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

Surveillance for the presence ofActinobacillus pleuropneumoniaeinfection in a population plays a central role in controlling the disease. In this study, a 4-plex fluorescent microbead-based immunoassay (FMIA), developed for the simultaneous detection of IgG antibodies to repeat-in-toxin (RTX) toxins (ApxI, ApxII, ApxIII, and ApxIV) ofA. pleuropneumoniae, was evaluated using (i) blood serum samples from pigs experimentally infected with each of the 15 knownA. pleuropneumoniaeserovars or withActinobacillus suis, (ii) blood serum samples from pigs vaccinated with a bacterin containingA. pleuropneumoniaeserovar 1, 3, 5, or 7, and (iii) blood serum samples from pigs with an unknownA. pleuropneumoniaeexposure status. The results were compared to those obtained in a previous study where a dual-plate complement fixation test (CFT) and three commercially available enzyme-linked immunosorbent assays (ELISAs) were conducted on the same sample set. On samples from experimentally infected pigs, the 4-plex Apx FMIA detected specific seroconversion to Apx toxins as early as 7 days postinfection in a total of 29 pigs inoculated with 14 of the 15A. pleuropneumoniaeserovars. Seroconversion to ApxII and ApxIII was detected by FMIA in pigs inoculated withA. suis. The vaccinated pigs showed poor humoral responses against ApxI, ApxII, ApxIII, and ApxIV. In the field samples, the humoral response to ApxIV and theA. pleuropneumoniaeseroprevalence increased with age. This novel FMIA (with a sensitivity of 82.7% and a specificity of 100% for the anti-ApxIV antibody) was found to be more sensitive and accurate than current tests (sensitivities, 9.5 to 56%; specificity, 100%) and is potentially an improved tool for the surveillance of disease and for monitoring vaccination compliance.