(1)H-NMR spectroscopy revealed Mycobacterium tuberculosis caused abnormal serum metabolic profile of cattle

Abstract

To re-evaluate virulence of Mycobacterium tuberculosis (M. tb) in cattle, we experimentally infected calves with M. tb andMycobacterium bovisvia intratracheal injection at a dose of 2.0×10(7) CFU and observed the animals for 33 weeks. The intradermal tuberculin test and IFN-γin vitro release assay showed that both M. tb and M. bovis induced similar responses. Immunohistochemical staining of pulmonary lymph nodes indicated that the antigen MPB83 of both M. tb and M. bovis were similarly distributed in the tissue samples. Histological examinations showed all of the infected groups exhibited neutrophil infiltration to similar extents. Although the infected cattle did not develop granulomatous inflammation, the metabolic profiles changed significantly, which were characterized by a change in energy production pathways and increased concentrations of N-acetyl glycoproteins. Glycolysis was induced in the infected cattle by decreased glucose and increased lactate content, and enhanced fatty acid β-oxidation was induced by decreased TG content, and decreased gluconeogenesis indicated by the decreased concentration of glucogenic and ketogenic amino acids promoted utilization of substances other than glucose as energy sources. In addition, an increase in acute phase reactive serum glycoproteins, together with neutrophil infiltration and increased of IL-1β production indicated an early inflammatory response before granuloma formation. In conclusion, this study indicated that both M. tb and M.bovis were virulent to cattle. Therefore, it is likely that cattle with M. tb infections would be critical to tuberculosis transmission from cattle to humans. Nuclear magnetic resonance was demonstrated to be an efficient method to systematically evaluate M. tb and M. bovi sinfection in cattle.

Figure 1. Experimental infection of calves with M. tbH37Rv, M.tb1458 of bovine origin and M. bovis induced IFN-γ production after in vitro stimulation with bovine PPD.

The IFN-γ concentrations, which were measured by BOVIGAM® ELISA kit and expressed as OD₆₃₀ values, fluctuated with the time course of infection. The OD₆₃₀ values at each timepoint were presented as mean ± SEM. On week 7PI, there were significant differences between the M. tbH37Rv- and M. tb1458-infected groups, while at week 15 PI, the difference were statistically significant between the M. tbH37Rv vs.1458, and M. tbH37Rv vs. M. bovis infected groups.

Figure 2. Experimental infection of calves with M. tbH37Rv, M.tb1458 of bovine origin, and M. bovis induced serum IgG production.

The IgG concentrations, which were measured by indirect ELISA and expressed as OD₆₃₀ values, apparently increased at the late stage of infection and were maintained for short time (weeks 28–29). The OD₆₃₀ values at each timepoint were presented as mean ± SEM.

Figure 3. Histopathological observations of the tissues.

An increase in neutrophil infiltration and lipofuscin-containing macrophages was observed in cattle lymph node tissue in groups infected with M. tbH37Rv, M. tb1458 of bovine origin, and M. bovis. The cells were stained with hematoxylin and eosin and observed via light microscopy. The black solid arrows indicate neutrophils and the double line arrow indicate lipofuscin-containing macrophages. Only B shows both cell types because it was difficult to find both co-existing in the same field.

Figure 4. Western blot analysis to detect the specific MPB83 monoclonal antibody.

(A) M, prestain marker; Lane 1, M. bovis; Lane 2, M. tb1458; Lane 3, H37Rv; Lane 4, M.graminis; Lane 5, M. avium; Lane 6, M. intracellulare; Lane7, M. smegmatisc; and Lane 8, MDBK cells. (B) M, prestain marker; Lane 1, H37Rv; Lane 2, M. paratuberculosis; Lane 3, E.coli from bovine origin; and Lane 4, Salmonella from bovine origin. The arrows indicate the MPB 83 protein.

Figure 5. Gross pathological observations of the calves.

The subcutaneous adipose tissues were very thick in the negative control group (A). The calves had thinner layers of subcutaneous adipose tissue and obviously gelatinous materials after infection with M.bovis (B), M. tbH37Rv (C), and M. tb 1458of bovine origin (D). Arrows indicate the gelatinous tissue material.

Figure 6. Immunohistochemical detection of a specific antigen against M. tb and M. bovis in lymph node tissues.

The antigen MPB83 for M. tb and M. bovis in the lymph node tissues of calves was observed after immunohistochemical staining. There were no positive cells in the negative control group (A). The MPB83-positive cells were stained brown in the groups infected with M. bovis (B), M. tbH37Rv (C), and M. tb 1458of bovine origin (D).

Figure 7. Serum IL-1β detection in all groups.

IL-1β concentrations were detected at weeks 7, 15, and 27 PI. in the H37Rv group, the serum IL-1β concentration was enhanced and there was significant difference between week 7 vs. 15 and week 7 vs. 27, while there was no significant increase in the other groups during the same periods.

Figure 8. Typical 600-MHz ¹H NMR CPMG spectra of plasma.

A–D, represent controls (A) and calves infected with M. bovis (B), H37Rv (C), and M. tb1458 (D), respectively. The spectral regions of δ 6.5–8.5 were expanded 16 times. Keys: 1, valine; 2, leucine; 3, isoleucine; 4, 3-hydroxybutyrate; 5, lactate; 6, alanine; 7, lysine; 8, acetate; 9, N-acetyl-glycoprotein; 10, glutamate; 11, glutamine; 12, methionine; 13, acetone; 14, acetoacetate; 15, pyruvate; 16, citrate; 17, creatine; 18, choline; 19, phosphorylcholine; 20, glycerylphosphorylcholine; 21, glucose; 22, fumarate; 23, tyrosine; 24, 1-methyl-histidine; 25, phenylalanine; 26, glycine; 27, formate; 28, lipid; 29, unsaturated fatty acid; 30, glucose and amino acids a-CH resonances; 31. Triacylglycerol; 32, myo-inositol; and 33, unknown.

Figure 9. OPLSDA comparison of plasma spectra between the control and infected groups (M. bovis, M. tbH37Rv, and M. tb1458).

In panels A, B, and C, the triangle (▴) represents the control, the black circle (•) represents M. bovisinfected group, the diamond (◊) represents the M. tbH37Rv-infected group, the asteric (*) represents the M. tb1458-infected group; In panels a, b, and c, the upward and downward peaks, respectively, denote the elevated and decreased metabolites in the infected calves with hot colored metabolites contributing more significantly to the class discrimination than the cold colored metabolites.

Figure 10. Metabolic changes induced by M. tb and M. bovis infection prompted a switch in the energy production pathways.

Our hypothesis states that the infection induced glycolysis, which led to a decrease in glucose concentration and a lactate accumulation. The decrease in cellular energy levels due to infection triggered lipid degradation. Then, the triacylglycerol and free fatty acids were used as a source of carbon and energy via β-oxidation. Meanwhile, ketone bodies were by-products that contributed to energy production via acetyl coenzyme A. Other energy sources included creatine. The amino acids may contribute to energy production by conversion of the intermediates of the TCA cycle or converted into glucose through gluconeogenesis. ↑ represents markedly increased metabolites, while ↓ representsdecreased metabolites.