Serological characterization of Actinobacillus pleuropneumoniae serotype 2 strains by using polyclonal and monoclonal antibodies

The antigenic differences between strains of serotype 2 of both biotypes I and II of Actinobacillus pleuropneumoniae were studied by using several serological techniques. Monoclonal antibodies (MAbs) against A. pleuropneumoniae biotype I serotype 2 were produced by fusion of spleen cells of BALb/c mice immunized with whole-cell (WC) suspension with SP2/O-Ag14 murine myeloma cells. Desirable MAbs were selected by enzyme-linked immunosorbent assay (ELISA) using WC as antigen. MAbs MK-7 and MK-10 identified multiple bands of lipopolysaccharide in Western-blot. Treatment of WC with proteinase K and sodium periodate indicated that both MAb binding sites were carbohydrates in nature. In both ELISA and Western-blot, MAbs MK-7 and MK-10 recognized only biotype I serotype 2 isolates. Neither MAb MK-7 nor MK-10 reacted with reference strains of remaining serotypes of A. pleuropneumoniae and other Gram-negative bacteria tested. The results obtained with various serological tests showed that strains of serotype 2 biotype I shared antigenic determinants with strain N-282 of serotype 2 biotype II, but not with strain N-273 of serotype 1 biotype II. It is suggested that data obtained from this study may be helpful in the development of specific serotyping and serodiagnostic reagents of A. pleuropneumoniae strains.     

Serodiagnosis of swine pleuropneumonia due to Actinobacillus pleuropneumoniae serotypes 7 and 4 using long-chain lipopolysaccharides.

A saline boiled extract (SBE), capsular polysaccharides (CPS) and long-chain lipopolysaccharides (LC-LPS) of Actinobacillus pleuropneumoniae serotype 7 have been evaluated in ELISA for the serodiagnosis of swine pleuropneumonia caused by this serotype. Mean optical densities (ODs) obtained with the 3 antigens using sera from negative herds as well as from animals experimentally and naturally exposed to A. pleuropneumoniae serotypes 7 or 4 were not statistically different. The positive ELISA reaction with anti-serotype 4 sera was unexpected with the CPS, which are supposed to be serotype-specific; LPS traces present in the CPS appeared to be responsible for this reaction. In addition, sera from animals exposed to A. pleuropneumoniae serotypes 5 or 10 presented cross-reactions with the SBE and the CPS, but not with the LC-LPS. Cross-reactions were mainly due to rough LPS, as shown by immunoblotting. The LC-LPS is easily obtainable and can be used for the detection of antibodies in animals infected with A. pleuropneumoniae serotypes 7 and 4.     

Detection of antibodies against Actinobacillus pleuropneumoniae serotypes 1, 2, 5 and 7 using the immunohistochemical staining.

Whole cells of Actinobacillus pleuropneumoniae (A. pleuropneumoniae) serotype 1, 2, 5 or 7 attached to fibrins were fixed in 10% neutral buffered formalin and embedded in paraffin. The sections on a slide glass were stained by the avidin-biotin complex immunoperoxidase (ABC) method. Test sera were applied to sections as primary antibodies. The serum antibodies against A.pleuropneumoniae (serotypes 1, 2, 5 and 7) were measured by the ABC method and complement fixation (CF) test. There was good correlation between the ABC and CF tests. The present results indicate that the immunohistochemical staining is as useful as the CF test for the detection and quantification of antibody in swine sera.     

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.     

Atypical Actinobacillus pleuropneumoniae isolates that share antigenic determinants with both serotypes 1 and 7.

In the present study, the characterization of 3 atypical isolates of Actinobacillus pleuropneumoniae is presented. Two isolates (1B and 27E) showed positive reactions in coagglutination, immunodiffusion, and indirect hemagglutination tests for serotypes 1 and 7, whereas the third isolate (26B) reacted with antisera to serotypes 1, 4, and 7. These atypical isolates of A. pleuropneumoniae possessed a capsular polysaccharide (CPS) antigenically related to serotype 1 as well as an O-chain lipopolysaccharide antigenically related to serotype 7 or to serotypes 4 and 7, as shown by the use of monoclonal antibodies. Results of toxin profile and virulence assays for mice and pigs showed them to be more related to A. pleuropneumoniae serotype 7 field isolates. All 3 isolates induced antibodies mainly against serotype 7/4 O-long-chain lipopolysaccharide (LC-LPS) and, to a lesser extent, to the CPS of serotype 1, in experimentally infected pigs. Diagnostic laboratories that use a LC-LPS-based enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of A. pleuropneumoniae infection in swine would probably diagnose herds infected with these atypical isolates as being infected by A. pleuropneumoniae serotypes 7 or 4, whereas those that use a CPS-based ELISA would probably consider them as infected by A. pleuropneumoniae serotype 1.     

Passive protection of piglets by monoclonal antibodies against experimental infection with Actinobacillus pleuropneumoniae

Mouse monoclonal antibodies 11C11 (an IgG) and 4A9 (an IgM), which combine with a superficial component of cells belonging, respectively, to serovars 1 or 3 of Actinobacillus pleuropneumoniae, were given intraperitoneally 24 hours before and intranasally one hour before two-week-old, colostrum-deprived piglets were exposed by the intranasal route to 10(9) viable cells of either strain Shope 4074 (serovar 1) or 2/10 (serovar 3). Compared with control piglets given phosphate buffered saline or the heterologous monoclonal antibody, this procedure conferred substantial protection against the development of peracute or acute pleuropneumonia. Protection against the experimental disease was somewhat less in other piglets to which monoclonal antibody 4A9 was given only by the intranasal route one hour before the organism was administered than in those given the antibody intraperitoneally 24 hours beforehand, although its effect was still significantly greater than in piglets given phosphate buffered saline only. These two monoclonal antibodies consequently offer means of investigating at the molecular level the pathogenesis of the disease associated with A pleuropneumoniae and the potential value of anti-idiotypes as immunising agents.     

Production and characterization of monoclonal antibodies to Actinobacillus pleuropneumoniae serotype 5b.

Monoclonal antibodies specific for capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of A. pleuropneumoniae serotype 5b were generated by hybridoma cells and selected by indirect ELISA of culture supernatants with purified and structurally defined LPS and CPS preparations and their synthetic conjugates. It was shown in this study that at least one monoclonal antibody, 3B4, presented 100% specificity and recognized all A. pleuropneumoniae serotype 5 field strains tested in a dot-ELISA assay.     

Serologic studies of Actinobacillus (Haemophilus) pleuropneumoniae strains of serotype-3 and their antigenic relationships with other A pleuropneumoniae serotypes in swine

Serotype-3 strains of Actinobacillus (Haemophilus) pleuropneumoniae possess epitopes shared with almost all other Actinobacillus serotypes. Common epitopes detected in particulate antigens were heat-labile and heat-stable and were of minor nature. Additional cross-reactive epitopes were detected in soluble and particulate antigens prepared from strains of serotypes 3, 6, and 8. Cross-reactions occurring between serotype-3 antigens and those of other serotypes were of 2 types: one associated mainly with 2-mercaptoethanol (2-ME)-sensitive IgM antibodies and the other associated mainly with 2-ME-resistant IgG antibodies. The cross-reactivity between serotypes 3, 6, and 8 was associated mainly with IgG antibodies, as shown by the results of 2-ME tube agglutination, 2-ME-indirect hemagglutination, and coagglutination tests. None of the tests was entirely satisfactory for serotyping serotype-3 isolates, mainly as a result of overlapping of type-specific antigenic determinants of serotypes 3, 6, and 8 in different combinations in the same strain. A combination of tests, using particulate and soluble antigens, may be necessary for typing of serotype-3 isolates.     

Serological studies of Actinobacillus (Haemophilus) pleuropneumoniae strains of serotype 6 and their antigenic relationship with other serotypes

Serological tests such as agglutination, coagglutination, precipitation and indirect haemagglutination were used to study the antigenic relationship of reference and field strains of Actinobacillus (Haemophilus) pleuropneumoniae of serotype 6 with reference strains of other serotypes. Both cell-associated particulate and cell-free soluble antigens prepared from unheated and heat-treated bacterial suspensions of reference and field strains of serotype 6 were used in the studies. Species-specific, common antigenic determinants associated mainly with heat-treated particulate antigens of serotype 6 were cross-reactive in tube agglutination tests with almost all the serotypes. The species-specific antigens were of a minor nature because the cross-reactivities were abolished in both 2-mercaptoethanol agglutination and coagglutination tests. Cell-free saline extracts of both unheated and heat-treated suspensions of serotype 6 strains possessed epitopes specific for serotypes 3, 5 and 8 in addition to their own specific determinants. The epitopes were dominant because the reactions of strains of serotype 6 with antisera against serotypes 3, 5 and 8 persisted in almost all the serological tests used. Serotype 6 strains were antigenically closer to serotype 8 than to serotypes 3 or 5. A combination of serological tests such as coagglutination followed by 2-mercaptoethanol tube agglutination and, or, immunodiffusion tests differentiated serotype 6 strains from those of other cross-reacting 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.     

Identification and characterization of fowlpox virus strains using monoclonal antibodies.

The use of 2 monoclonal antibodies (MAbs), P1D9 and P2D4, which recognize different fowlpox virus (FPV) antigens, for the identification and characterization of FPV strains was evaluated. Initially, the MAbs were used in conjunction with a dot blot assay that enabled FPV to be differentiated from the avian herpesvirus, infectious laryngotracheitis virus. Confirmation of the specificity of these MAbs was provided by the demonstration that only FPV antigens were recognized by a combination of both antibodies when used for immunoblotting proteins contained in various avipoxviruses. Later, an antigenic characterization of 11 FPV field isolates, 6 FPV vaccine strains, and 3 pigeonpox virus vaccines was performed by Western blotting with the individual MAbs. Whereas MAb P2D4 consistently recognized a protein with an apparent molecular weight of 60 kD, there was variability in the size of the antigen that was immunoreactive with the other MAb. For example, MAb P1D9 recognized an antigen of apparent molecular weight of 46 kD in all vaccine strains except 2 of FPV origin. In these exceptions, either only a 39-kD or both a 42- and 46-kD protein were immunoreactive. As for the field isolates, a 39-kD antigen was recognized in 8 of them, whereas a 42-kD antigen was detected in the remaining 3. Therefore, the more extensive immunoblotting technique may facilitate FPV strain differentiation, whereas routine diagnosis of fowlpox could be accomplished by using the MAb-based dot blot assay.     

Serodiagnosis of pleuropneumonia using enzyme-linked immunosorbent assay with capsular polysaccharide antigens of Actinobacillus pleuropneumoniae serotypes 1, 2, 5 and 7.

Capsular polysaccharide antigens of serotypes 1, 2, 5 and 7 of Actinobacillus pleuropneumoniae were used in enzyme-linked immunosorbent assays (ELISAs) to test sera from experimentally infected and field pigs. Specific reactions were found in sera of experimental pigs with antigens of serotypes 1, 5 and 7 whereas the serotype 2 antigen was cross-reactive. A 1:200 serum dilution was used for testing of 300 sera from 21 swine herds in southern Ontario. Cases of pleuropneumonia had occurred in 11 of these herds, but not in the others. The negative cut-off value was the mean optical density at 405 nm (OD405) + three standard deviations (SD) for 16 negative reference sera. Sera from four pigs naturally infected with Actinobacillus suis were tested and found to react to varying degrees with each of the antigens. Therefore a second cut-off value was determined as the mean OD405 + 2 SD for the A. suis sera. Sera which, in the ELISA produced OD readings above the latter cut-off were considered positive for antibodies to A. pleuropneumoniae; those which were lower than the former cut-off were considered negative. Readings between the two cut-off values may have been due to low positive titers or cross-reactivity, possibly with A. suis, and could not be used to predict pleuropneumonia. Of the pleuropneumonia-free herds, none had positive reactors to serotypes 5 or 7, whereas one and two herds had positive reactors to serotypes 1 and 2, respectively. Of the pleuropneumonia positive herds, six had positive reactors to serotype 1, one to serotype 2, four to serotype 5, and eight to serotype 7.     

猪胸膜肺炎放线杆菌血清7型25—4株感染及免疫攻毒试验

为了评价猪胸膜肺炎放线杆菌(APP)血清7型25-4株致病性和其灭活疫苗的免疫原性,本研究采用猪胸膜肺炎放线杆菌7型25-4株感染健康猪,每组感染剂量分别为106CFU、107CFU、108CFU,结果发现106CFU感染剂量就可以复制出典型的猪传染性胸膜肺炎病理变化;以该菌株制备的灭活疫苗免疫健康猪21 d后.用106CFU、107CFU 2个不同感染剂量进行强毒攻击,其对照组均发病,免疫组获得100%保护.     

Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes.

A high molecular-mass proteolytic enzyme of Actinobacillus pleuropneumoniae serotype 1, was purified from culture supernatants (CSN) by using DEAE-cellulose and sepharose-4B-gelatin chromatography. In 10% SDS-polyacrylamide gels copolymerized with porcine gelatin, the protease showed a single band of activity of > 200 kDa. However, minor molecular-mass proteolytic bands were observed when the protease was electrophoresed in the presence of either 5% beta-mercaptoethanol, 50 mM dithiothreitol, or 0.25 M urea. Furthermore, when the > 200-kDa purified protein was passed through a sucrose gradient, several bands with proteolytic activity were found: 62, 90, 190, and 540 kDa. The proteolytic activity was increased in the presence of calcium or zinc and was not affected after being heated at 90 degrees C for 5 min. Proteolytic activities were also observed in CSN from all A. pleuropneumoniae serotypes and biotypes. The purified protease hydrolyzed porcine IgA and IgG in vitro. In addition, by immunoblot the protease was recognized by serum of naturally infected pigs with serotypes 1 and 5, and by serum of pigs experimentally infected with serotypes 1, 2, 8, or 9. Serum of a pig vaccinated with CSN of a serotype 3 strain also recognized the protease, but not sera of pigs vaccinated with a bacterin (serotype 1). Proteins from CSN of all the serotypes, which were precipitated with 70% (NH4)2SO4, were recognized by a polyclonal antibody raised against the purified protease. Taken together these results indicate that an antigenic protease is produced in vivo by all the serotypes of A. pleuropneumoniae. The results indicate that proteases could have a role in the disease and in the immune response of pigs infected with A. pleuropneumoniae.     

The genetic organisation of the capsule biosynthesis region of Actinobacillus pleuropneumoniae serotypes 1, 6, 7, and 12

The aim of the present study was to investigate the organisation of the genes (cps) involved in biosynthesis the capsular polysaccharide (CPS) of Actinobacillus pleuropneumoniae serotypes 6, 7, and 12 and to compare these to the corresponding genes previously described in other A. pleuropneumoniae serotypes. In serotypes 6 and 7 the sequenced DNA regions comprised five and four open reading frames, respectively, designated cps6ABCDE and cps7ABCD, whereas the sequenced DNA region in serotype 12 comprised only two open reading frames designated cps12AB. At the amino acid level, CpsA, CpsB, and CpsC of A. pleuropneumoniae serotypes 2, 6, 7, and 8 contained a high degree of homology. At the amino acid level Cps6D revealed a high degree of homology to Cps8D, whereas Cps7D contained a high degree of homology to the Cps2D. The deduced gene product of the partially sequenced cps6E gene showed no homology to any deduced gene products of any cps genes of A. pleuropneumoniae investigated so far. None of the deduced gene products of the cps genes involved in encapsulation of A. pleuropneumoniae serotypes 2, 6, 7, and 8 revealed homology to the deduced gene products of the cps genes of serotypes 1, 5A, and 12. For some genes, a local homology was found to genes probably involved in teichoic acid synthesis in other bacteria. The results obtained revealed a high degree of homology among the genes involved in CPS biosynthesis for serotypes 2, 6, 7, and 8 and a different group of homologous cps genes for serotypes 1 and 12. In some serotype 7 strains, including the serotype 7 reference strain, WF83, the cps genes were not located adjacent to the genes responsible for CPS export (cpx), probably due to genetic rearrangements.     

The transferrin receptor of Actinobacillus pleuropneumoniae: quantitation of expression and structural characterization using a peptide-specific monoclonal antibody

When Actinobacillus pleuropneumoniae (A. pp) is grown under iron-restricted conditions in vitro, transferrin binding proteins (Tbps) are induced. The functional transferrin receptor of A. pp is composed of two outer membrane proteins (Tbp1 and Tbp2) and shows an exquisite specificity for porcine transferrin. This complex was studied using a monoclonal antibody (Mab 1.48) raised against a synthetic peptide corresponding to a hydrophilic domain of Tbp2 common to several A. pp serotypes. The antibody reacted specifically with a 60-70kDa Tbp2-antigen found in all serotypes of A. pp obtained from iron-restricted culture. It was found that Tbp2 was not expressed in iron replete medium by any serotype except serotypes 5a, 5b and 6 where a weak expression was seen. There was a weak expression of related antigens in Actinobacillus indolicus and Actinobacillus suis under iron-depleted conditions while no similar antigens were detected with the Mab in iron-starved Actinobacillus lignieresii, Actinobacillus porcinus, Actinobacillus minor, Haemophilus influenzae, and Haemophilus parasuis.Using an enzyme-linked immunosorbent assay (ELISA) based on the Mab 1.48, Tbp2 could be detected in both recombinant E. coli expressing Tbp2 and in wild type A. pp grown under iron restricted conditions. The subcellular location of Tbp2 in A. pp was studied by immunoelectron microscopy using the Mab 1.48. Interestingly, all antibody binding was found inside the A. pp cells, while Tbp2 expressed in recombinant E. coli was found both in the cytosol and on the outer membrane. These results indicate that the Mab 1.48-reactive epitope of Tbp2 is surface exposed when it is expressed without Tbp1 in E. coli while the inaccessibility of this epitope of Tbp2 in A. pp could be due to shading by the association between Tbp2 and Tbp1.     

Serological characterization of Haemophilus pleuropneumoniae (Actinobacillus pleuropneumoniae) strains and proposal of a new serotype: serotype 9

Ten strains of H. pleuropneumoniae isolated from 10 herd outbreaks of pleuropneumonia were studied by means of the slide agglutination test, the indirect haemaggluitiniation (IHA) test and by gel diffusion. The strains were antigenically homogeneous and serologically distinct from serotypes 1 through 8. It is therefore proposed to refer these strains to a new serotype: serotype 9, with strain CVJ 13261 as the type strain.In addition to the serotype-specific capsular antigens, capsular antigen of serotype 1 (strain 4074) could be demonstrated in the 10 strains by means of gel diffusion analyses.In cross protection studies it was shown that the antigenic determinants shared by serotypes 9 and 1 were unable to yield a sufficient protection against disease. Thus, parenteral immunization with a killed 6-h culture of serotype 9 did not afford an acceptable protection against challenge with serotype 1 since only 3 of the 5 vaccinates were protected. The reverse experiment showed that parenteral immunization with serotype 1 only protected 1 out of 4 vaccinates.     

Quantifying by monoclonal antibodies of specific IgG, IgM and IgA in the serum of minipigs experimentally infected with Actinobacillus pleuropneumoniae.

Fifteen minipigs were infected intratracheally with three different doses (10(8), 10(5) or 5 x 10(3) colony forming units) of the reference strains of serotypes 2 or 4 of Actinobacillus pleuropneumoniae, and three remained as controls. The titre of specific IgG, IgM and IgA in the serum was measured weekly for 15 weeks with an indirect ELISA using monoclonal antibodies specific for each isotype. IgG attained the highest titres, IgM lower and IgA the lowest, being only detected in four animals. Serotype 4 evoked significantly higher titres than serotype 2 (P < or = 0.01). In general the highest IgG titres were attained at four to six weeks after infection. Some of the minipigs were reinfected after seven weeks but this evoked an increased titre in only two instances.     

Serological characterization of Actinobacillus pleuropneumoniae strains and proposal of a new serotype: serotype 12

Until now 11 serotypes of Actinobacillus pleuropneumoniae have been described (Nicolet 1971, Gunnarsson 1980, Nielsen 1982, Rosendal & Boyd 1982, Nielsen & O’Connor 1984, Nielsen 1985, Kamp 1986). Recently a hitherto unrecognized serotype was isolated from 9 Danish outbreaks of pleuropneumonia in pigs. The origin of the strains is given in Table 1. From 3 herds the unrecognized serotype was found in 2 to 3 pigs submitted for necropsy at different times. The present study describes the serological properties of the 13 isolated strains.