Bacterial preparations, method for producing same, and their use as vaccines

FIELD OF THE INVENTION
The invention relates to novel bacterial preparations, methods for producing the preparations, and the use of the preparations as vaccines and to prepare antibodies.
BACKGROUND OF THE INVENTION
A family of toxins, referred to as the Repeats in Toxins (RTX), have been associated with infections caused by gram-negative organisms including E. coli, Proteus, Morganella, Pasteurelia, Actinobacillus, and Bordetella spp. (Menestrina G., et al, Toxicology 87 (1994) 249-267, and references referred to therein). The prototype toxin is Escherichia coli .alpha.-hemolysin which is a primary virulence factor of urinary infections, peritonitis, meningitis, and septicemia caused by virulent strains of E. coli. The RTX toxins share several common features which are discussed in the review by Welch, Mol. Microbiol. 5, 521, 1991.
The RTX toxins of Actinobacillus pleuropneumoniae play an important role in infections cause by strains of Actinobacillus pleuropneumoniae (J. Frey et al., in Bacterial Protein Toxins, Zbl.Bakt. Suppl. 24, Freer et al. (Eds)., Gustav Fischer, Stuttgart, Jena, N.Y., 1994). Actinobacillus pleuropneumoniae is the agent responsible for swine pleuropneumonia, a severe contagious disease which causes great economic losses (J. I. MacInnes and N. L Smart, Actinobacillus and Haemophilus in Pathogenesis of Bacterial Infections in Animals, 2nd ed. C. L. Gyles and C. O. Thoen, Iowa State University Press, Ames, Chapter 16). A. pleuropneumoniae is a gram-negative bacterium of the family Pasteurellaceae J. I. MacInnes and N. L Smart, supra). Twelve serotypes of the organism have been described by serotyping based on capsular polysaccharides (Nicolet, J. Can. Vet. J. 29:578-580, 1988 and Nielsen, R., cta. Vet. Scand. 27:453-455, 1986). Differences in virulence have been observed among the serotypes and a number of virulence factors have been considered responsible for the differences. These factors include capsular polysaccharides, liposaccharides, exotoxins and adhesion factors (J. I. MacInnes and N. L Smart, supra).
Two hemolytic RTX toxins, ApxI and ApXII, and one non-hemolytic RTX toxin, ApxIII have been identified in different A. pleuropneumoniae serotype strains J. Frey et al., supra) The toxins are secreted into the growth medium by A. pleuropneumoniae, they have different molecular masses, and they can be distinguished serologically by polyclonal and monoclonal antibodies (J. Frey et al., supra). It has been reported that A. pleuropneumoniae serotypes which produce a combination of two Apx toxins are more virulent than serotypes with one toxin alone (J. Frey et al., supra). The serotypes producing ApxI and ApxII are the most virulent (J. Frey et al., supra). No A. pleuropneumoniae strains have been identified which produce all three toxins J. Frey et al., supra).
RTX toxins have also been reported to be secreted in other bacterial strains including Bordetella pertussis (Betsou et al.1993 and CA 1,189,790) E. coli, Listeria, Moraxella, Pseudomonas, Staphylococcus, Vibrio (Nakai et al. 1983) and Neisseria meningitidis (Thompson et al.1993).
Vaccines which have been developed for preventing infections by A. pleuropneumoniae have been based on whole live cells, attenuated cells, lysates, culture supernatants, and extracts of A. pleuropneumoniae. Canadian Patent 1,189,790 describes a vaccine containing A. pleuropneumoniae cells, cell fragments, extracts and/or metabolites, and an adjuvant derived from Bordetella pertussis. Other proposed vaccines contain: (a) inactivated toxin of serotype I and optionally a toxin of another serotype (EP-A-420.743); (b) whole cell or sonicated whole cell components of a virulent strain obtained after passage in a host (WO9321951-A); (c) transferring binding protein, cytolysin and/or APP4 (WO9308283-A); (d) at least one immunogenic part of at least one cytolytic A. pleuropneumoniae protein prepared by recombinant DNA methods (Canadian Patent Application 2,045,950); (e) an iron-repressible outer membrane protein of molecular weight 105 kD (Canadian Patent Application 2,045,950); (f) outer membrane proteins having a major dominant antigenic protein component of 42 kD and a haemolysin of 105 kD and/or macrophage toxin of 120 kD (Canadian Patent Application No. 2,040,544); (g) inactivated toxin of serotype 1 of A. pleuropneumoniae which is obtained from culture supernatant (EP-420743); (h) extracellular proteinaceous materials from the culture medium of strains of at least two different serotypes of A. pleuropneumoniae. (EP-420743); or, (i) hemolysin antigen produced by recombinant techniques. Many of the known vaccines have limited effectiveness particularly against infection by heterologous serotypes.
Conventional formalin-killed bacterins provide limited protection against challenge with homologous serotypes of A. pleuropneumoniae and poor protection against heterologous serotypes (Neilson, 1984). In contrast, convalescent pigs are completely protected from challenge with homologous serotypes and significantly protected from disease from heterologous serotypes (Neilson, 1984).
Many of the antigenic components of A. pleuropneumoniae which have been investigated as potential vaccine candidates all fall short of affording complete cross-protection. Devenish et al. (1990) demonstrated homologous protection from challenge when gel-purified ApxI and ApxII cytolysins were used as a vaccine. However, others have reported less than complete protection with partially purified toxin vaccines, although the protection afforded is increased when these vaccines are enriched with other cellular components such as outer membrane proteins (OMPs) (Van den Bosch et al., 1992).
Live attenuated vaccines made from strains of A. pleuropneumoniae lacking in capsule production have been reported to protect against homologous challenge (Rosendal et al., 1990; Inzana et al., 1988). Results have not been reported with heterologous challenge. Live strains deficient in RTX toxin production have been reported to afford no protection against disease (Inzana et al., 1991). Jansen (1994) has concluded that both opsonization of A. pleuropneumoniae to enhance phagocytosis and neutralization of the RTX toxins are necessary for immune protection against disease.
SUMMARY OF THE INVENTION
The present inventors observed significant quantities of cell-associated RTX toxins when strains of A. pleuropneumoniae that are capable of producing RTX toxins, are cultured under certain conditions. Further, the whole cell protein composition of the cultures was found to substantially correspond to the whole cell protein profiles obtained from in vivo cells recovered at necropsy from the pleural fluid of infected swine. Thus, the in vitro preparation obtained by the present inventors mimicked the profiles of organisms from the lungs of swine exhibiting cross-protective immunity.
Vaccination with a bacterin prepared from heat-inactivated cultures having significant quantities of cell-associated RTX toxins, and adjuvanted with a double adjuvant system, resulted in significant protection of swine against challenge with homologous strains. Lung scores were found to be similar to those of a low-dose challenge group that were fully protected from disease. In contrast, there was 100% mortality in swine vaccinated with a commercial bacterin.
Broadly stated the present invention relates to a bacterial preparation comprising one or more isolated and purified strain of a microorganism which produces one or more RTX toxins, and which strain has at least one RTX toxin which is substantially cell-associated. Preferably, the microorganism is a pathogenic gram negative bacteria, particularly from the family Pasteurellaceae or Enterobacteriaceae. Most preferably, the microorganism is A. pleuropneumoniae, A. actinomycetemcomitans, A. suis, A. equuli, Pasteurella haemolytica, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Morganella morganii, or Bordetella pertussis.
In an embodiment of the invention, a bacterial preparation is provided comprising one or more isolated and purified strain of the same or different serotype of A. pleuropneumoniae which has one or more of ApxI, ApxII and ApxIII which are substantially cell-associated. In a preferred embodiment, the bacterial preparation comprises two or more isolated and purified strains of different serotypes of A. pleuropneumoniae which collectively have ApxI, ApxII and ApxIII which are substantially cell-associated.
The present invention also relates to methods for preparing the bacterial preparations of the invention. In one embodiment of the invention, a method is provided for preparing a bacterial preparation comprising selecting one or more strain of a microorganism which produces one or more RTX toxins; culturing the selected strain under suitable conditions so that the RTX toxin is substantially cell-associated.
In another embodiment of the invention, a bacterial preparation of the invention is prepared by selecting one or more strain of a microorganism which produces one or more RTX toxins, and treating the strain with a substance which interferes with the secretion of the RTX toxins.
The bacterial preparations of the invention may also be prepared by producing, using gene transfer techniques, a strain of a microorganism which has cell-associated RTX toxins.
The bacterial preparations of the invention may be used as vaccines for the prophylaxis and treatment of infectious diseases caused by strains of microorganisms which produce one or more RTX toxins. The bacterial preparations of the invention may also be used to prepare antibodies which may be used as a means of passive immunization.
The invention also relates to a method of preparing a vaccine against infectious diseases caused by a strain of a microorganism which produces one or more RTX toxins which comprises selecting a bacterial preparation which contains one or more of the strain of the microorganism, and which strain has at least one RTX toxin which is substantially cell-associated.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, reference is made herein to various publications, which are hereby incorporated by reference in their entirety. For ease of reference, a list of abbreviations, and the full citations of some of the publications cited and incorporated herein by reference, listed in alphabetical order by author, follow the Examples in the Detailed Description of the Invention.

DESCRIPTION OF THE DRAWINGS
The invention will be better understood with reference to the drawings in which:
FIG. 1 as depicted in FIGS. 1(a), 1(b), 1(c), 1(d) and 1(e), shows the nucleotide sequence of apxICA genes and the deduced amino acid sequences of ApxIC and ApxIA from GeEMBL Accession X73117;
FIG. 1B, as depicted in FIGS. 1B(a), 1B(b), 1B(c), 1B(d), 1B(e) and 1B(f), shows the nucleotide sequence of the apxIBD genes and the deduced amino acid sequences of ApxIB and ApxID from GenEMBL Accession X61112;
FIG. 2 as depicted in FIGS. 2(a), 2(b), 2(c), 2(d), 2(e) and 2(f), shows the nucleotide sequence of the apxIIA, apxIIB', and apxIIC genes and the deduced amino acid sequences of ApxIIA, ApxIIB, and ApxIIC from GenEMBL Accession X61111;
FIG. 2B as depicted in FIGS. 2B(a), 2B(b), 2B(c), 2B(d), 2B(e), 2B(f), 2B(g), 2B(h) and 2B(i), shows the nucleotide sequence of the apxIIIA, apxIIIB, apxIIIC, and apxIIID genes, and the deduced amino acid sequence of ApxIIIC; ApxIIIA, ApxIIIB, and ApxIIID from GenEMBL Accession L12145;
FIG. 3 shows the arrangement of the operon of the genes encoding the Apx toxins of the 12 reference serotypes of A.pleuropneumoniae from J. Frey et al., in Bacterial Protein Toxins, Zbl.Bakt. Suppl. 24, Freer et al. (Eds)., Gustav Fischer, Stuttgart, Jena, N.Y., 1994, FIG. 1;
FIG. 4A shows Coomassie-blue stained SDS-PAGE profiles of WC proteins of A. pleuropneumoniae serotype 1 grown in different media under various conditions;
FIG. 4B shows Coomassie-blue stained SDS-PAGE profiles of outer-membrane enriched fractions (B) of A. pleuropneumoniae serotype 1 grown in different media under various conditions;
FIG. 5A shows Western blots of WC proteins of A. pleuropneumoniae serotype 1 grown in various media probed with sera from a pig exposed to low-dose challenge of A. pleuropneumoniae CM5;
FIG. 5B shows Western blots of WC proteins of A. pleuropneumoniae serotype 1 grown in various media probed with sera from a pig hyperimmunized with purified cytolysin from CM5;
FIG. 5C shows Western blots of WC proteins of A. pleuropneumoniae serotype 1 grown in various media probed with sera from a pig immunized with commercial bacterin;
FIG. 6 shows a Western blot of OMPs of A. pleuropneumoniae serotype 1 grown in various media probed with sera from a pig exposed to low-dose challenge of A. pleuropneumoniae CM5;
FIG. 7 shows a Western blot of WC proteins of A. pleuropneumoniae CM5 probed with sera from naturally-infected swine;
FIG. 8 shows a Western blot of OMPs of Actinobacillus pleuropneumoniae CM5 probed with sera from naturally-infected swine;
FIG. 9 as depicted in FIGS. 9aand 9b, shows Western blots of WC proteins of A. pleuropneumoniae serotype 1 strain CM5 grown in RPMI (A); and in TYE (B); and WC proteins of A. pleuropneumoniae serotype 1 strain VSB68 in TSB (C); Western blots of outer-membrane proteins A. pleuropneumoniae serotype 1 strain CM5 grown in RPMI (D); in TYE (E); and Western blots of outer-membrane proteins A. pleuropneumoniae serotype 1 strain VSB68 grown in TSB (F);
FIG. 10 shows a Western blot of CYT from CM5 probed with sera from CDCD pigs;
FIG. 11A shows Western blots of WC proteins of A. pleuropneumoniae probed with serum from a pig hyperimmunized with gel-purified CYT;
FIG. 11B shows Western blots of WC proteins of A. pleuropneumoniae probed with serum from a pig exposed to low-dose challenge;
FIG. 12 shows the distribution of lung lesions of the CDCD pigs at necropsy;
FIG. 13 shows a Western blot of WC proteins of A. pleuropneumoniae probed with sera from CDCD pigs;
FIG. 14 shows a Western blot of CYT probed with sera from vaccinated pigs;
FIG. 15 shows the distribution of lung lesions of the SPF pigs at necropsy;
FIG. 16 as depicted in FIGS. 16a and 16b, shows Western blots of WC proteins (A and B) and CYT (C and D) probed with sera from vaccinated SPF pigs;
FIG. 17 shows Western Blots of WC proteins of CM5 (lane 1), transposon mutant #17 (lane 3), and transposon mutant #18 (lane 5), the corresponding culture supernatants (lanes 2, 4, and 6), and purified CYT (lane 7);
FIG. 18 shows WC preparations of transposon mutant #19 (lane 1), control transposon mutant #20 (i.e., not a BD mutant) (lane 3), serotype 3 strain BC181 (lane 5), the corresponding culture supernatants (lanes 2, 4 and 6), and purified CYT (lane 7); and
FIG. 19 shows WC preparations of CM5-JB, a low passage variant (lane 1), transposon mutant #17 (lane 3), and transposon mutant #18 (lane 5), the corresponding culture supernatants (lanes 2, 4, and 6), and purified CYT (lane 7).

DETAILED DESCRIPTION OF THE INVENTION
I. Characterization of the Bacterial Preparations
The bacterial preparations of the invention comprise one or more isolated and purified strains of a microorganism which produces one or more RTX toxins, and which strains have at least one RTX toxin which is substantially cell-associated. The preparations may additionally contain one or more strains of a different microorganism which produces one or more RTX toxins.
The microorganisms used in the preparations of the invention are selected from pathogenic Gram negative bacteria, particularly from the family Pasteurellaceae, such as A. pleuropneumoniae, P. haemolytica, Actinobacillus suis, and Actinobacillus actinomycetemcomitans. Other microorganisms which may be used in the preparations of the invention include Bordetella pertussis and microorganisms from the family Enterobacteriaceae including E. coli, Proteus mirabilis, Proteus vulgaris, and Morganella morganii, (Chang et al, 1989; Devenish et al, 1989; Gygi et al, 1990; Frey et al, 1993; Smits et al, 1991; Jansen et al, 1992).
The strains of the microorganism present in the bacterial preparations of the invention may be reference strains which may be obtained from researchers working in the field or from public depositories such as the American Type Culture Collection, Bethesda, Md. Serotype reference strains of A. pleuropneumoniae are described below. The strains may also be obtained from animals suffering from naturally occurring infections, they may be naturally-occurring mutant strains, or strains produced using gene transfer techniques. It will be appreciated that attenuated strains may be selected or produced for use in the bacterial preparations. The microorganism may be classified into strains or particular serotypes using generally known methods. For example, the classification of strains of A. pleuropneumoniae into particular serotypes is provided in Veterinary Microbiology 13 (1987), pages 249-257).
"RTX toxins" used herein refers to toxins of the family of homologous cytotoxins and exoproteins of which E. coli hemolysin (HlyA) is the prototype. RTX toxins are characterized by their ability to lyse target cells. The lytic activity is Ca.sup.2+ dependent and is caused by a colloid osmotic shock due to the formation of hydrophilic pores in the membranes of the target cells. The designation RTX for "repeats in toxin" is based on the presence in the cytolytic product of homologous glycine-rich repeat domains responsible for Ca.sup.2+ dependent binding to target cells and expression of lytic activity.
RTX toxins are also generally characterized by the organization of their operon. The operon generally consists of the following four genes: an activator gene (designated "C"), a structural toxin gene (designated "A"), and two secretion genes (designated "BD"). The four genes are typically arranged in the order CABD. The genetic arrangement of the genes encoding the Apx toxins of the 12 reference serotypes of A. pleuropneumoniae is shown in FIG. 3B. The gene profile and distribution may differ in different strains and some strains may not contain certain of the genes, for example the secretion genes.
The RTX toxins may also be characterized on the basis that they are secreted as an active toxin by the proteins encoded by the hemolysin BD genes of E. coli. RTX toxins also include toxins which have substantial sequence homology to known RTX toxins, in particular homologs of E. coli hemolysin (HlyA).
The RTX toxins have different target-cell specificities (Welch, 1991). For example, A. pleuropneumoniae cytolysins ApxI and ApxII hemolyse erythrocytes from pig, sheep, bovine and human and are cytolytic to porcine and rabbit neutrophils and porcine alveolar macrophages. Whereas the leukotoxin from P. haemolytica is specific for ruminant leukocytes.
Examples of RTX toxins are the ApxI, ApxII, and ApxIII toxins of A. pleuropneumoniae. ApxI, also known as hemolysin I or cytolysin I is strongly hemolytic and has an apparent molecular mass of 105 kDa (J. Frey et al., 1994a). Serotypes 1, 5a, 5b, 9, 10 and 11 of A. pleuropneumonia produce ApxI (J. Frey et al., 1994a). The structural apxIA gene contains three strongly hydrophobic domains and a domain with 13 glycine rich repeat nonapeptides J. Frey et al., 1994a). ApxI shows strong similarities to the Escherichia coli hemolysin and to a lesser extent to the Pasteurella haemolytica leukotoxin (J. Frey et al., 1994a). apxI contains the activator gene apxIC and the structural gene apxIA (J. Frey et al., 1994a). FIG. 1 shows the nudeotide sequence of the apxICA genes and the deduced amino acid sequences of ApxIC and ApxIA. FIG. 1B shows the nucleotide sequence of the apxIBD genes and the deduced amino acid sequences of ApxIB and ApxID. The nucleotide sequence of the apxICA genes is also shown in the Sequence Listing as SEQ. ID. NO. 3, and the deduced amino acid sequences of ApxIC and ApxIA are shown in the Sequence Listing as SEQ. ID. NOS. 1 and 2, respectively. The nudeotide sequence of the apxIB and apxID genes, and the deduced amino acid sequences of ApxIB and ApxID are shown in the Sequence Listing as SEQ. ID. NOS. 6, 4, and 5, respectively.
ApxII, also known as hemolysin II or cytolysin II, is a weakly hemolytic and weakly cytotoxic RTX-toxin which is produced by all serotypes of A. pleuropneumoniae except serotype 10 (J. Frey et al., 1994a). ApxII provides weak hemolytic character to serotypes which do not simultaneously produce ApxI (J. Frey et al., 1994a). ApxII has an apparent molecular weight by SDS gel electrophoresis of 103 kDa (J. Frey et al., 1994a). The structural gene for ApxII contains three strongly hydrophobic domains and a domain with 8 glycine rich repeated nonapeptides. ApxII is closely related to the Pasteurella haemolytica leukotoxin. FIG. 2 shows the nucleotide sequence of the apxIIA, apxIIB', and apxIIC genes and the deduced amino acid sequences of ApxIIA, ApxIIB, and ApxIIC. The nudeotide sequences of the apxIIA, apxIIB', and apxIIC genes, and the deduced amino acid sequences of ApxIIA, and ApxIIC, are also shown in the Sequence Listing as SEQ. ID. NOS. 9, 8, and 7, respectively.
ApxIII, also known as pleurotoxin or cytolysin III is non-hemolytic but strongly cytotoxic for alveolar macrophages and neutrophils (J. Frey et al., 1994a). ApxIII is produced by serotypes 2, 3, 4, 6, and 8. Its apparent molecular weight by SDS gel electrophoresis is 120 kDa (J. Frey et al., 1994a). The ApxIII gene contains three strongly hydrophilic domains and a region with 13 glycine rich repeat. The amino acid sequence of ApxIII deduced from the DNA sequence shows identity to APxI, ApxII, E. coli hemolysin and P. haemolytica leukotoxin (J. Frey et al., 1994a). FIG. 2B shows the nucleotide sequence of the apxIIIA, apxIIIB, apxIIIC, and apxIIID genes, and the deduced amino acid sequence of ApxIIIC, ApxIIIA, ApxIIIB, and ApxIIID. The nucleotide sequence of the apxIIIA, apxIIIB, apxIIIC, and apxIIID genes, and the deduced amino acid sequences of ApxIIIC, ApxIIIA, ApxIIIB, and ApxIIID, are also shown in the Sequence Listing as SEQ. ID. NOS. 14, 10, 11, 12, and 13, respectively.
Table 1 is a summary of the RTX toxin genes of A. pleuropneumoniae showing their designations and their synonyms. Table 2 shows the distribution of the RTX genes of the 12 serotype reference strains of A. pleuropneumoniae. The apx gene profile has been shown to be identical in serotype reference strains and field strains of A. pleuropneumoniae. The function of the four genes in the apx operon has also been demonstrated in trans-complementation experiments in E. coli and the helper genes for activation and secretion have been shown to be interchangeable functionally to some extent with the E. coli or Proteus vulgaris hlyC and hlyBD analogues.
Additional examples of RTX toxins are Pasteurella haemolytica leukotoxin, E. coli hemolysin, Proteus vulgaris hemolysin, A. actinomycetemcomitans leukotoxin, the RTX toxin encoded by the ash operon of A suis, A. equuli hemolysin, Proteus mirabilis hemolysin, Morganella morganii hemolysin, and Bordetella pertussis adenylate cyclase.
The RTX toxins are typically secreted into the culture medium and are recovered in culture supernatants. The ability of a strain of a microorganism to produce an RTX toxin(s) may be determined by assaying for RTX toxin(s) in the culture supernatant by measuring the ability to lyse erythrocytes from pig, sheep, bovine and human and/or kill lymphocytes, epithelial cells, T lymphocytes, and macrophages in vitro (Devenish et al, 1982 Infect. Immun. 60:2139-42), or by using polyclonal or monoclonal antibodies specific to an RTX toxin. For example, the presence of ApxI, ApxII, and/or ApxIII in culture supernatants may be assayed using a chemiluminescence assay as described in Dom, P. et al., Infect. Immunity 60:4328-434, 1992). Briefly, a preparation of porcine neutrophils is allowed to adhere to the walls of cuvettes such as abiolumcuvettes (Lumac, Landgraff, The Netherlands), a known concentration of a strain of the microorganism is added to the cuvettes, and the oxidative burst of the porcine neutrophils is measured as chemiluminescence. The chemiluminescence is typically measured for about 80 minutes using a luminometer (Lumicon, Hamilton, Bonaduz, Switzerland). Opsonized zymosan may be added after a first incubation and chemiluminescence is measured for about an additional 20 minutes. Failure of the zymosan to provoke chemiluminescence indicates that the neutrophils were killed.
In the bacterial preparations of the present invention the RTX toxins are substantially cell-associated. "Substantially cell-associated" used herein refers to the presence in the cell, or on the cell surface of the microorganism strain, of an amount of RTX toxin which is capable of inducing an immunogenic response. The cell association may involve transport across the cell membrane of the RTX toxin, or attachment of the RTX toxin molecule or precursors thereof to the cell membrane before secretion.
The presence of a RTX toxin on the cell surface may be determined for example, using the methods described herein by obtaining membrane enriched fractions of the bacterial preparation, and detecting the presence of the RTX toxins for example by Western Blotting using convalescent sera or polyclonal monospecific or monoclonal antibodies. Similarly, immunoblots of whole cell proteins extracted from the microorganism in the bacterial preparation may also be probed with convalescent sera or polyclonal monospecific or monoclonal antibodies.
In an embodiment of the invention a bacterial preparation is provided comprising one or more strains of different serotypes of A. pleuropneumoniae which have one or more of ApxI, ApxII and ApxIII which are substantially cell-associated. The serotype distribution of the RTX toxin genes of A. pleuropneumonae in serotypes 1 to 12 is shown in Table 2. Preferably, the preparation contains at least two different serotype strains which collectively have ApxI, ApxII, and ApxIII which are substantially cell-associated. Most preferably, the bacterial preparation contains two strains of serotypes 1 and 3 of A. pleuropneumoniae which have ApxI, ApxII and ApxIII substantially cell-associated.
Strains of A. pleuropneumoniae known from the literature such as ATCC 27088 (serotype 1), ATCC 27090, ATCC 33378 and ATCC 33377 (reference strains for serotypes 1-5), ATCC 33590 (reference strain for serotype 6), Shope 4071, S1536, S1421, M62, K17, Femo, WF83, 405, CVJ13261, 56153 (reference strains for serotypes 1 through 10), and 8329 (reference strain 12) may be used in this embodiment of the bacterial preparation of the invention. Serotype strains may also be field strains which are obtained from swine with naturally occurring pleuropneumonia (MacInnes et al. 1990) or they may be obtained from researchers working in the field such as researchers from the Dept. of Vet. Microbiology, University of Guelph, Guelph, Ontario, Canada. The strains may also be prepared using the gene transfer methods described herein.
Whole cell protein profiles of the bacterial preparations of the invention prepared using the methods described herein, show that the preparations are enriched in RTX toxins. For example, a 105 K cytolysin (ApxI) and/or a 103 K cytolysin (ApxII) are found in whole cell protein profiles from A. pleuropneumoniae preparations. The protein profiles also show that the preparations of the invention contain whole cell antigens substantially analogous to those found in vivo. The in vitro cultures which mimic the profiles of organisms recovered from natural infection should stimulate a protective immune response.
In another embodiment of the invention a bacterial preparation is provided comprising mutant strains prepared by gene transfer techniques which have one or more of ApxI, ApxII and ApxIII which are substantially cell associated. In a preferred embodiment, the bacterial preparation contains a A. pleuropneumoniae transposon mutant with an insertion in the apxIBD genes.
The microorganisms in the bacterial preparation which are prepared by gene transfer as described herein may additionally be modified to include other genes. For example, a gene encoding an immunogen or another toxin may be included rendering the preparation useful against additional immunogens/toxins.
II. Preparation of Bacterial Preparations
The present invention also relates to methods for preparing the bacterial preparations of the invention. In one embodiment of the invention, a method is provided for preparing a bacterial preparation comprising selecting one or more strains of a microorganism which produces one or more RTX toxins, and culturing the selected strain(s) under suitable conditions so that the RTX toxin is substantially cell-associated.
The microorganisms which are used to prepare the bacterial preparations of the invention are described above, and include without limitation, strains of A. pleuropneumoniae, E. coli, P. haemolytica, Actinobacillus suis, Actinobacillus actinomycetem-comitans, Bordetella pertussis, Proteus mirabilis, Proteus vulgaris, and Morganella morganii, which produce one or more RTX toxins. In a preferred embodiment, the microorganism is an A. pleuropneumoniae strain which produces one or more of ApxI, ApxII and ApxIII. The ability of the microorganism to produce an RTX toxin may be determined by assaying the culture media for the presence of the RTX toxin using known hemolytic and cytolytic tests some of which are described herein.
The strains are cultured in a medium which permits the RTX toxins to be cell-associated. In a preferred embodiment for preparing a bacterial preparation comprising A. pleuropneumoniae, the strains are cultured in a tryptone yeast extract medium. The medium generally has the following composition: 10 to 30 g of tryptone; 2.5 to 7.5 g of yeast extract, 2.5 to 9.0 g of NaCl, 0.4 to 1.15 g of KCl, 0.7 to 2.1 of Na.sub.2 HPO.sub.4 (10 mM), 0.2 to 0.7 of (KH.sub.2 PO.sub.4), 0.9 to 2.7 g of glucose, in dH.sub.2 O (in a final volume of about 1000 ml). The medium also contains about 1 ml of 10% (wt/vol) NAD per liter. The composition of a preferred medium for preparing bacterial preparations containing A. pleuropneumoniae strains comprises 5 to 15 g of tryptone; 3 to 7 g of yeast extract, 3 to 7 g of NaCl, 0.5 to 1.0 g of KCl, 1 to 2 g of Na.sub.2 HPO.sub.4 *(KH.sub.2 PO.sub.4); 1.0 to 2.0 g of glucose, in one liter of dH.sub.2 O. The composition of a most preferred medium for preparing bacterial preparations containing A. pleuropneumoniae strains is set forth in Table 3 (3). Equivalent conditions may also be produced by adjusting the concentrations of the elements of the medium and/or substituting one or more of the elements of the medium.
The strains of the microorganism are cultured overnight, seeded into the medium using a 10% inoculum, and maintained in log phase. The concentration of cells seeded into the medium and the culture conditions will be selected having regard to the nature of the microorganism and other factors known to a skilled artisan. By way of example, 10.sup.9 to 10.sup.12 cells of an overnight culture may be seeded into 100 ml of liquid medium and incubated with shaking at 50-200 rpm at 37.degree. C., for a period of 6 hours, for strains of A. pleuropneumoniae, This can be scaled up for industrial purposes using a 10% inoculum in a new medium. Scale-up is generally progressive for example, with 100 ml of inoculum seeded into 1 liter, 1 liter of inoculum seeded into 10 liters, etc. at 6 to 12 hour intervals. Following growth, cultures may be tested for purity, cell density, cell associated RTX toxin(s) and strain confirmation. The culture may optionally be inactivated as described below. Following inactivation, harvests may be tested for sterility, cell-associated RTX toxin(s), and density.
When the bacterial preparation contains more than one different strain of a microorganism, each strain may be individually cultured and optionally inactivated using the above described methods.
In another embodiment of the invention, the bacterial preparation is prepared by selecting one or more strains of a microorganism which produces one or more RTX toxins, and treating the strains with a substance which interferes with the secretion of the RTX toxins.
Substances which interfere with the secretion of an RTX toxin may be identified by treating a strain of a microorganism which produces one or more RTX toxins, with a substance which is suspected of interfering with the secretion of the RTX toxin, under conditions so that the RTX toxin(s) are capable of being secreted into the medium, and measuring the amount of RTX toxin in the medium or the amount of cell-associated RTX toxin. Microorganisms which may be used to identify interfering substances and methods for measuring the amount of RTX toxin in the medium or the amount of cell-associated RTX toxin are described herein.
An example of a substance which may interfere with the secretion of an RTX toxin is a nucleic acid sequence encoding the D and/or B transport genes inverted relative to their normal orientation for transcription i.e. antisense D and B nucleic acid molecules. Such antisense nucleic acid molecules may be chemically synthesized using naturally occurring nudeotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with RTX toxin mRNA or the RTX toxin gene. The antisense sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.
A nucleic acid molecule containing the antisense sequences may be introduced into the microorganism using conventional techniques, such as transformation, transfection, infection, conjugation and physical techniques such as electroporation. Suitable vectors or cloning vehicles for transferring the nucleic acid molecules are known in the art. Examples of suitable vectors are set out in Table 4.
The bacterial preparations of the invention may also be prepared by producing, using gene transfer techniques, strains which have cell-associated RTX toxins. Gene transfer methods can be applied to microorganisms which produce RTX toxins as described herein. For example, A. pleuropneumoniae strains which produce RTX toxins may be manipulated so that the RTX toxin(s) are cell-associated by targeted gene mutagenesis using allelic replacement, insertional inactivation, or deletion formation of the D and/or B transport genes, or other genes. En addition to the RTX genes, others genes may also be inserted into the bacterial preparation by gene transfer techniques. Other genes that may be added include genes encoding other antigens or other toxins to increase the immunogenicity of the bacterial preparation and/or to induce a response against other antigens.
Selection marker genes which may be used in insertional mutagenesis by transposition or by allelic replacement include antibiotic resistance genes. Examples of antibiotic resistance genes which may be used include: (a) the gene encoding aminoglycoside 3'phosphotransferase I activity from transposon Tn903 (Oka et al., J. Mol. Biol. 147:217-226, 1981) which confers kanamycin resistance in A. pleuropneumoniae (Tascon et al., J. Bacteriol. 175:5717-5722, 1993); (b) variants of transposon Tn5 confer high levels of resistance to kanamycin, bleomycin, and streptomycin to A. pleuropneumoniae (Willson, Can. J. Vet. Res. 54 Suppl. S73-S77, 1990); (c) the gene for gentamycin resistance (Gm.sup.r) from the IncP plasmid, R1033 (Hirsch et al., Gene 48:203-209, 1986) which confers very high resistance to gentamycin (100 .mu.g/ml) in A. pleuropneumoniae; (d) the Gm.sup.r gene from Tn1696 can also be expressed in A. pleuropneumoniae (West et al., 1995); (e) the chloramphenicol resistance gene from an A. pleuropneumoniae plasmid pYG10 (Lalonde and O''Hanley, Gene 85:243-246, 1989); (f) the type II Cm.sup.r gene from Shigella flexneri plasmid pS-a (Hedges and Datta, Nature 234:220-222, 1971) or Tn9 (West et al., 1995) which have been shown to confer chloramphenicol resistance to A. pleuropneumoniae; (g) ROB-1 beta-lactamase (bla) genes originating from P. haemolytica (Azad et al.,J Gen. Microbiol. 138:1185-1196, 1992; Craig et al., J. Gen. Microbiol. 135:2885-2890, 1989; and Livrelli et al, Antimicrob. Agents Chemother. 32:1282-1284, 1988) express ampicillin resistance in A. pleuropneumoniae, P. haemolytica, and P. multocida (West et al., 1995); (h) genes encoding dihydrophteroate synthase, which confers sulfonamide resistance to A. pleuropneumoniae, P. haemolytica, and P. multocida; (i) streptomycin kinase which confers streptomycin resistance to A. pleuropneumoniae, P. haemolytica, and P. multocida which are present on a P. haemolytica plasmid pYFC1 (Chang et al., Vet. Microbiol. 32:319-325, 1992); and (j) a Tc.sup.r gene originating from Streptococcus agalactiae which confers resistance to low concentrations of tetracycline (0.2 .mu.g/ml) in A. pleuropneumoniae (Jansen et al., Infect. Immun., 1995).
If the bacterial preparations are used to prepare vaccines, it is desirable to eliminate the antibiotic resistance for example, using media (e.g. Boekner media) to select for mutants which do not carry the selection marker gene. In the alternative, heavy metal resistance (e.g. mercury) may be used as a selectable marker.
Plasmid vectors which may be used to manipulate genes in microorganisms which produce one or more RTX toxins, in particular of the family Pasteurellaceae, include endogenous plasmids of A. pleuropneumoniae and P. haemolytica. Examples of endogenous plasmids of A. pleuropneumoniae and P. haemolytica are pYG53, pYG54 (Lalonde and O'Hanley., supra, 1989); pLS88 which contains the selection marker genes Sm.sup.r, Su.sup.r, and Km.sup.r, plasmid pYFC1 which contains the Sm.sup.r and Su.sup.r genes (Chang et al., Vet. Microbiol. 32:319-325, 1992); pJFF224-NX and PJFF224-XN (Frey et al, Res. Microbiol. 143:263-269, 1992); a native 4.3 kb P. haemolytica plasmid pPH33 (Azad et al., J. Gen. Microbiol. 138:1185-1196, 1992; Craig et al., J. Gen. Microbiol. 135:2885-2890, 1989); a 4.3 kb plasmid pPH843 from P. haemolytica and a derivative of it pAKA which has been constructed as a mobilizable suicide vector for gene replacements in P. haemolytica and P. multocida (Azad et al., Gene 145:81-85, 1994); pGZRS-18/19 (West et al., 1995); and, the cosmid plasmid pSW206 which is a derivative of the broad host range plasmid RK2 and contains the Km.sup.r gene from Tn903 for selection. The properties of some plasmids are summarized in Table 4.
Transposon inactivation by transposon mutagenesis is a useful tool for the genetic manipulation of microorgamsms and it may be used to produce a microorganism with cell-associated RTX toxins. A transposon mutagenesis procedure involving the use of a mobilizable suicide plasmid pLOF Km carrying a mini Tn10 transposon carrying the Km.sup.r gene from Tn903, and an IPTG inducible transposase located outside the mobile element (Herrero et al., J. Bacteriol. 172:6557-6567, 1990) provide transposon frequencies of approximately 10.sup.-5 in A. pleuropneumoniae (Tascon et al., J. Bacteriol. 175:5717-5722, 1993). Single, stable, and random insertions into the chromosome of A. pleuropneumonia serotypes 1 to 11 can be made with this system. This system has been used to isolate insertions in apxIA or apxIB of the A. pleuropneumoniae serotype 1 strain 4074 (Tascon et al., J. Bacteriol. 175:5717-5722, 1993; Tascon et al., Mol. Microbiol. 14:207-216, 1994).
The present inventors have prepared transposon mutants of A. pleuropneumoniae CM5 with insertions in the apxIBD genes (see Example 5). A suicide conjugative plasmid, pLOFKm, carrying a mini-Tn10 was used for the transposon mutagenesis as described by Tascon et al, Bacteriol. 175:5717-5722 (1993). The plasmid was mobilized from E. coli to A. pleuropneumoniae by biparental mating. Transconjugants were screened on blood agar plates, and non-haemolytic mutants were selected. The transposon insertion in the BD region in the mutants was found to interfere with the export of the ApxI toxin into the culture medium.
Allelic gene exchange using non-replicating or conditionally-replicating plasmids has been used widely for the mutagenesis of both procaryotes and eukaryotes. Allelic exchange can be used to create deletions of specific genes (e.g., the B and D genes), or parts of genes. Gene replacement systems are available for A. pleuropneumoniae and P. haemolytica. For example, an efficient system for targeted mutagenesis of A. pleuropneumoniae has been described by Jansen et al., Infect. Immun., 1995. The system is based on a conditionally-replicating thermosensitive, broad-host-range plasmid, pVE6063 (Maguin et al., J. Bacteriol. 174:5633-5638, 1992). The pVE6063 plasmid does not replicate in A. pleuropneumoniae and insertional mutagenesis occurs by homologous recombination. The apxBD genes inserted in pVE6063, may thus be deleted by replacement with either Cm.sup.r genes from S. aureus or by Tc.sup.r genes from S. agalactiae. The constructs may subsequently be introduced in A. pleuropneumoniae serotype 1 by electroporation.
Gene replacement in P. haemolytica may also be carried out using the system described by Tatum et al. (Appl. Environ. Microbiol. 60:2011-2016, 1994). In the system a cloned aroA gene is inactivated with a fragment containing P. haemolytica ampicillin resistance gene. The plasmid carrying the disrupted fragment is methylated using cloned PhaI and introduced into P. haemolytica by electroporation. Mutants with the disrupted aroA gene integrated into the chromosome may be obtained after repeated passage.
Plasmids may be transferred to species of the family Pasteurellaceae using electroporation and conjugation. Conditions for the electroporation can be established for each strain of microorganism. Conjugative mobilization of plasmids generally provides higher transfer rates than electroporation (Lalonde and O'Hanley. Gene 85:243246, 1989; Azad et al., Gene 145:81-85, 1994). The recipient strain needs to be modified so that the donor (usually E. coli) can be counterselected. This is generally done by screening for spontaneous nalidixic, rifampicin, or streptomycin resistant mutants or by introduction of an antibiotic resistance gene on a compatible plasmid to the recipient strain (Lalonde and O'Hanley, 1989, supra; Azad et al., 1994, supra). Virulent E.coli bacteriophages such as T4 and T7 may also be used for counterselection of the donor strains.
It will also be appreciated that the bacterial preparations of the invention may also be prepared using a strain which naturally has cell associated RTX toxin(s). For example, serotype 3 strains of A. pleuropneumoniae which do not contain the apxIB and D genes which are necessary for ApxII secretion, may be used to prepare A. pleuropneumoniae bacterial preparations.
Existing strains may be modified using the methods described above and conventional recombinant techniques known in the art, to produce a strain which has cell-associated RTX toxin(s). For example, a serotype 3 strain of A. pleuropneumoniae may be modified so that it does not secret ApxIII using the gene transfer techniques discussed above; and the strain may be modified to express ApxI by introducing the apxIC and apxB genes into the strain using conventional recombinant techniques. Thus, the resulting modified strain produces cell-associated ApxI, ApxII, and ApxIII. A modified serotype 1 strain which produces ApxI, ApxII, and ApxIII may also be constructed by modiying a serotype 1 strain using the gene transfer techniques discussed above so that it does not secret ApxI, and the apxIIIA and apxIIIC genes may be introduced into the strain using conventional recombinant techniques, so that the strain expresses ApxIII.
The composition of the bacterial preparations prepared by the above methods may be confirmed for example for A. pleuropneumoniae preparations by assaying for cell-associated ApxI, ApxII, and ApxIII as described herein. The whole cell protein profile of the bacterial preparation may also be determined by the methods described herein (See for example Examples 1 and 2). The bacterial preparations of the invention have a whole cell protein profile that substantially mimics the whole cell protein profile of in vivo cultures of A. pleuropneumoniae recovered from natural infection (See FIG. 9).
III. Utility of the Bacterial Preparations
The bacterial preparations may be used as vaccines for the prophylaxis and treatment of infectious diseases caused by microorganisms which produce RTX toxins. Infectious diseases caused by microorganisms which produce RTX toxins include swine pleuropneumonia caused by A. pleuropneumoniae; shipping fever in cattle caused by P. haemolytica; whooping cough caused by B. pertussis; in foals sleepy foal disease or joint ill (purulent nephritis, arthritis), in adult horses abortion, septicemia, nephritis, and endocarditis, arthritis in piglets and abortion and endocarditis in swine caused by A. equuli; septicemia in swine and horses, pneumonia, nephritis, and arthritis in swine, polyarthritis in horses and abortion in cattle caused by A. suis; urinary infections, peritonitis, meningitis, and septicemia caused by virulent strains of E. coli.; (acute juvenile) periodontitis caused by Actinobacillus acinetomycetemcomitans; and gastroenteritis caused by P. mirabilis or M. morganii. Thus, the vaccines can be intended for administration to humans and various other mammals, including bovines, equines, and swine.
Prior to formulating into a vaccine the strains of the microorganism in the preparation may be inactivated. The strains of the microorganism may be inactivated using heat (e.g. treatment for two hours at 60.degree. C.) or chemical agents, typically those commonly used for commercial vaccine preparations, following standard procedures known to persons skilled in the art. Chemical agents suitable for inactivating the bacterial preparations of the invention include .beta.-propiolactone (.beta.-Prone, Grand Laboratories Inc., Larchwood, Iowa.) or 0.1 M binary-ethyleneinine (BEI). Inactivation of the cultures may be confirmed for example, by plating multiple samples onto a medium such as CBA-NAD, and incubating the plates for 48 h at 37.degree. C. with 5% CO.sub.2.
It will also be appreciated that inactivation will not be necessary when the strains used in the bacterial preparation are attenuated.
The vaccine of the invention contains an immunologically effective amount of a bacterial preparation of the invention, for example between 5.times.10.sup.9 to 5.times.10.sup.10, preferably 1.times.10.sup.10 to 2.times.10.sup.10 cells per dosage unit. A dosage unit is typically 0.5 to 2 ml. The optimum amounts of the bacterial preparation depend on the nature of the infection against which protection is required, the characteristics of the mammals to be protected, and other factors known to persons skilled in the art.
In an embodiment of the invention, the vaccine contains a bacterial preparation comprising at least one A. pleuropneumoniae strain which produces an RTX toxin which is substantially cell-associated. The strains used in the bacterial preparation in the vaccines are selected based on the toxins produced by the different serotypes as shown in Table 2. A vaccine containing an A. pleuropneumoniae strain of serotypes 7 or 12 which has been treated so that the ApxII toxin is substantially cell-associated will provide protective immunity against infections by serotypes 7 or 12. A vaccine containing A. pleuropneumoniae strain serotype 10 which has been treated so that the ApxI toxin is cell-associated will provide protective immunity against infections by serotype 10. Similarly, a vaccine containing strains of serotypes 1, 5, 7, 10, 11, and 12 which have been treated so that the ApxI and ApxII toxins are cell-associated will provide protection against infection by serotypes 1, 5, 7, 10, 11, and 12. A vaccine containing serotypes 2, 3, 4, 6, 7, 8, and 12 which have been treated so that the ApxII and ApxIII toxins are cell-associated will provide protection against infection by serotypes 2, 3, 4, 6, 7, 8, and 12. A preferred vaccine of the invention contains strains which are capable of collectively producing ApxI, ApxII, and ApxIII. For example, the vaccine may contain strains of serotypes 1 and 3. This preferred vaccine should be effective against all pathogenic strains of A. pleuropneumoniae including the known serotypes set out in Table 2 as well as any still unknown serotypes, strains or mutants. If new strains produce different toxins a comparable approach may be employed. The vaccine may also contain strain(s) with cell-associated RTX toxins which have been prepared using the gene transfer and recombinant methods described herein.
In addition to the bacterial preparation, the vaccine may comprise an immunologically acceptable carrier such as aqueous diluents, suspending aids, buffers, excipients, and one or more adjuvants known in the art. Suitable adjuvants include aluminum hydroxide, Freunds adjuvant (complete or incomplete), bacteria such as Bordetella pertussis or E. coli or bacterium derived matter, immune stimulating complex (iscom), oil, sapronin, oligopeptide, emulsified paraffin-Emulsigen.TM. (MVP Labs, Ralston, Nebr.), L80 adjuvant containing AL(OH).sub.3 (Reheis, N.J.), Quil A (Superphos), or other adjuvants known to the skilled artisan. Preferably, the adjuvant is L80 adjuvant containing AL(OH).sub.3 (Reheis, N.J.) and Quil A (Superphos). The vaccine may also contain preservatives such as sodium azide, thimersol, gentamicin, neomycin, and polymyxin.
The vaccine may be a multivalent vaccine and additionally contain other immunogens related to other diseases in a prophylactically or therapeutically effective manner. For example, a vaccine containing a bacterial preparation comprising A. pleuropneumoniae strains of serotypes producing RTX toxins which are substantially cell associated, may also contain inactivated cultures of one or more of Haemophilus parasuis which causes polyserositis; Pasteurella multocida which contributes to the severity of A. pleuropneumoniae respiratory clinical signs and pathology; Bordetella bronchiseptica which causes atrophic rhinitis; and Erysipelothrix rhusiopathiae which causes erysipelas. Additional immunogens may also be added to the vaccine in a number of ways. In one example, the DNA encoding the immunogen may be incorporated into strains in the bacterial preparation using gene transfer techniques. In another example, the immunogen may be added in an immunologically effective amount directly to the preparation.
The vaccines of the invention may be administered in a convenient manner, such as intravenously, intramuscularly, subcutaneously, intraperitoneally, intranatally or orally. Preferably the vaccine is administered intramuscularly or subcutaneously. The vaccine may be administered in a liposome delivery system to allow for the slow sustained release of the immunogens.
The dosage will depend on the nature of the infection, on the desired effect and on the chosen route of administration, and other factors known to persons skilled in the art. For example, in respect of a vaccine containing an A. pleuropneumoniae strain which produces an RTX toxin which is substantially cell-associated, about 1 to 5 ml may be administered intramuscularly or subcutaneously to pigs 30 lbs. or over, and 0.5 to 1 ml for pigs under 30 lbs. A second vaccination is generally recommended two to three weeks later. Breeding females are generally given two doses. The initial dose may be administered about five weeks prior to farrowing, and the second dose 2 weeks to three weeks prior to farrowing. Revaccinations are generally given annually.
The bacterial preparations of the invention are also useful for preparing antibodies which may be used as a means of passive immunization. Within the context of the present invention, antibodies are understood to include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab').sub.2 and recombinantly produced binding partners. Antibodies may be prepared using conventional methods.
For example, polyclonal antibodies may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, various fowl, rabbits, mice, or rats. Briefly, the bacterial preparation is utilized to immunize the animal through intraperitoneal, intramuscular, intraocular, or subcutaneous injections, in conjunction with an adjuvant such as Freund complete or incomplete adjuvant. Following several booster immunizations, samples of serum are collected and tested for reactivity to the bacterial preparations. Particularly preferred polyclonal antisera will give a signal on one of these assays that is at least three times greater than background. Once the titer of the animal has reached a plateau in terms of its reactivity, larger quantities of antisera may be readily obtained either by weekly bleedings, or by exsanguinating the animal.
The following non-limiting examples are illustrative of the present invention:
EXAMPLES
The following materials and methods were utilized in the investigations outlined in the examples:
Bacterial Strains and Growth Conditions
Actinobacillus pleuropneumoniae CM5, a serotype 1 strain, was isolated from a pig with naturally occurring pleuropneumonia (Rosendal et al., 1981) and stored at -70.degree. C. in pleural fluid. A. pleuropneumoniae BC181, a serotype 3 strain, was isolated from a case of porcine pleuropneumonia in British Columbia, and stored frozen at -70.degree. C. in skim milk with 20% glycerol (Rosendal et al., 1981). Serotype 1 strain VSB68, was provided as a lyophilized culture by Langford/Cyanamid, Guelph, Ont. For use, cultures were streaked onto trypticase soy agar supplemented with 5% (vol/vol) heated sheep blood (chocolate agar) and 0.01% (wt/vol) nicotinamide adenine dinucleotide (CBA-NAD) and incubated for 24 h at 37.degree. C. with 5% CO.sub.2.
Cultures used in preparation of antigens for polyacrylamide gel electrophoresis (PAGE) and Western blotting (WB) experiments were grown in several different media under different conditions. A well defined commercial medium, RPMI (Table 3) supplemented with 10% fetal calf serum (FCS) was used. Tryptone yeast extract broth (TYE) (Table 3; O'Reilly, and Niven, 1985) was used to provide enriched growth conditions.
To provide oxygen-limited conditions, screw-capped 500 ml glass bottles (Corning) were filled to the top with medium prior to autoclaving and the caps tightened to limit dissolved oxygen after autoclaving. Cultures were incubated without agitation. Aerobic conditions were achieved by incubating cultures in 500 ml bottles partially filled with medium at 200 rpm on a rotary shaker.
Strains of A. pleuropneumoniae serotypes 1, 2, 5, 7, and 9 from a commercial bacterin were provided as individual, formalin-inactivated suspensions by Langford/Cyanamid.
Inactivation
Cultures were inactivated using one of three techniques. Six h cultures were heated to 6.degree. C. for 2 h and then cooled to 4.degree. C. or .beta.-propiolactone (.beta.-Prone, Grand Laboratories Inc., Larchwood, Iowa.) was added at the rate of 1 ml/1000 ml and the cultures incubated at 37.degree. C. and 100 rpm overnight. Alternatively, 0.1 M binary-ethylenimine (BEI) was added at the rate of 100 ml/1000 ml, and the cultures incubated at 37.degree. C., and 100 rpm overnight. The BEI was neutralized using 100 ml of 1 M sodium thiosulfate. Inactivation of the cultures was confirmed by plating multiple 1 ml samples onto CBA-NAD, and incubating the plates for 48 h at 37.degree. C. with 5% CO.sub.2. From each inactivated culture, 500 ml was stored at 4 to 7.degree. C. The remaining 500 ml from each culture was centrifuged at 15,000.times.g for 15 min at 4.degree. C. The supernatant was recovered and stored at 4.degree. C. The cell pellet was washed once in PBS. The cells were resuspended in 10 ml of PBS and stored frozen at -20.degree. C.
Pig Sera
Sera from pigs naturally infected with A. pleuropneumoniae serotypes 1, 5, and 7 were supplied by B. J. Fenwick, Manhattan, Kans. Serum from a pig immunized with gel-purified haemolysin/cytolysin from A. pleuropneumoniae serotype 1 was provided by J. Devenish (1989). Because ApxI and ApxII have very similar molecular weights, this antisera recognizes both ApxI and ApxII. Sera from pigs vaccinated with commercial bacterin, experimental bacterins, adjuvant, PBS, or low-dose challenged with A. pleuropneumoniae CM5, were obtained in the study described herein. Pigs which were low-dose challenged were exposed to 1.times.10.sup.6 cells on two separate occasions and treated with penicillin at the first sign of respiratory distress. These pigs were solidly immune to subsequent challenge.
Collection and Processing of Blood Samples
Blood was collected from the orbital sinus using 20 gauge needles. Alternately, blood was obtained from the anterior vena cava using 10 ml Vacutainer tubes and 20 gauge needles. The blood was allowed to clot at room temperature and the serum separated by centrifugation at 2000 rpm for 10 min in a Sorvall GLC-1 centrifuge (DuPont Ltd., Mississauga, Ont.). The serum was dispensed into plastic vials and stored at -20.degree. C. prior to use.
Preparation of Antigens
Outer membrane proteins (OMPs) were prepared by the method of Deneer and Potter (1989). Cells grown overnight on CBA-NAD were washed from plates and suspended in 1 ml sterile PBS. Approximately 100 .mu.l were then used to inoculate 10 ml of growth medium. The culture was incubated for 2 h and used to inoculate 1 liter of the same liquid medium. This culture was incubated overnight under appropriate conditions. Bacteria were harvested by centrifugation in 500 ml bottles at 7000 rpm for 20 min at 4.degree. C. in a Sorval RC5B centrifuge with a GS3 rotor (DuPont). The resulting pellets were washed by repeat centrifugation with PBS, pooled, and frozen at -20.degree. C. for overnight storage. The next day, the pellets were thawed, resuspended in 10 mM HEPES, pH 7.4 (at a rate of approximately 1 g wet weight cells to 18 ml), and sonicated until the solution cleared (about 10 min). Aggregates and unbroken cells were removed by centrifugation at 10,000 rpm for 4 to 5 min at 4.degree. C. in a SS34/SA600 rotor. The supernatants were incubated for 10 min at room temperature with 2% (vol/vol) Sarcosyl in 10 mM HEPES, and then centrifuged at 100,000.times.g for 60 min at 4.degree. C. in a Beckman XL-90 ultracentrifuge using an SW41 rotor. The supernatants were discarded and the pellets resuspended in 6.5 ml of 10 mM HEPES. An equal volume of 2% sarcosyl in 10 mM HEPES was added and the preparation covered with Parafilm and incubated at room temperature for 30 min with shaking. The preparations were then centrifuged at 100,000.times.g for 60 min at 4.degree. C. Pellets were resuspended in approximately 200 .mu.l pyrogen free water and stored at -20.degree. C. The protein content was determined using the Bio-Rad protein microassay kit (Bio-Rad, Richmond, Calif.). For electrophoresis, the preparations were diluted to 1 .mu.g/.mu.l in Laemmli's sample buffer and boiled for 5 min.
In vivo antigen was recovered from pleural fluid obtained at necropsy from pigs experimentally infected with A. pluroptneumoniae CM5. Fluid was centrifuged at 60.times.g for 20 min at 4.degree. C. The supernatant was centrifuged three more times as before. The resulting supernatant was centrifuged at 4.degree. C. to pellet the bacteria. Due to the amount of fibrin in the material, the quantity of cells recovered was very low. The purity of the washed pellet was determined by plating on CBA-NAD.
Polyacrylamide Gel Electrophoresis (PAGE)
A modification of the discontinuous buffer system of Laenli as described by Rodriquez and Tait (1983) was employed using the Bio-Rad Miniprotean II electrophoresis apparatus. The stacking gel contained 4% (wt/vol) acrylamide/0.1% (wt/vol) bis-acrylamide and the resolving gel contained 12% (wt/vol) acrylamide/0.4% (wt/vol) bis-acrylamide. Prior to electrophoresis, samples were solubilized by boiling for 5 min in SDS-PAGE sample buffer containing 20% (vol/vol) glycerol, 10% (vol/vol) 2-mercaptoethanol, 6% (wt/vol) sodium dodecyl sulfate (SDS), 0.125 M Tris (pH 6.8) and 0.1% (wt/vol) bromophenol blue. Electrophoresis was carried out for 45 min at 195 V with cooling. The gels were then used for immunoblotting, stained with Coomassie Brilliant Blue G250 (Bio-Rad) or silver stained using the Bio-Rad silver staining kit (Bio-Rad). High molecular weight, low molecular weight, or pre-stained molecular weight standards (Bio-Rad) were included in the gels as needed.
Immunoblotting
Proteins separated by SDS-PAGE were transferred to nitrocellulose (NC; Schleicher & Schuell, Inc., Keene, N.H.) or nylon (Biotrans; ICN Pharmaceuticals Inc., Irvine, Calif.) membranes using the Bio-Rad Mini Trans Blot blotting apparatus. Transfers were performed at 100 V for 1 h. After transfer, the membranes were blocked overnight in skim milk solution (BLOTTO-Table 3). Following two 15 min washes in Tween-Tris buffered saline (TTBS-Table 3) the membranes were incubated for 2 to 24 h at room temperature with various swine antisera diluted 1:100 in antibody buffer (Table 3). Various sera were compared on the same blot using a 16 channel slit-blotter (Miniblotter-16, Immunetics, Cambridge, Mass.). Following two washes in TTBS the blots were incubated for 1 h at room temperature with 1:1000 goat anti-swine-IgG (H&L)-alkaline phosphatase (Kirkegaard and Perry Laboratories Inc., Gaithersburg, Md.). Following two 5 min washes in Tris buffered saline (TBS), blots were developed using 5-bromo-4-chloro-3-indoyl phosphate and nitroblue tetrazolium colour development substrate (BCIP/NBT, Bio-Rad). Development was stopped by rinsing the blots in distilled water.
Experimental Bacterin Preparations
Growth Conditions--Trial 1
Lyophilized cultures of A. pleuropneumoniae CM5 (serotype 1), and BC181 (serotype 3) were plated onto 5% sheep blood agar with 0.01% (wt/vol) NAD (SBA-NAD) and incubated overnight at 37.degree. C. with 5% CO.sub.2. Isolated colonies of each strain were selected and seeded into separate 100 ml volumes of TYE with 0.01% NAD (TYE-NAD) pre-warmed to 37.degree. C. These were incubated overnight with shaking (100 rpm) at 37.degree. C. These seed cultures were inoculated into 1 liter volumes of TYE-NAD at 37.degree. C., and incubated for 6 h at 100 rpm. After incubation, cultures were plated for purity and heat-inactivated for 2 h at 60.degree. C. These cultures were used to make experimental bacterin A.
Growth Conditions--Trial 2
A. pleuropneumoniae VSB68, serotype 1 (Langford/Cyanamid) was used to prepare the experimental bacterins for this trial. Cultures were grown by two different methods. The first culture was grown according to the production methodology used to produce the commercial multi-strain bacterin. Overnight cultures of A. pleuropneumoniae from CBA-NAD plates were seeded into 100 ml of Tryptic Soy Broth (TSB) with 0.1% NAD (TSB-NAD) at 37.degree. C., and incubated with shaking at 100 rpm for 24 h. These overnight cultures were then seeded into 1 litre of TSB-NAD at 37.degree. C. and incubated as before for 24 h. Following incubation, the cultures were heat-inactivated for 2 h at 60.degree. C. Alternately, 24 h cultures from CBA-NAD plates were seeded into 100 ml of TYE-NAD broth and incubated for 24 h at 37.degree. C. with shaking at 100 rpm. This culture was then used to seed 1 litre of TYE-NAD at 37.degree. C. which was further incubated for 6 h at 37.degree. C. with shaking at 100 rpm. Following incubation the culture was heat-inactivated for 2 h at 60.degree. C. These cultures were used to make experimental bacterins B, C, D, and E.
Bacterin Composition--Trial 1
The antigens for bacterin A were blended to a final density of 1.times.10.sup.10 /2 ml dose of each strain. The bacterin was adjuvanted with L80 adjuvant (Langford/Cyanamid), containing Al(OH).sub.3 (Reheis), and Quil A (Superphos, Denrmark).
The composition of bacterin A is summarized in Table 5.
Bacterin Composition--Trial 2
Experimental bacterins B, C, D, and E were prepared from the heat-inactivated cultures of A. pleuropneumoniae VSB68. The culture from each medium (TYE or TSB) was used to prepare two bacterins at 6.times.10.sup.9 or 2.times.10.sup.10 /2 ml dose respectively. All bacterins were adjuvanted with AlOH, and Quil A. The composition of bacterins B, C, D, and E are summarized in Table 6.
Vaccination and Challenge with Bactrin Compositions
For each experiment, pigs were vaccinated intramuscularly with two 2 ml doses of vaccine or placebo, 21 days apart. Low-dose vaccination was performed by exposing the pigs to an aerosol of a culture containing approximately 10.sup.6 organisms/ml of A. pleuropneumoniae CM5A. Aerosol challenge was performed 14 days following the second vaccination using the following steps:
1. Challenge organism is obtained from pleural fluid of an experimentally infected pig, stored at -70.degree. C. (Serotype 1, strain CM5)
2. Subculture the organism overnight on chocolate blood agar with 0.1% NAD to determine purity.
3. Resuspend the growth in sterile PBS and adjust the OD.sub.625 =0.2
4. For challenge this suspension is further diluted 1/10-1/500 in PBS depending on the age and type of pig (CDCD vs SPF). (CFU=approx. 10.sup.6 /ml)
5. Place 75 ml of diluted suspension into the DeVilbiss nebulizer which is connected to the aerosol chamber containing the pigs.
6. Run the nebulizer for 10 minutes to fill the chamber. With the nebulizer off, allow a further 10 minutes for the pigs to inhale the aerosol.
Trial 1--Bacterin A
The pigs used in this experiment were from a litter of Caesarian-derived, colostrum-deprived (CDCD) piglets of a sow from the Arkell Research Station, University of Guelph. The piglets were hand reared in isolation, and fed Launch, a liquid milk replacement (Shur-Gain, Canada Packers Inc.), and Vital, a bovine colostrum supplement (Shur-Gain, Canada Packers Inc.), until they were able to eat solid swine rations. This litter was one of nine similar litters involved in a larger study examining humoral and cell-mediated immunity to A. pleuropneumoniae.
Pigs were assigned ad hoc to groups as follows: improved bacterin (5), commnercial bacterin (2), low-dose exposed (3), adjuvant controls (4), PBS controls (6). Starting at day 35, piglets in each treatment group were either low-dose exposed or injected IM with the bacterins, adjuvant or PBS. This was repeated 21 days later. Two weeks after the second treatment, all the piglets were challenged with an aerosol of A. pleuropneumoniae CM5 as described above. Blood samples were taken just prior to the first vaccination, the second vaccination, challenge, and euthanasia.
Trial 2--Bacterins B, C, D, and E
The pigs used in the second vaccination trial were derived from an SPF herd and obtained through Dr. C. Templeton (Orange Hill Farms, Ontario, Canada). Representative pigs from the herd were determined to be free of antibodies to A. pleuropneumoniae toxins by ELISA and susceptible to challenge with A. pleuropneumoniae CM5 in a preliminary challenge experiment.
Thirty pigs were placed ad hoc into three isolation rooms, and were then randomly assigned a treatment by drawing ear tags pre-labelled with a treatment group from a bag. Each group was vaccinated IM with 2 doses of vaccine or placebo, 21 days apart. Pigs were challenged with an aerosol of A. pleuropneumoniae CM5 two weeks after the second dose. Blood samples were taken immediately prior to the first and second vaccinations, prior to challenge, and at euthanasia. The pigs were weighed at first vaccination and those surviving to day 5 post-challenge were weighed again prior to euthanasia and necropsy.
All procedures involving animals were carried out in accordance with the guidelines outlined in the Canadian Council On Animal Care's "Guide to the Care and Use of Experimental Animals".
ELISA
Serum antibody titres to lipopolysaccharide (LPS), capsular polysaccharide (CPS), and haemolysin/cytolysin (CYT) were measured by ELISA according to the method of Bosse et al. (1992). Antigens were prepared as described previously (Bosse et al., 1992). To coat ELISA plates (Microwell 2-69620, Nunc), CPS was diluted to 0.125 .mu.g/ml and LPS was diluted to 0.625 .mu.g/ml in carbonate coating buffer. Plates were incubated at 4.degree. C. overnight with 100 .mu.l of diluted antigen per well. Flat bottom microtitre plates (Immulon II, Dynatech Laboratories) were coated with 100 .mu.l/well of a 1:2000 dilution of rabbit anti-CM5 CYT overnight at 4.degree. C. Wells were blocked with 5% normal rabbit serum in PBS-T for 1 h. Using rabbit anticytolysin antibody (Devenish et al., 1989), CYT was captured out of crude supernatant containing 800 hemolytic unit/ml by incubation with 100 .mu.l/well overnight at 4.degree. C. Plates were washed 3 times with 250 .mu.l PBS-T between each step.
Serum samples were diluted 1:50, 1:200, 1:800, and 1:3200 in TTBS. For the CYT ELISA, 5% normal rabbit serum was added to the diluent. Samples were tested in duplicate on opposing quadrants of each plate. Positive and negative control sera were included on all plates. Positive serum was obtained from a pig exposed to a low-dose challenge with A. pleuropneumoniae CM5.
Plates were incubated with serum dilutions (100 .mu.l per well) for 2 h at room temperature. After washing, plates were incubated for 2 h at room temperature with biotin-labelled mouse anti-swine IgG (H+L) (Zymed Laboratories, South San Francisco, Calif.), and then for 30 min with 50 .mu.l/well of 1:8000 stepavidin-peroxidase (Zymed Laboratories, South San Francisco, Calif.).
The hydrolysis of H.sub.2 O.sub.2 by bound peroxidase was determined in the presence of ABTS (Boehringer Mannheim Biochemical, Indianapolis, Ind.) at a wavelength of 405 nm with a microplate reader (Bio-Tek Instruments, Winooski, Vt.). The absorbance of the 1:200 dilution of the positive control serum was monitored until the OD reached 1.000 at which time the plates were read.
The mean OD of duplicate wells for each serum dilution were calculated and these data were used to calculate least squares mean OD for each treatment group (General linear models procedure, the SAS system, Kelwig and Council, 1979).
Example 1
Variation of Cellular Antigens as a Function of Growth Conditions
The expression of WC and outer-membrane protein antigens of A. pleuropneumoniae CM5 grown under different conditions in different media was examined. Cells were grown under aerobic or anoxic conditions. In vivo antigen, recovered from the lungs of infected pigs, was compared. Coomassie blue-stained polyacrylamide gels of WC and outer membrane protein enriched fractions are shown in FIG. 4A and FIG. 4B.
The following should be noted in FIGS. 4A and 4B: Molecular weight markers (lane 1), WC proteins of strain CM5 grown under anoxic conditions in RPMI (lane 2), CM5, RPMI, aerobic (lane 3), CM5, TYE, anoxic (lane 4), CM5, TYE, aerobic (lane 5), strain VSB68, TSB, aerobic (lane 6), CM5 in vivo (lane 7). The positions of the 35, 45, 47, and 72K bands are indicated with arrows. The sizes of the molecular weight markers (in K) are indicated on the left; (B) Molecular weight markers (lane 1), Outer-membrane protein-enriched fractions of strain VSB68, TSB, aerobic (lane 2), CM5, TYE, aerobic (lane 3), CM5, RPMI, anoxic (lane 4), CM5, RPMI, aerobic (lane 5). The positions of the 17, 26, 32, 40, 42 and 72K bands are indicated with arrows. The sizes of the molecular weight markers (in K) are indicated on the left.
In FIG. 4A, the PAGE profiles of WC preparations derived from RPMI (aerobic and anoxic) (lanes 2 and 3), TYE (aerobic and anoxic) (lanes 4 and 5), TSB (bacterin) (lane 6) and in vivo cultures (lane 7) are shown. There were major protein bands at 47K, 45K, and 37K. An additional major protein band was seen at 72K in WC samples from the RPMI-grown cultures (lanes 2 and 3). This band was not as evident in the WC samples grown in the other media. The TSB WC gel profile (lane 6) showed significant smearing of bands in the high molecular weight range and individual bands were difficult to discern.
In FIG. 4B, PAGE profiles of TSB (bacterin) (lane 2), TYE (lane 3), and RPMI (lane 4 and 5) derived OMP samples are shown. Major OMPs were detected at 17K, 26K, 32K, 40K, 42K and 72K in the RPMI and TYE grown samples (lanes 3, 4, and 5). Unfortunately, the OMP yields from bacterin and in vivo cultures were too low to make definitive comparisons possible.
In order to determine whether there were other differences in antigen expression of A. pleuropneumoniae due to medium and growth conditions, the protein profiles were investigated by immunoblotting. Sera collected from swine which had been vaccinated with a commercial bacterin, low-dose challenged, or mock-vaccinated, were used (Table 7). The sera were used to probe Western blots of WC fractions of A. pleuropneumoniae serotype 1 from TSB, TYE, RPMI and in vivo cultures (FIGS. 5A to 5C).
FIGS. 5A to 5C show Western blots of WC proteins of A. pleuropneumoniae serotype 1 grown in various media probed with sera from a pig exposed to low-dose challenge of A. pleuropneumoniae CM5 (A), or sera from a pig hyperimmunized with purified cytolysin from CM5 (B) (Devenish and Rosendal, 1989), or sera from a pig immunized with commercial bacterin (C). Note the following in the Figures: WC proteins of strain VSB68 grown in TSB aerobically (lane 1), strain CM5 grown in TYE, aerobic (lane 2), CM5, TYE, anoxic (lane 3), CM5, RPMI (lane 4), in vivo (lane 5). The positions and sizes of the molecular weight markers (in K) are indicated on the right.
FIG. 5A shows a Western blot of WC preparations probed with sera from low-dose challenged pigs. An antigen of an approximate molecular weight of 105K, which appeared as a doublet, was detected in cells grown in TYE (lane 2) but, not in the other media (lanes 3 and 4), nor in in vivo grown cells (lane 5). When hyperinmmune pig serum raised against gel-purified CM5 cytolysin was used to probe an identical blot, a similar band was seen in the TYE grown WC preparation but not in the other samples (FIG. 5B). Serum from a pig vaccinated with a commercial A. pleuropneumoniae bacterin failed to recognize this antigen, but did detect antigens of approximate molecular weights of 17K, 32K, 34K, and 40K in all the preparations (FIG. 5C).
A similar result was seen when immunoblots of WC proteins of A. pleuropneumoniae strain BC181, serotype 3, grown in TYE broth were probed with the same sera. The sera from low-dose exposed pigs recognized a unique band at 105K as well as an additional band at approximately 120K. These bands were not recognized by the sera from bacterin vaccinates or controls (Data not shown).
FIG. 6 shows a Western blot of OMPs of A. pleuropneumoniae serotype 1 grown in various media probed with sera from a pig exposed to low-dose challenge of A. pleuropneumoniae CM5. Note the following in the Figure: OMPs of strain VSB68 grown in TSB aerobically (lane 1), strain CM5, TYE, aerobic (lane 2), CM5, TYE, anoxic (lane 3), CM5, RPMI, aerobic (lane 4), CM5, RPMI anoxic (lane 5), in vivo (lane 6). The positions and sizes of the molecular weight markers (in K) are indicated on the right.
In FIG. 6, sera from low-dose challenged pigs was used to probe blots of OMPs derived from cultures grown in the various media. No major differences in reactivity patterns were seen between OMP profiles from cells grown in TYE or RPMI. As mentioned above, it was not possible to adequately compare blotting profiles from bacterin and in vivo preparations due to the poor recovery of OMPs from the available material.
These experiments confirm that the expression of protein antigens of A. pleuropneumoniae can be altered by changing growth conditions (Deneer and Potter, 1989; Niven et al., 1989; O'Reilly et al., 1991).
In these experiments, in vivo cells recovered at necropsy from the pleural fluid of infected pigs were used as a standard for comparison of WC protein profiles of cells grown in vitro. Of the cultures studied, TYE grown cells had a WC protein profile most similar to that of the cells grown in vivo. On the other hand, RPMI and TSB WC profiles were quantitatively different from the in vivo standard. There were only minor quantitative differences in patterns from anoxic or aerobic paired samples grown in the same medium.
It was not possible to recover sufficient quantities of OMP-enriched fractions of in vivo and bacterin-derived cells to make a definitive comparison. Although there appeared to be minor quantitative differences in expression of major OMPs, no major differences in OMP pattern was seen between samples derived from different medium or growth conditions.
Of particular significance in this study was the finding that 103K and 105K proteins that were cell-associated could be detected in cells harvested from log-phase TYE broth culture. This confirms the work of Fedorka-Cray and others (1989) who determined that hemolysin and/or cytolysin is not only secreted into the culture medium but is also cell-associated. The nature of this association is not known, possibly involving transport across the cell membrane or attachment of the toxin molecule or precursor proteins before secretion.
This study indicates that the quantities of cell-associated cytolysin produced in culture are affected by the growth medium. Immunoblotting showed that cells from RPMI or TSB (bacterin) cultures were deficient in cell-associated cytolysin as compared to TYE cultures. Also, the expression of other cellular antigens was influenced by the growth medium. WC protein profiles of organisms grown in TYE broth were nearly identical to the in vivo standard. Therefore, TYE would be a preferred growth medium to produce a conventional WC bacterin which is enriched in cytolysin protein and contains WC antigens analogous to those found in vivo.
Example 2
Identification of Major Cellular Antigens Recognized by Immune and Control Sera
To correlate the expression of key antigens in vitro to the recognition of those antigens by protected or susceptible animals, WC antigens, OMPs (outer membrane proteins) and partially purified cytolysins from A. pleuropneumoniae serotype 1 were separated by polyacrylamide gel electrophoresis and were examined by immunoblotting using sera from swine naturally exposed to A. pleuropneumoniae serotypes 1, 5, and 7. The sera used are listed in Table 8.
As well, these antigens were probed with sera from swine exposed to low-dose challenge, vaccinated with a commercial bacterin or control groups vaccinated with adjuvant or PBS placebo vaccines (Table 9).
Naturally Infected Animals
FIG. 7 shows Western blots of WC antigen preparations from A. pleuropneumoniae strain CM5 grown in TYE probed with sera from animals naturally infected with serotypes 1, 5, and 7. Note in FIG. 7, Serotype 1: serum #1438, #1458, and #1467 (lanes 1 to 3); Serotype 5: serum #5777, #5781, and #5682 (lanes 4 to 6); Serotype 7: serum #5816, #5818, and #5819 (lanes 7 to 9); Serum from a pig hyperimmunized with gel-purified cytolysin from CM5 (lane 10). The positions and sizes of the molecular weight markers (in K) are indicated on the right.
As shown in FIG. 7, all the sera recognized a predominant reactive band at 105K. However, the band which was apparent with serotype 1 and 5 antisera appeared as a distinct doublet (lanes 1 to 6), while the band recognized by serotype 7 antisera was a single line (lanes 7 to 9).
When the same sera were used to probe blots of OMPs of CM5, distinct differences were seen. FIG. 8 shows a Western blot of OMPs of Actinobacillus pleuropneumoniae CM5 probed with sera from swine naturally infected with serotype 1, serum #1438, #1458 and #1467 (lanes 1 to 3); serotype 5, serum #5777, #5781 and #5782 (lanes 4 to 6); serotype 7, serum #5816, #5818, and #5819 (lanes 7 to 9); Serum from a pig hyperimmunized with gel-purified cytolysin from CM5 (lane 10). The positions and sizes (in K) of the molecular weight markers is shown on the right.
FIG. 8 shows that sera from the homologous serotype, 1, recognized the majority of OMP bands (anes 1 to 3). Sera from serotype 5 recognized fewer bands at 72K and 106K (lanes 4 to 6), while sera from serotype 7 did not seem to recognize any of the OMPs of serotype 1 Lanes 7 to 9).
Vaccinated Animals
FIG. 9, Panels A to F show Western blots of WC proteins of A. pleuropneumoniae serotype 1 strain CM5 grown in RPMI (A), CM5, TYE, (B), VSB68, TSB (C), and outer-membrane proteins from CM5 grown in RPMI (D), CM5, TYE (E), VSB68, TSB (F). The blots were probed with swine sera from low-dose vaccinated animals #A4-3, #A9-3, #B14-3, and #B15-3 (lanes 1 to 4), commercial bacterin vaccinated animals #A3-3, #A11-3, #B19-3, #B20-3, and #B21-3 (lanes 5 to 9), PBS control animals #A10-3, and #B16-3 (lanes 10 to 11), adjuvant control animals #A6-3, #A8-3, #B8-3, and #B22-3 (lanes 12 to 15). The positions and sizes (in K) of the molecular weight markers are shown on the right.
FIG. 9, Panel A, lanes 1 to 4, 5 to 9, 10 to 11, and 12 to 15, respectively show the results of the studies where sera from pigs exposed to a low-dose challenge, vaccinated with a commercial A. pleuropneumoniae bacterin, or PBS, or adjuvant, were used to probe WCs proteins from strain CM5 grown in RPMI. Aside from minor variations between individual pigs, the sera from the bacterin and low-dose vaccinates recognized the same pattern of WC proteins, while control sera from PBS and adjuvant treated animals reacted only weakly. However, when the same sera were used to probe WC proteins from cells grown in TYE medium, a significant difference in the antigen recognition pattern was evident (FIG. 9, Panel B). An intensely stained doublet at approximately 105K was recognized by the low-dose sera (FIG. 9, Panel B, lanes 1 to 4) but not by any other sera. Immunoblots of WC proteins from VSB68 grown in TSB (bacterin) were poorly reactive when probed with the same sera (FIG. 9, Panel C). No apparent differences in reactivity were detected with any of the sera.
Sera from low-dose (lanes 1 to 4) and bacterin (lanes 5 to 9) vaccinated animals reacted similarly in blots of OMPs from CM5 grown in RPMI (FIG. 9, Panel D). Wide, densely stained bands were seen at 17K and 32K, as well as bands at 40-42K, 72K and 106K. All the sera, including control sera, reacted with a band at approximately 55K. In contrast, when Western blots of OMPs from CM5 grown in TYE were probed with sera from low-dose, commercial bacterin vaccinates and controls, some differences were seen (FIG. 9, Panel E). Sera from low-dose vaccinates recognized 4 or more OMPs at 72K, 42K, 40K and 26K (lanes 1 to 4), while sera from bacterin vaccinates recognized only lower molecular weight OMPs at 40K and 26K (lanes 5 to 9). Control sera were non-reactive. None of the sera reacted well with OMPs from VSB68 grown in TSB (bacterin) (FIG. 9, Panel F). Except for weak reactions with the 32K OMP band shown by sera from low-dose (lanes 1 to 4) and bacterin (lanes 5 to 9) vaccinates, the sera were non-reactive.
In order to confirm the identity of the 105K bands seen in the blots of the TYE grown cells, the same sera were used to probe a partially purified cytolysin preparation from CM5. FIG. 10 shows a Western blot of CYT from CM5 probed with sera from CDCD pigs exposed to low-dose challenge of A. pleuropneumoniae CM5 (lanes 1 to 4), vaccinated with a commercial bacterin (lanes 5 to 9), vaccinated with PBS (lanes 10 and 11), vaccinated with adjuvant (lanes 12 to 14), CYT positive control sera (lane 15). The positions and sizes (in K) of the molecular weight markers is shown on the right. As shown in FIG. 10, similar 105K bands were recognized by the sera from the low-dose vaccinates (lanes 1 to 4), but not with the sera from commercial bacterin (lanes 5 to 9), PBS (lanes 10 and 11), or adjuvant vaccinated animals (lanes 12 to 14).
Inactivation
The effect of the inactivation procedure on the reactivity of WC antigens from serotype 1 and serotype 3 grown in TYE was compared by immunoblotting. FIG. 11A and FIG. 11B show Western blots of WC proteins of A. pleuropneumoniae probed with serum from a pig hyperimmunized with gel-purified CYT (FIG. 11A), or serum from a pig exposed to low-dose challenge (FIG. 11B). Note the following in the Figures: Strain BC181 serotype 3 (lanes 1 to 4), strain CM5 serotype 1 (lanes 5 to 8). Formalin inactivated (lane 1 and 5), beta-propiolactone inactivated (lanes 2 and 6), binary-ethyleneimine inactivated (lanes 3 and 7), heat-inactivated (lanes 4 and 8). The positions and sizes (in K) of the molecular weight markers is shown on the right.
As shown in FIG. 11, only minor variations in the reactivity of the major 105K band were seen when cells inactivated with the chemical agents formaldehyde, BPL and BEI (lanes 1 to 3 and 5 to 7) were compared to heat-inactivated preparations (lanes 4 and 8).
In North America, the three serotypes of A. pleuropneumoniae most commonly causing disease are 1, 5, and 7. Kamp et al., (1991) confirmed that these three serotypes produce a 103K cytolysin, ApxII, and that serotypes 1 and 5 produce a 105K cytolysin, ApxI. Sera from pigs naturally exposed to the individual serotypes were used to identify the key antigens in these serotypes. Immunoblotting experiments confirmed that IgG antibodies to the ApxI and ApxII cytolysins of A. pleuropneumoniae serotype 1 were common and conspicuous in these sera.
There was less consistency in the antigens recognized in OMP-enriched preparations. Homologous sera reacted well, however sera from serotype 5 infected animals detected fewer OMP antigens of serotype 1, while sera from serotype 7 infected animals were non-reactive. This is in contrast to cross-reactivity seen with serotype 1 to 8 hyperimmune rabbit sera (MacInnes and Rosendal, 1987). It may be that less virulent serotypes such as serotype 7 do not stimulate as strong an immune response as the more virulent serotypes.
There are consistant differences in the reactivity of sera from low-dose infected animals as compared to bacterin vaccinates. The sera from animals vaccinated with the commercial bacterin lacked the antibodies to the ApxI and ApxII cytolysins whereas sera from animals that had recovered from infection had high levels of anti-cytolysin antibodies. Also, the response to commercial vaccine appeared to favour production of antibodies to lower molecular weight antigens as seen in the immunoblots (FIG. 7C, lanes 1 to 5).
In general, the quality of the commercial antigen was poor. In the blotting experiments, protein bands were consistantly diffuse and fuzzy, independent of the antibody used to probe the blots. This could be the result of the use of formalin as an inactivating agent. The chemical cross-linking of proteins likely interferes with electrophoresis, transfer, and antibody binding.
These experiments highlight a consistent difference in antibody response between animals infected with A. pleuropneumoniae and animals vaccinated with a commercial inactivated-WC bacterin. The response of infected animals to the cytolysins of A. pleuropneumoniae is likely a marker for protective immunity, in contrast to the lack of response to these proteins by vaccinated animals.
Therefore, the protective efficacy of a WC bacterin may be improved if the level of immunogenic cytolysin could be increased. The evaluation of experimental bacterins based on TYE grown cell is described below.
Example 3
Vaccination of Pigs with Experimental Bacterins
One of the objectives of the investigations described herein was to improve the protection afforded by a conventional WC A. pleuropneumoniae bacterin. By evaluating the expression of cellular antigens in various media under different growth conditions and comparing the response patterns of protected and susceptible pigs, a method of culturing and inactivating A. pleuropneumoniae to produce and conserve important antigens was developed. Experimental bacterins were prepared (as described above) and used to vaccinate CDCD and conventional pigs in two separate challenge experiments. The protection afforded by the vaccines was evaluated by measuring clinical signs, gross pathological examination, bacteriological culture, and by serological evaluation of blood samples collected throughout the trials.
Trial One--Caesarian Derived, Colostrum Deprived Pigs
All of the piglets receiving adjuvant placebo treatment and four of the five pigs given PBS were euthanized with severe clinical signs within 24 h after challenge. The remaining PBS-treated pig was euthanized at day 10. The group receiving the conventional bacterin were all euthanized within 24 h of challenge. One of the pigs exposed to a low-dose of A. pleuropneumoniae developed severe clinical disease and had to be euthanized prior to challenge. This pig was excluded from the study. The remaining low-dose piglets and the piglets receiving Bacterin A all survived challenge.
The lung scores of the pigs at necropsy are presented in Table 10 and the lung lesions are depicted in FIG. 12. In FIG. 12, the treatment groups are as follows (A) adjuvant, (B) PBS, (C) bacterin A, (D) low-dose challenge, (E) commercial bacterin. The low-dose and experimental vaccine groups had similar, very low lung lesion scores with means of 0.5 and 0.4 respectively. In contrast, the pigs vaccinated with the commercial bacterin had lesions similar to those of PBS or adjuvant control groups. The means for these groups were 5.5, 4.0, and 6.0 respectively.
Statistically there was no significant difference between the mean lesion scores of the low-dose and bacterin A treatment groups (p=0.5). Also, the mean lesion score for the commercial bacterin group was significantly different from those of the low-dose and bacterin A treatment groups, but was not significantly different from adjuvant and PBS control groups.
Western blot analysis of WC proteins from A. pleuropneumoniae CM5 probed with the sera from the various treatment groups is presented in FIG. 13. In particular, FIG. 13 shows a Western blot of WC proteins of A. pleuropneumoniae probed with sera from CDCD pigs vaccinated with adjuvant (lanes 1 to 4), PBS (lane 5), low-dose challenge (lanes 6 and 7), Bacterin A (lanes 8 to 12), commercial bacterin (lanes 13 and 14), and CYT positive control serum (lane 15). The positions and sizes (in K) of the molecular weight markers are shown on the right.
As shown in FIG. 13, the sera from two low-dose vaccinates recognized prominant bands at 105K, 72K, 45K, 32K and 17K (lanes 6 and 7). The sera from the experimental bacterin-treated pigs had nearly identical profiles to those of the low-dose sera except there was a less intense reaction at 105K (lanes 8 to 12). The antigen recognition of the sera from pigs vaccinated with commercial bacterin was extremely poor (lanes 13 and 14), and not very different to that seen with the sera from the PBS (lane 5) and adjuvant treated (lanes 1 to 4) control animals.
An immunoblot of purified CM5 CYT probed with sera from the vaccinated pigs in Trial 1 is shown in FIG. 14. The low-dose pig sera recognized the 105K and 103K antigen band (lanes 1 and 2), as did the sera from the pigs given experimental bacterin A (lanes 5 to 8). In contrast, sera from pigs given the commercial bacterin did not react with the CYT from CM5 (lanes 3 and 4). The haemolysin control is shown in lane 9. The positions and sizes (in K) of the molecular weight markers are shown on the right in FIG. 14.
ELISA titres of the sera are presented in Table 12. The low-dose group had a significant response to CYT (0.800), as did the Bacterin A pigs (0.264). The response of the pigs vaccinated with commercial bacterin (0.069) was not significantly different from that of the PBS or adjuvant control groups. Except for the Bacterin A pigs, the response to LPS and CPS was not signifcant.
Trial Two--SPF Pigs
In a second trial, commercially-reared SPF pigs, 4 weeks of age were vaccinated with experimental bacterin preparations B, C, D, and E. Table 13 summarizes lung scores, lung/heart weight ratios, lung culture results, and weight gain or loss for the animals surviving to day 5. The distribution of lung lesions is pictured in FIG. 15. (In FIG. 15, the treatment groups shown are as follows (A) adjuvant, (B) PBS, (C) bacterin B, (D) bacterin C, (E) bacterin D, (F) bacterin E.) All 5 pigs that received adjuvant showed severe clinical signs within 24 h post-challenge and were euthanized. Three of five PBS vaccinated animals also showed severe clinical signs within 24 h post-challenge and were euthanized. The remaining two PBS-treated animals developed severe respiratory distress at day 2 and day 4 respectively and were euthanized.
Of the four vaccine treatment groups, all five pigs in groups B and C survived and were euthanized 5 days post-challenge. One animal in group D developed significant respiratory symptoms at day 1 and was euthanized. However, at post-mortem lung lesions were less remarkable than those of the control animals. The four remaining animals in group D survived to day 5. In group A, three of five animals developed disease and were euthanized between days 1 and 3. The remaining two pigs survived to day 5.
Statistical analysis of mean lung scores and lung/heart weight ratio showed similar results. There was a significant difference between mean scores for the adjuvant and PBS treatment groups compared to all four bacterin treatment groups. There was no significant difference in mean lung lesion or lung/heart ratio scores of any of the bacterin treatment groups. The control groups were not significantly different from each other.
Table 14 summarizes the serological ELISA results for CYT, LPS and CPS. In contrast to the results of trial 1, all of the animals receiving bacterin treatments responded significantly to LPS and CPS, in addition to CYT, as compared to the PBS and adjuvant treatment groups.
There was no difference in response to LPS between bacterin groups B, C, and E. Bacterin D induced an LPS response that was statistically significantly better than that of bacterin C, but not significantly different from that of bacterins B and E. Exactly the same pattern of significance was seen when response to CPS was compared.
Statistical analysis of CYT responses showed a slightly different response. The CYT titre to bacterin D was significantly higher than that of bacterins B, C and E. The response to bacterin E was better than the response to bacterin C, but not significantly different than the response to bacterin B. There was no difference in the CYT responses to bacterins B and C.
Western blots of WC and column-purified cytolysins probed with post vaccination sera from these pigs are presented in FIG. 16. In particular, FIG. 16 shows Western blots of WC proteins (A and B) and CYT (C and D) probed with sera from vaccinated SPF pigs. Note the following in the Figure: WC proteins probed with sera from pigs vaccinated with adjuvant (A, lanes 1 to 5), bacterin B (A, lanes 6 to 10) bacterin C (A, lanes 11 to 15), bacterin D(B, lanes 1 to 5), bacterin E (B, lanes 6 to 10), PBS (B lanes 11 to 15). CYT probed with sera from pigs vaccinated with CYT positive control (C and D, lane 1) adjuvant (C, lanes 2 to 5), bacterin B (C, lanes 6 to 10), bacterin C (C, lanes 11 to 15), bacterin D (D, lanes 2 to 6), bacterin E (D, lanes 7 to 11), PBS (C, lanes 12 to 15).
As expected, sera from pigs vaccinated with PBS (Panel B, lanes 11 to 15; Panel D, lanes 12 to 15) or adjuvant (Panel A, lanes 1 to 5; Panel B, lanes 2 to 5) gave almost no reaction with WC or CYT blots. Sera from pigs vaccinated with the experimental bacterins, B (Panel A, lanes 6 to 10), C (Panel A, lanes 11 to 15), D (Panel B, lanes 1 to 5), and E (Panel B, lanes 6 to 10), gave similar reactions with blots of WC protein. Differences were most obvious in the blots of CYT probed with these sera. In these blots, sera from bacterin D (Panel D, lanes 2 to 6) recognized the 105K bands that correspond to the cytolysins of CM5. Also, the sera from animals vaccinated with bacterin E (Panel D, lanes 7 to 11), also recognized these 105K bands, although more weakly.
For many years it has been reported in the literature that, at best, commerdal WC bacterins provide only partial, serotype-specific protection against morbidity and mortality in swine following infection with A. pleuropneumoniae (Neilson, 1984; Fedorka-Cray et al., 1990). In contrast, animals that recover from natural infection have solid cross-protective immunity.
In vaccine trial 1, experimental bacterin A containing heat-killed, TYE-grown cells and the proprietary adjuvant L80, was effective in providing protection against experimental challenge with the homologous strain. The mean lung score for the bacterin A group was not statistically different from that of the low-dose challenge group in which clinical disease was not observed. In comparison, the commercial bacterin was completely ineffective in providing protection against disease. The mean lung score for the commercial bacterin group was not statistically different from those of the PBS or adjuvant treated groups. This result is consistent with previous experimental results obtained with the commercial bacterin in five separate litters of CDCD pigs tested in separate experiments (Furesz, S. and B. Mallard, University of Guelph, unpublished data).
ELISA titres of sera from protected animals showed that bacterin A induced a marked anti-cytolysin response in vaccinated animals, but no titres to LPS or CPS. Immunoblots confirmed that sera from the protected animals recognized the 103K and 105K cytolysins and that these cytolysins were not recognized by sera from control animals or pigs that received the commercial vaccine. The association of anti-CYT response with protection is similar to the findings in previous studies showing that IgG response to CYT is necessary for protection (Rycroft et al., 1991; Bhatia et al., 1991; Inzana et al., 1991; Fedorka-Cray et al., 1990).
In addition to the changes of growth medium and inactivation methodology, the experimental bacterin used in the first trial differed from the commercial bacterin in several other ways. There were only two serotypes of A. pleuropneumoniae in the experimental vaccine, compared with four serotypes in the commercial product. The cell concentration in the experimental vaccine was higher than that of the commercial preparation, and the experimental vaccine contained an adjuvant which differed from the one in the commercial bacterin. Some, or all of these additional factors may have been important in the increased efficacy of the experimental vaccine over the commercial product.
The rationale for including both serotype 1 and 3 strains in bacterin A was the fact that these serotypes together produce all 3 A. pleuropneumoniae RTX toxins. Recent work by Frey et al. (1994, submitted for publication) showed that serotype 3 strains of A. pleuropneumoniae do not contain the apxIB and D genes which encode proteins that are responsible for ApxII secretion in other serotypes.
The second trial was designed to limit the number of variables between vaccines by eliminating serotype, antigen concentration, inactivation and adjuvant variables in the hopes of identifying which factor(s) were most important in improving bacterin performance. In vaccine trial 2, SPF pigs, determined to be susceptible to challenge with A. pleuropneumoniae, were used to compare monovalent experimental bacterins prepared from the serotype 1 strain of A. pleuropneumoniae used in the commercial bacterin. Low and high antigen content bacterins were prepared using either the TSB-grown, heat-kllled cells prepared using production methodology for the commercial bacterin, or TYE-grown, heat-killed cells prepared using the production methodology described for bacterin A.
Compared to controls, all four vaccines provided significant protection against aerosol challenge with a different serotype 1 isolate. In contrast to the findings of trial 1, serum ELISA titres to the four experimental preparations showed significant responses to LPS and CPS in addition to CYT. Since the SPF animals used in this experiment were conventionally-reared pigs, it is possible that they had some previous exposure to organisms with cross-reacting antigens that allowed them to produce an anamnestic response to LPS and CPS. In contrast to the commercial bacterin-treated group, all of the animals in the four experimental vaccine groups had a significant immunity to challenge. This is perhaps accounted for by the use of a more effective adjuvant in this group of vaccines as compared to the commercial bacterin. As shown by the ELISA data, all animals had IgG antibody responses to LPS, CPS, and CYT. The response to CYT was significantly better in the TYE bacterin groups than in the TSB bacterin groups, while the responses to LPS and CPS were only significantly different between high dose TSB and low-dose TYE groups.
Antibody responses to high and low-doses of antigen were not significantly different for LPS and CPS within bacterin types. However, antibody response to CYT was significantly better in the lower dose TYE bacterin group compared to the high dose TYE bacterin group. This suggests that there may be an optimum dose of CYT which is titratable or alternately that antigen/adjuvant ratios were not optimum in the higher dose antigen vaccines.
Example 4
PROTOCOL RE HETEROLOGOUS CHALLENGE
The test animals which will be used in this study are conventionally raised specific pathogen free swine from a high health status herd, 4-5 weeks of age, and free of antibody to A. pleuropneumoniae. Three or more groups of 10-15 animals will be used in the study. The animals ar identified by numbered ear tags. The animals are fed conventional swine ration without antibiotics. The swine will be given 2 doses of each test preparation (A. pleuropneumoniae experimental bacterin, adjuvant placebo, and PBS) administered intramuscularly 3-4 weeks apart. The animals will be weighed at second vaccination and at post mortem. At post mortem heart and lung weights are recorded for lung/body weight ratios. Blood samples are taken prior to the start of vaccinations at each vaccination, prior to challenge and at post mortem for serological analysis. Animals are observed several times daily prior to vaccination. Animals are also observed closely for several hours post-vaccination for adverse reactions such as anaphylaxis, vomiting, anorexia, diarrhea, lameness, fever, or malaise. Post challenge animals are observed at least every 3 hours for signs of disease. At post mortem, lung lesions are recorded and scored according to a standardized scoring system. The "least squared means" analysis statistical method will be employed to analyze serological scores and clinical scores.
Example 5
Creation of BD Mutants
A suicide conjugative plasmid, pLOFKm, carrying a mini-Tn10 was used for transposon mutagenesis of A. pleuropneumoniae CM5 as described by Tascon et al, Bacteriol. 175:5717-5722 (1993). The plasmid was mobilized from E. coli to A. pleuropneumoniae by biparental mating. Transconjugants were screened on blood agar plates, and non-hemolytic mutants were selected. Four non-hemolytic mutants were selected for further analysis.
Southern Blot Analysis of Non-hemolytic Mutants
Chromosomal DNA was extracted from the transposon mutants, digested with restriction enzymes, separated by electrophoresis on 0.7% agarose gels, and transferred to nylon membranes using conventional procedures (Sambrook et al. Molecular doning--A Laboratory Manual 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, (1989)). The membranes were probed with a PCR-generated 1.4 kb fragment which included part of the BD gene region, and with the mini-Tn10. The probes were labelled using the non-radioactive DNA detection kit from Boehringer Mannheim. Hybridisations were performed under stringent conditions (Sambrook et al. supra).
Southern blots of chromosomal DNA from the parent strain and the mutants digested with BgIII, EcoRI, and XhoI, and probed with the BD region probe, demonstrated transposon insertion in the BD region. This was indicated by an increase in size of the probe-reactive fragments in the mutants, when compared to the corresponding fragments in the parent strain. The presence of a single copy of the transposon in the mutants was confirmed by probing XhoI digests of chromosomal DNA with the mini-Tn10.
Western Blots
Transposon mutants with insertions in the apxIBD genes export little or no CYT and contain 105K cell-associated CYT. Western blots of WC, culture supernatant, and column-purified hemolysin probed with antibodies made to gel-purified CYT from strain CM5 are presented in FIGS. 17 to 19. The cultures were grown in TYE.
FIG. 17 shows WC proteins of CM5 (lane 1), transposon mutant #17 (lane 3), and transposon mutant #18 (lane 5), the corresponding culture supernatants (lanes 2, 4, and 6), and purified CYT (lane 7); FIG. 18 shows WC preparations of transposon mutant #19 (lane 1), control transposon mutant #20 (i.e., not a BD mutant) (lane 3), serotype 3 strain BC181 (lane 5), the corresponding culture supernatants (lanes 2, 4 and 6), and purified CYT (lane 7); FIG. 19 shows WC preparations of CM5-JB, a naladixic acid resistant variant used to make the transposon (lane 1), transposon mutant #17 (lane 3), and transposon mutant #18 (lane 5), the corresponding culture supernatants (lanes 2, 4, and 6), and purified CYT (lane 7). The transposon insertion in the BD region interferes with the export of the toxin into the culture medium as evidenced in little or no toxin detected in the culture supernatants from the mutants.
Having illustrated and described the principles of the invention in a preferred embodiment, it should be appreciated to those skilled in the art that the invention can be modified in arrangement and detail without departure from such principles. We claim all modifications coming within the scope of the following claims.
TABLE 1______________________________________RTX toxins of A. pleuropneumoniae. Molecular Toxin Weight Synonyms Activities______________________________________ApxI 105K HIyI, ClyI Strongly haemolytic Strongly cytotoxic ApxII 103K HlyII, ClyII, App, Cyt Weakly haemolytic Moderately cytotoxic ApxIII 120K Ptx, ClyIII, Mat Strongly cytotoxic______________________________________
TABLE 2______________________________________Distribution of the RTX toxin genes of A. pleuropneumoniae in serotypes 1 to 12. SerotypeGenes 1 2 3 4 5 6 7 8 9 10 11 12______________________________________apxICA + - - - + - - - + + + - apxIBD + + - + + + + + + + + + apxIICA + + + + + + + + + - + + apxIIICA - + + + - + - + - - - - apxIIIBD - + + + - + - + - - - -______________________________________
TABLE 3______________________________________Media______________________________________1) Trypticase Soy Broth pancreatic digest of casein 17 g NaCl 5.0 g papaic digest of soybean meal 3.0 g K.sub.2 HPO.sub.4 glucose 2.5 g dH.sub.2 O qs 1 l mix and dispense autoclave 2) RPMI NaCl 6.0 g NaHCO.sub.3 2.0 g D-Glucose 2.0 g Na.sub.2 HPO.sub.4..sub.7 H.sub.2 O 1.5 g KCL 0.4 g L-Glutamine 0.3 g L-Arginine 0.2 g Ca(NO.sub.3).sub.2..sub.4 H.sub.2 O 0.1 g MgSO.sub.4..sub.7 H.sub.2 O 0.1 g L-Asparagine 0.05 g L-Cystine 0.05 g L-Isoleucine 0.05 g L-Leucine 0.05 g L-Lysine-HCl 0.04 g i-Inositol 0.035 g L-Serine 0.03 g L-Aspartic acid 0.02 g L-Glutamic acid 0.02 g L-Hydroxyproline 0.02 g L-Proline 0.02 g L-Threonine 0.02 g L-Tyrosine 0.02 g L-Valine 0.02 g L-Histidine 0.015 g L-Methionine 0.015 g L-Phenylalanine 0.015 g Gycine 0.01 g L-Tryptophan 5.0 mg Phenol Red 5.0 mg Choline Chloride 3.0 mg Glutathione 1.0 mg p-Aminobenzoic acid 1.0 mg Folic acid 1.0 mg Nicotinamide 1.0 mg Pyridoxine.HCl 1.0 mg Thiamine.HCl 1.0 mg D-Calcium pantothenate 0.25 mg Biotin 0.20 mg Riboflavin 0.20 mg Vitamine B.sub.12 5.0 ug 3) TYE Reference Niven and O'Reilly - M.sup.c Gill Tryptone (Difco) 20 g Yeast extract (Difco) 5 g NaCl (10 mM) 5.84 g KCl (10 mM) 0.75 g Na.sub.2 HPO.sub.4 (10 mM) 1.42 g *(KH.sub.2 PO.sub.4) 0.46 g Glucose (10 mM) 1.80 g dH.sub.2 O 950 ml to pH 7.4 with KOH (not NaOH) dH.sub.2 O QS. 1000 ml autoclave add 1 ml of 10% (wt/vol) NAD per liter 4) 5% Sheep Blood Agar with NAD Columbia agar base (Difco) 44 g ddH2O qs 1 l autoclave cool to 45.degree. C. add defibrinated sheep blood 50 ml add 10% NAD 10 ml aseptically pour into petri plates 5) CBA with NAD Columbia agar base (Difco) 44 g ddH2O qs 1 l autoclave cool to 55.degree. C. add defibrinated sheep blood 50 ml heat to 65.degree. C. for 30 min cool to 45.degree. C. add 10% NAD 10 ml aseptically pour into petri plates 6) BLOTTO non-fat dry milk powder (Carnation) 25 g antifoam A 0.05 ml thimerosal 50 mg PBS QS 500 ml 7) TBS Trizma base 4.84 g NaCl 58.48 g dH.sub.2 O 1.5 L pH to 7.5 with 1M HCl dH.sub.2 O QS to 2 L 8) TTBS add 0.5 ml Tween-20 to 1 liter TBS 9) Antibody Buffer add 2 g gelatin to 200 ml TTBS 10) Western BIot Transfer Buffer pH 8.3 Trizma base 3.03 g glycine 14.4 g dH.sub.2 O 500 ml methanol 200 ml dH.sub.2 O QS to 1 L______________________________________
TABLE 4______________________________________ Type and Source of Cloning Vectors Antibiotic and Plasmids Properties and Origin Resistance______________________________________pAKA16 Mobilizable shuttle vector from 4.3 Ap.sup.r kb P. Haemolytica plasmid (pHP834) pIIP843, with MCS and lacZ' pAKA19 Mobilizable suicide vector from Ap.sup.r pAKA16, with oriV of pBR322 (pHP834) pAKA22 Mobilizable suicide vector from Ap.sup.r pAKA16, with oriV of R6K ( pir (pHP834) dependent) pGZRS-1 Endogenous 4.3 kb plasmid from A. Smr pleuropneumoniae pGZRS-18/19 Shuttle vector from pGZRS-1, with Ap.sup.r MCS and lacZ' (Tn3).sup.1) pGZRS-38/39 Shuttle vector from pGZRS-1, with Kmr MCS and lacZ' (Tn9O3) pJFF224-NX/XN Mobilizable broad host range Qnr expression shuttle vectors, based on p.sup.S-a RSF1010 replicon, with MCS and promoter cassette (phage T4 gene 32) pLOF/Km Mobilizable transposon delivery Km.sup.r plasmid carrying mini-Tn10/Km (Tn903) with inducible transposase (oriV of R6K, pir dependent) pLS88 Endogenous H. ducrei plasmid, used Sm.sup.r for various Pasteurellacea as Su.sup.r shuttle plasmid Km.sup.r All are from pLS88 pVE6063 Thermosenstitive replicon in E. coli Dependent and in other bacteria, but does not upon insert replicate in A. pleuropneumoniae. resistance used Creates double recombination for creation of events. Can be used for targeted knockout insertional mutagenesis. mutations pYG53 (Mobilizable?) Shuttle vector with Cm.sup.r MCS, based on A. (pYG10) pleuropneumoniae plasmid pYG10 pYG54 Mobilizable shuttle vector from Cm.sup.r pYG53, containing oriT of RK2 (pYG10)______________________________________ Cloning vectors and plasmids for transposon and gene replacement delivery systems for HAP bacteria. MCS: multiple cloning site .sup.1) : with A. pleuropneumoniae promoter
TABLE 5______________________________________Composition of Bacterin A, Trial 1. Component Volume______________________________________Strain CM5 heat inactivated 1.2 .times. 10.sup.10 /ml 208 ml Strain BC181 heat inactivated 2.15 .times. 10.sup.10 /ml 116 ml CM5 supernatant 63.5 ml L80 adjuvant 110 ml thimerosal 2% 2.5 ml______________________________________
TABLE 6______________________________________Composition of Bacterins B, C, D, and E, Trial 2. Experimental BacterinComponent B C D E______________________________________Strain VSB68 TSB (24 h) 32 ml 108 ml 1.86 .times. 10.sup.10 /ml Strain VSB68 TYE (6 h) 28 ml 93 ml 2.44 .times. 10.sup.10 /ml PBS 113 ml 37 ml 117 ml 52 ml L80 adjuvant 54 ml 54 ml 54 ml 54 ml thimerosol 2% 1 ml 1 ml 1 ml 1 ml______________________________________
TABLE 7______________________________________Swine sera used in comparative blotting experiments to examine antigen expression and recognition. Serum (pig) # Litter Treatment Group______________________________________ 4 A low-dose 9 A low-dose 14 B low-dose 15 B low-dose 3 A bacterin 11 A bacterin 19 B bacterin 20 B bacterin 21 B bacterin 10 A PBS 16 B PBS 6 A adjuvant 8 A adjuvant 18 B adjuvant 22 B adjuvant______________________________________ low dose = low dose challenge with strain CM5 bacterin = vaccinated with a commercial bacterin PBS = mockvaccinated with PBS adjuvant = mockvaccinated with TSB + AlOH placebo vaccine
TABLE 8______________________________________Sera of swine exposed to A. pleuropneumoniae serotypes 1, 5, and 7. Serum # Serotype Source______________________________________1438 1 B. J. Fenwick, Manhattan, Kansas 1458 1 B. J. Fenwick, Manhattan, Kansas 1467 1 B. J. Fenwick, Manhattan, Kansas 5777 5 B. J. Fenwick, Manhattan, Kansas 5781 5 B. J. Fenwick, Manhattan, Kansas 5782 5 B. J. Fenwick, Manhattan, Kansas 5816 7 B. J. Fenwick, Manhattan, Kansas 5818 7 B. J. Fenwick, Manhattan, Kansas 5819 7 B. J. Fenwick, Manhattan, Kansas______________________________________
TABLE 9______________________________________Sera of CDCD swine exposed to low-dose challenge or vaccinated with a commercial bacterin, PBS, or adjuvant. Serum # Treatment Group______________________________________A4-3 low dose A4-3 low-dose A9-3 low-dose B14-3 low-dose B15-3 bacterin A3-3 bacterin A11-3 bacterin B19-3 bacterin B20-3 bacterin B21-3 PBS A10-3 PBS B16-3 adjuvant A6-3 adjuvant A8-3 adjuvant B8-3 adjuvant______________________________________ low dose = low dose challenge with strain CM5 bacterin = vaccinated with a commercial bacterin PBS = mockvaccinated with PBS adjuvant = mockvaccinated with TSB + AlOH placebo vaccine
TABLE 10______________________________________Lung Score System Lesion Distribution Score______________________________________No visible lesions 0 Minimal focal lesions 1 Consolidation/hemorrhage/adhesions: 0-25% of lung 2 25-50% of lung 3 50-75% of lung 4 75-100% of lung 5 pleural effusion 1 fibrin adhesions 1 tracheal froth 1 pericarditis 1 Total score 9______________________________________ Coding of lesions on the affected lung: 1 = haemorrhage 2 = consolidation 3 = nodules 4 = fibrin tags
TABLE 11______________________________________Summary of lung scores of Caesarean derived-colostrum deprived pigs at necropsy. - Removal Day Mean Post-challe Lung Lung Pig # Treatment nge Score Score______________________________________126 adjuvant D1 8 110 adjuvant D1 4 116 adjuvant D1 8 104 adjuvant D1 4 6.0 112 bacterin D1 5 106 bacterin D1 6 5.5 127 expt. bacterin A D22 0 128 expt. bacterin A D3# 1 119 expt. bacterin A D22 0 118 expt. bacterin A D3# 0 117 expt. bacterin A D22 1 0.4 105 low-dose D22 1 113 low-dose D22 0 0.5 130 PBS D10 1 115 PBS D1 5 135 PBS D1 5 111 PBS D1 5 114 PBS D1 6 129 PBS D1 2 4.0______________________________________ *lung score out of 9 # sacrificed early for lunglavage experiment PBS = phosphate buffered saline bacterin = commercial product
TABLE 12______________________________________Mean antibody responses (OD.sub.405) of the CDCD pigs to CYT, LPS, and CPS prior to challenge. CYT LPS CPSTreatment group PRE POST PRE POST PRE POST______________________________________Bacterin A 0.045 0.264 0.002 0.190 0.001 0.017 Commercial 0.040 0.069 0.0 0.007 0.0 0.000 Bacterin Low-dose 0.057 0.800 0.005 0.008 0.0 0.008 PBS 0.020 0.077 0.002 0.014 0.0 0.009 Adjuvant 0.084 0.052 0.0 0.015 0.0 0.018______________________________________ PRE = prevaccination bleed POST = immediately prior to challenge
TABLE 13______________________________________Summary of outcome of challenge of SPF pigs with A. pleuropneumoniae serotype 1 strain, CM5. Weight Removal Lung/ Treatment Gain Lung Mean Day Heat Culture Pig # Group (Loss) Score Lung Score Ratio Result______________________________________ 36 adjuvant ND 4 D1 3.0 + 37 adjuvant ND 9 D1 4.3 + 38 adjuvant ND 9 D1 5.1 + 39 adjuvant ND 9 D1 4.6 + 40 adjuvant ND 9 8.0 D1 5.2 + 101 bacterin B ND 5 D1 1.3 + 102 bacterin B 0 0 D5 1.6 - 103 bacterin B ND 5 D2 3.8 + 104 bacterin B (9) 2 D5 1.4 - 105 bacterin B ND 4 3.2 D3 1.2 + 106 bacterin C 2 0 D5 1.5 - 107 bacterin C 0 1 D5 1.4 - 108 bacterin C (2) 3 D5 1.4 + 109 bacterin C (1) 3 D5 1.3 + 110 bacterin C 0 3 2.0 D5 1.5 - 111 bacterin D (12) 4 D5 1.2 ND 112 bacterin D (13) 4 D5 1.2 + 113 bacterin D (2) 0 D5 1.5 - 114 bacterin D (1) 0 D5 1.5 + 115 bacterin D 6 0 1.8 D5 1.6 - 116 bacterin E 2 2 D5 1.4 + 117 bacterin E 6 0 D5 1.5 - 118 bacterin E (4) 4 D5 1.3 + 119 bacterin E (5) 4 D5 1.2 + 120 bacterin E ND 5 3.0 D1 2.2 + 121 PBS ND 9 D1 2.9 + 122 PBS ND 9 D2 6.2 + 123 PBS ND 9 D1 4.7 + 124 PBS ND 6 D1 1.7 + 125 PBS ND 9 8.4 D4 1.1 +______________________________________ ND = not done + = A. pleuropneumoniae cultured B = TSB low antigen C = TSB high antigen D = TYE low antigen E = TYE high antigen
TABLE 14______________________________________Least-squared mean antibody responses (OD.sub.405) of the SPF pigs to CYT, LPS and CPS. Treatment CYT LPS CPSgroup Pre Post Pre Post Pre Post______________________________________Bacterin B 0.116 0.371 0.003 0.814 0.005 0.788 Bacterin C 0.154 0.321 0.007 0.559 0.008 0.617 Bacterin D 0.109 0.567 0.006 0.962 0.003 0.956 Bacterin E 0.126 0.445 0.007 0.744 0.006 0.818 PBS 0.083 0.057 0.003 -0.003 0.004 -0.001 Adjuvant 0.129 0.145 0.001 0.003 0.002 0.001______________________________________ Bacterin B = TSBgrown, low antigen concentration Bacterin C = TSBgrown, high antigen concentration Bacterin D = TYEgrown, low antigen concentration Bacterin E = TYEgrown, high antigen concentration Pre = prevaccination bleed Post = immediately prior to challenge
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ABBREVIATIONS
A.sub.405 --absorbance at 405 nm
AlOH--aluminum hydroxide
BEI--binary-ethyleneimine
BPL--.beta.-propiolactone
CBA--chocolate blood agar
CBA-NAD--chocolate blood agar with 0.1% (wt/vol) NAD
CDCD--caesarean derived, colostrum deprived
CPS--capsular polysaccharide
CYT--cytolysin
.degree.C--degrees Celsius
ddH.sub.2 O--double distilled water
FCS--fetal calf serum
g--grams
.times.g--times gravity
h--hours
IM--intramuscularly
LPS--lipopolysaccharide
M--molar
mg--milligram
min--minutes
ml--milliliter
mM--millimolar
NaCl--sodium chloride
NAD--nicotinamide adenine dinucleotide
ng--nanogram
OD--optical density
OMP--outer-membrane protein
PAGE--polyacrylamide gel electrophoresis
PBS--phosphate buffered saline
qs--quantum sufficit
rpm--revolutions per minute
SDS--sodium dodecyl sulfate
SPF--specific pathogen free
Tris--tris(hydroxymethyl)aminomethane
TBS--Tris buffered saline
TSB-NAD--trypficase soy broth with 0.01% (wt/vol) NAD
TTBS--Tween 20-Tris buffered saline
TYE-NAD--tryptone-yeast extract with 0.01% (wt/vol) NAD
.mu.g--microgram
.mu.l--microliter
V--volts
vol--volume
WC--whole cell
wt--weight
__________________________________________________________________________# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 14 - - - - (2) INFORMATION FOR SEQ ID NO:1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 172 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: circular - - (ii) MOLECULE TYPE: protein - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Actinobacill - #us pleuropneumoniae - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: - - Met Ser Lys Lys Ile Asn Gly Phe Glu Val Le - #u Gly Glu Val AlaTrp 1 5 - # 10 - # 15 - - Leu Trp Ala Ser Ser Pro Leu His Arg Lys Tr - #p Pro Leu Ser Leu Leu 20 - # 25 - # 30 - - Ala Ile Asn Val Leu Pro Ala Ile Glu Ser As - #n Gln Tyr Val Leu Leu 35 - # 40 - # 45 - - Lys Arg Asp Gly Phe Pro Ile Ala Phe Cys Se - #r Trp Ala Asn Leu Asn 50 - # 55 - # 60 - - Leu Glu Asn Glu Ile Lys Tyr Leu Asp Asp Va - #l Ala Ser Leu Val Ala 65 - #70 - #75 - #80 - - Asp Asp Trp Thr Ser Gly Asp Arg Arg Trp Ph - #e Ile Asp Trp Ile Ala 85 - # 90 - # 95 - - Pro Phe Gly Asp Ser Ala Ala Leu Tyr Lys Hi - #s Met Arg Asp Asn Phe 100 - # 105 - # 110 - - Pro Asn Glu Leu Phe Arg Ala Ile Arg Val As - #p Pro Asp Ser Arg Val 115 - # 120 - # 125 - - Gly Lys Ile Ser Glu Phe His Gly Gly Lys Il - #e Asp Lys Lys Leu Ala 130 - # 135 - # 140 - - Ser Lys Ile Phe Gln Gln Tyr His Phe Glu Le - #u Met Ser Glu Leu Lys 145 1 - #50 1 - #55 1 -#60 - - Asn Lys Gln Asn Phe Lys Phe Ser Leu Val As - #n Ser 165 - # 170 - - - - (2) INFORMATION FOR SEQ ID NO:2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1022 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: circular - - (ii) MOLECULE TYPE: protein - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Actinobacill - #us pleuropneumoniae - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: - - Met Ala Asn Ser Gln Leu Asp Arg Val Lys Gl - #y Leu Ile Asp SerLeu 1 5 - # 10 - # 15 - - Asn Gln His Thr Lys Ser Ala Ala Lys Ser Gl - #y Ala Gly Ala Leu Lys 20 - # 25 - # 30 - - Asn Gly Leu Gly Gln Val Lys Gln Ala Gly Gl - #n Lys Leu Ile Leu Tyr 35 - # 40 - # 45 - - Ile Pro Lys Asp Tyr Gln Ala Ser Thr Gly Se - #r Ser Leu Asn Asp Leu 50 - # 55 - # 60 - - Val Lys Ala Ala Glu Ala Leu Gly Ile Glu Va - #l His Arg Ser Glu Lys 65 - #70 - #75 - #80 - - Asn Gly Thr Ala Leu Ala Lys Glu Leu Phe Gl - #y Thr Thr Glu Lys Leu 85 - # 90 - # 95 - - Leu Gly Phe Ser Glu Arg Gly Ile Ala Leu Ph - #e Ala Pro Gln Phe Asp 100 - # 105 - # 110 - - Lys Leu Leu Asn Lys Asn Gln Lys Leu Ser Ly - #s Ser Leu Gly Gly Ser 115 - # 120 - # 125 - - Ser Glu Ala Leu Gly Gln Arg Leu Asn Lys Th - #r Gln Thr Ala Leu Ser 130 - # 135 - # 140 - - Ala Leu Gln Ser Phe Leu Gly Thr Ala Ile Al - #a Gly Met Asp Leu Asp 145 1 - #50 1 - #55 1 -#60 - - Ser Leu Leu Arg Arg Arg Arg Asn Gly Glu As - #p Val Ser Gly SerGlu 165 - # 170 - # 175 - - Leu Ala Lys Ala Gly Val Asp Leu Ala Ala Gl - #n Leu Val Asp Asn Ile 180 - # 185 - # 190 - - Ala Ser Ala Thr Gly Thr Val Asp Ala Phe Al - #a Glu Gln Leu Gly Lys 195 - # 200 - # 205 - - Leu Gly Asn Ala Leu Ser Asn Thr Arg Leu Se - #r Gly Leu Ala Ser Lys 210 - # 215 - # 220 - - Leu Asn Asn Leu Pro Asp Leu Ser Leu Ala Gl - #y Pro Gly Phe Asp Ala 225 2 - #30 2 - #35 2 -#40 - - Val Ser Gly Ile Leu Ser Val Val Ser Ala Se - #r Phe Ile Leu SerAsn 245 - # 250 - # 255 - - Lys Asp Ala Asp Ala Gly Thr Lys Ala Ala Al - #a Gly Ile Glu Ile Ser 260 - # 265 - # 270 - - Thr Lys Ile Leu Gly Asn Ile Gly Lys Ala Va - #l Ser Gln Tyr Ile Ile 275 - # 280 - # 285 - - Ala Gln Arg Val Ala Ala Gly Leu Ser Thr Th - #r Ala Ala Thr Gly Gly 290 - # 295 - # 300 - - Leu Ile Gly Ser Val Val Ala Leu Ala Ile Se - #r Pro Leu Ser Phe Leu 305 3 - #10 3 - #15 3 -#20 - - Asn Val Ala Asp Lys Phe Glu Arg Ala Lys Gl - #n Leu Glu Gln TyrSer 325 - # 330 - # 335 - - Glu Arg Phe Lys Lys Phe Gly Tyr Glu Gly As - #p Ser Leu Leu Ala Ser 340 - # 345 - # 350 - - Phe Tyr Arg Glu Thr Gly Ala Ile Glu Ala Al - #a Leu Thr Thr Ile Asn 355 - # 360 - # 365 - - Ser Val Leu Ser Ala Ala Ser Ala Gly Val Gl - #y Ala Ala Ala Thr Gly 370 - # 375 - # 380 - - Ser Leu Val Gly Ala Pro Val Ala Ala Leu Va - #l Ser Ala Ile Thr Gly 385 3 - #90 3 - #95 4 -#00 - - Ile Ile Ser Gly Ile Leu Asp Ala Ser Lys Gl - #n Ala Ile Phe GluArg 405 - # 410 - # 415 - - Val Ala Thr Lys Leu Ala Asn Lys Ile Asp Gl - #u Trp Glu Lys Lys His 420 - # 425 - # 430 - - Gly Lys Asn Tyr Phe Glu Asn Gly Tyr Asp Al - #a Arg His Ser Ala Phe 435 - # 440 - # 445 - - Leu Glu Asp Thr Phe Glu Leu Leu Ser Gln Ty - #r Asn Lys Glu Tyr Ser 450 - # 455 - # 460 - - Val Glu Arg Val Val Ala Ile Thr Gln Gln Ar - #g Trp Asp Val Asn Ile 465 4 - #70 4 - #75 4 -#80 - - Gly Glu Leu Ala Gly Ile Thr Arg Lys Gly Se - #r Asp Thr Lys SerGly 485 - # 490 - # 495 - - Lys Ala Tyr Val Asp Phe Phe Glu Glu Gly Ly - #s Leu Leu Glu Lys Glu 500 - # 505 - # 510 - - Pro Asp Arg Phe Asp Lys Lys Val Phe Asp Pr - #o Leu Glu Gly Lys Ile 515 - # 520 - # 525 - - Asp Leu Ser Ser Ile Asn Lys Thr Thr Leu Le - #u Lys Phe Val Thr Pro 530 - # 535 - # 540 - - Val Phe Thr Ala Gly Glu Glu Ile Arg Glu Ar - #g Lys Gln Thr Gly Lys 545 5 - #50 5 - #55 5 -#60 - - Tyr Glu Tyr Met Thr Glu Leu Phe Val Lys Gl - #y Lys Glu Lys TrpVal 565 - # 570 - # 575 - - Val Thr Gly Val Gln Ser His Asn Ala Ile Ty - #r Asp Tyr Thr Asn Leu 580 - # 585 - # 590 - - Ile Gln Leu Ala Ile Asp Lys Lys Gly Glu Ly - #s Arg Gln Val Thr Ile 595 - # 600 - # 605 - - Glu Ser His Leu Gly Glu Lys Asn Asp Arg Il - #e Tyr Leu Ser Ser Gly 610 - # 615 - # 620 - - Ser Ser Ile Val Tyr Ala Gly Asn Gly His As - #p Val Ala Tyr Tyr Asp 625 6 - #30 6 - #35 6 -#40 - - Lys Thr Asp Thr Gly Tyr Leu Thr Phe Asp Gl - #y Gln Ser Ala GlnLys 645 - # 650 - # 655 - - Ala Gly Glu Tyr Ile Val Thr Lys Glu Leu Ly - #s Ala Asp Val Lys Val 660 - # 665 - # 670 - - Leu Lys Glu Val Val Lys Thr Gln Asp Ile Se - #r Val Gly Lys Arg Ser 675 - # 680 - # 685 - - Glu Lys Leu Glu Tyr Arg Asp Tyr Glu Leu Se - #r Pro Phe Glu Leu Gly 690 - # 695 - # 700 - - Asn Gly Ile Arg Ala Lys Asp Glu Leu His Se - #r Val Glu Glu Ile Ile 705 7 - #10 7 - #15 7 -#20 - - Gly Ser Asn Arg Lys Asp Lys Phe Phe Gly Se - #r Arg Phe Thr AspIle 725 - # 730 - # 735 - - Phe His Gly Ala Lys Gly Asp Asp Glu Ile Ty - #r Gly Asn Asp Gly His 740 - # 745 - # 750 - - Asp Ile Leu Tyr Gly Asp Asp Gly Asn Asp Va - #l Ile His Gly Gly Asp 755 - # 760 - # 765 - - Gly Asn Asp His Leu Val Gly Gly Asn Gly As - #n Asp Arg Leu Ile Gly 770 - # 775 - # 780 - - Gly Lys Gly Asn Asn Phe Leu Asn Gly Gly As - #p Gly Asp Asp Glu Leu 785 7 - #90 7 - #95 8 -#00 - - Gln Val Phe Glu Gly Gln Tyr Asn Val Leu Le - #u Gly Gly Ala GlyAsn 805 - # 810 - # 815 - - Asp Ile Leu Tyr Gly Ser Asp Gly Thr Asn Le - #u Phe Asp Gly Gly Val 820 - # 825 - # 830 - - Gly Asn Asp Lys Ile Tyr Gly Gly Leu Gly Ly - #s Asp Ile Tyr Arg Tyr 835 - # 840 - # 845 - - Ser Lys Glu Tyr Gly Arg His Ile Ile Ile Gl - #u Lys Gly Gly Asp Asp 850 - # 855 - # 860 - - Asp Thr Leu Leu Leu Ser Asp Leu Ser Phe Ly - #s Asp Val Gly Phe Ile 865 8 - #70 8 - #75 8 -#80 - - Arg Ile Gly Asp Asp Leu Leu Val Asn Lys Ar - #g Ile Gly Gly ThrLeu 885 - # 890 - # 895 - - Tyr Tyr His Glu Asp Tyr Asn Gly Asn Ala Le - #u Thr Ile Lys Asp Trp 900 - # 905 - # 910 - - Phe Lys Glu Gly Lys Glu Gly Gln Asn Asn Ly - #s Ile Glu Lys Ile Val 915 - # 920 - # 925 - - Asp Lys Asp Gly Ala Tyr Val Leu Ser Gln Ty - #r Leu Thr Glu Leu Thr 930 - # 935 - # 940 - - Ala Pro Gly Arg Gly Ile Asn Tyr Phe Asn Gl - #y Leu Glu Glu Lys Leu 945 9 - #50 9 - #55 9 -#60 - - Tyr Tyr Gly Glu Gly Tyr Asn Ala Leu Pro Gl - #n Leu Arg Lys AspIle 965 - # 970 - # 975 - - Glu Gln Ile Ile Ser Ser Thr Gly Ala Phe Th - #r Gly Asp His Gly Lys 980 - # 985 - # 990 - - Val Ser Val Gly Ser Gly Gly Pro Leu Val Ty - #r Asn Asn Ser Ala Asn 995 - # 1000 - # 1005 - - Asn Val Ala Asn Ser Leu Ser Tyr Ser Leu Al - #a Gln Ala Ala 1010 - # 1015 - # 1020 - - - - (2) INFORMATION FOR SEQ ID NO:3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3762 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: circular - - (ii) MOLECULE TYPE: DNA (genomic) - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Actinobacill - #us pleuropneumoniae - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: - - AGATTAATGA GCGATATTGT TATAAAATCA TAATGTAAAC CTCATTTGTA AT -#GAATTGGT 60 - - AAATTATATA AATAATCAAA AAACTTACTT TTTTTTATTT TTATCGGTAA GT -#ATTTACAA 120 - - TCAAGTCAGA CAAACGGCAA TATTGTTATA AATCTGGGGG GATGAATGAG TA -#AAAAAATT 180 - - AATGGATTTG AGGTTTTAGG AGAGGTGGCA TGGTTATGGG CAAGTTCTCC TT -#TACATCGA 240 - - AAGTGGCCGC TTTCTTTGTT AGCAATTAAT GTGCTACCTG CGATTGAGAG TA -#ATCAATAT 300 - - GTTTTGTTAA AGCGTGACGG TTTTCCTATT GCATTTTGTA GCTGGGCAAA TT -#TGAATTTG 360 - - GAAAATGAAA TTAAATACCT TGATGATGTT GCCTCGCTAG TTGCGGATGA TT -#GGACTTCC 420 - - GGCGATCGTC GATGGTTTAT AGATTGGATA GCACCGTTCG GAGACAGTGC CG -#CATTATAC 480 - - AAACATATGC GAGATAACTT CCCGAATGAG CTGTTTAGGG CTATTCGAGT TG -#ATCCGGAC 540 - - TCTCGAGTAG GGAAAATTTC AGAATTTCAT GGAGGAAAAA TTGATAAGAA AC -#TGGCAAGT 600 - - AAAATTTTTC AACAATATCA CTTTGAATTA ATGAGTGAGC TAAAAAATAA AC -#AAAATTTT 660 - - AAATTTTCAT TAGTAAATAG CTAAGGAGAC AACATGGCTA ACTCTCAGCT CG -#ATAGAGTC 720 - - AAAGGATTGA TTGATTCACT TAATCAACAT ACAAAAAGTG CAGCTAAATC AG -#GTGCCGGC 780 - - GCATTAAAAA ATGGTTTGGG ACAGGTGAAG CAAGCAGGGC AGAAATTAAT TT -#TATATATT 840 - - CCGAAAGATT ATCAAGCTAG TACCGGCTCA AGTCTTAATG ATTTAGTGAA AG -#CGGCGGAG 900 - - GCTTTAGGGA TCGAAGTACA TCGCTCGGAA AAAAACGGTA CCGCACTAGC GA -#AAGAATTA 960 - - TTCGGTACAA CGGAAAAACT ATTAGGTTTC TCGGAACGAG GCATCGCATT AT -#TTGCACCT 1020 - - CAGTTTGATA AGTTACTGAA TAAGAACCAA AAATTAAGTA AATCGCTCGG CG -#GTTCATCG 1080 - - GAAGCATTAG GACAACGTTT AAATAAAACG CAAACGGCAC TTTCAGCCTT AC -#AAAGTTTC 1140 - - TTAGGTACGG CTATTGCGGG TATGGATCTT GATAGCCTGC TTCGTCGCCG TA -#GAAACGGT 1200 - - GAGGACGTCA GTGGTTCGGA ATTAGCTAAA GCAGGTGTGG ATCTAGCCGC TC -#AGTTAGTG 1260 - - GATAACATTG CAAGTGCAAC GGGTACGGTG GATGCGTTTG CCGAACAATT AG -#GTAAATTG 1320 - - GGCAATGCCT TATCTAACAC TCGCTTAAGC GGTTTAGCAA GTAAGTTAAA TA -#ACCTTCCA 1380 - - GATTTAAGCC TTGCAGGACC TGGGTTTGAT GCCGTATCAG GTATCTTATC TG -#TTGTTTCG 1440 - - GCTTCATTCA TTTTAAGTAA TAAAGATGCC GATGCAGGTA CAAAAGCGGC GG -#CAGGTATT 1500 - - GAAATCTCAA CTAAAATCTT AGGCAATATC GGTAAAGCGG TTTCTCAATA TA -#TTATTGCG 1560 - - CAACGTGTGG CGGCAGGCTT ATCCACAACT GCGGCAACCG GTGGTTTAAT CG -#GTTCGGTC 1620 - - GTAGCATTAG CGATTAGCCC GCTTTCGTTC TTAAATGTTG CGGATAAGTT TG -#AACGTGCG 1680 - - AAACAGCTTG AACAATATTC GGAGCGCTTT AAAAAGTTCG GTTATGAAGG TG -#ATAGTTTA 1740 - - TTAGCTTCAT TCTACCGTGA AACCGGTGCG ATTGAAGCGG CATTAACCAC GA -#TTAACAGT 1800 - - GTGTTAAGTG CGGCTTCCGC AGGTGTTGGG GCTGCTGCAA CCGGCTCATT AG -#TCGGTGCG 1860 - - CCGGTAGCAG CTTTAGTTAG TGCAATCACC GGTATTATTT CAGGTATTTT AG -#ATGCTTCT 1920 - - AAACAGGCAA TCTTCGAACG AGTTGCAACG AAATTAGCGA ATAAGATTGA CG -#AATGGGAG 1980 - - AAAAAACACG GTAAAAACTA TTTTGAAAAC GGTTATGACG CCCGCCATTC CG -#CATTCTTA 2040 - - GAAGATACCT TTGAATTGTT ATCACAATAC AATAAAGAGT ATTCGGTAGA GC -#GTGTCGTT 2100 - - GCTATTACGC AACAGCGTTG GGATGTCAAT ATCGGTGAAC TTGCCGGCAT TA -#CTCGCAAA 2160 - - GGTTCTGATA CGAAAAGCGG TAAAGCTTAC GTTGATTTCT TTGAAGAAGG AA -#AACTTTTA 2220 - - GAGAAAGAAC CGGATCGTTT TGATAAAAAA GTGTTTGATC CGCTTGAAGG TA -#AAATCGAC 2280 - - CTTTCTTCAA TTAACAAAAC CACTTTATTG AAATTTGTTA CGCCGGTCTT TA -#CCGCAGGT 2340 - - GAAGAGATTC GTGAGCGTAA GCAAACCGGT AAATACGAAT ATATGACCGA AT -#TATTCGTT 2400 - - AAAGGTAAAG AAAAATGGGT GGTAACCGGT GTGCAGTCAC ATAATGCGAT TT -#ATGACTAT 2460 - - ACGAATCTTA TCCAATTAGC GATAGATAAA AAAGGTGAAA AACGTCAAGT GA -#CCATTGAA 2520 - - TCTCATTTGG GTGAGAAAAA TGATCGTATA TATCTTTCAT CCGGTTCATC TA -#TCGTATAT 2580 - - GCGGGTAACG GACATGATGT AGCATATTAC GATAAAACCG ATACAGGTTA CT -#TAACATTT 2640 - - GACGGACAAA GTGCACAGAA AGCCGGTGAA TATATTGTCA CTAAAGAACT TA -#AAGCTGAT 2700 - - GTAAAAGTTT TAAAAGAAGT GGTTAAAACT CAGGATATTT CAGTTGGAAA AC -#GCAGTGAA 2760 - - AAATTAGAAT ATCGTGATTA TGAGTTAAGC CCATTCGAAC TTGGGAACGG TA -#TCAGAGCT 2820 - - AAAGATGAAT TACATTCTGT TGAAGAAATT ATCGGTAGTA ATCGTAAAGA CA -#AATTCTTT 2880 - - GGTAGTCGCT TTACCGATAT TTTCCATGGT GCGAAAGGCG ATGATGAAAT CT -#ACGGTAAT 2940 - - GACGGCCACG ATATCTTATA CGGAGACGAC GGTAATGATG TAATCCATGG CG -#GTGACGGT 3000 - - AACGACCATC TTGTTGGTGG TAACGGAAAC GACCGATTAA TCGGCGGAAA AG -#GTAATAAT 3060 - - TTCCTTAATG GCGGTGATGG TGACGATGAG TTGCAGGTCT TTGAGGGTCA AT -#ACAACGTA 3120 - - TTATTAGGTG GTGCGGGTAA TGACATTCTG TATGGCAGCG ATGGTACTAA CT -#TATTTGAC 3180 - - GGTGGTGTAG GCAATGACAA AATCTACGGT GGTTTAGGTA AGGATATTTA TC -#GCTACAGT 3240 - - AAGGAGTACG GTCGTCATAT CATTATTGAG AAAGGCGGTG ATGATGATAC GT -#TATTGTTA 3300 - - TCGGATCTTA GTTTTAAAGA TGTAGGATTT ATCAGAATCG GTGATGATCT TC -#TTGTGAAT 3360 - - AAAAGAATCG GAGGAACACT GTATTACCAT GAAGATTACA ATGGGAATGC GC -#TCACGATT 3420 - - AAAGATTGGT TCAAGGAAGG TAAAGAAGGA CAAAATAATA AAATTGAAAA AA -#TCGTTGAT 3480 - - AAAGATGGAG CTTATGTTTT AAGCCAATAT CTGACTGAAC TGACAGCTCC TG -#GAAGAGGT 3540 - - ATCAATTACT TTAATGGGTT AGAAGAAAAA TTGTATTATG GAGAAGGATA TA -#ATGCACTT 3600 - - CCTCAACTCA GAAAAGATAT TGAACAAATC ATTTCATCTA CGGGTGCATT TA -#CCGGTGAT 3660 - - CACGGAAAAG TATCTGTAGG CTCAGGCGGA CCGTTAGTCT ATAATAACTC AG -#CTAACAAT 3720 - - GTAGCAAATT CTTTGAGTTA TTCTTTAGCA CAAGCAGCTT AA - # - #3762 - - - - (2) INFORMATION FOR SEQ ID NO:4: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 707 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Actinobacill - #us pleuropneumoniae - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: - - Met Asp Phe Tyr Arg Glu Glu Asp Tyr Gly Le - #u Tyr Ala Leu Thr Ile 1 5 - # 10 - # 15 - - Leu Ala Gln Tyr His Asn Ile Ala Val Asn Pr - #o Glu Glu Leu Lys His 20 - # 25 - # 30 - - Lys Phe Asp Leu Glu Gly Lys Gly Leu Asp Le - #u Thr Ala Trp Leu Leu 35 - # 40 - # 45 - - Ala Ala Lys Ser Leu Glu Leu Lys Ala Lys Gl - #n Val Lys Lys Ala Ile 50 - # 55 - # 60 - - Asp Arg Leu Ala Phe Ile Ala Leu Pro Ala Le - #u Val Trp Arg Glu Asp 65 - #70 - #75 - #80 - - Gly Lys His Phe Ile Leu Thr Lys Ile Asp As - #n Glu Ala Lys Lys Tyr 85 - # 90 - # 95 - - Leu Ile Phe Asp Leu Glu Thr His Asn Pro Ar - #g Ile Leu Glu Gln Ala 100 - # 105 - # 110 - - Glu Phe Glu Ser Leu Tyr Gln Gly Lys Leu Il - #e Leu Val Ala Ser Arg 115 - # 120 - # 125 - - Ala Ser Ile Val Gly Lys Leu Ala Lys Phe As - #p Phe Thr Trp Phe Ile 130 - # 135 - # 140 - - Pro Ala Val Ile Lys Tyr Arg Lys Ile Phe Il - #e Glu Thr Leu Ile Val 145 1 - #50 1 - #55 1 -#60 - - Ser Ile Phe Leu Gln Ile Phe Ala Leu Ile Th - #r Pro Leu Phe PheGln 165 - # 170 - # 175 - - Val Val Met Asp Lys Val Leu Val His Arg Gl - #y Phe Ser Thr Leu Asn 180 - # 185 - # 190 - - Val Ile Thr Val Ala Leu Ala Ile Val Val Le - #u Phe Glu Ile Val Leu 195 - # 200 - # 205 - - Asn Gly Leu Arg Thr Tyr Ile Phe Ala His Se - #r Thr Ser Arg Ile Asp 210 - # 215 - # 220 - - Val Glu Leu Gly Ala Arg Leu Phe Arg His Le - #u Leu Ala Leu Pro Ile 225 2 - #30 2 - #35 2 -#40 - - Ser Tyr Phe Glu Asn Arg Arg Val Gly Asp Th - #r Val Ala Arg ValArg 245 - # 250 - # 255 - - Glu Leu Asp Gln Ile Arg Asn Phe Leu Thr Gl - #y Gln Ala Leu Thr Ser 260 - # 265 - # 270 - - Val Leu Asp Leu Met Phe Ser Phe Ile Phe Ph - #e Ala Val Met Trp Tyr 275 - # 280 - # 285 - - Tyr Ser Pro Lys Leu Thr Leu Val Ile Leu Gl - #y Ser Leu Pro Phe Tyr 290 - # 295 - # 300 - - Met Gly Trp Ser Ile Phe Ile Ser Pro Ile Le - #u Arg Arg Arg Leu Asp 305 3 - #10 3 - #15 3 -#20 - - Glu Lys Phe Ala Arg Gly Ala Asp Asn Gln Se - #r Phe Leu Val GluSer 325 - # 330 - # 335 - - Val Thr Ala Ile Asn Thr Ile Lys Ala Leu Al - #a Val Thr Pro Gln Met 340 - # 345 - # 350 - - Thr Asn Thr Trp Asp Lys Gln Leu Ala Ser Ty - #r Val Ser Ala Gly Phe 355 - # 360 - # 365 - - Arg Val Thr Thr Leu Ala Thr Ile Gly Gln Gl - #n Gly Val Gln Phe Ile 370 - # 375 - # 380 - - Gln Lys Val Val Met Val Ile Thr Leu Trp Le - #u Gly Ala His Leu Val 385 3 - #90 3 - #95 4 -#00 - - Ile Ser Gly Asp Leu Ser Ile Gly Gln Leu Il - #e Ala Phe Asn MetLeu 405 - # 410 - # 415 - - Ser Gly Gln Val Ile Ala Pro Val Ile Arg Le - #u Ala Gln Leu Trp Gln 420 - # 425 - # 430 - - Asp Phe Gln Gln Val Gly Ile Ser Val Thr Ar - #g Leu Gly Asp Val Leu 435 - # 440 - # 445 - - Asn Ser Pro Thr Glu Ser Tyr Gln Gly Lys Le - #u Ala Leu Pro Glu Ile 450 - # 455 - # 460 - - Lys Gly Asp Ile Thr Phe Arg Asn Ile Arg Ph - #e Arg Tyr Lys Pro Asp 465 4 - #70 4 - #75 4 -#80 - - Ala Pro Val Ile Leu Asn Asp Val Asn Leu Se - #r Ile Gln Gln GlyGlu 485 - # 490 - # 495 - - Val Ile Gly Ile Val Gly Arg Ser Gly Ser Gl - #y Lys Ser Thr Leu Thr 500 - # 505 - # 510 - - Lys Leu Ile Gln Arg Phe Tyr Ile Pro Glu As - #n Gly Gln Val Leu Ile 515 - # 520 - # 525 - - Asp Gly His Asp Leu Ala Leu Ala Asp Pro As - #n Trp Leu Arg Arg Gln 530 - # 535 - # 540 - - Val Gly Val Val Leu Gln Asp Asn Val Leu Le - #u Asn Arg Ser Ile Arg 545 5 - #50 5 - #55 5 -#60 - - Asp Asn Ile Ala Leu Ala Asp Pro Gly Met Pr - #o Met Glu Lys IleVal 565 - # 570 - # 575 - - His Ala Ala Lys Leu Ala Gly Ala His Glu Ph - #e Ile Ser Glu Leu Arg 580 - # 585 - # 590 - - Glu Gly Tyr Asn Thr Ile Val Gly Glu Gln Gl - #y Ala Gly Leu Ser Gly 595 - # 600 - # 605 - - Gly Gln Arg Gln Arg Ile Ala Ile Ala Arg Al - #a Leu Val Asn Asn Pro 610 - # 615 - # 620 - - Lys Ile Leu Ile Phe Asp Glu Ala Thr Ser Al - #a Leu Asp Tyr Glu Ser 625 6 - #30 6 - #35 6 -#40 - - Glu His Ile Ile Met Arg Asn Met His Gln Il - #e Cys Lys Gly ArgThr 645 - # 650 - # 655 - - Val Ile Ile Ile Ala His Arg Leu Ser Thr Va - #l Lys Asn Ala Asp Arg 660 - # 665 - # 670 - - Ile Ile Val Met Glu Lys Gly Gln Ile Val Gl - #u Gln Gly Lys His Lys 675 - # 680 - # 685 - - Glu Leu Leu Ala Asp Pro Asn Gly Leu Tyr Hi - #s Tyr Leu His Gln Leu 690 - # 695 - # 700 - - Gln Ser Glu 705 - - - - (2) INFORMATION FOR SEQ ID NO:5: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 478 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Actinobacill - #us pleuropneumoniae - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: - - Met Lys Thr Trp Leu Met Gly Leu Tyr Glu Ph - #e Phe Gln Arg Tyr Lys 1 5 - # 10 - # 15 - - Thr Val Trp Thr Glu Ile Trp Lys Ile Arg Hi - #s Gln Leu Asp Thr Pro 20 - # 25 - # 30 - - Asp Arg Glu Lys Asp Glu Asn Glu Phe Leu Pr - #o Ala His Leu Glu Leu 35 - # 40 - # 45 - - Ile Glu Thr Pro Val Ser Lys Lys Pro Arg Le - #u Ile Ala Tyr Leu Ile 50 - # 55 - # 60 - - Met Leu Phe Leu Phe Leu Ala Leu Val Ile Se - #r Ile Val Ser His Val 65 - #70 - #75 - #80 - - Glu Ile Val Ala Thr Ala Thr Gly Lys Leu Al - #a Phe Ser Asp Arg Ser 85 - # 90 - # 95 - - Lys Glu Ile Lys Pro Ile Glu Asn Ala Leu Va - #l Lys Glu Ile Phe Val 100 - # 105 - # 110 - - Gln Asp Gly Gln Phe Val Glu Lys Asp Gln Le - #u Leu Leu His Leu Thr 115 - # 120 - # 125 - - Ala Leu Gly Ala Asp Ala Asp Gln Gln Lys Th - #r Lys Ser Ser Leu Ser 130 - # 135 - # 140 - - Leu Thr Lys Leu Glu Arg Tyr Arg Tyr Glu Il - #e Leu Leu Glu Ala Val 145 1 - #50 1 - #55 1 -#60 - - Ala Ala Asp Arg Leu Pro Leu Ile Glu Leu Th - #r Lys Asp Glu PheLys 165 - # 170 - # 175 - - His Ala Thr Glu Glu Asp Lys Thr Arg Ile Ar - #g Tyr Leu Ile Thr Glu 180 - # 185 - # 190 - - Gln Phe Glu Ala Trp Gln Lys Gln Lys Tyr Gl - #n Lys Glu Leu Ala Leu 195 - # 200 - # 205 - - Gln Arg Arg Glu Ala Glu Lys Gln Thr Val Le - #u Ala Asn Ile Arg Lys 210 - # 215 - # 220 - - Tyr Glu Gly Ile Ser Arg Val Glu Asn Glu Ar - #g Leu Lys Asp Leu Lys 225 2 - #30 2 - #35 2 -#40 - - Lys Leu Phe Asn Ser Lys Ser Thr Ser Lys Hi - #s Asp Val Leu ThrGln 245 - # 250 - # 255 - - Glu Asn Arg His Ile Glu Ala Val Asn Glu Le - #u Ala Val Tyr Lys Ser 260 - # 265 - # 270 - - Arg Leu Asn Glu Val Glu Ser Asp Leu Arg Gl - #n Ala Lys Glu Glu Ile 275 - # 280 - # 285 - - His Leu Ile Thr Gln Leu Phe Arg Ala Asp Il - #e Leu Glu Lys Leu Lys 290 - # 295 - # 300 - - Gln Asn Val Glu Ala Glu Lys Gln Leu Ser Le - #u Glu Leu Glu Lys Asn 305 3 - #10 3 - #15 3 -#20 - - Glu Gln Arg Gln Ile Ala Ser Val Ile Arg Al - #a Pro Val Ser GlyThr 325 - # 330 - # 335 - - Val Gln Gln Leu Lys Thr His Thr Val Gly Gl - #y Val Val Thr Thr Ala 340 - # 345 - # 350 - - Glu Thr Leu Met Val Ile Ala Pro Glu Asp As - #p Val Leu Glu Val Thr 355 - # 360 - # 365 - - Ala Leu Ile Gln Asn Lys Asp Ile Gly Phe Il - #e Glu Val Gly Gln Asp 370 - # 375 - # 380 - - Ala Val Ile Lys Val Glu Thr Phe Pro Tyr Th - #r Arg Tyr Gly Tyr Leu 385 3 - #90 3 - #95 4 -#00 - - Met Gly Lys Val Lys Asn Ile Thr Leu Glu Al - #a Ile Glu His ProGln 405 - # 410 - # 415 - - Leu Gly Leu Val Phe Asn Ser Ile Ile Ser Il - #e Asp Arg Lys Thr Leu 420 - # 425 - # 430 - - Ser Gly Lys Asp Gly Lys Glu Ile Glu Leu Gl - #y Ser Gly Met Ser Val 435 - # 440 - # 445 - - Thr Ala Glu Ile Lys Thr Gly Glu Arg Ser Va - #l Ile Ser Tyr Leu Leu 450 - # 455 - # 460 - - Ser Pro Leu Glu Glu Ser Val Ser Glu Ser Le - #u Arg Glu Arg 465 4 - #70 4 - #75 - - - - (2) INFORMATION FOR SEQ ID NO:6: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5120 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Actinobacill - #us pleuropneumoniae - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: - - TCGTCATATC ATTATTGAGA AAGGCGGTGA TGATGATACG TTATTGTTAT CG -#GATCTTAG 60 - - TTTTAAAGAT GTAGGATTTA TCAGAATCGG TGATGATCTT CTTGTGAATA AA -#AGAATCGG 120 - - AGGAACACTG TATTACCATG AAGATTACAA TGGGAATGCG CTCACGATTA AA -#GATTGGTT 180 - - CAAGGAAGGT AAAGAAGGAC AAAATAATAA AATTGAAAAA ATCGTTGATA AA -#GATGGAGC 240 - - TTATGTTTTA AGCCAATATC TGACTGAACT GACAGCTCCT GGAAGAGGTA TC -#AATTACTT 300 - - TAATGGGTTA GAAGAAAAAT TGTATTATGG AGAAGGATAT AATGCACTTC CT -#CAACTCAG 360 - - AAAAGATATT GAACAAATCA TTTCATCTAC GGGTGCATTT ACCGGTGATC AC -#GGAAAAGT 420 - - ATCTGTAGGC TCAGGCGGAC CGTTAGTCTA TAATAACTCA GCTAACAATG TA -#GCAAATTC 480 - - TTTGAGTTAT TCTTTAGCAC AAGCAGCTTA AGATAGTTAT TTTTAGATGA TA -#AATAGCAA 540 - - TCCTATATAT ATTAGGTGTG TAGGATTGCT ATTTTATTTA TGGAGGAGCA AA -#TGGATTTT 600 - - TATCGGGAAG AAGACTACGG ATTATACGCA CTGACGATTT TAGCCCAGTA CC -#ATAATATT 660 - - GCTGTAAATC CGGAAGAACT AAAACATAAA TTCGACCTTG AAGGAAAAGG CT -#TAGATCTA 720 - - ACCGCTTGGC TATTAGCCGC AAAATCATTA GAACTTAAAG CAAAACAAGT AA -#AAAAAGCG 780 - - ATTGATCGTT TGGCGTTTAT CGCACTACCG GCACTTGTAT GGCGAGAAGA CG -#GTAAACAT 840 - - TTTATTTTGA CTAAAATTGA TAATGAAGCA AAAAAATATT TAATTTTTGA TT -#TGGAAACG 900 - - CATAATCCTC GCATTTTGGA ACAAGCGGAA TTCGAGAGCT TATACCAAGG AA -#AACTGATT 960 - - TTAGTTGCAT CAAGAGCTTC CATCGTAGGT AAGCTGGCAA AGTTTGACTT CA -#CTTGGTTT 1020 - - ATACCGGCGG TAATTAAGTA TCGTAAGATT TTTATTGAAA CGTTAATTGT TT -#CAATTTTT 1080 - - TTGCAAATTT TCGCACTAAT TACACCGCTT TTTTTCCAAG TCGTGATGGA TA -#AAGTCTTG 1140 - - GTACACCGAG GTTTTTCAAC CTTAAATGTG ATTACGGTGG CATTAGCGAT CG -#TCGTGCTG 1200 - - TTTGAAATTG TGCTAAACGG TTTACGTACC TATATTTTTG CGCATAGTAC CA -#GCCGTATT 1260 - - GATGTGGAGT TGGGAGCAAG ATTATTCAGA CATTTATTAG CACTCCCAAT CT -#CTTATTTT 1320 - - GAAAATCGTC GAGTCGGCGA TACGGTGGCT CGTGTACGAG AACTCGATCA AA -#TTCGTAAC 1380 - - TTCTTAACCG GGCAGGCACT TACTTCCGTG TTGGATTTAA TGTTTTCCTT TA -#TCTTCTTT 1440 - - GCAGTGATGT GGTATTACAG CCCTAAACTT ACTCTTGTGA TTTTAGGCTC GT -#TACCGTTT 1500 - - TATATGGGGT GGTCGATTTT TATCAGCCCT ATTTTACGTC GCCGTTTAGA TG -#AAAAATTC 1560 - - GCACGTGGTG CGGACAATCA GTCATTCTTA GTGGAATCGG TGACTGCAAT CA -#ATACGATT 1620 - - AAAGCGTTGG CGGTTACCCC TCAAATGACT AATACCTGGG ATAAGCAATT AG -#CCAGCTAT 1680 - - GTATCGGCGG GATTCCGTGT AACCACATTA GCTACTATCG GACAGCAAGG TG -#TACAATTT 1740 - - ATTCAAAAAG TCGTGATGGT TATTACCTTA TGGCTAGGCG CACATTTAGT GA -#TTTCAGGC 1800 - - GATTTAAGTA TCGGACAATT AATCGCATTT AATATGTTAT CCGGTCAAGT GA -#TTGCACCG 1860 - - GTGATTCGTT TAGCGCAACT TTGGCAAGAT TTCCAACAAG TGGGAATTTC GG -#TAACGCGT 1920 - - TTAGGTGATG TTTTAAACTC TCCGACCGAG AGCTATCAAG GAAAATTGGC GT -#TACCGGAA 1980 - - ATTAAAGGCG ATATTACCTT CCGTAATATA CGCTTCCGCT ACAAACCGGA TG -#CGCCGGTG 2040 - - ATTTTAAATG ATGTGAATTT ATCGATTCAG CAAGGTGAAG TGATCGGTAT CG -#TAGGACGT 2100 - - TCAGGCTCAG GGAAGAGCAC CTTAACGAAA TTAATTCAAC GTTTTTATAT TC -#CGGAAAAC 2160 - - GGTCAGGTAT TAATAGATGG GCATGATTTA GCATTGGCGG ATCCGAACTG GC -#TACGTCGT 2220 - - CAAGTCGGGG TGGTATTACA AGATAACGTA CTATTAAATC GTAGTATTCG AG -#ATAATATT 2280 - - GCCTTAGCGG ATCCGGGTAT GCCAATGGAA AAAATTGTCC ATGCGGCAAA AT -#TAGCCGGC 2340 - - GCACATGAAT TTATTTCTGA ATTGCGTGAG GGATATAACA CGATTGTTGG TG -#AGCAAGGT 2400 - - GCGGGGCTAT CTGGCGGGCA ACGCCAACGT ATTGCGATTG CACGCGCTTT GG -#TGAATAAC 2460 - - CCGAAAATCT TAATTTTTGA TGAAGCGACC AGCGCATTAG ATTATGAATC CG -#AGCATATC 2520 - - ATCATGCGCA ATATGCACCA GATTTGTAAA GGGAGAACGG TAATTATCAT TG -#CACACCGT 2580 - - TTATCTACGG TAAAAAATGC CGACCGTATT ATTGTGATGG AAAAAGGTCA GA -#TTGTGGAA 2640 - - CAAGGTAAGC ATAAAGAGCT GCTTGCTGAT CCAAACGGCT TATATCACTA CT -#TACACCAA 2700 - - TTACAATCGG AATAGGAGGA CTTATGAAAA CATGGCTAAT GGGTTTATAT GA -#GTTTTTCC 2760 - - AACGCTATAA AACGGTTTGG ACGGAGATCT GGAAAATTCG TCATCAATTG GA -#TACGCCGG 2820 - - ATCGAGAAAA GGATGAAAAT GAATTTTTAC CTGCACACTT AGAGCTGATT GA -#AACACCGG 2880 - - TGTCAAAAAA ACCGAGATTG ATCGCTTATT TAATTATGCT GTTCCTATTT TT -#GGCATTAG 2940 - - TTATTTCAAT TGTCAGTCAC GTAGAAATTG TGGCGACCGC AACGGGTAAA TT -#AGCGTTTA 3000 - - GCGACCGTAG CAAAGAAATT AAGCCGATTG AAAACGCCTT GGTTAAAGAA AT -#CTTTGTGC 3060 - - AAGACGGACA ATTTGTTGAG AAAGATCAGT TGCTGTTACA CTTGACCGCA TT -#GGGAGCCG 3120 - - ATGCGGATCA ACAAAAAACC AAAAGTTCGT TATCGCTGAC TAAATTGGAA CG -#TTATCGTT 3180 - - ATGAAATTTT ATTAGAGGCG GTTGCGGCGG ATAGGTTGCC GCTCATTGAA CT -#GACAAAGG 3240 - - ATGAATTTAA ACATGCTACG GAAGAAGATA AAACCAGAAT TCGCTATTTG AT -#CACCGAGC 3300 - - AATTTGAAGC TTGGCAAAAG CAAAAGTATC AAAAAGAATT AGCTTTGCAA CG -#TAGAGAAG 3360 - - CAGAAAAACA AACGGTTCTA GCTAATATTC GTAAATATGA GGGAATCAGT CG -#AGTTGAAA 3420 - - ATGAAAGATT AAAAGATCTT AAAAAATTAT TTAATTCGAA ATCGACTTCT AA -#ACATGATG 3480 - - TCTTGACTCA AGAAAATCGT CATATCGAAG CGGTAAATGA GTTGGCGGTG TA -#TAAATCTC 3540 - - GGTTGAATGA AGTGGAAAGT GACTTACGTC AAGCCAAAGA GGAAATACAT TT -#AATAACTC 3600 - - AGTTGTTTAG AGCCGATATT CTGGAGAAGT TGAAACAAAA TGTTGAAGCG GA -#GAAACAGC 3660 - - TTTCGCTCGA ATTAGAAAAA AATGAGCAGC GTCAAATTGC TTCGGTGATT CG -#TGCGCCGG 3720 - - TTTCCGGTAC GGTTCAGCAA CTTAAAACCC ATACGGTAGG CGGCGTCGTG AC -#GACTGCCG 3780 - - AAACCTTGAT GGTAATTGCT CCGGAAGATG ATGTTTTAGA GGTAACGGCG TT -#AATTCAAA 3840 - - ATAAGGATAT CGGTTTTATC GAGGTCGGTC AGGATGCGGT GATTAAAGTA GA -#AACTTTTC 3900 - - CTTATACTCG TTACGGCTAT TTAATGGGTA AAGTAAAAAA TATCACGCTG GA -#AGCCATCG 3960 - - AACATCCGCA ACTCGGTCTA GTTTTTAACT CGATTATTTC TATTGATAGA AA -#AACTTTAT 4020 - - CCGGCAAAGA CGGCAAAGAA ATTGAACTTG GATCAGGTAT GAGTGTGACG GC -#GGAAATTA 4080 - - AAACTGGAGA ACGTAGCGTT ATTAGTTATT TACTCAGTCC GTTGGAAGAA TC -#CGTTTCGG 4140 - - AGAGTTTAAG AGAACGCTAA AGCAGATAAA ACAAGCGGCC ATATTTTCTT AC -#TTTTTTGC 4200 - - AAAAAACGTA TGAAATATGA CCGCTTGTCG TTTGTAAAAG ACTATTTATT TA -#CAATAATT 4260 - - TTAGCACCGT TAGAAAATAC GATCTGACGA GCTTCAAATT GAGCGGAGAG CT -#GTGCTTGC 4320 - - GGGTTTAGAA ATACGGCTTG TGCTTCTTGC GGTAAGTCTG AAACCGGTAC GC -#AAAGGCAA 4380 - - GTTCCGCCGT GGTTTGGCGT TTTAAGTTAT CTTTAAAGGT AACGGGCGCA TC -#TTGCGTGA 4440 - - GGATAACTTT ATCATTGTAA ACATAGTTTA CCGCCCATTG AACGATACGA AT -#ATTGCGTT 4500 - - TGGTTTTATT TTCAATACTG TATTTAAAGC TAACCATCGG CTGCCCTTCT TT -#ATTTTTAG 4560 - - CCAATTCATA ACCGAAAAAA CGTAACCCGA TACTGTCATT AAATTGTTTA AG -#GCGTTTTT 4620 - - CTTTAGCCGA AAGAGGTGCA TTTTTCGTTA CTGATTTATG TTCAACCGTC GG -#TTGAATTT 4680 - - TATTGCCTTC AGCTTGAGCA TTAAACGCTA AAAAGAATGA TGCTACCGCC GT -#GCTAAGTA 4740 - - ATTTAATGTG TTTCATAATT CACCTCGTAA TGAGAGCTAA AAGCCGACTT GA -#TATATTAC 4800 - - GCTATATATT GTCAGATTTA CGGCACAGTT GCAATGACCG CATAACCGTC CG -#ATTCGGCA 4860 - - ATAATCTCGA CTTGGCTTTC CGCCGCAATG AAAATCGCTT CGCCTTGTTG GA -#GATAAATG 4920 - - GACTCTTCAC CGAGGTCGAT ATAGATACTG CCTTTCATCA CCAATAAGAT AC -#TTGCACAG 4980 - - TCGGCCGTAA AGTTTTCTTC GTCAAATGCG TTGAATTGCA TATGTTGCAA TG -#CAAAATCT 5040 - - TTCGCTTCAG GCGTCGGATA AAGATGAATG AAACCGTCGT TTTCTTGATA AG -#GCGGAATA 5100 - - ACTTCGGGGT AATCGGGCGA - # - # 512 - #0 - - - - (2) INFORMATION FOR SEQ ID NO:7: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 159 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Actinobacill - #us pleuropneumoniae - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: - - Met Leu Lys Asn Asp Phe Asn Val Leu Gly Gl - #n Ile Ala Trp Leu Trp 1 5 - # 10 - # 15 - - Ala Asn Ser Pro Met His Arg Asn Trp Ser Va - #l Ser Leu Leu Met Lys 20 - # 25 - # 30 - - Asn Val Ile Pro Ala Ile Glu Asn Asp Gln Ty - #r Leu Leu Leu Val Asp 35 - # 40 - # 45 - - Asp Gly Phe Pro Ile Ala Tyr Cys Ser Trp Al - #a Lys Leu Thr Leu Glu 50 - # 55 - # 60 - - Ser Glu Ala Arg Tyr Val Lys Asp Thr Asn Se - #r Leu Lys Ile Asp Asp 65 - #70 - #75 - #80 - - Trp Asn Ala Gly Asp Arg Ile Trp Ile Ile As - #p Trp Ile Ala Pro Phe 85 - # 90 - # 95 - - Gly Asp Ser Ser Leu Leu Tyr Lys His Met Ar - #g Gln Arg Phe Pro Tyr 100 - # 105 - # 110 - - Asp Ile Gly Arg Ala Ile Arg Ile Tyr Pro Se - #r Lys Lys Asp Thr Gly 115 - # 120 - # 125 - - Lys Ile Ile Tyr Leu Lys Gly Gly Lys Ile Th - #r Lys Lys Val Ala Glu 130 - # 135 - # 140 - - Lys Thr Phe Leu Gln Tyr Glu Gln Glu Leu Il - #e Thr Ala Leu Gln 145 1 - #50 1 - #55 - - - - (2) INFORMATION FOR SEQ ID NO:8: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 956 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Actinobacill - #us pleuropneumoniae - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: - - Met Ser Lys Ile Thr Leu Ser Ser Leu Lys Se - #r Ser Leu Gln Gln Gly 1 5 - # 10 - # 15 - - Leu Lys Asn Gly Lys Asn Lys Leu Asn Gln Al - #a Gly Thr Thr Leu Lys 20 - # 25 - # 30 - - Asn Gly Leu Thr Gln Thr Gly His Ser Leu Gl - #n Asn Gly Ala Lys Lys 35 - # 40 - # 45 - - Leu Ile Leu Tyr Ile Pro Gln Gly Tyr Asp Se - #r Gly Gln Gly Asn Gly 50 - # 55 - # 60 - - Val Gln Asp Leu Val Lys Ala Ala Asn Asp Le - #u Gly Ile Glu Val Trp 65 - #70 - #75 - #80 - - Arg Glu Glu Arg Ser Asn Leu Asp Ile Ala Ly - #s Thr Ser Phe Asp Thr 85 - # 90 - # 95 - - Thr Gln Lys Ile Leu Gly Phe Thr Asp Arg Gl - #y Ile Val Leu Phe Ala 100 - # 105 - # 110 - - Pro Gln Leu Asp Asn Leu Leu Lys Lys Asn Pr - #o Lys Ile Gly Asn Thr 115 - # 120 - # 125 - - Leu Gly Ser Ala Ser Ser Ile Ser Gln Asn Il - #e Gly Lys Ala Asn Thr 130 - # 135 - # 140 - - Val Leu Gly Gly Ile Gln Ser Ile Leu Gly Se - #r Val Leu Ser Gly Val 145 1 - #50 1 - #55 1 -#60 - - Asn Leu Asn Glu Leu Leu Gln Asn Lys Asp Pr - #o Asn Gln Leu GluLeu 165 - # 170 - # 175 - - Ala Lys Ala Gly Leu Glu Leu Thr Asn Glu Le - #u Val Gly Asn Ile Ala 180 - # 185 - # 190 - - Ser Ser Val Gln Thr Val Asp Ala Phe Ala Gl - #u Gln Ile Ser Lys Leu 195 - # 200 - # 205 - - Gly Ser His Leu Gln Asn Val Lys Gly Leu Gl - #y Gly Leu Ser Asn Lys 210 - # 215 - # 220 - - Leu Gln Asn Leu Pro Asp Leu Gly Lys Ala Se - #r Leu Gly Leu Asp Ile 225 2 - #30 2 - #35 2 -#40 - - Ile Ser Gly Leu Leu Ser Gly Ala Ser Ala Gl - #y Leu Ile Leu AlaAsp 245 - # 250 - # 255 - - Lys Glu Ala Ser Thr Glu Lys Lys Ala Ala Al - #a Gly Val Glu Phe Ala 260 - # 265 - # 270 - - Asn Gln Ile Ile Gly Asn Val Thr Lys Ala Va - #l Ser Ser Tyr Ile Leu 275 - # 280 - # 285 - - Ala Gln Arg Val Ala Ser Gly Leu Ser Ser Th - #r Gly Pro Val Ala Ala 290 - # 295 - # 300 - - Leu Ile Ala Ser Thr Val Ala Leu Ala Val Se - #r Pro Leu Ser Phe Leu 305 3 - #10 3 - #15 3 -#20 - - Asn Val Ala Asp Lys Phe Lys Gln Ala Asp Le - #u Ile Lys Ser TyrSer 325 - # 330 - # 335 - - Glu Arg Phe Gln Lys Leu Gly Tyr Asp Gly As - #p Arg Leu Leu Ala Asp 340 - # 345 - # 350 - - Phe His Arg Glu Thr Gly Thr Ile Asp Ala Se - #r Val Thr Thr Ile Asn 355 - # 360 - # 365 - - Thr Ala Leu Ala Ala Ile Ser Gly Gly Val Gl - #y Ala Ala Ser Ala Gly 370 - # 375 - # 380 - - Ser Leu Val Gly Ala Pro Val Ala Leu Leu Va - #l Ala Gly Val Thr Gly 385 3 - #90 3 - #95 4 -#00 - - Leu Ile Thr Thr Ile Leu Glu Tyr Ser Lys Gl - #n Ala Met Phe GluHis 405 - # 410 - # 415 - - Val Ala Asn Lys Val His Asp Arg Ile Val Gl - #u Trp Glu Lys Lys His 420 - # 425 - # 430 - - Asn Lys Asn Tyr Phe Glu Gln Gly Tyr Asp Se - #r Arg His Leu Ala Asp 435 - # 440 - # 445 - - Leu Gln Asp Asn Met Lys Phe Leu Ile Asn Le - #u Asn Lys Glu Leu Gln 450 - # 455 - # 460 - - Ala Glu Arg Val Val Ala Ile Thr Gln Gln Ar - #g Trp Asp Asn Gln Ile 465 4 - #70 4 - #75 4 -#80 - - Gly Asp Leu Ala Ala Ile Ser Arg Arg Thr As - #p Lys Ile Ser SerGly 485 - # 490 - # 495 - - Lys Ala Tyr Val Asp Ala Phe Glu Glu Gly Gl - #n His Gln Ser Tyr Asp 500 - # 505 - # 510 - - Ser Ser Val Gln Leu Asp Asn Lys Asn Gly Il - #e Ile Asn Ile Ser Asn 515 - # 520 - # 525 - - Thr Asn Arg Lys Thr Gln Ser Val Leu Phe Ar - #g Thr Pro Leu Leu Thr 530 - # 535 - # 540 - - Pro Gly Glu Glu Asn Arg Glu Arg Ile Gln Gl - #u Gly Lys Asn Ser Tyr 545 5 - #50 5 - #55 5 -#60 - - Ile Thr Lys Leu His Ile Gln Arg Val Asp Se - #r Trp Thr Val ThrAsp 565 - # 570 - # 575 - - Gly Asp Ala Ser Ser Ser Val Asp Phe Thr As - #n Val Val Gln Arg Ile 580 - # 585 - # 590 - - Ala Val Lys Phe Asp Asp Ala Gly Asn Ile Il - #e Glu Ser Lys Asp Thr 595 - # 600 - # 605 - - Lys Ile Ile Ala Asn Leu Gly Ala Gly Asn As - #p Asn Val Phe Val Gly 610 - # 615 - # 620 - - Ser Ser Thr Thr Val Ile Asp Gly Gly Asp Gl - #y His Asp Arg Val His 625 6 - #30 6 - #35 6 -#40 - - Tyr Ser Arg Gly Glu Tyr Gly Ala Leu Val Il - #e Asp Ala Thr AlaGlu 645 - # 650 - # 655 - - Thr Glu Lys Gly Ser Tyr Ser Val Lys Arg Ty - #r Val Gly Asp Ser Lys 660 - # 665 - # 670 - - Ala Leu His Glu Thr Ile Ala Thr His Gln Th - #r Asn Val Gly Asn Arg 675 - # 680 - # 685 - - Glu Glu Lys Ile Glu Tyr Arg Arg Glu Asp As - #p Arg Phe His Thr Gly 690 - # 695 - # 700 - - Tyr Thr Val Thr Asp Ser Leu Lys Ser Val Gl - #u Glu Ile Ile Gly Ser 705 7 - #10 7 - #15 7 -#20 - - Gln Phe Asn Asp Ile Phe Lys Gly Ser Gln Ph - #e Asp Asp Val PheHis 725 - # 730 - # 735 - - Gly Gly Asn Gly Val Asp Thr Ile Asp Gly As - #n Asp Gly Asp Asp His 740 - # 745 - # 750 - - Leu Phe Gly Gly Ala Gly Asp Asp Val Ile As - #p Gly Gly Asn Gly Asn 755 - # 760 - # 765 - - Asn Phe Leu Val Gly Gly Thr Gly Asn Asp Il - #e Ile Ser Gly Gly Lys 770 - # 775 - # 780 - - Asp Asn Asp Ile Tyr Val His Lys Thr Gly As - #p Gly Asn Asp Ser Ile 785 7 - #90 7 - #95 8 -#00 - - Thr Asp Ser Gly Gly Gln Asp Lys Leu Ala Ph - #e Ser Asp Val AsnLeu 805 - # 810 - # 815 - - Lys Asp Leu Thr Phe Lys Lys Val Asp Ser Se - #r Leu Glu Ile Ile Asn 820 - # 825 - # 830 - - Gln Lys Gly Glu Lys Val Arg Ile Gly Asn Tr - #p Phe Leu Glu Asp Asp 835 - # 840 - # 845 - - Leu Ala Ser Thr Val Ala Asn Tyr Lys Ala Th - #r Asn Asp Arg Lys Ile 850 - # 855 - # 860 - - Glu Glu Ile Ile Gly Lys Gly Gly Glu Arg Il - #e Thr Ser Glu Gln Val 865 8 - #70 8 - #75 8 -#80 - - Asp Lys Leu Ile Lys Glu Gly Asn Asn Gln Il - #e Ser Ala Glu AlaLeu 885 - # 890 - # 895 - - Ser Lys Val Val Asn Asp Tyr Asn Thr Ser Ly - #s Asp Arg Gln Asn Val 900 - # 905 - # 910 - - Ser Asn Ser Leu Ala Lys Leu Ile Ser Ser Va - #l Gly Ser Phe Thr Ser 915 - # 920 - # 925 - - Ser Ser Asp Phe Arg Asn Asn Leu Gly Thr Ty - #r Val Pro Ser Ser Ile 930 - # 935 - # 940 - - Asp Val Ser Asn Asn Ile Gln Leu Ala Arg Al - #a Ala 945 9 - #50 9 - #55 - - - - (2) INFORMATION FOR SEQ ID NO:9: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4731 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Actinobacill - #us pleuropneumoniae - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: - - CTTAACCATT ACAGAACGTT GGTACAAAAA ATTTTACAGG AAAATGATGG AT -#AGTCCTTA 60 - - ACAAAAATTA ATGTTTTATT TCCTATAAAA CATCCGACCA GTATTATTTT TG -#ATTAAAAA 120 - - AAGAACAAAC AGATCATGAC AAACGTTTGC CTTGTTTTCC TTCACAAAAA TA -#TTATGGTT 180 - - TTTTATTTAG AATAAATTAT CTATATTCAT TTTTTAGGGA ATGGGAGGGA TG -#ATGCTAAA 240 - - AAATGATTTT AACGTATTGG GACAAATTGC TTGGTTATGG GCAAATTCTC CA -#ATGCACCG 300 - - AAATTGGTCA GTTTCACTGT TAATGAAGAA TGTTATTCCT GCAATTGAAA AT -#GACCAATA 360 - - TTTGTTACTA GTTGATGATG GTTTTCCTAT TGCATATTGC AGTTGGGCCA AA -#TTAACTCT 420 - - AGAGAGTGAG GCTCGCTATG TAAAGGACAC CAATTCATTA AAAATAGATG AT -#TGGAATGC 480 - - AGGAGATCGT ATATGGATCA TTGATTGGAT TGCCCCATTC GGGGATTCAT CT -#CTATTGTA 540 - - TAAACATATG AGACAACGTT TTCCATACGA TATTGGAAGG GCAATTAGAA TC -#TATCCTAG 600 - - CAAAAAAGAT ACTGGAAAAA TCATATATTT AAAAGGAGGA AAAATAACAA AA -#AAAGTAGC 660 - - TGAAAAGACA TTTCTTCAGT ATGAGCAAGA GTTAATAACA GCTCTACAAT AA -#TATCTTTA 720 - - AATGATCAAT TATATAAAGG AGACTCTTTT ATGTCAAAAA TCACTTTGTC AT -#CATTAAAA 780 - - TCGTCCTTAC AACAAGGATT GAAAAATGGG AAAAACAAGT TAAATCAAGC AG -#GTACAACA 840 - - CTGAAGAATG GTTTAACTCA AACTGGTCAT TCTCTACAGA ATGGGGCTAA AA -#AATTAATC 900 - - TTATATATTC CTCAAGGCTA TGATTCGGGT CAAGGAAATG GAGTTCAAGA TT -#TAGTTAAA 960 - - GCTGCTAATG ATTTAGGTAT TGAAGTATGG CGAGAAGAAC GCAGCAATTT GG -#ACATTGCA 1020 - - AAAACTAGCT TTGATACAAC TCAGAAAATT CTAGGTTTTA CTGATAGAGG AA -#TTGTATTA 1080 - - TTTGCACCTC AGCTAGATAA TTTATTAAAG AAGAATCCTA AAATTGGCAA TA -#CATTAGGA 1140 - - AGTGCTTCTA GCATCTCACA AAATATAGGT AAAGCCAATA CTGTATTAGG TG -#GTATTCAA 1200 - - TCTATTTTAG GATCTGTTTT ATCTGGAGTA AATCTGAATG AATTACTTCA AA -#ATAAAGAT 1260 - - CCTAATCAAT TAGAACTTGC AAAAGCAGGG CTAGAACTGA CTAATGAATT AG -#TTGGTAAT 1320 - - ATTGCTAGCT CGGTGCAAAC TGTAGATGCA TTTGCAGAAC AAATATCTAA AC -#TAGGTTCA 1380 - - CATTTACAGA ATGTGAAAGG ATTAGGAGGA TTGAGTAATA AATTACAAAA TC -#TACCAGAT 1440 - - CTAGGAAAAG CAAGTTTAGG TTTGGACATT ATCTCTGGTT TACTTTCTGG AG -#CATCTGCA 1500 - - GGTCTCATTT TAGCAGATAA AGAGGCTTCA ACAGAAAAGA AAGCTGCCGC AG -#GTGTAGAA 1560 - - TTTGCTAACC AAATTATAGG TAATGTAACA AAAGCGGTCT CATCTTACAT TC -#TTGCCCAA 1620 - - CGAGTCGCTT CAGGTTTGTC TTCAACTGGT CCTGTCGCTG CATTAATCGC AT -#CTACAGTT 1680 - - GCACTAGCTG TTAGCCCTCT TTCATTCTTA AATGTAGCTG ATAAGTTTAA AC -#AAGCTGAT 1740 - - TTAATCAAAT CATATTCTGA ACGCTTCCAA AAATTAGGAT ATGATGGAGA TC -#GTTTATTA 1800 - - GCTGATTTTC ACCGTGAGAC AGGAACTATT GATGCTTCTG TAACAACAAT TA -#ACACTGCT 1860 - - TTAGCAGCTA TCTCCGGTGG AGTTGGAGCT GCAAGCGCGG GTTCTCTAGT CG -#GAGCTCCA 1920 - - GTTGCGTTAC TCGTTGCTGG TGTTACGGGA CTTATTACAA CTATTCTAGA AT -#ATTCTAAA 1980 - - CAAGCCATGT TTGAACATGT TGCAAATAAG GTTCATGACA GAATAGTTGA AT -#GGGAGAAA 2040 - - AAACATAATA AAAACTATTT TGAGCAAGGT TATGATTCTC GTCATTTAGC TG -#ATTTACAA 2100 - - GACAATATGA AGTTTCTTAT CAATTTAAAT AAAGAACTTC AGGCTGAACG CG -#TAGTAGCT 2160 - - ATTACCCAAC AAAGATGGGA TAACCAAATT GGAGACCTAG CGGCAATTAG CC -#GTAGAACG 2220 - - GATAAAATTT CCAGTGGAAA AGCTTATGTG GATGCTTTTG AGGAGGGGCA AC -#ACCAGTCC 2280 - - TACGATTCAT CCGTACAGCT AGATAACAAA AACGGTATTA TTAATATTAG TA -#ATACAAAT 2340 - - AGAAAGACAC AAAGTGTTTT ATTCAGAACT CCATTACTAA CTCCAGGTGA AG -#AGAATCGG 2400 - - GAACGTATTC AGGAAGGTAA AAATTCTTAT ATTACAAAAT TACATATACA AA -#GAGTTGAC 2460 - - AGTTGGACTG TAACAGATGG TGATGCTAGC TCAAGCGTAG ATTTCACTAA TG -#TAGTACAA 2520 - - CGAATCGCTG TGAAATTTGA TGATGCAGGT AACATTATAG AATCTAAAGA TA -#CTAAAATT 2580 - - ATCGCAAATT TAGGTGCTGG TAACGATAAT GTATTTGTTG GGTCAAGTAC TA -#CCGTTATT 2640 - - GATGGCGGGG ACGGACATGA TCGAGTTCAC TACAGTAGAG GAGAATATGG CG -#CATTAGTT 2700 - - ATTGATGCTA CAGCCGAGAC AGAAAAAGGC TCATATTCAG TAAAACGCTA TG -#TCGGAGAC 2760 - - AGTAAAGCAT TACATGAAAC AATTGCCACC CACCAAACAA ATGTTGGTAA TC -#GTGAAGAA 2820 - - AAAATTGAAT ATCGTCGTGA AGATGATCGT TTTCATACTG GTTATACTGT GA -#CGGACTCA 2880 - - CTCAAATCAG TTGAAGAGAT CATTGGTTCA CAATTTAATG ATATTTTCAA AG -#GAAGCCAA 2940 - - TTTGATGATG TGTTCCATGG TGGTAATGGT GTAGACACTA TTGATGGTAA CG -#ATGGTGAC 3000 - - GATCATTTAT TTGGTGGCGC AGGCGATGAT GTTATCGATG GAGGAAACGG TA -#ACAATTTC 3060 - - CTTGTTGGAG GAACCGGTAA TGATATTATC TCGGGAGGTA AAGATAATGA TA -#TTTATGTC 3120 - - CATAAAACAG GCGATGGAAA TGATTCTATT ACAGACTCTG GCGGACAAGA TA -#AACTGGCA 3180 - - TTTTCGGATG TAAATCTTAA AGACCTCACC TTTAAGAAAG TAGATTCTTC TC -#TCGAAATC 3240 - - ATTAATCAAA AAGGAGAAAA AGTTCGTATT GGGAATTGGT TCTTAGAAGA TG -#ATTTGGCT 3300 - - AGCACAGTTG CTAACTATAA AGCTACGAAT GACCGAAAAA TTGAGGAAAT TA -#TTGGTAAA 3360 - - GGAGGAGAAC GTATTACATC AGAACAAGTT GATAAACTGA TTAAGGAGGG TA -#ACAATCAA 3420 - - ATCTCTGCAG AAGCATTATC CAAAGTTGTG AATGATTACA ATACGAGTAA AG -#ATAGACAG 3480 - - AACGTATCTA ATAGCTTAGC AAAATTGATT TCTTCAGTCG GGAGCTTTAC GT -#CTTCCTCA 3540 - - GACTTTAGGA ATAATTTAGG AACATATGTT CCTTCATCAA TAGATGTCTC GA -#ATAATATT 3600 - - CAATTAGCTA GAGCCGCTTA ATATTCAAAT CATAGCAATC CTATGGTGTA AA -#TTATAGGA 3660 - - TTGTTATTTT TTTAAAGGAG AAGTTATGGA ACCCAATAAA AATAAGGATC TT -#GGTTTAGC 3720 - - TGTAGAAAAT CAAACCTAAT CTGACAGTTC CCGTTTAAAA TTACCGTGTC TG -#TCAGATTA 3780 - - ATTTGAGCTT AAATTCTTTT CTGCCCAAAT CCGTTTTCCA TCAAGTAATG TT -#GCCATCGG 3840 - - TGTTCTGCCA CAGCACACTT TTCCTTGATG TGTTCGATGG TGATTATAAT AC -#ATTCATCT 3900 - - AAATCAGCTT GTAATGTCGC TAAATCCGTA TATATTTTCT TCCTAAATGC GA -#CTTGGTAA 3960 - - AATTCTTGTA AGATAGTCTT ATGAAAACGT TCACAGATAC CATTCGTCTG TG -#GATGCTTC 4020 - - ACTTTCGTTT TAGTATGCTC TATGTCATTT ATCGCTAAAT AAAGCTCATA AT -#CGTGATTT 4080 - - TCCACTTTGC CACAATATTC ACTGCCACGG TCGGTGAGAA TACGCAACAT CG -#GTAATCCT 4140 - - TGGGCTTCAA AGAACGGCAG TACTTTATGA TTGAGCATAT CTGCAGCGGC AA -#TTGCGGTT 4200 - - TTCATTGTGT AGAGCTTTGC AAAAGCAACC TTACTATAAG TATCAACAAA TG -#TTTGCTGA 4260 - - TAAATGCGTC CAACACCTTT TAAATTACCT ACATAAAAGG TATCTTGTGA AC -#CTAAATAG 4320 - - CCCGGATGAG CGGTTTCAAT TTCTCCACTC GATATATCAT CCTCTTTCTT AC -#GTTCTAGG 4380 - - GCTTGGACTT GACTTTCATT TAGAATAATG CCTTTCTCAG CCACTTCTTT CT -#CTAGTGCA 4440 - - TTTAAACGCT GTTTAAAGTT AGTAAGATTA TGACGTAGCC AAATGGAACG AA -#CACCACCG 4500 - - GCTGAAACAA ACACACCTTG CTTGCGAAGT TCGTTACTCA CTCGAACTTG TC -#CGTAAGCT 4560 - - GGAAAATCTA GAGCAAATTT TACAACAGCT TGCTCAATGT GCTCGTCTAC TC -#GATTTTTG 4620 - - ATATTCGGTA CCCGACGAGT TTGCTTAAGT AATGCTTCAA CACCGCCTTG CG -#CTACGGCT 4680 - - TGTTGATAGC GATAGAATGT ATCTCGGCTC ATTCCCATCG CTTTACAAGC T - # 4731 - - - - (2) INFORMATION FOR SEQ ID NO:10: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 173 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Actinobacill - #us pleuropneumoniae - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: - - Met Ser Tyr Lys Asn Val Lys Asn Leu Thr As - #p Asp Phe Thr Thr Leu 1 5 - # 10 - # 15 - - Gly His Ile Ala Trp Leu Trp Ala Asn Ser Pr - #o Leu His Lys Glu Trp 20 - # 25 - # 30 - - Ser Ile Ser Leu Phe Thr Lys Asn Ile Leu Pr - #o Ala Ile Gln His Asp 35 - # 40 - # 45 - - Gln Tyr Ile Leu Leu Met Arg Asp Glu Phe Pr - #o Val Ala Phe Cys Ser 50 - # 55 - # 60 - - Trp Ala Asn Leu Thr Leu Thr Asn Glu Val Ly - #s Tyr Val Arg Asp Val 65 - #70 - #75 - #80 - - Thr Ser Leu Thr Phe Glu Asp Trp Asn Ser Gl - #y Glu Arg Lys Trp Leu 85 - # 90 - # 95 - - Ile Asp Trp Ile Ala Pro Phe Gly Asp Asn As - #n Thr Leu Tyr Arg Tyr 100 - # 105 - # 110 - - Met Arg Lys Lys Phe Pro Asn Glu Val Phe Ar - #g Ala Ile Arg Val Tyr 115 - # 120 - # 125 - - Pro Gly Ser Thr Glu Ala Lys Ile Ile His Va - #l Gln Gly Gly Gln Ile 130 - # 135 - # 140 - - Asn Lys Phe Thr Ala Lys Lys Leu Ile Gln Gl - #n Tyr Gln Glu Glu Leu 145 1 - #50 1 - #55 1 -#60 - - Ile Gln Val Leu Asn Asn His Lys Lys Ile Va - #l Arg Gly 165 - # 170 - - - - (2) INFORMATION FOR SEQ ID NO:11: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1049 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Actinobacill - #us pleuropneumoniae - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: - - Met Ser Thr Trp Ser Ser Met Leu Ala Asp Le - #u Lys Lys Arg AlaGlu 1 5 - # 10 - # 15 - - Glu Ala Lys Arg Gln Val Lys Lys Gly Tyr As - #p Val Thr Lys Asn Gly 20 - # 25 - # 30 - - Leu Gln Tyr Gly Val Ser Gln Ala Lys Leu Gl - #n Ala Leu Ala Ala Gly 35 - # 40 - # 45 - - Lys Ala Val Gln Lys Tyr Gly Asn Lys Leu Va - #l Leu Val Ile Pro Lys 50 - # 55 - # 60 - - Glu Tyr Asp Gly Ser Val Gly Asn Gly Phe Ph - #e Asp Leu Val Lys Ala 65 - #70 - #75 - #80 - - Ala Glu Glu Leu Gly Ile Gln Val Lys Tyr Va - #l Asn Arg Asn Glu Leu 85 - # 90 - # 95 - - Glu Val Ala His Lys Ser Leu Gly Thr Ala As - #p Gln Phe Leu Gly Leu 100 - # 105 - # 110 - - Thr Glu Arg Gly Leu Thr Leu Phe Ala Pro Gl - #n Leu Asp Gln Phe Leu 115 - # 120 - # 125 - - Gln Lys His Ser Lys Ile Ser Asn Val Val Gl - #y Ser Ser Thr Gly Asp 130 - # 135 - # 140 - - Ala Val Ser Lys Leu Ala Lys Ser Gln Thr Il - #e Ile Ser Gly Ile Gln 145 1 - #50 1 - #55 1 -#60 - - Ser Val Leu Gly Thr Val Leu Ala Gly Ile As - #n Leu Asn Glu AlaIle 165 - # 170 - # 175 - - Ile Ser Gly Gly Ser Glu Leu Glu Leu Ala Gl - #u Ala Gly Val Ser Leu 180 - # 185 - # 190 - - Ala Ser Glu Leu Val Ser Asn Ile Ala Lys Gl - #y Thr Thr Thr Ile Asp 195 - # 200 - # 205 - - Ala Phe Thr Thr Gln Ile Gln Asn Phe Gly Ly - #s Leu Ala Glu Asn Ala 210 - # 215 - # 220 - - Lys Gly Leu Gly Gly Val Gly Arg Gln Leu Gl - #n Asn Ile Ser Gly Ser 225 2 - #30 2 - #35 2 -#40 - - Ala Leu Ser Lys Thr Gly Leu Gly Leu Asp Il - #e Ile Ser Ser LeuLeu 245 - # 250 - # 255 - - Ser Gly Val Thr Arg Ser Phe Ala Leu Arg As - #n Lys Asn Ala Ser Thr 260 - # 265 - # 270 - - Ser Thr Lys Val Ala Ala Gly Phe Glu Leu Se - #r Asn Gln Val Ile Gly 275 - # 280 - # 285 - - Gly Ile Thr Lys Ala Val Ser Ser Tyr Ile Le - #u Ala Gln Arg Leu Arg 290 - # 295 - # 300 - - Ala Gly Leu Ser Thr Thr Gly Pro Ala Ala Al - #a Leu Ile Ala Ser Ser 305 3 - #10 3 - #15 3 -#20 - - Ile Ser Leu Ala Ile Ser Pro Leu Ala Phe Le - #u Arg Val Ala AspAsn 325 - # 330 - # 335 - - Phe Asn Arg Ser Lys Glu Ile Gly Glu Phe Al - #a Glu Arg Phe Lys Lys 340 - # 345 - # 350 - - Leu Gly Tyr Asp Gly Asp Lys Leu Leu Ser Gl - #u Phe Tyr His Glu Ala 355 - # 360 - # 365 - - Gly Thr Ile Asp Ala Ser Ile Thr Thr Ile Se - #r Thr Ala Leu Ser Ala 370 - # 375 - # 380 - - Ile Ala Ala Gly Thr Ala Ala Ala Ser Ala Gl - #y Ala Leu Val Gly Ala 385 3 - #90 3 - #95 4 -#00 - - Pro Ile Thr Leu Leu Val Thr Gly Ile Thr Gl - #y Leu Ile Ser GlyIle 405 - # 410 - # 415 - - Leu Glu Phe Ser Lys Gln Pro Met Leu Asp Hi - #s Val Ala Ser Lys Ile 420 - # 425 - # 430 - - Gly Asn Lys Ile Asp Glu Trp Glu Lys Lys Ty - #r Gly Lys Asn Tyr Phe 435 - # 440 - # 445 - - Glu Asn Gly Tyr Asp Ala Arg His Lys Ala Ph - #e Leu Glu Asp Ser Phe 450 - # 455 - # 460 - - Ser Leu Leu Ser Ser Phe Asn Lys Gln Tyr Gl - #u Thr Glu Arg Ala Val 465 4 - #70 4 - #75 4 -#80 - - Leu Ile Thr Gln Gln Arg Trp Asp Glu Tyr Il - #e Gly Glu Leu AlaGly 485 - # 490 - # 495 - - Ile Thr Gly Lys Gly Asp Lys Leu Ser Ser Gl - #y Lys Ala Tyr Val Asp 500 - # 505 - # 510 - - Tyr Phe Gln Glu Gly Lys Leu Leu Glu Lys Ly - #s Pro Asp Asp Phe Ser 515 - # 520 - # 525 - - Lys Val Val Phe Asp Pro Thr Lys Gly Glu Il - #e Asp Ile Ser Asn Ser 530 - # 535 - # 540 - - Gln Thr Ser Thr Leu Leu Lys Phe Val Thr Pr - #o Leu Leu Thr Pro Gly 545 5 - #50 5 - #55 5 -#60 - - Thr Glu Ser Arg Glu Arg Thr Gln Thr Gly Ly - #s Tyr Glu Tyr IleThr 565 - # 570 - # 575 - - Lys Leu Val Val Lys Gly Lys Asp Lys Trp Va - #l Val Asn Gly Val Lys 580 - # 585 - # 590 - - Asp Lys Gly Ala Val Tyr Asp Tyr Thr Asn Le - #u Ile Gln His Ala His 595 - # 600 - # 605 - - Ile Ser Ser Ser Val Ala Arg Gly Glu Glu Ty - #r Arg Glu Val Arg Leu 610 - # 615 - # 620 - - Val Ser His Leu Gly Asn Gly Asn Asp Lys Va - #l Phe Leu Ala Ala Gly 625 6 - #30 6 - #35 6 -#40 - - Ser Ala Glu Ile His Ala Gly Glu Gly His As - #p Val Val Tyr TyrAsp 645 - # 650 - # 655 - - Lys Thr Asp Thr Gly Leu Leu Val Ile Asp Gl - #y Thr Lys Ala Thr Glu 660 - # 665 - # 670 - - Gln Gly Arg Tyr Ser Val Thr Arg Glu Leu Se - #r Gly Ala Thr Lys Ile 675 - # 680 - # 685 - - Leu Arg Glu Val Ile Lys Asn Gln Lys Tyr Al - #a Val Gly Lys Arg Glu 690 - # 695 - # 700 - - Glu Thr Leu Glu Tyr Arg Asp Tyr Glu Leu Th - #r Gln Ser Gly Asn Ser 705 7 - #10 7 - #15 7 -#20 - - Asn Leu Lys Ala His Asp Glu Leu His Ser Va - #l Glu Glu Ile GlySer 725 - # 730 - # 735 - - Asn Gln Arg Asp Glu Phe Lys Gly Ser Lys Ph - #e Arg Asp Ile Phe His 740 - # 745 - # 750 - - Gly Ala Asp Gly Asp Asp Leu Leu Asn Gly As - #n Asp Gly Asp Asp Ile 755 - # 760 - # 765 - - Leu Tyr Gly Asp Lys Gly Asn Asp Glu Leu Ar - #g Gly Asp Asn Gly Asn 770 - # 775 - # 780 - - Asp Gln Leu Tyr Gly Gly Glu Gly Asp Asp Ly - #s Leu Leu Gly Gly Asn 785 7 - #90 7 - #95 8 -#00 - - Gly Asn Asn Tyr Leu Ser Gly Gly Asp Gly As - #n Asp Glu Leu GlnVal 805 - # 810 - # 815 - - Leu Gly Asn Gly Phe Asn Val Leu Arg Gly Gl - #y Lys Gly Asp Asp Lys 820 - # 825 - # 830 - - Leu Tyr Gly Ser Ser Gly Ser Asp Leu Leu As - #p Gly Gly Glu Gly Asn 835 - # 840 - # 845 - - Asp Tyr Leu Glu Gly Gly Asp Gly Ser Asp Ph - #e Tyr Val Tyr Arg Ser 850 - # 855 - # 860 - - Thr Ser Gly Asn His Thr Ile Tyr Asp Gln Gl - #y Lys Ala Ser Asp Ser 865 8 - #70 8 - #75 8 -#80 - - Asp Lys Leu Tyr Leu Ser Asp Leu Ser Phe As - #p Asn Ile Leu ValLys 885 - # 890 - # 895 - - Arg Val Asn Asp Asn Leu Glu Phe Arg Ser As - #n Asn Asn Ser Asn Ser 900 - # 905 - # 910 - - Gly Val Leu Thr Ile Lys Asp Trp Phe Lys Gl - #y Gly Asn Ser Tyr Asn 915 - # 920 - # 925 - - His Lys Ile Glu Gln Ile Val Asp Lys Asn Gl - #y Arg Lys Leu Thr Ala 930 - # 935 - # 940 - - Gly Asn Leu Gly Asn Asn Phe His Asp Thr Gl - #n Gln Ala Ser Ser Leu 945 9 - #50 9 - #55 9 -#60 - - Leu Lys Asn Val Thr Gln Glu Gln Asn Glu Se - #r Asn Leu Ser SerLeu 965 - # 970 - # 975 - - Lys Thr Glu Leu Gly Lys Ile Ile Thr Asn Al - #a Gly Asn Phe Gly Val 980 - # 985 - # 990 - - Ala Lys Gln Gly Asn Thr Gly Ile Asn Thr Al - #a Ala Leu Asn Asn Glu 995 - # 1000 - # 1005 - - Val Asn Lys Ile Ile Ser Ser Ala Asn Thr Ph - #e Ala Thr Ser Gln Leu 1010 - # 1015 - # 1020 - - Gly Gly Ser Gly Met Gly Thr Leu Pro Ser Th - #r Asn Val Asn Ser Met 1025 1030 - # 1035 - # 1040 - - Met Leu Gly Asn Leu Ala Arg Ala Ala 1045 - - - - (2) INFORMATION FOR SEQ ID NO:12: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 711 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Actinobacill - #us pleuropneumoniae - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: - - Met Glu Ser Gln Met Pro Phe Asn Glu Lys Il - #e Asp Tyr Gly Leu His 1 5 - # 10 - # 15 - - Ala Leu Val Ile Leu Ala Gln Tyr His Asn Va - #l Ala Val Asn Pro Glu 20 - # 25 - # 30 - - Glu Val Lys His Lys Phe Asp Leu Asp Gly Ly - #s Gly Leu Asp Leu Val 35 - # 40 - # 45 - - Ala Trp Leu Leu Ala Ala Lys Ser Leu Glu Le - #u Lys Val Lys Arg Val 50 - # 55 - # 60 - - Lys Lys Ser Ile Glu Arg Leu Pro Phe Ile Hi - #s Leu Pro Ala Leu Ile 65 - #70 - #75 - #80 - - Trp Arg Asp Asp Gly Gln His Val Ile Leu Me - #t Lys Ile Asp Thr Gln 85 - # 90 - # 95 - - Thr Asn Arg Tyr Leu Ile Phe Asp Leu Glu Gl - #u Arg Asn Pro Lys Val 100 - # 105 - # 110 - - Leu Ser Ala Ala Glu Phe His Glu Ile Phe Gl - #n Gly Gly Met Ile Leu 115 - # 120 - # 125 - - Ile Thr Ser Arg Ala Ser Ile Met Gly Gln Le - #u Ala Lys Phe Asp Phe 130 - # 135 - # 140 - - Thr Trp Phe Ile Pro Ala Val Ile Lys Tyr Ar - #g Lys Ile Phe Val Glu 145 1 - #50 1 - #55 1 -#60 - - Thr Ile Ile Val Ser Ile Phe Leu Gln Leu Ph - #e Ala Leu Ile ThrPro 165 - # 170 - # 175 - - Leu Phe Phe Gln Val Val Met Asp Lys Val Le - #u Val His Arg Gly Phe 180 - # 185 - # 190 - - Ser Thr Leu Asn Val Ile Thr Val Ala Leu Se - #r Val Val Val Ile Phe 195 - # 200 - # 205 - - Glu Ile Val Leu Ser Gly Leu Arg Thr Tyr Il - #e Phe Ser His Ser Thr 210 - # 215 - # 220 - - Ser Arg Ile Asp Val Glu Leu Gly Ala Lys Le - #u Phe Arg His Leu Leu 225 2 - #30 2 - #35 2 -#40 - - Ala Leu Pro Ile Ser Tyr Phe Glu Asn Arg Ar - #g Val Gly Asp ThrVal 245 - # 250 - # 255 - - Ala Arg Val Arg Glu Leu Asp Gln Ile Arg As - #n Phe Leu Thr Gly Gln 260 - # 265 - # 270 - - Ala Leu Thr Ser Val Leu Asp Leu Leu Phe Se - #r Phe Ile Phe Phe Ala 275 - # 280 - # 285 - - Val Met Trp Tyr Tyr Ser Pro Lys Leu Thr Il - #e Val Ile Leu Leu Ser 290 - # 295 - # 300 - - Leu Pro Cys Tyr Ile Ala Trp Ser Ile Phe Il - #e Ser Pro Ile Leu Arg 305 3 - #10 3 - #15 3 -#20 - - Arg Arg Leu Asp Glu Lys Phe Ala Arg Asn Al - #a Asp Asn Gln SerPhe 325 - # 330 - # 335 - - Leu Val Glu Ser Val Ser Ala Ile Asp Thr Il - #e Lys Ala Leu Ala Val 340 - # 345 - # 350 - - Thr Pro Gln Met Thr Asn Ile Trp Asp Lys Gl - #n Leu Ala Ser Tyr Val 355 - # 360 - # 365 - - Ser Ala Asp Phe Arg Val Thr Val Leu Ala Th - #r Ile Gly Gln Gln Gly 370 - # 375 - # 380 - - Val Gln Leu Ile Gln Lys Thr Val Met Ile Il - #e Asn Leu Trp Leu Gly 385 3 - #90 3 - #95 4 -#00 - - Ala His Leu Val Ile Ser Gly Asp Leu Ser Il - #e Gly Gln Leu IleThr 405 - # 410 - # 415 - - Phe Asn Met Leu Ser Gly Gln Val Ile Ala Pr - #o Val Val Arg Leu Ala 420 - # 425 - # 430 - - Gln Leu Trp Gln Asp Phe Gln Gln Val Gly Il - #e Ser Ile Thr Arg Leu 435 - # 440 - # 445 - - Gly Asp Val Leu Asn Ser Pro Thr Glu Asn Ty - #r Gln Gly Lys Leu Ser 450 - # 455 - # 460 - - Leu Pro Glu Ile Phe Gly Asp Ile Ala Phe Ly - #s His Ile Arg Phe Arg 465 4 - #70 4 - #75 4 -#80 - - Tyr Lys Pro Asp Ala Pro Ile Ile Leu Asp As - #p Val Asn Leu SerVal 485 - # 490 - # 495 - - Lys Gln Gly Glu Val Ile Gly Ile Val Gly Ar - #g Ser Gly Ser Gly Lys 500 - # 505 - # 510 - - Ser Thr Leu Thr Lys Leu Leu Gln Arg Phe Ty - #r Ile Pro Glu Asn Gly 515 - # 520 - # 525 - - Gln Val Leu Ile Asp Gly His Asp Leu Ala Le - #u Ala Asp Pro Asn Trp 530 - # 535 - # 540 - - Leu Arg Arg Gln Ile Gly Val Val Leu Gln As - #p Asn Val Leu Leu Asn 545 5 - #50 5 - #55 5 -#60 - - Arg Ser Ile Arg Asp Asn Ile Ala Leu Thr As - #p Pro Ser Met SerMet 565 - # 570 - # 575 - - Glu Arg Val Ile Tyr Ala Ala Lys Leu Ala Gl - #y Ala His Asp Phe Ile 580 - # 585 - # 590 - - Ser Glu Leu Arg Glu Gly Tyr Asn Thr Ile Va - #l Gly Glu Leu Gly Ala 595 - # 600 - # 605 - - Gly Leu Ser Gly Gly Gln Arg Gln Arg Ile Al - #a Ile Ala Arg Ala Leu 610 - # 615 - # 620 - - Val Asn Asn Pro Arg Ile Leu Ile Phe Asp Gl - #u Ala Thr Ser Ala Leu 625 6 - #30 6 - #35 6 -#40 - - Asp Tyr Glu Ser Glu His Ile Ile Met Gln As - #n Met Gln Lys IleCys 645 - # 650 - # 655 - - His Gly Arg Thr Val Ile Ile Ile Ala His Ar - #g Leu Ser Thr Val Lys 660 - # 665 - # 670 - - Asn Ala Asp Arg Ile Ile Val Met Glu Lys Gl - #y His Ile Val Glu Gln 675 - # 680 - # 685 - - Gly Lys His Asn Gln Leu Leu Glu Asn Glu As - #n Gly Leu Tyr Tyr Tyr 690 - # 695 - # 700 - - Leu Asn Gln Leu Gln Ser Asn 705 7 - #10 - - - - (2) INFORMATION FOR SEQ ID NO:13: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 477 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Actinobacill - #us pleuropneumoniae - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: - - Met Lys Leu Trp Ile Leu Gly Leu Gly Glu Ph - #e Phe Gln Arg Tyr Arg 1 5 - # 10 - # 15 - - Asn Ile Trp Arg Glu Ile Trp Lys Ile Arg Ly - #s Gln Leu Asp Thr Pro 20 - # 25 - # 30 - - Ala Arg Gln Lys Asp Glu Asn Glu Phe Leu Pr - #o Arg His Leu Glu Leu 35 - # 40 - # 45 - - Ile Glu Thr Pro Ile Ser Lys Lys Pro Arg Le - #u Ile Ala Tyr Leu Ile 50 - # 55 - # 60 - - Met Leu Phe Leu Phe Leu Ala Ile Val Ile Se - #r Ile Ile Ser Lys Val 65 - #70 - #75 - #80 - - Glu Ile Val Ala Ser Ala Thr Gly Lys Leu Va - #l Phe Ser Gly His Ser 85 - # 90 - # 95 - - Lys Glu Ile Lys Pro Ile Glu Asn Ala Leu Va - #l Lys Asp Ile Phe Val 100 - # 105 - # 110 - - Lys Asp Gly Gln Phe Val Glu Lys Gly Gln Le - #u Leu Leu Asn Leu Thr 115 - # 120 - # 125 - - Ala Leu Gly Cys Asp Ala Asp Lys Gln Lys Th - #r Lys Val Ser Leu Gly 130 - # 135 - # 140 - - Leu Glu Arg Leu Asp Gly Tyr Arg Tyr Lys Se - #r Leu Leu Tyr Ser Ile 145 1 - #50 1 - #55 1 -#60 - - Glu His Asn Arg Leu Pro Leu Leu Asp Phe As - #n Gln Ala Asp PheAsp 165 - # 170 - # 175 - - Ser Val Gln Glu Glu Asp Lys Thr Gly Ala Ar - #g His Leu Ile Thr Glu 180 - # 185 - # 190 - - Gln Phe Glu Thr Trp Gln Lys Gln Lys Tyr Gl - #n Lys Glu Leu Ala Tyr 195 - # 200 - # 205 - - Gln Arg Lys Gln Ala Glu Lys Gln Thr Val Le - #u Ala Asn Ile Arg Lys 210 - # 215 - # 220 - - Tyr Glu Ser Ala Ser Arg Ile Glu Lys Glu Ly - #s Leu Ser Asp Leu Lys 225 2 - #30 2 - #35 2 -#40 - - Lys Leu Tyr Asp Val Lys Ser Ile Ser Lys Hi - #s Glu Leu Leu AlaGln 245 - # 250 - # 255 - - Glu Asn Arg Tyr Val Glu Ala Ser Asn Glu Le - #u Ser Val Tyr Gln Ser 260 - # 265 - # 270 - - His Leu Lys Glu Val Glu Ser Asp Leu Leu Ly - #s Ala Gln Glu Asp Leu 275 - # 280 - # 285 - - Lys Leu Val Thr Gln Leu Phe Lys Ser Asp Il - #e Leu Glu Lys Leu Gln 290 - # 295 - # 300 - - Gln Asn Ile Gln Arg Glu Lys Gln Leu Thr Le - #u Glu Leu Glu Lys Asn 305 3 - #10 3 - #15 3 -#20 - - Glu Gln Arg Gln Leu Ala Ser Ile Ile Arg Al - #a Pro Val Ser GlyThr 325 - # 330 - # 335 - - Val Gln Gln Leu Lys Thr His Thr Lys Gly Gl - #y Val Val Thr Thr Ala 340 - # 345 - # 350 - - Glu Thr Leu Met Val Ile Ala Pro Glu Asp As - #p Val Leu Glu Val Ser 355 - # 360 - # 365 - - Ala Leu Ile Gln Asn Lys Asp Val Gly Phe Va - #l Glu Ile Gly Gln Glu 370 - # 375 - # 380 - - Ala Val Ile Lys Val Glu Thr Phe Pro Tyr Th - #r Arg Tyr Gly Tyr Leu 385 3 - #90 3 - #95 4 -#00 - - Tyr Gly Lys Val Lys Thr Ile Thr Leu Asp Al - #a Ile Glu His ProGln 405 - # 410 - # 415 - - Leu Gly Leu Val Phe Asn Ser Ile Ile Glu Il - #e Asn Lys Lys Thr Leu 420 - # 425 - # 430 - - Thr Asp Gly Asp Lys Glu Ile Gln Leu Gly Se - #r Gly Met Ser Val Ile 435 - # 440 - # 445 - - Ala Glu Ile Lys Thr Gly Glu Arg Ser Val Il - #e Ser Phe Leu Leu Ser 450 - # 455 - # 460 - - Pro Leu Glu Glu Ser Ile Thr Glu Ser Leu Ar - #g Glu Arg 465 4 - #70 4 - #75 - - - - (2) INFORMATION FOR SEQ ID NO:14: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7721 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Actinobacill - #us pleuropneumoniae - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: - - TCTTGAATAA TAGTTGGTTT TGTAGATATT CTTTTAATAT CAAACAACTA TT -#GTTATTTG 60 - - TCTGAGTGTA GATATGTAGC ATTGTGTATT TCTTTATTTA CAACTCTAAT CT -#TAATCTAA 120 - - AAAGATTTCT ATATTTTCTT TGTAAGAAAT TTTGTTAAAA TCCGACTAAC TA -#TATAATTA 180 - - ACGGTTCTTA AAGTGGATAA ATAATAAAAT TATGAGTTAT AAAAATGTTA AA -#AATTTAAC 240 - - AGATGATTTT ACAACTTTAG GGCATATCGC TTGGTTGTGG GCTAATTCTC CG -#TTACATAA 300 - - GGAGTGGTCT ATCTCTTTGT TTACTAAGAA TATTTTGCCA GCCATTCAAC AT -#GATCAATA 360 - - TATTTTACTT ATGCGAGATG AGTTCCCTGT AGCGTTTTGT AGTTGGGCAA AT -#TTAACGTT 420 - - AACTAATGAA GTGAAGTATG TACGTGATGT GACGTCATTG ACTTTTGAAG AT -#TGGAATTC 480 - - AGGAGAACGA AAATGGTTGA TCGATTGGAT TGCGCCATTT GGGGATAACA AT -#ACGCTTTA 540 - - TAGATATATG CGTAAAAAAT TTCCTAATGA AGTATTCCGG GCCATTCGAG TA -#TATCCTGG 600 - - TTCTACAGAA GCGAAAATCA TTCATGTTCA AGGAGGACAA ATTAATAAAT TT -#ACAGCTAA 660 - - AAAATTAATA CAACAATATC AGGAAGAACT TATTCAAGTT CTTAACAATC AC -#AAAAAAAT 720 - - TGTAAGAGGA TAAAATATGA GTACTTGGTC AAGCATGTTA GCCGACTTAA AA -#AAAAGGGC 780 - - TGAAGAAGCC AAAAGACAAG TTAAAAAAGG CTACGATGTA ACTAAAAATG GT -#TTGCAATA 840 - - TGGGGTGAGT CAAGCAAAAT TACAAGCATT AGCAGCTGGT AAAGCCGTTC AA -#AAGTACGG 900 - - TAATAAATTA GTTTTAGTTA TTCCAAAAGA GTATGACGGA AGTGTTGGTA AC -#GGTTTCTT 960 - - TGATTTAGTA AAAGCAGCTG AGGAATTAGG CATTCAAGTT AAATATGTTA AC -#CGTAATGA 1020 - - ATTGGAAGTT GCCCATAAAA GTTTAGGTAC CGCAGACCAA TTCTTGGGTT TA -#ACAGAACG 1080 - - TGGACTTACT TTATTTGCAC CGCAACTAGA TCAGTTCTTA CAAAAACATT CA -#AAAATTTC 1140 - - TAACGTAGTG GGCAGTTCTA CTGGTGATGC AGTAAGTAAA CTTGCTAAGA GT -#CAAACTAT 1200 - - TATTTCAGGA ATTCAATCTG TATTAGGTAC TGTATTAGCA GGTATTAATC TT -#AATGAAGC 1260 - - TATTATTAGT GGCGGTTCAG AGCTCGAATT AGCTGAAGCT GGTGTTTCTT TA -#GCCTCTGA 1320 - - GCTCGTTAGC AATATTGCTA AAGGTACAAC AACAATAGAT GCTTTCACTA CA -#CAAATCCA 1380 - - GAACTTTGGG AAATTAGCGG AAAATGCTAA AGGGTTAGGT GGTGTTGGCC GC -#CAATTACA 1440 - - GAATATTTCA GGTTCTGCAT TAAGCAAAAC TGGATTAGGT TTGGATATTA TC -#TCAAGCTT 1500 - - ACTTTCAGGA GTAACTCGAA GTTTTGCTTT ACGGAATAAG AATGCTTCAA CA -#AGCACTAA 1560 - - AGTTGCTGCT GGCTTTGAAC TCTCAAATCA AGTAATTGGT GGTATTACGA AA -#GCAGTATC 1620 - - AAGCTATATT CTTGCACAGC GTTTACGTGC TGGTTTATCA ACGACAGGTC CT -#GCTGCAGC 1680 - - ACTAATTGCG TCTAGTATTT CTTTAGCAAT CAGTCCATTG GCGTTTTTAC GT -#GTAGCTGA 1740 - - TAATTTTAAT CGTTCTAAAG AAATTGGCGA ATTTGCTGAA CGTTTCAAAA AA -#TTGGGCTA 1800 - - TGACGGCGAT AAACTACTTT CAGAGTTTTA TCACGAAGCT GGTACTATTG AT -#GCCTCAAT 1860 - - TACTACAATT AGTACAGCAC TTTCTGCTAT CGCAGCTGGA ACGGCCGCCG CG -#AGTGCAGG 1920 - - TGCATTAGTT GGCGCACCAA TTACTTTGTT GGTTACTGGT ATCACAGGAT TA -#ATTTCTGG 1980 - - TATTTTAGAG TTCTCTAAAC AACCAATGTT AGATCATGTT GCATCGAAAA TT -#GGTAACAA 2040 - - AATTGACGAA TGGGAGAAAA AATACGGTAA AAATTACTTC GAGAATGGCT AT -#GATGCTCG 2100 - - TCATAAAGCT TTCTTAGAAG ATTCATTCTC ATTATTGTCT AGTTTTAATA AA -#CAATATGA 2160 - - AACTGAAAGA GCTGTTTTAA TTACACAACA ACGTTGGGAT GAATATATTG GC -#GAACTTGC 2220 - - GGGTATTACT GGTAAAGGTG ACAAACTCTC TAGTGGTAAG GCGTATGTAG AT -#TACTTTCA 2280 - - AGAAGGTAAA TTATTAGAGA AAAAACCTGA TGACTTTAGC AAAGTAGTTT TC -#GATCCAAC 2340 - - TAAGGGCGAA ATTGATATTT CAAATAGCCA AACGTCAACG TTGTTAAAAT TT -#GTTACGCC 2400 - - ATTATTAACA CCAGGTACAG AGTCACGTGA AAGAACTCAA ACAGGTAAAT AT -#GAATATAT 2460 - - CACGAAGTTA GTTGTAAAAG GTAAAGATAA ATGGGTTGTT AATGGCGTTA AA -#GATAAAGG 2520 - - TGCCGTTTAT GATTATACTA ATTTAATTCA ACATGCTCAT ATTAGTTCAT CA -#GTAGCACG 2580 - - TGGTGAAGAA TACCGTGAAG TTCGTTTGGT ATCTCATCTA GGCAATGGTA AT -#GACAAAGT 2640 - - GTTCTTAGCT GCGGGTTCCG CAGAAATTCA CGCTGGTGAA GGTCATGATG TG -#GTTTATTA 2700 - - TGATAAAACC GATACAGGTC TTTTAGTAAT TGATGGAACC AAAGCGACTG AA -#CAAGGGCG 2760 - - TTATTCTGTT ACGCGCGAAT TGAGTGGTGC TACAAAAATC CTGAGAGAAG TA -#ATAAAAAA 2820 - - TCAAAAATAT GCTGTTGGTA AACGTGAAGA AACCTTGGAA TATCGTGATT AT -#GAATTAAC 2880 - - GCAATCAGGT AATAGTAACC TAAAAGCACA TGATGAATTA CATTCAGTAG AA -#GAAATTGG 2940 - - AAGTAATCAG AGAGACGAAT TTAAAGGTAG TAAATTCAGA GATATTTTCC AT -#GGTGCCGA 3000 - - TGGTGATGAT CTATTAAATG GTAATGATGG GGATGATATT CTATACGGTG AT -#AAAGGTAA 3060 - - CGATGAGTTA AGAGGTGATA ACGGTAACGA CCAACTTTAT GGTGGTGAAG GT -#GATGACAA 3120 - - ACTATTAGGA GGTAATGGCA ATAATTACCT CAGTGGTGGT GATGGCAATG AT -#GAGCTTCA 3180 - - AGTATTAGGC AATGGTTTTA ATGTGCTTCG TGGCGGTAAA GGCGATGATA AA -#CTTTATGG 3240 - - TAGCTCAGGT TCTGATTTAC TTGATGGTGG AGAAGGTAAT GATTATCTAG AA -#GGAGGCGA 3300 - - TGGTAGCGAT TTTTATGTTT ATCGTTCCAC TTCAGGTAAT CATACTATTT AT -#GATCAAGG 3360 - - TAAAGCTAGC GATTCAGATA AGCTATATTT GTCAGATCTT TCTTTTGATA AT -#ATTTTAGT 3420 - - TAAAAGGGTT AACGATAACC TTGAGTTTAG AAGCAATAAT AACAGTAATA GT -#GGTGTGCT 3480 - - TACGATCAAG GACTGGTTCA AAGGCGGCAA TAGTTACAAT CATAAAATTG AA -#CAAATTGT 3540 - - TGATAAAAAT GGTAGAAAAT TGACAGCTGG GAATTTAGGA AATAACTTCC AT -#GATACTCA 3600 - - ACAAGCTAGT AGTTTACTTA AAAATGTTAC ACAAGAACAA AATGAAAGCA AT -#TTATCTTC 3660 - - ACTTAAAACT GAATTAGGTA AAATTATTAC TAATGCAGGT AATTTTGGTG TG -#GCAAAACA 3720 - - AGGTAATACT GGAATCAATA CAGCTGCCTT GAACAATGAA GTGAATAAAA TC -#ATTTCTTC 3780 - - TGCTAATACC TTTGCTACTT CACAATTGGG TGGCTCAGGG ATGGGAACAT TA -#CCATCAAC 3840 - - GAATGTAAAT TCAATGATGC TAGGTAACCT AGCTAGAGCA GCTTAATCAT CT -#GCAATAAT 3900 - - CAATAGCAAT CCTATGGTTA TTCTAGGATT GCTATTTTAT TTATGGAGTC AC -#AAATGCCT 3960 - - TTTAACGAAA AAATAGATTA CGGATTACAT GCATTGGTAA TTCTCGCGCA AT -#ATCACAAT 4020 - - GTTGCCGTAA ACCCTGAAGA GGTAAAACAT AAATTTGATC TTGATGGCAA AG -#GATTGGAT 4080 - - CTTGTTGCTT GGTTATTAGC AGCAAAATCA TTAGAATTAA AAGTCAAACG AG -#TAAAAAAG 4140 - - AGTATTGAGC GTTTACCATT TATTCATCTT CCTGCTTTAA TCTGGCGAGA TG -#ATGGTCAA 4200 - - CACGTTATTT TGATGAAAAT TGACACCCAA ACTAACCGTT ACCTTATTTT TG -#ACTTAGAA 4260 - - GAACGAAACC CTAAAGTACT AAGTGCGGCT GAATTTCACG AAATTTTTCA AG -#GTGGTATG 4320 - - ATTCTTATTA CTTCACGAGC TTCTATTATG GGGCAATTGG CGAAGTTTGA TT -#TCACTTGG 4380 - - TTTATCCCCG CAGTAATTAA ATACCGTAAA ATTTTTGTAG AAACTATTAT TG -#TTTCTATT 4440 - - TTTTTGCAGC TTTTTGCACT AATTACTCCC TTATTTTTCC AAGTTGTGAT GG -#ATAAAGTT 4500 - - CTTGTCCATC GTGGATTTTC TACACTTAAT GTTATCACGG TTGCATTATC TG -#TAGTGGTT 4560 - - ATCTTTGAAA TTGTATTAAG CGGTCTACGG ACTTATATAT TTTCCCATAG CA -#CTAGCCGA 4620 - - ATTGATGTAG AACTTGGTGC AAAATTATTT CGTCACTTGT TAGCGTTACC TA -#TTTCTTAT 4680 - - TTCGAAAATA GACGTGTAGG TGACACAGTT GCTCGAGTAC GAGAATTGGA TC -#AAATACGC 4740 - - AATTTTTTAA CAGGTCAGGC ACTTACCTCT GTATTAGATC TCTTATTCTC TT -#TTATTTTC 4800 - - TTTGCAGTGA TGTGGTATTA CAGCCCAAAA CTAACTATTG TGATTTTACT TT -#CATTACCT 4860 - - TGTTATATCG CATGGTCAAT ATTTATTAGC CCAATATTAC GTCGTCGTCT AG -#ATGAAAAA 4920 - - TTTGCTCGTA ATGCTGATAA TCAATCTTTT TTAGTTGAAT CTGTTTCTGC AA -#TAGACACG 4980 - - ATCAAGGCTC TTGCTGTAAC ACCTCAAATG ACAAATATTT GGGATAAACA GT -#TAGCAAGT 5040 - - TATGTATCAG CAGATTTTAG AGTGACAGTA TTGGCAACTA TTGGACAGCA AG -#GTGTACAA 5100 - - CTTATCCAAA AAACAGTAAT GATAATTAAT TTATGGTTAG GTGCACATTT AG -#TAATTTCA 5160 - - GGGGATCTTA GCATTGGACA ATTAATTACT TTTAATATGC TTTCAGGACA AG -#TTATTGCA 5220 - - CCTGTAGTTC GTTTAGCACA ATTGTGGCAA GACTTTCAAC AAGTAGGAAT TT -#CTATTACA 5280 - - CGATTGGGAG ATGTCTTAAA TTCACCTACA GAAAATTATC AAGGTAAGCT TT -#CACTACCA 5340 - - GAAATCTTTG GGGATATCGC ATTTAAACAT ATTCGCTTTC GCTATAAGCC CG -#ATGCTCCA 5400 - - ATCATTTTAG ATGATGTAAA TTTATCGGTT AAACAGGGGG AAGTTATTGG GA -#TAGTAGGA 5460 - - CGTTCAGGTT CAGGTAAAAG TACTCTCACT AAATTATTAC AACGTTTTTA TA -#TTCCGGAA 5520 - - AATGGCCAAG TATTGATTGA TGGTCACGAT CTTGCGCTTG CTGATCCTAA TT -#GGTTACGT 5580 - - CGTCAAATTG GTGTTGTTTT ACAAGATAAT GTGTTATTAA ACCGTAGTAT TC -#GCGATAAT 5640 - - ATCGCACTCA CTGATCCAAG CATGTCTATG GAACGTGTTA TCTATGCGGC AA -#AATTAGCA 5700 - - GGGGCACATG ATTTTATTTC TGAATTACGT GAAGGTTACA ATACTATTGT AG -#GAGAGCTT 5760 - - GGTGCAGGCT TATCTGGTGG ACAACGTCAA CGGATTGCTA TTGCACGAGC TT -#TAGTCAAT 5820 - - AACCCTAGGA TTTTGATTTT TGATGAGGCG ACAAGTGCAT TAGATTATGA AT -#CTGAACAT 5880 - - ATCATTATGC AAAATATGCA AAAAATCTGC CATGGACGGA CAGTAATCAT TA -#TTGCCCAC 5940 - - CGTCTTTCTA CAGTAAAAAA TGCGGATCGC ATTATTGTTA TGGAAAAGGG AC -#ATATTGTA 6000 - - GAGCAAGGTA AACATAACCA ATTACTGGAA AATGAAAATG GACTCTATTA TT -#ACCTCAAC 6060 - - CAACTACAAT CAAATTAAGG TGAAACAACA TGAAGTTATG GATTCTAGGA CT -#TGGGGAAT 6120 - - TTTTTCAACG TTATCGTAAT ATTTGGCGTG AAATATGGAA AATCCGCAAA CA -#ATTAGATA 6180 - - CCCCAGCAAG ACAAAAAGAT GAAAACGAAT TTTTGCCTCG GCATTTAGAG TT -#AATTGAGA 6240 - - CACCTATTTC AAAAAAGCCA CGGCTGATCG CTTATTTGAT AATGCTATTT CT -#ATTTTTAG 6300 - - CTATTGTAAT TTCCATTATT AGTAAAGTAG AAATTGTTGC TAGTGCTACA GG -#TAAGTTGG 6360 - - TATTTAGTGG ACATAGTAAA GAAATAAAGC CTATTGAGAA TGCTTTAGTA AA -#AGACATTT 6420 - - TTGTTAAAGA TGGACAATTT GTTGAAAAAG GACAATTATT ATTAAATCTC AC -#CGCACTTG 6480 - - GCTGCGATGC AGACAAACAA AAAACTAAAG TATCGTTAGG ATTGGAAAGA TT -#AGATGGTT 6540 - - ACCGATATAA GTCATTGTTA TATAGCATTG AACACAATAG ATTACCTTTA TT -#GGATTTTA 6600 - - ACCAAGCTGA TTTTGATTCT GTTCAGGAAG AAGATAAGAC TGGCGCACGT CA -#TTTAATTA 6660 - - CCGAACAATT TGAGACTTGG CAAAAACAAA AATATCAGAA GGAATTAGCG TA -#TCAACGTA 6720 - - AACAAGCTGA AAAACAAACA GTATTAGCAA ATATCCGTAA ATATGAAAGC GC -#TAGTCGTA 6780 - - TTGAAAAGGA GAAATTAAGT GATTTAAAAA AATTATATGA TGTAAAGTCT AT -#TTCTAAGC 6840 - - ATGAGTTGTT AGCACAAGAA AATAGATATG TTGAAGCTAG TAATGAATTG TC -#TGTTTATC 6900 - - AATCTCATCT CAAAGAAGTA GAAAGTGACT TGCTTAAAGC ACAAGAAGAT TT -#AAAGCTTG 6960 - - TTACTCAATT ATTTAAGAGT GATATTTTGG AAAAACTACA GCAAAATATA CA -#ACGCGAAA 7020 - - AGCAGCTCAC TTTAGAACTT GAGAAAAATG AACAACGTCA ATTAGCCTCT AT -#CATTAGGG 7080 - - CGCCAGTATC AGGCACAGTC CAACAATTAA AAACTCATAC TAAAGGTGGC GT -#AGTAACTA 7140 - - CTGCAGAAAC CTTAATGGTC ATTGCTCCTG AGGATGACGT GTTGGAAGTA AG -#TGCTTTAA 7200 - - TTCAAAACAA AGATGTTGGT TTTGTTGAAA TTGGACAGGA AGCAGTTATT AA -#AGTGGAAA 7260 - - CTTTTCCCTA CACAAGATAT GGTTATCTCT ATGGAAAAGT AAAAACTATT AC -#TCTTGATG 7320 - - CTATTGAGCA CCCTCAGCTT GGTTTAGTTT TCAATTCTAT TATTGAGATT AA -#CAAGAAAA 7380 - - CATTAACAGA TGGTGATAAA GAAATTCAAT TAGGTTCTGG AATGAGCGTT AT -#TGCAGAAA 7440 - - TTAAAACAGG AGAACGCAGT GTTATCAGTT TCCTACTCAG TCCATTAGAA GA -#ATCTATTA 7500 - - CTGAAAGTCT AAGAGAACGT TAATTATCTC TTCTAAATTA AGCAAATATA TA -#ACTTTTGT 7560 - - AAAAACGTTA TTTAAGGAGA GTTGCTAATA GAAGTTAAAA TATCTATTAG CA -#ACTATATT 7620 - - ATCTCTTTGA GCTATTTTTA GCTTCTTTAG AAGTTAGAGA TTTTTAGATA TT -#CATAATAT 7680 - - ATGAAACTAT TTGCTGATCT AATTTAAAAC TAAAATCTAG A - # - # 7721__________________________________________________________________________

Number	Date	Country
1189790		CAX
2040544		CAX
2045950		CAX
A-420743		EPX
WO9308283		WOX
WO9321951		WOX

Bacterial preparations, method for producing same, and their use as vaccines

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