Cross-reactivity and antigenic heterogeneity among Actinobacillus pleuropneumoniae strains of serotypes 4 and 7.

Actinobacillus pleuropneumoniae strains of serotypes 4 and 7 were studied for their antigenic properties by means of agglutination, coagglutination, indirect hemagglutination, immunodiffusion, and counterimmunoelectrophoresis tests. Strains of serotype 4 showed cross-reactivity with those of serotype 7 in various serological tests. Serotype 7 strains were antigenically heterogeneous and shared common antigens with several other serotypes. By using boiled whole-cell saline extract as the antigen in the immunodiffusion test, serotype 7 strains could be divided into four subgroups. Subgroup I strains did not have antigens in common with other serotypes, whereas subgroup II strains had antigens in common with serotype 4; subgroup III strains had antigens in common with serotype 10, and subgroup IV had antigens in common with serotypes 1, 9, and 11. The indirect hemagglutination test using unheated whole-cell saline extract as the antigen detected serotype-specific activity. Quantification of serotype-specific and group-specific antigens by coagglutination and immunodiffusion tests was found useful for identifying strains that belonged to serotype 4 or 7.     

Analysis of major antigens of Haemophilus (Actinobacillus) pleuropneumoniae and related organisms.

Outer membrane protein (OMP)-enriched extracts and whole-cell protein preparations of Haemophilus (Actinobacillus) pleuropneumoniae and related organisms were examined by polyacrylamide gel electrophoresis and immunoblotting. Both the OMP-enriched and whole-cell protein profiles of Actinobacillus suis, A. pleuropneumoniae (NAD-independent biovar), A. lignieresii, and Pasteurella haemolytica were very similar to those of H. pleuropneumoniae serotypes 1 to 8. Antisera prepared against H. pleuropneumoniae typically recognized three major OMP antigens with approximate molecular weights of 17,000 (17K), 32K, and 42K in immunoblots of H. pleuropneumoniae serotypes 1 to 8, Actinobacillus spp., and P. haemolytica. Antisera prepared against Actinobacillus spp. and Haemophilus sp. "minor group" also recognized these 17K, 32K, and 42K antigens. Using absorbed sera, we demonstrated that the 17K antigen had an epitope (or epitopes) common to all the gram-negative organisms examined, including Escherichia coli. The 32K and 42K antigens had epitopes common to members of the family Pasteurellaceae but, in the case of the 32K antigen, also contained unique epitopes. These results provide a basis for understanding the lack of specificity of serodiagnostic tests for H. pleuropneumoniae infection and provide another line of evidence for the association of H. pleuropneumoniae with the genus Actinobacillus.     

Molecular cloning and characterization of a hemolysin gene from Actinobacillus (Haemophilus) pleuropneumoniae.

This article describes the molecular cloning and expression of a hemolysin gene from a serotype 1 strain of Actinobacillus pleuropneumoniae. The hemolysin was a thermolabile protein with an apparent molecular weight of 29,500 (29.5K hemolysin). Unlike expression of the recently described 105K hemolysin of A. pleuropneumoniae (J. Frey and J. Nicolet, FEMS Microbiol. Lett. 55:41-46, 1988), expression of this hemolysin was not regulated by Ca2+. Antiserum prepared against the 105K hemolysin did not neutralize the activity of the 29.5K hemolysin; conversely, antiserum prepared against the 29.5K hemolysin did not neutralize the activity of the 105K hemolysin. The hemolytic activity was not neutralized with antisera against hemolytic Escherichia coli, Streptococcus agalactiae, or purified streptolysin O, but antisera prepared against recombinants containing the 29.5K gene and convalescent pig sera abrogated hemolytic activity. Although hemolytic activity could be detected in several strains of E. coli K-12 and in minicells expressing several different constructs encoding the 29.5K hemolysin, we could not rigorously exclude the possibility that the gene which we have isolated encodes a regulator of hemolytic activity rather than a hemolysin per se.     

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.     

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.     

Quantitation of serotype-specific and cross-reacting group-specific antigens by coagglutination and immunodiffusion tests for differentiating Actinobacillus (Haemophilus) pleuropneumoniae strains belonging to cross-reacting serotypes 3, 6, and 8.

There are strong cross-reactions among strains of Actinobacillus pleuropneumoniae belonging to serotypes 3, 6, and 8. Various serological tests were used to differentiate these serotypes from each other. Tube agglutination, coagglutination, and indirect hemagglutination tests were not sufficiently sensitive to differentiate strains of serotypes 3, 6, and 8. However, higher antibody titers were obtained with a 2-mercaptoethanol agglutination test in homologous rabbit antisera. Absorption of immune sera with homologous and heterologous serotypes as well as quantitative estimation of antigenic activity in the unheated and heat-treated bacterial cell suspensions of reference strains with rabbit homologous and heterologous antisera revealed serotype-specific and cross-reacting group-specific antigens. Usually, serotype-specific antigens were major and dominant over group-specific antigens. The coagglutination test could be used quantitatively to measure the ratio of serotype-specific and group-specific antigens with rabbit hyperimmune sera against serotypes 3, 6, and 8. The highest antigen content for a particular serotype reflected serotype-specific antigen. For strains showing equal amounts of antigen for two or more serotypes in the coagglutination test, the immunodiffusion test with boiled cell-saline extract as the antigen and rabbit antisera against whole-cell suspensions of serotypes 3, 6, and 8 clearly revealed the serotype-specific antigen. It is suggested that coagglutination and immunodiffusion tests could be used successfully to determine the exact serotype of strains belonging to serotypes 3, 6, and 8.     

Effect of iron restriction on the outer membrane proteins of Actinobacillus (Haemophilus) pleuropneumoniae.

The outer membrane protein profile of Actinobacillus (Haemophilus) pleuropneumoniae grown under iron-restricted and iron-replete conditions was studied by polyacrylamide gel electrophoresis and immunoblotting. A virulent serotype 1 isolate synthesized a novel protein with an apparent molecular weight of 105,000 (105K) and increased the synthesis of a 76K protein under iron-restricted conditions. Both proteins were synthesized within 15 min of establishment of iron-restricted conditions. Proteins of equivalent molecular weights could also be induced by iron restriction in serotype 2, 3, 4, 5, and 7 isolates of A. pleuropneumoniae. Convalescent-phase sera from serotype 1-infected pigs contained antibodies which recognized both the 105K and 76K proteins from all six serotypes examined, indicating that these proteins were expressed in vivo and were immunologically conserved. Cells expressing the 105K and 76K proteins also displayed an enhanced ability to bind Congo red and hemin, suggesting that one or both of these proteins functioned to acquire complexed iron during in vivo growth.     

Production of Apx toxins by field strains of Actinobacillus pleuropneumoniae and Actinobacillus suis.

The three Apx toxins of Actinobacillus pleuropneumoniae have potential value for use in vaccines and diagnostic tests which will be species specific instead of serotype specific, provided that the Apx toxins are species specific and all field strains produce these toxins. We examined 114 A. pleuropneumoniae field strains and found that they secreted either ApxI, ApxII, ApxI and ApxII, or ApxII and ApxIII and secreted no other cytolytic activities. However, proteins similar to ApxI and ApxII were also produced by Actinobacillus suis.     

Identification of a maltose-inducible major outer membrane protein in Actinobacillus (Haemophilus) pleuropneumoniae

The addition of maltose to the growth media of Actinobacillus pleuropneumonia (serotype 1) resulted in the induction of an outer membrane protein (OMP) with a molecular mass of 42 kDa. This protein had porin-like properties in that it was peptidoglycan-associated and was resistant to proteolysis by trypsin. A pleuropneumoniae expressing the 42 kDa OMP were unable to bind lambda phage. Similar proteins were also induced in A. pleuropneumoniae isolates representing serotypes 2 to 7 with the exception of serotype 4; however, not all isolates of any given serotype expressed a maltose-inducible OMP. Western immunoblotting using convalescent antiserae against the serotype 1 A. pleuropneumoniae indicated that the 42 kDa OMP was expressed in vivo and was cross-reactive with the maltose-inducible OMPs from other serotypes.     

Comparison of the cytolysin II genetic determinants of Actinobacillus pleuropneumoniae serotypes.

Cytolysins (Cly) I, II, and III are toxins secreted by Actinobacillus pleuropneumoniae. These toxins are thought to play an important role in the pathogenesis of porcine pleuropneumonia. ClyI and ClyII are RTX toxins and in general these toxins are encoded by operons consisting of four genes, C, A, B, and D. Our group recently cloned the C and A genes of the ClyII operon (clyIICA) of serotype 9. We found that this ClyII operon is truncated and lacked intact B and D genes (clyIIBD). B and D genes of the ClyI operon (clyIBD) were present however in serotype 9. In this study we analyzed the ClyII operons of the reference strains of the 12 A. pleuropneumoniae serotypes and compared them with the ClyII operon of serotype 9. We focused on (i) the presence, (ii) the sequence similarity, and (iii) the genomic environment of the clyIICA genes. The presence of the clyIICA sequences was studied by hybridization analysis of genomic DNA. The sequence similarity was studied by restriction fragment analysis on polymerase chain reaction-amplified DNA. The genomic environment was compared by analysis of the sequences that are located 3' of the clyIICA genes. We demonstrated that the clyIICA genes (i) are present in the reference strains of all serotypes, except serotype 10, (ii) have a high degree of sequence similarity, and (iii) are not contiguous with intact clyIIBD genes. We conclude that the organization and nucleotide sequence of the ClyII operons of A. pleuropneumoniae are very similar. We also studied the presence of clyIBD sequences and found them to be present in the reference strains of all serotypes, except serotypes 3 and 6. Thus, in most serotypes the clyIBD genes may complement the absent clyIIBD genes.     

Structures and sugar compositions of lipopolysaccharides isolated from seven Actinobacillus pleuropneumoniae serotypes.

Highly purified lipopolysaccharide (LPS) preparations obtained from seven Actinobacillus pleuropneumoniae strains representative of seven different serotypes were used to determine the structure and monosaccharide composition of the polysaccharide components of each lipopolysaccharide. An indication of the structure of each LPS was obtained by procedures that included sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining and gel chromatographic fractionation of acetic acid-hydrolyzed LPS. The polysaccharide components of the LPSs were analyzed by gas-liquid chromatography. The LPSs of the strains of serotypes 2, 4, and 7 were of the smooth type, and those of the strains of serotypes 3 and 6 were of the rough type; the LPSs of the strains of serotypes 1 and 5 could be considered semirough. Rhamnose was present only in the O polysaccharide of the smooth-type and semirough-type LPSs, whereas galactose was present only in the O polysaccharide of the smooth-type LPS and in the core oligosaccharides of the rough-type and semirough-type LPSs. Glucoheptose and mannoheptose were present in the core oligosaccharides of all the LPSs except for the strain of serotype 3, in which only mannoheptose was detected. N-Acetylglucosamine was detected only in the O polysaccharides of the strains of serotypes 1 and 5.     

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.     

Serotype-related differences in production and type of heat-labile hemolysin and heat-labile cytotoxin of Actinobacillus (Haemophilus) pleuropneumoniae.

Reference strains of serotypes 1 to 12 of Actinobacillus (Haemophilus) pleuropneumoniae were cultured in Eagle minimal essential medium with 10% Serum Plus. Culture supernatants were examined for cytotoxicity to alveolar macrophages and for the ability to hemolyze sheep erythrocytes. All strains except the reference strain of serotype 6 produced cytotoxin, whereas only serotypes 1, 5, 9, 10, and 11 produced hemolysin. Both cytotoxin and hemolysin appeared to be heat labile. Antisera were raised against cytotoxin- and hemolysin-containing culture supernatants of serotypes 1 to 11. Cross-neutralization studies revealed that the hemolysins were serologically homogeneous. In contrast, four serologically different cytotoxins were distinguished. One cytotoxin was produced by serotypes 1, 5, 9, and 11, and a second was produced by serotypes 2, 3, 4, and 8. A third cytotoxin was produced by serotypes 7 and 12; this cytotoxin was related to the cytotoxins of serotypes 1, 2, 4, 5, 9, and 11. A fourth cytotoxin, produced by serotype 10, was related to the cytotoxin of serotypes 1, 5, 9, and 11. Seventy field strains belonging to serotypes 2, 3, 7, 8, 9, and 11 were also tested for production of cytotoxin and hemolysin. All strains belonging to serotypes 9 and 11 produced hemolysin and cytotoxin, whereas all strains of serotypes 2, 3, 7, and 8 produced only cytotoxin. Hemolysins and cytotoxins of both the field strains and the corresponding serotype reference strains were comparably neutralized. These findings strongly suggest that the observed differences in production and type of hemolysin and cytotoxin were related to serotype and not to strain.     

RTX toxin genotypes and phenotypes in Actinobacillus pleuropneumoniae field strains.

Actinobacillus pleuropneumoniae serotype reference strains and 204 A. pleuropneumoniae field strains representing all 12 serotypes and both biovars 1 and 2, obtained from laboratories from various countries worldwide, were analyzed for the presence of the toxin genes apxIC, apxIA, apxIB, apxID, apxIIC, apxIIA, apxIIIC, apxIIIA, apxIIIB, and apxIIID by DNA-DNA hybridization with specific gene probes. Expression of the toxins ApxI, ApxII, and ApxIII was assessed by immunoblot analysis with monoclonal antibodies. The results show that the patterns of apx genes and those of the expressed Apx toxins in biovar 1 field strains are the same as those of the genes and toxins of corresponding serotype reference strain. We found only three strains which had certain apx genes missing compared with the genes in their serotype reference strains. Analysis of the expression of the three toxins showed that nearly all strains expressed their apx genes and produced the same Apx toxins as their serotype reference strain. We found only one strain that did not produce ApxI, although it contained the apxICABD genes, and one strain which did not express ApxII but which contained apxIICA. Several field strains which initially showed that their serotype did not correspond to the apx gene profile of the reference strain and which had an unexpected virulence for the given serotype revealed that their initial serotyping was erroneous. We show that the apx gene profiles are inherent to a given serotype. The method cannot differentiate between all 12 serotypes. However, it allowed us to distinguish five groups of toxin gene patterns which showed pathological, toxicological, and epidemiological significance. None of the biovar 2 strains contained apxIII genes. The apxI and apxII genes in the biovar 2 strains, however, were the same as those found in the serotype reference strains of biovar 1.     

Detection and identification of Actinobacillus pleuropneumoniae serotypes 1, 2, and 8 by multiplex PCR

Multiplex PCR assays were developed to identify Actinobacillus pleuropneumoniae serotypes 1, 2, and 8. Primers designed for the conserved capsular polysaccharide (CP) export region amplified a 489-bp DNA fragment from all serotypes. Primers specific to the CP biosynthesis regions of serotypes 1, 2, and 8 amplified fragments of 1.6 kb, 1.7 kb, and 970 bp from only their respective serotypes.     

Hemolysin patterns of Actinobacillus pleuropneumoniae.

The secreted hemolytic activities produced by the reference strains and field isolates of the 12 serotypes and 2 subtypes of Actinobacillus pleuropneumoniae were analyzed. Serotype 1 produced a Ca2(+)-inducible hemolysin, which was previously characterized as a 105-kilodalton protein and was named hemolysin I (HlyI). Serotypes 2, 4, 6, 7, and 8 produced a different hemolytic activity that was not inducible by Ca2+ but required this ion for its activity. The hemolytic activity produced by these serotypes was much weaker than that found in serotype 1 and was not neutralized by rabbit antibodies against HlyI. It was, however, neutralized by serum from pigs that were experimentally infected with a serotype 2 strain and was called hemolysin II (HlyII). Serotypes 5a, 5b, 9, 10, and 11 produced both HlyI and HlyII. In these strains, HlyI was the major contributor to the hemolytic activity. The remaining serotypes, 3 and 12, produced a very weak hemolytic activity, which was not further analyzed. Immunoblot analysis of the culture supernatants from all 12 serotypes with rabbit polyclonal antibodies directed against HlyI revealed reactions with a protein in the 105-kilodalton size range for all serotypes, indicating that HlyI and HlyII might be serologically related. Strains producing active HlyI seem to belong to serotypes that are generally considered to be virulent types and that are frequently isolated from pigs in severe pleuropneumonia outbreaks.     

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.     

Morphology and ultrastructure of oral strains of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

Selected human oral and nonoral strains of the genera Actinobacillus and Haemophilus were examined by transmission and scanning electron microscopy. The strains examined were morphologically identical to recognized Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus. By transmission electron microscopy, the cells were typically gram negative in morphology, with several strains possessing some extracellular ruthenium red-staining polymeric material. Numerous vesicular structures, morphologically identical to lipopolysaccharide vesicles, were seen to originate from and be continuous with the surface of the outer membrane. Large numbers of these vesicles were also found in the external environment. Scanning electron microscopic observations revealed that both actinobacilli and haemophili possessed surface projections and an amorphous surface material which connected and covered adjacent cells.     

Molecular characterization of a protective outer membrane lipoprotein (OmlA) from Actinobacillus pleuropneumoniae serotype 1.

An expression library was constructed from an Actinobacillus pleuropneumoniae serotype 1 clinical isolate using a plasmid vector. The library was screened with serum raised against the culture supernatant of this strain. One Escherichia coli transformant which also reacted with convalescent serum was isolated and found to express a protein with an electrophoretic mobility of approximately 50,000. The A. pleuropneumoniae-derived DNA encoding the protein was localized and characterized by nucleotide sequence analysis and primer extension mapping. One open reading frame of 1,095 bases was detected and confirmed by TnphoA insertion mutagenesis. It encoded a protein with a calculated molecular mass of 40 kDa which was lipid modified and present in the outer membrane and in membrane blebs of A. pleuropneumoniae. This protein was designated as outer membrane lipoprotein A (OmlA), and the encoding gene as omlA. Southern blotting under low-stringency conditions revealed the presence of hybridizing sequences in all A. pleuropneumoniae type strains, and a specific serum detected a homologous protein in serotypes 2, 8, 9, 11, and 12 type strains. Pigs immunized with this recombinant protein preparation were protected from death in an aerosol challenge experiment with an A. pleuropneumoniae serotype 1 isolate.