Interleukin 8 and Pentaxin (C-Reactive Protein) as Potential New Biomarkers of Bovine Tuberculosis

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 (bTB_PCR-P) and M. bovis-infected cattle that were nested PCR negative (bTB_PCR-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 bTB_PCR-P (n = 20), bTB_PCR-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 bTB_PCR-P from bTB_PCR-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 bTB_PCR-P from bTB_PCR-N.

Keywords: CRP; IL-8; biomarker; bovine tuberculosis; proteomics.

FIG 1

Experimental design work flow. Proteomics of serum and PPD-B-stimulated plasma from PCR-positive M. bovis-infected cattle (bTB_PCR-P), PCR-negative M. bovis-infected cattle (bTB_PCR-N), and uninfected cattle (NC) were identified using the iTRAQ-2D LC-MS/MS technology and then validated by parallel reaction monitoring (PRM) and ELISAs.

FIG 2

Levels of SAA, CRP, AGP, and TF in bTB_PCR-P, bTB_PCR-N, and NC. (A) SAA levels in serum. (B) CRP levels in PPD-B-stimulated plasma. (C) AGP levels in serum. (D) AGP levels in PPD-B-stimulated plasma. (E) TF levels in serum. (F) TF levels in PPD-B-stimulated plasma. Whole blood was collected from 21 bTB_PCR-P, 21 bTB_PCR-N, and 19 NC and stimulated with PPD-B for 24 h. Serum and plasma were harvested to measure the levels of SAA, CRP, AGP, and TF using commercial kits. Significant differences in protein levels between bTB_PCR-P, bTB_PCR-N, and NC were determined using a one-way ANOVA (Kruskal-Wallis test) followed by Dunn’s multiple-comparison test. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

FIG 3

Cytokine levels in bTB_PCR-P, bTB_PCR-N, and NC. (A) IL-8 induced by PPD-B. (B) IL-8 induced by CE. (C) IL-8 induced by PBS. (D) Concentrations of IL-8, IFN-γ, IP-10, and IL-17A induced by PPD-B. (E) Concentrations of IL-8, IFN-γ, IP-10, and IL-17A induced by PBS. Whole blood was obtained from 21 bTB_PCR-P, 21 bTB_PCR-N, and 19 NC and then stimulated with PPD-B, CE, or PBS for 24 h. Serum and plasma were harvested to measure cytokine levels using commercial kits. Significant differences in protein concentrations were determined using a one-way ANOVA (Kruskal-Wallis test) followed by Dunn’s multiple-comparison test. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.

FIG 4

Levels of IL-8 and CRP induced by proteins unrelated to M. bovis in bTB_PCR-P, bTB_PCR-N, and NC. (A) IL-8 induced by different stimuli. (B) CRP induced by different stimuli. Samples were obtained from five bTB_PCR-P, five bTB_PCR-N, and five NC. Whole blood was collected and stimulated with PPD-B, CE, PET, BSA, or PBS for 24 h. Plasma was harvested to measure cytokine levels using commercial kits. Significant differences between protein concentrations were determined using a two-way ANOVA followed by Bonferroni posttests. ***, P < 0.0001.