall be taken to refer to the number of identical residues between said sequences as determined using any standard algorithm known to those skilled in the art. For example, nucleotide sequences may be aligned and their identity calculated using the BESTFIT programme or other appropriate programme of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America (Devereaux et al, 1984).

Preferably, a homologue of the protein-encoding region of a flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN gene hybridizes under at least medium stringency conditions to the non-coding strand of said gene, even more preferably under high stringency conditions to the non-coding strand of said gene.

For the purposes of defining the level of stringency, a low stringency is defined herein as being a hybridisation and/or a wash carried out in 6xSSC buffer, 0.1% SDS at 28°C. A moderate stringency is defined herein as being a hybridisation and/or washing carried out in 2xSSC buffer, 0.1% SDS at a temperature in the range WO 02/038594 PCT/AU01/01462 -43- 0 C to 65 0 C. A high stringency is defined herein as being a hybridisation and/or wash carried out in 0.1xSSC buffer, 0.1% SDS, or lower salt concentration, and at a temperature of at least 65°C. Reference herein to a particular level of stringency encompasses equivalent conditions using wash/hybridization solutions other than SSC known to those skilled in the art.

Generally, the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and/or wash. Those skilled in the art will be aware that the conditions for hybridisation and/or wash may vary depending upon the nature of the hybridisation membrane or the type of hybridisation probe used. Conditions for hybridisations and washes are well understood by one normally skilled in the art. For the purposes of clarification of the parameters-affecting hybridisation between nucleic acid molecules, reference is found in pages 2.10.8 to 2.10.16. of Ausubel et al.

(1987), which is herein incorporated by reference.

As used herein, a "nucleic acid molecule in an expressible format" is a proteinencoding region of a nucleic acid molecule placed in operable connection with a promoter or other regulatory sequence capable of regulating expression in the vaccine vector system.

Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. In the present context, the term "promoter" is also used to describe a recombinant, synthetic or fusion molecule, or derivative which confers, activates or enhances the expression of a nucleic acid molecule to which it is operably connected, and which encodes the immunogenic polypeptide. Preferred promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or to alter the WO 02/038594 PCT/AU01/01462 -44spatial expression and/or temporal expression of the said nucleic acid molecule.

Placing a nucleic acid molecule under the regulatory control of, "in operable connection with", a promoter sequence means positioning said molecule such that expression is controlled by the promoter sequence. Promoters are generally, but not necessarily, positioned 5' (upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, the gene from which the promoter is derived.

Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene. As is known in the art, some variationin this distance can be accommodated without loss of promoter function.

Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.

The prerequisite for producing intact polypeptides in bacteria such as E. coli is the use of a strong promoter with an effective ribosome binding site. Typical promoters suitable for expression in bacterial cells such as E. coli include, but are not limited to, the lacz promoter, temperature-sensitive AL or AR promoters, T7 promoter or the IPTG-inducible tac promoter. A number of other vector systems for expressing the nucleic acid molecule of the invention in E. coli are well-known in the art and are described, for example, in Ausubel et al (1987) or Sambrook et al (1989). Numerous plasmids with suitable promoter sequences for expression in bacteria and efficient ribosome binding sites have been described, such as for example, pKC30 (k: Shimatake and Rosenberg, 1981); pKK173-3 (tac: Amann and Brosius, 1985), pET-3 (T7: Studier and Moffat, 1986); the pBAD/TOPO or pBAD/Thio-TOPO series of vectors containing an arabinose-inducible promoter (Invitrogen, Carlsbad, CA), the latter of which is designed to also produce fusion proteins with thioredoxin to enhance solubility of the expressed protein; the pFLEX series of expression vectors (Pfizer WO 02/038594 PCT/AU01/01462 Inc., CT, USA); or the pQE series of expression vectors (Qiagen, CA), amongst others. Typical promoters suitable for expression in viruses of eukaryotic cells and eukaryotic cells include the SV40 late promoter, SV40 early promoter and cytomegalovirus (CMV) promoter, CMV IE (cytomegalovirus immediate early) promoter amongst others.

Means for introducing the isolated nucleic acid molecule or a genetic construct comprising same into a cell for expression of the immunogenic component of the vaccine composition are well-known to those skilled in the art. The technique used for a given organism depends on the known successful techniques. Means for introducing recombinant DNA into animal cells include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation and microparticle bombardment such as by using DNAcoated tungsten or gold particles (Agracetus Inc., WI, USA) amongst others.

The immunogenic component of a vaccine composition as contemplated herein exhibits excellent therapeutic activity, for example, in the treatment and/or prophylaxis of PPE when administered in an amount which depends on the particular case. For example, for recombinant polypeptide molecules, from about 0.5 ug to about 20 mg may be administered, preferably from about 1 yg to about 10 mg, more preferably from about 10 Ag to about 5 mg, and most preferably from about 50 yg to about 1 mg equivalent of the immunogenic component in a volume of about 1ml to about For DNA vaccines, a preferred amount is from about 0.1 /g/ml to about 5 mg/ml in a volume of about 1 to about 5 ml. The DNA can be present in "naked" form or it can be administered together with an agent facilitating cellular uptake in liposomes or cationic lipids). The important feature is to administer sufficient immunogen to induce a protective immune response. The above amounts can be administered as stated or calculated per kilogram of body weight. Dosage regime can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation. Booster administration may also be required.

WO 02/038594 PCT/AU01/01462 -46- The vaccine of the present invention can further comprise one or more additional immunomodulatory components such as, for example, an adjuvant or cytokine molecule, amongst others, that is capable of increasing the immune response against the immunogenic component. Non-limiting examples of adjuvants that can be used in the vaccine of the present invention include the RIBI adjuvant system (Ribi Inc., Hamilton, MT, USA), alum, mineral gels such as aluminium hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as, for example, Block co-polymer (CytRx, Atlanta GA, USA),QS-21 (Cambridge Biotech Inc., Cambridge MA, USA), SAF-M (Chiron, Emeryville CA, USA), AMPHIGEN" adjuvant, Freund's complete adjuvant; Freund's incomplete adjuvant; and Saponin, QuilA or other saponin fraction, monophosphoryl lipid A, and Avridine lipid-amine adjuvant. Other immunomodulatory agents that can be included in the vaccine include, for example, one or more cytokines, such as interferon and/or interleukin, or other known cytokines- Non-ionic surfactants such as, for example, polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether may also be included in the vaccines of the present invention.

The vaccine composition can be administered in a convenient manner such as by oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or by implantation using slow release technology), provided that a sufficient degree of the immunogenicity of the immunizing antigen is retained for the purposes of eliciting an immune response in the animal being treated. Depending on the route of administration, the immunogenic component may be required to be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate it, such as those in the digestive tract.

The vaccine composition may also be administered parenterally or intraperitoneally.

Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, or in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.

Alternatively, the vaccine composition can be stored in lyophilised form to be rehydrated with an appropriate vehicle or carrier prior to use.

WO 02/038594 PCT/AUO1/01462 -47- Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be fluid to the extent that easy syringability exists, unless the pharmaceutical form is a solid or semi-solid such as when slow release technology is employed. In any event, it must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms.

The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use-of-a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents such as, for example,, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents such as, for example,, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption such as, for example,, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter-sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which'contains the basic dispersion medium and the required other ingredients selected from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

WO 02/038594 PCT/AU01/01462 -48- The present invention extends to vaccine compositions which confer protection against infection by one or more isolates or sub-types of L. intracellularis including those that belong to the same serovar or serogroup as L. intracellularis. The vaccine composition preferably also confers protection against infection by other species of the genus Lawsonia or other microorganisms related thereto, as determined at the nucleotide, biochemical, structural, physiological and/or immunointeractive level; the only requirement being that said other species or other microorganism expresses a polypeptide which is immunologically cross-reactive to a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, or a homologue, analogue or derivative of any one or more of said polypeptides as described herein. For example, such related microorganisms may comprise genomic DNA which is at least about 70% identical overall to the genomic DNA of L. intracellularis as determined using standard genomic DNA hybridisation and analysis techniques.

The terms "serogroup" and "serovar" relate to a classification of microorganisms which is based upon serological typing data, in particular data obtained using agglutination assays such as the microscopic agglutination test (MAT). Those skilled in the art will be aware that serovar and serogroup antigens are a mosaic on the cell surface and, as a consequence there will be no strict delineation between bacteria belonging to a serovar and/or serogroup. Moreover, organisms which belong to different species may be classified into the same serovar or serogroup because they are indistinguishable by antigenic determination. As used herein, the term "serovar" means one or more Lawsonia strains which are antigenically-identical with respect to antigenic determinants produced by one or more loci. Quantitatively, serovars may be differentiated from one another by cross-agglutination absorption techniques. As used herein, the term "serogroup" refers to a group of Lawsonia spp. whose members cross-agglutinate with shared group antigens and do not cross-agglutinate with the members of other groups and, as a consequence, the members of a serogroup have more or less close antigenic relations with one another by simple cross-agglutination.

WO 02/038594 PCT/AU01/01462 -49- The present invention thus clearly extends to vaccine compositions for the treatment and/or prophylaxis of animals, in particular, vaccine compositions for the treatment and/or prophylaxis of porcine and/or avian species, against any bacterium belonging to the same serovar or serogroup as L. intracellularis. Preferably, such organisms will express a polypeptide homologue, analogue or derivative of a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides.

The present invention extends further to vaccine compositions capable of conferring protection against a "genetic variant" of L. intracellularis, the only requirement being that said variant expresses a polypeptide which is immunologically cross-reactive to a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides. Genetic variants of L. intracellularis can be developed by mutation, recombination, conjugation or transformation of L.

intracellularis or may occur naturally. It will be known to a person skilled in the art how to produce such derivatives.

In a particularly preferred embodiment, the vaccine composition of the invention is intended for or suitable for the prophylaxis and/or treatment of infection in a porcine or avian animal and more preferably, for prophylaxis and/or treatment of a porcine animal for infection by L. intracellularis.

Accordingly, the present invention clearly extends to the use of the immunogenic polypeptide of the invention or a DNA or RNA molecule encoding the same, according to any one of the preceding embodiments or as exemplified herein in the preparation of a medicament for the treatment and/or prophylaxis of PPE in animals, particularly porcine or avian animals.

The invention further extends to a method of treatment and/or prophylaxis of PPE in an animal such as an avian or porcine animal, said method comprising administering the vaccine composition or the immunogenic polypeptide of the invention or a DNA WO 02/038594 PCT/AU01/01462 or RNA molecule encoding the same, as described or exemplified herein to said animal for a time and under conditions sufficient for an immune response to occur thereto. Preferably, in the case of administration of a vaccine composition, the immune response to the immunogen is a protective immune response.

Those skilled in the art will recognise the general applicability of the invention in vaccinating animals other than porcine and avian animals against L. intracellularis and/or related microorganisms. In the general application of the vaccine of the present invention, the only prerequisite is that the animal on which protection is conferred is capable of being infected with L. intracellularis and/or a related microorganism thereto and that, in the case of a related microorganism to L.

intracellularis, said related microorganism expresses a B-cell or T-cell epitope which mimics or cross-reacts with the polypeptide component of the-vaccire-composition described herein. Animals which may be protected by the vaccine of the present invention include, but are not limited to, humans, primates, companion animals cats, dogs), livestock animals pigs, sheep, cattle, horses, donkeys, goats), laboratory test animals mice, rats, guinea pigs, rabbits) and captive wild animals kangaroos, foxes, deer). The present invention also extends to the vaccination of birds such as poultry birds, game birds and caged birds.

The present invention further extends to combination vaccines comprising an effective amount of a first immunogenic component comprising a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, or a homologue, analogue or derivative thereof as described herein, or a DNA or RNA molecule encoding the same, combined with an effective amount of a second immunogenic component comprising one or more other antigens capable of protecting a porcine animal, or bird, against either Lawsonia spp. or another pathogen that infects and causes disease in said animal. The second immunogenic component is different from the first immunogenic component and is preferably selected from the group consisting of the L. intracellularis FIgE, hemolysin, OmpH, SodC, flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides and homologues, analogues or derivatives thereof. The present invention clearly WO 02/038594 PCT/AU01/01462 -51extends to DNA vaccines and vaccine vectors which express said first immunogenic component and said second immunogenic component.

It is within the scope of the invention to encompass vaccine compositions comprising multimeric and polymeric forms of any one or more of the immunogenic polypeptides described herein, such as tandem arrays of homologous amino acid sequences, or, altematively, tandem arrays of heterologous immunogenic repeats of amino acid sequences. The present invention extends further to nucleic acid molecules encoding such polymeric forms.

The isolated or recombinant polypeptide of the invention, or an immunologicallyequivalent homologue, analogue or derivative thereof is also useful for the preparation of immunologically--interactive molecules which are useful in the diagnosis of infection-of arr animal by Lawsonia spp., in particular by L. intracellularis or a related organism thereto.

As used herein, the term "immunologically interactive molecule" includes antibodies and antibody derivatives and functional equivalents, such as a Fab, or a SCAB (single-chain antibody), any of which optionally can be conjugated to an enzyme, radioactive or fluorescent tag, amongst others. The only requirement of such immunologically interactive molecules is that they are capable of binding specifically to the immunogenic polypeptide of the present invention as hereinbefore described.

Accordingly, a further aspect of the invention extends to an immunologically interactive molecule which is capable of binding to a polypeptide selected from the group consisting of: a polypeptide which comprises an amino acid sequence which has at least about 60% sequence identity overall to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; (ii) a polypeptide which comprises an amino acid sequence which has at least about 60% sequence identity overall to an amino acid sequence encoded by L. intracellularis DNA contained within a plasmid selected from the WO 02/038594 PCT/AU01/01462 -52group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 gInH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); (iii) a polypeptide which comprises at least about 5 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; (iv) a polypeptide which comprises at least about 5 contiguous amino acids of an amino acid sequence encoded by L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); a polypeptide which comprises an amino acid sequence encoded by a nucleotide sequence having at least about 60% sequence identity overall to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; (vi) a polypeptide which comprises an amino acid sequence encoded by a nucleotide sequence having at least about 60% sequence identity overall to the nucleotide sequence of L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fiR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); (vii) a polypeptide encoded by at least about 15 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5,7, 9,11, 13,15, and 17; (viii) a polypeptide encoded by at least about 15 contiguous nucleotides of WO 02/038594 PCT/AU01/01462 -53a nucleotide sequence of L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); and (ix) a homologue, analogue or derivative of any one of to (viii) which mimics a B-cell or T-cell epitope of Lawsonia spp.

In a preferred embodiment, the immunologically interactive molecule is an antibody that binds specifically to one or more epitopes of a polypeptide selected from the group consisting of flhB, fliR, ntrC, ginH, motA, 'motB, tlyC, ytfM, and ytfN polypeptides. More preferably, the immunologically interactive molecule binds specifically to one or more epitopes of a polypeptide from a causative agent of PPE, such as, for example, L. intracellularis.

Conventional methods can be used to prepare the immunologically interactive molecules. For example, by using a polypeptide immunogen of the present invention, polyclonal antisera or monoclonal antibodies can be made using standard methods.

For example, a mammal, a mouse, hamster, or rabbit) can be immunized with an immunogenic form of the polypeptide of the present invention which elicits an antibody response in the mammal. Techniques for conferring immunogenicity on a polypeptide include conjugation to carriers, or other techniques well known in the art.

For example, the polypeptide can be administered in the presence of adjuvant or can be coupled to a carrier molecule, as known in the art, that enhances the immunogenicity of the polypeptide. The progress of immunization can be monitored by detection of antibody titres in plasma or serum. Standard ELISA or other immunoassay can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained and, for example, IgG molecules corresponding to the polyclonal antibodies can be isolated from the antisera.

WO 02/038594 PCT/AU01/01462 -54- To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an animal immunised with a polypeptide of the present invention and fused with myeloma cells by standard somatic cell fusion procedures, thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art, for example, the hybridoma technique originally developed by Kohler and Milstein (1975), as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., 1983), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985), and screening of combinatorial antibody libraries (Huse et al., 1989). Hybridoma cells can be isolated and screened immunochemically for production of antibodies that are specifically reactive with the polypeptide and monoclonal antibodies isolated therefrom.

As with all immunogenic compositions for eliciting antibodies-the immunogenically effective amounts of the peptides of the invention must-be determined empirically.

Factors to be considered include the immunogenicity of the native polypeptide, whether or not the polypeptide will be complexed with or covalently attached to an adjuvant or carrier protein or other carrier, the route of administration for the composition, intravenous, intramuscular, subcutaneous, etc., and the number of immunizing doses to be administered. Such factors are known in the vaccine art and it is well within the skill of immunologists to make such determinations without undue experimentation.

The term "antibody" as used herein, is intended to include fragments thereof which are also specifically reactive with a polypeptide that mimics or cross-reacts with a Bcell or T-cell epitope of the L. intracellularis polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides.

Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments.

WO 02/038594 PCT/AU01/01462 It is within the scope of this invention to include any secondary antibodies (monoclonal, polyclonal or fragments of antibodies), including anti-idiotypic antibodies, directed to the first mentioned antibodies discussed above. Both the first and second antibodies can be used in detection assays or a first antibody can be used with a commercially available anti-immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of a polypeptide which mimics, or cross-reacts with a B-cell or T-cell epitope of a L. intracellularis polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides.

The antibodies described herein are useful for determining B-cell or T-cell epitopes of a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, andytfN-polypeptides, such as, for example, by testing the ability of synthetic peptides to cross-react immunologically with said polypeptide or to elicit the production of antibodies which cross-react with said polypeptide. Using methods described herein, polyclonal antibodies, monoclonal antibodies or chimeric monoclonal antibodies can also be raised to peptides which mimic or cross-react with a B-cell or T-cell epitope of a L. intracellularis polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides.

More particularly, the polyclonal, monoclonal or chimeric monoclonal antibodies can be used to detect the polypeptide of the invention and/or any homologues, analogues or derivatives thereof, in various biological materials. For example, they can be used in an ELISA, radioimmunoassay, or histochemical test. In other words, the antibodies can be used to test for binding to a polypeptide of the invention or to a homologue, analogue or derivative thereof, in a biological sample to diagnose the presence of L.

intracellularis therein.

Accordingly, a further aspect of the invention provides a method of diagnosing infection of an animal by L. intracellularis or a related microorganism thereto, said method comprising the steps of contacting a biological sample derived from said animal with an immunologically interactive molecule which is capable of binding to WO 02/038594 PCT/AU01/01462 -56a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, or a hdmologue, analogue or derivative thereof, for a time and under conditions sufficient for an antigen:antibody complex to form, and detecting said complex formation.

According to this embodiment of the present invention, the immunologically interactive molecule is preferably an antibody molecule prepared against a L.

intracellularis polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, or an analogue or derivative thereof.

If the biological sample being tested contains one or more epitopes of a polypeptide selected from the group consisting of flhB, fliR, ntrC, gInH, motA, motB, tlyC, ytfM, and ytfN polypeptides, or an immunologically cross-reactive homologue, analogue or derivative thereof, it will give a positive binding result to the immunologically interactive molecule.

Preferably, the biological sample is derived from a porcine or avian host of the pathogen L. intracellularis or a related microorganism thereto, and includes an appropriate tissue or fluid sample from the animal.

Preferred biological samples are derived from the ileum, caecum, small intestine, large intestine, whole serum or lymph nodes of the porcine or avian host animal being tested. Alternatively or in addition the biological test sample may comprise faeces or a rectal swab derived from the animal.

To distinguish L. intracellularis from other microorganisms resident in the gut or other organ of an animal, the antibodies should not be prepared against highly-conserved epitopes of the L. intracellularis polypeptide, such as, for example, those amino acid sequences of at least 5 amino acids in length which are conserved between L.

intracellularis and a microorganism which is present in the gut or other organ of an animal in respect of which diagnosis is sought such as, for example, E.coli.

WO 02/038594 PCT/AU01/01462 -57- Conventional immunoassays can be used to perform this embodiment of the invention. A wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These, of course, include both single-site and two-site or "sandwich" assays of the non-competitive types, as well as the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target. It will be readily apparent to the skilled technician how to modify or optimise such assays to perform this embodiment of the present invention, and all such modifications and optimisations are encompassed by the present invention.

In one altemrnative embodiment, the present invention contemplates a method of identifying whether or not an animal has suffered from a past infection, or is currently infected with L. intracellularis or a related microorganism thereto, said methodcomprising contacting blood or serum derived from said animal with the immunogenic polypeptide of the invention for a time and under conditions sufficient for an antigen:antibody complex to form, and detecting said complex formation. This embodiment differs from the embodiment described supra in that it relies upon the detection of circulating antibodies against L. intracellularis or related organism in the animals blood or serum which are present as a consequence of a past or present infection by this pathogen. However, it will be apparent to those skilled in the art that the principle of the assay format is the same. As with other embodiments of the invention referred to supra, conventional immunoassays can be used. Persons skilled in the art will readily be capable of varying known immunoassay formats to perform the present embodiment. This embodiment of the invention can also utilise derivatives of blood and serum which comprise immunologically interactive molecules such as, for example, partially-purified IgG or IgM fractions and buffy coat samples, amongst others. The preparation of such fractions will also be known to those skilled inthe art.

A further aspect of the present invention provides an isolated nucleic acid molecule which comprises a sequence of nucleotides that encodes, or is complementary to a nucleic acid molecule that encodes a polypeptide selected from the group consisting WO 02/038594 PCT/AU01/01462 -58of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, including any and all genes selected from the group consisting of flhB, fliR, ntrC, glhH, motA, motB, tlyC, ytfM, and ytfN genes as defined hereinabove.

In a preferred embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide that is immunologically cross-reactive with L.

intracellularis or other causative agent of PPE, wherein said nucleotide sequence is selected from the group consisting of: a nucleotide sequence having at least about 60% sequence identity overall to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1,3, 5, 7, 9, 11, 13, 15, and 17; (ii) a nucleotide sequence having at least about 60% sequence identity overall to L. intracellularis DNA contained within-a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); (iii) a nucleotide sequence which comprises at least about 15 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; (iv) a nucleotide sequence which comprises at least about 15 contiguous nucleotides of L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); a nucleotide sequence which hybridizes under at least low stringency conditions to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,15, and 17 or a complementary nucleotide sequence thereto; WO 02/038594 PCT/AU01/01462 -59- (vi) a nucleotide sequence which hybridizes under at least low stringency conditions to L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motAB); NM00/16480 (plasmid tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); and (vii) a homologue, analogue or derivative of any one of to (vi) which encodes a polypeptide which mimics a B-cell or T-cell epitope of Lawsonia spp.

For the present purpose, a "homologue" of a nucleotide sequence shall be taken to refer to an isolated nucleic acid molecule which encodes .a polypeptide that is immunologically cross-reactive to a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, but which includes one or more nucleotide substitutions, insertions, deletions, or rearrangements.

An "analogue" of a nucleotide sequence set forth herein shall be taken to refer to an isolated nucleic acid molecule which encodes a polypeptide which is immunologically cross-reactive to a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, but which includes one or more non-nucleotide constituents not normally present in said isolated nucleic acid molecule, such as, for example, carbohydrates, radiochemicals including radio nucleotides, reporter molecules such as, but not limited to biotin, DIG, alkaline phosphatase or horseradish peroxidase, amongst others.

A "derivative" of a nucleotide sequence set forth herein shall be taken to refer to any isolated nucleic acid molecule which contains at least about 60% nucleotide sequence identity to 15 or more contiguous nucleotides present in the nucleotide sequence of a gene selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN genes.

WO 02/038594 PCT/AU01/01462 Generally, a flhB, fliR, ntrC, gInH, motA, motB, tlyC, ytfM, or ytfN gene may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or insertions. Nucleotide insertional derivatives include 5' and 3' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides or nucleotide analogues. Insertional nucleotide sequence variants are those in which one or more nucleotides or nucleotide analogues are introduced into a predetermined site in the nucleotide sequence of the gene, although random insertion is also possible with suitable screening of the resulting product being performed. Deletional nucleotide sequence variants are characterised by the removal of one or more nucleotides from the gene. Substitutional nucleotide sequence variants are those in which at least one nucleotide in the gene sequence has been removed and a different nucleotide or nucleotide analogue inserted in its place. In a preferred embodiment, such substitutions are selected based on the degeneracy of the genetic code, as known in the art, with the resulting substitutional variant encoding the amino acid sequence of a flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN polypeptide.

Preferred homologues, analogues and derivatives of a flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN gene comprise a sequence of nucleotides which has at least about 80%identity, even more preferably at least about 90% identity, and yet still more preferably at least about 95% identity to said gene.

In determining whether or not two nucleotide sequences fall within these percentage limits, reference is made to the description supra of methods for conducting a sideby-side comparison or multiple alignment of nucleotide sequences.

Alternatively or in addition, preferred homologues, analogues and derivatives of a flhB, fliR, ntrC, gInH, motA, motB, tlyC, ytfM, or ytfN gene comprise a sequence of nucleotides which hybridizes under at least moderate stringency conditions and to the nucleotide sequence of said gene, or to a nucleic acid fragment comprising at least about 20 contiguous nucleotides in length derived therefrom, and even more preferably, under high stringency conditions to said gene, or to said nucleic acid WO 02/038594 PCT/AU01/01462 -61fragment. For the purposes of defining the level of stringency, reference is made to the description hereinabove of hybridization stringencies.

In a more preferred embodiment, such a nucleotide sequence encodes a polypeptide that is immunologically cross-reactive with L. intracellularis or other causative agent of PPE.

In a particularly preferred embodiment, the isolated nucleic acid molecule of the present invention comprises or consists of a nucleotide sequence selected from the group consisting of: a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1,3, 5, 7, 9, 11, 13, 15, and 17; (ii) a nucleotide sequence of the L. intracellularis DNA contained within a deposited plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); (iii) a nucleotide sequence that encodes the same polypeptide as or (ii), wherein said polypeptide is selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides; and (iv) a nucleotide sequence that is complementary to or (ii) or (iii).

The present invention clearly encompasses genetic constructs comprising the subject nucleic acid molecule in an expressible format suitable for the preparation of a recombinant immunogenic polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, such as for use in recombinant univalent or polyvalent recombinant vaccines.

In such cases, the nucleic acid molecule will be operably connected to a promoter sequence which can thereby regulate expression of said nucleic acid molecule in a WO 02/038594 PCT/AU01/01462 -62prokaryotic or eukaryotic cell as described supra.

The genetic construct optionally further comprises a terminator sequence. The term "terminator" refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. A "terminator" is a nucleotide sequence, generally located within the 3'-non-translated region of a gene or mRNA, comprising a polyadenylation signal to facilitate the post-transcriptional addition of a polyadenylate sequence to the 3'-end of a primary mRNA transcript. Terminator sequences may be isolated from the genetic sequences of bacteria, fungi, viruses, animals and/or plants. Terminators active in animal cells are known and described in the literature.

In a preferred embodiment, the genetic construct can be a cloning or expression vector, as known in the art, such as a plasmid, cosmid, or phage, comprising a nucleic acid molecule of the present invention, and host cells transformed or transfected therewith. In a non-limiting embodiment, the vector is a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN).

The genetic constructs of the present invention are particularly useful for producing the immunogenic component of the vaccine composition described herein or for use in a DNA vaccine.

A range of genetic diagnostic assays to detect infection of an animal by L.

intracellularis or a related microorganism can be employed using the nucleic acid molecule described herein such as, for example, assays based upon the polymerase chain reaction (PCR) and nucleic acid hybridisation. All such assays are contemplated in the present invention.

WO 02/038594 PCT/AU01/01462 -63- Accordingly, a still further aspect of the invention provides a diagnostic method of detecting L. intracellularis or related microorganism in a biological sample derived from an animal subject, said method comprising the steps of hybridising one or more probes or primers derived from a nucleotide sequence of a flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN gene as defined hereinabove, or a homologue, analogue or derivative thereof, to a DNA or RNA molecule present in said sample and then detecting said hybridisation using a detection means.

As used herein, the term "probe" refers to a nucleic acid molecule which is capable of being used in the detection of a gene selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN genes. Probes may comprise DNA (single-stranded or double-stranded) or RNA riboprobes) or analogues thereof.

The term "primer" refers to a probe as hereinbefore defined which is further capable of being used to amplify a nucleotide sequence from L. intracellularis or a related microorganism thereto in a PCR.

Preferred probes and primers include fragments of a gene selected from the group consisting of flhB, fliR, ntrC, gInH, motA, motB, tlyC, ytfM, and ytfN genes, including synthetic single-stranded DNA or RNA molecules of at least about 15 nucleotides in length.

Preferably, probes and primers according to this embodiment will comprise at least about 20 contiguous nucleotides in length from a gene selected from the group consisting of flhB, fliR, ntrC, gInH, motA, motB, tlyC, yffM, and ytfN genes, even more preferably at least about 25 contiguous nucleotides, still even more preferably at least about 50 contiguods nucleotides, and even more preferably at least about 100 nucleotides to about 500 nucleotides in length from said gene. Probes and primers comprising the full-length gene or a complementary nucleotide sequence thereto are also encompassed by the present invention.

Probes or primers can comprise inosine, adenine, guanine, thymidine, cytidine or WO 02/038594 PCT/AU01/01462 -64uracil residues or functional analogues or derivatives thereof that are capable of being incorporated into a polynucleotide molecule, provided that the resulting probe or primer is capable of hybridising under at least low stringency conditions to a gene selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN genes, or is at least about 60% identical to one strand of said gene.

The biological sample according to this aspect of the invention includes any organ, tissue, cell or exudate which contains or is likely to contain L. intracellularis or a nucleic acid derived therefrom. A biological sample can be prepared in a suitable solution such as, for example, an extraction buffer or suspension buffer. The present invention extends to the testing of biological solutions thus prepared, the only requirement being that said solution at least comprises a biological sample as described herein.

The diagnostic assay of the present invention is useful for the detection of L.

intracellularis or a microorganism which is related thereto which expresses a polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides.

The present invention clearly contemplates diagnostic assays which are capable of both genus-specific and species-specific detection. Accordingly, in one embodiment, the probe or primer, or a homologue, analogue or derivative thereof, comprises DNA capable of being used to detect multiple Lawsonia spp. In an alternative embodiment, the probe or primer or a homologue, analogue or derivative thereof comprises DNA capable of being used to distinguish L. intracellularis from related microorganisms.

Less-highly conserved regions within the flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN genes are_particularly useful as species-specific probes and/or primers for the detection of L. intracellularis and very closely related species.

Furthermore, the diagnostic assays described herein can be adapted to a genusspecific or species-specific assay by varying the stringency of the hybridisation step.

WO 02/038594 PCT/AU01/01462 Accordingly, a low stringency hybridisation can be used to detect several different species of Lawsonia in one or more biological samples being assayed, while a high stringency hybridisation can be used to distinguish L. intracellularis from such other species.

The detection means according to this aspect of the invention may be any nucleic acid-based detection means such as, for example, nucleic acid hybridisation techniques or paper chromatography hybridisation assay (PACHA), or an amplification reaction such as PCR, or nucleic acid sequence-based amplification (NASBA) system. The invention further encompasses the use of different assay formats of said nucleic acid-based detection means, including restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), single-strand chain polymorphism (SSCP), amplification-and-mismatch detection (AMD), interspersed repetitive sequence polymerase chain reaction (IRS-PCR), inverse polymerase chain reaction (iPCR), in situ polymerase chain reaction and reverse transcription polymerase chain reaction (RT-PCR), amongst others.

Where the detection means is a nucleic acid hybridisation technique, the probe can be labelled with a reporter molecule capable of producing an identifiable signal a radioisotope such as 32 P or 35S, or a biotinylated molecule). According to this embodiment, those skilled in the art will be aware that the detection of said reporter molecule provides for identification of the probe and that, following the hybridisation reaction, the detection of the corresponding nucleotide sequences in the biological sample is facilitated. Additional probes can be used to confirm the assay results obtained using a single probe.

A variation of the nucleic acid hybridisation technique contemplated by the present invention is the paper chromatography hybridisation assay (PACHA) described by Reinhartz et al. (1993) and equivalents thereof, wherein a target nucleic acid molecule is labelled with a reporter molecule such as biotin, applied to one end of a nitrocellulose or nylon membrane filter strip and subjected to chromatography under the action of capillary or other forces an electric field) for a time and under WO 02/038594 PCT/AU01/01462 -66conditions sufficient to promote migration of said target nucleic acid along the length of said membrane to a zone at which a DNA probe is immobilised thereto such as, for example, in the middle region. According to this detection format, labelled target nucleic acid comprising the Lawsonia spp. nucleotide sequences complementary to the probe will hybridise thereto and become immobilised in that region of the membrane to which the probe is bound. Non-complementary sequences to the probe will diffuse past the site at which the probe is bound. The target nucleic acid may comprise a crude or partially-pure extract of DNA or RNA or, alteratively, an amplified or purified DNA. Additional variations of this detection means which utilise the nucleotide sequences described herein are clearly encompassed by the present invention.

Wherein the-detection-means is a RFLP, nucleic acid derived from the biological sample, in particular DNA, is digested with one or more restriction endonuclease enzymes and the digested DNA is subjected to electrophoresis, transferred to a solid support such as, for example, a nylon or nitrocellulose membrane, and hybridised to a probe optionally labelled with a reporter molecule as hereinbefore defined.

According to this embodiment, a specific pattern of DNA fragments is displayed on the support, wherein said pattem is preferably specific for a particular Lawsonia spp., to enable the user to distinguish between different species of the bacterium.

Wherein the detection means is an amplification reaction such as, for example, a polymerase chain reaction or a nucleic acid sequence-based amplification (NASBA) system or a variant thereof, one or more nucleic acid primer molecules of at least contiguous nucleotides in length derivable from a gene selected from the group consisting of flhB, fliR, ntrC, gInH, motA, motB, tlyC, ytfM, and ytfN genes is hybridised to nucleic acid derived from a biological sample, and nucleic acid copies -of the FIgE-encoding genetic sequences in said.sample, or a part or fragment thereof, are enzymically-amplified.

Those skilled in the art will be aware that there must be a sufficiently high percentage of nucleotide sequence identity between the primers and the sequences in the WO 02/038594 PCT/AU01/01462 -67biological sample template molecule to which they hybridise the "template molecule"). As stated previously, the stringency conditions can be selected to promote hybridisation.

Preferably, each primer is at least about 95% identical to a region of a gene selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN genes in the template molecule to which it hybridises.

Those skilled in the art will also be aware that, in one format, PCR provides for the hybridisation of non-complementary primers to different strands of the template molecule, such that the hybridised primers are positioned to facilitate the 3' synthesis of nucleic acid in the intervening region, under the control of a thermostable DNA polymerase enzyme. As a consequence, PCR provides an advantage over other detection means in so far as the nucleotide sequence in the region between the hybridised primers may be unknown and unrelated to any known nucleotide sequence.

In an alternative embodiment, wherein the detection means is AFLP, the primers are selected such that, when nucleic acid derived from the biological sample, in particular DNA, is amplified, different length amplification products are produced from different Lawsonia spp. The amplification products can be subjected to electrophoresis, transferred to a solid support such as, for example, a nylon or nitrocellulose membrane, and hybridised to a probe optionally labelled with a reporter molecule as hereinbefore described. According to this embodiment, a specific pattern of amplified DNA fragments is displayed on the support, said pattern optionally specific for a particular Lawsonia ssp., to enable the user to distinguish between different species of the bacterium in much the same way as for RFLP analysis.

The technique of AMD facilitates, not only the detection of Lawsonia spp. DNA in a biological sample, but also the determination of nucleotide sequence variants which differ from the primers and probes used in the assay format. Wherein the detection means is AMD, the probe is end-labelled with a suitable reporter molecule and mixed WO 02/038594 PCT/AU01/01462 -68with an excess of the amplified template molecule. The mixtures are subsequently denatured and allowed to renature to form nucleic acid "probe:template hybrid molecules" or "hybrids", such that any nucleotide sequence variation between the probe and the temple molecule to which it is hybridised will disrupt base-pairing in the hybrids. These regions of mismatch are sensitive to specific chemical modification using hydroxylamine (mismatched cytosine residues) or osmium tetroxide (mismatched thymidine residues), allowing subsequent cleavage of the modified site using piperidine. The cleaved nucleic acid may be analysed using denaturing polyacrylamide gel electrophoresis, followed by standard nucleic acid hybridisation as described supra, to detect the Lawsonia-derived nucleotide sequences. Those skilled in the art will be aware of the means of end-labelling a genetic probe according to the performance of the invention described in this embodiment.

According to this embodiment, the use of a single end-labelled probe allows unequivocal localisation of the sequence variation. The distance between the point(s) of sequence variation and the end-label is represented by the size of the cleavage product.

In an alternative embodiment of AMD, the probe is labelled at both ends with a reporter molecule, to facilitate the simultaneous analysis of both DNA strands.

Wherein the detection means is RT-PCR, the nucleic acid sample comprises an RNA molecule which is a transcription product of Lawsonia-derived DNA or a homologue, analogue or derivative thereof. As a consequence, this assay format is particularly useful when it is desirable to determine expression of one or more Lawsonia genes.

According to this embodiment, the RNA sample is reverse-transcribed to produce the complementary single-stranded DNA which is subsequently amplified using standard procedures.

Variations of the embodiments described herein are described in detail by McPherson et al. (1991).

WO 02/038594 PCT/AU01/01462 -69 The present invention clearly extends to the use. of any and all detection means referred to supra for the purposes of diagnosing Lawsonia spp. and in particular L.

intracellularis infection in animals.

The amplification reaction detection means described supra can be further coupled to a classical hybridisation reaction detection means to further enhance sensitivity and specificity of the inventive method, such as by hybridising the amplified DNA with a probe which is different from any of the primers used in the amplification reaction.

Similarly, the hybridisation reaction detection means described supra can be further coupled to a second hybridisation step employing a probe which is different from the probe used in the first hybridisation reaction.

A further aspect of the invention provides an isolated probe or primer derived from a gene selected from the group consisting of flhB, fiR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN genes. Preferably, the probe or primer of the invention comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 19 to SEQ ID NO: 68 or a complementary nucleotide sequence thereto.

The present invention does not extend to any nucleic acid or polypeptide of Camplylobacter or Helicobacter that was disclosed publicly before the filing date or priority date of this application, or otherwise takes priority over the instant application, and which is homologous to a nucleotide sequence or amino acid sequence of Lawsonia spp. disclosed herein.

The present invention is further described with reference to the following non-limiting Examples.

EXAMPLE 1 Molecular Cloning of Lawsonia intracellularis genes Isolation of DNA and construction of DNA libraries L. intracellularis DNA was purified from pig intestinal mucosa isolated from the ileum WO 02/038594 PCT/AU01/01462 70 of pigs experimentally infected with L. intracellularis. DNA purification from homogenized intestinal mucosa was performed according to the method of Nollau et al. (1996); or alternatively, by phenol extraction and sodium acetate-ethanol precipitation of DNA.

To facilitate cloning of L. intracellularis gene sequences, several genomic libraries were constructed. These libraries were specifically modified by ligation of a known sequence (Vectorette II

T

Genosys Biotechnologies, Inc., The Woodlands, TX) to the and ends of restricted DNA fragments. Vectorette T libraries were constructed by separately digesting aliquots of L. intracellularis-infected pig mucosal DNA extract with restriction endonucleases Hindlll, EcoRI, Dral or Hpal at 37°C overnight. The reaction was then spiked with additional fresh restriction enzyme and adjusted to 2 mM ATP, 2 mM DTT final concentration. Vectorette T tailing was carried out by addition of T 4 DNA Ligase (1 unit) plus 3 pMol of the appropriate compatible Vectorette T M linker (Hindill VectoretteTM: Hindlll-digested DNA; EcoRl: EcoRI digested DNA; Blunt: Dral-, Hpal- digested DNA). The mixture was incubated for three cycles, each cycle consisting of 20°C for 60 min; followed by 37°C for min, to complete the tailing reaction. Reaction volumes were then adjusted to 200 1l with water, and reactions were stored at WO 02/038594 PCT/AU01/01462 -71- EXAMPLE 2 Expression of the YtfN and YtfM genes of L. Intracellularis i) Isolation of a C-terminal fragment of ytfN gene by genome walking The complete sequence of the L. intracellularis YffN gene was determined from genomic DNA and is set forth herein as, SEQ ID NO: 17. Based upon the 2,035 bp sequence obtained for the amino terminal portion of the ytfN gene fragment, oligonucleotide primer KWK-Li-YtfN-4C (SEQ ID NO:29) was designed and synthesized (Life Technologies; Rockville, MD). This oligonucleotide binds within the 3'-region of the YffN gene in the L. intracellularis chromosome to allow amplification of DNA downstream of the existing gene fragment. For polymerase chain amplification, primer KWK-Li-YtfN-4C (SEQ ID NO:29) was used in combination with a Vectorette T M specific oligonucleotide primer (ER70; SEQ ID NO:108) in 50 1l reactions containing 1x PCR Buffer II (Perkin Elmer, Foster City, CA), 2.0 mM MgCI 2 250 pjM each deoxy-NTP, 50 pMol each primer, and 2.5 U AmpliTaq T M Gold (Perkin Elmer) thermostable polymerase. Reactions were performed with 1 pl of the Vectorette T libraries as DNA template. Amplification was carried out as follows: denaturation (94°C, 9 min); 40 cycles of denaturation (94 0 C, 30 sec), annealing 0 C, 30 sec), and polymerization (72°C, 4.0 min); this was followed by a final extension at 72°C for 7 minutes.

The amplified products were visualized by separation on a 1.0% agarose gel (Sigma; St. Louis, Mo). Screening of the Hpal library by PCR resulted in amplification of a fragment approximately 1.5 kb in length. The PCR product was purified using a QIAquick T M PCR Purification kit (Qiagen; Valencia, CA) and cloned into the TA cloning site of pCR2.1-TOPO (Invitrogen; Carlsbad, CA); the ligated product was transformed into Max Efficiency E. coli DH5a cells (Life Technologies; Rockville, MD).

Sequence analysis of the cloned fragment failed to identify a termination codon for ytfN. Therefore, oligonucleotide- primer KWK-Li-YtfN-12C (SEQ ID NO:21) was designed and synthesized to be used in a second round of PCR amplification using the Vectorette libraries. The above-mentioned conditions for amplification were used, and products were visualized by agarose gel electrophoresis. A fragment approximately 1.6 kb in length was amplified from the Dral library. The PCR product WO 02/038594 PCT/AU01/01462 -72was purified using a QIAquick PCR Purification kit, cloned into pCR2.1-TOPO, and subsequently transformed into Max Efficiency E. coli DH5a cells. Sequence analysis of the fragment identified a terminal TAA codon indicating the end of the ytfN gene.

ii) Determination of genomic sequence of complete ytfN gene Results from the preliminary sequencing described above were used to design oligonucleotide primers for the specific amplification of two overlapping ytfN gene fragments directly from L. intracellularis chromosomal DNA. These products encompass the entire ytfN gene and were sequenced directly in an attempt to avoid introduction of sequence artifacts due to mutations which might arise during PCR amplification and subsequent cloning steps. To obtain the first of the two fragments, PCR amplifications were carried out in triplicate and contained 100 pMol of primers YtfN-D (SEQ. ID NO:45) and YtfN-U (SEQ. ID NO:46), 100 ng purified chromosomal DNA, 1x PC2 buffer (Ab Peptides; St. Louis, MO), 200 uM each dNTP, 15 U KlenTaqi (Ab Peptides) and 0.3 U cloned Pfu (Stratagene; La Jolla, CA) thermostable polymerases in a 100 /l final sample volume. Conditions for amplification consisted of denaturation (94°C, 9 min), followed by 40 cycles of denaturation (94 0 C, 30 sec), annealing (60 0 C, 30 sec), and polymerization (72*C, min), and a final extension at 72°C for 7 min. To obtain the second (overlapping) fragment, PCR amplifications were carried out in triplicate as described above, except that primers KWK-Li-YtfN-12C (SEQ ID NO:21) and KWK-LI-YtfN-15N (SEQ ID NO:24) were used. Conditions for amplification consisted of denaturation (940C, 9 min), followed by 40 cycles of denaturation (94°C, 30 sec), annealing (55°C, sec), and polymerization (72 0 C, 2.5 min), and a final extension at 72 0 C for 7 min.

Following amplification, each set of triplicate samples was pooled and the specific product from each was purified (QIAquick T PCR Purification kit). Both purified DNA Sfragments were then subjected to direct sequence analysis using DyeDeoxy termination reactions on an ABI automated DNA sequencer (Lark Technologies Inc., Houston, TX).

Synthetic oligonucleotide primers (SEQ ID NOs:21, 24, 26-38, 43-46, 51-53, and WO 02/038594 PCT/AU01/01462 -73were used to sequence both DNA strands of the amplified products.

The ytfN ORF extends from nucleotides 1-4149 of SEQ ID NO:17 and encodes a 1382 amino acid protein (SEQ ID NO:18), having a theoretical molecular weight of 150,887 Daltons. The sequence of the amino terminus of the encoded protein resembles a prokaryotic signal sequence (von Heijne, 1985; Nielsen, et al., 1997), although the precise site of cleavage is not presently known. The ytfN ORF was compared against existing nucleotide and protein databases using the Basic Local Alignment Search Tool (BLAST) programs (Altschul, et al., 1990). The entry with which it shared the greatest homology was a hypothetical 40.5 kDa protein from Zymomonas mobilis. The second-most-significant homologous sequence identified was a YtfN homolog from Neisseria meningitidis.

iii) Cloning of recombinantytfM gene into expression vectors For the purpose of recombinant protein expression, both the ytfM and ytfN genes or fragments thereof were cloned without the sequences encoding their respective signal peptides.

The ytfM gene was amplified from L. intracellularis chromosomal DNA using oligonucleotide primers RA202-b (SEQ ID NO: 50) and RA201-b (SEQ ID NO: 49).

For polymerase chain amplification, triplicate 50 ul reactions were set up with eachcontaining 100 ng of chromosomal DNA as template, lx PC2 buffer, 200 JM each dNTP, 50 pMol each primer, 7.5 U KlenTaql and 0.15 U cloned Pfu thermostable polymerases. Amplification was carried out as follows: denaturation (94°C, 9 min); 40 cycles of denaturation (94°C, 30 sec), annealing (60°C, 30 sec), and polymerization (72°C, 2.5 min), followed by a final extension at 72°C for 7 minutes. Following amplification, the samples were purified (QIAquick M

PCR

-Purification kit)-and pooled. The purified PCR product was cloned directly into the TA cloning site of both pBAD-TOPO and pBAD/Thio-TOPO (Invitrogen). The ligated products were transformed into Max Efficiency E. coli DH5a cells. The predicted amino terminal sequence of the encoded protein expressed from pBAD-TOPO:YtfM would consist of the vector-encoded sequence MGSGSGDDDDKLALLTM (SEQ ID WO 02/038594 PCT/AU01/01462 -74- NO: 61) followed immediately by the sequence ATSITTS (SEQ ID NO: 62) beginning at Alanine-24 of the YtfM ORF (SEQ ID NO:16). A clone containing the appropriate plasmid was identified, and purified plasmid was isolated from a small-scale broth culture using a QIAprep Spin Miniprep kit (Qiagen). This plasmid was transformed into E. coli BL21 (Novagen; Madison, WI) and BL21-CodonPlus-RIL cells (Stratagene); a clone was identified in each strain that contained the appropriate plasmid.

The predicted amino terminal sequence of the encoded fusion protein expressed from pBAD/Thio-TOPO:YtfM would consist of the thioredoxin protein and a 15 amino acid residue linker followed immediately by the sequence ATSITTS (SEQ ID NO: 62) beginning at Alanine-24 of the YtfM ORF (SEQ ID NO:16). A clone containing the appropriate plasmid was identified, and purified plasmid was isolated from a smallscale broth culture using a QIAprep Spin Miniprep kit. This plasmid was transformed into E. coi BL21 and BL21-CodonPlus-RIL cells; a clone was identified in each strain that contained the appropriate plasmid.

For cloning into pET-30a, the purified PCR product encoding YtfM was digested with BamHI and Ncol, then purified using a QIAquick T M PCR Purification kit. pET-30a was also digested with BamHI and Ncol; the linearized plasmid was purified using a JETsorb' T kit (Genomed; Frederick, MD) prior to ligation. The ligated product was transformed into Max Efficiency E. coli DH5a cells. The predicted amino terminal sequence of the encoded fusion protein expressed from pET-30a:YtfM would consist of MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAM (SEQ ID NO: 63) encoded by the vector, followed by the sequence ATSITTS (SEQ ID NO: 62) beginning at Alanine-24 of the YtfM ORF (SEQ ID NO:16). A clone containing the appropriate plasmid was identified, and purified plasmid was isolated from a smallscale broth culture using a QIAprep Spin Miniprep kit. This plasmid was transformedinto E. coli BL21(DE3) (Novagen) and BL21-CodonPlus(DE3)-RIL cells (Stratagene); a clone was identified in each strain that contained the appropriate plasmid.

The ytfM gene was also amplified from L. intracellularis chromosomal DNA by PCR WO 02/038594 PCT/AU01/01462 amplification using oligonucleotide primers RA200 (SEQ ID NO: 47) and RA201 (SEQ ID NO: 48). Duplicate 50 yl reactions were set up each containing 100 ng of chromosomal DNA as template, lx PC2 buffer, 200 pM each dNTP, 50 pMol each primer, 7.5 U KlenTaql and 0.15 U cloned Pfu thermostable polymerases.

Amplification was carried out as follows: denaturation (94°C, 9 min); 30 cycles of denaturation (94*C, 30 sec), annealing (60°C, 30 sec), and polymerization (72*C, 2 min), followed by a final extension at 72°C, for 7 minutes. Following amplification, the samples were purified (QIAquick T PCR Purification kit) and pooled. The purified PCR product was cloned directly into the TA cloning site of pCR2.1-TOPO. The ligated product was transformed into Max Efficiency E. coli DH5ca cells. A clone containing the appropriate plasmid was identified, propagated, and plasmid DNA was isolated using a QIAprep Spin Miniprep kit. Following digestion of the plasmid with EcoRI, a fragment corresponding to bp 437 of SEQ ID NO:15 to the EcoRI site in the MCS of pCR2.1-TOPO was purified using a JETsorb T M kit. pET-30a was also digested with EcoRI, and purified using a QIAquick PCR T Purification kit. The two fragments were ligated and transformed into Max Efficiency E. coil DH5a cells. The predicted amino terminal sequence of the encoded fusion protein would consist of MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGS (SEQ ID NO: 64) encoded by the vector followed by the sequence EFNLSKG (SEQ ID NO: 65) beginning at Aspartate-146 of the YtfM ORF (SEQ ID NO:16). A clone containing the plasmid with the gene fragment inserted in the proper orientation was identified, and purified plasmid was isolated from a small-scale broth culture using a QIAprep.

Spin Miniprep kit. This plasmid was transformed into E. coli BL21-CodonPlus(DE3)- RIL cells; a clone was identified that contained the appropriate plasmid.

iv) Cloning of recombinant ytfN gene into expression vectors The 5' half of the ytfN gene, excluding that encoding the signal sequence, was amplified from L. intracellularis chromosomal DNA using oligonucleotide primers RA205-b (SEQ ID NO: 53) and RA204-b (SEQ ID NO: 52). For polymerase chain amplification, triplicate 100 pl reactions were set up each containing 100 ng of chromosomal DNA as template, lx PC2 buffer, 200 pM each dNTP, 100 pMol each primer, 15 U KlenTaqi and 0.3 U cloned Pfu thermostable polymerases.

WO 02/038594 PCT/AU01/01462 -76- Amplification was carried out as follows: denaturation (94°C, 9 min); 40 cycles of denaturation (94*C, 30 sec), annealing (60°C, 30 sec), and polymerization (72*C, 4 min), followed by a final extension at 72°C for 7 minutes. Following amplification, the samples were purified (QlAquick T PCR Purification kit) and pooled. The purified PCR product was cloned directly into the TA cloning site of both pBAD-TOPO and pBAD/Thio-TOPO (Invitrogen). The ligated products were transformed into Max Efficiency E. coli DH5a cells. The predicted amino terminal sequence of the encoded protein expressed from pBAD-TOPO:YtfN would consist of the vector-encoded sequence MGSGSGDDDDKLALGHM (SEQ ID NO: 66) followed immediately by the sequence RTSTGIA (SEQ ID NO: 67) beginning at Arginine-33 of the YtfN ORF (SEQ ID NO:18). A clone containing the appropriate plasmid was identified, and purified plasmid was isolated from a small-scale broth culture using a QIAprep Spin Miniprep kit. This plasmid was transformed into E. coli BL21-CodonPlus-RIL cells; a clone was identified that contained the appropriate plasmid.

The predicted amino terminal sequence of the encoded protein expressed from pBAD/Thio-TOPO:YtfN would consist of the thioredoxin protein and a 15 amino acid linker followed immediately by the sequence RTSTGIA (SEQ ID NO: 67) beginning at Arginine-33 of the YtfN ORF (SEQ ID NO:18). A clone containing the appropriate plasmid was identified, and purified plasmid was isolated from a small-scale broth culture using a QIAprep Spin Miniprep kit. This plasmid was transformed into E. coli BL21-CodonPlus-RIL cells; a clone was identified that contained the appropriate plasmid.

For cloning into pET-30a, the purified PCR product was digested with BamHI and Ndel and extracted using a QlAquick PCR Purification kit. The linearized plasmid was purified using a JETsorb T kit prior to ligation. The ligated product was transformed into Max Efficiency E. coli DH5a cells. The predicted amino terminal sequence of the protein expressed from pET-30a:YtfN would consist of Met encoded by RA205-b (SEQ ID NO: 53) followed by the sequence RTSTGIA (SEQ ID NO: 67) beginning at Arginine-33 of the YtfN ORF (SEQ ID NO:18). A clone containing the appropriate plasmid was identified, and purified plasmid was isolated from a small- WO 02/038594 PCT/AU01/01462 -77scale broth culture using a QIAprep Spin Miniprep kit. This plasmid was transformed into E. coli BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP cells (Stratagene); a clone was identified in each strain that contained the appropriate plasmid.

Utilizing oligonucleotide primers RA205-b (SEQ ID NO: 53) and KWK-Li-YtfN-Bglll-3' (SEQ ID NO:39), an approximate 1kb fragment encoding the N-terminal portion of YtfN, excluding the signal sequence, was amplified by PCR. Triplicate 50 pl reactions were set up each containing 100 ng of chromosomal DNA as template, 1x PC2 buffer, 200 pM each dNTP, 50 pMol each primer, 7.5 U KlenTaql and 0.15 U cloned Pfu thermostable polymerases. Amplification was carried out as follows: denaturation (94 0 C, 9 min); 40 cycles of denaturation (94 0 C, 30 sec), annealing 0 C, 30 sec), and polymerization (72°C, 1 min); followed by a final extension at 72°C for 7 minutes. Following amplification, the samples were purified (QIAquick

T

PCR Purification kit) and pooled. The purified PCR product was cloned directly into the TA cloning site of both pBAD-TOPO and pBAD/Thio-TOPO. The ligated products were transformed into Max Efficiency E. coi DH5a cells. The predicted amino terminal sequence of the protein expressed from pBAD-TOPO would consist of the vector-encoded sequence MGSGSGDDDDKLALGHM (SEQ ID NO: 66) followed immediately by the sequence RTSTGIA (SEQ ID NO: 67) beginning at Arginine-33 of the YtfN ORF (SEQ ID NO:18); the protein would terminate with lsoleucine-332 of ytfN (SEQ ID NO:18). A clone containing the appropriate plasmid was identified, and purified plasmid was isolated from a small-scale broth culture using a QIAprep Spin Miniprep kit. This plasmid was transformed into E. coli BL21-CodonPlus-RIL cells; a clone was identified that contained the appropriate plasmid.

The predicted amino terminal sequence of the encoded fusion protein expressed from pBAD/Thio-TOPO:YtfN would consist of the thioredoxin protein and a 15 amino acid linker followed immediately by the sequence RTSTGIA (SEQ ID NO: 67) beginning at Arginine-33 of the YtfN ORF (SEQ ID NO:18); again, the polypeptide would terminate with lsoleucine-332 of ytfN (SEQ ID NO:18). A clone containing the appropriate plasmid was identified, and purified plasmid was isolated from a small- WO 02/038594 PCT/AU01/01462 -78scale broth culture using a QIAprep Spin Miniprep kit. This plasmid was transformed into E. coil BL21-CodonPlus-RIL cells; a clone was identified that contained the appropriate plasmid.

In order to generate a construct consisting of the above-described 1 kb to Bgll site) ytfN fragment in pET30a, the plasmid pET-30a:YtfN containing the 5' half of ytfN amplified using RA205-b (SEQ ID NO: 53). and RA204-b (SEQ ID NO: 52) was digested with Bgll and BamHI, thus excising the gene sequence downstream of the Bgll site within ytfN. The fragment containing the vector and ytfN sequence up to the Bgll site was purified using a JETsorbm kit, and the remaining fragment was religated and transformed into E. coli Max Efficiency DH5a cells. The predicted amino terminal sequence of the protein expressed would consist of Met encoded by RA205-b (SEQ ID NO: 53) followed by the sequence RTSTGIA (SEQ ID NO: 67) beginning at Arginine-33 of the YtfN ORF (SEQ ID NO:18). The polypeptide would terminate with lsoleucine-332 of SEQ ID NO:18, followed by a C-terminal extension consisting of DPNSSSVDKLAAALEHHHHHH (SEQ ID NO: 68) encoded by the vector. A clone containing the appropriate plasmid was identified, and purified plasmid was isolated from a small-scale broth culture using a QIAprep Spin Miniprep kit (Qiagen). This plasmid was transformed into E. coil BL21(DE3) and BL21- CodonPlus(DE3)-RIL cells; a clone was identified in each strain that contained the appropriate plasmid. Stocks of the clone containing this plasmid in E. coil BL21 (DE3) were frozen at -80 0

C.

v) Expression of Recombinant YtfN Polypeptide Frozen working stock of the E. coli BL21 (DE3) transformant harboring containing the 5' portion of ytfN up to the Bgfll site (eg. corresponding to amino acids 33-332 of the encoded YtfN protein) was thawed and seeded at a 1:5000 dilution in RWLDM/G vi defined medium [K 2

HPO

4 (6 KH 2

PO

4 (3 (NH 4 2

SO

4 (5 g/L), NaCI (2 0.2 mL CaCI 2 (15 0.4 mL FeCI 3 .6H 2 0 (5 0.4 ml MgS04.7H 2 0 (480 ZnCI2 (6.5 MnSO 4

.H

2 0 (12 Na 2 MoO 4 .2H 2 0 (5 CuSO 4 CoCI 2 .6H 2 0 (2 H 3

BO

3 (0.5 and 37% HCI Kanamycin was also added to a concentration of 25 .g/ml to maintain the expression plasmid. The P\OPERUMSMSPECIFICATIONS\I4810-02.DOC- 2/2/OS -79culture was grown under fed-batch (50% glycerol) in a 5 liter working volume BioFlow 3000 fermentor (New Brunswick Scientific; Edison, NJ) at 37 0 C until A 625 was 2.9.

At this time, IPTG was added to 0.1 mM, and culture samples were collected at 0, 2.25, and 3 hours post induction to monitor expression of recombinant YtfN (see Figure). The primary culture was maintained 28 hours post inoculation then immediately chilled, and wet cells were collected by centrifugation and stored at 0 C. Data showing expression of ytfN protein are presented in Figure 1.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

S. 15 The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

*g ooo** ooo o 0~ •go• WO 02/038594 PCT/AU01/01462

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EDITORIAL NOTE APPLICATION NUMBER 14810/02 The following Sequence Listing pages 1 to 56 are part of the description. The claims pages follow on pages 83 to 96.

WO 02/038594 WO 02/38594PCT/AU01/01462 SEQUENCE LISTING <110> Australian Pork Limited AND Agriculture Victoria Services Pty Ltd AND Pfizer Products Inc.

<120> Novel therapeutic compositions for treating infection by Lawsonia spp.

<130> <150> AU PR1381 <151> 2000-11-10 <150> US 60/249596 <151> 2000-11-17 <160> 68 <170> Patentln Ver. <210> 1 <211> 622 <212> DNA <213> Lawsonia intraceliularis <220> <221> CDS <222> .(621) <400> 1 atg tct Met Ser 1 gat gat ccc agt aaa aca gag aaa gca acc ccg aaa cga cgt Asp Asp Pro Ser Lys Thr Glu Lys Ala Thr Pro Lys Arg Arg cag gas gct cgt tct gs ggg agt gtc cct aaa tca gaa Gin Glu Ala Arg Ser Glu Gly Ser Val Pro Lys Ser Glu gag gtt Glu Val aaa gca ttg Lys Ala Leu act act gca gca Thr Thr Ala Ala atg ctg ggg ctt Met Leu Gly Leu att tat tca Ile Tyr Ser ggc gta atg gga cgt cat ttt gaa aca att ttc tac tat att ttt aca Phe Glu Thr Ile Phe Tyr Tyr Ile Phe Thr Gly Val s0 Met Gly Arg His gaa tca tt~t cgg ttt gag gtt aca gca cag tca gta tat gct tta ttt Glu Ser Phe Arg Phe Giu Val Thr Ala Gin Ser Val Tyr Ala Leu Phe WO 02/038594 WO 02/38594PCT/AUOI/01462 att tat gtt gct caa gag Ile Tyr Val Ala Gin Giu ata gct att tta ttg atg Ile Ala Ile Le~i Leu Met 90 cca ata tta ctt Pro Ile Leu Leu caa gtt ggt gca Gin Vai Gly Ala 110 ttt att gct gtt Phe Ile Ala Vai 100 acg gca tgg att Thr Aia Trp Ile tta cgt gta ILeu Arg Vai tta tgg act aca aag gtt ttt Leu Trp Thr Thr Lys Val Phe 115 ttt aaa tgg agt Phe Lys Trp Ser gct tct caa caa Ala Ser Gin Gin ttt aat.ata Phe Aen Ile ata aaa ggg Ile Lys Giy 130 ttg aaa gga atg ttt Leu Lye Gly Met Phe 135 aca ctt gtt cga Thr Leu Val Arg ctt Leu 145 tta cgt agt tta Leu Arg Ser Leu gtt caa gta att gtt ata ggt att gtt cca Val Gin Vai Ile Val Ile Gly Ile Vai Pro 150 155 atg att ata aaa gga gag ttt tca aac ttt tta cca tta Met Ilie Ile Lys Giy GlU Phe Ser Aen Phe Leu Pro Leu 16S 170 tat tat Tyr Tyr 175 agt cct tca ggt gtg gca Ser Pro Ser Giy Vai Aia 180 gtt tta tat acg cta att Val Leu Tyr Thr Leu Ilie 195 gat tat atg ctt aat aca Asp Tyr Met Leu Asn Thr 185 gga ata gta ctt Giy Ile Val Leu 190 gtc gca gat c Val Ala Asp 205 cct atg Pro Met 200 aca att att gca Thr Ile Ile Ala <210> 2 <211> 207 <212> PRT <213> Lawsonia intraceilularis <400> 2 Met Ser Asp Asp Pro 5cr Lys Thr 1 5 Gin Giu Aia Arg Ser Giu Gly Ser Giu Lye Ala Thr Pro Lye Arg Arg 10 1s Vai Pro Lye Ser Glu Giu Vai Thr WO 02/038594 WO 02/38594PCT/AU01/01462 Lys Ala tLeu Thr Thr Ala Ala Gly Met 40 Gly -Glu Ile Val Met so Ser Phe Tyr Val Phe Ile Ala Leu Trp Thr 115 Ilie Lys Gly 130 Leu Leu Arg 145 Met Ile Ile Gly Arg Arg Phe Ala Gin Val Thr 100 Thr Lys Leu Lys Ser Leu Lys Gly His Phe Giu 55 Giu Val Thr 70 Giu Ile Ala Ala Trp Ile Val Phe Lys 120 Gly Met Phe 135 Val Gin Val 150 Glu Phe Ser Ala Asp Tyr Ile Pro Met 200 -3- Leu Gly Leu Ala Ile Phe Tyr Tyr Gin Ser Val Tyr Leu Leu Met Pro 90 Leu Arg Vai G.in Lys Trp Ser Lys i2 Ser Gin Gin Thr 140 Val Ile Gly Ile 155 Phe Leu Pro Leu 170 Leu Asn Thr Gly Ile Ile Ala Val 205 Ile Tyr Ile Phe Ala Leu Ile Leu Val Gly 110 Phe Asn Ser Val 165 Pro Ser Gly Vai 180 Leu Tyr Thr Leu 195 <210> 3 <211> 789 <212> DNA <213> Lawsonia intracellularis <220> <221> CDS <222> <400> 3 atg aat tta ttt aat ttt gat cct agt atg ttt ctt agt ttt tta ctt 48 WO 02/038594 WO 02/38594PCT/AUOI/01462 -4- Met ABn Leu Phe Aen Phe Asp Pro Ser Met Phe Leu Ser Phe Leu Leu aca ttt tta Cgt att agt get gtC tta, Phe Leu Arg Ilie Ser Vai Val Leu .25 ate gat ggt ttt cct aat aeg tea aaa Ile Asp Gly Phe Pro Asn Met Leu Lys ttt atg ctt cct Phe Met Leu Pro gca. tca ata gct Ala Ser Ile Ala ett tt Cct Phe Phe Ser cte ate cta Leu Ile Leu ace ata Thr Ile gtt cCC egg ggg cgt cCC cCC cCC Val Leu Trp Gly Arg Leu Ser Leu 55 eca gga Ser Gly aca caa. aeg cca.

Thr Gin Met Pro 9c9 cat CCt tCC gat Ala His Pro Phe ASP gta ttg tta atc Val Leu Leu Ile agc gag gte ttt Ser Giu Val Phe ggt att gta etg ggg cCC gcg gta aac tCC tcC ttt gca gga Giy Ile Val Leu. Gly Leu Ala Vai Asn Phe Phe Phe Aia Giy gct ggg gga Ala Gly Giy att cte gCe Ile Leu Ala aca caa Thr Gin 105 atg ggg ttt Met Gly Phe aca atg att acg Thr Mee Ile Thr 110 CCC gca gac cca. tta ace Leu Aia Asp Pro Leu Thr 115 ggt aac Gly Asn 120 acc aca gge CCC Thr Thr Gly Phe gca cat CCC Ala His Phe cCC Cat Leu Tyr 130 atg gte gct aca eta get ttt cCC gCt ctt aat ggc cat teg Met Val Aia Thr Leu Val Phe Leu Ala Leu Asn Gly His Leu tCt CCC ata aaa gct Phe Leu Ile LYe Aia 145 aca tat act tt aaa aeg get cca. gca Thr Tyr Thr Phe Lye Met Val Pro Ala 155 gga Giy 160 gga CCC gtt gea -aga gaa att tea. ttg agt gaa~Ctt ctt aat Giy Leu Val Val Arg Giu Ile Leu Leu Ser Giu Leu Leu Asn atg gca Met Ala 17S ggg aeg att eec Gly Met Ile Phe 180 get ttt gcc tea cat gte gcg gca cCa Val Phe Ala Leu His Vai Ala Ala Pro 185 get atg eca Val Met Ser 190 576 WO 02/038594 WO 02/38594PCT/AU01/01462 gct ctt ttt Ala Leu Phe 195 tta gta gag Leu Val Glu atc tct tta Ile Ser Leu 200 gga ctt atg Gly Leu Met gca aga gct gct Ala Axg Ala Ala 205 aaa att ggt gta Lys Ile Gly Val cct cag att cat att atg gaa gtt gga ttt cct Pro Gin Ile His Ile Met Glu Val Gly Phe Pro 672 gga ttt ttt ttc att Gly Phe Plie Phe Ile 225 tat cga ttt att gca Tyr Arg Phe Ile Ala cta tta ttt act Leu Leu Phe Thr tta tca aaa gaa Leu Ser Lys Glu ggc cta gag gga Gly Leu Giu Gly ttt ttt aac tta Phe Phe Asn Leu ctt act Leu Thr 255 gta atg ggt Val Met Gly agt gga aaa tag Ser Gly Lys 260 <210> 4 <c211> 262 <212> PRT <213> Lawsonia intraceliularis <400> 4 Met Asn 1 Leu Phe Asn Phe Asp Pro Ser Met Phe Leu Ser Phe Ijeu Leu Thr Phe Leu Arg Ile Ser Val Val Leu Phe Met Leu Pro Phe Phe Ser Ile Asp Gly Phe Pro Asn Met Leu Lys Ala Ser Ile Ala Leu Ile Leu Thr Ile Val Leu Trp Gly Arg Leu Ser Leu Ser Gly Thr Gin Met Pro Ala His Pro Phe Asp Leu Val Leu Leu Ile Ile Ser Glu Val Phe Leu Ile Gin Gly Ile Val Leu Gly Leu Ala Val Asn Phe Phe Phe Ala Gly 85 90 WO 02/038594 WO 02/38594PCT/AU01/01462 -6- Ala Gly Gly Giu Ile Leu Ala Thr Gin Met Gly Phe Thr Met Ile Thr inn lOS 110 Leu Ala Asp Pro Leu Thr Gly Asn Thr Thr Gly Phe Ile Ala His Phe 125 teu Tyr Met Val Ala Thr Leu Val Phe Leu Ala Leu Asn Gly His Leu 130 135 140 Phe Lev Ile Lys Ala Phe Thr Tyr Thr Phe Lys Met Val Pro Ala Gly 145 I50 155 160 Gly Leu Vai Val Arg Glu Ilie Leu Leu 165 Glu Leu Leu Asn Met Ala 175 Gly Met Ile Phe Vai Phe Ala Leu His Val Ala Ala Pro 180 185 Val Met Ser 190 Arg Ala Ala Ala Lev Phe 195 Leu Val Glu Ile Ser Leu Giy Leu Met 200 Pro Gin Ile His Ile Met Glu 210 215 Val Gly Phe Pro Val 220 Lys Ile Gly Val Gly Phe 225 Phe Phe Ile Giy Leu Leu Phe Thr Ile Leu Ser Lys Glu Tyr Arg Phe Ile Ala Gly Leu Glu Gly 245 Val Met Gly Ser Gly Lys 260 <210> c211> 1371 <212> DNA <213> Lawsonia intraceliularis Phe Phe Asn Leu Leu Thr 255 <220> <221> <222>

CDS

(1)..(1368) <400> atg tca gca cgt ata ctt att ata gat gat gaa gac tct att aga ttt 48 Met Ser Ala Arg Ile Leu Ile Ile Asp Asp Giu Asp Ser Ile Arg Phe WO 02/038594 WO 02/38594PCT/AU01/01462 ca ttg aaa. gga att ttt gaa gat gag ggc cat gaa gtt tta gaa aga 96 Ser Leu Lys Gly Ile Phe Giu Asp Giu Gly His Giu Val Leu Glu Arg gct tca gca gaa gaa gga ctt aag tgt gtt Ala Ser Ala Glu Giu Gly Leu Lys Cys Val gat gta gag tct cca gat 144 Asp Val Glu Ser Pro Asp ctt gtt ttt Leu Val Phe ctt gat att tgg ctt cct ggg atg gat ggt ctt atg gct Leu Asp Ile Trp Leu Pro Gly Met Asp Giy Leu Met Ala 192 tta.

Leu gac cat att cag gct ctt cat cag gaa tta cct gtt att Asp His Ile Gin Ala Leu His Gin Giu Leu Pro Val Ile atg att Met Ile caa. ggt Gin Gly tca ggt cat gcc aca att gaa act gct gta aca gct atc cgt Ser Giy His Ala Thr Ile Giu Thr Ala Val Thr Ala Ile Arg gct tat gat Ala Tyr Asp att gas sag cct Ile Giu Lys Pro tct ttg gas *aas Ser Leu Giu Lys gtc ctt att Val Leu Ile 110 336 aca gct aat ags gct ata ga Thr Ala Asn Arg Ala Ile Glu 115 gts aga tts aga agg gsa aac aa Val Arg Leu Arg Arg Giu Asn Lys 125 384 tts cta Leu Leu 130 cgt act gta tta cct gag gag agt gag ttt ata gga cag tct Arg Thr Val Leu Pro Glu Giu Ser Glu Phe Ile Gly Gin Ser 432 cct gtt atc tta aaa Pro Val Ile Leu Lys aaa agt tta tta tca cag gtc gct cca Lys Ser Leu Leu Ser Gin Val Ala Pro 155 gat gct tgg gts cta ctt aca gga gag aat ggt sca ggt aaa Asp Ala Trp -Val Leu Leu Thr Gly Glu Asn Gly Thr Gly Lys gct gca caa gca ttg cac aas. gga Ala Ala Gin Ala Leu His Lys Gly 180 tca cga tat caa Ser Arg Tyr Gin aaa. cca ttt Lys Pro Phe 190 WO 02/038594 WO 02/38594PCT/AU01/01462 ata gct gtt aat Ile Ala Val Asn 195 tge gct gct atc cct Cys Ala Ala Ile Pro 200 gaa gaa ttg att Glu Giu Leu Ile 205 gaa agc gaa Giu Ser Giu cta tt Leu Phe 210 ggt cat gaa aaa ggg 9CC ttt act ggt gcc gat gct tct cyt Gly His Glu Lys Gly Ala Phe Thr Gly Ala Asp Ala Ser Arg 215 220 gca.

Ala 225 ggt cgt tee gag Gly Arg Phe Giu teg gca Leu Ala 230 cat aaa. gga aca tta ttt ctt His Lys Gly Thr Leu Phe Leu 235 gat ga Asp Giu 240 att ttg Ile Leu 255 ata gga gat atg age eta. aas aca caa Ilie Gly Asp Met Ser Leu Lys Thr Gin 245 gca aaa Ala Lys 250 att ttg cgt Ile Leu Arg caa gas caa Gin Giu Gin gat gta aga Asp Val Arg 275 ttt gaa aaa. at Phe Glu Lys Ile ggt age get aga Gly Ser Vai Arg 265 ast aag aat ctt Asn Lys Asn Leu act ate ass gt Thr Ilie Lys Val 270 gtt aet gca gca Val Ile Ala Ala gas gac 9cC Giu Asp Ala 285 agc gat Ser Asp 290 gga. aca. tC cgt Gly Thr Phe Arg gat teg tat Cat Asp Leu Tyr Tyr eta cga get gt Leu Arg Val Val cca Pro 305 eeg cat ctt ccc Leu His Leu Pro ctt cgt gaa cgt Leu Arg Glu Arg tct gae att gag Ser Asp Ile Giu tea eta aae agg Leu Leu Aen Arg geg ate cag teg Val Ile Gln Leu aaa cgt Cat aga Lys Arg Tyr Arg 960 1008 1056 ccg cct ate Pro Pro Ile tea gat gag gtc Ctc cct gta ttg aaa Leu Asp Giu Val Phe Pro Val. Leu Lys 345 caa Cat tgt Gin Tyr Cys 350 cga aeg get Arg Met Val tgg cca ggg sat Trp Pro Giy Asn 355 aga gaa eta rg Giu Leu 360 ctt aae tee gta Leu Asn Phe Val 1104 ate ctc tat tca ggg sag aaa gta ege teg aca gat ccc sag gta aaa Ile Leu Tyr Ser Gly Lye Lys Val Cys Leu Thr Asp Pro Lye Val Lye 1152 WO 02/038594 PCT/AU01/01462 370 375 age Ser 385 aat tta aaa tat Asn Leu Lys Tyr tta ccc Leu Pro 390 aag aaa ttt Lys Lys Phe tct tcc Ser Ser 395 cat tat aac His Tyr Aen 1200 ctt ccc gat ata Leu Pro Asp Ile ttt tta act gaa Phe Leu Thr Glu 420 ttt aac cag get aaa ata get ttt gaa Phe Asn Gin Ala Lye Ile Ala Phe Glu 410 1248 aaa tta cat get Lys Leu His Ala caa gga aat att Gin Gly Asn Ile acc cga tta Thr Arg Leu 430 aag cta aaa Lys Leu Lye 1296 gca gaa get att gga ctt gaa Ala Glu Ala Ile Gly Leu Glu 435 agt tat tta tat aga Ser Tyr Leu Tyr Arg 445 1344 age tat Ser Tyr 450 ggt att tat ctg tct gag tga Gly Ile Tyr Leu Ser Glu 455 1371 <210> 6 <211> 456 <212> PRT <213> Lawsonia intracellularis <400> 6 Met Ser Ala 1 Arg Ile Leu Ile Ile Asp Asp Glu Asp Ser Ile Arg Phe Ser Leu Lys Gly Ile Phe Glu Asp Glu Gly His Glu Val Leu Glu Arg Ser Pro Asp Ala Ser Ala Glu Glu Gly Leu Lys Cys Val Asp 40 Val Glu Leu Val Phe Leu Asp Ile Trp Leu Pro Gly Met Asp Gly Leu Met Ala S 50 .55 Leu Asp His Ile Gin Ala Leu His Gin Glu Leu Pro Val Ile Met Ile Ser Gly His Ala Thr Ile Glu Thr Ala Val Thr Ala Ile Arg Gin Gly WO 02/038594 WO 02/38594PCT/AUOI/01462 Ala Tyr Asp Phe 100 Thr Ala Asn Arg 115 Leu Leu Arg Thr 130 Pro Val Ile Leu 145 Asp Ala T-p Val Ala Ala Gin Ala 180 Ilie Ala Val Asn 195 Leu Phe Gly His 210 Ala Gly Arg Phe 225 Ile Gly Asp Met Gin Glu Gln Cys 260 Asp Val Arg Val 275 Ser Asp Gly Thr 290 Pro Leu His Leu 305 Ile Glu Ala Ile Val Leu Lys Phe iso Leu Leu 165 Leu His Cys Ala Giu Lys Giu Leu 230 Ser Leu 245 Phe Glu Ile Ala Phe Arg Pro Pro 310 Leu beu Ser 155 Giu Asn Gly 170 Ser Ser Arg 185 Pro Glu Glu Phe Thr Gly Lys Gly Thr 235 Gin Ala Lys 250 Gly Ser Val 265 Asn Lys Asn Leu Tyr Tyr Glu Arg Asp 315 Leu Leu Asn Arg Val Ile Gin Leu Ser Lye Arg Tyr Arg 330 Arg Glu 335 WO 02/038594 WO 02/38594PCT/AUO 1/0 1462 11 Pro Pro Ile Phe Leu Asp Giu Val Phe Pro Val Leu Lys Gin Tyr Cys 350 Trp Pro Gly Asn 355 Val Arg Giu Leu Asn Phe Val Giv Arg Met Vai 365 Ile Leu 370 Tyr Ser Gly Lys Val Cys Leu Thr Asp 380 Ser Ser 395 Pro Lye Val Lys Ser 385 Asn Leu Lys Tyr Pro Lys Lys Phe His Tyr Asn Leu Pro Asp Ile Asp Phe Asn Gin Ala Lys 405 410 Ile Ala Phe Giu Pro Lys 415 Phe Leu Thr Aia Giu Ala 435 Giu Lys Leu His Ala Tyr Gin Giy Asn Ile 420 425 Ile Gly Leu Giu Arg Ser Tyr Leu Tyr Arg 440 445 Thr Ar9 Leu 430 Lys Leu Lys Ser Tyr 450 Gly Ile Tyr b.eu Ser Giu 455 <210> 7 <211> 412 <212> DNA <213> Laweonia intraceilularis c220> -c221> CDS <222> ,c400> 7 aaa caa att gat ata Lys Gin Ile Asp Ile atc att agt ggg gct acg ata act ctt gaa cgt 48 Ile Ile Ser Giy Ala Thr Ile Thr Leu Giu Arg aat ctt caa gtc aat ttt tct aac cca tac cat Asn Leu Gin Vai Asn Phe Ser Asn Pro Tyr His 25 gtc ctg gct aat gca aaa aaa gtt aaa ggg atg Val Leu Ala Aen Ala Lys Lye Val Lye Giy Met 35 40 caa aca get att Gin Thr ASP Ile aag ttt cce caa Lye Phe Pro Gin gac 144 Asp WO 02/038594 WO 02/38594PCT/AUOI/01462 12 ttt aat Phe Asn aaa cct gaa qtt ata gtt gct ata cgt aat ggt agt aca gtt Lys Pro Glu Val Ile Val Ala Ile Arg Asn Gly Ser Thr Val att act cct gca aag caa ctt ctt cct aaa gca tct ttt aga ctc Ile Thr Pro Ala Lys Gin ],eu Leu Pro Lys Ala Ser Phe Arg Leu ttt Phe gat gat gaa gtt gca tct ata aaa gat gta gaa tct gga caa Asp Asp Giu Val Ala Ser Ile Lys Asp Val Giu Ser Gly Gin s0 tca cat Ser His ata tta tta gct Ile Leu Leu Ala 100 tca gca cca tta cca gcg att caa gct Ser Ala Pro Leu Pro Ala Ile Gin Ala 105 ata aac tca Ile Asn Ser 110 cat caa tct His Gin Ser aat ggc aac ctt att cgt tta Aen Gly Asn Leu Ile Arg Leu 115 gat ace Asp Thr 120 ctc ccc att Leu Pro Ile gta gga ttt gca Val Gly Phe Ala 130 ata aag aag gga gat c Ile Lys Lys Gly Asp 135 <210> 8 <211> 137 <212> PRT <213> Lawsonia intraceilularis <400> 8 Lys Gin Ile Asp Ile Ile Ile Val Gly Ala Thr Ile Thr Xjeu Giu Arg Asn Leu Gin Val Asn Phe Ser Aen Pro Tyr His Gin Thr Asp Ile Giu Pro Gin Asp Vai Leu Ala Asn Ala Lys Lys Val Lys Gly Met Lys Phe Asn Lys Pro Giu Val Ile Val Ala Ile Arg Asn Gly Ser Thr Val Ile Thr Pro Ala Lys Gin Leu Leu Pro Lys Ala Ser Phe Arg Leu rs s 70 WO 02/038594 WO 02/38594PCT/AUO 1/0 1462 13- Asp Asp Giu Val Ala Ser Ile Lys Asp Val 90 Giu Ser Gly Gin Ser His Ile Leu Leu Ala Ser Ala Pro Leu 100 Ala Ile Gin Aia Ile Asn Ser 110 Thr His Gin Ser 125 Asn Gly Asn 115 Leu Ile Arg Leu Thr Leu Pro Ile Val Gly 130 Phe Ala Ile Lys Lys Gly Asp 135 <210> 9 <211> 849 <212> DNA <213> Laweonia intraceliuiaris <220> <221> CDS <222> <400> 9 stg tat Met Tyr 1 att att att gga tac ttt att gtt att gct tcc att Ile Ile Ile Gly Tyr Phe Ile Val Ile Ala Ser Ile att gga Ile Gly ggc tac ctt stg gct a ggg aat ctt gct tta ctc ttt Gly Tyr Leu Met Ala Lye Gly Asn taeu Ala Leu lieu Phe 25 gaa ctt gtt atc att att. ggg gca gca tta ggt gct ttt Giu Leu Val Ilie Ile Ile Gly Ala Ala Leu Giy Ala Phe caa cct gca Gin Pro Ala ttt gct tca Phe Ala Ser cag acg Gin Thr so aaa tat tca ttt act ctg gtc att aaa ast tta tca cac att Lays Tyr Ser Phe Thr Leu Val Ilie Lys Aen Leu Ser His Ile t tt.

Phe ggc gat cca aac agt aca aaa ata aaa tac ctt gas aca ctt Gly Asp Pro Asn Ser Thr Lys Ile Lys Tyr Leu Giu Thr Leu 240 ctt ctc tat gga Leu Leu Tyr Gly ttc tta aaa atg aat aga gaa ggt gtc Phe Leu Lye Met Asn Arg Giu Gly Val att agt Ile Ser WO 02/038594 WO 02/38594PCT/AU01/01462 -14ata gaa agt gat Ile Giu Ser Asp 100 ata gaa aaa cct gaa tca agt cct atc ttt agt aaa Ile G1u Lys Pro Giu Ser Ser Pro Ile Phe Ser Lys 105 110 tac cct aca att gta aaa gat act aaa gtt gtt gcc Tyr Pro Thr Ile Val Lys Asp Thr Lys Vai Val Ala 115 120 att gca gat Ile Ala Asp aca tta Thr Ijeu 130 cga gtt tat Arg Val Tyr ctg aca Leu Thr 135 ggt gca cca gaa Giy Ala Pro Glu 140 gat ata gat aac Asp Ile Asp Asn ctc Leu 145 atg gaa tct Met Glu Ser gac atg Asp Met 150 agc cat Ser H-is 165 aaa att aca cac gaa Lys Ile Thr His Glu 155 gaa gaa tta tta Glu Glu Leu Leu gca cat tcc atc Ala His Ser Ile atg gca gag Met Ala Giu cta cca gga atg Leu Pro Giy Met gtt gct gca Val Ala Ala cct cca gaa Pro Pro Glu 195 tta ggt gtt gtt Leu Gly Val Val acc atq gga aaa Thr Met Gly Lys att aat Ile Asn 190 gtc ctt ggg cat Val Leu Gly His att gga gca gct Ile Gly Ala Ala ttg gtt ggt Leu Val Gly 205 624 ttt ata Phe Ile 210 ggt att ctt ttc tgt tat ggt ttt ttt Gly Ile Leu Phe Cys Tyr Gly Phe Phe 215 gga cct atg ggt tca Gly Pro Met Gly Ser 220 aag Lye 225 ctt gaa acc tct Leu Glu Thr Ser gas gas gca cat Glu Glu Ala His tat tat aat tcc Tyr Tyr Asn Ser aaa gas gct gtt Lys Giu Ala Val gta gaa tat ggs Val Glu Tyr Gly 260 gct gct atc cga Ala Ala Ile Arg tct aca cca atg Ser Thr Pro Met aga cgt gcc ata Arg Arg Ala Ile cct aat aca ttt cgt cca tca ttt Pro Asn Thr Phe Arg Pro Ser Phe 265 270 tcg gaa atg gaa gaa cgt cta as aca gga tas WO 02/038594 WO 02/38594PCT/AUO 1/0 1462 Ser Giu Met Giu Giu Arg Leu Lys Thr Gly 275 280 <210> <211> 282 <212> PRT <213> Lawsonia intraceilularis <400> Met Tyr Ile 1 Gly Tyr Leu Giu Leu Val Gin Thr Lys so Phe Gly Asp LeU Leu Tyr Ile Giu Ser Tyr Pro Thr 115 Thr Leu Arg 130 Leu Met Giu 145 Aia His Ser Val Ala Ala Ile Gly Tyr 5 Ala Lys Gly Ile Ile Gly Ser Phe Thr 55 Asn Ser Thr 70 Leu Phe Leu Ile Giu Lys Val Lys Asp Tyr Leu Thr 135 Asp Met Lys 150 Ser His Met 165 Leu Gly Val Phe Ilie Val Ile Ala Ser Ile Ile Gly 10 Asn Leu Ala Ala Leu Val Lys Ile Lys Met Pro Glui 105 Thr LYS 120 Thr Gly Ile Thr Ala Giu Val Ile 185 Leu Leu Gly Ala Lys Asn Tyr Leu 75 Arg Giu Ser Pro Val Ala Pro Glu 140 Giu Giu 155 Leu Pro Met Gly Pro Ala Ala Ser His Ile Leu Ala Ile Ser Ser Lys Ala Asp Asp Asn Leu Pro 160 Gly Ile 175 Asn Glu WO 02/038594 WO 02/38594PCT/AU01/01462 -16- Pro Pro Glu 195 Val Leu Gly His Tyr 200 Ile Gly Ala Ala Val Gly Thr Phe Ile Gly Ile Leu 210 Phe Cys 215 Ala Glu 230 Tyr Gly Phe Phe Pro Met Gly Ser Leu Glu Thr Ser Glu Ala His Tyr Tyr Aen Ser Lys Glu Ala Val Ala Ala Ala Ile Arg 245 Ser Thr Pro Met Ile Ala .255 Val Glu Tyr Ser Glu Met 275 Arg Arg Ala Ile Aen Thr Phe Arg Pro Ser Phe 270 Glu Glu Arg Leu Lys Thr Gly 280 <210> 11 <211> 717 <212> DNA <213> Lawsonia. intracellularis <220> <221> CDS <222> <400> 11 atg tct ggc tca tgg aaa gtg gct tat gca gac ttt gtt Met Ser Gly Ser Trp Lys Val Ala Tyr Ala Asp Phe Val 1 5 10 aca. gat Thr Ala atg gct ttc ttt cta ctg atg tgg att ctt gca. atg Met Ala Phe Phe Leu Leu Met Trp Ile Leu Ala Met aca ccc cct Thr Pro Pro gtt aaa. gaa.

Val Lye Glu ggt ctt gct gca. tat ttt tct tca tct gat gct aca. ttt Gly Leu Ala Ala Tyr Phe Ser Ser Ser Asp Ala Thr Phe aaa, aca Lys Thr cct gat agt tcg cca atc tct aac Pro Asp Ser Ser Pro Ile Ser Aen 55 aat cct Aen Pro ctt atc aac caa.

Leu Ile Asn Gin ata gat aaa ctt gat act cga caa tta aaa att aat gaa aca gaa caa 24 240 WO 02/038594 WO 02/38594PCT/AU01/01462 -17- Ile Asp Lys Leu Asp Thr Arg Gin Leu Lys Ile Asn Giu Thr 70 75 tct cat tat gct ctt gct aat aaa tta aaa aaa atg tta atg Ser His Tyr Ala Leu Ala Asn Lys Leu Lys Lys Met Leu Met Glu Gin gct gat Ala Asp.

gct atc cca Ala Ilie Pro tta tta cgt Leu Leu Arg 115 ctt aca ccc Leu Thr Pro 130 tca gca aca gga ata agt gct gac gat Ser Ala Thr Giy Ile Ser Ala Asp Asp 105 gtt ggt gta Vai Gly Val 110 gta aat tct aat Val Asn Ser Asn tcc acg Ser Thr 120 ttt ttt cct ggt aca gca act Phe Phe Pro Giy Thr Ala Thr 125 gaa ggg aaa Giu Gly Lys gtt atg gga act gtt tta gcc gtt ctc Val Met Gly Thr Val Leu Ala Val Leu 140 cgt gaa tat aat ctt Arg Giu Tyr Asn Leu 145 ctt gtg ata cgt Leu Val Ile Arg cat gct gat att His Ala Asp Ile gaa ata aca aaa ggc agc cct ttt gct tct aac tgg gaa ctt Giu Ile Thr Lys Gly Ser Pro Phe Ala Ser Asn Trp Giu Leu tca gga Ser Gly 175 gct cgt gca Ala Arg Ala gct tca cga Ala Ser Arg 195 gca gct gca cag tat ctt gta gag cac Ala Ala Ala Gin Tyr Leu Val Giu His ggg ata aag Giy Ile Lys 190 att cgc tct Ile Arg Ser gta gga Val Gly 200 tat gca gat aca aga cct cta gaa Tyr Ala Asp Thr Arg Pro Leu Giu 205 cct agt Pro Ser 210 tct cct gaa gga agt aca aaa aat cgt Ser Pro Giu Gly Ser Thr Lys Asn Arg 215 cgt ata Arg Ile 220 gaa ttc tat Giu Phe Tyr ca-t cgg cca gaa His Arg Pro Giu atg tct tat ggc M~et Ser Tyr Gly gtt-gta tat taa tag Val Val T1yr 235 <210>. 12 <211> 237 WO 02/038594 WO 02/38594PCT/AUOI/0 1462 -18- <212> PRT <213> Lawsonia intracellularis <400> 12 Met Ser Gly Ser Tr 1 Met Ala Phe Phe Le p Lys 5 ~u Leu Val Lys Lys Thr 50 Ilie Asp Ser His Gly Leu Ala Val Ala Met Trp Ala Tyr Pro Ile 55 Arg Gin Asp Ser Leu Asp Ala Leu Ala Ile Pro Gin 100 Leu Leu Arg Val 115 Leu Thr Pro Giu Ser 130 Ala Asn Lys Ala Thr Gly Ser Aen Ser 120 Lye Lys Val 135 Tyr Leu Val 150 Ser Pro Phe Ala Ala Gin Ser Val Gly 200 Leu Ile 105 Thr Met Ile Ala Tyr 185 Tyr Ala Asp Phe 10 Leu Ala Met Ser Ser Ser Aen Asn Pro Lys Ile Asn 75 Lys Lye Met 90 Ser Ala Asp Phe Phe Pro Gly Thr Val 140 Arg Gly His 155 -Ser Aen Trp 170 Leu Val Glu Ala Asp Thr Val Thr Ala Met Thr Pro Pro Giu Asp Ala Thr Phe Leu Ile Asn Gin Glu Thr Giu Gin Leu Met Ala Asp Asp Val Gly Val 110 Gly Thr Ala Thr 125 Leu Ala Val Leu Ala Asp Ile Gly 160 Giu Leu Ser Gly 175 His Gly Ile Lys 190 Arg Pro Leu Glu 205 Ile Glu Phe Tyr Arg 145 Giu Ala Ala Glu Tyr Asn Leu Thr Lye Gly 165 Ala Ala Ala 180 Arg Ile Arg 195 Pro Ser Ser 210 Pro Glu Gly Ser Thr Lye Aen Arg Arg 215 220 WO 02/038594 WO 02/38594PCT/AUOI/01462 -19- Phe His Arg Pro Giu Val Met Ser Tyr Gly Val Val Tyr 225 230 235 <210> 13 <211> 1047 <212> DNA <213> LaWsonia irtraceilularis <220> <221> CDS <222> (1.044) <400> 13 atg ata atc Met Ile Ile 1 ctt eta gga. act get tte Leu Leu Gly Thr Val Phe att get ctt atc Ile Val Leu Ile tta tgc eca atg atg gaa gct gCC ata tac tcC atc cct Leu Cys Ser Met Met Glu Ala Ala Ile Tyr Ser Ile Pro att act tat Ile Thr Tyr aaa ctC tat Lys Leu Tyr att gaa cac Ile Giu His ctt cgt gaa cag gga agc aaa aaa gga Leu Arg Glu Gin Giy Ser Lys Lys Gly tat tta Tyr Leu cat age aat att gat cag ccC att aca gcc gta eta ata ttg His Ser Asn Ile Asp Gin Pro Ile Thr Ala Val Leu Ile Leu act ata gca aat act 9CC gga 9CC 9CC ctt gCC gga gca ate Thr Ile Ala Asn Thr Ala Gly Ala Ala Leu Ala Gly Ala Ile aca aca aca cCC cat gaa tcC act sag Thr Thr Thr Leu His Glu Ser Thr Lys cct tc CCC Pro Phe Pile 90 gca gca aec Ala Aia Ile acc ttg ctt att eta gcC CCC ggg gaa att ata Thr Leu Leu Ile LeU Ala Phe Gly Glu Ile Ile 100 105 ccC aaa aca cta ggt Pro Lys Thr Leu Gly 110 get gct tac tCC aaa cgt att 9cC ata att cC ctt aat ccC ctc tcC Val Ala Tyr Ser Lys Arg Ile Aia Ile Ile Leu Leu Asn Pro Leu Ser 115 120 125 WO 02/038594 WO 02/38594PCT/AUOI/01462 20 att ctt ata Ile Leu Ile 130 gtt act tta aaa ccc ctt att atq Val Thr Leu Lays Pro Leu Ile Met 135 ctt tca agc tac tta Leu Ser Ser Tyr Leu 140 aca cga ctt Thr Arg Leu 145 tca cct Ser Pro 150 cga aaa cgt cct aca gtt aca gaa gat Arg Lys Arg Pro Thr Val Thr Glu Asp 155 gac Asp 160 atc cgt gca ctt aca agt ctt tcc aga gag tct ggt cgt att Ilie Arg Ala Leu Thr Ser Leu Ser Arg Glu Ser Gly Arg Ile aag cca Lys Pro 175 tat gaa gaa Tyr Giu Glu gtc ata aaa aat atc ctt agt ctt gat Val Ile Lys Asn Ile Leu Ser Leu Asp 185 tta ass. tat Leu Lys Tyr 190 ctt cat gs Leu His Glu 576 gct cat gsa att Ala His Giu Ile 195 atg act ccc Met Thr Pro act atg gtc ttt Thr-Met Val Phe 624 aac ctt Asn Leu 210 act gtc tct gaa Thr Val Ser Glu tat agc aac ccc aaa ata tgg aac tat Tyr Ser Asn Pro Lys Ile Trp Asn Tyr 220 672 agt cgc atc cct act Ser Arg Ile Pro Thr gga gaa aat sac gaa gac att act Gly Giu Asn Asn Giu Asp Ile Thr 235 ggc att Gly Ile 240 aca gaa Thr Glu 255 atc cas cga tat gaa att gga cga tat Ile Gin Arg Tyr Giu Ile Gly Arg Tyr 245 acc aat gga ga Thr Aen Gly Giu 768 ass as ctt tta gaa att atg caa Lys Lye Leu Leu Gu Ile met Gin 260 caa act gta gat cat tta ctt ctt Gin Thr Val Asp His Leu Leu Leu 275 280 gca ass. ttt gtc Ala Lays Phe Val ctt gaa agt Leu Giu Ser 270 aga caa cat Arg Gin His gca ttt tta gsa gas Ala Phe Leu Giu Giu 285 ctt ttt Leu Phe 290 att gta ctt gat Ile Vai Leu Asp tat ggg gga tta tct ggt gtt gtt tcc Tyr Giy Gly LeU~ Ser Gly Vai Val Ser 300 tta gaa gat gta tta gaa act atg ctt gga aga gaa att gtt gat gas Leu Giu Asp Val Leu Glu Thr met Leu Gly Arg Glu Ile Val Asp Glu WO 02/038594 WO 02/38594PCT/AUOI/01462 -21- 310 agt gat aca aca cct gat ctt aga gca Ser Asp Thr Thr Pro Asp Leu Arg Ala 325 gca aaa aaa aga cat agt Ala Lys Lys Arg His Ser 335 1008 gca tta atc Ala Leu Ile aat aat aaa. aat Aen Asn Lys Asn ctt tta aaa taa.

Leu Leu Lys 1047 <210> 14 <211> 348 <212> PRT <213> Lawsonia intracellularis <400>. 14 Met Ile Ile Leu Leu Gly Thr Vai Phe Leu Ile Val Leu Ile Ser Ala 1 5 10 Leu Cys Ser Met Met Giu Ala Ala Ile Tyr Ser Ile Pro 25 Ile Glu His Leu Arg Giu Gin Gly Ser Lys Lys Gly Glu Ile Thr Tyr Lys Leu Tyr Tyr Leu so His Ser Asn Ile Asp Gin Pro Ile Thr Ala Vai Leu Ile Leu Asn Thr Ile Ala Asn Thr Aia Gly Ala Ala Leu Ala Gly Ala Ile Ala Ile Leu Thr Thr Thr Leu His Glu Ser Thr Lye Pro Phe Phe Ala Ala Thr Leu Leu Ilie Leu Ala Phe Gly Giu Ile Ile Pro 100 105 Lys Thr Leu Gly 110 Val Ala Tyr 115 Ser Lys Arg Ilie Ala Ile 120 Ile Leu Leu Asn Pro Leu Ser 125 Ile Leu 13 0 Ile Val Thr Leu Lye Pro Leu Ile Met Leu Ser Ser Tyr Leu 135 140 Thr Arg Leu Val Ser Pro 145 150 Arg Lys Arg Pro Thr Val Thr Giu Asp WO 02/038594 WO 02/38594PCT/AUO1/01462 22 Ile Arg Ala Leu Thr Ser Leu Ser D Tyr Ala Asn Ser 225 Ile Lye Gin Leu Leu 305 Ser Ala Glu Glu His Glu 195 Leu Thr 210 Arg Ile Gin Arg Lys Leu Thr Val 275 Phe Ile 290 Glu Asp Val Ile Met Thr Val Ser Giu Pro Thr Tyr 230 Tyr Glu Ile 245 Leu Giu Ile 260 Asp His Leu Val Leu Asp Arg Giu Ser 170 Ile Leu Ser 185 Thr Met Val Ser Aen Pro Asn Aen Giu 235 Tyr Met Thr 250 Pro Ala Lye 265 Ala Phe Leu Gly Gly Leu tLeu Gly Arg 315 Ala Leu Ala 330 Thr Leu Leu 345 Gly Arg Leu Asp Phe Ser 205 Lye Ile 220 Asp Ile Asn Gly Phe Vai Giu Giu 285 Ser Gly 300 Giu Ile Lye Lye Lys Ile Lys Pro 175 Leu Lys Tyr 190 Leu His Glu Trp Asn Tyr Thr Gly Ile 240 Giu Thr Glu 255 Leu Giu Ser 270 Arg Gin His Val Val Ser Val Asp Giu 320 Arg His Ser 335 Leu Glu Val Leu Leu 280 Glu Tyr 295 Thr Met Leu Arg Lye Aen Asp Thr Leu Ile <210> <211> .1812..

<2i2> DNA .<213> Laweonia intraceilularis <220> <221> CDS <222> (1)..(1809) WO 02/038594 WO 02/38594PCT/AUOI/01462 23 <400> atg caa aaa gta tgt tat ttt ttt ctt Met Gin Lys Val Cys Tyr Phe Phe Leu 1 5 acc ttt ttc tac Thr Phe Phe Tyr ttt ttc Phe Phe is ata aca gaa aat tat ctc ttt gct aca tca att acc act Ile Thr Giu Asn Tyr Leu Phe Ala Thr Ser Ile Thr Thr tcc aca att Ser Thr Ile aac caa caa cat ata gca tat aca gtt act ttt acc tct cca gaa aat Asn Gin Gin His Ile Ala Tyr Thr Val Thr Phe Thr Ser Pro Giu Asn cct aat Pro Asn ctt gca aca gag atg gaa aca cat agt Lieu Ala Thr Glu Met Giu Thr His Ser tta gta aag ctt Leu Vai Lys Lieu gca Ala aat caa tct tta gat agt aaa ata ggt tta aat tta cgt gtt Asn Gin Ser Leu Asp Ser Lys Ile Giy Lieu Asn Lieu Arg Vai aaa Lys gas. gat ata agt aca gca caa aaa att ctt gac tcg aat ggt Giu Asp le'Ser Thr Ala Gin Lys Ile Leu Asp Ser Asn Giy tat tat Tyr Tyr agt gga agt gtc gag gga aag att gac tgg cag acg aac Ser Giy Ser Val Giu Giy Lys Ilie Asp Trp Gin Thr Asn cct att agt Pro Ile Ser 110 336 atc caa atc caa ttt aaa cca aat gta caa tat aaa ata sat aca ata Ile Gin Ile Gin Phe Lys Pro Asn Vai Gin Tyr Lys Ile Asn Thr Ile 115 120 125 cat atc caa tac ctt gat agt gaa ctt gca tat ctc cct ctt too tta His Ile Gin Tyr Leu AsP Ser Giu Leu Aia Tyr Leu Pro Leu Ser Leu 432 gaa Giu 145 gaa ttc aat Giu.Phe Asn ctc tct Leu- Ser.

150 aaa ggt aat cct gct ctt gct gtt aat Lys Giy Asn Pro Aia Leu Ala Val Asn 155 atc Ile 160 cta tcc tct gta agt agc ctc atg caa tat ata cat aat aat Lieu -Ser Ser Val Ser Ser Lieu Met Gin Tyr Ile His Asn Asn gga tat Gly Tyr 175 16517 170 WO 02/038594 WO 02/38594PCT/AU01/01462 24 cca tta 9CC Pro Leu Ala ata aaa aaa act Ile Lys Lys Thr caa tac ata att aat cgg atg gat Gin Tyr Ile Ile Asn Arg Met Asp 185 190 tat aca ttt Tyr Thr Phe 195 atg ggt aaa Met Gly Lys 210 gat att gat tta Asp Ile Asp Leu ata aga caa gga Ile Arg Gin Gly tta ctc cat Leu Leu His gta caa cct.

Val Gin Pro cat aat ctc aat His Asn Leu Asn tca aca ata ttc Ser Thr Ile Phe cta Leu 225 aat aaa att. gct Aen Lye Ilie Ala tgg aag gaa gga Trp Lys Giu Gly gta tgg aac Val Trp Aen aat gca Aen Ala 240 720 ctc ctt gat tct tat cga aca cgg ctt Leu Leu Asp Ser Tyr Arg Thr Arg Lau 245 caa aca ggc ctt ttc Gin Thr Gly Leu Phe 255 tct ata act Ser Ile Th~r tct ata ga Ser Ile Giu 275 ggc tta caa Gly Leu Gin 290 sat cca agy sat Asn Pro Arg Asn aaa gaa cas Lye Giu Gin aat ggt aac Aen Giy Asn 270 ctt gtt gca Leu Vai Ala tac tct tct Tyr Ser Ser aca gaa Thr Giu 280 gat caa Asp Gin 295 9CC cct cca agg Ala Pro Pro Arg att agt ggt Ile Ser Gly ggt att ggt Gly Ile Gly cgt 999 act tgg Arg Gly Thr Trp gaa Giu 305 cat cga aat gtt His Arg Asn Val ggt sat ggs gaa ctt ttt cgt ata aca Giy Aen Gly Giu Leu Phe Arg Ile Thr 315 gca Ala 320 960 1008 cca ata agt Pro Ile Ser cgs gat Arg Asp 325 gat cas aaa att atg gca aac ttc cas Asp Gin Lye Ile Met Ala Aen Phe Gin 330 ass cca Lys Pro 335 9cc ttt ggc cgt cca Ala Phe Gly Arg Pro 340 aat caa tca Asn Gin Ser att agt gaa gca Ile Ser Glu Ala caa ctt. aaa Gin Leu Lys 350 1056 ass gas aat aca aaa agt tac aaa caa cas ctt gca tct att gct tta Lys Giu Asn Thr Lys Ser Tyr Lye Gin Gin Leu Ala Ser Ile Ala Leu 1104 WO 02/038594 WO 02/38594PCT/AUO1/01462 25 gga att gaa Gly Ile Giu 370 cga caa ttt Arg Gin Phe aat aga cgt tgg ttt ggt agt age agt ctt Asn Arg Arg Trp Phe Giy Ser Ser Ser Leu 375 380 atg gat gat cga gat tct ata aaa aaa ata Met Asp Asp Arg Asp Ser Ile Lys Lys Ile 395 400 1152 gtt gat aca gga Val Asp Thr Giy 1200 ttt act ett ttt ggc ate ccc tta tea Phe Thr Leu Phe Giy Ile Pro Leu Ser 405 aca agg gat agt Thr Arg Asp Ser 1248 1296 gat cet ett aat ect ate caa gga ace aaa get ace Asp Pro Leu Aen Pro Ile Gin Gly Thr L~ys Aia Thr 420 425 tta eat gtt act Leu Asn Vai Thr 430 ect tat att Pro Tyr Ile 435 ttt gat ttt Phe Asp Phe 450 ggt aaa tat aaa Giy Lye Tyr Lys aaa aag Lye Lys 440 att ttg act tte cgt agt egg Ile Leu Thr Leu Arg Ser Arg 1344 age ttt tac ate gac gtt ctt aae Ser Phe Tyr Ile Asp Vai Ljeu Lys 455 ggg aaa ctt ate Giy Lye Leu Ile 1392 ttg get Leu Ale 465 aac eea ata Aen Lye Ile ate ggt tee etc eta ggg aaa get eta Ile Giy Ser lieu Leu Giy Lye Asp Ile 475 1440 eec tat cet gee ate eta egg ttt tat Aen Tyr Pro Aia Ile Leu Arg Phe Tyr 485 ggg ggt ggt ggt Giy Giy Gly Giy agt gta Ser Vai 495 14 88 aga ggg tat Arg Giy Tyr get att gge Aia Ile Giy 515 gee tat Asp Tyr 500 cee tea ttg gga cee aaa aat Gin Ser Leu Giy Pro Lys Asn 505 aaa tat ggg gat Lys Tyr Giy Asp 1536 gga ett tet ttt Giy Leu Ser Phe act ett egt ttt gee tta ega tta Thr Ile Ser Phe Giu Leu Arg Leu 525 1584 eee ata Lye Ile 530 ace gee tee att Thr Giu Ser Ile att gtg cee att Ile Val Pro Ile tgg atg ggg gaa Trp Met Giy Giu 1632 WO 02/038594 WO 02/38594PCT/AUO 1/0 1462 26 tat tta cga aaa Tyr Leu Arg Lys 545 aaa aat ttc Lys Asn Phe 550 ctg act tta Leu Thr Leu aaa tca ata tat Lys Ser Ile Tyr 1680 ggg gta ggc Gly Val Gly cta cga tat tat Leu Arg Tyr Tyr agt ttt gcc ccc ata Ser Phe Ala Pro Ile 575 1728 tta gat ata gca act cca ctt Leu Asp Ile Ala Thr Pro.Leu 580 caa gat Gin Asp 585 aga agc cat Arg Ser His aat aaa cac ttt Aan Lys His Phe 590 1776 caa ctt tat Gin Leu Tyr 595 att agt att ggg caa gca ttc taa tga Ile Ser Ile Gly Gin Ala Phe 600 1812 <210> 16 <211>. 602 <212> PRT <213> Lawsonia intraceilularis <400> 16 Met Gin Lys Val Cys Tyr Phe Phe Leu 1 5 Thr Phe Phe Tyr Phe Phe Ile Thr Giu Aen Tyr Leu Phe Ala Thr Ser Ile Thr Thr Ser Thr Ile Asn Gin Gin His Ile Ala Tyr Thr Vai Thr Phe Thr Ser Pro Giu Asn Pro Asn Leu Ala Thr Giu Giu Thr His Ser Giu ILeu Val Lys Leu Ala Asn Gin Ser Leu Asp Ser Lys Ile Gly Asn Leu Arg Val Giu Asp Ile Ser Thr Ala Gin Lys Ilie Asp Ser Asn Gly Tyr Tyr Ser Giy Ser Val Giu Gly Lys Ilie Asp Trp Gin Thr Asn Pro Ile Ser 110 Ile Aen Thr Ile 125 Ile Gin Ile 115 Gin Phe Lys Pro Asn Val 120 Gin Tyr Lys WO 02/038594 WO 02/38594PCT/AU01/01462 27 His Ile Gin Tyr Leu Asp Ser Glu Leu 130 Giu Glu Phe 145 Leu Ser Ser Pro Leu Ala Tyr Thr Phe 195 Met Gly Lys 210 Leu Aen Lys 225 Leu Leu Asp Ser Ile Thr Ser Ile Giu 275 Gly Leu Gin 290 Giu His Arg 305 Pro Ile Ser Ala Phe Giy 135 Ser Lys Giy Asn 150 Ser Leu Met Gin Lye Lys Thr Gin 185 Asp Leu Val Ile 200 Pro Gin His Asn 215 Thr Trp Lys Giu Ile Val Gin Ile Ala 230 Ser Tyr Arg 245 Leu Asn Pro 260 ILeu Val Ala Ala Tyr Leu 140 Pro Ala Leu 155 Tyr Ile His 170 Tyr Ile Ile Arg Gin Gly Leu Asn Ile 220 Gly Arg Val 235 Gin Gin Thr 250 Lys Giu Gin Pro Pro Arg Ile Oly Ala 300 Giu ILeu Phe 315 Met Ala Asn 330 Ile Ser Giu Pro Leu Ser Leu Ala Val Aen Ile 160 Asn Asn Gly Tyr 175 Asn Arg Met Asp 190 Pro Leu Leu His 205 Ser Thr Ile Phe Trp Asn Aen Ala 240 Gly Leu Phe Ser 255 Asn Gly Asn Thr 270 Thr Ile Ser Gly 285 Arg Gly Thr Trp Arg Ile Thr Ala 320 Phe Gin Lys Pro 335 Ala Gin Leu Lys 350 Ser Ser Val Phe 310 Asp Asp 325 Pro Asn Thr Arg Leu Arg Asn Gin 265 Thr Giu Ala 280 Asp Gin Gly 295 Gly Asn Gly Gin Lys Ilie Gin Ser Ljeu 345 Lys Giu Aen 355 Thr Lys Ser Tyr Lye 360 Gin Gin Leu Ala Ser 36S Ile Ala Leu WO 02/038594 WO 02/38594PCT/AUOI/01462 28 Gly Ile Glu Arg Gin Phe Aen Arg Arg Trp Phe Gly Ser Ser Ser Leu 370 Ser Val 385 Phe Thr Asp Pro Pro Tyr Phe Asp 450 Leu Ala 465 Asn Tyr Arg Gly Ala Ile Lys Ile 530 Tyr Leu 545 Gly Val LeU Asp Thr Gly Phe Gly 405 Asn Pro 420 Giy Lys Ser Phe Lys Ile Ala Ilie 485 Asp Tyr 500 Gly Leu Giu Ser Lys Lys Leu Gly Ala Thr Phe 390 Ile Ile Tyr Tyr Ala 470 Leu Gin Ser Ile Aen 550 Leu Pro 375 Met Asp Asp Arg Asp 395 Leu Ser Ile Thr Arg 410 Gly Thr Lys Ala Thr 425 Lys Lys Ile Leu Thr 440 Asp Vai Leu Lye Thr 460 Gly Ser Leu Leu Giy 475 Phe Tyr Ala Giy Gly 490 Leu Gly Pro Lye Aen 505 Ser Thr Ile Ser Phe 520 Ile Vai Pro Ile Tvr Ile Lys Lye Asp Ser Ser 415 Leu Asn Val 430 Leu Arg Ser 445 Giy Lys Leu Lye Asp Ile Gly Gly Ser 495 Lys Tyr Giy sio Giu Leu Arg 525 Trp Met Giy Ser Ile Tyr Ala Pro Ile 575 Aen Lys His Leu Thr Leu Ser His 580 Gin Leu Tyr Ilie 595 <210> 17 Ser Ile Gly Gin Ala Phe 600 WO 02/038594 WO 02/38594PCT/AUO1/01462 29 <211> 4149 <c212> DNA.

<213> Lawsonia intraceliularis 4220> <221> <222>

CDS

(1)..(4146) <400> 17 atg aat aac Met Aen Asn 1 gca ttt acg Ala Phe Thr aca aaa ata ctt tct aag tta ctc tat acc ctc tta gga Thr Lys Ile Lev Ser Lys Leu Leu Tyr Thr Leu Leu Giy tta ttt tLeu Phe tta gga ctt att att aca ggc att ILeu Gly Leu Ile Ile Thr Gly Ile 25 ctt ttt ata Leu Phe Ile cgg acc tct Arg Thr Ser aoa ggc att got tgg att aaa aat sos gtt tot tot tta Thr Gly Ile Ala Trp Ile Lys Aen Thr Val Ser Ser Leu ctt cas Leu Gin s0 caa caa gga att ata cta caa gta tct tca att att gga cca Gin Gin Gy Ile Ile Leu Gin Val Ser Ser Ile Ile Gly Pro 192 ttc cca gas oaa att Phe Pro Giu Gin Ile att. sat gaa ctt ago ctt agt gat gtg Ile Asn Giu Leu Ser Leu Ser Asp Val 75 sat Aen gga act tac ott aca ata tct sac tta gaa atc caa tca aac Giy Thr Tyr Leu Thr Ile Ser Aen Leu Giu Ile Gin Ser Asn tta tgg I eu Trp got tta ttc Ala Lev Phie ggt osa ott gaa att ctg tot ttt gas Gly Gin Leu Giu Ile Leu Ser Phe Giu 105 ott aat gat Leu Asn Asp 110 aaa toa tot Lys Ser Ser Ott gta tta tat ogo tta 000 toa aat aat sat cta Leu Val Leu Tyr Arg Leu Pro Ser Aen Asn Aen Leu aca agt Thr Ser 130 ttt gtg tta Oct Phe Vai Lev Pro ata tca ttt gat tta aot coa tgg tgg Ile Ser Phe Asp Leu Thr Pro Trp Trp 140 act gsa oat att cgt att osa aso ato oat att sac sat sos oaa ott WO 02/038594 WO 02/38594PCT/AU01/01462 Thr Giu His Ile Arg Ilie Gin Asn Ile Hie Ile Asn Asn Thr 145 150 155 tcc tct gat att ata ggt att cca ttg gta tta tcc ctt gag Ser Ser Asp le Ile Gly Ile Pro Leu Val Leu Ser Leu Giu Gin Leu 160 ggt gat Giy Asp 175 ggt aca tta aca aat tgg aat gga aca ttt' caa cta tcc Giy Thr Leu Thr Ann Trp Ann Gly Thr Phe Gin Leu Ser 180 185 tct tct aac Ser Ser Asn 190 aaa aca aaa Lys Thr Lys 195 att ata gga acg Ile Ile Gly Thr cgt tac caa ggg, aat aag aca caa Arg Tyr Gin Giy Asn Lys Thr Gin 205 ttt ttt gaa tat gtt cat Phe Phe Giu Tyr Val His 210 aca. cgg ata gta Thr Arg Ile Vai cta gag ata. gac Leu Giu Ile Asp 0 gta gct gat Val Ala Asp aaa aag Lys Lys 230 tca tat aat aat agt atc ctt gaa caa Ser Tyr Asn Asn Ser Ile Leu Giu Gin 235 cta cat tta cac ctt tct att tat cct. gaa cat aat aga Leu His Lieu His Leu Ser Ile Tyr Pro Giu His Ann Arg 245 250 att atc tta Ile Ile Leu 255 cac tca tta His Ser Leu att gaa gta Ile Giu Val 275 cta gct Leu Ala 260 gaa tat ggt agc tgg tta ctt Giu Tyr Giy Ser Trp Leu LeV 265 aca tca gaa agt Thr Ser Giu Ser 270 tct aat gag caa Ser Asn Giu Gin tta aaa gga Leu Lys Gly 280 aat att tta tta aaa tat Ann Ile Leu Leu Lys Tyr 285 aat gga Asn Gly 290 gaa gct act cat Giu Ala Thr His ctt cct ata aaa Leu Pro Ile Lys ctt aac tca tca Leu Asn Ser Ser 912 att acc Ilie Thr 305 ctc agt ggc tca cta aat aaa cct aat ttt agt ata caa Leu Ser Gly Ser Leu Asn Lys Pro Asn Phe Ser Ile Gin 960 aca tta cct Thr Leu Pro gaa att Giu Ile 325 aac att aca aaa aac Asn Ile Thr Lys Aen 330 ata ata gat ctt Ile Ile Asp Leu 1008 WO 02/038594 WO 02/38594PCT/AU0I/01462 gaa ctt gtt Glu Leu Val aca tct ggg Thr Ser Gly 355 att aat Ile Asn 340 cta gga ctt ttc tct act cac ict Leu Giy Leu Phe Ser Thr His Ser 345 gat att Asp Ile 350 1056 aca att aca gta cag gga gaa act ata ThrIle Thr Val Gin Gly Glu Thr Ile 360 aat agt att Asn Ser Ile 1104 ctt tcc Leu Ser 370 agt gca gtt gat Ser Ala Val Asp ata gcc tct aca Ile Ala Ser Thr aca aca cat aca att Thr Thr His Thr Ile 380' 1152 acc Thr 385 tta gag cat gca.

Leu Glu His Ala tta. aca tct cca Leu Thr Ser Pro atg cat ttt tcc Met His Phe Ser 1200 1248 tct gga gaa Ser Gly Giu ttt aat Phe Asn 405 agt. ctt. cta gga Ser Ijeu Leu Gly atc gat gca aac Ile Asp Ala Asn ggt aat act cca Gly Asn Thr Pro 420 gat ctt act ggg Asp Leu Thr Gly 435 act ctt agt ata Thr Leu Ser Ile tct tct ctt ctt Ser Ser Leu Leu gga cta. cct Gly Leu Pro 430 1296 caa agt aac att act ata gga tta cac cgt caa ggg Gin Ser Asn Ile Thr Ile Giy Leu His Arg Gin Gly 440 445 1344 tct, tcc Ser Ser 450 tct tca ata. gaa gga aca gca act gtc Ser Ser Ile Giu Gly Thr Ala Thr Val 455 ctt aat aat atg Leu Asn Asn Met 1392 aac Asn 465 tgg gga gta caa gca tta cag ggg aca tta ggt gat aat gca Trp Giy Val Gin Ala Leu Gin Gly Thr Leu Gly Asp Asn Ala 470 475 1440 1488 cta agt gga ata tat aat tta act ccc ata gac tgg tct Leu Ser Giy Ile Tyr Asn ILeu Thr Pro Ile Asp Trp Ser 485 490 att tct Ile Ser 495 ggc ctt Gly Leu 510 aac aaa ttg aaa tta aca gca aag Asn Lys Leu Lys Leu Thr Ala Lys 500 gtt tat gct gaa Val Tyr Ala Glu 1536 aat ttt caa aaa aaa tac ata gat agc tct ata aat ctt ata att cct 18 15B4 WO 02/038594 WO 02/38594PCT/AUOI/01462 32 Asn Phe Gin 515 Lys Lys Tyr IleAsp Ser Ser Ilie Aen Leu Ile Ile Pro 520 525 aac ctt Asn Leu 530 cag eta ata get Gin Leu Ile Ala cet ata tct Pro Ile Ser gga gag Giy Giu 540 tct ata Ser .Iie 555 tta caa tee tta Leu Gin Ser Leu att Ile 545 aca gtg tet gga Thr Vai Ser Giy ctt gac 9ea ect Leu Asp Aia Pro gaa age aaa.

Giu Ser Lys 1632 1680 1728 ttt tca tca caa Phe Ser Ser Gin ace tgg aat geg Thr Trp Asn Aia eaa ctt aat aat Gin Leu Asn Asn ect caa Pro Gin 575 etc ata ata Leu Ile Ile ata aca etc Ile Thr Leu 595 act act caa tet Thr Thr Gin Ser tee tct geg att 5cr Ser Ala Ile aaa ggt aat Lys Gly Asn 590 aee ttt tea Thr Phe Ser teg get gag eca Ser Ala Giu Pro tea tet gag gca Ser Ser Giu Ala 1776 1824 1872 agt aat ser Asn 610 tgg gga ate Trp Gly Ile eta. ect Leu Pro 615 aeg gaa ata Thr Giu Ile eta gta Leu Val 620 gaa aaa att ata Giu Lys Ile Ile gga Gly 625 aat ata tta gga Asn Ile Leu Gly gta aat ett gat ggt aat Val Asn Leu Asp Gly Asn 630 635 att aaa ata aea Ile Lys Ile Thr 1920 aaa gat tac ett Lys Asp Tyr Leu aat ggt gat att Asn Gly Asp Ile gea gaa gtt cag Ala Giu Val Gin 1968 aaa gat att Lys Asp Ile ata aaa ata Ile Lys Ilie 675 aac ata ttg caa Asn Ile Leu Gin cet att aga Pro Ile Arg ggt tea gea tea Gly Scr Ala Ser 670 2016 cag ttt gat eea aag Gin Phe Asp Pro Lys 680 aat caa eaa tgt att tet act eaa Asn Gin Gin Cys Ile Ser Thr Gin 685 2064 tgg caa tta aaa aat tte ata tta ggt aat aat Gly Asn Asn aat gta act act Asn Val Thr Thr 2112 Trp Gin Leu Lys 690 Asn Phe Ile Leu 695 WO 02/038594 WO 02/38594PCT/AUO1/01462 -33aaa gga aga gca Lys Gly Arg Ala gat aca ata caa ctt cat aag aat cct Asp Thr Ile Gin Leu His Lys Asn Pro 710 715 att ggt gct ggt aca tat gaa gac ttt Ile Gly Ala Gly Thr Tyr Giu Asp Phe 730 aca att Thr Ile 720 caa tgg Gin Trp 735 2160 gct ctc tct tca Ala Leu Ser Ser 2208 aca caa 999 Thr Gin Giy tta gac ata aaa Leu Asp Ile Lys aca tta aaa aat Thr Leu Lys Asn ttt aat agt Phe Asn Ser 750 ttt caa aca Phe Gin Thr ass. ata aat Lys le Asn 755 ata gca gga caa Ile Ala Giy Gin act gta aac gca Thr Val Asn Aia 2256 2304 2352 2400 aat ctt ttt gaa aaa Asn Leu Phe Giu Lys 770 att caa aaa aat ata Ile Gin Lys Asn Ile 785 aat att Asn Ile 775 gga att Giy Ile 790 aat ata act act Asn Ile Thr Thr aat tta aaa aat Asn Leu Lys Aen aag ctc ctt Lys Leu Leu cca ata aaa att Pro Ile Lys Ile gtc tca cct caa Val Ser Pro Gin caa ttt gtt Gin Phe Val 805 ctt sat aac tgt tca cta Leu Aen Aen Cys Ser Leu 810 gca att Ala Ile 815 caa cga Gin Arg 830 2448 cca tct gga Pro Ser Gly aat gct aat Asn Ala Asn 835 ttt agt ata Phe Ser Ile 850 att aca act gat ata tat gtt act cct Ile Thr Thr Asp Ile Tyr Vai Thr Pro 825 2496 gca atc att aaa Ala Ile Ile Lye gtt tca ctt ctc tct ttc caa cca Vai Ser Leu Leu Ser Phe Gin Pro 845 2544 ctt ctt cct Leu Leu Pro gga aat ata sat Gly Aen Ile Asn cac ata aca ctt His Ile Thr Leu 2592 aca gga ata Thr Giy Ile 865 cct agt aaa cct assa gga aca Pro Ser Lye Pro Lye Gly Thr 870 tca ttt gat at Ser Phe Asp Ilie 2640 aac ata cat tat cca agg cca aat cca tca ata gca aac tta cat gta 28 2668 WO 02/038594 WO 02/38594PCT/AUOI/01462 34 Asn Ile His Tyr Pro Arg 885 Pro Asn Pro Ser Ile Ala Asn Leu His Val 890 895 gaa ggg gaa att ata tct tet ect aac aat ata tgt aaa Giu Gly Glu Ilie Ile Ser Ser Pro Asn Aan Ile Cys Lys ett aat gca Leu Amn Ala 910 caa gea aca Gin Ala Thr 2736 2784 ace eta aca gaa aaa aaa gag ect ata ect ata Thr Leu Thr Giu Lys Lys Giu Pro Ile Pro Ile tea ata Ser Ile 925 ete eet Leu Pro 930 ttt gag tte aea gaa aac aat ate eet atg eta tet aaa atg Phe Glu Phe Thr Giu Asn Asn Ile Pro Met Leu Ser Lys Met 2832 eet ttt tet gee Pro Phe Ser Ala ate aag tgg act gga ata tta gat aea Ile Lys Trp Thr Gly Ile Leu Asp Thr 955 2880 tgg aaa etc att Trp Lys Leu Ilie ett act gat tac Leu Thr Asp Tyr atg get ggg aat Met Ala Giy Asn gga tet Gly Ser 975 2928 tta gat get tet ctt tet ggg act tta gat agt eca Leu Asp Ala Ser Leu 980 Ser Gly Thr ata aea aca ett tet aat get aae Ile Thr Thr Leu Ser Asn Ala Asn 995 1000 Leu Asp Ser Pro 985 ttt eaa gat etc Phe Gin Asp Leu tta cag gte ttt Leu Gin Val Phe 1020 gat ggt aaa caa Asp Gly Lys Gin 1035 aea tat gea att Thr Tyr Ala Ile 990 tee ett ggt ett Ser Leu Gly Leu 1 005 tet aat aga ate Ser Aen Arg Ile ggt agt ata eaa Gly Ser Ile Gin 1040 3024 3072 2976 tae tta Tyr Leu 1010 tee eat Ser His 1025 aat ate aat get Aen Ile Aen Ala l01s eaa get aea gea Gin Ala Thr Ala 1030 3120 ett att ggt aat att ggc tea tet aaa gaa eac ttt ect ttg Leu Ile Gly Asn Ile Gly Ser Ser Lye Glu His Phe Pro Leu tet att Ser Ile L055 3168 1045 1050 aat gge tee ttt aea aae ett get eea tta eaa cgt Aan Gly Ser Phe Thr Asn Leu Ala Pro Leu Gin Arg aaa gae eta agt Lye Asp Leu Ser 1070 3216 1060 1065 WO 02/038594 PCT/AU0I/01462 ctt aca ctt Leu Thr Leu 1075 tca gga gca gct act ctt gaa gga aca tta aaa cag tct Ser Sly Ala Ala Thr Leu Glu Sly Thr Leu Lye Gin Ser 1080 1085 3264 gaa gtt Slu Val 1090 gaa ggg Glu Sly 1105 aaa ggc gat att gtt.att aac Lys Gly Asp Ile Val Ile Asn 1095 caa ggc gaa ttt Sin Sly Slu Phe 1100 caa ctt act Sin Leu Thr 3312 tta acc agt aat att cca act ctt aat gta gtt gat Leu Thr Ser Aen Ile Pro Thr Leu Aen Val Val Asp 1110 1115 agc act Ser Thr 1120 cct acc Pro Thr 1135 3360 3408 caa caa caa Sin Sin Sin aat aca aag acc aaa aaa gct acc tat caa caa Asn Thr Lye Thr Lye Lye Ala Thr Tyr Sin Sin 1125 1130 tta tct att Leu Ser Ile atg ttt gaa Met Phe Slu 1155 gcg tta agt Ala Leu Ser L140 atc ccg aat cgt ttt ttt gtc cgt agt Ile Pro Aen Arg Phe Phe Val Arg Ser 1145 1150 3456 agt gag tgg gga Ser Glu Trp Gl) L ggg aac cta r Sly Asn Leu 1160 act att aac Thr Ile Aen 1165 aaa gtc ata Lye Val Ile 3504 3552 aca agt Thr Ser 1170 cct gtt att aca Pro Val Ile Thr gga gca cta act Sly Ala Leu Thr L175 tct ata Ser Ile 1180 age gga aat ttt Arg Sly Aen Phe aat tta cta Asn Leu Leu 1185 tca gga tca Ser Sly Ser gga aaa caa ttt tct ctt gct Sly Lye Sin Phe Ser Leu Ala 1190 1 aaa Lye L195 agt aca eta tca ttt Ser Thr Ile Ser Phe 1200 3600 3648 gtt cca cca aac cca Val Pro Pro Aen Pro 1205 cta ctc Leu Leu 1210 aat att tct tta ace tat Asn Ile Ser Leu Thr Tyr 1215 att aaa ggt aca act agt Ile Lye sly Thr Thr Ser 1230 tca tca cct Ser Ser Pro tct att aca Ser Ile Thr 1220 gct ate ggc ett Ala Ile Sly Ile 1225 3696 eat cct aet att act ttt Aen Pro Aen Ile Thr Phe 1235 tca agt aca cca cct tte cct caa get gaa Ser Ser Thr Pro Pro Leu Pro ln Asp lu 3744 1240 1245 ata gtt tcc caa gtt ctt ttt ggt aea agc tea cea agt ctt agc agg 3792 WO 02/038594 WO 02/38594PCTIAU01/01462 -36- Ilie Val Ser Gin Val Leu Phe Gly Lys Ser Ser Gin Ser Leu Ser Arg 1250 1255 1260 ata caa'gcc ats caa ctt gct Ilie Gin Ala Ile Gin Leu Ala 1265 1270 caa gas tta gca aac tta Gin Glu Leu Ala Asn Leo 1275 aca gga ttt Thr Gly Phe 1280 3840 aat act gga agt atg aat ASn Thr Gly Ser Met Asn 1285 tta gat ata ctt agc tta Leu Asp Ile Leu Ser Leu 1300 tcc aac tca aac gat caas Ser Asn Ser Asn Asp Gin 1315 ttc cta aca aat att cga cag Phe Leo Thr Aen Ile Arg Gin 1290 aca tta Thr Leo 1295 3888 ggg aca act tct sat aga as gcc sat Gly Thr Thr Ser Aen Arg Lys Ala Asn 1305 1310 3936 gaa gat Glu Asp 1320 atc cct gtt ata gaa cta ggt Ilie Pro Val Ile Giu Leo Gly 1325 ggt gtt gaa caa agt tat tta Gly Val Giu Gin Ser Tyr Leu 1340 3984 ass tat Lys Tyr 1330 gat agt Asp Ser 1345 att aca gac act gtt tat gtt Ile Thr Asp Thr Val Tyr Val 1335 sat gat act ggg gca aga ata Asri Asp Thr Gly Ala Arg Ile 1350 4032 4080 tca gtt gaa ctt gca Ser Val Giu Leu Ala 1355 aat Asn 1360 ttt sat ctt gas ggt aga Phe Aen Leo Giu Gly Axg 1365 acs ggg act Thr Gly Thr cas tat agt gag ata Gin Tyr Ser Giu Ile 1370 ggt att Gly Ile 1375 4128 sat tgg ass ass gat tat tas Asn Trp Lys Lys Asp Tyr 1380 <210> 18 <211> 1382 <212> PRT <213> Lawsonia intraceliularis <400> 18 Met Aen Asn Thr Lys Ile Leo Ser Lys Leo Leu TIyr Thr Leo Leo Gly 1 5 10 Ala Phe Thr Leo Phe Leo Gly Leu Ile Ile Thr Gly Ile Leo Phe Ile 4149 WO 02/038594 PCT/AU01/01462 -37- Arg Leu Phe 65 Gly Ala Leu Thr Thr 145 Ser Gly Lye Phe Ser 225 Leu Thr Ser Gin Gin Pro Glu Thr Tyr Leu Phe Val Leu 115 Ser Phe 130 Glu His Ser Asp Thr Leu Thr Lys 195 Phe Glu 210 Val Ala His Leu Ile Ala Ile Ile 55 Thr Ile 70 Ile Ser Gin Leu Leu Pro Pro His 135 Ile Gln 150 Gly Ile rrp Ile Lye 40 Leu Gln Val Asn Giu Leu Asn Leu Glu Giu Ile Leu 105 Ser Asn Aen 120 Ile Ser Phe Asn Ile His Pro Leu Val 170 Gly Thr Phe 185 Leu Arg Tyr 200 Thr Arg Ile Tyr Asn Asn Tyr Pro Glu 250 Gly Ser Trp 265 Asn Thr Val Ser Ser Leu Ser Ser Ser Leu Ile Gin Ser Phe Asn Leu Asp Leu 140 Ile Asn 155 Leu Ser Gln Leu Gin Gly Val Thr 220 Ser Ile 235 His Asn Gly Pro Val Asn Asn Leu Trp Leu Asn Asp 110 Lye Ser See Pro Trp Trp Thr Gin Leu 160 Giu Gly Asp 175 Ser Ser Asn 190 Lye Thr Gln Giu Ile Asp Asn Trp Asn Ile Giy Thr Tyr Vai His Asp His Gin Pro 240 Ile Leu 255 His Ser Leu Ala Giu Tyr Leu Leu Thr Ser Giu Ser 270 WO 02/038594 PCT/AU01/01462 -38- Gly Asn Ile lie Asn Ile 305 Thr Glu Thr Leu Thr 385 Ser Gly Asp Ser Asn 465 Leu Glu Val Ser Asn 275 Gly Glu Ala Thr 290 Thr Leu Ser Gly Leu Pro Glu Ile 325 Leu Val Ile Asn 340 Ser Gly Thr Ile 355 Ser Ser Ala Val 370 Leu Glu His Ala Gly Glu Phe Asn 405 Asn Thr Pro Thr 420 Leu Thr Gly Gin 435 Ser Ser Ser Ile 450 Trp Gly Val Gin Glu Gin Leu Lys His Gln 295 Ser Leu 310 Asn Ile Leu Gly Thr Val Asp Ile 375 Thr Leu 390 Ser Leu Leu Ser Leu Pro Ile Lys Lys 300 Asn Lys Pro Asn Phe 315 Thr Lys Asn Ile Ile 330 Leu Phe Ser Thr His 345 Gin Gly Glu Thr Ile 360 Ile Ala Ser Thr Thr 380 Thr Ser Pro Glu Met 395 Leu Gly Asn Ile Asp 410 Ile Phe Ser Ser Leu 425 Ile Thr Ile Gly Leu 440 Thr Ala Thr Val Ser 460 Gin Gly Thr Leu Gly 475 Thr Pro Ile Asp Trp 490 Leu Leu 285 Leu Asn Ser Ile Lye Tyr Ser Ser Gin Met .320 Gin Thr 335 Ile Leu Ser Ile Thr Ile Ser Leu 400 Leu Lye 415 Leu Pro Gin Gly Asn Met Ala Thr 480 Ser Leu 495 Ser Asn Glu Gly 455 Ala Leu 470 Asn Leu 'Tyr 485 Ser Gly Ile Asn Lys Leu Leu Thr Ala Lye Val Tyr Ala Glu Gly Leu Ile 510 WO 02/038594 WO 02/38594PCT/AUOI/01462 Asn Asn Ile 545 Gin Lys Lys 515 Gin Leu Ilie Val Ser Gly 'u Phe Leu Ile Ser Giy 625 Lys Lys Ile Trp Ile 705 Ala- Thr Ser Ser Gin Ile Ile Thr 580 Thr Leu Ser 595 Asn Trp Giy Ile Leu Asp Tyr Leu Ile Leu Pro 615 Giy Val Aen 630 Ile Aen Gly 645 Asn Ile Leu Phe Asp Pro Asn Phe Ile 695 Ala-Asp Thr 710 Lys Ile Giy 725 Leu Asp Ile Asp Ser 520 Pro Ile Asp Ala Aen Ala Ser Ser 585 Aia Ser 600 Thr Giv Leu Asp Asp Ile Gin Ile 665 Lys Asn 680 Leu Giy Ile Gin Ala Gly Lys Gly 745 -39- Ser Ilie Asn Ser Gly Giu 540 Pro Ser Ile 555 Leu Gin Leu 570 Ser Ser Ala Ser Giu Ala Ile Leu Val 620 Gly Asn Ile 635 Ile Ala Giu 650 Pro Ile Arg Gin Gin Cys Aen Asn Phe 700 Leu His Lye 715 Thr Tyr Giu 730 Thr Leu Lys Leu Ile Ile Pro 525 Leu Gin Ser Leu Giu Ser Lye Ile 560 Asn Aen Pro Gin 575 Ile Lye Gly Asn 590 Leu Thr Phe Ser 605 Giu Lys Ile Ile Lys Ile Thr Lye 640 Val Gin Ser Trp 655 Gly Ser Ala Ser 670 Ile Ser Thr Gin 685 Asn Vai Thr Thr Asn Pro Thr Ile 720 Asp Phe Gin Trp 735 Aen Phe Asn Ser 750 Lys Ilie Asn Ilie Ala Giy Gin Thr Thr Vai Aen Ala Aen Phe Gin Thr WO 02/038594 WO 02/38594PCT/AU01/01462 40 755 Asn Leo Phe 770 Ile Gin Lye 785 Val Ser Pro Giu Asn Gin Pro Ser Giy Thr 820 Asn Ala Aen Ala 835 Lays Asn Ile Gly 790 Gin Phe 805 Ile Thr Ile Ile Leu Pro Ser Lye Phe Ser 850 Thr Gly 865 Aen Ile Glu Gly Thr Leu Leu Pro 930 Arg Pro 945 Trp Lys Leu Asp Ile Leu Ile Pro 760 Ie Aen Ile Thr 7.75 Ile Lye Leo Leu Val Leo Asn Asn 810 Thr Asp Ile Tyr 825 Lys Giu Vai Ser 840 Gin Gly Asn Ile 855 Pro Lys Gly Thr Pro Asn Pro Ser 690 Ser Pro Asn Aen 905 Giu Pro Ile Pro 920 Giu Asn Ann Ile 935 Ile Lye Trp, Thr Thr Asp Tyr Ile 970 Giy Thr Leu Asp 985 Thr Leu Aen 780 Gin Pro Ile 795 Cye Ser Leo Val. Thr Pro Leu Leu Ser 845 Ann Gly His 860 Leu Ser Phe 875 Ile Ala Aen Ile Cys Lys Ile Ser Ile 925 Pro Met Leo 940 Giy Ile Leo 955 Met Ala Giy Leo Lye Lye Ile Ala Ile Leu 815 Gin Arg Leo 830 Phe Gin Pro Ile Thr Leu His Tyr Pro 885 Gu Ile Ile Ile Leo 880 His Val 895 Aen Ala Ala Thr Lys Met Thr Leo 960 Gly Ser 975 Ala Ile Asp Aen Le Ilie Ala Ser 980 Ser Pro Thr Tyr 990 Ile Thr Thr Leo Ser Asn Ala Asn Phe Gin Asp Leu Ser Leo Gly Leo 995 1000 1005 WO 02/038594 PCT/AU01/01462 -41- Lys Leu Gin Val Phe 1020 Tyr Leu Glu Asn Ile Asn Ala 1010 1015 Ser His Ile Gin Ala Thr Ala 1025 1030 Leu Ile Gly Asn Ile Gly Ser 1045 Asn Gly Ser Phe Thr Asn Leu 1060 Leu Thr Leu Ser Gly Ala Ala 1075 Glu Val Lys Gly Asp Ile Val 1090 1095 Glu Gly Leu Thr Ser Asn Ile 1105 1110 Gin Gin Gin Asn Thr Lys Thr 1125 Leu Ser Ile Ala Leu Ser Ile 1140 Met Phe Glu Ser Glu Trp Gly 1155 Thr Ser Pro Val Ile Thr Gly 1170 1175 Asn Leu Leu Gly Lys Gln Phe 1185 1190 Ser Gly Ser Val Pro Pro Asn Ser Asn Arg Ile Ser Asp Gly Lys 1035 Ser Lye Glu His 1050 Ala Pro Leu Gin 1065 Thr Leu Glu Gly 1080 Ile Asn Gin Gly Gin Gly Ser Ile 1 Phe Pro Leu Ser 1055 Arg Lys Asp Lev 1070 Thr Leu Lys Gin 1085 Glu Phe Gin Leu 1100 Gin .040 Ile Ser Ser Thr Pro Thr Leu Lys Lys Ala 1130 Pro Asn Arg 1145 Gly Asn Leu 1160 Ala Leu Thr Ser Leu Ala Pro Leu Leu 1210 Ile Gly Ile 1225 Asn Val Val Asp Ser Thr 1115 1120 SThr Tyr Gln Gin Pro Thr 1135 Phe Phe Val Arg Ser Ser 1150 Thr Ile Asn Lys Val Ile 1165 SSer Ile Arg Gly Asn Phe 1180 SLys Ser Thr Ile Ser Phe 1195 1200 SAsn Ile Ser Leu Thr Tyr 1215 Ile Lys Gly Thr Thr Ser 1230 1205 Ser Ser Pro Ser Ile 1220 Thr Ala Asn Pro Asn Ile Thr Phe Ser Ser Thr Pro Pro Leu Pro Gln Asp Glu 1235 1240 1245 WO 02/038594 WO 02/38594PCT/AUO 1/0 1462 42 Ile Val Ser Gin Val Leu Phe Gly Lys Ser Ser Gin 1250 1255 1260 Ile Gin Ala Ile Gin Leu Ala Gin Giu Leu Ala Aen 1265 1270 1275 Ser Leu Ser Arg Leu Thr Gly Phe 1280 Asn Thr Gly Ser Met Asn Phe 1285 Leu Thr Aen Ile 1290 Arg Gin Thr Leu Gly 1295 Leu Asp Ilie Leu Ser ILeu Gly Thr Thr Ser Asn Arg Lys Ala Aen Thr 1310 1300 1305 Ser Asn Ser Aen Asp 1315 Gin Ile Glu 1320 Asp Ile Pro Vai Ile 1325 Giu Leu Gly Lys Tyr Ile Thr 1330 Asp Thr Vai 1335 Tyr Val Gly Val Glu Gin Ser Tyr Leu 1340 Asp Ser Asn Asp Thr Gly Ala 1345 1350 Arg Ile Ser Vai Glu Leu Ala 1355 Pro Asn 1360 Phe Asn Leu Giu Giy Arg Thr Gly Thr Gin Tyr Ser Glu Ile Gly Ile 1365 1370 1375 Asn Trp Lys Lys Asp Tyr 1380 <210> 19 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> 19 catattcaag gtacagcatc tgatgg <210> <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: WO 02/038594 WO 02/38594PCT/AUOI/01462 43 Oligonucleotide probe/primer <400> ctcctttaca aaccttgctc c <210> 21 c211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer.

<400> 21 gctcatctaa agaacacttt cc 22 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <c220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> 22 caaggtagta tacaacttat tgg <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> 23 gacctaagtc ttacactttc agg <210> 24 WO 02/038594 WO 02/38594PCT/AUOI/01462 -44- <211> 24 <212> DNA <213> Artificial Sequence c220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> 24 gtattaatac tacattagtt gacg <210> c211> 21 <212> DNA c213> Artificial Sequence <220> <223>- Description of Artificial Sequence: Oligonucleotide probe/primer <400> ggataataat ggaaaaagtg 9 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> 26 caagcaatgc ctgtagaggt cc <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 01 igonucleotide probe/primer WO 02/038594 PCT/AUOI/01462 45 <400> 27 aagaatgcct gtaataataa gtcc 24 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence 220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> 28 ttggggaatc ctacctacg 19 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> 29 tattaggagt aaatcttgat g 21 <210> <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 01 igonucleot ide probe/primer 400> gcaggacaaa caactgtaaa cg 22 <210> 31 <211> 22 WO 02/038594 WO 02/38594PCT/AUOI/01462 46 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide. probe/primer <400> 31 gaggaagaag tatactaaat 99 <210> 32 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> 32 tgttggacta tctaaagtcc <:210> 33 <211> 22 <212> DlL <213> Ar'tificial sequence <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> 33 ctattgatgg atttggcctt 99 22 <210> 34 <211> 20

DNA

<213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <212> WO 02/038594 PCT/AU01/01462 47 <400> 34 gtgctggtac atatgaagac <210> <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> ttcatcacct tctattacag <210> 36 <211> <212>*DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> 36 ggaaactatt tcatcttgag <210> 37 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> 37 attaggtgca agttcaactg <210> 38 <211> <212> DNA WO 02/038594 WO 02/38594PCT/AUOI/01462 48 <213> Artificial Sequence* <220> <223> Description of Artificial Sequence: 01 igonucleotide probe/primer <400> 38 tttagatagt aatgatactg <210> 39 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> 39 ttattatatt atgttttttg taatgttaat ttcagg <210> <211> <212> <213> <220> <223> 28

DNA

Artificial Sequence Description of Artificial Sequence: 01 igonucleotide probe/primer <400> gacatatgaa taacacaaaa atactttc <210> 41 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> 41 WO 02/038594 WO 02/38594PCT/AUOI/01462 49 gaggatcctc tagagttaat caaact9tat ttttattgat 9 <210> 42 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> 42 gacatatgcg gacctctaca ggcattgctt g <210> 43 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> 43 gatcaggtag tccaagaaga gaag <210> 44 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> 44 ttggaggatc ctctagagtt atcaggttgt aattgttcca gatgg <210> <211> 21 <212> DN~ <213> Ar'tificial Sequence WO 02/038594 PCT/AU01/01462 <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> ccttggttaa taacaatatc g 21 <210> 46 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide probe/primer <400> 46 caactccact tcaagataga age 23 <210> 47 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligo <400> 47 gaccatggaa aaagtatgtt atttttttc 29 <210> 48 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligo <400> 48 gaggatcctc tagagttaga atgcttgccc aatact 36 <210> 49 <211> 39 <212> DNA WO 02/038594 PCT/AU01/01462 -51- <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligo <400> 49 ttggaggatc ctctagagtt agaatgcttg cccaatact 39 <210> <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligo <400> ttgaccatgg ctacatcaat taccacttcc ac 32 <210> 51 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligo <400> 51 ggacatatga ataacacaaa aatactttc 29 <210> 52 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligo <400> 52 ttggaggatc ctctagagtt aatcaaactg tatttttatt gatg_ 44 <210> 53 <211> 32 <212> DNA <213> Artificial Sequence WO 021038594 WO 02/38594PCT/AU01/01462 -52- <220> <223> Description of Artificial Sequence: oligo <400> 53 ggacatatgc ggacctctac aggcattgct tg 32 <210> 54 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligo <400> 54 tgaggattat taagttggag <210> <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligo <400> gcatgcaacc ttaacatctc <210> 56 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligo <400> 56 tttctgatgt aagtaaccag <210> 57 <211> <212> DNA <213> Artificial Sequence <220> WO 02/038594 PCT/AU01/01462 -53- <223> Description of Artificial Sequence: oligo <400> 57 tctgcccata tcaagtggac <210> 58 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligo <400> 58 ggaacatttc aactatcctc <210> 59 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligo <400> 59 gtaaggtaag ttccattcac <210> <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligo <400> caacgtggat ccgaattcaa gcttc <210> 61 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide WO 02/038594 WO 02/38594PCT/AUOI/01462 54 <400> 61 Met Gly Ser Gly Ser Gly Asp Asp Asp Asp Lys Leu Ala Leu Leu Thr 1 5 10 Met <210>. 62 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 62 Ala Thr Ser Ile Thr Thr Ser <210> 63 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 63 Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gin His Met Asp 25 Ser Pro Asp Leu. Gly Thr Asp Asp 40 Asp Asp Lys Ala Met <210> 64 <211> <212> PRT <213> Artificial Sequence <220> WO 02/038594 PCT/AUOI/01462 55 <223> Description of Artificial Sequence: Peptide <400> 64 Met His

I

His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gin His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp 40 Asp Lys Ala Met Ala Asp Ile Gly Ser <210> <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> Glu Phe Asn Leu Ser Lys Gly <210> <211> <212> <213> 66 17

PRT

Artificial Sequence Description of Artificial Sequence: Peptide <220> <223> <400> 66 Met Gly Ser Gly Ser Gly Asp Asp Asp Asp Lys Leu Ala Leu Gly His Met <210> 67 WO 02/038594 WO 02/38594PCT/AUOI/01462 -56- <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 67 Arg Thr Ser Thr Gly Ile Ala <210> 68 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial sequence: Peptide <400> 68 Asp Pro Asn Ser Ser Ser Val Asp Lye Leu Ala Ala Ala Leu Glu His 1 5 10 His His His His His

Claims (37)

1. An isolated or recombinant immunogenic polypeptide comprising a Lawsonia spp. polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, wherein said polypeptide is capable of conferring a protective immune response against Lawsonia spp. when administered to an avian or porcine animal.

2. The isolated or recombinant immunogenic polypeptide of claim 1 capable of eliciting the production of antibodies against Lawsonia spp. when administered to an avian or porcine animal.

3. The isolated or recombinant immunogenic polypeptide of claim 1 wherein the Lawsonia spp. is L. intracellularis. S 15 4. The isolated or recombinant immunogenic polypeptide of claim 2 wherein the Lawsonia spp. is L. intracellularis. An isolated or recombinant polypeptide selected from the group consisting of: a polypeptide of Lawsonia spp. which comprises an amino acid sequence 20 which has at least about 70% sequence identity overall to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; (ii) a polypeptide of Lawsonia spp. which comprises an amino acid sequence which has at least about 70% sequence identity overall to an amino acid sequence encoded by Lawsonia intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); (iii) a polypeptide which comprises at least about 5 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NOs: P:\OPER'JMS\SPECIFICA71ONS\1481I0-02.DOC 2/2/05 84 2, 4,6, 8, 10, 12, 14, 16, and 18; (iv) a polypeptide which comprises at least about 5 contiguous amino acids of an amino acid sequence encoded by Lawsonia intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NMOO/16476 (plasmid pGTE#1 glnH); NMOO/16477 (plasmid pGTE#2 flhB); NMOO/1 6478 (plasmid pGTE#3 MiR); NMOO/1 6479 (plasmid pGTE#4 motAIB); NM0O/16480 (plasmid pGTE#5 tlyC); NMO0/16481 (plasmid pGTE#6 ntrC); NMOO/16482 (plasmid pGTE#7 ytfM); and NMV01/23286 (plasmid pGTE#8 yffN); and a polypeptide which comprises an amino acid sequence encoded by a nucleotide sequence of Lawsonia spp. having at least about 80% sequence identity overall to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5,7, 9,11, 13,15, and 17; (vi) a polypeptide which comprises an amino acid sequence encoded by a nucleotide sequence of Lawsonia spp. having at least about 80% sequence identity overall to the nucleotide sequence of Lawsonia intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NMOO/1 6476 (plasmid pGTE#1 glnH); NM0O/16477 (plasmid pGTE#2 flhB); NM0O/16478 (plasmid pGTE#3 MiR); NMOO/16479 (plasmid pGTE#4 motAIB); NMO0/16480 (plasmid pGTE#5 tlyC); NMOO/16481 (plasmid :pGTE#6 ntrC); NMOO/16482 (plasmid pGTE#7 ytfM); and NMO11/23286 (plasmid pGTE#8 yffN); (vii) a polypeptide encoded by at least about 15 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5,7, 9,11, 13, 15,andl17; (viii) a polypeptide encoded by at least about 15 contiguous nucleotides of a nucleotide sequence of Lawsonia intracellulanis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NMOO/16476 (plasmid pGTE#1 glnH); NMO0/16477 (plasmid pGTE#2 fl hB); NMOO/16478 (plasmid pGTE#3 fliR); NM0O/1 6479 (plasmid pGTE#4 motAIB); NMOO/1 6480 (plasmid pGTE#5 tlyC); NMOO/16481 (plasmid pGTE#6 ntrC); NMOO/16482 (plasmid pGTE#7 ytfM); and NMO11/23286 (plasmid pGTE#8 ytfN); and P:\OPERVMS\SPECIFICATnONS\1481-02.DOC 2//OS (ix) a homologue, analogue or derivative of any one of to (vii) which mimics a B-cell or T-cell epitope of Lawsonia spp., capable of conferring a protective immune response against Lawsonia spp. when administered to a porcine or avian animal.

6. The isolated or recombinant polypeptide of claim 5 capable of eliciting the production of antibodies against Lawsonia spp. when administered to a porcine or avian animal.

7. The isolated or recombinant polypeptide of claim 5 wherein the Lawsonia spp. is Lawsonia intracellularis.

8. The isolated or recombinant polypeptide of claim 6 wherein the Lawsonia spp. is Lawsonia intracellularis.

9. The isolated or recombinant polypeptide of claim 5 comprising an amino acid sequence selected from the group consisting of: an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; and S 20 (ii) an amino acid sequence encoded by Lawsonia intracellularis DNA :contained within a deposited plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 gInH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 :"(plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN), capable of inducing a protective immune response against Lawsonia intracellularis when administered to a porcine or avian animal.

10. The isolated or recombinant polypeptide of claim 9 capable of eliciting the production of antibodies against Lawsonia intracellularis when administered to an avian or porcine animal. P:\OPER\JMS\SPECIFICATIONS\14810-02.DOC- 2t/OS -86-

11. A vaccine composition for the prophylaxis or treatment of infection of an animal by Lawsonia spp., said vaccine composition comprising an immunogenic component which comprises the isolated or recombinant immunogenic polypeptide according to claim 1 in combination with one or more carriers, diluents or adjuvants suitable for veterinary or pharmaceutical use.

12. The vaccine composition according to claim 11 wherein the Lawsonia spp. is Lawsonia intracellularis.

13. The vaccine composition according to claim 11 wherein the immunogenic component comprises an isolated or recombinant polypeptide having an amino acid sequence selected from the group consisting of: an amino acid sequence selected from the group consisting of SEQ 15 ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; and an amino acid sequence encoded by Lawsonia intracellularis DNA contained within a deposited plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 S0 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 20 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 yffN).

14. The vaccine composition of claim 11, wherein the immunogenic component is a recombinant polypeptide which is capable of conferring a protective immune response against Lawsonia spp. when administered to a porcine or avian animal, expressed in a cell that has been transfected with a vector comprising a nucleotide sequence selected from the group consisting of: a protein-encoding nucleotide sequence having at least about sequence identity overall to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; (ii) a protein-encoding nucleotide sequence having at least about P:OPERVMS\SPECW1CATIONSkI481 2.DOC M105 -87- sequence identity overall to the protein-encoding sequence of Lawsonia intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); (iii) a protein-encoding nucleotide sequence which comprises at least about contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; (iv)a protein-encoding nucleotide sequence which comprises at least about contiguous nucleotides of the protein-encoding sequence of Lawsonia intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); 15 NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); .NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); a protein-encoding nucleotide sequence which hybridizes under at least 20 moderate stringency conditions to the complement of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,15, and .17; (vi)a protein-encoding nucleotide sequence which hybridizes under at least moderate stringency conditions to the non-coding strand of Lawsonia 25 intracellularis DNA contained within a p plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); and (vii) a homologue, analogue or derivative of any one of to (vi) which encodes a polypeptide which mimics a B-cell or T-cell epitope of Lawsonia spp. P:\OPER\JMS\SPECIFICAONS\14810-02DOC 2/2/OS -88- A combination vaccine composition for the prophylaxis or treatment of infection of an animal by Lawsonia spp., said vaccine composition comprising: a first immunogenic component which comprises the isolated or recombinant polypeptide having according to claim 1; (ii) a second immunogenic component different from said first immunogenic component and comprising a polypeptide selected from the group consisting of the Lawsonia intracellularis FIgE, hemolysin, OmpH, SodC, flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides; and (iii)one or more carriers, diluents or adjuvants suitable for veterinary or pharmaceutical use.

16. A vaccine vector that comprises, in an expressible form, an isolated nucleic acid molecule comprising a nucleotide sequence which encodes a polypeptide capable 15 of conferring a protective immune response against Lawsonia spp. when administered to a porcine or avian animal, selected from the group consisting of: a protein-encoding nucleotide sequence having at least about sequence identity overall to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; 20 (ii) a protein-encoding nucleotide sequence having at least about sequence identity overall to the protein-encoding sequence of Lawsonia intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); 25 NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); (iii) a protein-encoding nucleotide sequence which comprises at least about contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; (iv)a protein-encoding nucleotide sequence which comprises at least about contiguous nucleotides of the protein-encoding sequence of Lawsonia P:\OPER\MSSPECIFICATIONSI 4810-02.DOC 2/2/O5 -89- intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); a protein-encoding nucleotide sequence which hybridizes under at least moderate stringency conditions to the complement of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; (vi)a protein-encoding nucleotide sequence which hybridizes under at least moderate stringency conditions to the non-coding strand of Lawsonia intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); 15 NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); and (vii) a homologue, analogue or derivative of any one of to (vi) which encodes a 20 polypeptide which mimics a B-cell or T-cell epitope of Lawsonia spp.

17. The vaccine vector of claim 16 wherein the immunogenic polypeptide is expressed by a process comprising: placing an isolated nucleic acid molecule in an expressible format, said 25 nucleic acid molecule comprising the coding region of a gene selected from the group consisting of flhB, fliR, ntrC, gInH, motA, motB, tlyC, ytfM, and ytfN genes, or a protein-encoding homologue, analogue or derivative thereof; (ii) introducing the isolated nucleic acid molecule of in an expressible format into a suitable vaccine vector; and (iii) incubating or growing the vaccine vector for a time and under conditions sufficient for expression of the immunogenic component encoded by said nucleic acid molecule to occur. P:\OPER\JMS\SPECIFlCATIONS\14S -02DOC 2/2OS

18. The vaccine vector of claim 16 wherein the Lawsonia spp. is L. intracellularis.

19. An isolated polyclonal antibody or a monoclonal antibody molecule that binds specifically to a Lawsonia spp. polypeptide selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, or a homologue, analogue or derivative of any one or more of said polypeptides. The isolated polyclonal antibody or a monoclonal antibody molecule of claim 19 wherein the polypeptide or derivative thereof is capable of conferring a protective immune response against Lawsonia spp. when administered to a porcine or avian animal, and comprises an amino acid sequence selected from the group consisting of: an amino acid sequence of Lawsonia sp. which has at least about S 15 sequence identity overall to a sequence selected from the group consisting of SSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; (ii) an amino acid sequence of Lawsonia sp. which has at least about sequence identity overall to a sequence encoded by L. intracellularis DNA Scontained within a plasmid selected from the group consisting of AGAL 20 Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); (iii) an amino acid sequence which comprises at least about 5 contiguous amino acids of a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; (iv)an amino acid sequence which comprises at least about 5 contiguous amino acids of a sequence encoded by L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); P:\OPER\IMSSPECIFCAONSl 4810O-02.DOC 2/205 -91 NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); an amino acid sequence which is encoded by a nucleotide sequence having at least about 80% sequence identity overall to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; (vi) an amino acid sequence which is encoded by a nucleotide sequence having at least about 80% sequence identity overall to the nucleotide sequence of L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); (vii) an amino acid sequence which is encoded by at least about 15 contiguous ,nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; an amino acid sequence which is encoded by at least about 15 contiguous nucleotides of a nucleotide sequence of L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NMI01/23286 (plasmid pGTE#8 ytfN); and (ix) a homologue, analogue or derivative of any one of to (viii) which mimics a B-cell or T-cell epitope of Lawsonia spp.

21. A method of diagnosing infection of a porcine or avian animal by Lawsonia intracellularis or a microorganism that is immunologically cross-reactive thereto, said method comprising the steps of contacting a biological sample derived from said P:\OPERUMS\SPECIFICATIONS 4810-02. DOC 2/2/O5 -92- animal with the antibody molecule of claim 19 for a time and under conditions sufficient for an antigen:antibody complex to form, and then detecting said complex formation.

22. The method of claim 21 wherein the biological sample comprises whole serum, lymph nodes, ileum, caecum, small intestine, large intestine, faeces or a rectal swab derived from a porcine animal.

23. A method of identifying whether or not a porcine or avian animal has suffered from a past infection, or is currently infected, with Lawsonia intracellularis or a microorganism that is immunologically cross-reactive thereto, said method comprising contacting blood or serum derived from said animal with the immunogenic polypeptide of claim 1 for a time and under conditions sufficient for an antigen:antibody complex to form and then detecting said complex formation.

24. An isolated nucleic acid molecule which consists of a nucleotide sequence encoding a Lawsonia spp. polypeptide selected from the group consisting of flhB, SfliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN, wherein said polypeptide is capable of conferring a protective immune response against Lawsonia spp. when 20 administered to a porcine or avian animal. 0*0* 0* 0

25. The isolated nucleic acid molecule according to claim 24 comprising a sequence of nucleotides which encodes a polypeptide capable of conferring a 0 protective immune response against Lawsonia spp. when administered to a porcine or avian animal selected from the group consisting of: a nucleotide sequence having at least about 80% sequence identity overall to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; (ii) a nucleotide sequence having at least about 80% sequence identity overall to L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); P:\OPER\JMS'SPECIFICATIONS\14810-02. DOC 2/2/0 93 NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and; (iii) a nucleotide sequence which comprises at least about 15 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; (iv)a nucleotide sequence which comprises at least about 15 contiguous nucleotides of L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); a nucleotide sequence which hybridizes under at least moderate stringency 15 conditions to a nucleotide sequence selected from the group consisting of SEQ SID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17 or a complementary nucleotide sequence thereto; (vi)a nucleotide sequence which hybridizes under at least moderate stringency conditions to L. intracellularis DNA contained within a plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); and (vii) a homologue, analogue or derivative of any one of to (vi) which encodes a polypeptide which mimics a B-cell or T-cell epitope of Lawsonia spp.

26. The isolated nucleic acid molecule of claim 25 comprising a nucleotide sequence selected from the group consisting of: a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1,3, 5, 7, 9, 11, 13, 15, and 17; (ii) a nucleotide sequence of the L. intracellularis DNA contained within a P:\OPER\JMS\SPECIFICATIONS\I4810-O2.DOC- 2/2/05 -94- deposited plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); (iii) a nucleotide sequence that encodes the same polypeptide as a nucleotide sequence of or wherein said polypeptide is selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides; and (iv)a nucleotide sequence that is complementary to or (ii) or (iii).

27. The isolated nucleic acid molecule of claim 26 consisting of the protein- encoding region of or (ii).

28. A method of detecting Lawsonia intracellularis or related microorganism in a biological sample derived from a porcine or avian animal subject, said method S' comprising the steps of hybridising one or more probes or primers to said sample and then detecting said hybridisation using a detection means, wherein said probes or primers are derived from a Lawsonia spp. gene selected from the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN genes. 4 S

29. The method of claim 28 wherein the biological sample comprises whole serum, lymph nodes, ileum, caecum, small intestine, large intestine, faeces or a rectal swab derived from a porcine animal. The method of claim 28 wherein the detection means comprises any nucleic acid based hybridisation or amplification reaction.

31. A probe or primer comprising a nucleotide sequence selected from the group consisting of: any one of SEQ ID NOs: 19 to 68; and P:\OPERVMS\S PECFICAT1ONS\l4810-02.DOC 2/2/5O (ii) a complementary nucleotide sequence to

32. A plasmid selected from the group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 gInH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN).

33. A recombinant vector capable of replication in a host cell, wherein said vector comprises the isolated nucleic acid of claim 24.

34. A recombinant vector capable of replication in a host cell, wherein said vector comprises the isolated nucleic acid of claim

35. A recombinant vector capable of replication in a host cell, wherein said vector S* comprises the isolated nucleic acid of claim 26. .36. A recombinant vector capable of replication in a host cell, wherein said vector S 20 comprises the isolated nucleic acid of claim 27.

37. An isolated host cell comprising the recombinant vector of claim 33.

38. The host cell of claim 37 wherein said host cell is a bacterium.

39. An isolated host cell comprising the recombinant vector of claim 34. The host cell of claim 39 wherein said host cell is a bacterium.

41. An isolated host cell comprising the recombinant vector of claim

42. The host cell of claim 41 wherein said host cell is a bacterium. P \OPER\JMS\SPECIFICATIONS\14810-02.DOC 2/205 -96-

43. An isolated host cell comprising the recombinant vector of claim 36.

44. The host cell of claim 43 wherein said host cell is a bacterium. Dated this 2 nd day of February 2005 Agriculture Victoria Services Pty. Ltd., Australian Pork Limited AND Pfizer Products, Inc. By their Patent Attorneys Davies Collison Cave o