Purification and partial characterization of the capsular polymer of Haemophilus pleuropneumoniae serotype 5

The capsular polymer (CP) of Haemophilus pleuropneumoniae serotype 5 was purified, and its chemical composition was analyzed. Radioimmunoassay experiments showed that the maximum amount of CP could be obtained from broth cultures of bacteria in the late stationary phase, rather than from bacteria washed off agar plates. The CP was precipitated from culture supernatant with 5 mM hexadecyltrimethylammonium bromide (Cetavlon) and solubilized with 0.4 M NaCl. Ninety percent of the CP in the culture supernatant was precipitated with Cetavlon, although some material remained insoluble after NaCl extraction. The CP was further purified by phenol extraction, ultracentrifugation, and Sepharose CL-4B gel filtration. The Kav of the CP from Sepharose CL-4B chromatography was 0.33. The CP preparation contained 85% hexosamine, 12% hexose, 3% phosphate, 0.17% protein, 0.20% nucleic acid, and 0.01% endotoxin. Thin-layer chromatography, an amino acid analyzer, and a glucose oxidase colorimetric kit were used to identify the sugar components of the hydrolyzed CP as glucosamine and glucose. Analysis of the native CP by 13C nuclear magnetic resonance indicated that amino, N-acetyl, and carboxyl groups were present and that the CP was a disaccharide.     

Virulence properties and protective efficacy of the capsular polymer of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5.

The role of the capsule of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5 in bacterial virulence, and the protective efficacy of antibody to serotype 5 capsule was investigated. Encapsulated H. pleuropneumoniae serotype 5 were resistant to killing by complement and antibody to capsule or somatic antigens, whereas a noncapsulated mutant was sensitive to killing by the alternative complement pathway alone. Antiserum to whole H. pleuropneumoniae serotype 5 bacteria or monospecific antiserum to capsule was capable of opsonizing bacteria of the homologous serotype for phagocytosis by swine polymorphonuclear leukocytes but was not opsonic for a heterologous serotype. An immunoglobulin M monoclonal antibody to the serotype 5 capsule was not opsonic for any serotype. Mice were protected against lethal, intranasal challenge with the homologous or heterologous serotype after immunization with live encapsulated or noncapsulated bacteria, but not after immunization with killed bacteria, lipopolysaccharide, or a capsule-protein conjugate vaccine. The protection induced by immunization with live bacteria was transferred to nonimmune, syngeneic mice by serum but not by spleen cells. Nonimmune pigs passively immunized with monospecific swine serum to capsule were protected from lethal infection but not from development of hemorrhagic lung lesions, whereas pigs passively immunized with swine antiserum to live bacteria did not develop severe respiratory lesions. Thus, the capsule of H. pleuropneumoniae serotype 5 was inhibitory to the bactericidal activity of serum and was antiphagocytic. Antibody to the capsule was opsonic but was not fully protective.     

Morphological and biochemical comparison of virulent and avirulent isolates of Haemophilus pleuropneumoniae serotype 5.

Capsular structure and biochemical composition varied between two isolates (virulent and avirulent) of Haemophilus pleuropneumoniae serotype 5. The presence of capsule was determined by transmission electron microscopy with glutaraldehyde-osmium, ruthenium red, alcian blue, and phosphotungstic acid staining procedures. The virulent isolate of H. pleuropneumoniae had a distinct, adherent capsule. The avirulent isolate had a fragile, easily removed capsule. Capsular material (CM) and a lipopolysaccharide (LPS) were isolated from each bacterial isolate and were compared biochemically and biologically. CM from both isolates contained carbohydrates, no detectable protein, and no detectable to trace amounts of lipid A. Each LPS contained heptose, hexose, galactose, glucosamine, 2-keto-3-deoxyoctonate, and lipid A. Biological responses to CM and LPS from both isolates were demonstrated in the proclotting enzyme of Limulus polyphemus amebocyte lysate activation and in serological cross-reactions by immunofluorescence and immunodiffusion precipitation. The virulent isolate contained approximately 10 mg of LPS per g more on an original dry weight basis than the avirulent isolate. LPS from the virulent isolate contained approximately 13 times more galactose than LPS from the avirulent isolate. The differences of capsular structure and biochemical composition may contribute to the role of CM in porcine H. pleuropneumoniae infections.     

Identification and characterization of a DNA region involved in the export of capsular polysaccharide by Actinobacillus pleuropneumoniae serotype 5a.

Actinobacillus pleuropneumoniae synthesizes a serotype-specific capsular polysaccharide that acts as a protective barrier to phagocytosis and complement-mediated killing. To begin understanding the role of A. pleuropneumoniae capsule in virulence, we sought to identify the genes involved in capsular polysaccharide export and biosynthesis. A 5.3-kb XbaI fragment of A. pleuropneumoniae serotype 5a J45 genomic DNA that hybridized with DNA probes specific for the Haemophilus influenzae type b cap export region was cloned and sequenced. This A. pleuropneumoniae DNA fragment encoded four open reading frames, designated cpxDCBA. The nucleotide and predicted amino acid sequences of cpxDCBA contained a high degree of homology to the capsule export genes of H. influenzae type b bexDCBA, Neisseria meningitidis group B ctrABCD, and, to a lesser extent, Escherichia coli K1 and K5 kpsE and kpsMT. When present in trans, the cpxDCBA gene cluster complemented kpsM::TnphoA or kpsT::TnphoA mutations, determined by enzyme immunoassay and by restored sensitivity to a K5-specific bacteriophage. A cpxCB probe hybridized to genomic DNA from all A. pleuropneumoniae serotypes tested, indicating that this DNA was conserved among serotypes. These data suggest that A. pleuropneumoniae produces a group II family capsule similar to those of related mucosal pathogens.     

Evaluation of slide agglutination and ring precipitation tests for capsular serotyping of Haemophilus pleuropneumoniae.

Rapid slide agglutination (RSA), quantitative plate agglutination, slow tube agglutination (STA), and ring precipitation (RP) tests were performed on 200 isolates of Haemophilus pleuropneumoniae by using the type sera produced in rabbits against five known serotype strains and one strain 202. RSA and RP tests both yielded the same results as those by STA. None of the agglutination procedures could be used for serotyping isolates that autoagglutinated in saline. The RP test was successfully used for serotyping such strains. The specificity of the RSA and RP tests was confirmed by cross-absorption studies. All of the isolates except two had strong serotype-specific activities. The most common serotype isolated in Quebec was serotype 1, followed by serotypes 5 and 2. None of the isolates belonged to serotypes 3 and 4. Only two isolates were found to be untypable; they could possibly belong to serotype(s) not yet defined. The RSA and RP tests may be at least as reliable as the STA test, but easier to perform, less expensive, and much more rapid than any of the other methods reported. Of all the procedures studied by us, the RP test proved to be the method of choice for serotyping H. pleuropneumoniae; hence, it should replace the STA test for serotyping H. pleuropneumoniae.     

Haemophilus pleuropneumoniae serotyping.

A total of 126 Haemophilus strains isolated from porcine pneumonia were serotyped, using the indirect fluorescent-antibody technique. Of these, 103 were successfully typed within the recognized scheme of serotypes 1 to 5. Eleven strains were antigenically similar but were different from other strains of H. pleuropneumoniae or H. parasuis. These strains are proposed as serotype 7. Eight strains were not identified as serotype 1 when serum against strain Shope 4074 was used, but their identity as type 1 strains was concluded on the basis of complete cross-titrations, using unabsorbed and absorbed sera and indirect fluorescent-antibody and agglutination tests. The type-specific antigen of these strains may have been masked by an additional antigen. A similar situation was believed to exist for four strains which belonged to serotype 5 but did not react with serum against strain K17 (reference strain) in the indirect fluorescent-antibody test. Strain Femø was antigenically different from other H. pleuropneumoniae or H. parasuis strains and was proposed as serotype 6, thus replacing the "minor group" (represented by strain 202), which is of uncertain taxonomical status within the genus Haemophilus.     

Immune responses to the lipopolysaccharides and capsular polysaccharides of Haemophilus pleuropneumoniae in convalescent and immunized pigs.

The immunologic responses to a smooth-type lipopolysaccharide (LPS) (HpS-LPS), a rough-type LPS (HpR-LPS), and a capsular-enriched polysaccharide preparation (HpC-PS) purified from Haemophilus pleuropneumoniae were determined in pigs immunized with a commercial H. pleuropneumoniae cellular vaccine, in pigs experimentally infected with H. pleuropneumoniae, in control pigs, and in immunized rabbits. The ability of the preparations to induce lymphocyte blastogenesis and B-cell activation was determined in the pigs and compared with the responses induced by the LPS of Escherichia coli O111:B4 and the LPS of Salmonella minnesota Re595. All the LPS preparations acted to induce proliferation of peripheral blood lymphocytes (PBL) from all pigs. The blastogenic response of PBL from H. pleuropneumoniae-infected pigs to HpS-LPS and HpR-LPS was significantly (P less than 0.05) greater than that of PBL from immunized and control pigs. HpC-PS did not induce a blastogenic response in the PBL of control pigs but did in PBL from H. pleuropneumoniae-infected pigs and to a greater degree in immunized pigs. An increase in the response of PBL to the S. minnesota LPS occurred only in the H. pleuropneumoniae-infected pigs. Significantly more (P less than 0.05) immunoglobulin-secreting cells (ISC) were induced in a reverse hemolytic plaque assay by stimulation with HpS-LPS and HpC-PS of PBL isolated from pigs infected with H. pleuropneumoniae than of PBL from immunized pigs. Increasing the number of T cells increased the number of ISC induced by HpS-LPS in control and immunized pigs, but not in convalescent pigs. The presence of macrophages reduced activation of ISC by HpS-LPS in control pigs and to a lesser degree in immunized pigs, whereas in H. pleuropneumoniae-infected pigs macrophages enhanced the induction of ISC by HpS-LPS. In immunized pigs, macrophages acted to inhibit the ability of HpC-PS to induce ISC. Serologic studies indicate that HpC-PS contains strain- and serotype-specific antigens; that HpS-LPS has both serotype-specific and cross-reacting species-specific antigens; and that HpR-LPS does not contain detectable serotype-specific antigens but does have both non species- and species-specific antigens. These studies show that the serotype-specific protection provided by immunization of pigs with an H. pleuropneumoniae cellular vaccine is principally the result of immunity to capsular antigens and that a weak cellular immune response occurs as compared with that induced by infection with H. pleuropneumoniae.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the antigenic lipopolysaccharide O chain and the capsular polysaccharide produced by Actinobacillus pleuropneumoniae serotype 13

Serotyping of Actinobacillus pleuropneumoniae, the etiologic agent of porcine pleuropneumonia, is important for epidemiological studies and for the development of homologous vaccine cell preparations. The serology is based on the specific chemical structures of capsular polysaccharides (CPSs) and lipopolysaccharide (LPS) antigenic O-polysaccharide moieties (O-PSs), and knowledge of these structures is required for a molecular-level understanding of their serological specificities. The structures of A. pleuropneumoniae serotype 1 to 12 CPSs and O-PSs have been elucidated; however, the structures associated with three newly proposed serotypes (serotypes 13, 14, and 15) have not been reported. Herein we described the structures of the antigenic O-PS and CPS of A. pleuropneumoniae serotype 13. The O-PS of the A. pleuropneumoniae serotype 13 LPS is a polymer of branched tetrasaccharide repeating units composed of l-rhamnose, 2-acetamido-2-deoxy-d-galactose, and d-galactose residues (1:1:2). By use of hydrolysis, methylation, and periodate oxidation chemical methods together with the application of one- and two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy and mass spectrometry, the structures of the O chain and CPS were determined. The CPS of A. pleuropneumoniae serotype 13 was characterized as a teichoic-acid type polymer. The LPS O antigen was identical to the O-PS produced by A. pleuropneumoniae serotype 7. The CPS has the unique structure of a 1,3-poly(glycerol phosphate) teichoic acid type I polymer and constitutes the macromolecule defining the A. pleuropneumoniae serotype 13 antigenic specificity.     

Detection and Identification of Actinobacillus pleuropneumoniae Serotype 5 by Multiplex PCR

Serotyping of Actinobacillus pleuropneumoniae is based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection of A. pleuropneumoniae and identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide of A. pleuropneumoniae serotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains of A. pleuropneumoniae tested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 10² CFU. The A. pleuropneumoniae serotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of other A. pleuropneumoniae serotypes would expand the diagnostic and epidemiologic capabilities of this assay.     

Clonal diversity in Haemophilus pleuropneumoniae.

Genetic diversity among 135 isolates of nine serotypes of Haemophilus pleuropneumoniae recovered from pigs with pleuropneumonia or other invasive diseases in 14 countries was estimated by multilocus enzyme electrophoresis, which detects allelic variation in structural genes. Thirty-two multilocus genotypes (electrophoretic types [ETs]) were distinguished on the basis of allele profiles at 15 enzyme loci, and 36 distinctive combinations of ET and serotype were identified. The recovery of isolates with identical properties in widely separated geographic regions and over a 20-year period indicated that the population structure of H. pleuropneumoniae is clonal. Isolates of the same ET generally shared the same serotype and electrophoretic pattern of the outer membrane proteins, but some ETs were represented by isolates of several different serotypes, outer membrane protein patterns, or both. On average, the genetic diversity among ETs of the same serotype was 56% of the total genetic diversity in the species. Isolates of serotype 1, which are unusually pathogenic, belong to a distinctive group of clones that are closely related to clones marked by serotype 9.     

Cloning and Mutagenesis of a Serotype-Specific DNA Region Involved in Encapsulation and Virulence of Actinobacillus pleuropneumoniae Serotype 5a: Concomitant Expression of Serotype 5a and 1 Capsular Polysaccharides in Recombinant A. pleuropneumoniae Serotype 1

A DNA region involved in Actinobacillus pleuropneumoniae serotype 5 capsular polysaccharide (CP) biosynthesis was identified and characterized by using a probe specific for the cpxD gene involved in CP export. The adjacent serotype 5-specific CP biosynthesis region was cloned from a 5.8-kb BamHI fragment and an 8.0-kb EcoRI fragment of strain J45 genomic DNA. DNA sequence analysis demonstrated that this region contained four complete open reading frames, cps5A, cps5B, cps5C, and cps5D. Cps5A, Cps5B, and Cps5C showed low homology with several bacterial glycosyltransferases involved in the biosynthesis of lipopolysaccharide or CP. However, Cps5D had high homology with KdsA proteins (3-deoxy-d-manno-2-octulosonic acid 8-phosphate synthetase) from other gram-negative bacteria. The G+C content of cps5ABC was substantially lower (28%) than that of cps5D and the rest of the A. pleuropneumoniae chromosome (42%). A 2.1-kb deletion spanning the cloned cps5ABC open reading frames was constructed and transferred into the J45 chromosome by homologous recombination with a kanamycin resistance cassette to produce mutant J45-100. Multiplex PCR confirmed the deletion in this region of J45-100 DNA. J45-100 did not produce intracellular or extracellular CP, indicating that cps5A, cps5B, and/or cps5C were involved in CP biosynthesis. However, biosynthesis of the Apx toxins, lipopolysaccharide, and membrane proteins was unaffected by the mutation. Besides lack of CP biosynthesis, and in contrast to J45, J45-100 grew faster, was sensitive to killing in precolostral calf serum, and was avirulent in pigs at an intratracheal challenge dose three times the 50% lethal dose (LD₅₀) of strain J45. At six times the J45 LD₅₀, J45-100 caused mild to moderate lung lesions but not death. Electroporation of cps5ABC into A. pleuropneumoniae serotype 1 strain 4074 generated strain 4074(pJMLCPS5), which expressed both serotype 1 and serotype 5 CP. However, serotype 1 capsule expression was diminished in 4074(pJMLCPS5) in comparison to 4074. The recombinant strain produced significantly less total CP (serotypes 1 and 5 CP combined) in log phase (P = 0.0012) but significantly more total CP in late stationary phase than 4074 (P < 0.0001). In addition, strain 4074(pJMLCPS5) caused less mortality and bacteremia in pigs and mice following respiratory challenge than strain 4074, indicating that virulence was affected by diminished capsule production. These results emphasize the importance of CP in the serum resistance and virulence of A. pleuropneumoniae.Actinobacillus pleuropneumoniae is an encapsulated, gram-negative bacterium that causes swine pleuropneumonia, a frequently fatal and highly contagious respiratory disease. There are 12 recognized serotypes of A. pleuropneumoniae that vary in virulence and geographic predominance (33). The capsular polysaccharide (CP) is responsible for serotype specificity and is required for virulence (19, 20). Nonencapsulated A. pleuropneumoniae mutants obtained by chemical mutagenesis have been shown to be effective vaccine candidates in that they are safe and highly protective (20). However, not all such mutants are stable, and the nature of the mutation(s) is unknown. Hence, characterization of the genes involved in CP export and biosynthesis would be desirable.We have previously reported cloning and sequencing an A. pleuropneumoniae DNA region involved in export of the CP of serotype 5a. This region consists of four genes, designated cpxABCD, which had a high degree of homology to the group II capsule export genes of Haemophilus influenzae type b (bexDCBA), Neisseria meningitidis group B (ctrABCD), and to a lesser extent, Escherichia coli K5 (kpsE and kpsMT) (54). This homology suggested that A. pleuropneumoniae also synthesized a group II capsule, whose organization predicted that the biosynthesis region would be upstream of cpxDCBA (12). We now report cloning and sequencing of four genes upstream of the cpx CP export region that correspond to capsular biosynthesis genes. A mutant with a deletion in three of these genes by allelic exchange was incapable of synthesizing CP and was avirulent in pigs. trans expression of these three genes in serotype 1 resulted in a chimeric strain producing both serotype 1 and 5 CP. However, expression of serotype 5 CP genes resulted in diminished production of serotype 1 CP, as well as diminished virulence in pigs and mice.     

Cytolysins of Actinobacillus pleuropneumoniae serotype 9.

Cytolysin I (ClyI) and cytolysin II (ClyII), which are present in the culture supernatant of Actinobacillus pleuropneumoniae serotype 9, are thought to play an important role in the pathogenesis of pig pleuropneumonia. The purpose of this study was to clone and characterize the genetic determinants of these cytolysins. Cloning was accomplished by the screening of DNA libraries for the presence of cytolytic activity and for the presence of DNA sequences homologous to leukotoxin DNA of Pasteurella haemolytica. Both genetic determinants were found to be members of the RTX cytotoxin family. The ClyII determinant was characterized in more detail. It appeared that ClyII more closely resembled the leukotoxin of P. haemolytica than the alpha-hemolysin of Escherichia coli. The ClyII amino acid sequence was identical to a hemolysin gene sequence of A. pleuropneumoniae serotype 5; this finding indicates that the latter gene also codes for ClyII and not for ClyI, as has previously been suggested. The genetic organization of the ClyII determinant differed from the genetic organization of other RTX determinants. Genes responsible for secretion of ClyII were not contiguous with the toxin gene. Instead, secretion genes were present elsewhere in the genome. These secretion genes, however, belong to the ClyI operon. This indicates that the secretion genes of the ClyI operon are responsible for secretion of ClyI and ClyII.     

Determination of antigenic specificity and relationship among Haemophilus pleuropneumoniae serotypes by an indirect hemagglutination test.

Serological properties of antigens extracted from strains of Haemophilus pleuropneumoniae belonging to seven different serotypes were investigated. Antisera were prepared in rabbits against Formalin-treated whole cell suspensions as well as autoclaved cell suspensions. Saline and heat extracts and their alcohol precipitate antigens of H. pleuropneumoniae were used in the indirect hemagglutination test. All the antigens used were easily adsorbed directly onto sheep erythrocytes. Saline extract antigen showed maximum type specificity. Heating of the whole cell suspension revealed the cross-reactive minor antigenic determinants. Thus, the heat extract preparations had both type-specific and species-specific antigens. It is suggested that the indirect hemagglutination test may be useful for both serotyping and serodiagnosis of H. pleuropneumoniae infections in pigs.     

Efficacy of a cell extract from Actinobacillus (Haemophilus) pleuropneumoniae serotype 1 against disease in swine.

We partially characterized a cell extract (CE) from Actinobacillus pleuropneumoniae serotype 1 and used the CE to test the efficacy of secreted proteins against disease. Secreted products from 4-h culture supernatants were precipitated with 20% polyethylene glycol. Analysis of the CE indicated the presence of protein, endotoxin, and carbohydrate. Hemolytic activity to bovine erythrocytes and cytotoxic activity to porcine mononuclear leukocytes was also demonstrated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the CE from a 4-h culture showed a major band at 110 kilodaltons (kDa), while a CE of a 26-h culture indicated the presence of a number of additional proteins, including the 110-kDa protein. The 110-kDa protein was also identified as a glycoprotein by periodic acid-Schiff and silver staining. A single band precipitated against convalescent-phase pig antiserum when the polyethylene glycol precipitate was used in an Ouchterlony plate. Vaccination with CE conferred greater protection against challenge with the homologous serotype than either a commercial bacterin or an outer membrane protein vaccine. Hemolysin-neutralizing titers were higher both pre- and postchallenge in the group vaccinated with the CE compared with in all other groups. We believe that this demonstrates the importance of secreted factors in protection against disease and suggests that the 110-kDa protein is an important immunogen.     

Cloning and characterization of a protective outer membrane lipoprotein of Actinobacillus pleuropneumoniae serotype 5.

The gene encoding an outer membrane lipoprotein (omlA) of Actinobacillus pleuropneumoniae serotype 5 was cloned, and the protein was expressed in Escherichia coli. One open reading frame of 1,104 bp was detected that encoded a protein (OmlA) with a predicted molecular mass of 40 kDa. A comparison with the omlA gene and the corresponding protein of A. pleuropneumoniae serotype 1 (G.-F. Gerlach, C. Anderson, S. Klashinsky, A. Rossi-Kampos, A.A. Potter, and P.J. Wilson, Infect. Immun. 61:565-572, 1993) revealed that the nucleic acid sequences had an overall sequence identity of 62.9% and the deduced amino acid sequences showed a sequence agreement of 57.3%. Both proteins were antigenically distinct. In a Western blot (immunoblot) analysis using a specific antiserum against A. pleuropneumoniae serotype 5 OmlA, a homologous protein was detected in the reference strains of A. pleuropneumoniae serotypes 5A, 5B, and 10. Pigs immunized with this recombinant protein were protected from death in an aerosol challenge experiment with an A. pleuropneumoniae serotype 5 isolate.     

Outer membrane protein profiles of Haemophilus pleuropneumoniae.

Outer membrane protein profiles of Haemophilus pleuropneumoniae were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cells were disrupted by sonication, and outer membrane-enriched fractions were prepared by differential centrifugation and selective solubilization of the inner membrane with sodium N-lauroyl sarcosinate. Colony type, growth medium, time of harvest, and in vitro or in vivo passage had no appreciable effect on the protein profiles of the strains examined. Seven patterns were distinguished among the reference strains of the nine capsular serotypes. These patterns were based on the mobility of the major outer membrane proteins migrating in the 39,000- to 44,000-molecular-weight region of the gel, a 16K to 16.5K protein, and a heat-modifiable 29K protein. Strains of serotypes 1 and 9 had identical outer membrane protein profiles, as did strains of serotypes 2 and 6. The reference strains of the remaining five serotypes each had a distinct pattern. The outer membrane protein profiles of 95 field isolates belonging to serotypes 1, 5, 7, and 9 from swine in the midwestern United States were determined and compared with the reference patterns. The results indicate that the population of H. pleuropneumoniae is clonal, with three predominant clones distinguished by both serotype and outer membrane protein profile responsible for the majority of H. pleuropneumoniae disease occurring in swine in the United States.     

Vaccine potential of Haemophilus pleuropneumoniae oligosaccharide-tetanus toxoid conjugates.

Oligosaccharides of smooth-type lipopolysaccharide (LPS) and oligosaccharides of rough-type LPS were isolated from Haemophilus pleuropneumoniae and conjugated to tetanus toxoid by reductive amination. The antigenic and immunogenic characteristics of the oligosaccharides, the oligosaccharide-tetanus toxoid conjugates, and the LPS of H. pleuropneumoniae were determined by passive hemagglutination, enzyme-linked immunosorbent assay, and inhibition enzyme-linked immunosorbent assay with antisera produced by immunization of rabbits and pigs. The findings were compared with the immunologic response induced by immunization of pigs with an H. pleuropneumoniae whole-cell vaccine and by infection of pigs with viable H. pleuropneumoniae. The results show that conjugation of isolated oligosaccharides of H. pleuropneumoniae to tetanus toxoid improves the immunogenicity of the oligosaccharides without significantly altering their antigenic character. These findings extend the understanding of the immunobiology of H. pleuropneumoniae infection in pigs and suggest the potential of purified oligosaccharides as vaccines to prevent porcine pleuropneumonia.     

Characterization of porcine plasma ficolins that bind Actinobacillus pleuropneumoniae serotype 5B

Ficolins are collagenous lectins implicated in resistance to infection by some micro-organisms with surfaces containing N-acetylglucosamine residues. Pigs have two known ficolin genes, alpha and beta, but ficolins in pig plasma appear in various electrophoretic forms, the origin and function of which are unclear. In this investigation the forms of ficolin in pig plasma that bind to the pneumonic pathogen Actinobacillus pleuropneumoniae serotype 5b (APP5) were compared with affinity-purified porcine plasma ficolins. APP5-bound ficolins consisted of two distinct multimeric (non-denatured) forms composed of subunits that migrated as multiple 38,40 and 42 kDa forms (apparent MW) with pI range 5.2-6.0. The APPS-binding forms of ficolin were consistent with ficolin alpha and were indistinguishable from affinity-purified plasma ficolins by peptide mass fingerprint analysis. Subunit bands separated by 2D-PAGE were consistent with porcine ficolin alpha. Affinity-purified plasma ficolins had a major subunit mass of 35081 Da by MALDI-TOF MS. Affinity-purified ficolins digested with N-glycosidase F retained APP5-binding activity and migrated faster as a single band of 38 kDa. These studies indicate that ficolin alpha is the major APPS-binding form in porcine plasma and suggest that N-glycosylation contributes to the apparent electrophoretic heterogeneity of porcine ficolins but is not required for APP5-binding activity.     

Optimization and standardization of an enzyme-linked immunosorbent assay protocol for serodiagnosis of Actinobacillus pleuropneumoniae serotype 5.

An indirect enzyme-linked immunosorbent assay protocol has been optimized with special emphasis given to assay standardization and quality control. Technical aspects such as choice of a microplate, antigen immobilization, buffer composition, optimal screening dilution of sera, and kinetics of the enzymatic reaction were studied and evaluated in order to design a standard protocol offering maximal analytical sensitivity and specificity, as well as to obtain minimal within- and between-plate variability. Among the 27 plates tested, the Nunc 475-094 and 269-620 immunoplates were found to be the best in terms of high positive-to-negative ratio and low variability. No significant differences in antigen immobilization were found by using buffers of various compositions or pHs; however, the presence of magnesium ions (Mg2+; 0.02 M) resulted in a twofold increase in nonspecific background. An optimal screening dilution of sera was established at 1:200. A 1-h incubation period for test serum was found to be optimal. Maximum enzymatic activity for peroxidase was obtained by adjusting both substrate (H2O2) and hydrogen donor [2,2' -azinobis(3-ethylbenz-thiazoline sulfonic acid)] concentrations to 4 and 1 mM, respectively. To control between-plate variability, a timing protocol was adopted. Within-plate variability was also controlled by using a sample placement configuration pattern. Sliding scales were determined by repeated testing of a cross section of samples to set acceptance limits for both within- and between-plate variability. These limits were used in a quality control program to monitor assay performance. The results obtained suggest that this standardized protocol might be useful in the serodiagnosis of Actinobacillus pleuropneumoniae serotype 5.     

Staphylococcus aureus strains that express serotype 5 or serotype 8 capsular polysaccharides differ in virulence

Most isolates of Staphylococcus aureus produce a serotype 5 (CP5) or 8 (CP8) capsular polysaccharide. To investigate whether CP5 and CP8 differ in their biological properties, we created isogenic mutants of S. aureus Reynolds that expressed CP5, CP8, or no capsule. Biochemical analyses of CP5 and CP8 purified from the isogenic S. aureus strains were consistent with published structures. The degree of O acetylation of each polysaccharide was similar, but CP5 showed a greater degree of N acetylation. Mice challenged with the CP5(+) strain showed a significantly higher bacteremia level than mice challenged with the CP8(+) strain. Similarly, the CP5(+) strain survived preferentially in the bloodstream and kidneys of infected mice challenged with a mixed inoculum containing both strains. The enhanced virulence of the CP5(+) strain in vivo correlated with its greater resistance to in vitro killing in whole mouse blood. Likewise, in vitro opsonophagocytic killing assays with human neutrophils and sera revealed greater survival of the Reynolds (CP5) strain, even though the kinetics of opsonization by C3b and iC3b was similar for both the CP5(+) and CP8(+) strains. Electron micrographs demonstrated C3 molecules on the cell wall beneath the capsule layer for both serotype 5 and 8 strains. Purified CP5 and CP8 stimulated a modest oxidative burst in human neutrophils but failed to activate the alternative complement pathway. These results indicate that CP5 and CP8 differ in a number of biological properties, and these differences likely contribute to the relative virulence of serotype 5 and 8 S. aureus in vivo.