Identification of New Mycobacterium bovis antigens and development of a multiplexed serological bead-immunoassay for the diagnosis of bovine tuberculosis in cattle

Abstract

Serological assays for bovine tuberculosis diagnosis require the use of multiple Mycobacterium bovis specific antigens to ensure the detection of infected animals. In the present study, identification and selection process of antigens, based on data from published proteomic studies and involving the use of bioinformatics tools and an immuno-screening step, was firstly performed for identifying novel antigens that elicit an antibody response in M. bovis infection. Based on this approach, a panel of 10 M. bovis antigens [with known relevance (MPB70, MPB83, MPB70/83, and ESAT6/CFP10) and novel (Mb1961c, Mb1301c, Mb3871, Mb1403, Mb0592, and PE25/PPE41)] were constructed and thenused to develop a new multiplexed serological assay based on Luminex technology. The performance of the Luminex-bTB immunoassay was evaluated using sera from cattle with known tuberculosis status. Among the proteins whose ability to detect bovine tuberculosis was evaluated for the first time, PE25/PPE41 and Mb1403, but not Mb3871, showed good detection capacity. Following multiple antigen combination, the final Luminex-bTB immunoassay included seven antigens (MPB70, MPB83, MPB70/83, ESAT6/CFP10, PE25/PPE41, Mb1403, and Mb0592) and showed better global performance than the immunoassay using the four usual antigens (MPB70, MPB70/83, MPB83 and ESAT6/CFP10). The specificity and sensitivity values were, respectively, of 97.6% and 42.8% when the cut-off of two-positive antigens was used to classify samples as positive. With the use of the more-restrictive criterion of three-positive antigens, the specificity increased to 99.2% but the sensitivity decreased to 23.9%. The analysis of antigen profiles generated with the Luminex-bTB immunoassay showed that mainly serodominant proteins were recognized in samples from infected cattle. The detection of Mb1961c and Mb1301c appeared to be associated with presumed false-positive results. Moreover, sera from cattle originating from bTB-outbreaks but having inconclusive or negative skin test results were identified as positive by the Luminex-bTB immunoassay and showed an antigen pattern associated with M. bovis infection. The Luminex-bTB immunoassay including seven antigens may be useful as adjunct test for the detection of M. bovis–infected herds, and different cut-offs could be applied according to the bovine tuberculosis epidemiological context.

The Presence of esat-6 and cfp10 and Other Gene Orthologs of the RD 1 Region in Non-Tuberculous Mycobacteria, Mycolicibacteria, Mycobacteroides and Mycolicibacter as Possible Impediments for the Diagnosis of (Animal) Tuberculosis

“…This has been a great concern, as these protein products have been broadly used as markers in the cell-mediated immunodiagnosis of both human and animal TB, since they were thought to be specific to members of the MTBC [ 6 , 10 , 25 ]. As such, other proteins have been investigated for use as markers for the detection of CMI and humoral responses or as a combination of both for the diagnosis of (animal) TB [ 3 , 16 ].…”

“…The M. bovis and M. tuberculosis Esx-1 encodes 23 genes, namely esxA to esxW , which occur in pairs, and the singleton esxQ , related to the WXG100 family of proteins, which is characterized by a size of ~100 amino acids and the presence of a Trp-Xaa-Gly (W-X-G) motif [ 2 ]. The role of the Esx-1 secretion system in virulence as well as immunogenicity has been well described [ 3 ]. The WXG100 family of proteins are among the most immunodominant antigens recognised by the animal and human immune system [ 4 ].…”

“…Since (animal) TB diagnosis by CMI is complicated by the cross-reactivity of the immunodominant proteins with NTM orthologs, antibody-based assay development has therefore become a subject of research. Proteins such as MBP70 and MBP83, which are well-documented candidate antigens for humoral response; MBP63, which has been shown to induce both CMI and humoral response; and PE25 and PE41, which also form a dimer, and have been used as fusion antigens, have been evaluated as candidate antigens for recognition by Th2 lymphocytes in humoral immune response assays or in a combination of both CMI and humoral assays, including TB Enflerplex, Luminex technology and multi antigen print immune assay (MAPIA) [ 3 ]. Thus, there has been more of a focus on multiplex antigen assay development for antibody- and CMI-based diagnostics, as well as on the combination of both assays.…”

“…Thus, there has been more of a focus on multiplex antigen assay development for antibody- and CMI-based diagnostics, as well as on the combination of both assays. A greater number of antigens may provide increased sensitivity, but specificity may be reduced due to cross reactions [ 3 ]. There has also been more of a focus on fusion of the ESAT 6/CFP 10 antigens with other antigens for improved sensitivity and specificity of CMI- and humoral-based assays, with these two antigens deemed important in (animal) TB diagnosis [ 16 ]…”