Microbial Pathogenesis 1991 ; 11 : 305-316

Mini-review Virulence properties of Actinobacillus pleuropneumoniae Thomas J . Inzana Veterinary Microbiology Research Laboratories, Department of Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, U .S .A .

Introduction Actinobaci//us (Haemophilus) pleuropneumoniae is the etiologic agent of pleuropneumonia in swine. This bacterium is a fermentative, hemolytic, Gram-negative coccobacillus . Two biotypes are recognized : biotype 1 requires nicotinamide adenine dinucleotide (NAD or V factor) for growth, and biotype 2 is NAD- independent' Organisms currently recognized as A . pleuropneumoniae were previously classified as Haemophilus pleuropneumoniae (biotype 1), or Pasteurella haemolytica-like (biotype 2) . The agent was reclassified from these two species into the genus Actinobacillus on the basis of DNA-DNA homology and other biochemical characteristics .' At least 12 serotypes of biotype 1 and 2 serotypes of biotype 2 are currently recognized worldwide , 2 '3 but only a few serotypes predominate in any given geographic location . This organism was first described from cases of swine pleuropneumonia in 1957, ° and its prevalence has since been rising .' The onset of clinical disease is manifested by severe respiratory distress, e •' leading to acute death in 24 to 48 h, or a chronic persistent infection . The lung lesions are predominately necrotic and hemorrhagic, with fibrinous adhesions .'-10 The severity of disease depends upon the immune status of the herd . Actinobacillus pleuropneumoniae is highly contagious and may spread rapidly in a non-immune herd, often as a result of the introduction of an immune carrier animal . Commercially available, killed vaccines provide some protection, but are clearly inadequate . 9-11 An extensive review of pleuropneumonia and its etiologic agent was published by Sebunya and Saunders in 1983 ." Since then information on the taxonomic classification, antigenic properties, and virulence factors of A . pleuropneumoniae has undergone extensive growth and revision . This article is not intended to examine all the literature on A . pleuropneumoniae (i .e . diagnosis, vaccines, treatment, etc .) . Rather, current knowledge of the virulence factors that enable the organism to survive in vivo, that are thought to be responsible for clinical disease, and the immunoprotective properties of these factors will be reviewed .

Extracellular toxins Cells and culture supernatant from A . pleuropneumoniae are toxic to red blood cells, lung macrophages, lymphocytes, and various other cells . 12-19 Strains have been 0882-4010/91/110305+12 $03 .00/0

© 1991 Academic Press Limited



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identified that are cytotoxic, but not hemolytic by hemolysin assay ." , " Four distinct types of cytotoxic activity, 15 and three distinct types of hemolytic activity 12 have been identified . Most, if not all, of the hemolytic and cytotoxic activity in A . p/europneumoniae culture supernatant is highly labile, is inactivated by heat, formalin, and proteolytic enzymes, and is produced predominately in mid-log phase ." - " The extracellular hemolytic activity is thought to be responsible for the hemorrhagic and necrotic lesions characteristic of pleuropneumonia . 20 The mechanism of action of at least one hemolysin purified by Lalonde et al .," is by formation of pores in red blood cells and phospholipid membranes . The strongest hemolytic activity is due to a Ca t '-inducible hemolysin that has been purified from serotype 1 strain 4074, and identified as a 105 kDa, labile protein referred to as Hlyl .22,23 Hlyl activity has been identified in serotypes 1, 5a, 5b, and 9-11 . 12 Recently, Hlyl has been shown to be capable of binding 45 Ca 2+ , probably due to the glycine-rich domain present in this toxin (see below), and may indicate that Ca t ' is also required for this toxin's activity . 23 a A second, weaker type of hemolytic activity has been identified in serotypes 2 and 4-11 that requires calcium for activity, but not for induction and is referred to as Hlyll . A third type of hemolytic activity is present in serotypes 3 and 12, but is too weak to characterize . 12 Hlyl is strongly immunogenic, and is a predominant antigen recognized by convalescent field sera or sera from experimentally infected pigs . Antibodies to Hlyl strongly cross-react with a similar molecular mass protein from all 12 serotypes, 12 and from A . rossii and A . suis by immunoblotting, but do not cross-react with Escherichia co/i a-hemolysin or P. haemo/ytica leukotoxin . 24 However, anti-Hlyl only neutralizes the hemolytic activity of other serotypes that produce Ca t+ -inducible hemolysins (serotypes 1, 5a, 5b, 9-11 ) . 12 In contrast, Devenish et a/. 25 reported that polyclonal and monoclonal antibodies (mAb) to a 104 kDa hemolysin of serotype 1 recognize a similar molecular mass protein in immunoblots from serotypes 1-12, and also to taxon 'Minor group', P. haemo/ytica, and a-hemolysin producing E. coil; only polyclonal antibodies reacted with a protein from A . suis . Cross-reactivity also occurs when hemolysin from heterologous A . pleuropneumoniae serotypes and P. haemolytica leukotoxin is reacted with mAb and polyclonal antibodies to a 110 kDa hemolysin of serotype 5 . 26 Therefore, each type of hemolytic activity is apparently due to protein(s) that are antigenically related and of similar size . Neutralization studies and reactivity of antibody with hemolysin varies among the different serotypes and species, however, suggesting that the proteins are similar, but not identical .' 2,15,24,25 Thus, studies that have focused on measuring hemolytic and cytotoxic activity or immunological cross-reactivity have led to confusion due to the numerous serotypes and multiple toxins present in this organism . Purification, gene cloning, and sequence analysis of these hemolysins and their genes have helped to clarify the identity of these toxic activities . The hemolysins of A . pleuropneumoniae are members of the RTX (repeat in toxin) family of hemolysins/cytotoxins, which include E. co/i a-hemolysin, P . haemo/ytica leukotoxin, and many others . The E. coil a-hemolysin is coded for by an 8 kb cluster of four genes required for synthesis, activation, and export of the hemolysin . The structural gene, h/yA, is downstream from the gene required for activation (h/yC) of the hemolysin, and is upstream from two genes required for export, h/yB and h/yD . Export of the a-hemolysin requires a 53-amino acid, glycine-rich carboxyl terminus that is coded for by hlyA . 27 HIyl of serotype 1 has been purified, the p/ has been estimated to be 4 .3, 13 and the structural gene (h/y/A) has been cloned . 28 Biosynthesis of active Hlyl from the cloned h/y/A gene in E. coil requires complementation in trans with plasmids containing the E. co/i h/yC gene for activation of the hemolysin from the precursor prohemolysin . Secretion of Hlyl produced in E. coli requires the E. coli



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x-hemolysin secretion genes (hlyB and hlyD) . 28 The structure of the protein derived from the DNA sequence analysis of h/ylA indicates the presence of three hydrophobic regions in the N-terminal region, 13 glycine-rich domains in the second half of the protein, and a hydrophilic C-terminal region, confirming that Hlyl is related to E. co/i x-hemolysin . 23a The Hlyll hemolysin of serotype 2, which does not produce Hlyl, 12 is a 105 kDa protein that requires Ca 2' as a co-factor for hemolytic activity . Antibodies to Hlyll, prepared by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), neutralize the extracellular hemolytic activity of culture supernatant . Besides being of similar molecular mass, Hlyll also cross-reacts immunologically with Hlyl . This explains why antibodies to Hlyl react with a protein of similar molecular mass from all 12 serotypes . However, DNA : :DNA hybridization by Southern blotting with h/y/A indicates that hly//A codes for a different protein ; there is at least 30% heterology between hly/A and hlyl/A . 29 A 110 kDa hemolysin has been purified from a serotype 5 strain by mAb-affinity chromatography, 30 and the p/ of this hemolysin is 5 .3 . 31 The structural and activation genes (appCA) for a serotype 5 110 kDa hemolysin have also been cloned and sequenced by Chang et al. 32 using a DNA probe containing the P. haemolytica lktCA (leukotoxin) genes . The appC and appA genes are 66 .2 and 78 .2% homologous with the P. haemolytica lktC and /ktA genes, respectively, based on the predicted amino acid sequence . In contrast, sequence analysis of the hly/A gene from serotype 1 shows only 42% homology with the hemolysin structural gene of serotype 5, 41 % homology with the P. haemolytica leukotoxin gene, and 56% homology with the E. co/i xhemolysin gene . 23 a The hemolysin of serotype 5, therefore, is more closely related to the P. haemolytica leukotoxin than the E. co/i x-hemolysin, while the reverse is true of Hlyl of serotype 1 . Recently, the genes encoding the serotype 1, Ca"-inducible hemolysin, 28 and the genes encoding the serotype 5 hemolysin cloned by Chang et al .,32 have been found to be distinct showing no cross-hybridization at high stringency (M . Sirois and R . Levesque, personal communication) . The genes required for export of the serotype 5 hemolysin (appBD) have recently been cloned and sequenced by Chang et al ." Of interest is that immediately downstream from the appA gene are remnants of RTX B and A pseudogenes, and the functional appBD genes are located at least 8 kb from the appCA genes . Furthermore, while the appA gene is most closely related to the P . haemolytica leukotoxin gene, the appAx pseudogene is more closely related to the E. co/i h/yA gene . 33 Smits et al. 34 have used a P. haemolytica leukotoxin gene and a hemolysin-neutralizing mAb, presumably directed to Hlyll (see below), as a probe to clone a hemolysin gene that codes for a 103 kDa protein of serotype 9 . The DNA sequence of the cloned hemolysin gene of serotype 9 was almost identical to the appCA genes of serotype 5 cloned by Chang et a/ . 32 These results would suggest that the gene cloned from serotype 5 may be Hlyll, and that some serotypes or strains of A . pleuropneumoniae produce two RTX hemolysins . There is still no direct evidence establishing the role of hemolysins in the pathogenesis of pleuropneumonia . However, Inzana eta/. 31 have recently isolated several chemicallyinduced mutants of A . pleuropneumoniae serotype 5 strain J45 that lack extracellular hemolytic or cytotoxic activity . Detailed analysis of phenotypic properties indicated that at least one of the mutants lacks an extracellular, Ca 2+ -inducible, 110 kDa hemolysin, but contains small amounts of active intracellular hemolysin . Other phenotypic properties of the mutant [i .e . lipopolysaccharide (LPS) and protein electrophoretic profiles, capsule content, and growth rate] are identical to those of the parent . Furthermore, several of the mutants are incapable of causing disease or



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lesions in the lungs of swine or mice challenged with 10 times the lethal dose of the parent . Mice or swine immunized with the non-hemolytic mutant are not protected against lethal challenge with the parent, even though antibodies to somatic components (but not neutralizing hemolytic antibodies) are present . In other studies, Fedorka-Cray et a/ . 35 reported that vaccination of pigs with a serotype 1 cell extract, consisting predominately of a 110 kDa protein, induces high neutralizing hemolytic activity and confers greater protection against challenge with the homologous serotype than a commercial bacterin or outer membrane protein vaccine . Three adjuvant-supplemented subcutaneous immunizations and one intravenous immunization with a 104 kDa protein purified from serotype 1 by preparative SDS-PAGE, prevent mortality and reduce lung lesions in pigs challenged with the homologous serotype . 36 These results indicate that one or more hemolysins are required for A . pleuropneumoniae to cause disease, and that antibodies to hemolysin(s) substantially contribute to protection . A 104 kDa protein has been described from serotype 1 that is hemolytic for bovine red blood cells, and cytotoxic for porcine and bovine endothelial cells . Cytotoxicity of bacteria-free culture supernatant can be neutralized, at least in part, by antiserum to the purified 104 kDa protein . 37 A distinct protein cytotoxin has now been identified in serotype 2 strains that is heat-labile, and is 120 kDa in size . 38,3 ' The toxin was identified by Kamp et a/ . by immunoblotting with neutralizing mAb to the cytotoxin, 38 and by Rycroft et a/ . by isolating mutants lacking hemolysin (Hly - ) and/or cytotoxic activity following chemical mutagenesis. 39 Mutants lacking hemolytic, but not cytotoxic, activity lack a protein of 109 kDa and contain the 120 kDa protein in culture supernatant . Mutants also lacking cytotoxic activity do not produce the 120 kDa protein . Antiserum to the Hly - mutants is reactive with the 120 kDa, but not the 109 kDa, protein by immunoblotting ; the antiserum neutralizes cytotoxic, but not hemolytic activity . Cytotoxic activity and the 120 kDa protein are also found in other strains of serotypes 2, 3 and 4, but is non-existent or very weak in the serotype 5 strains examined . Kamp et al. have produced three mAb following immunization of mice with culture filtrates of serotypes 2 or 9 . One mAb inhibits the cytotoxic activity of serotypes 2-4 and 8, and reacts with a protein of about 120 kDa from culture filtrate of the same serotypes; this protein has strong cytotoxic activity, but not hemolytic activity . A second mAb inhibits hemolytic and cytotoxic activity of serotypes 1, 5, 9-11, and reacts with a protein of about 105 kDa from culture supernatant of the same serotypes ; this protein has strong cytotoxic and hemolytic activity . The third mAb inhibits hemolytic and cytotoxic activity of serotypes 7 and 12, and also the hemolytic, but not cytotoxic, activity of serotypes 2-4 and 8 . This mAb reacts with a protein of approximately 103 kDa in culture filtrates of all serotypes except serotype 10, and is associated with weak hemolytic and moderate cytotoxic activity . Based on the differences in strength of hemolytic activity and the distribution pattern of the associated proteins compared to the patterns reported by Frey and Nicolet, 12 the investigators postulate that the 105 kDa protein is Hlyl, the 103 kDa protein is Hlyll, and the 120 kDa protein is a cytotoxin . 39a A co-hemolysin that produces a CAMP-like effect in the presence of Staphylococcus a-toxin has been identified in serotype 1 as a protein with an apparent molecular mass of 27 kDa ; its gene has been cloned in E. co/i. 40 A similar protein with a calculated molecular mass of 29 .5 kDa has also been cloned from serotype 1 by Lian et al .," and is assumed to be the same protein since the restriction maps of the two DNA clones are identical . The relationship of this CAMP-like hemolysin to hemolytic activity or pathogenesis is not yet known . Antibodies to the serotype 1 CAMP-like hemolysin do not react with the Hlyl, but do cross-react with the Streptococcus agalactiae protein B CAMP factor ."



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A heat-stable hemolytic and cytotoxic carbohydrate (not associated with endotoxin) has been identified in the cell-free culture supernatant of a serotype 2 strain grown in chicken infusion broth and chicken serum .42, 43 However, this substance has not been identified from other serotypes or by other investigators, and no additional information has been reported on this substance . In summary, two distinct RTX protein hemolysins (Hlyl and Hlyll) have thus far been identified in A . pleuropneumoniae that are of similar molecular mass, and crossreact immunologically . One or both hemolysins are present in different serotypes or strains . Hlyl is a stronger hemolysin than Hlyll, and their induction and/or activity may be influenced differently by Ca t + . The hemolysins are also cytotoxic . In serotypes 1 and 5, one or both hemolysins are important for virulence and immunoprotection . A distinct cytotoxin is present in some, but not all, serotypes/strains of A . pleuropneumoniae, but the relative role of this toxin in pathogenesis is not known . Thus, additional work needs to be done to clarify if the toxins produced by A . pleuropneumoniae are serotype- or strain-specific, and the role of each toxin in virulence and immunoprotection compared to other factors .

Capsules On a clear medium (e .g . brain heart infusion agar supplemented with NAD), colonies of A . pleuropneumoniae are iridescent due to capsule production ." The capsules of A . pleuropneumoniae serotypes 1-7 have been purified by precipitation from culture supernatant with hexadecyltrimethylammonium bromide (Cetavlon), followed by extraction with NaCl and phenol, and ultracentrifugation or column chromatography ." ," The composition and structure of the capsules of all 12 serotypes have been elucidated .45-52 Each is composed of repeating oligosaccharide units, teichoic acid polymers joined by phosphate diester bonds, or oligosaccharide polymers joined through phosphate bonds . 52 The capsules are negatively charged due to phosphate or carboxylic acid residues ; some are partially O-acetylated . 52 The capsule is responsible for serotype specificity in A . pleuropneumoniae, 53 and as for most bacterial capsules, is biologically inert . Purified capsule fails to activate the complement cascade (C . Melnik and T . J . Inzana, unpublished data), and does not demonstrate toxic activity by dermal Shwartzman reaction, pyrogenic activity, chick embryo mortality, 54 or blastogenesis of swine peripheral blood lymphocytes ." The capsule is the primary component that protects the bacterium from host defenses . Encapsulated A . pleuropneumoniae of serotypes 1-3, 5 and 7 are known to be resistant to bactericidal killing by antibody and complement, even in hyperimmune rabbit or swine serum . 56,5' However, serotypes 2 and 3 have been reported to be killed by normal human serum, but not a serotype 5 strain ." Non-capsulated mutants of serotype 5 are effectively killed by normal serum from swine, humans, rabbits, and guinea pigs (C . Melnik and T . J . Inzana, unpublished data) . 56 These results suggest that the presence and type of capsule are important in resistance to complement-mediated killing in the presence or absence of specific antibody . In normal serum (containing active complement), A . pleuropneumoniae is also resistant to phagocytosis by polymorphonuclear leukocytes, but is effectively opsonized in the presence of antibody to capsule . Phagocytosis of non-capsulated mutants of serotype 5 also requires opsonization with antibody." Antibodies to capsule can protect the host against lethal disease, but the protection is inadequate against infection and chronic disease The limited protection provided by killed vaccines is also serotype-specific and therefore may, in part, be attributed to antibodies to capsule . 55,5s Certain serotypes apparently are more virulent than others ." Variations in virulence



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may be related to the composition and structure of the capsule, or the amount of capsule adherent to the cell . Jacques et al ." used electron microscopy to show that more virulent serotypes have larger, more adherent capsules . However, only one or two strains of each serotype were examined . A strain of serotype 5 with a less adherent capsule has been shown to be less virulent than another strain of the same serotype with a more adherent capsule ; quantitative differences in the amount of LPS between the two strains, however, have also been noted .62 Variation in adherence of the capsule of different serotypes and/or strains has also been confirmed by Steffens et al. 13 As noted by Komal and Mittal,64 at least some strains of serotypes 1, 5, 9-11 are more virulent than strains of serotypes 2-4, 6-8, and 12 . However, some strains of serotype 5 are of low virulence, while some strains of serotypes 3 and 7 are highly virulent . Furthermore, other properties have been noted to vary among different serotypes, such as the structure of the LPS 0 side chains, 65 and the number and patterns of hemolysins and cytotoxins . 12 .15 Thus, it is likely that variation in virulence between serotypes is due to the presence of several factors . While it is clear that the capsule is required for virulence in A . p/europneumoniae, the molecular mechanism by which capsules of different serotypes contribute to virulence is not yet well known . Strains of serotypes 1 and 5 that have been passed in vitro and selected for loss or deficiency in capsule production are less virulent in pigs .56,66 than their parent strains Non-capsulated mutants that have been isolated in the author's laboratory following chemical mutagenesis are highly stable, contain all the toxins and phenotypic properties of the parent strain (except encapsulation), and cause no mortality or lung lesions in pigs following intratracheal challenge with two times the LD 75 of the parent (T . J . Inzana, J . Todd and H . Veit, unpublished data) . Lipopolysaccharide The composition of the carbohydrate moiety of LPS from most Gram-negative respiratory pathogens (e .g . Haemophilus sp) differs from that of enteric bacteria by lack of 0 side chains . In A . pleuropneumoniae, however, a pattern of smooth, partially rough, or rough LPSs (terminology used to refer to LPS with 0 side chains, a limited number of 0 side chains, or no 0 side chains, respectively) have been reported for different serotypes . For instance, serotypes 2, 4, and 7 have been reported to be smooth, serotypes 1 and 5 as partially rough, and serotypes 3 and 6 as rough ." However, smooth LPS has been reported from a serotype 5 strain . 54 Structural studies of the 0 side chains of each serotype by Altman et al. and Perry et al. 12,1"--12 have helped to clarify these confusing reports, particularly with serotype 5 . Each serotype is associated with a unique LPS, which differs primarily by the structure of the 0 side chain .12,6' All serotypes have a distinct core and 0 side chain, but the linear length of the 0 side chain may vary among different strains (such as serotype 5), resulting in variations of smooth, partially rough, or rough within a serotype, which can be demonstrated by polyacrylamide gel electrophoresis . 72 The composition and structure of the LPS 0 side chains is specific to each serotype, but all are composed of the common hexose derivatives glucose, galactose, rhamnose, N-acetylglucosamine, and N-acetylgalactosamine . Glucose, heptose, and 3-deoxy-o-manno octulosonic acid are present in the core oligosaccharide of serotypes 1-12 . 65 The lipid A component consists predominately of C 14 ;0 , C1 6 :o, and 3-hydroxy C 14 .0 fatty acids, and makes up only about 9 .2% of the total LPS . The 3-deoxy-D-manno octulosonic acid component appears to be C-4- and C-S-substituted and requires hydrolysis with 4 M HCI to be reactive in colorimetric assays ." Various epitopes of A . pleuropneumoniae LPS may be serotypespecific, cross-reactive among serotypes within the species, and cross-reactive with



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other species . 55 Although the 0 side chains are distinct for each serotype, enough similarity exists in LPS epitopes between some serotypes (e .g . 1, 9, and 11) to allow substantial cross- reactivity in serological assays . 52 The biologic activity of A . pleuropneumoniae endotoxin is similar to that of other Gram-negative bacteria : purified LPS induces a positive dermal Shwartzman reaction and blastogenic response of peripheral blood lymphocytes, clots lysates of Limulus 5a,55 amoebocytes, is pyrogenic, lethal for chick embryos, and activates the alternative complement pathway (C . Melnik and T . J . Inzana, unpublished data) . In vivo, purified A . pleuropneumoniae LPS can induce an interstitial or multifocal to consolidating pneumonia in the lungs of mice and pigs with inflammatory cell infiltration . The severity of the lesions correlates with the dose of LPS administered . However, the necrotic and hemorrhagic lesions typical of pleuropneumonia are absent from animals given pure LIPS ." It is likely that LPS acts synergistically to intensify the lesions caused by other toxins . Belanger et a/. 73 reported that 83% of serotypes with a smooth LPS (e .g . serotypes 2 and 7) adhere in large numbers to porcine tracheal rings, whereas 80% of serotypes with a partially rough LPS (e .g . serotypes 1 and 5) adhere poorly . Furthermore, purified smooth LPS blocks adherence to tracheal rings indicating that the LPS 0 side chains, at least in part, may be a specific adherence factor for colonization of the porcine upper respiratory tract by serotypes with a smooth LPS . Adequate protection against disease in mice or swine has not been produced following immunization with pure LPS . 5s,74 Immunization of pigs with an E. co/i rough mutant (J5) provides protection similar to that of an A . p/europneumoniae killed vaccine ." However, whether cross-reacting antibodies to core LPS or other factors are responsible for the limited protection is not known . Non-pyrogenic vaccines consisting of the oligosaccharide moiety of A . pleuropneumoniae LPS conjugated to tetanus toxoid induce higher LPS titers than pure LPS, whole cells, or a commercial vaccine ." The protection afforded by this conjugate vaccine has not yet been reported . Thus, LPS probably contributes to the pathogenicity of A . p/europneumoniae, but other factors are required for the full spectrum of disease and protective immunity . Outer membrane proteins Three to five major proteins are present in the outer membrane of strains of A . pleuropneumoniae serotypes 1-9 that have been examined, as well as 10-20 minor proteins (some of which may be contaminating inner membrane proteins) ." - " The electrophoretic protein profiles are very stable and are not affected by variations in the growth medium, growth phase at harvest, or in vitro or in vivo passage . However, the method of outer membrane protein preparation does affect the protein electrophoretic profile ." The major proteins are of molecular mass 39-44 kDa (the size of most porin proteins in other Gram-negative bacteria), 16-16 .5 kDa, and 9 kDa ; the latter being a heat-modifiable protein . The major outer membrane protein profiles of serotypes 1 and 9 are identical, as are the profiles of serotypes 2 and 6 . Major outer membrane protein profiles of field isolates of serotypes 1 and 5 are similar, but not identical, to the reference strains ; protein profiles of field isolates of serotype 7 are identical to the reference strain .'' Serotypes 1-5 and 7 produce novel proteins of about 105 and about 76 kDa under iron restricted conditions . 79,80 Furthermore, antibodies in sera from swine convalescing from serotype 1 infection react with these proteins from each of the serotypes examined, indicating that these proteins are produced in vivo and are immunologically conserved ." As reported by Niven et al ." A . pleuropneumoniae can grow on iron-restricted media in the presence of porcine transferrin, but not bovine or human transferrin, and the



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bacteria do not produce siderophores . Proteins of 105 and 56 kDa have been recovered by an affinity-isolation procedure, using biotinylated porcine transferrin and strepavidin agarose, and have been confirmed to be porcine-specific transferrin receptors ." The capability of these proteins to bind porcine transferrin to obtain iron from the host, but not transferrin of other species, helps to explain the host specificity of this organism for pigs . Another outer membrane protein of 42 kDa is found in strains of serotypes 1-3, and 5-7 when maltose is added to the growth medium . 82 Not all strains within a serotype produce the 42 kDa protein, however, and its properties are similar to those of the maltose-inducible porin of E. co/i . Convalescent swine sera contain antibodies to this 42 kDa protein that react with a maltose-inducible protein from other serotypes, indicating that this protein is antigenically conserved and produced in vivo . Antibodies in convalescent sera of pigs challenged with a serotype 5 strain recognize most of the major outer membrane proteins, as well as some high molecular mass (>94 kDa) minor proteins . Adsorption of sera with whole cells removes antibodies specific for 45, 49 .5, 66 .5, and >94 kDa proteins, indicating that these proteins are exposed on the cell surface . Furthermore, antibodies to some serotype 5 outer membrane proteins cross-react with proteins of other serotypes . 53,a3 A study by Maclnnes and Rosendal 76 demonstrated that A . pleuropneumoniae outer membrane proteins are similar to proteins of other Actinobaci/lus sp . and P . haemo/ytica . Antisera raised to serotypes 1-8 react most strongly with three major outer membrane proteins of the homologous serotype, but also cross-react with similar proteins of other serotypes, Actinobacillus sp . and P . haemolytica . A 17 kDa antigen appears antigenically conserved in all Gram-negative bacteria examined, including E. co/i. Thus, both LPS and outer membrane protein antigens are likely to cross-react between serotypes of A . p/europneumoniae and between species, and may be responsible for the lack of specificity in sensitive diagnostic tests that utilize either crude preparations for antigen or antiserum to whole cells . Immunization of pigs with proteinase K-treated outer membranes significantly reduces the severity and number of pigs with lung lesions following intranasal challenge, compared to immunization with untreated outer membranes or periodatetreated outer membranes . However, not all the proteins are eliminated by proteinase K treatment, and the antigens responsible for the improved protection have not been identified ." It has not been reported if antibodies to any outer membrane proteins are protective against lethal challenge .

Other factors A . p/europneumoniae is extremely host-specific and does not cause natural disease in animals other than pigs . Although the mouse has been used as a model for the acute form of the disease, a true infection is not established and it is likely that challenged mice die due to the effects of one or more toxins .", " Host specificity is probably due to the organism's limited capability to obtain iron only from porcine transferrin (see above), and possibly by specific adherence factors . In addition to the role of LPS 0 side chains in the adherence of some, but not all, serotypes of A . p/europneumoniae (see above), conventional fimbriae or pili may also be important . Utrera and Pijoan have noted the presence of fimbriae in 45% of fresh field isolates of A . p/europneumoniae . 85 However, following subculture of fimbriated strains on media lacking blood, all strains lose fimbriae by the third passage . Pilus-like structures have also been identified on A . p/europneumoniae cells phagocytized by polymorphonuclear Ieukocytes . 56 Therefore, the phenotypic expression of adherence factors, such as pili, may be induced by conditions in vivo, and lost upon subculture in vitro where they are not needed .



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Hemagglutination is a property of some, but not all, isolates of A . pleuropneumoniae, and is not specific for blood cells of a given animal species . In fact, seven different patterns of hemaggIutination have been reported based on the animal species of blood cells agglutinated . Hemagglutination does not seen to be mediated by pili or a hydrophobic surface ." Small plasmids (1 .7-7 MDa) have been identified in strains of A . pleuropneumoniae that are resistant to sulfonamides, streptomycin, ampicillin, and chloramphenicol . 87-89 Such plasmids likely code for enzymes such as fJ-lactamase and chloramphenicol acetyl transferase . Many strains, however, lack any plasmids, particularly if they are not antibiotic resistant . 89 There is no evidence to date to indicate that plasmids in A . pleuropneumoniae code for any virulence properties .

Future perspectives During the last 5 years information on the number and role of factors that contribute to the virulence of A . pleuropneumoniae, and the pathogenesis of pleuropneumonia, has grown tremendously. Most of these advances have been obtained through the application of traditional methods of biochemistry, immunology, microbiology, cell biology, and mutagenesis . As of yet, only a few of the genes that control these virulence factors have been cloned and sequenced . In order to enhance understanding of the role of virulence factors in pathogenesis and immunoprotection, an improved understanding of the genes coding for these primary (protein) and secondary (carbohydrate) factors is needed . In addition, the contribution of each system of host immunity (mucosal, humoral, and cellular) in prevention of disease due to these factors needs to be clarified . Once such progress is made, a new generation of improved vaccines can be developed to adequately control this important disease .

Work from the author's laboratory was supported, in part, by grants from the U .S . Department of Agriculture, HATCH, the Virginia Center for Innovative Technology, and the National Pork Producers Council .

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