A Bacterial Enginnered Glycoprotein as a Novel Antigenic Target for Diagnosis of Bovine Brucellosis

ANDRÉS E. CIOCCHINI; REY SERANTES D.A.; MELLI LJ; IWASHKIW JA; ELENA S.; FRANCO C.; NICOLA A.M.; FELDMAN M. F.; COMERCI D. J; UGALDE JE

Chicago

Conferencia; 66th Annual Brucellosis Research Conference; 2013

Brucellosis Research Society

Brucellosis is a highly contagious zoonosis that affects livestock and human beings. Due to its heterogeneous and poorly specific symptoms, diagnosis of brucellosis always requires laboratory confirmation. Diagnosis of the disease based exclusively on Brucella isolation presents many limitations; therefore, laboratory diagnosis very often relies on detecting specific serum antibodies. Here we present the first recombinant glycoprotein antigen for diagnosis of bovine brucellosis. Since the humoral immune response to ??smooth?? brucellae is dominated by antibodies to the O-polysaccharide portion of the Brucella smooth lipopolysaccharide (sLPS) and because Yersinia enterocolitica O:9 and Brucella abortus share an almost identical O-polysaccharide structure, we explored the application of a recombinant Y. enterocolitica O:9-polysaccharide-protein conjugate (OAg-AcrA) in the diagnostics of bovine brucellosis. OAg-AcrA was produced in Y. enterocolitica O:9 cells co-expressing the N-linked protein glycosylation OTase PglB and the protein acceptor AcrA of Campylobacter jejuni. Introduction of PglB and AcrA in Y. enterocolitica resulted in the transfer of the N-formylperosamine O-polysaccharide from its lipid carrier to AcrA and the resulting glycoprotein was purified from the periplasm by affinity chromatography. An indirect immunoassay based on the detection of anti O-polysaccharide IgG antibodies in bovine samples was developed coupling OAg-AcrA to magnetic beads or ELISA plates. As a proof of concept, we analyzed serum, whole blood and milk samples obtained from non-infected, experimentally infected and vaccinated animals included in a vaccination/infection trial performed in our laboratory. Obtained data demonstrate that with this antigen it is possible to clearly differentiate infected from non-infected and vaccinated animals using samples of different nature, and indicate that the detected antibody response is specifically directed towards the O-polysaccharide moiety of the glycoconjugate. To validate the assay, more than 900 serum and milk samples obtained from naturally infected and vaccinated animals from Argentina were tested, and a receiver-operating characteristic (ROC) analysis was performed. Based on this analysis, the area under the ROC curve for the test was 0.9996 (95% CI, 0.9989 to 1.000) and the optimum cutoff value was 22.9 %, which resulted in a diagnostic sensitivity and specificity of 98.9 and 99.5 %, respectively. A cutoff value of 18.4 % resulted in a diagnostic sensitivity of 100% and a diagnostic specificity of 97.6 %. Finally, a cutoff value of 28.8 % resulted in a test sensitivity of 94.6% and a test specificity of 100%. Our results demonstrate that glycoconjugate-based assays are highly accurate for diagnosis of bovine brucellosis and we propose the OAg-AcrA antigen as a new diagnostic tool for diagnosis of brucellosis caused by ??smooth?? brucellae.