A Bacterial Glycoengineered Antigen for Improved Serodiagnosis of Porcine Brucellosis

Abstract

dBrucellosis 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 (glycoiELISA). 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. B rucella spp. are Gram-negative facultative intracellular bacteria responsible for brucellosis in humans and animals. Animal brucellosis has a major economic impact because the infection causes abortions and stillbirths and reduces fertility in herds, while brucellosis in humans is a debilitating disease characterized by fever, sweating, and pain (1, 2). Brucella suis is the etiological agent of porcine brucellosis and one of the main human brucellosis pathogens, together with Brucella melitensis and Brucella abortus. There are five biovars of B. suis, with biovars 1, 2, and 3 being responsible for porcine brucellosis (3). B. suis biovars 1 and 3, endemic in Asia and America, are highly zoonotic and cause serious reproductive problems in pigs (infertility, abortion, and orchitis) and a serious disease in humans. Biovar 2 is restricted to Europe, where it represents an emerging problem with a high economic impact in pig farms and is less pathogenic for humans.In the absence of an effective porcine brucellosis vaccine, control of the disease in pigs depends exclusively on detection and slaughter of infected animals. The gold standard method for confirmation of the infection is isolation of the pathogen; however, the slow growth of brucellae in primary cultures (up to 7 days), the risk involved in their handling, and the poor sensitivity of the meth...

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