Abstract
Bovine tuberculosis (bTB) is primarily caused by infection with Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex. The airborne route is considered the most common for transmission of M. bovis, and more than 15% of cattle with bTB shed the Mycobacterium, which can be detect by nested PCR to amplify mycobacterial mpb70 from a nasal swab from a cow. To screen for cytokines fostering early and accurate detection of bTB, peripheral blood mononuclear cells were isolated from naturally M. bovis-infected, experimentally M. bovis 68002-infected, and uninfected cattle, then these cells were stimulated by PPD-B, CFP-10-ESAT-6 (CE), or phosphate-buffered saline (PBS) for 6 h. The levels of interferon gamma (IFN-γ), IFN-γ-induced protein 10 (IP-10), IL-6, IL-12, IL-17A, and tumor necrosis factor alpha mRNA were measured using real-time PCR. To explore the cytokines associated with different periods of M. bovis infection, cattle were divided into three groups: PCR-positive, PCR-negative, and uninfected using the tuberculin skin test, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, IFN-γ release assay (IGRA), CFP-10/ESAT-6 (CE)-based IGRA, and nested PCR. The expression of IP-10, IL-17A, and IFN-γ proteins induced by PPD-B, CE, or PBS was detected by ELISA. The results showed that levels of PPD-B-stimulated IL-17A and IP-10 (mRNA and protein), and CE-induced IP-10 (mRNA and protein) were significantly higher in cattle naturally or experimentally infected with M. bovis than in those that were uninfected. The levels of PPD-B- or CE-induced IL-17A and IP-10 (protein) could be used to differentiate M. bovis-infected calves from uninfected ones for 6 to 30 weeks post-infection, whereas PPD-B- and CE-induced IP-10 and IL-17A mRNA expression could be used to differentiate M. bovis-infected calves from uninfected ones between 6 and 58 weeks post-infection. However, CE-induced IL-17A (protein) was not a reliable indicator of M. bovis infection in cattle that were confirmed positive for infection by nested PCR. Furthermore, the levels of PPD-B- or CE-induced IP-10 and IL-17A protein were lower than IFN-γ in M. bovis-infected cattle. Therefore, IL-17A and IP-10 protein are not suitable biomarkers for bTB. Antigen-induced IP-10 mRNA should be analyzed further for their potential to be used in the diagnosis of bTB.
Highlights
Bovine tuberculosis is primarily caused by infection with Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex (MTC) that includes M. tuberculosis, M. bovis, M. africanum, M. microti, M. caprae, Mycobacterium pinnipedii, M. mungii, and M. canetti [1, 2]
To screen for cytokines related to M. bovis infection, 10 naturally M. bovis-infected and five uninfected cows were determined by TST, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, interferon gamma (IFN-γ) release assay (IGRA), and CE-based IGRA
The levels of IFN-γ, IFN-γinduced protein 10 (IP-10), IL-6, IL-17A, and TNF-α mRNA were significantly higher in both PPD-B- and CE-stimulated peripheral blood mononuclear cells (PBMCs) from M. bovis-infected cows than from uninfected cows (Figures 1A,B), whereas the levels of IL-12 mRNA were significantly higher only in PPD-B-stimulated PBMCs from M. bovisinfected cows relative to those in uninfected cows (Figure 1A)
Summary
Bovine tuberculosis (bTB) is primarily caused by infection with Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex (MTC) that includes M. tuberculosis, M. bovis, M. africanum, M. microti, M. caprae, Mycobacterium pinnipedii, M. mungii, and M. canetti [1, 2]. The traditional bTB diagnostic methods are the tuberculin skin test (TST) and interferon gamma (IFN-γ) release assay (IGRA). Both TST and IGRA are based on the detection and comparison of cell-mediated responses induced by bovine purified protein derivatives (PPD-B) and avian purified protein derivatives (PPD-A) [8]. PPD-A is used in IGRA to exclude environmental Mycobacterium infections, it has failed to detect some M. bovis-infected cattle in bTB- and paratuberculosis- coprevalent dairies. Considering that neither TST nor IGRA can differentiate between stages in the progression of bTB, a nested PCR assay based on the amplification of a fragment of mpb was established and used to detect mycobacteria in milk, nasal exudates, and bronchoalveolar lavage (BAL) fluid [12]
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