Differential gene expression segregates cattle confirmed positive for bovine tuberculosis from antemortem tuberculosis test-false positive cattle originating from herds free of bovine tuberculosis

Abstract

Antemortem tests for bovine tuberculosis (bTB) currently used in the US measure cell-mediated immune responses against Mycobacterium bovis. Postmortem tests for bTB rely on observation of gross and histologic lesions of bTB, followed by bacterial isolation or molecular diagnostics. Cumulative data from the state of Michigan indicates that 98 to 99% of cattle that react positively in antemortem tests are not confirmed positive for bTB at postmortem examination. Understanding the fundamental differences in gene regulation between antemortem test-false positive cattle and cattle that have bTB may allow identification of molecular markers that can be exploited to better separate infected from noninfected cattle. An immunospecific cDNA microarray was used to identify altered gene expression (P ≤ 0.01) of 122 gene features between antemortem test-false positive cattle and bTB-infected cattle following a 4-hour stimulation of whole blood with tuberculin. Further analysis using quantitative real-time PCR assays validated altered expression of 8 genes that had differential power (adj P ≤ 0.05) to segregate cattle confirmed positive for bovine tuberculosis from antemortem tuberculosis test-false positive cattle originating from herds free of bovine tuberculosis.

Figure 1

Number of genes from microarray analysis that were differentially expressed (P ≤ 0.01) within single antemortem test-false positive (SFP), double antemortem test-false positive (DFP), and bTB-infected (bTB) groups of cattle. The RNA used for microarray analysis was harvested after a 4-hour stimulation of whole blood with tuberculin, and comparison of gene expression levels was with a reference pool of mRNA harvested from the blood of healthy cattle after 4-hour stimulation with tuberculin. The number of genes for each group of cattle that showed increased expression (solid box) or decreased expression (shaded box) relative to the reference pool of RNA is indicated by the figure in the boxes.

Figure 2

Numbers of gene features identified from analysis of microarray data that were differentially expressed (P ≤ 0.01) among single antemortem test-false positive (SFP), double antemortem test-false positive (DFP), and bTB-infected (bTB) groups of cattle. The numbers of genes common to or unique for the groups of cattle are shown in a Venn diagram.

Figure 3

The relative gene expression levels compared with the reference pool of RNA from healthy cattle, as determined by qPCR assays, for the single antemortem test-false positive (SFP, shaded box), double antemortem test-false positive (DFP, solid box), and bTB-infected (bTB, clear box) groups of cattle. Gene expression levels (in log₂ fold change) were calculated using the published mathematical algorithm [37] in which the reference pool of RNA was set as baseline (0 value at Y-axis) and used as the calibrator. Statistically significant differences were determined using Student's t-test and are shown at P ≤ 0.05 (*) and P ≤ 0.01 (**). The error bars represent the standard error of the mean expression level for a group of cattle. (a) Genes with increased expression in all groups of cattle. (b) Genes with decreased expression in all groups of cattle.

Figure 4

Venn diagrams showing the statistically significant (adj P ≤ 0.05) differentially expressed genes that were unique to or common among (a) single antemortem test-false positive (SFP), double antemortem test-false positive (DFP), and bTB-infected (bTB) groups of cattle in initial analysis of 30 cattle and (b) single antemortem test-false positive (SFP), double antemortem test-false positive non-bTB-exposed (DFP-non-ex), and bTB-infected (bTB) groups of cattle after removal of bTB-exposed cattle from the DFP group. The gene expression levels were determined by qPCR assay, where each animal was calibrated relative to the reference pool of RNA from healthy cattle; differential expression between 2 groups of cattle (i.e., X versus Y) was determined using ANOVA analysis.