Use of recombinant antigens to detect antibodies against Mycoplasma suis, with correlation of serological results to hematological findings

Abstract

Porcine eperythrozoonosis is a disease with worldwide distribution caused by the unculturable hemotrophic bacterium Mycoplasma suis. Current serological testing utilizes crude M. suis antigens purified from the blood of experimentally infected pigs. These antigens show high variability and are restricted to specialized laboratories. We evaluated a novel serological assay based on two recombinant M. suis antigens (rMSG1 and rHspA1). Antigen specificity was proven by means of sera raised against nonhemotrophic mycoplasma and other relevant bacteria. Using experimental and convalescent-phase sera, rMSG1 and rHspA1 enzyme-linked immunosorbent assays (ELISAs) demonstrated sensitivities, specificities, and predictive values (94.0 to 100.0%) equal to or higher than those of the M. suis whole-cell ELISA. Field samples from 120 weaning piglets grouped by quantitative PCR results were used to evaluate the diagnostic capability of the new ELISA systems in comparison to that of the whole-cell ELISA. Assuming a 100.0% specificity of the PCR, the whole-cell ELISA, rHspA1 ELISA, and rMSG1 ELISA showed specificities of 84.8%, 83.8%, and 90.6% and sensitivities of 61.5%, 74.0% and 58.1%, respectively. Cohen's kappa coefficients comparing the recombinant ELISAs to the whole-cell ELISA indicate moderate to substantial agreement. The detection of anti-MSG1 and/or anti-HspA1 antibodies in pigs was significantly correlated with decreased hematocrit, erythrocyte numbers, and hemoglobin concentrations, indicating that a single seropositive result is connected with clinical and etiological significance. In conclusion, rMSG1 and rHspA1 are sensitive and specific serological and infection markers which are for the first time used independently of animal experiments. They are especially fit to be used in routine diagnosis, pathogenesis studies, and large-scale epidemiological investigations.

FIG. 1.

(A) SDS-PAGE of recombinant MSG1 and HspA1, showing the purity after affinity chromatography preparation. (B and C) Immunoblot analysis of recombinant MSG1 and HspA1; blots were detected with a serum pool of experimentally M. suis-infected pigs (B) or with a serum pool of M. suis-negative pigs (C). MW, molecular mass standard; Ec, E. coli control preparation.

FIG. 2.

Serological results for M. suis-positive pigs (circles; experimentally infected and diseased pigs) and control pigs (blank triangles; mock-infected and healthy, PCR-negative pigs). Results are shown for both recombinant antigens (rMSG1, 60 ng/well; rHspA1, 30 ng/well) in comparison to the M. suis whole-cell (Ms) ELISA. Adsorbance (OD₄₀₅₎ values are shown for reactions with serum samples diluted 100-fold. Cutoff values for each antigen are indicated as dashed line.

FIG. 3.

Representative antibody kinetics for one experimental pig, using three different ELISAs, is presented. Sequential sera from an M. suis-infected pig collected pre- and postinoculation were incubated with blood-derived Ig-depleted M. suis antigen (Ms ELISA) or the recombinant protein rMSG1 or rHspA1. Net optical densities (OD₄₀₅) were measured. During acute PE (weeks 4 and 7, indicated by arrows), low levels of ELISA reactivities with the M. suis antigen and both recombinant proteins were monitored.