Actinobacillus (Haemophilus) pleuropneumoniae serotype-8 isolates and their antigenic relationships with other A pleuropneumoniae serotypes

Antigenic relationship of Actinobacillus (Haemophilus) pleuropneumoniae serotype-8 isolates with other serotypes was studied, using tube agglutination, with and without 2-mercaptoethanol, indirect hemagglutination with and without 2-mercaptoethanol, ring precipitation, coagglutination, and immunodiffusion tests. Serotype-8 isolates possessed serotype-specific, group-specific common antigens cross-reactive with serotypes 3 and 6 and species-specific common antigens cross-reactive with other serotypes. Absorption studies were done to study the antigenic relationship of serotype 8 with serotypes 3 and 6. Rabbit antisera against whole-cell (WC) suspensions of reference strains of serotypes 3, 6, and 8 were used for absorption studies with WC and boiled WC suspensions of homologous and heterologous serotypes. Unabsorbed and absorbed sera were tested for antibodies against WC and boiled WC antigen preparations of serotype 8, using various serotests. Absorption studies revealed that serotype-8 strains possessed 2 main types of epitopes, one of which was serotype-specific and did not have cross-reactivity with other serotypes. The second type of epitopes was group specific and was cross-reactive with serotypes 3 and 6.     

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.     

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.     

Biochemical and serological properties of Actinobacillus pleuropneumoniae biotype 2 strains isolated from swine

The biochemical and serological properties of 21 strains of Actinobacillus pleuropneumoniae biotype 2 isolated from haemorrhagic necrotic pleuropneumonia of swine were examined. For serologic typing, the indirect haemagglutination (IHA) and the double gel-diffusion tests were used. On the basis of their soluble surface antigens, our A. pleuropneumoniae biotype 2 isolates could be assigned to two proposed serotypes. Serotype 1 comprised 11 strains and serotype 2 comprised 10 strains. All strains contained two surface antigen components. In the strains belonging to serotype 1, one of the antigens was identical with the serotype-specific antigen of Pasteurella haemolytica T4. Both antigens of serotype 2 strains proved to be type-specific. Four strains received from Switzerland, including the holotype strain of A. pleuropneumoniae biotype 2, and three strains isolated from swine in the G.D.R. belonged to serotype 2. Both the double gel diffusion and the IHA tests detected a 2-way cross-reaction between biotype 1, serotype 2 and biotype 2, serotype 2 strains of A. pleuropneumoniae, which could be eliminated using cross-absorbed sera.     

Serological characterization of Actinobacillus pleuropneumoniae biotype 2 strains isolated from pigs in two Danish herds

Eight Actinobacillus pleuropneumoniae biotype 2 strains were isolated in pure culture from lungs of pigs originating from two Danish herds with growing and finishing pigs. The antigenic properties were studied by indirect haemagglutination (IHA) and immunodiffusion (ID) tests using soluble surface antigens and by enzyme-linked immunosorbent assays (ELISA) using capsular enriched fractions and LPS. In all tests the strains proved antigenically homogeneous and serologically distinct from the known biotype 1 and 2 serotypes. Thus, the strains represent a new serotype which is provisionally proposed as biotype 2 serotype 14.     

PCR specific for Actinobacillus pleuropneumoniae serotype 3

Serotypes 3 and 8 of Actinobacillus pleuropneumoniae, the aetiological agent of porcine pleuropneumonia, have been reported to predominate in the UK. Direct serotyping of isolates of the organism is typically determined by the immunological reactivity of rabbit serum to its surface polysaccharides, but the method has limitations, for example, cross-reactions between serotypes 3, 6 and 8. This study describes the development of a serotype 3-specific pcr, based on the capsule locus, which can be used in a multiplex format with the organism's specific gene apxIV. The pcr test was evaluated on 266 strains of A pleuropneumoniae and 121 strains of other organisms, including all the major respiratory bacterial pathogens of pigs. The test was highly specific and sensitive and should be useful for differentiating strains of serotypes 3, 6 and 8, and in seroprevalence and epidemiological surveys in regions where serotype 3 is prevalent, such as the UK.     

Effect of heat treatment on the surface antigens of Haemophilus pleuropneumoniae

Coagglutination and ring precipitation tests were used to study the effect of heat on the surface antigens of Haemophilus pleuropneumoniae strains employing the reference strains belonging to serotypes 1 to 7 and field isolates belonging to serotypes 1, 2, 3, 5, 6 and 7. By immunising rabbits with formalin-fixed whole-cell suspension, antibodies were obtained which sensitised Cowan I Staphylococcus aureus to coagglutinate antigen preparations which had not been heated, or heated at 56 degrees C, or boiled or autoclaved. Similar positive reactions were obtained with the ring precipitation test. Heating the cultures at 56 degrees C for one hour was best for exposing the most potent serotype-specific antigens in all the strains studied. All the reference strains and most of the field isolates possessed the thermostable type specific antigens which could withstand autoclaving for one hour. However, many field isolates belonging to serotype 1 did not possess this antigen. The apparent antigenic heterogeneity of serotype 1 strains based on the presence or absence of these thermostable antigens could be valuable in epidemiological investigations. It was shown that most potent serotype-specific antigens are present as freely diffusible material on the surface layer of the bacterial cells, which could easily be removed by washing in saline solution. Well washed bacterial cells devoid of surface materials are poor antigens. It is recommended that test strains should not be heated above 56 degrees C for serotyping because higher temperatures are liable to destroy the capsular antigen of some strains and render the culture untypeable.     

Genotypic prevalence of apx1V in Actinobacillus pleuropneumoniae field isolates.

A total of 90 Actinobacillus pleuropneumoniae field strains from pigs were serotyped by slide agglutination and analyzed for the presence of the apxIV gene by polymerase chain reaction. Of the 90 isolates serotyped, serotypes 2 (47 isolates) and 5 (25 isolates) were the most common, followed by serotype 6 (10 isolates). Three isolates belonged to serotype 7, and 5 isolates could not be typed. All 90 A. pleuropneumoniae field isolates tested carried the apxIV gene. This gene is species specific rather than serotype specific. Therefore, the ApxIV toxin has potential value for use both in vaccines and in diagnostic tests.     

Development and evaluation of an enzyme-linked immunosorbent assay for bovine antibody (IgG) to Pasteurella haemolytica

The sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) for bovine IgG serum antibody to Pasteurella haemolytica was compared with that of an indirect hemagglutination (IHA) test. Pasteurella haemolytica serotypes were grown in a chemically defined cell culture medium, and soluble antigens released into the growth medium were used in the ELISA and IHA test. An ELISA with serotype-1 antigen consistently detected antibody in sera that were positive by IHA test (correlation, 99%). Sera reacting with serotype-1 ELISA antigens also reacted with ELISA antigens prepared from other serotypes. Although ELISA titers averaged 5 log2 units higher than IHA titers, plots of titers determined by the 2 methods were approximately linear. Titer increases detected in paired serum samples by either test were similar. The ELISA was more sensitive than was the IHA in detecting colostral IgG antibody in serum of newborn calves. The ELISA uses a simple, stable antigen preparation and detects antibody to P haemolytica serotypes that commonly infect cattle.     

Actinobacillus pleuropneumoniae in Thai pig herds. Prevalence of serum antibodies and relation to performance

The aim of the study was to investigate the prevalence of Actinobacillus pleuropneumoniae infections in market weight pigs in Thailand. ELISA systems employing purified lipopolysaccharide antigens were used to detect antibodies in 549 serum samples collected from pigs of 22 herds. Relevant cut-off values were established from three herds defined seronegative. Serum antibodies were detected to all serotypes except serotype 10. Almost 60% of the samples were seropositive to at least one serotype and 45% of the pigs were seropositive to more than one serotype. Antibodies to the cross-reacting serotypes 1, 9 or 11 were found in 29% of the pigs. Other common serotypes included the cross-reacting serotypes 3, 6 or 8 (26% seropositive pigs) and serotype 5a (also 26%). Antibodies to serotypes 2, 5b and 12 were low in prevalence (<10%). Three herds were regarded to be seronegative and six to have a low pathogen load with respect to the prevalence of seropositive pigs. The remaining 13 herds had a high incidence of pigs with antibodies to A. pleuropneumoniae, dominated by serotypes 1-9-11 and 5a (n = 6), serotypes 3-6-8, and 5a (n = 4) or 1-9-11, 3-6-8, 5a and 4-7 (n = 3). A low pathogen load with respect to A. pleuropneumoniae, as well as small herd size and age-segregated rearing, tended to improve the performance of growers.     

Use of monoclonal antibodies to identify outer membrane antigens of Actinobacillus species

Three monoclonal antibodies (LG17, LG30, and LG33) were used to identify outer membrane antigens of Actinobacillus sp (As8C isolate) cultured from the epididymides of an infected ram lamb. Specificity of the 3 antibodies to As8C antigens was determined by use of bacterial agglutination, the enzyme-linked immunosorbent assay, and the indirect fluorescent antibody test. Results of immunoelectron microscopy confirmed that each antibody was specific for epitopes on As8C outer membrane antigens. Evaluation by use of enzyme-linked immunoelectrotransfer blot indicated that target antigens for LG17 and LG33 antibodies had molecular weights of 10 kilodaltons and 43 kilodaltons, respectively. Multiple-band staining was observed with the LG33 antibody. The target antigen for the LG30 antibody could not be discerned by use of enzyme-linked immunoelectrotransfer blot. For each of the 3 monoclonal antibodies, enzyme-linked immunosorbent assay titers were obtained for Actinobacillus seminis, A actinomycetemcomitans, and 10 field isolates of Actinobacillus spp. Target antigens for LG17 and LG30 antibodies occurred infrequently or were absent on these bacteria. However, the target antigen for the LG33 antibody was shared by Actinobacillus seminis, A actinomycetemcomitans, and the 10 field isolates of Actinobacillus spp, indicating some diversity of outer membrane antigens between isolates.     

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.     

Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxIVA gene and by ribotyping

During the period of 2001-2003, a total of 591 Actinobacillus pleuropneumoniae field isolates from the Czech Republic were serotyped with a high occurrence of cross-reactions. The cross-reactions were observed in 416 isolates. Most frequently, in 401 isolates (67.9%), cross-reactions with antisera specific for serotypes 9, 11, and/or 1 were observed. Two additional molecular methods, ribotyping and restriction analysis of PCR amplified apxIVA gene (PCR-REA), were therefore used for detailed characterisation of A. pleuropneumoniae. In this subsequent analysis, reference strains representing serotypes 1-12 and 25 field isolates showing the most frequent serotype cross-reactions were examined. PCR-REA enabled all reference strains to be distinguished except for the strains of serotypes 9 and 11. Ribotyping distinguished all reference strains except two pairs of serotypes: 3 versus 6, and 9 versus 11, respectively. Field isolates with serotype cross-reactivity 9, 11, and/or 1 could not be differentiated by either of these methods.     

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.     

Biochemical typing of Actinobacillus pleuropneumoniae

A study was conducted to evaluate the possibility of using biochemical differences among strains of a given serotype of Actinobacillus pleuropneumoniae as epidemiological markers, to rapidly identify the source of infection in herds affected with swine pleuropneumonia. Out of 38 different biochemical and physiological tests performed on a total of 67 strains belonging to serotypes 1 and 5 of A. pleuropneumoniae, three fermentation tests, glycerol, lactose and raffinose, allowed the classification of serotype 1 strains into 6 phenotypic groups and serotype 5 strains into 4 of these groups. Groups II and III were exclusively composed of serotype 1 strains, whereas the majority of strains in groups I and IV belonged to serotypes 1 and 5 respectively, the latter comprising almost all the serotype 5 studied.     

Study of antigenic heterogeneity among Actinobacillus pleuropneumoniae serotype 7 strains

Actinobacillus pleuropneumoniae serotype 7 strains were studied for their antigenic heterogeneity using rabbit polyclonal hyperimmune sera against all the known twelve reference strains of A. pleuropneumoniae and a battery of different serological tests such as coagglutination (COA), immunodiffusion (ID), indirect hemagglutination (IHA), counterimmunoelectrophoresis (CIE), rapid dot-ELISA (RDE), serum soft-agar (SSA) and growth agglutination (GA). Reference serotype 7 strain (WF83) showed cross-reactivity with reference serotype 1B strain but not with other serotypes. Field serotype 7 strains showed cross-reactivities with serotypes 1A, 1B, 4, 9, 10, and 11 in COA, ID, and CIE tests, but not in IHA test. Two field strains of serotype 7 (90-3182 and 86-1411) which appeared to be different from the typical serotype 7 strains were selected for further antigenic characterization by SDS-PAGE, Western blot, and Tricine SDS-PAGE assays, and identified as serotypes 1 and 7, respectively. For serotyping atypical strains, it is suggested to use Western blot assay as a confirmatory test to identify serotype-specific capsular and somatic antigens.     

Capsular polysaccharide antigens for detection of serotype-specific antibodies to Actinobacillus pleuropneumoniae.

Capsular polysaccharides (CPS) of serotypes 1, 2, 5 and 7 of Actinobacillus (Haemophilus) pleuropneumoniae were obtained from 18 h culture supernatants by precipitation with hexadecyltrimethyl-ammonium bromide (Cetavlon) followed by extraction with sodium chloride and reprecipitation in ethanol. These crude extracts, and portions purified further by phenol extraction to remove contaminating proteins, were evaluated as antigens for the detection of serotype-specific antibodies to A. pleuropneumoniae in sera from immunized rabbits and swine by an enzyme-linked immunosorbent assay. The crude extracts reacted strongly with homologous antisera, but except for serotype 1 showed considerable cross-reactivity with antisera to other serotypes. Phenol extraction greatly improved the serospecificity of the antigens from serotypes 1, 7 and, to a lesser extent, 5. The serotype 2 CPS antigen showed poor reactivity following phenol extraction, and did not appear as useful for detection of serotype-specific antibodies.     

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.     

An Actinobacillus pleuropneumoniae PCR typing system based on the apx and omlA genes--evaluation of isolates from lungs and tonsils of pigs

The genetic variability of a gene coding for an outer membrane lipoprotein (omlA) was used to develop a PCR typing system for Actinobacillus pleuropneumoniae. Sequence differences in the middle region of the gene divided the A. pleuropneumoniae serotypes in five distinct groups. Group I included serotypes 1, 9, 11 and 12 (omlA l), Group II consisted of serotypes 2 and 8 (omlA II), Group III included serotypes 3, 6 and 7 (omlA III), Group IV (omlA IV) consisted of serotype 4 and Group V of serotypes 5a, 5b and 10 (omlA V). The sequence differences were utilized to construct PCR primers specific for each group, except of Group IV, as the amplicon of serotype 4 could be separated from Group III by size. Together with a PCR apx typing system, the omlA PCR typing system could discriminate the majority of A. pleuropneumoniae serotypes of biovar 1 except of serotypes 1, 9 and 11 and serotypes 2 and 8. The PCR typing system was tested on 102 field strains of A. pleuropneumoniae isolated from lungs of diseased pigs. The serotyping results of the investigated field strains were in agreement with the apx and omlA gene patterns found in the reference strains of the bacteria, with the exception of the omlA gene of five strains of serotype 8. To examine the apx and omlA gene pattern of tonsil isolates, the PCR typing system was tested on a total of 280 A. pleuropneumoniae field strains isolated from tonsils of pigs. Agreement between serotyping and DNA typing was found in 96% of the isolates using the apx gene patterns and in 89% of the isolates using the omlA gene. The same serotype specific apx/omlA gene pattern was thus found in the majority of the tonsil isolates and in isolates from diseased lungs. Most of the differences in the omlA gene were found in 18 tonsil isolates of serotype 12. The omlA/apx PCR typing system described in the present study makes it possible to determine the type specificity of the majority of A. pleuropneumoniae isolates by simple PCR technique and enables phenotype independent characterization of isolates non-typable by serotyping.     

Blocking enzyme-linked immunosorbent assay for detection of antibodies against Actinobacillus pleuropneumoniae serotype 6 in pig serum

A blocking enzyme-linked immunosorbent assay (ELISA) detecting antibodies against Actinobacillus pleuropneumoniae (Ap) serotype 6 was developed. The blocking ELISA was based on the inhibition of a polyclonal antibody raised against Ap serotype 6. Purified lipopolysaccharide from Ap serotype 6 was used as antigen. The blocking ELISA was tested against sera from pigs experimentally infected with the 12 serotypes of Ap biotype 1. Cross-reaction with serotypes 3 and 8 but not with other serotypes was observed. The sensitivity and specificity of the test on a herd level were evaluated with sera from herds naturally infected with serotypes 2, 6, 8 or 12 and with sera from herds free of infection with any Ap serotype. The blocking ELISA showed a high herd sensitivity (1.00 (0.79-1.00)) and specificity (0.97 (0.93-0.99)).