Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the Apx toxins of Actinobacillus pleuropneumoniae

The reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae express one or two of three different RTX exotoxins designated Apx I, Apx II and Apx III. The toxins are important virulence factors. In the present study, ELISAs with purified Apx I, Apx II and Apx III, respectively, as antigen were evaluated as candidates for serological diagnosis of Actinobacillus pleuropneumoniae infection in pigs. The pigs were inoculated with biotype 1, serotypes 1-12, and biotype 2, serotype 14, respectively. A strong humoral antibody response was seen to all the three antigens in most pigs irrespective of the serotype used for inoculation. However, titers to the exotoxins secreted by the serotype used for inoculation were generally highest. The results show that toxin proteins of Actinobacillus pleuropneumoniae are antigenically related and that a correlation between serotype and secretion of exotoxin is not revealed serologically in the ELISA test.     

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.     

An atypical biotype I Actinobacillus pleuropneumoniae serotype 13 is present in North America

Atypical Actinobacillus pleuropneumoniae serotype 13 strains present in North America are described here for the first time. Different from serotype 13 strains described in Europe, North America strains are biotype I and antigenically related to both, serotypes 13 and 10. Chemical and structural analysis of the capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of a representative strain revealed that the CPS is almost identical to that of the reference strain of serotype 13, having a slightly higher degree of glycose O-acetylation. However, it produces an O-PS within the LPS antigenically and structurally identical with that of the reference strain of A. pleuropneumoniae serotype 10. The O-PS was characterized as a homopolymer of 1,2 linked β-D-galactofuranosyl residues, a structure unrelated to that of the O-PS produced by the reference strain of serotype 13. Strains from Canada and United States are antigenically, phenotypically and genotypically similar. Animals infected by one of these strains induced antibodies that were detected by a LPS-based ELISA diagnostic test using either the homologous antigen or that of serotype 10. Based on the LPS and toxin profile, these strains might be misidentified as A. pleuropneumoniae serotype 10.     

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.     

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.     

Antigenic analysis of feline coronaviruses with monoclonal antibodies (MAbs): preparation of MAbs which discriminate between FIPV strain 79-1146 and FECV strain 79-1683.

We prepared 31 monoclonal antibodies (MAbs) against either FIPV strain 79-1146 or FECV strain 79-1683, and tested them for reactivity with various coronaviruses by indirect fluorescent antibody assay (IFA). Sixteen MAbs which reacted with all of the 11 strains of feline coronaviruses, also reacted with canine coronavirus (CCV) and transmissible gastroenteritis virus (TGEV). In many of them, the polypeptide specificity was the recognition of transmembrane (E1) protein of the virus. We succeeded in obtaining MAbs which did not react with eight strains of FIPV Type I viruses (showing cell-associated growth) but reacted with FIPV Type II (79-1146, KU-1) and/or FECV Type II (79-1683) (showing non-cell associated growth). These MAbs also reacted with CCV or TGEV. These MAbs recognized peplomer (E2) glycoprotein, and many antigenic differences were found in this E2 protein. These results suggest that FIPV Type II and FECV Type II viruses are antigenically closer to TGEV or CCV than to FIPV Type I viruses. Furthermore, the MAb prepared in this study has enabled discrimination between FIPV strain 79-1146 and FECV strain 79-1683, which was thought to be impossible by the previous serological method.     

Evaluation of an enzyme-linked immunosorbent assay for serological surveillance of infection with Actinobacillus pleuropneumoniae serotype 5 in pig herds

An indirect enzyme-linked immunoassay for serological surveillance of infection of pigs with Actinobacillus pleuropneumoniae (Ap) serotype 5 was developed. The antigen used was prepared from Ap serotype 5b strain L20. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the antigen contained high molecular weight lipopolysaccharide (LPS) and presumably also capsular polysaccharide (CP). The Ap serotype 5 ELISA was tested using sera from pigs experimentally infected with the 12 different Ap serotypes of biotype 1 and with sera from herds naturally infected with Ap serotypes 5, 6, 7 and 12. Cross-reactions were shown in one pig from a herd naturally infected with Ap serotype 7 and in one pig from a herd naturally infected with Ap serotype 12. The herd sensitivities of the Ap5 ELISA and a complement fixation test (CFT) were both estimated to 1.0, on the basis of serum samples from six herds naturally infected with Ap serotype 5. The herd specificities of both tests were estimated to 0.98, based on serum samples from 123 pig herds (10 samples from each herd) from the Danish specific pathogen-free (SPF) programme for pig production.     

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.     

PCR-based identification of serotype 2 isolates of Actinobacillus pleuropneumoniae biovars I and II

A genetic typing method utilizing PCR for the identification of Actinobacillus pleuropneumoniae serotype 2 isolates has been developed based on the in vitro amplification of a 1.4 kb DNA segment of the serotype 2 capsular polysaccharide genes cps2AB. The assay was tested with all serotype reference strains and a collection of 92 different A. pleuropneumoniae strains of all 15 serotypes of both biovars I and II, originating from 18 different countries worldwide. The cps2 based PCR identified the serotype 2 reference strain and all 12 serotype 2 collection strains contained in this set. DNA was not amplified from the remaining A. pleuropneumoniae reference and collection strains, indicating the PCR assay was highly specific. Furthermore, the PCR method detected all 31 A. pleuropneumoniae serotype 2 field isolates from diseased pigs that were identified in parallel as serotype 2 by agar gel diffusion. The serotype 2 PCR assay proved to be highly specific and reliable for the identification of serotype 2 isolates of A. pleuropneumoniae.     

Evaluation of a monoclonal antibody-based approach for the selection of foot-and-mouth disease (FMD) vaccine strains

Foot-and-mouth disease (FMD) virus exists as seven serotypes within which are numerous variants necessitating careful selection of vaccine strains. Currently, a serological assay system based on the use of polyclonal vaccine antisera is widely used for this selection. However, inherent variability in the matching antisera used makes the tests poorly reproducible and difficult to interpret. In this study, we have explored the possibility of replacing or supplementing the polyclonal antibody (PAb)-based method with one based on use of monoclonal antibodies (MAb). Panels of MAbs raised against two serotype O vaccine strains were examined for reactivity with 22 field viruses, isolated over a 10-year period between 1991 and 2001. Antigenic site 2 was found to comprise more than one epitope. The sequence variation in capsid protein VP2 harbouring antigenic site 2 was analysed and the amino acid residues at positions 79 and 134 appeared to greatly influence the binding of site 2 MAbs. Prediction of antigenic match based on MAb reactivity did not correlate closely with the results of a PAb-based "gold-standard" method and it was concluded that a wider panel of MAbs are needed that recognise all protective epitopes present on the surface of FMD virus together with a better understanding of those epitopes which are important in conferring protection.     

Monoclonal antibodies against surface antigens of Pasteurella multocida strain P-1059

Four monoclonal antibodies (MAbs) were developed against serotype 3:A, P-1059 strain of Pasteurella multocida. Enzyme-linked immunosorbent assays were used to screen those hybridomas producing antibodies to either a surface protective (2.5 S) or lipopolysaccharide (LPS) antigen. MAbs 6EE11, D7H10, E11E3, and C11H2 were positive against 2.5 S antigen, and two of them, E11E3 and C11H2, were positive for the LPS antigen. MAbs 6EE11 and D7H10 reacted with a major protein band of molecular weight of 35,500, whereas E11E3 and C11H2 recognized a band with a molecular weight of 12,500 of the 2.5 S antigen. Treatment of the 2.5 S antigen with periodic acid abolished epitopes reacting with E11E3 but not with 6EE11. MAb 6EE11 did not recognize any band in Western blot after proteinase K treatment of the 2.5 S antigen, whereas antibody activity of E11E3 did not change. MAb 6EE11 reacted with serotypes 3, 4, 9, 10, 11, 12, and with M-9 strains in the immunofluorescence test. MAb E11E3 was positive only with serotype 3 or 10 strains, excluding M-9 strain. Electron microscopic studies with P-1059 strain indicated that antigens binding to 6EE11 and/or E11E3 were present in the capsule.     

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.     

Identification of Actinobacillus pleuropneumoniae strains of serotypes 7 and 4 using monoclonal antibodies: demonstration of common LPS O-chain epitopes with Actinobacillus lignieresii

Monoclonal antibodies (Mabs) against Actinobacillus pleuropneumoniae serotype 7 were produced and characterized. Three Mabs directed against surface polysaccharides were selected. One of the Mabs was directed against a capsular polysaccharide epitope (CPS) of A. pleuropneumoniae serotype 7 whereas two other Mabs reacted with different epitopes of the LPS O-chain. One of the latter reacted with the reference strain of serotype 7 and the other one with serotypes 7 and 4. These three Mabs were used to test, by Dot-ELISA, 508 field strains of A. pleuropneumoniae. None of the strains belonging to other serotypes different from serotypes 4 and 7 were positive with the Mabs. Used in combination, the CPS and one of the LPS O-chain directed Mabs were shown to be suitable for serotyping since they detected 100% of serotype 7 strains. In this study, we confirm for the first time that A. pleuropneumoniae serotype 4 is present in North America. Finally, both O-chain specific Mabs also reacted with the O-chain of Actinobacillus lignieresii. The cross-reactivity between the two species was confirmed using sera from pigs experimentally infected with A. pleuropneumoniae serotype 7 and A. lignieresii, using immunoblotting and ELISA. This is the first report of a specific cross-reactivity between the LPS of these bacterial species.     

Comparative virulence of porcine Haemophilus bacteria.

The virulence of strains of Haemophilus pleuropneumoniae serotype 1, 2, 3, 7 and strains of the "minor-group" and Haemophilus parasuis were compared by inoculating specific pathogen-free pigs into the lower airways with specified doses of bacteria. Haemophilus pleuropneumoniae, strain W, serotype 1, given in 1 X 10(8) colony-forming units, produced a lethal acute pleuropneumonia in four pigs. Nonlethal localized pulmonary necrosis was induced in four groups of two pigs given 1 X 10(7), 1 X 10(6), 1 X 10(5) and 1 X 10(4) respectively of the same strain. Two groups of four pigs developed chronic lesions when inoculated with 1 X 10(7) colony-forming units of H. pleuropneumoniae, strain Shope 4074, serotype 1 and 1 X 10(7) colony-forming units of H. pleuropneumoniae, strain WF83, serotype 7, respectively. Of 20 pigs given 1 X 10(8) colony-forming units of strain 1536, serotype 2, two died of acute pleuropneumonia and 18 had lesions of pulmonary necrosis or abscessation and pleuritis. A dose of 4 X 10(9) colony-forming units of strain BC181, serotype 3, induced pulmonary necrosis similar to the lesions in pigs given 10(7) colony-forming units or less of strain W, serotype 1, suggesting that the serotype 3 strain is less virulent. No clinical signs, but focal areas of pulmonary fibrosis and pleural adhesions were induced in four pigs inoculated with 4 X 10(9) colony-forming units of the "minor-group" strain 7ATS. Similarly, four pigs inoculated with "minor-group" strain 33PN did not show clinical signs, but had focal necrotic and fibrotic pulmonary lesions and pleural adhesions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of two monoclonal antibodies for serotyping Haemophilus paragallinarum

Two monoclonal antibodies (MAbs) were evaluated for their ability to serotype 108 isolates of Haemophilus paragallinarum. One MAb (E5C12D10) was raised against a Page serovar A strain and the other (F2E6) against a Page serovar C strain. In both dot blot and hemagglutination-inhibition tests, MAb E5C12D10 recognized the type strains of Page serovar A and Kume serovars A-1, A-2, A-3, and A-4. MAb F2E6 recognized the type strains of Page serovar C and Kume serovars C-1, C-2, and C-3. Neither antibody recognized the type strains of Page serovar B or Kume serovars B-1 and C-4. When evaluated with 97 field isolates in a dot blot test, the MAbs serotyped 81 isolates, which was better than agglutinin typing by the Page scheme (69 isolates serotyped). The field isolates that did not react with the MAbs were either Page serovar B/Kume serovar B-1 (three isolates), Page serovar C/Kume serovar C-4 (12 isolates), or nontypable by either the Page or Kume scheme (one isolate).     

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.     

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.     

Characterization of monoclonal antibodies against avian reovirus S1133 protein sigmaA synthesized in Escherichia coli

Monoclonal antibodies (MAbs) were prepared against avian reovirus S1133 protein sigmaA (esigmaA) synthesized in Escherichia coli. MAbs were characterized and used to develop a diagnostic test. Ten MAbs were selected for competitive binding assay following coupling with horseradish peroxidase. The results indicated that these MAbs delineated two epitopes I and II of esigmaA. An immuno-dot binding assay was used to detect the effect of denaturation on antibody recognition of the epitopes. All MAbs bound to esigmaA in its native form. After denaturation by boiling in SDS and 2-mercaptoethanol, the binding of MAbs recognizing epitope I was fully abolished. However, the reactivity of MAbs recognizing epitope II was not affected. MAbs 31 and 32, recognizing epitopes I and II, respectively, were selected for the cross-reactivity to heterologous reovirus strains. The results suggest that the two epitopes are highly conserved among these virus strains. A MAb capture enzyme-linked immunosorbent assay (ELISA) procedure was developed using MAbs 32 and 31 to detect reovirus protein sigmaA in samples from tendon tissues of infected bird and chicken embryo fibroblast (CEF) cell cultures. Avian reovirus sigmaA antigens in tendon specimens were detected from the inoculated birds as early as 2 days post-inoculation (PI), approximated a peak at 7 days PI, and maintained this until 16 days PI, then decreased gradually. A clear difference in absorbance values between the tendon samples of the avian reovirus- and mock-infected birds is obtained. Positive results were also obtained from avian reovirus-infected CEF and from the tendon tissues of naturally infected broilers. These results indicated that the MAb capture ELISA is a useful methods for the detection of avian reovirus from chickens suspected to have avian reovirus infections.