ABSTRACT
Actinobacillus pleuropneumoniae (serotype 5) long-chain lipopolysaccharides (LC-LPS) may be used as the antigen for ELISA serodiagnosis of pig pleuropneumonia. A method was developed in order to augment the recovery of this antigen compared to the previously described method. In liquid culture, A pleuropneumoniae was shown to produce 2.4 times more cells than in solid medium. LC-LPS could be recovered by phenol extraction of the crude extract. Only 1 additional phenol extraction was required to produce LC-LPS of the same quality as the one obtained with solid-medium-grown cells, as revealed by SDS-PAGE and ELISA using reference sera. From the same volume of crude extract, approximately 8 times more antigen was present in the aqueous phase of the phenol extraction.
Subject(s)
Actinobacillus pleuropneumoniae/immunology , Lipopolysaccharides/isolation & purification , Animals , Electrophoresis, Polyacrylamide Gel/veterinary , Enzyme-Linked Immunosorbent Assay/veterinary , Serologic Tests/methods , SwineABSTRACT
A saline extract of boiled-formalinized whole cells of Actinobacillus pleuropneumoniae serotype 1 reference strain (Shope 4074) has been previously used as the antigen in an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of swine pleuropneumonia. Phenol extraction of this crude extract permitted the recovery of LPS with long O-chains in the aqueous phase. This antigen was shown to be specific for serotypes 1, 9 and 11 as they all possess structurally similar O-chains. Immunoblotting was used to identify the fraction present in the crude extract of strain 4074 responsible for cross-reactions observed in ELISA with a serum raised against a serotype 3 strain of A. pleuropneumoniae. The specific reactions in ELISA were shown to be associated with long O-chain LPS and the cross-reactions to LPS with short O-chains. LPS seem to be an important antigen for A. pleuropneumoniae serotype 1 as all homologous sera tested reacted with it. This antigen is easily recovered from the crude extract and it can be used in minute amounts (1-6 micrograms) for ELISA serodiagnosis of pig pleuropneumonia.
Subject(s)
Actinobacillus Infections/veterinary , Actinobacillus pleuropneumoniae/immunology , Lipopolysaccharides/immunology , Pleuropneumonia/veterinary , Swine Diseases/diagnosis , Actinobacillus Infections/diagnosis , Animals , Antigens, Bacterial/immunology , Antigens, Bacterial/isolation & purification , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay/veterinary , Immune Sera/immunology , Immunoblotting , Lipopolysaccharides/isolation & purification , Pleuropneumonia/diagnosis , Sensitivity and Specificity , SwineABSTRACT
Long chain lipopolysaccharides (LC-LPS) of Actinobacillus pleuropneumoniae serotype 5 have been evaluated and compared with a crude boiled extract (CBE) in ELISA for the serodiagnosis of swine pleuropneumonia caused by this serotype. The mean optical density (OD) obtained with the LC-LPS in ELISA using sera from negative herds as well as from animals experimentally and naturally exposed to A. pleuropneumoniae serotype 5 was not significantly different from that obtained with the CBE. However, sera from animals exposed to serotypes of A. pleuropneumoniae other than serotype 5 presented a significantly lower mean OD (P < 0.05) when the LC-LPS was used. As a consequence, it was demonstrated that a high percentage of non-specific cross-reactions were eliminated, without losing specificity. The specificity and the sensitivity of the LC-LPS- and CBE-ELISA were evaluated using two different cut-off values (the OD plus two and three standard deviations) (SD) obtained from 593 sera from negative herds. The LC-LPS appeared a more suitable antigen than the CBE, since the sensitivity and the specificity (obtained with both thresholds) were statistically improved (P < 0.01). A threshold of 0.244 (mean OD plus three SD) for the LC-LPS-ELISA seemed more suitable, since a sensitivity of 79% and a specificity of 97% was achieved. Nevertheless, it may be advisable to keep a buffer range (OD between 0.194 and 0.243) and to consider sera presenting values within this range as suspicious. In the present study, the complement fixation test presented a high specificity (97%) and a very low sensitivity (47%). A herd with animals presenting ELISA positive and CFT negative results in serology, along with the absence of suggestive lesions should not be considered as a non-infected herd.
Subject(s)
Actinobacillus Infections/veterinary , Actinobacillus pleuropneumoniae/isolation & purification , Antibodies, Bacterial/blood , Lipopolysaccharides/immunology , Pleuropneumonia/veterinary , Swine Diseases , Actinobacillus Infections/diagnosis , Actinobacillus pleuropneumoniae/classification , Actinobacillus pleuropneumoniae/immunology , Animals , Antibody Specificity , Complement Fixation Tests , Cross Reactions , Enzyme-Linked Immunosorbent Assay/methods , Pleuropneumonia/diagnosis , Sensitivity and Specificity , Serologic Tests , Serotyping , SwineABSTRACT
A saline extract of boiled-formalinized whole cells from a local strain (81-750; Quebec, Canada) of Actinobacillus pleuropneumoniae, serotype 5b was used as an antigen in an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of swine pleuropneumonia. Characterization of this crude extract was done and proteins, neutral sugars, hexosamines, and 2-keto-3-deoxyoctonate (KDO) were evaluated. On phenol extraction of the crude extract a serotype-specific antigen of polysaccharidic nature was recovered from the aqueous phase. This antigen was characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie blue, silver and Schiff stainings. Immunoblots were done using sera of experimentally infected pigs that showed serotype specificity and cross-reactivity. Overall, the results indicate that the O-chain of lipopolysaccharides is a specific antigen that could be used in ELISA for the serodiagnosis of serotype 5 of A. pleuropneumoniae.
