Serologic Detection of Actinobacillus pleuropneumoniae in Swine by Capsular Polysaccharide-Biotin-Streptavidin Enzyme-Linked Immunosorbent Assay

Abstract

Serologic detection of Actinobacillus pleuropneumoniae infections in swine have been problematic due to antigenic cross-reactivity of Apx toxins, lipopolysaccharide, and outer membrane proteins between A. pleuropneumoniae serotypes and other bacterial species. To maximize serologic specificity and sensitivity, we developed an assay that uses highly purified A. pleuropneumoniae capsular polysaccharide (CP) conjugated to biotin, which is then bound to streptavidin-coated enzyme-linked immunosorbent assay (CP-BS-ELISA) plates. This assay was used to test a panel of 240 serum samples from pigs prior to challenge, after challenge with bacterial species other than A. pleuropneumoniae, or after challenge with A. pleuropneumoniae serotype 1, 5, or 7. Overall assay results for the individual sera tested were reproducible on the same day and on separate days. The sensitivity of the assay was 100% by ELISAs with biotin-CPs of serotypes 1 and 7 and 87.5% by ELISAs with biotin-CP of serotype 5. Specificity was 100% by ELISAs with biotin-CPs of serotypes 1 and 5 and 94.5% by ELISAs with biotin-CP of serotype 7. The biotin-CPs of at least three A. pleuropneumoniae serotypes could be combined for use in a screening assay to detect antibodies to CPs from strains of different serotypes. In conclusion, the CP-BS-ELISA proved to be a serotype-specific and species-specific assay with high sensitivity for the identification of pigs exposed to A. pleuropneumoniae.

Simultaneous Detection of Antibodies against Apx Toxins ApxI, ApxII, ApxIII, and ApxIV in Pigs with Known and Unknown Actinobacillus pleuropneumoniae Exposure Using a Multiplexing Liquid Array Platform

“…The CFT (19), currently required for importation of live animals into the People's Republic of China, is laborious, timeconsuming, and expensive to use. Among the different ELISAs, those targeting ApxIV toxin to quantify circulating levels of the antibody to A. pleuropneumoniae (20), those targeting ApxI, ApxII, or ApxIII for the detection of specific antibodies to Apx exotoxins (21), those targeting capsular polysaccharides of A. pleuropneumoniae to identify antibodies against groups of its serovars (22), and serovar-specific ELISAs based on long-chain lipopolysaccharides (23) have been widely used and offer better sensitivities and specificities than the CFT. It has also been demonstrated that ELISAs utilizing different antigens or that are performed under different assay conditions may have conflicting results and cross-reactions between A. pleuropneumoniae serovars and other bacterial species, and this may be problematic (2,3).…”

Isolation and genetic characterization of an Actinobacillus pleuropneumoniae serovar K12:O3 strain

“…In order to examine this hypothesis, full genome sequencing would be helpful. In practical application, the use of a CPSbased enzyme-linked immunosorbent assay (ELISA) 4,12 would probably diagnose herds infected with the atypical isolate as infected by A. pleuropneumoniae serovar 12, whereas the use of an LPS-based ELISA 7-9 would probably identify them as infected by serovars 3 or 15. In conclusion, the antigenic and genetic study of strain QAS106 in the current study revealed the presence of an A. pleuropneumoniae K12:O3 strain expressing a new combination of CPS and LPS O-PS antigens in Japan.…”

Use of an Inhibition Enzyme-Linked Immunosorbent Assay for Quantification of Capsular Polysaccharide or Proteins in Vaccines

“…The direct CP ELISA was done using streptavidin-coated plates (Nunc A/S, Roskilde, Denmark) and biotin-labeled CP as described previously (12). All incubations were at 37°C for 1 h, and all washes were done five times with PBS-0.5% Tween 20.…”

“…CPs from serotypes 1, 2, 3, 4, and 7 were conjugated to biotin following oxidation with sodium metaperiodate to generate aldehyde groups (24), followed by reaction with biotin-LC-hydrazide according to the manufacturer's instructions (Pierce Chemical Co., Rockford, IL). Biotin-LC-hydrazide was coupled directly to the carboxyl groups of 3-deoxy-D-manno-2-octulosonic acid in the serotype 5 CP with ethyl-dimethylaminopropyl-carbodiamide HCl (EDC; Bio-Rad Laboratories, Richmond, CA) as previously described (12). The biotin-CP ELISA was done as previously described (12).…”

“…Biotin-LC-hydrazide was coupled directly to the carboxyl groups of 3-deoxy-D-manno-2-octulosonic acid in the serotype 5 CP with ethyl-dimethylaminopropyl-carbodiamide HCl (EDC; Bio-Rad Laboratories, Richmond, CA) as previously described (12). The biotin-CP ELISA was done as previously described (12).…”