Identification of Mycoplasma suis antigens and development of a multiplex microbead immunoassay

Abstract

The aims of the current study were to identify Mycoplasma suis antigens and develop a multiplex microbead immunoassay (MIA). A M. suis-expression library was screened for immunogens using sera from infected pigs. Based on bioinformatics, putative antigens were identified within positive inserts; gene fragments were expressed and purified as polyhistidine fusion proteins, and immunoreactivity was confirmed by Western blot. Selected antigens were used to develop a MIA. Sera from noninfected and infected pigs were used to set the median fluorescent intensity (MFI) cutoffs and as positive controls, respectively. Assay specificity was tested using sera from pigs seropositive for other pathogens (2 different pigs seropositive for each pathogen). Samples from 51 field pigs and 2 pigs during the course of acute (pig 1) and chronic (pig 2) infections were tested using MIA, indirect hemagglutination assay (IHA), and quantitative polymerase chain reaction (qPCR). Sixteen reactive plaques (52 genes) were detected. A heat-shock protein (GrpE), a nicotinamide adenine dinucleotide-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPN), and 4 proteins from paralogous gene families (PGFs) were identified as antigens by Western blot. While GrpE, GAPN, and 1 PGF protein were strong antigens, the others were not suitable as MIA targets. A MIA using GrpE, GAPN, and the strongly reactive PGF protein was developed. Cross-reactivity with sera from pigs infected with Mycoplasma hyopneumoniae, Porcine circovirus-2, Porcine parvovirus, Porcine reproductive and respiratory syndrome virus, and Porcine respiratory coronavirus with this MIA was not observed. Pig 2 was consistently positive by MIA and qPCR, whereas pig 1, initially negative, seroconverted before becoming qPCR positive. Only 2 samples (from pig 1) were IHA positive. Five (9.8%) field samples were qPCR positive and 40 (78.43%) were positive for all 3 MIA antigens; however, all were IHA negative. In summary, the MIA is specific and more sensitive than qPCR and IHA, providing simultaneous evaluation of antibody response to M. suis antigens.