Actinobacillus Pleuropneumoniae Serotype Research Articles

Pulmonary Lesions in Mice Inoculated with Actinobacillus pleuropneumoniae Hemolysin and Lipopolysaccharide

Non-Swiss albino (CF1) male mice were used as a model to study the effects of specific toxic components of Actinobacillus pleuropneumoniae on the respiratory system. Mice were divided into five groups of ten each and were inoculated by the intranasal route with whole-cell Actinobacillus pleuropneumoniae serotype 1, cell-free culture supernatant fluid (CFCSF) containing hemolysin protein, purified lipopolysaccharide (LPS), heat-treated CFCSF, or phosphate-buffered saline solution. Pulmonary lesions were evaluated at 6 and 12 hours after inoculation. Mice inoculated with whole cells, CFCSF, and LPS developed severe purulent bronchiolitis and alveolitis. Focal pulmonary necrosis was also observed in these three groups but was most consistent in the CFCSF-inoculated mice. Ultrastructurally, lungs from mice inoculated with whole cells, LPS, and CFCSF were characterized by severe degenerative changes in type I and type II pneumocytes. Alveolar spaces contained cellular debris and fibrin. Endothelial cells were swollen, and selected pulmonary capillaries were occluded with platelets and fibrin. Infiltrating neutrophils were often swollen, vacuolated, and degranulated. Although inoculation with relatively large numbers of A. pleuropneumoniae are required to kill mice, the mouse lung appears quite sensitive to the toxic components produced by these bacteria. The elaboration of these two toxic components by A. pleuropneumoniae may be responsible for the characteristic pulmonary inflammatory and necrotic lesions observed in infected swine.

Blocking enzyme-linked immunosorbent assay for detection of antibodies against Actinobacillus pleuropneumoniae serotype 8

A blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against Actinobacillus pleuropneumoniae serotype 8 is described. The test is based upon a competitive reaction between the serum to be tested and polyclonal rabbit antiserum prepared against serotype 8 and absorbed with whole cells of serotypes 3 and 6. The blocking ELISA proved able to distinguish animals infected with serotype 8 from those infected with any other serotype of A. pleuropneumoniae including the antigenically closely related serotypes 3 and 6. Positive results were not observed with field sera from serotype 6 infected herds or from herds known to be free from A. pleuropneumoniae infection.

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.

Antibodies against Actinobacillus pleuropneumoniae serotype 2 in mucosal secretions and sera of infected pigs as demonstrated by an enzyme-linked immunosorbent assay

Mucosal and serum antibodies against Actinobacillus pleuropneumoniae serotype 2 were demonstrable in an ELISA which used whole bacterial cells as antigen. Following experimental infection of pigs with A pleuropneumoniae serotype 2, specific antibodies against the bacteria were demonstrated in saliva and fluid obtained by bronchoalveolar lavage. There was a good correlation between the IgA antibody titre in this fluid and serum. As a result of natural infection, IgA antibodies were also demonstrable in the saliva at an early stage of infection when no serum antibodies were detectable. Later in the infection, when antibodies were present in the serum, salivary IgA antibodies were undetectable. The data suggest that the mucosal IgA antibody response arises before the systemic response. Measuring mucosal antibodies in saliva or nasal or bronchoalveolar fluid might therefore open the possibility of identifying pigs infected with A pleuropneumoniae at an early stage of infection.

HlyIA-like sequence present in a genetic locus with low homology to appA in Actinobacillus pleuropneumoniae serotype 10.

Using the appA gene encoding Actinobacillus pleuropneumoniae-RTX-toxin II (ApxII) as a probe, Southern hybridization demonstrated the presence of appA-sequence in all 12 serotypes except serotype 10. Serotype 10 strains had a genetic locus with low homology to appA (tentatively designated Lha). DNA sequence analysis revealed that Lha contained a part of hlyIA gene encoding a 105-kDa hemolysin ApxI. Southern hybridization demonstrated the presence of Lha (hlyIA) sequence not only in serotype 10 but also in serotypes 1, 5a, 5b, 9, and 11, which had strong hemolytic activity with sheep red blood cells and were highly virulent for mice. These results suggest that ApxI may be associated with the virulence phenotype of the organism.

Use of passive immunization for the production of monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 1,6 and 12

Hyperimmune sera from BALB/c mice immunized intraperitoneally (IP) with Actinobacillus pleuropneumoniae serotype 2 were used for passive intraperitoneal (i.p.) immunization of BALB/c mice. The immunized mice were subsequently immunized i.p. with a mixture of A. pleuropneumoniae serotypes 1, 6 and 12. Numerous monoclonal antibodies specific for serotypes 1, 6 and 12 were obtained. Using this immunization scheme antibodies can be obtained against specific antigens from closely related bacteria.

Characterization of monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5

SUMMARY Two monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5, designated as 5MAb-l and 5MAb-6, were characterized. Enzyme-linked immunosorbent assay-inhibition tests with whole-cell antigens obtained from strains of serotype 1 through 12 of A pleuropneumoniae revealed that 5 MAb-1 bound to only serotype-5 strains. The epitope recognized by 5MAb-l was a carbohydrate that was sensitive to periodate oxidation and resided on the structure of β-l,6-linked d-galactose in an O-antigen polysaccharide of serotype-5 lipopolysaccharide. Analysis of these results revealted that the O-antigen polysaccharide of lipopolysaccharide was 1 of the antigenic determinants responsible for the serotype specificity of A pleuropneumoniae. On the other hand, 5MAb-6 reacted with strains of serotype 1 through 10 in varying degrees and its epitope was located on polypeptides sensitive to proteinase K. In an immunoblotting analysis, 5MAb-6 reacted with 2 polypeptide bands, with molecular weights of approximately 41,500 and 28,000, in the outer membrane protein-rich fraction obtained from strains of serotype 1 through 10. These results indicated that outer membrane proteins from several serotype strains of A pleuropneumoniae possessed common antigenic determinants.

Immunization of pigs against Actinobacillus pleuropneumoniae with two recombinant protein preparations

Two Actinobacillus pleuropneumoniae serotype 7 antigens were expressed in Escherichia coli and tested for their protective efficacy in an experimental pig model. The antigens used were a fusion protein containing the carboxy-terminal 70% of the ± 103 kDa cytolysin and a full length 60 kDa protein which has been shown previously to bind transferrin. Pigs were immunized twice with 25 μg of either or both preparations. All pigs developed a strong humoral immune response comparable to that induced by infection. Upon challenge with an A. pleuropneumoniae serotype 7 strain, all immunized groups were less affected by the disease and showed significantly lower mortality than the controls ( p < 0.1). Pigs receiving both antigens had a tendency to recover faster than the controls or animals which were vaccinated with only one antigen. Protection was serotype-specific since no cross-protection was detected following challenge with an A. pleuropneumoniae serotype 1 strain. A dose-response experiment using the single antigens at 200, 50 or 12.5 μg per dose showed no difference in protection among the groups.

Identification of the cross-reacting antigen among Actinobacillus pleuropneumoniae strains of serotype 1, 9 and 11 by use of monoclonal antibodies.

Two monoclonal antibodies (MAbs), lMAb-1 and lMAb-5, against Actinobacillus pleuropneumoniae serotype 1 were obtained. In enzyme-linked immunosorbent assay-inhibition tests with whole cell antigens obtained from serotype 1 to 12 strains of A. pleuropneumoniae, lMAb-1 reacted to only a serotype 1, strain 4074. The epitope recognized by lMAb-1 was a carbohydrate sensitive to periodate oxidation and resided on capsular polysaccharide (CP) of A. pleuropneumoniae serotype 1. On the other hand, lMAb-5 reacted with serotype 1, 9 and 11 strains at the same degree and its epitope was found to be located on O-polysaccharide of serotype 1, 9 or 11 lipopolysaccharide (LPS). These results showed that CP was one of the serotype-specific antigens of A. pleuropneumoniae, and that O-polysaccharide of LPS obtained from serotype 1, 9 or 11 strain was the cross-reacting antigen among these strains.

Isolation and molecular characterization of spontaneously occurring cytolysin-negative mutants of Actinobacillus pleuropneumoniae serotype 7

Actinobacillus pleuropneumoniae serotype 7 strains are shown to spontaneously lose cytolytic activity with a frequency of approximately 10(-4). The phenotypic change is associated with the loss of approximately 8.5 kbp of chromosomal DNA. A genomic fragment encoding the cytolysin and its flanking sequences was cloned and characterized. Also, the corresponding truncated fragment was cloned from a spontaneous mutant. Comparison of the two clones allowed the definition of the excision site. The ends of the excised fragment are composed of 1,201 bp long direct identical repeats, possibly facilitating the genotypic change by homologous recombination. In accordance with this hypothesis, one repeat is conserved in the spontaneous mutant. Each repeat contains one open reading frame preceded by a Shine-Dalgarno consensus sequence, and the ends of each repeat contain 26-bp complementary sequences with four mismatches.

Cross-reactivity between Actinobacillus pleuropneumoniae serotypes comparing different antigens and serological tests

Several antigens were prepared from suspensions of reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae (saline extract [ SE], capsular extract [ CE], whole cell suspension [ WCS], boiled extract [ BE], and autoclaved cell antigen [ ACA]). The cross reactions between each antigen and the antisera against reference strains of the other 11 serotypes were compared by using the complement fixation test, ELISA and the indirect haemagglutination test. ACA produced the most cross reactions, which, in some serotypes, took place in all the antisera tested. BE produced fewer cross reactions, but these were more abundant than those obtained with SE, CE or WCS. The least cross reactivity occurred with SE in the indirect haemagglutination test. This test is, therefore, the most reliable method for serotyping field strains of A pleuropneumoniae.

Calcium binds to and is required for biological activity of the 104-kilodalton hemolysin produced by Actinobacillus pleuropneumoniae serotype 1

Actinobacillus pleuropneumoniae serotype 1, strain Shope 4074, was grown on agar medium containing 10 mM Ca2+ and under Ca2+ limiting conditions by addition of 2 and 5 mM EGTA to the growth medium. Hemolysis of washed bovine erythrocytes was observed from the culture grown in Ca2+ excess but not from the two cultures where Ca2+ was chelated from the growth medium by using EGTA. However, the hemolytic activity of these latter two cultures could be restored if 10 mM Ca2+ was added to the dilution buffer. This restored hemolytic activity could be neutralized with a rabbit homologous polyclonal antiserum specific for the 104-kDa hemolysin of serotype 1 but not with preimmune serum from the same rabbit. Stained SDS-PAGE gels and immunoblots showed the 104-kDa protein hemolysin in fractions from each of the three growth conditions. Thus, the restored hemolytic activity was associated with the 104-kDa protein in the three cultures. In addition, the 104-kDa protein, when electroblotted onto nylon membranes, bound 45Ca, indicating that the molecule has binding sites for Ca2+. The results indicate that Ca2+ is required for the biological activity of the 104-kDa hemolysin of A. pleuropneumoniae serotype 1.

Grouping of Actinobacillus pleuropneumoniae strains of serotypes 1 through 12 on the basis of their virulence in mice

Variations in virulence among strains of different serotypes of Actinobacillus pleuropneumoniae were detected on intraperitoneal or intranasal inoculation in mice. In general, strains of serotypes 1, 5, 9, 10 and 11 were found to be highly virulent and those of serotypes 2, 3, 4, 6, 7, 8 and 12 to be less virulent. However, a few strains of serotype 5 caused low mortality in mice while some strains of serotype 3 and 7 were found to be highly virulent. Highly virulent strains of A. pleuropneumoniae were invasive and appeared in the blood within 3 to 6 h of intranasal inoculation. The type specific antigen as detected by the coagglutination test was distributed in lungs, liver, heart and spleen after intraperitoneal inoculation whereas it was mostly concentrated in the lungs after intranasal inoculation. Lowest concentration of boiled whole-cells suspension of A. pleropneumoniae showing limulus amebocyte lysate activity was variable and independent of the serotype. Mortality caused by boiled whole cell suspension was also variable and serotype independent.

Structural characteristics of the antigenic capsular polysaccharides and lipopolysaccharides involved in the serological classification of Actinobacillus (Haemophilus) pleuropneumoniae strains

The detailed structures of the specific capsular polysaccharides and cellular lipopolysaccharides of the 12 known serotypes of Actinobacillus (Haemophilus) pleuropneumoniae are presented and their serological relationships are discussed together with their significance in the control of swine pleuropneumonia.

Chronic pleuritis in pigs for slaughter: an epidemiological study of infectious and rearing system-related risk factors

A representative sample of 4800 slaughter pigs was taken at a large Danish abattoir. Blood samples were obtained, chronic pleuritis (CP) lesions were recorded and routine slaughter information (meat inspection data, sex, weight and herd identification) was noted. All sera were examined for specific antibodies against Actinobacillus pleuropneumoniae serotype 2 and 6 (AP2, AP6), Hemophilus parasuis and Influenza suis. A questionnaire was forwarded to the herds supplying pigs to the sample requesting information about the production system, herd size and vaccination programs. A total of 623 herds supplied pigs to the sample, yielding an average of 7.3 pigs per herd. Of the herd owners, 513 responded to the questionnaire. CP was detected in 28.0% of all carcasses. The serological survey revealed positive titers for AP2 and AP6 in 27.6 and 10.3%, for H. parasuis in 40.3% and for I. suis in 49.9% of the pigs, respectively. Corrected for confounding factors in a multiple logistic regression model, adjusted odds ratios for the serological risk factors with the strongest association with CP were AP2 = 9.21, AP6 = 1.72 and I. suis = 1.25. Atrophic rhinitis, decreasing weight at slaughter (reflecting age differences) and increasing herd size were also significantly associated with an increase in the risk of CP. H. parasuis, male (as opposed to female), fattening herd (relative to sow herd) and conventional herd (relative to specific pathogen-free) were not associated with an increased risk of CP. This study demonstrated that AP2 is probably the factor with the largest impact on the frequency of CP in Danish pigs for slaughter. It was calculated that 44% of all CP lesions in the sample could be attributed to this infection. However, I. suis is also of importance, mainly because of its high prevalence. It was estimated that 11% of all CP lesions could be explained by I suis.

Structural analysis of the lipopolysaccharide of Actinobacillus (Haemophilus) pleuropneumoniae serotype 10

The antigenic O-chain of the lipopolysaccharide produced by Actinobacillus (Haemophilus) pleuropneumoniae serotype 10 was shown by chemical and magnetic resonance methods to be a unique linear unbranched homopolymer of exclusively 1,2-linked beta-D-galactofuranose residues.

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.

Studies on the interactions of two different serotypes of Actinobacillus pleuropneumoniae

In vitro and in vivo interactions of various field strains of Actinobacillus pleuropneumoniae of serotypes 1, 2, 5 and 7 were studied. There was no influence of one serotype over the other when strains belonging to two serotypes were cultivated together in vitro. In vivo interactions showed predominance of serotype 1 over other serotypes when a strain of serotype 1 was inoculated together with a strain of serotype 2 or 5 in mice. Serotype 1 strain remained predominant irrespective of whether it was inoculated before or after the inoculation of serotype 2 strain. The mortality caused by the inoculation of two strains was higher than the mortality caused by a single strain. Early mortality was observed on inoculation of strains of serotype 2, 5 or 7 along with a strain of serotype 1. Both serotypes could be detected in the blood on cultural examination of mice infected with mixed serotypes.

Production and characterization of monoclonal antibodies against Actinobacillus (Haemophilus) pleuropneumoniae serotype 2.

Four hybridoma cell lines producing monoclonal antibodies (MAbs) against Actinobacillus (Haemophilus) pleuropneumoniae were established by fusion of mouse myeloma and spleen cells obtained from mice immunized with a serotype 2, strain SH-15. Enzyme-linked immunosorbent assay-inhibition tests with antigens obtained from 12 serotype strains of A. pleuropneumoniae and 9 other gram-negative bacteria showed that all the MAbs bound to only serotype 2 strains of A. pleuropneumoniae. The epitopes recognized by the MAbs were located on a carbohydrate moiety of lipopolysaccharide (LPS) of the organism, which was sensitive to periodate oxidation. In immunoblotting analyses of LPS obtained from A. pleuropneumoniae serotype 2, all the four MAbs reacted specifically with the characteristic ladder bands of LPS detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that O-antigen side chains of the LPS are one of the antigenic determinants responsible for the serotype-specificity of A. pleuropneumoniae.

Isolation of the Actinobacillus pleuropneumoniae haemolysin gene and the activation and secretion of the prohaemolysin by the HlyC, HlyB and HlyD proteins of Escherichia coli

The gene encoding the c. 105 kD secreted haemolysin protein of the porcine pathogen Actinobacillus pleuropneumoniae serotype 1 has been isolated by screening a lambda gt11 expression library in Escherichia coli with antiserum raised against the wild-type protein. A derivative recombinant DNA pJFF702 expressed the hlylA haemolysin gene from the pUC19 lac promoter but the resulting haemolysin I protein remained within the E. coli cell and was haemolytically inactive. Export of the intracellular A. pleuropneumoniae prohaemolysin out into the medium was achieved by the presence in trans of the E. coli haemolysin secretion genes hlyB and hlyD, and high levels of intracellular haemolytic activity were attained similarly by the E. coli post-translational haemolysin activator gene, hlyC. Southern hybridization of A. pleuropneumoniae parental DNA nevertheless indicated only a low degree of nucleotide sequence identity to the haemolysin structural and secretion genes hlyA and hlyB of E. coli. The data show that despite substantial nucleotide sequence divergence the A. pleuropneumoniae serotype 1 haemolysin determinant is closely related to that which is dispersed throughout other Gram-negative human and animal pathogens.