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

Abstract

c Surveillance for the presence of Actinobacillus pleuropneumoniae infection 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) of A. pleuropneumoniae, was evaluated using (i) blood serum samples from pigs experimentally infected with each of the 15 known A. pleuropneumoniae serovars or with Actinobacillus suis, (ii) blood serum samples from pigs vaccinated with a bacterin containing A. pleuropneumoniae serovar 1, 3, 5, or 7, and (iii) blood serum samples from pigs with an unknown A. pleuropneumoniae exposure 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 15 A. pleuropneumoniae serovars. Seroconversion to ApxII and ApxIII was detected by FMIA in pigs inoculated with A. suis. The vaccinated pigs showed poor humoral responses against ApxI, ApxII, ApxIII, and ApxIV. In the field samples, the humoral response to ApxIV and the A. pleuropneumoniae seroprevalence 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.

Detection of Actinobacillus pleuropneumoniae through Duplex PCR Based on ApxIA and ApxIVA Genes

“…Apx exotoxins secreted by Actinobacillus pleuropneumoniae are toxic factors that deeply impact the pathogenesis of porcine pleuropneumonia. Former studies reported that ApxIVA is produced by all the 15 serotypes of APP and is highly specific to APP (Wei et al, 2012;Giménez-Lirola et al, 2014). Due to the specificity of the ApxIVA gene to the Actinobacillus pleuropneumoniae strains, the detection assays based on this gene have been widely explored for the identification of the Actinobacillus pleuropneumoniae strains (Schaller et al, 2001;Dreyfus et al, 2004;Turni and Blackall, 2007).…”

“Pathotyping” Multiplex PCR Assay for Haemophilus parasuis: a Tool for Prediction of Virulence

“…The presence of multiple isolates of H. parasuis, particularly in URT samples (27)(28)(29), is one of the major challenges in designing a prospective molecular tool for determining virulence. Current surveillance methods for other pathogens such as Actinobacillus pleuropneumoniae operate on an individual isolate level, where the detection of the bacterium initiates a more thorough investigation at the single-colony level to look for the presence of toxins (30,31). We see this pathotyping mPCR as a useful tool for surveillance as a preventative measure.…”

Padronização da técnica de nanopartícula de ouro não modificada (AuNPs) para detecção de Actinobacillus pleuropneumoniae em pulmões de suínos

“…O contágio geralmente ocorre após inalação de aerossóis ou contato direto, o microrganismo inalado coloniza o tecido pulmonar aderindo-se ao muco, proteínas e a células hospedeiras com posterior multiplicação no local (Chiers et al 2010). O monitoramento do status da doença em rebanhos, o controle e a erradicação são de fundamental importância devido a características de contágio rápido deste agente, para impedir que animais portadores sejam introduzidos em rebanhos saudáveis (Tremblay et al 2013).Testes indiretos são muito utilizados (Machado et al 2001, Shin et al 2011, Eamens et al 2012a,b, GimenezLirola et al 2014,para detecção simultânea de anticorpos para toxinas com sensibilidade de 82,7% e especificidade de 100% (Gimenez-Lirola et al 2014). São descritos testes de ELISA monovalentes e polivalentes para sorotipos, com sensibilidade e especificidade variando de 88,3% a 96,6% (Machado et al 2001).…”

“…Testes indiretos são muito utilizados (Machado et al 2001, Shin et al 2011, Eamens et al 2012a,b, GimenezLirola et al 2014,para detecção simultânea de anticorpos para toxinas com sensibilidade de 82,7% e especificidade de 100% (Gimenez-Lirola et al 2014). São descritos testes de ELISA monovalentes e polivalentes para sorotipos, com sensibilidade e especificidade variando de 88,3% a 96,6% (Machado et al 2001).…”