Diagnostic performance of serological tests for swine brucellosis in the presence of false positive serological reactions

https://doi.org/10.1016/j.mimet.2015.02.001Get rights and content

Highlights

  • False positive serological reactions pose problems in swine brucellosis surveillance.

  • Cytosoluble O/PS free protein extracts (CP) were used for differential serodiagnosis.

  • Diagnostic performance of Brucella CP, R-LPS and S-LPS based tests was assessed.

  • I-ELISA using either CP or R-LPS antigens resulted in poor diagnostic performance.

  • Gel immunodifussion with CP and S-LPS can be used at herd level to solve FPSR problem.

Abstract

Swine brucellosis caused by Brucella suis biovar 2 is an emerging disease in Europe. Currently used diagnostic tests for swine brucellosis detect antibodies to the O-polysaccharide (O-PS) of Brucella smooth lipopolysaccharide (S-LPS) but their specificity is compromised by false-positive serological reactions (FPSRs) when bacteria carrying cross-reacting O-PS infect pigs. FPSRs occur throughout Europe, and the only tool available for a specific B. suis diagnosis is the intradermal test with Brucella protein extracts free of O-PS or S-LPS. Using sera of 162 sows naturally infected by B. suis biovar 2, 406 brucellosis-free sows, and 218 pigs of brucellosis-free farms affected by FPSR, we assessed the diagnostic performance of an indirect ELISA with rough LPS (thus devoid of O-PS) and of gel immunodiffusion, counterimmunoelectrophoresis, latex agglutination and indirect ELISA with O-PS free proteins in comparison with several S-LPS tests (Rose Bengal, complement fixation, gel immunodiffusion and indirect ELISA). When adjusted to 100% specificity, the sensitivity of the rough LPS ELISA was very low (30%), and adoption of other cut-offs resulted in poor specificity/sensitivity ratios. Although their specificity was 100%, the sensitivity of protein tests (ELISA, latex agglutination, counterimmunoelectrophoresis, and gel immunodiffusion) was only moderate (45, 58, 61 and 63%, respectively). Among S-LPS tests, gel immunodiffusion was the only test showing acceptable sensitivity/specificity (68 and 100%, respectively). Despite these shortcomings, and when the purpose is to screen out FPSR at herd level, gel immunodiffusion tests may offer a technically simple and practical alternative to intradermal testing.

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.

References (58)

  • K. Nielsen et al.

    Validation of the fluorescence polarization assay as a serological test for the presumptive diagnosis of porcine brucellosis

    Vet. Microbiol.

    (1999)
  • K. Nielsen et al.

    Serological discrimination by indirect enzyme immunoassay between the antibody response to Brucella sp. and Yersinia enterocolitica O:9 in cattle and pigs

    Vet. Immunol. Immunopathol.

    (2006)
  • K. Nielsen et al.

    Validation of a second generation competitive enzyme immunoassay (CELISA) for the diagnosis of brucellosis in various species of domestic animals

    Vet. Immunol. Immunopathol.

    (2008)
  • U. Riber et al.

    Cell-mediated immune responses differentiate infections with Brucella suis from Yersinia enterocolitica serotype O:9 in pigs

    Vet. Immunol. Immunopathol.

    (2007)
  • F.A. Stuart et al.

    Experimental Brucella abortus infection in pigs

    Vet. Microbiol.

    (1987)
  • V. Thibodeau et al.

    Development of an ELISA procedure to detect swine carriers of pathogenic Yersinia enterocolitica

    Vet. Microbiol.

    (2001)
  • R. Adone et al.

    Antigenic, immunologic and genetic characterization of rough strains B. abortus RB51, B. melitensis B115 and B. melitensis B18

    Plos One

    (2011)
  • B. Alonso-Urmeneta et al.

    Evaluation of lipopolysaccharides and polysaccharides of different epitopic structures in the indirect enzyme-linked immunosorbent assay for diagnosis of brucellosis in small ruminants and cattle

    Clin. Diagn. Lab. Immunol.

    (1998)
  • V. Aragón et al.

    Characterization of Brucella abortus and Brucella melitensis native haptens as outer membrane O-type polysaccharides independent from the smooth lipopolysaccharide

    J. Bacteriol.

    (1996)
  • P.C. Baldi et al.

    Humoral immune response against lipopolysaccharide and cytoplasmic proteins of Brucella abortus in cattle vaccinated with B. abortus S19 or experimentally infected with Yersinia enterocolitica serotype 0:9

    Clin. Diagn. Lab. Immunol.

    (1996)
  • J.M. Blasco et al.

    Efficacy of different Rose Bengal and complement fixation antigens for the diagnosis of Brucella melitensis infection in sheep and goats

    Vet. Rec.

    (1994)
  • J.M. Blasco et al.

    Evaluation of allergic and serological tests for diagnosing Brucella melitensis infection in sheep

    J. Clin. Microbiol.

    (1994)
  • R.A. Bowden et al.

    Simultaneous expression of smooth and rough phase properties related to lipopolysaccharide in a strain of Brucella melitensis

    J. Med. Microbiol.

    (1993)
  • C.C. Chukwu

    Differentiation of Brucella abortus and Yersinia enterocolitica serotype 09 infections in cattle: the use of specific lymphocyte transformation and brucellin skin tests

    Vet. Quart.

    (1987)
  • A. Cloeckaert et al.

    O-chain expression in the rough Brucella melitensis strain B115: induction of o-polysaccharide specific monoclonal antibodies and intracellular localization demonstrated by immunoelectron microscopy

    J. Gen. Microbiol.

    (1992)
  • A. Cloeckaert et al.

    Use of recombinant BP26 protein in serological diagnosis of Brucella melitensis infection in sheep

    Clin. Diagn. Lab. Immunol.

    (2001)
  • M. Corrente et al.

    Development of a western blotting assay to discriminate Brucella spp. and Yersinia enterocolitica O:9 infections in sheep

    New Microbiol.

    (2004)
  • M.J. De Miguel et al.

    Development of a selective culture medium for primary isolation of the main Brucella species

    J. Clin. Microbiol.

    (2011)
  • R. Díaz

    Estudio de las relaciones antigénicas entre Yersinia enterocolítica serotipo O:9 y otras especies bacterianas gram-negativas

    Microbiol. Esp.

    (1974)
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