Research Article: Evaluation of the comparative accuracy of the complement fixation test, Western blot and five enzyme-linked immunosorbent assays for serodiagnosis of glanders

Date Published: April 5, 2019

Publisher: Public Library of Science

Author(s): Mandy Carolina Elschner, Karine Laroucau, Harisankar Singha, Bhupendra Nath Tripathi, Muhammad Saqib, Ian Gardner, Sheetal Saini, Subodh Kumar, Hosny El-Adawy, Falk Melzer, Iahtasham Khan, Praveen Malik, Carola Sauter-Louis, Heinrich Neubauer, Axel Cloeckaert.


Glanders is a zoonotic contagious disease of equids caused by Burkholderia (B.) mallei. Serodiagnosis of the disease is challenging because of false-positive and false-negative test results. The accuracy of the complement fixation test (CFT) which is prescribed for international trade by the World Organisation for Animal Health (OIE), five ELISAs and a Western blot (WB) were compared for serodiagnosis of glanders using sera from 3,000 glanders-free and 254 glanderous equids. Four ELISA tests are based on recombinant antigens (TssA, TssB, BimA and Hcp1), the IDVet ELISA is based on a semi-purified fraction of B. mallei and WB makes use of a purified LPS-containing B. mallei-antigen. Sensitivity and specificity of tests were estimated using cut-off values recommended by the test developers. The WB and all ELISAs, except BimA, were significantly more specific than the CFT. ELISAs based on TssA, TssB, and BimA antigens had significantly lower sensitivity compared to CFT while the sensitivities of the Hcp1-ELISA, the IDVet-ELISA and the WB did not differ significantly from that of the CFT. Given their comparable sensitivities and specificities, the CFT (98.0%, 96.4%), the WB (96.8%, 99.4%), the Hcp1-ELISA (95.3%, 99.6%) and the IDVet-ELISA (92.5%, 99.5%) should be further developed to meet OIE requirements.

Partial Text

Glanders is an infectious zoonotic disease of equids, caused by the gram-negative bacterium Burkholderia (B.) mallei. Although the disease has been eradicated in most European and North American countries in the last century, there are still outbreaks throughout the South American and Asian continents [1–5]. The disease is mainly chronic in horses and latently-infected animals pose a high risk of reintroduction of the infection into glanders-free countries [6]. Therefore, trade restrictions with animals and products from endemic regions or outbreak areas are mandatory.

The frequency of combinations of the 4 serologic test results (positive/negative) in infected and non-infected populations is shown in Table 2.

In the present study, five ELISAs, a WB and the CFT were comparatively evaluated for the detection of antibodies against B. mallei in equid serum. The diagnostic accuracy of the CFT was analyzed following the guidance in the OIE manual [7]. The specificity of tests depends on cross-reacting organisms which can vary from biotope to biotope [28]. Therefore, serum samples were analyzed separately from 3 different geographical regions (“South-America” batch, “Europe” batch, “Asia” batch), but no significant differences in specificity were found. Based on all 3,000-tested serum samples in this study, it was shown, that the five ELISAs and the WB were more specific than the CFT. Previously published reports showed the utility of ELISA techniques regarding the improvement of specificity in comparison to CFT. The specificity of ELISA using either recombinant BimA antigen or TssB protein were 98.9% and 100%, respectively [14, 18]. The WB was previously shown to have a specificity of 100% [9]. Another study showed the IDVet-ELISA to have excellent specificity (98.9%) in a preliminary validation [22]. However, the estimates were based on lower sample numbers than considered adequate by OIE.