Abstract
Bovine tuberculosis (bTB) is caused by Mycobacterium bovis During the early stage of infection, greater than 15% of M. bovis-infected cattle shed mycobacteria through nasal secretions, which can be detected by nested PCR. To compare the differences in the protein profiles of M. bovis-infected cattle that were nested PCR positive (bTBPCR-P) and M. bovis-infected cattle that were nested PCR negative (bTBPCR-N) and to screen for biomarkers that will facilitate the early and accurate detection of bTB, we investigated the protein expression profiles of serum and bovine purified protein derivative (PPD-B)-stimulated plasma among bTBPCR-P (n = 20), bTBPCR-N (n = 20), and uninfected cattle (NC; n = 20) by iTRAQ labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2D LC-MS/MS). After comprehensive analysis, we selected 15 putative differentially expressed serum proteins and 15 plasma proteins for validation by parallel reaction monitoring (PRM) with the same cohort used in the iTRAQ analysis. Four serum and five PPD-B-stimulated proteins were confirmed in follow-up enzyme-linked immunosorbent assays. PPD-B-stimulated interleukin 8 (IL-8) displayed the potential to differentiate M. bovis-infected cattle from NC, with an area under the curve (AUC) value of 0.9662, while PPD-B-stimulated C-reactive protein (CRP) displayed the potential to differentiate bTBPCR-P from bTBPCR-N, with an AUC value of 1.00. Finally, double-blind testing with 244 cattle indicated that the PPD-B-stimulated IL-8 test exhibited good agreement with traditional tests (κ > 0.877) with a >90% relative sensitivity and a >98% relative specificity; the PPD-B-stimulated CRP test displayed good agreement with nested PCR (κ = 0.9117), with an observed 94% relative sensitivity and 97% relative specificity. Therefore, the PPD-B-stimulated IL-8 and CRP tests could be used to detect bTB and to differentiate bTBPCR-P from bTBPCR-N.
Highlights
Bovine tuberculosis is caused by Mycobacterium bovis
Considering that TSTs and interferon gamma (IFN-␥) release assays (IGRAs) cannot distinguish the state of M. bovis infection, an mpb70-based nested PCR assay was established for detecting mycobacteria in milk and colostrum [9]
Based on an initial screening of serum proteins related to M. bovis infection, 82 naturally M. bovis-infected cattle and 39 NC were identified by TST, CFP-10/ESAT-6/TB10.4-based skin test (CET-ST), IGRA, and CFP-10 –ESAT-6 (CE)-based IGRA
Summary
During the early stage of infection, greater than 15% of M. bovis-infected cattle shed mycobacteria through nasal secretions, which can be detected by nested PCR. To compare the differences in the protein profiles of M. bovis-infected cattle that were nested PCR positive (bTBPCR-P) and M. bovis-infected cattle that were nested PCR negative (bTBPCR-N) and to screen for biomarkers that will facilitate the early and accurate detection of bTB, we investigated the protein expression profiles of serum and bovine purified protein derivative (PPD-B)-stimulated plasma among bTBPCR-P (n ϭ 20), bTBPCR-N (n ϭ 20), and uninfected cattle (NC; n ϭ 20) by iTRAQ labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2D LC-MS/ MS). M. bovis can infect wild animals and humans via an airborne route, and greater than 15% of M. bovis-infected cattle shed the mycobacteria through nasal secretions, especially during the early stages of infection [4]
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