BACKGROUND OF THE INVENTION Swine dysentery is a highly contagious diarrheal disease of growing and finishing pigs which is caused by the strong P-hemolytic, anaerobic, oxygen tolerant spirochete bacterium, Serpulina hyodysenteriae (Harris et al., 1972a; Harris et al., 1972b; Kinyon et al., 1977; Taylor et al., 1980; Taylor and Alexander, 1971; Harris and Lysons, 1992; Stanton et al., 1991; Stanton, 1992). Following colonization of the colonic mucosal surface, S. hyodysenteriae causes epithelial cell necrosis which results in severe mucohemorrhagic diarrhea (Harris et al., 1972a; Kinyon et al., 1977; Whipp et al., 1978).

Affected pigs have decreased weight gain, lower feed efficiency, and 30 percent of these animals may die because of severe dehydration (Harris and Lysons, 1992). The spirochete is transmitted by ingestion of contaminated feces, and on farms where the disease is enzootic, it is perpetuated by the addition of previously unexposed pigs. Using healthy pigs as sentinels, a carrier status has been shown to occur for up to 90 days following recovery from clinical swine dysentery (Songer and Harris, 1978; Fisher and Olander, 1981). These subclinically infected carrier pigs are important biological vectors of S. hyodysenteriae on infected farms, and also can transmit swine dysentery to naive pigs on farms free of the disease.

When introduced into a farm free of swine dysentery, S. hyodysenteriae quickly becomes established in the pig population and the environment, requiring continuous medication at a cost of up to approximately $8.84 per animal (Duhamel et al., 1993).

Financial losses result not only because of the cost of medication but also because of

additional labor, reduced feed efficiency, lower weight gain, animal death, loss of sales of breeding stock, and depopulation of the farm when necessary. The economic losses to Iowa pork producers associated with swine dysentery were estimated a decade ago to be approximately $2.4 million monthly (Owen, 1987).

Although the causative agent of swine dysentery has been known for nearly 25 years, no effective measures, other than medication of sick animals and sanitation of premises have been implemented to control the disease (Duhamel et al. 1993; Harris and Lysons, 1992). Careful management and continuous medication can help controlling the disease; however, recurrence of swine dysentery after withdrawal of drug therapy is common. Progressive failure of control measures resulting from drug resistance and reinfection of medicated pigs from environmental contamination emphasizes the short term value of chemotherapy alone for the control of swine dysentery. The negative view that consumers of pork are taking toward long term medication with residue-causing chemicals is an additional reason to support alternative control strategies for this disease.

The pathogenic mechanisms involved in S. hyodysenteriae colonization and development of colonic and cecal lesions have not been completely elucidated. Several virulence factors have been proposed, including chemotactic-regulated motility, adhesion, lipooligosaccharide, endotoxin, hemolysin and cytotoxin (Binek et al., 1986; Charon et al., 1992; Kennedy et al., 1988; Knoop et al., 1979; Lysons et al., 1991; Milner and Sellwood, 1994; Nuessen et al., 1983; terHuurne and Gaastra, 1995; terHuurne et al., 1992; Witter and Duhamel, 1996; Sacco et al., 1996). Some of these factors are assumed to be involved in the initial association of the spirochetes with the mucosal surface of the lower intestine of the pig (Kennedy et al., 1988). Motility of S. hyodysenteriae is by means of eight to thirteen periplasmic flagella (Harris and Lysons, 1992; Charon et al., 1992). Specific disruption of the flagellar genes, designated flaAl and flaB1, using gene replacement, results in mutant strains with morphologically intact flagella, but with altered motility (Rosey et al., 1995; Rosey et al., 1996). However, the role of altered motility in the pathogenesis of S. hyodysenteriae infection of a murine model of swine dysentery has not been completely elucidated. Similarly, adhesion has been implicated as a virulence factor in S. hyodysenteriae infection (Knoop et al., 1979; Binek et al., 1986; Bowden et al, 1989), but no specific adhesin has been identified. Production of a strong p-hemolysis by S. hyodysenteriae grown on blood agar plates correlates with

virulence in mice and pigs (Kinyon et al., 1977; Whipp et al., 1978). High concentrations of hemolysin and cytotoxin are present in cell free supernatant (CFS) from S. hyodysenteriae incubated in a defined medium (Lemcke and Burrows, 1982; Witters and Duhamel, 1996). The hemolysin and cytotoxin produced by S. hyodysenteriae may be important virulence factors involved in the pathogenesis of swine dysentery (Kent et al., 1988; Knoop, F. C., 1981 ; Muir et al., 1992; Wilcock and Olander, 1979). This is because epithelial damage similar to that seen in the natural disease has been reproduced by inoculation of pig gut loops with CFS from S. hyodysenteriae (Lysons et al., 1991 ; Bland et al., 1995). The native hemolysin produced by S. hyodysenteriae in CFS has been extensively studied (Dupont et al., 1994; Kent et al., 1988; Knoop, F. C., 1981 ; Muir et al., 1992; Saheb et al., 1981a ; Saheb et al., 1981b ; Saheb et al., 1980; Zhang et al., 1995; Witters and Duhamel, 1996). To date three distinct putative S. hyodysenteriae hemolysin genes, designated, lyB and tlyC, have been cloned and sequenced (Muir et al., 1992; terHuurne et al., 1994). Inactivation of the S. hyodysenteriae tlyA gene, encoding a 26.9- k-Da protein with hemolytic and cytotoxic activities for various cell cultures, caused a decrease in hemolysin activity in vitro (terHuurne et al., 1992) and decreased colonization and virulence in a murine model of swine dysentery (terHuurne et al., 1992) and in challenge-exposed pigs (Hyatt et al., 1994). However, this mutant was not completely avirulent and regained hemolytic and cytotoxic activities after several passages in vitro (terHuurne and Gaastra, 1995).

Specific disruption of S. hyodysenteriae genes by electroporation-mediated gene replacement or allelic exchange has been successful with at least two separate genetic systems, namely the tlyA gene encoding a putative hemolysin (terHuurne et al., 1992) and the flaA1 and the flaB1 and flaA1/flaB1 dual inactivation of the genes respectively encoding flagellar sheath and core proteins (Rosey et al., 1995; Rosey et al., 1996). This system of genetic exchange depends on homologous recombination between a known chromosomal gene sequence and its homologous disrupted counterpart placed in a suitable delivery vector. Selectable markers, such as kanamycin and chloramphenicol acetyltransferase, are suitable for identification of S. hyodysenteriae mutants with one or more specific gene deletions. These advances in molecular genetic of spirochetes have allowed studies of virulence mechanisms involved in the pathogenesis of swine dysentery.

It has been shown that if pigs are allowed to recover naturally from swine dysentery, without medication, they are immune to rechallenge for up to 17 weeks post- recovery (Joens et al, 1979; Olson, 1974). Despite these results, immunoprophylaxis using a S. hyodysenteriae whole-cell bacterin (Hy-Guardg, Haver, Mobay Corporation, Shawnee, KS) has not provided the anticipated results (Parizek et al., 1985). Vaccination with this whole-cell product was shown to provide less than 50 percent protection against development of clinical disease in challenge-exposed pigs, as compared to controls. As a result, this product is no longer commercially available. Since vaccines are not currently available for prevention of swine dysentery, transmission of the disease is prevented by strict biosecurity, including surgically-derived replacement stock. When pigs of unknown status are used as replacement stock, quarantine and medication with various therapeutic compounds known to be effective against S. hyodysenteriae are provided prior to commingling these animals with pigs free of swine dysentery.

Based on a review of the literature, it is fair to conclude that none of the currently available methods for prevention of swine dysentery are completely adequate.

Development of a safe and efficacious vaccine offers an attractive alternative to continuous medication of animals with expensive residue-causing antibiotics; however, it is conceivable that development of protective immunity to swine dysentery will require development of sufficient titers of neutralizing antibodies to virulence factors produced by enteropathogenic spirochetes during infection. It is therefore an object of the present invention to provide a a live attenuated S. hyodysenteriae vaccine in order to provide such protective immunity. Such vaccine could reduce the severity of the disease on farms where the disease is enzootic and cannot be controlled with antimicrobial therapy alone.

SUMMARY OF THE INVENTION The virulence attenuated S. hyodysenteriae mutant strains according to the present invention are provided by inactivating a gene associated with virulence. Foreign DNA, such as a DNA sequence conferring antimicrobial resistance, is placed into the virulence associated gene sequence by homologous recombination. One immunopositive clone from an expression plasmid library of S. hyodysenteriae genomic DNA, constructed in Escherichia coli, was identified and the DNA insert from that clone hybridized with HindIII digested genomic DNA of S. hyodysenteriae serotypes 1 through 7. Based on the DNA sequence of the cloned S. hyodysenteriae gene, a kanamycin resistance cassette was

inserted into a unique PstI site of the gene in pUC18, and isogenic mutants of S hvodysenteriae were generated by gene replacement. When examined in a murine model of swine dysentery, the mutants had significantly reduced virulence. Hence, the gene was designated virulence-associated gene A (virA) and the S. hvodvsenteriae virA mutants with attenuated virulence phenotypes are potential live attenuated vaccines for prevention of swine dysentery.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts the nucleotide sequence and deduced amino acid sequence of the Serpulina hyodvsenteriae virA gene open reading frame (ORF) (SEQ ID NO : 1). A putative ribosomal binding site (RBS) is underlined. A putative amino acid leader cleavage sequence is shadowed at positions 1 to 16 of the ORF. The PstI restriction enzyme site which was used for the insertion of the kanamycin cassette is indicated.

FIG. 2 depicts determination of virA gene copy number using Southern blot hybridization of [a-32P] dCTP labeled EcoRI-PstI digested fragment (bp 710-1939) of pRED3C6 (virA gene) as a probe. Serpulina hyodysenteriae chromosomal DNA was digested with each restriction enzyme, electrophoresed in a 0.8 percent agarose gel and transferred to a nylon membrane. Lane 1 is molecular size markers (in kilobases); Lanes 2-4, chromosomal digestion with PvuII, Alwl and ScaI, respectively.

FIG. 3 depicts a schematic illustration of a preferred method of targeted disruption of Serpulina hyodysenteriae virA gene by homologous recombination according to the present invention. The kanamycin antibiotic gene cassette is indicated by double stippled bars, solid bars represent the virA open reading frame targeted for disruption by homologous recombination, single stippled bars represent flanking sequences. Horizontal arrows denote the orientation of the antibiotic and virA genes. The orientation and approximate binding sites of oligonucleotide primers for PCR amplification and the predicted products in base pairs are indicated by arrowheads. Restriction endonuclease sites used during cloning and Southern blot hybridizations are indicated. Abbreviations for restriction enzyme sites are as follows: A, AlwI ; E, EcoRI ; H, HindIII ; P, PstI ; S, ScaI.

FIG. 4 shows an electrophoretic analysis of PCR amplified products derived from Serpulina hyodysenteriae parent strain and virA mutant. Primer sets used to generate these products are schematically represented in FIG. 3 were reacted with the respective genomic DNA templates as follows: Lane 1: Molecular size standard (1-kb DNA ladder,

GIBCO-BRL); Lane 2: [REP1, REP2] virA mutant; Lane 3: [REP1, REP2] B204 parent virA ; Lane 4: [REK1, REP2] B204 parent virA ; Lane 5: [REK1, REP2] virA mutant.

FIG. 5 shows hemolysin, lactic dehydrogenase (LDH) and cytotoxin (MTT) titers of cell-free supernatants from Serpulina hyodysenteriae parent strain B204 and virA mutant strain 1 B2R, as determined respectively on the basis of hemoglobin release by red blood cells, and LDH release and MTT-dye reduction by peripheral blood lymphocytes obtained from 5-week-old pigs. Error bars indicate standard errors of the means.

FIG. 6 shows mean longitudinal crypt length (LCL) of cecal crypt columns of C3H/HeN mice fed a define diet (Teklad Diet TD 85420; Harlan Teklad) and orally inoculated with either sterile medium, Serpulina hyodysenteriae parent strain B204, or virA mutant strain 1B2R. Each bar represents the percentage mean LCL of three S. hvodysenteriae-inoculated and three sterile medium-inoculated mice, with asterisks (*) indicating significant (P < 0.05) differences between each group.

FIG. 7 shows mean longitudinal crypt length (LCL) of cecal crypt columns of C3H/HeN mice fed a conventional diet (Purina Lab Chow; Purina Mills) and orally inoculated with either sterile medium, Serpulina hyodysenteriae parent strain B204, or virA mutant strains 1B2R or A1CB. Each bar represents the percentage mean LCL of three S. hyodysenteriae-inoculated and three sterile medium-inoculated mice, with asterisks (*) indicating significant (P < 0.05) differences between each group.

DESCRIPTION OF THE INVENTION We have described a clone, designated pRED3C6, containing a DNA sequence unique to S. hyodysenteriae (Elder et al., 1994; Nucleotide Sequence and Methods for Detection of Serpulina hvodvsenteriae, Serial No. 08/252,492, filed June 1,1994).

Briefly, genomic DNA from S. hyodysenteriae was isolated and digested with a restriction enzyme. Restriction fragments were size fractionated on a sucrose gradient.

Fragments between 4-and 9-kb were ligated to enzyme-digested and phophatase-treated pUC18 DNA (R. A. Kunkle, Ph. D. dissertation, University of Nebraska, 1993).

Meanwhile, a mouse monoclonal antibody (MAb), designated 1 OG6/G10, producing IgM antibodies that reacted with cell-free supernatant antigens from S. hyodysenteriae isolate B204, was identified. The procedures for production and identification of MAb 10G6/G10 are as described (Elder et al., 1994). The recombinant clones produced by transformation of Escherichia coli DH5a with S. hyodysenteriae DNA fragments ligated

into the plasmid vector pUC 18 were screened for antigenic expression by colony immunoblotting with the MAb 10G6/G10. One immunopositive clone, designated pRED3C6, was identified by Robert O. Elder on December 12,1991, based on development of a dark purple precipitate (R. O. Elder, M. S. dissertation, University of Nebraska, 1993). Southern blot analyses of genomic DNA obtained from S. hyodysenteriae serotypes 1 through 7, S. innocens, S. pilosicoli, and Treponema succinifaciens, with the 2.3-kb fragment from pRED3C6 revealed that the cloned sequence was unique to S. hvodvsenteriae. Further analyses of genomic DNA from spirochetes and intestinal bacteria belonging to other taxons by specific PCR amplification of the cloned sequence confirmed that it was unique to S. hyodysenteriae.

This discovery provides further evidence that the nucleotide and deduced amino acid sequence of this unique DNA fragment of S. hyodysenteriae had no overall homologies with any other spirochetal or bacterial sequences in the GenBank, but had an open reading frame encoding a protein with an N-terminal sequence motif suggestive of a signal sequence for signal peptidase 11 cleavage; a characteristic of gram-negative bacterial lipoproteins. This would suggest that the product of this gene may be post- translationally modified and might be membrane-associated or destined for export outside of the spirochete. The S. hvodvsenteriae-specific DNA sequence has been found in all known S. hyodysenteriae serotypes and serogroups (Elder et al., 1994; personal observations). To determine the role of this gene in the pathogenesis of swine dysentery, S. hyodysenteriae mutant strains were produced by specific gene replacement/inactivation. Inactivation of this unique gene appeared to upregulate hemolysin and cytotoxin production by S. hyodysenteriae ; isogenic mutants of S. hyodysenteriae displayed significantly higher in vitro hemolysin and cytotoxin titers, as determined by quantitative measurements of these activities in cell free supernatant.

However, when the same S. hyodysenteriae mutant strains were tested in a well characterized laboratory animal model for swine dysentery (Joens and Glock, 1979; Mysore and Duhamel, 1994; Nibbelink and Wannemuehler, 1992), they had significantly attenuated virulence phenotypes in vivo compared with the S. hyodysenteriae parent strain, as determined by quantitative measurements of cecal lesions. Some virulence factors involved in colonization of the colon, including chemotaxis-regulated motility and adherence might be altered in the mutant strains. However, the recovery rates of the S.

hyodysenteriae parent strains were not different from that of the mutant strains, suggesting that colonization of the mouse cecum by the S. hyodysenteriae mutants was not affected. Therefore, based on the molecular composition of this unique DNA fragment and the altered virulence phenotype of gene disrupted mutants, this novel gene was designated virulence-associated gene A (virA), making the S. hyodvsenteriae virA mutants potential live attenuated vaccines for prevention of swine dysentery.

Serpulina hyodysenteriae strain B204 serotype 2 was obtained from J. M. Kinyon, College of Veterinary Medicine, Iowa State University, Ames. The Escherichia coli DH5cs was purchased from a commercial source (GIBCO-BRL, Gaithersburg, Md.).

Propagation and isolation of genomic DNA from S. hyodysenteriae and E. coli DH5a were as previously described (Elder, et al. 1994). Bacteria, plasmids, and oligonucleotides used are presented in Table 1.

TABLE 1 Bacteria, plasmids and oligodeoxyribonucelotides.

Strain, plasmid or Relevant characteristics, derivation Reference or source oligonucleotide Escherichia coli DHSaTM F- 80dlacZOMlS A (lacZYA-argF) U169 Gibco-BRL deoR recAl endAI phoA hsaR17 (rK, mK) supE44'th)-l gyrA96 relAl S. hyodysenteriae B204 Serotype 2 (ATCC 31212) Laboratory stock Pig-passage virulent isolate 28Y experimental challenge in swine 1B2R Homologous recombination of pRED3C6Qkan This study at B204 chromosomal virA locus ID2A Homologous recombination of pRED3C6Qkan This study at B204 chromosomal virA locus A I CB Homologous recombination of pRED3C6Qkan This study at B204 chromosomal virA locus Plasmids pUC4K Plasmid containing the drug resistance gene Pharmacia Biotech from transposon Tn903 conferring resistance to kanamycin pRED3C6 pUC18 with a 2,332-bp HindIII chromosomal Elder et al., 1994 fragment containing virA pRED3C6Qkan pRED3C6 with kan gene insertion at PstI This study site (FIG. 1) Oligonucleotides REP1 5'-ACTGCATGTGCTTCTTCAAACC-3' [yirA, This study (+) bp341] a REP2 5'-AAGGTCTGTTTCCGCCTGTACC-3'This study [virA, (-) bp776] a REK1 5'-CAACAAAGCCGCCGTCCC-3'This study [kan, (+) bp650] a The target gene, strand and hybridization region are indicated for each oligonucleotide in brackets.

Nucleotide sequence analyses of the insert DNA from pRED3C6 at The Center for Biotechnology, DNA Sequencing Core Research Facility of the University of Nebraska-Lincoln, revealed a recombinant 2.3-kb DNA fragment with a single putative open reading frame (ORF) of 1098 bases (bp 291 to 1401; FIG. 1). Comparison of the

virA gene sequence with available sequences in the GenBank revealed no significant nucleotide or deduced amino acid sequence identity to other bacterial taxons. The ORF was predicted to encode a single protein composed of 367 amino acid residues with a predicted molecular weight of approximately 40.5-kDa (FIG. 1). The deduced protein had an isoelectric point of 5.38 and an overall charge of-5. Data base searches, using the MOTIFS program of the GCG, identified a putative leader signal cleavage sequence (MEYGMNGPNYSTVTAC) at amino acids 1 to 16, which would be cleaved by a specific lipoprotein signal peptidase (signal peptidase II). The putative signal peptidase recognition site underlined in FIG. 1 shows the cysteine residue where a glyceride-fatty acid lipid could be attached. This motif sequence has been identified in several gram- negative bacteria (Hayashi and Wu, 1990). During processing of the precursor protein, three fatty acid residues are covalently linked to the N-terminal cystine (Hantke and Braun, 1973). These lipoproteins can be membrane-associated or exported to the outside of the cell. An alanine residue at position 2 of the putative mature VirA lipoprotein (FIG.

1) indicate a Cys-Ala motif similar to that of Bacillus lichenifonnis P-lactamase, a lipoprotein secreted into the medium (Luirink et al., 1987) and the outer membrane- associated Osp B lipoprotein of Borrelia burgdorferi (Hayashi and Wu, 1990). A similar motif also has been identified in a 16-kDa surface-exposed lipoprotein common to many S. hyodysenteriae strains (Thomas et al., 1992; Thomas and Sellwood, 1993; Turner et al., 1995). This protein, designated smpA, has been characterized and the nucleotide sequence of the smpA gene has been determined (Thomas and Sellwood, 1993).

Convalescent-phase swine sera have been shown to contain antibodies that recognize SmpA. Comparisons of the sequence of the virA gene with the smpA gene revealed only 66 percent DNA homology and 44 percent and 24 percent amino acid similarity and identity, respectively. A potential ribosome-binding site (GGA) 10 bp upstream of the ATG start codon of the virA gene ORF (FIG. 1). No eubacterial related putative promoter sequences were found 5'to the virA gene ORF start codon.

Sequencing data, deduced amino acid sequence and database searching were done with the Genetics Computer Group package version 8.0 (September 1994).

To determine the number of copies of the virA gene in S. hyodysenteriae, an EcoRI-PstI fragment of the insert DNA from pRED3C6 (bp 712-1939) was radiolabeled and hybridized with S. hyodysenteriae genomic DNA digested with the restriction

enzymes AlwI, Bell. BglII, HaeIII, MboI, PvuII, ScaI and SI using Southern blotting, as previously described (Elder et al., 1994). The restriction enzymes AlwI, Bell. BglI, MboI and SI, which do not cut within the insert DNA, hybridized with the radiolabeled 1229-bp EcoRI-PstI digested fragment of the virA gene producing single bands of 4.1, 20.1,23.0,7. 2 and 18.1-kb, respectively. The hybridization pattern after digestion of S. hyodysenteriae genomic DNA with the restriction enzymes HaeIII, PvuII and ScaI, which cut only once within the insert DNA, produced bands of 6.2 and 24.5,2.3 and 3.5-kb, and 3.0 and 1 0. 4-kob, respectively (FIG. 2). These results confirmed that only one copy of the virA gene was present in S. hyodysenteriae strain B204.

For construction of S. hyodysenteriae virA mutants, the 1.3-kb Kanamycin Resistance GenBlock (Pharmacia, Piscataway, N. J.) from transposon Tn903 was digested with the restriction enzyme PstI. The GenBlock was inserted into the unique PstI site (bp 712) located within the open reading frame (ORF) of pRED3C6 S. hyodysenteriae DNA fragment (FIG. 3). The resulting plasmid derivative, designated pRED3C6Qkan, was used for transformation of S. hyodysenteriae by electroporation, as described by Rosey et al. (1995). Briefly, 250 ml of mid-logarithmic phase cultures were harvested by centrifugation at 10,000 g for 15 min. and resuspended in 1/10 volume of chilled 0.5 M sucrose. The cells were incubated on ice for 20 min., harvested by centrifugation and resuspended in 1/200 volume of cold 0.5 M sucrose.

The resulting cell suspensions were placed on ice until electroporation. For electroporation, 100 al of S. hyodysenteriae competent cells, in prechilled tubes, was mixed with 500 ng of pRED3C6Qkan. A control sample was prepared using 500 ng of pUC 18 DNA. Each preparation was transferred to 0.2-cm electroporation cuvettes (Bio- Rad, Hercules, Calif.) and pulsed with the pulse controller set at 2.0 kV, 25 tF and 200 ohms of resistance. These conditions resulted in field strengths of 20 kV/cm and time constants of 3.7 to 4.3 ms. Immediately after pulsing, the cells were transferred to anaerobic Hungate tubes containing 5 ml of PRAS and 4 percent fetal bovine serum.

After recovering for 8 to 16 hours at 37°C, selection of transformants was accomplished by adding 400 ug/ml of kanamycin to each culture. The cultures were incubated for an additional 12 hours, before plating onto trypticase soy agar containing 5 percent sheep red blood cells, 400 g/ml of kanamycin (TSABK) and incubated anaerobically in the Gas Pak Anaerobic System (Gibco) for 3 to 5 days. Single colony transformants were

isolated by stabbing the agar in areas of growth, using a 1.0 ml pipette and transferred to Hungate tubes containing 5 ml of PRAS medium, 400 llm/ml of kanamycin and 4 percent fetal bovine serum. Tubes with visible growth were aliquoted and frozen at-80°C. A negative control S. hyodysenteriae parent was electroporated with pUC18 DNA or no DNA and plated onto TSABK. An S. hyodysenteriae in vitro passage parent control strain was subjected to electroporation without DNA and plated onto TSAB without kanamycin. A total of 50 kanamycin resistant transformants were identified after electroporation and recovery on selective medium. Chromosomal gene replacement was confirmed by purifying genomic DNA from S. hyodysenteriae in vitro passage parent strain and eight randomly selected S. hyodYsenteriae virA mutants and performing genotypic characterization by PCR and Southern blot hybridization. On the basis of DNA sequence analyses of pRED3C6, several oligonucleotide primer sets were designed and synthesized (Integrated DNA Technologies, INC. Coralville, IA), for PCR amplification of regions encompassing the S. hyodYsenteriae virA gene and inserted antibiotic resistance marker. Purified genomic DNA of S. hyodysenteriae in vitro passage parent strain and virA mutants were amplified using a hot start PCR as described by the manufacturer (GeneAmp PCR System 480, Perkin Elmer, Norwalk, Conn.) in a total volume of 75 pl containing 4 mM MgCl2 ; 1X of PCR buffer; 0.2 mM of each dATP, dCTP, dGTP, dTTP (Perkin-Elmer); 75 pmol of primers; and 1.5 U of Tag DNA polymerase (Perkin-Elmer) in sterile filtered autoclaved water.

Initial denaturing was for 3 min. at 94°C, followed by 35 cycles (60 s at 94°C, 60 s at 57°C, 120 s at 72°C). The amplified products were visualized in 1.5 percent agarose gels ran at 3 V/cm and stained with ethidium bromide. The nucleotide sequence, target locus, and binding sites for each primer are presented in Table 1 and schematically illustrated in FIG. 3. Analyses of S. hyodYsenteriae virA mutants by PCR amplification confirmed that the virA gene disrupted by insertion of the Kanamycin Resistance GenBlock had recombined with the chromosomal virA gene causing specific gene disruptions in the mutants (FIG. 4). Three of the eight mutants shown to have the virA mutant genotype by PCR, and designated 1B2R, 1D2A and A1CB, were selected for Southern blot hybridization, as previously described (Elder et al., 1994). Briefly, purified genomic DNA from the S. hyodysenteriae in vitro passage parent strain and virA mutants were digested with the restriction endonuclease AccI, subjected to agarose gel

electrophoresis, transferred to nylon membranes and hybridized with [a-32P] dCTP- labeled S. hyodysenteriae virA gene probe prepared using a random oligonucleotide labeling kit (Pharmacia), as previously described (Elder et al., 1994). The membrane was exposed to X-OMAT AR Cronex radiograph film (Eastman Kodak Company, Rochester, N. Y.) in a cassette with lightning plus intensifying screens (DuPont, Wilmington, Del.) at-70°C. Probe DNA was spiked with a [« _32p] dCTP radiolabeled 1-kb ladder (Gibco) to allow direct radiographic detection of DNA mass standards following hybridization.

The virA probe hybridized with a 3.3-kb fragment of the S. hyodysenteriae parent strain and with a 4.6-kb fragment of the virA mutants. A kan gene probe hybridized with 1.0- kb and 1.8-kb fragments of genomic DNA obtained from the virA mutants digested with the restriction enzyme HindIII, but not with HindIII digested genomic DNA from the S. hyodysenteriae parent strain. These results are consistent with the predicted location of the kanamycin cassette and specific disruption of the virA gene in the mutant strains.

Cell-free supernatants (CFS) from S. hyodysenteriae in vitro passage parent strain and virA mutant strain 1B2R were produced in three separate experiments, as previously described (Dupont et al., 1994; Witters and Duhamel, 1996). The hemolysin and cytotoxin activities of each CFS were determined in duplicate using porcine red blood cells (RBC) and porcine peripheral blood lymphocytes (PBL), respectively, as previously described (Witters and Duhamel, 1996).

Results were expressed as hemolysin, lactic dehydrogenase (LDH) and cytotoxin (MTT) titers. The in vitro hemolysin and cytotoxin titers of CFS obtained from S. hyodysenteriae mutant were significantly (P < 0.0001) higher than the titers of CFS obtained from the S. hyodysenteriae parent strain; the virA mutant had average hemolysin and LDH titers of 796.4 and 365.6, respectively, compared with 426.7 and 146.1 with the S. hyodysenteriae parent strain, respectively (FIG. 5). Similarly, the virA mutant average MTT titer was 398.2 compared to 104.9 with the S. hyodysenteriae parent (FIG. 5). In separate experiments, the hemolysin and cytotoxin titers of CFS from the virA mutant strains 1D2A and A1CB were compared with that of the S. hyodysenteriae in vitro passage parent strain. The CFS of the virA mutants 1D2A and A1CB had significantly (P < 0.0001) higher hemolysin and cytotoxin titers than the titers of CFS obtained from the S. hyodysenteriae in vitro passage parent strain. The hemolysin and cytotoxin titers

of CFS from the virA mutant strains 1B2R, 1D2A and AICB were not significantly (P < 0.953) different from each other.

Two separate in vivo trials were conducted to determine the virulence of S. hyodysenteriae virA mutants in a murine model of swine dysentery. In both experiments conventional 8-week-old C3H/HeN female mice, obtained from a commercial supplier (Sasco Co., Omaha, Neb.), were randomly allocated to three treatment groups consisting of three mice each housed in separate cages in accordance with approved guidelines of the University's Institutional Animal Care and Use Committee (IACUC# 95-08-044). In the first trial, mice were fed a define diet shown to increase the susceptibility of conventional mice to S. hyodysenteriae infection (Nibbelink and Wannemuehler, 1992; Teklad Diet TD 85420; Harlan Teklad, Madison, Wis.). After a seven days adaption period to the diet, the feed was removed for 24 hours and the mice were inoculated twice intragastrically at eight hours intervals with 0.5 ml of either sterile medium (Group 1 negative control), or medium containing 7.8 (first inoculum) and 8.4 (second inoculum) X 108 S. hyodysenteriae in vitro passage parent strain/ml (Group 2), or 8.9 (first inoculum) and 8.7 (second inoculum) X 108 virA mutant strain lB2R/ml (Group 3).

Feeding was resumed four hours after the second inoculation. The mice were euthanized on post-inoculation days (PID) 3,7 and 14. The second trial was similar to the first trial, except that the mice were fed a conventional diet (Purina Lab Chow; Purina Mills, Inc., St. Louis, Mo) and they were euthanized on PID 3,7,14,21 and 28.

Mice in each group were inoculated as in the first trial with 0.5 ml of either sterile medium (Group 1 negative control), 7.5 (first inoculation) and 9.1 (second inoculation) X 108 S. hyodYsenteriae in vitro passage parent strain/ml (Group 2), 9.6 (first inoculation) and 8.7 (second inoculation) X 108 virA mutant strain lB2R/ml (Group 3), or 7.5 (first inoculation) and 9.1 (second inoculation) X 108 virA mutant strain AlCB/ml (Group 4).

After exposure to CO2, the mice were immediately processed for necropsy, as previously described (Mysore and Duhamel, 1994; Zhang et al., 1995). Cecal gross changes were recorded and sections of ceca were collected for histopathologic and bacteriologic examinations. For histopathologic examination, transverse and longitudinal sections of the cecum from each mouse were placed in PLP fixative (1 mM sodium m-periodate, 75 mM lysine, and 2 percent paraformaldehyde in 37 mM phosphate buffer, pH 7.4; McLean and Nolsane, 1974), embedded in paraffin, sectioned at 5 pm and stained with

hematoxylin and eosin. Each section was assigned a number code and examined by light microscopy. The longitudinal crypt length (LCL) of five well-oriented cecal crypt columns in each section of cecum from each mouse was measured at 40X magnification, using a video-imaging morphometric system (PC VISION plus frame grabber version of JAVA, Jandel Scientific, Corte Madera, Calcif.), as described (Mysore and Duhamel, 1994; Zhang et al., 1995). From these measurements, mean LCL was obtained for the cecum of each mouse. Mean SEM LCL of mice from the same treatment group euthanized on the same PID were calculated. Data were reported as percentage difference in mean LCL between three S. hyodysenteriae-inoculated and three sterile medium- inoculated mice for each weekly sample collection.

In trial 1, significant (P < 0.05) differences in the mean LCL were present between mice inoculated with either sterile medium or virA mutant strain 1B2R and the S. hyodysenteriae parent strain-inoculated mice on PID 14. No significant differences in the mean LCL were found between mice inoculated with sterile medium and mice inoculated with the S. hyodysenteriae parent strain or the virA mutant strain 1B2R on PID 3 and 7 (FIG. 6). In the second trial, in which mice were fed a conventional diet, macroscopic cecal changes consisting of edematous thickening of the cecal wail and replacement of the normal cecal contents by mucus was present in mice inoculated with the S. hyodysenteriae parent strain on PID 21.

Significantly (P < 0.05) different mean LCL were present between mice inoculated with S. hyodysenteriae parent strain and mice inoculated with sterile medium or virA mutant strains 1B2R and A1CB on PID 21 and 28 (FIG. 7). No significant differences in the mean LCL of mice inoculated with sterile medium or virA mutant strains were found at any time from PID 3 to 28. At necropsy, the cecal mucosa and cecal contents from each mouse were cultured, using a selective medium for S. hyodysenteriae, as described (Zhang et al., 1995). The parent S. hyodysenteriae strain and the virA mutants were identified on the basis of the pattern and intensity of p-hemolysis.

Additionally, the identity of representative S. hyodysenteriae isolates obtained from infected mice in both trials was confirmed based on the results of PCR amplification of purified DNA, using S. hyodysenteriae virA gene-specific primers, as previously described (Elder et al., 1994) with modification to include primers specifically designed for identification of the mutant strains (Table 1). Bacteriologic examinations yielded S.

hyodysenteriae by culture from the ceca of mice inoculated with either S. hyodysenteriae parent strain or S. hyodysenteriae virA mutants at all sampling times post-inoculation in both trials. Representative isolates of S. hyodysenteriae parent strain and virA mutants obtained from the ceca of infected mice in both trials corresponded to the inoculum, using strain-specific PCR amplification. Serpulina hyodysenteriae was not isolated from the ceca of any of the mice in the sterile medium-inoculated groups.

Hemolysin and cytotoxin titers were compared, using the SAS System by Split Plot Analysis with strain differences as the main plot factor and treatment differences as a subplot factor. Whether differences in titers could be accounted because of variations between blood donor pigs was also examined, using the same variables. For each analysis, hemolysin and cytotoxin titers from each duplicate sample were used without averaging. To determine whether the lesions in the ceca of mice inoculated with the virA mutants were different from those present in the ceca of mice inoculated with the parent S. hyodysenteriae or sterile medium, mean LCL SEM of mice fed each diet were subjected to ANOVA for each sampling, using the Student-Newman-Keuls test (Zhang et al., 1995). In each analysis, differences between titers or groups were considered significant if the P value was < 0.05.

The vaccine according to the present invention comprises a live S. hyodysenteriae virA mutant according to the present invention in a physiological solution such as phosphate buffered saline or other suitable medium. The vaccine may be administered orally, intramuscularly, subcutaneously, or intravenously.

The method for increasing the resistance of an animal to dysentery infection according to the present invention comprises administering to an animal the live attenuated vaccine according to the present invention. The dosage may range from 1 x 10'to 5 x 10"cells per dose and is preferably from 1 x 109 to 2 x 109 cells per dose.

The dosage regimen may include one or more administrations, with multiple inoculations being administered from 1 to about 14 days apart. The vaccine administration may be provided in conjunction with other antigens capable of eliciting an immune response, either by multiple administrations or by combination in a single formulation. Such antigens include killed cells of a strain of S. hvodvsenteriae, such as a virulent strain, for example, B204 or B234. Other antigens include S. hyodysenteriae hemolysin, cytotoxin, outer membrane proteins and lipopolysaccharides, for example.

Antigenic proteins may be harvested and/or purified from cell cultures or may be produced by recombinant techniques.

The description above should not be construed as limiting the scope of the invention, but as merely providing illustrations to some of the presently preferred embodiments of this invention. In light of the above description and examples, various other modifications and variations will now become apparent to those skilled in the art without departing from the spirit and scope of the present invention as defined by the appended claims. Accordingly, the scope of the invention should be determined solely by the appended claims and their legal equivalents.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Duhamel, Gerald E.

Elder, Robert (ii) TITLE OF INVENTION: Attenuated Serpulina hyodysenteriae Mutant for Prevention of Swine Dysentery (iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: SUITER AND ASSOCIATES, PC (B) STREET: 11516 NICHOLAS STREET, SUITE 205 (C) CITY: OMAHA (D) STATE: NE (E) COUNTRY: USA (F) ZIP : 68154 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: 3.5 INCH HIGH DENSITY FLOPPY DISK (B) COMPUTER: IBM PC COMPATIBLE (C) OPERATING SYSTEM: WINDOWS 95 (D) SOFTWARE: WORDPERFECT 7 (SAVED IN ASCII (DOS) FORMAT) (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 60/030,662 (B) FILING DATE: 12-NOV-1996 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: SUITER, SEAN PATRICK (B) REGISTRATION NUMBER: 34,260 (C) REFERENCE/DOCKET NUMBER: UNL-97-7-2 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 402-496-0300 (B) TELEFAX: 402-334-0333

(2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2322 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : 1 AAGCTTTACC AGTTGAGGGC GACTATTATT CTGATAAAAA AATGTTAAGA 51 AGATTAGACC CTTTTATTAA TTTTGGAATA TATGCCGCTC ATCATGCATT 101 TAAGCAGGCT GGTATAGAAC CGAAAACAGG CTTTGATCCT TTAAGAGCCG 151 GTTGTGTTCT TGGTAGCGGT ATTGGCGGTA TGACTACTCT TTTATCTAAC RBS 201 CATCAAGTTT TACTTAATGA CGGACCTGGC AGAGTATCAC CTTTCTTTGT 251 ACCTATGCAA ATAATCAATA TGACACCTGG ATTAATATCT ATGGAATATG M E Y G 301 GTATGAACGG ACCTAACTAC AGTACAGTTA CTGCATGTGC TTCTTCAAAC M N G P N Y S T V T A C A S S N 351 CACTCTATAG GTTTAGGTTA TAAATATATT AAAGATAATG AAGCTGATAT H S I G L G Y K H I K D N E A D I 401 TATGGTAGTT GGAGGTTCTG AAGCTACTAT AAATCCTCTT ACTATAGCTG M V V G G S E A T I N P L T I A G 451 GTTTCAATAA TGCTAGAGCT TTATCTACTA GAAATGATGA TCCTGCTAAA F N N A R A L S T R N D D P A K 501 GCATCAAGAC CTTTTGATAA AGGAAGAGAC GGACTTGCTA TAGCCAGATA A S R P T D K G R D G L A I A R Y 551 TTTAATAAAA AATGGCTATG ATGTAAAAAT ATATATCACA GGAAATCTTG L I K N G Y D V K I Y I T G N L D PstI 601 ACAGAGTTAA TAAAGATACC TACTCTAACT TTAATATATT AAAATCTATG R V N K D T Y S N F N I L K S M 651 AATATAGATA TTAATTATTT AGGAAGCGAA GAAGATGCCA TATCAGCTGC N I D I N Y L G S. E E D A I S A A 701 AGAAAATATA GAAAGAAAAT CAATAGTATT AGATTCATTA TTTGGTACAG E N I E R K S I V L D S L F G T G 751 GCGGAAACAG ACCTTTAGAA GGAATACAAA AAGCTCTTAT AGATAGTTTG G N R P L E G I Q K A L I D S L 801 AATAAATTAG ATGTTCTTAG AATAGCAATA GATATACCTT CAGGATTAGC N K L D V L R I A I D I P S G L A 851 TTCAAAAATA AATGATAATG ACAATGTATA TACTTGTTTT AAAGCACATG S K I N D N D N V Y T C F K A H E 901 AAACATATAC TATATGCTTC GCTAAAGATA TATTCTTTTT ATACAGAACA T Y T I C F A K D I F F L Y R T 951 AGAGAATATA TAGGAAAATT ATTCATAATA AAATCAATAT TCCCAGATGA R E Y I G K L F I I K S I F P D E 1001 AATATTAGAT AATTGGGGAT ATAAAGCTAA ACTTATAGAT TATAATGAAA I L D N W G Y K A K L I D Y N E K 1051 AAATAAATAT AAATAGAAAC TCTCTATACA GCAAAAGAGA ACAAGGAATG I N I N R N S L Y S K R E Q G M

1101 CTTGCTATAG TAGCAGGAAG TGATAATTAT ATAGGGGCTG CTGTTCTAGC L A I V A G S D N Y I G A A V L A 1151 TGTAAATGCT GCTTATAGAT TGGGTGTAGG ATACATAAGA TTATATGTAC V N A A Y R L G V G Y I R L Y V P 1201 CTAAAGGCAT AATAAAAAAT ATAAGAGATG CCATAATGCC TTCTATGCCT K G I I K N I R D A I M P S M P 1251 GAAATTGTTA TTATAGGAGT TGGAGAAGAA AATCAAAAAT TCTTCACAGA E I V I I G V G E E N Q K F F T E 1301 AAATGACATT GAAATAGTAA ATGATATTAA TAAAAGCGAT GCTTGTATAA N D I E I V N D I N K S D A C 1351 TAGGTTCTGG TATAGGCAGA GATTTGTCTA CAGAATTTTT GTAAATACTA G S G I G R D L S T E F L * 1401 TATTAAAGCA AATAAATATA CCTACTGTTA TTGATGCTGA TGCTTTATAT 1451 TTAATGTTTG AAAGCACTCT TAATGAACTT AATAATAATT TTATAATCAC 1501 TCCTCATATA TATGAATTTG AAAAACTTAC ACAGATAAAT CATATAGAGG 1551 TTTTAGAAAA TCCTTATCAG GCATTATTAA TATACAGAGA AAAAACTAAT 1601 GCCTCAATAG TATTAAAAGA TGCTGTAAGT TTCCTAATGC ATGAAAATGA 1651 TATATATATA AATTATAACC CTAGAGAATC TATGGGGAAA GCAGGTATGG 1701 GTGATGTTTT TGCTGGATTT ATAGGTGCTT TGCTCGCTAG AAAACTAAAT 1751 ATATTAGATG CTTCAAAACT AGCATTGATA ATACAGGCTA AATCTTTTAA 1801 TATATTATCA AAAAAATTCG GAAATGATTA TATTCAGCCT AAAGATTTGG 1851 CAAATATTTC ATATAAAATA CTAAAAAGGA TATAAATTTG CCTAGAGAAG 1901 TTTACGACCC TAAACAAAAA GAATTAGAAT TCTACGCTAA AAGAGAGGTA 1951 AAGCCCCCTG CTCCTAAAAG AGAGGTAAGC ATATTTGCTA GAAGATGGTT 2001 TATGTTTTTA TACGGAACTT TCCTCACATT AGTTGTAATT GGTATGCTTT 2051 TATATAAAAA AGGATTCTTT AATAATATAC CATTATTTGA AGCTTTAAAG 2101 CCTAAAACAG ATGTTATAGT AAAAATTAAT AATGCTGAAT TCGTTAATGA 2151 TGCAGTAATT ACAACTATAG AACTCGAAAA TTCAAATTAT ACTAATTCTG 2201 AAAGTATAGA AACACTAAGA AGTTATTTTT CATTGTACAA AAATAGAAAA 2251 TTAATATTTA CAGGCAATCG TTCTTTTAAT AATATAAGAT TCCCAGTAGG 2301 TCAGAGAATA GGATTCAAGC TT 2322