Diagnostic performance of serological tests for swine brucellosis in the presence of false positive serological reactions
Introduction
Swine brucellosis is an important infectious disease caused by three Brucella suis biovars. Biovars 1 and 3, endemic in Asia and America, are highly zoonotic, causing serious reproductive problems in pigs and a grave disease in humans (Olsen et al., 2012). B. suis biovar 2 is presently restricted to Europe, where it represents an emerging problem causing abortions, infertility and a high economic impact in pig farms. However, in contrast to biovars 1 and 3, biovar 2 is very rarely a cause of human brucellosis, and it has been isolated exclusively from a few immune-compromised patients (EFSA, 2009). In Europe, this infection affects mostly outdoor pig breeding systems, being in all likelihood a spill-over of brucellosis of wild boars and European hares, two known wildlife reservoirs of B. suis biovar 2 (Garin-Bastuji et al., 2000, Godfroid and Kasbohrer, 2002, EFSA, 2009). Despite this, Europe is currently considered free from swine brucellosis, and surveillance performed only for trade and semen production. Tests detecting antibodies to the O-polysaccharide (O-PS) moiety of Brucella smooth lipopolysaccharide (S-LPS), such as the Rose Bengal (RBT), Serum Agglutination (SAT), complement fixation (CFT), Fluorescence Polarization and indirect or competitive enzyme-linked immunosorbent assays (iELISA and cELISA, respectively) are recommended for this purpose (EFSA, 2009, OIE, 2012). However, in addition to validation issues (Muñoz et al., 2012), these O-PS tests lack specificity to discriminate the false positive serological reactions (FPSRs) caused by bacteria sharing O-PS epitopes with Brucella S-LPS. This FPSR problem is increasingly common in Europe, results in costly quarantines and retesting schedules, and is one of the most important causes of swine trade restrictions (Jungersen et al., 2006, EFSA, 2009). However, a sensitive bacteriological diagnosis for confirmation requires a thorough examination of necropsy samples (Olsen et al., 2012), and it is not practical for individual diagnosis in large animal populations.
Several serological tests have been investigated to solve the FPSR problem, including both cELISA and iELISA with S-LPS, as well as iELISA with an extract of rough (R) brucellae (Weynants et al., 1996, Nielsen et al., 2006, McGiven et al., 2012). The results of these investigations show that serial and cumbersome testing schedules would be necessary to increase the specificity of the diagnosis and that no test is fully specific to differentiate brucellosis from FPSR in pigs. On the other hand, a skin test with the protein-rich brucellin obtained from rough Brucella melitensis B115 is suitable for discriminating FPSR in pigs (EFSA, 2009, Dieste-Pérez et al., 2014). B. melitensis B115 is a spontaneous R mutant that, in addition to other genetic defects, carries a frame shift mutation in the O-PS export ABC system (Godfroid et al., 2000, Adone et al., 2011). Thus, although unable to synthesize S-LPS, B115 accumulates O-PS internally (Cloeckaert et al., 1992). Indeed, this O-PS is a potential cause of specificity problems in the FPSR context (Dieste-Pérez et al., 2014) and, in fact, it was reported that injection of B115 brucellin causes a rise in agglutinating antibodies to this antigen in pigs experimentally infected with Brucella (Stuart et al., 1987). Although this potential problem has been recently solved by the use of genetically defined Brucella R mutants blocked in O-PS biosynthesis (Dieste-Pérez et al., 2014), skin testing requires two veterinary visits and is thus somewhat cumbersome and expensive. Therefore, we have also investigated the usefulness in the FPSR context of serological tests with antigens lacking the O-PS. Here, we report the results obtained with R-LPS and the protein rich cytosolic fraction of a genetically defined R mutant in comparison with S-LPS tests.
Section snippets
Bacterial strains and growth conditions for antigen extraction
Brucella abortus tn5::per is a R mutant (obtained by transposon mutagenesis from S B. abortus 2308 virulent strain) that has been thoroughly characterized in a previous work (Monreal et al., 2003). As per encodes the perosamine synthase (Godfroid et al., 1998), the tn5 insertion prevents the synthesis of perosamine, the only sugar in the Brucella O-PS (for a review of O-PS synthesis, see González et al., 2008). Consequently, the LPS of this R mutant is truncated and devoid of O-PS. B. abortus
Results
Table 1 summarizes the results obtained with the different tests and antigens. The S-LPS tests showing the highest diagnostic sensitivities were the RBT (98.15% and 93.21%, modified and standard procedures, respectively) and the commercial S-LPS iELISA (95.06%). The better diagnostic sensitivity of the modified RBT was, however, accompanied by a decrease in specificity (90.32%), making this RBT modification unsuitable for swine brucellosis. Both CFT and S-LPS GD provided similar but only
Discussion
Because of the existence of FPSR and of the severe impact that an official declaration of a swine brucellosis outbreak would have on international trade, all seropositive pigs should be confirmed as truly infected. Yet, bacteriological diagnosis, presently the only unequivocal test, is impractical for routine use. Although the origin of the problem has not been fully investigated, Y. enterocolitica O:9 is considered the main cause of FPSR in pigs in the EU (Thibodeau et al., 2001, EFSA, 2009).
Conclusion
Considering their moderate to high combined sensitivity (86.4%), and the perfect (100%; CI = 98.3–100, for CP GD) or close to perfect (96.8%; CI = 93.5–98.7, for S-LPS GD) specificity of immunoprecipitation tests in either the brucellosis free or FPSR populations, these simple assays could be used in parallel on RBT (or S-LPS iELISA) positive samples as a practical protocol for a serological screening of FPSR in pigs. The skin test with O-PS free cytosoluble extracts could be then used for a
Acknowledgments
This research was supported by a PhD grant (L. Dieste-Pérez) from the Aragón Government (2011–2015) and by the following projects: INIA project RTA2011-00103-00-00, MICIN project AGL2011-30453-C04 and consolidated group from Aragón Government A14. We wish to acknowledge A. Delgado, S. Serrano and M. Uriarte for the excellent technical assistance.
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