Metabolomic Changes in Naturally MAP-Infected Holstein-Friesian Heifers Indicate Immunologically Related Biochemical Reprogramming

Abstract

Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), causes weight loss, diarrhoea, and reduced milk yields in clinically infected cattle. Asymptomatic, subclinically infected cattle shed MAP bacteria but are frequently not detected by diagnostic tests. Herein, we compare the metabolite profiles of sera from subclinically infected Holstein-Friesian heifers and antibody binding to selected MAP antigens. The study used biobanked serum samples from 10 naturally MAP-infected and 10 control heifers, sampled monthly from ~1 to 19 months of age. Sera were assessed using flow infusion electrospray-high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high-throughput, sensitive, non-targeted metabolite fingerprinting. Partial least-squares discriminant analyses (PLS-DA) and hierarchical cluster analysis (HCA) of the data discriminated between naturally MAP-infected and control heifers. In total, 33 metabolites that differentially accumulated in naturally MAP-infected heifers compared to controls were identified. Five were significantly elevated within MAP-infected heifers throughout the study, i.e., leukotriene B4, bicyclo prostaglandin E2 (bicyclo PGE2), itaconic acid, 2-hydroxyglutaric acid and N6-acetyl-L-lysine. These findings highlight the potential of metabolomics in the identification of novel MAP diagnostic markers and particular biochemical pathways, which may provide insights into the bovine immune response to MAP.

Keywords: MAP antigens; Mycobacterium avium subsp. paratuberculosis; antibody; eicosanoids; inflammation; metabolomics.

Figure 1

Partial least-squares discriminant analysis (PLS-DA) for naturally MAP-infected and control heifers in the (a) negative ionization and (b) positive ionization modes. The light red and green ellipses represent 95% confidence intervals.

Figure 2

Variable importance in projection (VIP) score plots based on two components from the partial least-squares discriminant analysis (PLS-DA) of metabolites differentially expressed in naturally MAP-infected and control heifers in the (a) negative ionization mode m/z and (b) positive ionization mode m/z.

Figure 3

Hierarchical cluster analysis (HCA) of the major metabolite changes differentiating naturally MAP-infected from control heifers between ~1 month and 19 months of age in the (a) negative ionization and (b) positive ionization mode; C = control heifer, NP = naturally MAP-infected heifer.

Figure 4

Box and whisker plots of metabolites which displayed minimal overlapping between naturally MAP-infected and control heifers, aged ~1 month to 19 months. (a) Leukotriene B4; (b) bicyclo-PGE2; (c) itaconic acid; (d) N6-acetyl-L-lysine (e) 2-hydroxyglutaric acid. Blue boxplots = naturally MAP-infected heifers, green boxplots = control heifers.

Figure 5

(a) Expression of WAG31 in naturally MAP-infected and control heifers between ~1 month and 19 months of age and (b) expression of MAP0211, MAP0946, WAG31, TOP4 and MAP1693 in naturally MAP-infected heifers between ~1 month and 19 months of age.

Figure 6

A heatmap of the Pearson’s correlation coefficients produced by comparing metabolites significantly affected by natural MAP infections and MAP-related antigens, whereby −0.4 < correlation co-efficient > 0.4. Positive correlations are shown in red, negative correlations are shown in blue. A = LPA (18:2 (9Z,12Z)/0:0), B = p-cresol, C = Hippuric acid, D = Eicosapentaenoic acid, E = Phytanic acid, F = WAG31, G = TOP4, H = MAP1693, I = MAP0211, J = MAP0946.