Serological assays are inexpensive, easy-to-perform diagnostic tests which are applied in a great range of microbiological routine laboratories. Their overall quality depends on the choice and high class of the antigens used for antibody detection. All serological M. suis
assays described formerly have the intrinsic disadvantage of employing complex and undefined M. suis
antigens obtained from the peripheral blood of experimentally infected pigs that contains uncontrollable amounts of porcine components (e.g., porcine Igs) (1
). Serological assays based on recombinant antigens are the only possibility for circumventing these drawbacks. Recombinant protein-based serological tests may achieve high sensitivity and specificity because of the high concentration of immunoreactive antigens and the lack of nonspecific moieties found in whole-cell preparations from the blood of experimentally infected pigs. An ideal antigen would be a principal target of the host immune response (serodiagnostic markers of infection), expressed by M. suis
during acute and chronic disease and conserved among M. suis
isolates. In addition, differentiation between autoreactive serological reactions directed against porcine constituents and agent-specific seroreactivities should be easily possible given the fact that the type and intensity of the immune response (M. suis
-specific versus autoreactive) obviously play a major role in the course of the hemoplasma infection and the development of disease (5
In previous studies, we identified two M. suis
proteins with such features, i.e., HspA1 and MSG1 (5
). Within the scope of this study, we evaluated serological assays based on these two proteins expressed in E. coli
. Purity and seroreactivity of both recombinant proteins were proven in SDS-PAGE and immunoblot studies. The analytical specificity of the two recombinant antigens was confirmed by testing rabbit and pig sera induced against nonhemotrophic mycoplasma species of a different origin but related to M. suis
and against other microorganisms with clinical relatedness. We did not detect any cross-reactivity in immunoblot and ELISA analyses in any of the aforementioned cases.
The investigation of sera from experimentally infected pigs with the novel recombinant ELISA demonstrated high specificities and sensitivities and a high agreement with the whole-cell M. suis
ELISA. In addition, the kinetics of the immune response in the course of experimentally induced PE was confirmed using rHspA1 and rMSG1. Typically, during acute clinical PE, a derailment of the M. suis
-specific antibody response was accompanied by peaking levels of autoreactive IgG antibodies (5
). The derailment of M. suis
-specific antibodies was also observed using recombinant proteins as antigens. Therefore, the problem of detecting M. suis
-specific antibodies during clinical acute PE was not resolved by means of using recombinant antigens. This phenomenon is obviously to be traced back not to the antigen used but to a derailment of the immune response during acute PE (5
Our results demonstrate that both antigens are suitable for diagnostic assays. In comparison, rHspA1 showed a higher sensitivity with a lower specificity, whereas rMSG1 showed a higher specificity combined with a lower sensitivity. Therefore, an assay combining both antigens could prove to be a good alternative: in our study, the parallel application of both antigens to classify pigs as positive or negative enhanced the sensitivity. Moreover, this antigen combination is considered to be appropriate because investigations of the M. suis
-specific immune response in the course of infection revealed a very heterologous antibody profile by means of the detected antigens (5
). Nevertheless, the combination of two immunodominant M. suis
antigens (HspA1 and MSG1) allowed us to detect both acutely and chronically infected pigs.
A reference test (gold standard) for the detection of antibodies directed against M. suis
with which the ELISA results could be compared is not available. Therefore, the establishment of the infectious status of the pigs was performed on the basis of quantitative PCR results. Negative sera were collected from experimental pigs free of M. suis
as tested by PCR. Positive sera were collected from pigs infected experimentally that tested positive for infections (PCR and clinical investigation). The examination of these well-defined groups by means of both recombinant ELISAs yielded high specificities and sensitivities (94.0 to 100.0%). To evaluate the diagnostic applicability, field sera from slaughtered pigs were grouped by investigating the corresponding anticoagulated blood specimens using real-time PCR (8
). In the group of PCR-positive piglets, the ratio of serologically positive piglets was significantly higher (58.3 to 75.0%) than that in the group of PCR-negative piglets (13.3 to 26.6%). When the results of recombinant ELISAs were compared to those of the whole-cell ELISA, we observed considerable levels of agreement (80.0% and 82.5%; kappa values, 0.52 and 0.71, respectively). It must be noted that the use of both recombinant ELISA results as a diagnostic criterion means that the two tests are not conditionally independent (both tests measure anti-M. suis
antibodies). Combinatorial testing with rMSG1 and rHspA1 as diagnostic antigens revealed a correlation of sensitivity of 0.09 (CI, 95%; range, 0.043 to 0.145) and the highest sensitivity (80.0% positive in the group of PCR-positive animals).
The significance of a positive serological result with rHspA1 or/and rMSG1 as antigens could be proved by a significant correlation between the result of the serology and the clinical hematological parameters, i.e., pigs showing antibodies against M. suis
also revealed significantly lowered values of packed cell volume, hemoglobin, and RBC numbers. The absence of significant differences in erythrocytic indices (mean cellular volume, mean cellular hemoglobin, and mean cellular hemoglobin concentration) are indicative for a normochromic, normocytic anemia due to intravascular hemolysis. However, at this point, we do not know whether intra- or extravascular hemolysis predominates during M. suis
), and the analysis of hemolysis mechanisms during M. suis
infections should be the goal of further studies. In addition, we discovered a greater ratio of pigs with bacterial loads higher than 105
per ml in the group of seropositive piglets.
Remarkably, antibodies against M. suis were detected in approximately 20% of the piglets in the PCR-negative group. At first sight, these results seem to speak in favor of a false-positive serology. However, all sera were reanalyzed using immunoblots against whole-cell M. suis antigens and against the recombinant proteins, and the seropositivity could thus be confirmed in practically all cases (data not shown). Therefore, it is obvious that our serological assay enables the detection of seropositive animals after clearance of the infection or the detection of chronic low-grade infections which cannot be recognized by PCR or by microscopy. These carrier pigs are of major economic significance, as well as being important with regard to epidemiology, surveillance, and the eradication of PE. Altogether, the new M. suis serological assays are a very valuable completion of our diagnostic possibilities for M. suis infections, especially in addition to PCR.
To date, no serological test for PE has achieved integration into routine diagnostic laboratories. The serological assays currently available are inadequate for routine work because they can be carried out only in those few laboratories able to propagate M. suis in splenectomized pigs and therefore able to prepare diagnostic antigens. Our new recombinant ELISA will contribute to rectifying this problem. The robustness of our serological assays, reflected by the low intra- and interassay coefficients of variation of 6.5 to 8.2% and 20.6%, respectively, provide further evidence for this. Arguments against the application of recombinant antigens in serology are protein stability, lack of sensitivity due to only a few antigens of the agent being used, and E. coli impurities which can be traced back to insufficient purification procedures. Within the scope of our work, the stability of the recombinant proteins proved to be very high (approximately 6 months at −80°C). We determined a similar sensitivity in comparison to results with whole-cell antigens, and as mentioned, our rHspA1 and rMSG1 antigens were quite pure. Nevertheless, an adsorption of the investigated sera with E. coli was unavoidable. Because an adsorption of the prediluted sera (1:25) over a period of about 2 h proved to be sufficient to remove cross-reacting antibodies, it should be possible to perform this additional task in routine laboratories.
In summary, this article describes for the first time the development of a serodiagnostic assay for PE based on antigens produced in the laboratory. The use of the recombinant proteins MSG1 and HspA1 has important advantages, i.e., (i) improved specificity, sensitivity, reproducibility, and reliability of PE serodiagnosis, (ii) replacement of animal experiments necessary to propagate M. suis for antigen production, (iii) the possibility of extensive prevalence and surveillance studies determining the actual impact of M. suis infection in pig populations, and (iv) studies on the connection between the M. suis immune response and PE pathogenesis.