Identification ofMycoplasma suisantigens 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.

An updated roadmap for MERS-CoV research and product development: focus on diagnostics

“…A syndromic approach involves testing for pathogens based on a syndrome such as fever or acute respiratory distress; a shift from individual tests to multiplex panels can quickly identify or eliminate likely pathogens from a single specimen. For analysis of circulating reservoirs, multiplex microbead-based immunoassays have been used to detect IgG antibodies for multiple pathogens 98 99. Multiplex, syndromic panels that include MERS-CoV have been demonstrated using PCR-based panels including MERS-CoV, showing similar limits of detection to single assays 89 100 101.…”

RNA-Seq based transcriptome of whole blood from immunocompetent pigs (Sus scrofa) experimentally infected with Mycoplasma suis strain Illinois

“…Sham-inoculated pigs (pig #1, pig #2 and pig #3) were consistently negative on qPCR for the extent of the experiment. B Serum antibodies were measured using recombinant GrpE (rGrpE) protein in a microbead immunoassay (MIA) [ 30 ]. + = positive, − = negative, based on established MFI (median fluorescence intensity) cut-off.…”

“…The animals were monitored daily (minimum twice a day) for direct observation of behavior (BAR status ) and body temperature. Blood was collected through the anterior venous cava vein and added to EDTA tubes every 3–4 days for monitoring infection by qPCR [ 20 ], hematocrit, and detection of specific antibodies for M. suis recombinant GrpE (rGrpE) antigen using microbead immunoassay (MIA) [ 30 ]. Pigs were considered seropositive when their median fluorescence intensity (MFI) crossed the cut-off defined as the mean plus 3 standard deviation of the pre-inoculation MFIs (day 0) for all animals.…”

Frequency of infection with Mycoplasma suis in gestating sows using qPCR on ten commercial French herds, and impact of the infection on clinical, haematological and biochemical parameters

“…For a long time, direct reading of a stained blood smear was the only available diagnostic method. This diagnostic limitation was a constraint for estimating its prevalence at regional and national level [5,9,15,19]. The introduction of quantitative polymerase chain reaction (qPCR) tests has made it much easier to detect the bacterium.…”