A Bacterial Glycoengineered Antigen for Improved Serodiagnosis of Porcine Brucellosis

Abstract

Brucellosis is a highly zoonotic disease that affects animals and human beings. Brucella suis is the etiological agent of porcine brucellosis and one of the major human brucellosis pathogens. Laboratory diagnosis of porcine brucellosis mainly relies on serological tests, and it has been widely demonstrated that serological assays based on the detection of anti O-polysaccharide antibodies are the most sensitive tests. Here, we validate a recombinant glycoprotein antigen, an N-formylperosamine O-polysaccharide-protein conjugate (OAg-AcrA), for diagnosis of porcine brucellosis. An indirect immunoassay based on the detection of anti-O-polysaccharide IgG antibodies was developed coupling OAg-AcrA to enzyme-linked immunosorbent assay plates (glyco-iELISA). To validate the assay, 563 serum samples obtained from experimentally infected and immunized pigs, as well as animals naturally infected with B. suis biovar 1 or 2, were tested. A receiver operating characteristic (ROC) analysis was performed, and based on this analysis, the optimum cutoff value was 0.56 (relative reactivity), which resulted in a diagnostic sensitivity and specificity of 100% and 99.7%, respectively. A cutoff value of 0.78 resulted in a test sensitivity of 98.4% and a test specificity of 100%. Overall, our results demonstrate that the glyco-iELISA is highly accurate for diagnosis of porcine brucellosis, improving the diagnostic performance of current serological tests. The recombinant glycoprotein OAg-AcrA can be produced in large homogeneous batches in a standardized way, making it an ideal candidate for further validation as a universal antigen for diagnosis of "smooth" brucellosis in animals and humans.

FIG 1

OAg-AcrA recombinant glycoprotein as a novel antigen for the diagnosis of porcine brucellosis. Six animals were intramuscularly inoculated with 5 × 10⁶ CFU of B. suis 1330, and serum samples were obtained before the infection and 16 and 60 days postinfection. (A) Glyco-iELISA analysis of the samples. The reactivity values are relative to the positive control included in each assay run. (B) Western blot analysis of the serum samples obtained before infection (Pre) and 60 days postinfection (Post). For animal 1, the analyzed sample corresponded to that obtained at 16 days postinfection. NG, nonglycosylated AcrA; G, glycosylated AcrA (OAg-AcrA). The positions of molecular mass standards (in kDa) are indicated on the left. The arrows on the right indicate the migration positions of nonglycosylated AcrA.

FIG 2

Dot plot and ROC analysis of glyco-iELISA results. (A) Dot plot analysis. Serum samples obtained from positive (POS) and negative (NEG) animals were tested by glyco-iELISA as indicated in Materials and Methods. The POS group included serum samples obtained from culture-positive and serologically positive (by at least two different serological tests) animals. The NEG group included samples obtained from animals coming from herds with a history of brucellosis (NEG exposed); herds without any history of the disease (NEG unexposed); and herds from Canada, a brucellosis-free country (NEG free country). The mean and standard deviation for each group are indicated: POS, 1.43 ± 0.33; NEG exposed, 0.21 ± 0.09; NEG unexposed, 0.13 ± 0.05; NEG free country, 0.14 ± 0.04. ***, P < 0.0001; Mann-Whitney test. (B) TG-ROC plot of the results. ROC analysis was carried out using as a positive reference samples the sera of the POS group and as a negative reference samples of the sera of the three NEG groups described for the dot plot in panel A. The dotted and dashed lines in the dot plot and TG-ROC plots indicate the cutoff values for which maximal diagnostic Se or Sp, respectively, were achieved. The two cutoff values represent the bounds of an intermediate range (IR) of reactivity values (shaded areas). (Inset) Se and Sp values obtained for the two cutoff values. The cutoff value that concurrently optimizes Se and Sp coincides with the cutoff value for which maximal Se was achieved (0.56; dotted line).