Production and characterization of monoclonal antibodies to serotype specific antigens of Haemophilus pleuropneumoniae serotype 2

Mouse monoclonal antibodies were produced to Haemophilus pleuropneumoniae bacteria of the most common serotype 2. 11 hybridoma cultures were recovered that produced antibodies with moderate to strong reactivity to the antigen. 10 of these antibodies were specific to isolated capsular antigens from H. pleuropneumoniae of serotype 2, while one antibody reacted with capsular antigens from bacteria of all 8 serotypes. One hybridoma producing antibodies with a titre of 1:1000 was cloned and the antibody specificity studied further. The binding of this antibody (1F3) to whole bacteria, and capsular extracts isolated at different temperatures indicate that the antibody is specific for a thermostable polysaccharide antigen present in the cellular capsule of H. pleuropneumoniae of serotype 2.     

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.     

Characterization of monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5.

Two monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5, designated as 5MAb-1 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-1 was a carbohydrate that was sensitive to periodate oxidation and resided on the structure of beta-1,6-linked D-galactose in an O-antigen polysaccharide of serotype-5 lipopolysaccharide. Analysis of these results revealed 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.     

Use of monoclonal antibodies for classifying Actinobacillus (Haemophilus) pleuropneumoniae

The serological typing (by enzyme-linked immunosorbent assay) of 119 isolates of Actinobacillus (Haemophilus) pleuropneumoniae (representing in varying numbers the 12 serovars of this taxon) by monoclonal antibodies derived from the reference strains of serovars 1 to 5 in general correlated reasonably with the serotype previously established for these strains by conventional procedures employing polyclonal antisera. However, where there were reasonable numbers of isolates representing a given serovar to provide a decision, there was no instance where the correlation between the monoclonal and the polyclonal antibody was in complete accord. In addition, some of the differences between monoclonal and polyclonal antibody binding with some isolates suggest that the distribution of the serotype-specific antigens within the taxon may be even more complex than has previously been supposed.     

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.     

Binding patterns of five monoclonal antibodies to Actinobacillus (Haemophilus) pleuropneumoniae

Five monoclonal antibodies were obtained after immunising mice with superficial antigens of three strains representing serovars 1 to 3 of Actinobacillus (Haemophilus) pleuro-pneumoniae. When tested in ELISA against the standard strains representing serovars 1 to 10, the monoclonal antibody raised against the standard strain of serovar 1 reacted only with that strain. Of the three monoclonal antibodies raised against the standard strain of serovar 3, one reacted with serovars 3 and 8 only, another with serovar 7 only and the third with the strains representing serovars 7, 9 and 10. The monoclonal antibodies produced with the serovar 2 strain also reacted with a wide spectrum of strains, representing serovars 7 to 10.     

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.     

Use of monoclonal antibodies to serotype-specific antigens of Haemophilus pleuropneumoniae serotype 2 in passive immunization

The prophylactic value of mouse monoclonal antibodies to the pig pathogen Haemophilus pleuropneumoniae was studied. Approximately 250 mg of purified mouse monoclonal antibody specific to capsular antigens of H pleuropneumoniae serotype 2 was given IV to five 9-week-old pigs. Five additional pigs from the same litter served as controls. On the following day, all pigs were given a lethal dose (5 x 10(9)) of H pleuropneumoniae serotype 2 into the trachea. Four controls and 1 pig that was given antibodies died within 24 hours. The surviving 5 pigs developed typical signs of pleuropneumonia. After 6 days, the pigs were euthanatized and their respiratory tracts were examined for pathologic changes. All 5 pigs had pathologic changes, but they were less severe in the 4 pigs that had been given antibodies, compared with those in the control pig.     

Identification and partial characterization of the hemolysin (HlyII) of Actinobacillus pleuropneumoniae serotype 2

The secreted hemolytic activity produced by Actinobacillus pleuropneumoniae serotype 2 reference strain is thermolabile, inactivated by proteinase K and requires Ca2+ as cofactor for its hemolytic activity. Purification of the hemolytic activity resulted in a fraction containing two proteins, one of 105 kDa and one of 125 kDa. These two proteins could be further separated by preparative SDS polyacrylamide gel electrophoresis. This purification step, resulted in loss of the hemolytic activity. Polyclonal antibodies were made against each of these proteins in rabbits. Neutralization experiments showed that antibodies made against the 105 kDa protein could neutralize the hemolytic activity produced by A. pleuropneumoniae serotype 2, while antibodies made against the 125 kDa protein were unable to neutralize the hemolytic activity. The 105 kDa protein therefore, is the hemolysin of A. pleuropneumoniae serotype 2, known as HlyII. This protein is closely related immunologically to the hemolysin I (HlyI) from A. pleuropneumoniae serotype 1. DNA::DNA hybridization experiments performed by the Southern blot method using the cloned structural gene of HlyI from A. pleuropneumoniae serotype 1 demonstrate that the structural genes of the two hemolysins (hlyIA and hlyIIA) are different and show at least 30% heterology. This confirms that HlyI and HlyII are two different proteins, although they have a very similar molecular weight and show strong immunological cross reactions.     

Production, characterization and protective effect of monoclonal antibodies to Haemophilus paragallinarum serotype A.

Monoclonal antibodies (mAbs) to Haemophilus paragallinarum serotype A were obtained by fusion of murine myeloma cells (P3-X63-Ag8-U1) and spleen cells from BALB/c mice immunized with whole cells of strain 221. Enzyme linked immunosorbent assay with whole cells was used to show that the monoclonal antibodies are specific for serotype A of H. paragallinarum. Four monoclonal antibodies indicated hemagglutination-inhibition (HAI) activity against serotype A; their titers were 10(4)-10(5). By western blotting, two of these monoclonal antibodies reacted with a protein of molecular weight 39,000. Chickens treated with mAbs possessing HAI activity survived without clinical signs of infection. No challenge strain was isolated from these chickens, indicating that four mAbs with HAI activity suppressed growth of the challenge strain in the nasal cavity, whereas mAbs without HAI activity showed no passive protective effect. These results demonstrated that HI antibodies contributed to protection, and strongly suggest that hemagglutinin (HA) antigen, especially the epitopes which were recognized by these mAbs are important for protective immunity in chickens.     

Detection of antibodies against Actinobacillus pleuropneumoniae serotype 5 using an inhibition enzyme immunoassay.

An inhibition enzyme immunoassay (EIA) for detection of antibodies against A. pleuropneumoniae serotype 5 (App-5) in pig sera, based on the inhibition of the binding of an App-5 specific monoclonal antibody was established. The monoclonal antibody (MAb 210-F11) was found to be directed against an epitope on the O-chain of App-5 LPS. In the inhibition EIA, highly purified App-5 LPS was used to coat microtitre plates. Serial dilutions of pig sera were added to the plates prior to the addition of the MAb 210-F11. The degree of binding of App-5 antibodies from pig sera was determined as the percentage inhibition of the MAb 210-F11. Pig serum from specific pathogen free (SPF) herds, from experimentally infected animals, and from acutely and chronically infected herds were tested. A serum dilution of 1/30 was found to be optimal, when using 50% inhibition as the discriminating inhibition percentage. No cross-reactivity was observed with serum from pigs infected with other App serotypes or bacteria isolated from the respiratory tract, such as A. suis and H. parasuis. The inhibition EIA will be used for surveillance of App-5 antibodies in SPF and conventional herds.     

Production and characterization of two serotype independent monoclonal antibodies against foot-and-mouth disease virus

Two foot-and-mouth disease virus (FMDV) monoclonal antibodies (mAbs) were produced from mice immunized with either FMDV serotype A, subunit (12S) or FMDV serotype O, whole virus (140S). Both mAbs (F1412SA and F21140SO) recognized all seven serotypes of FMDV in a double antibody sandwich (DAS) ELISA, suggesting that the binding epitopes of the two mAbs are conserved between serotypes. These mAbs are IgG1 isotype and contain kappa light chains. In order to define the mAb binding epitopes, the reactivity of these mAbs against trypsin-treated and denatured FMDV were examined using an indirect ELISA. The binding site of the mAb, F1412SA is trypsin sensitive and the epitope is linear. Both ELISA and Western blot results suggested that the polypeptide VP2 contributed to the immunodominant site. This mAb showed reactivity to VP2 peptide (DKKTEETTILEDRIL). The mAb, F21140SO, recognized an epitope which is trypsin resistant and discontinuous. This mAb binding to FMDV is dependent on conformational structures of intact viral (140S) or subunit (12S) particle, since it failed to recognize any viral protein in Western blot. This conformational and highly conserved epitope is the first identified epitope among all seven FMDV serotypes. Because the use of mAbs increases the specificity, accuracy and efficiency of diagnostic tests compared to polyclonal antisera, these two mAbs with different specificities are suitable for type-independent diagnosis of FMDV, such as DAS ELISA, or could be adapted to immuno-chromatographic or flow-through rapid test.     

Serology of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5 strains: establishment of subtypes a and b

The serological characteristicis of 14 strains assigned to serotype 5 were examined by the slide agglutination test, the indirect haemagglutination (IHA) test and by gel diffusion.All the strains possessed identical capsular antigenic determinants of polysaccharide (PS) nature and lipopolysaccharide (LPS) nature. However, based upon a capsular antigenic determinant of PS nature the strains could be divided in two subtypes. It is therefore proposed to refer to the H. pleuropneumoniae strains of serotype 5 to two subtypes: subtype a with strain K17 as the subtype strain and subtype b with strain L20 as the subtype strain.     

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.     

Characterization and classification of Actinobacillus (Haemophilus) pleuropneumoniae plasmids.

Actinobacillus (Haemophilus) pleuropneumoniae plasmids were characterized and classified. They were isolated from A pleuropneumoniae strains different in serotype, year isolated, or location from which isolated. Six of 8 plasmids encoded streptomycin (Sm) and sulfonamide (Su) resistance (SmSu). One of the other plasmids, pVM105, encoded ampicillin (Ap) resistance and another, pHM0, encoded no drug resistance. All SmSu plasmids were transferred to Escherichia coli strains by transformation. Among them, pABO and pMS260 were 8.1 kb and incompatible with each other; they were stable in E coli. The other SmSu plasmids, pHM1, pVM104, pVM106, and pKD25, were 4.3 kb and did not replicate stably in E coli. The former SmSu plasmids were mobilized in E coli strains by a plasmid RP4, which belonged to incompatibility (Inc) group P, but the latter plasmids were not. Further, each 8.1-kb SmSu plasmid and each 4.3-kb plasmid had the same respective restriction pattern. These results indicated that there were at least 2 types of SmSu plasmids in A pleuropneumoniae. The 2 types were classified in 2 groups: H1(pMS260 and pABO) and H2(pHM1, pVM104, pVM106, and pKD25). The H1 and H2 plasmids belonged to different Inc groups, and H2 plasmids belonged to a different Inc group from that of pHMO and pVM105.     

猪胸膜肺炎放线杆菌蛋白酶的分离纯化及特性分析

将血清7型猪胸膜肺炎放线杆菌(APP)山东分离株接种含0.2%NAD的LB液体培养基,37℃培养48 h后,经硫酸铵盐析、DEAE-纤维素离子交换层析、葡聚糖凝胶分子筛层析,从其培养上清液中分离纯化了1种蛋白酶.SDS-聚丙烯酰胺凝胶电泳显示,APP蛋白酶含有相对分子质量约为45 000的亚单位.以酪蛋白为底物测得该酶的最适PH值为7.5.最适温度为45℃;该酶对热有一定的稳定性,80℃加热30 min仍保留部分活性;乙二胺四乙酸(EDTA)可抑制其活性,而苯甲基磺酰氟(PMSF)对其无影响.     

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.