Serum Metabolomic Profiles of Paratuberculosis Infected and Infectious Dairy Cattle by Ambient Mass Spectrometry

Abstract

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis [Johne's disease (JD)], a chronic disease that causes substantial economic losses in the dairy cattle industry. The long incubation period means clinical signs are visible in animals only after years, and some cases remain undetected because of the subclinical manifestation of the disease. Considering the complexity of JD pathogenesis, animals can be classified as infected, infectious, or affected. The major limitation of currently available diagnostic tests is their failure in detecting infected non-infectious animals. The present study aimed to identify metabolic markers associated with infected and infectious stages of JD. Direct analysis in real time coupled with high resolution mass spectrometry (DART-HRMS) was, hence, applied in a prospective study where cohorts of heifers and cows were followed up annually for 2–4 years. The animals' infectious status was assigned based on a positive result of both serum ELISA and fecal PCR, or culture. The same animals were retrospectively assigned to the status of infected at the previous sampling for which all JD tests were negative. Stored sera from 10 infected animals and 17 infectious animals were compared with sera from 20 negative animals from the same herds. Two extraction protocols and two (-/+) ionization modes were tested. The three most informative datasets out of the four were merged by a mid-level data fusion approach and submitted to partial least squares discriminant analysis (PLS-DA). Compared to the MAP negative subjects, metabolomic analysis revealed the m/z signals of isobutyrate, dimethylethanolamine, palmitic acid, and rhamnitol were more intense in infected animals. Both infected and infectious animals showed higher relative intensities of tryptamine and creatine/creatinine as well as lower relative abundances of urea, glutamic acid and/or pyroglutamic acid. These metabolic differences could indicate altered fat metabolism and reduced energy intake in both infected and infectious cattle. In conclusion, DART-HRMS coupled to a mid-level data fusion approach allowed the molecular features that identified preclinical stages of JD to be teased out.

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

“…Few studies have used omic techniques to study MAP, particularly metabolomics, despite their ability to demonstrate the dynamics of infection and highlight potential diagnostic biomarkers. Previous studies examining the metabolomics of MAP using NMR and DART-HRMS highlighted significant energy deficits alongside altered fat metabolism in experimentally and MAP-infected cattle [19,20]. In this study, we observed five metabolites which discriminated between naturally MAP-infected and control heifers consistently from 1 to 19 months of age (Figure 4); clearly, these metabolites will require validation with further experimentation.…”

“…This was partially in contrast to previous studies. Tata et al [20] reported an increase in creatine/creatinine in MAP-infected and infectious cattle but a decrease in urea within infectious cattle. However, de Buck et al [19] demonstrated a significant increase in urea but a decrease in creatinine within MAP-inoculated cattle.…”

“…Previous sera-based studies of experimentally inoculated Holstein-Friesian calves using 1 H nuclear magnetic resonance (NMR) spectrometry showed metabolite changes suggestive of energy deficits as well as elevated lipid metabolism and protein turnover [19]. Likewise, Holstein cattle of mixed ages which were categorized as infectious or infected and analysed using direct analysis in real time coupled with high-resolution mass spectrometry (DART-HRMS), showed altered fat metabolism and decreased energy intake, compared to control cattle [20]. However, as with many other omic biomarkers [12,13], none have been commercially developed into a diagnostic method.…”

Combination of vibrational spectroscopy, mass spectrometry and chemometrics in an innovative food chemistry laboratory of the Italian health authority and research organization for animal health and food safety: Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe)

“…In the field of clinical chemistry, we have defined the characteristic profiles of sera from cows affected by paratuberculosis to accelerate the early diagnosis of this bovine pulmonary disease. 8 In regards to routine microbial diagnostics, we have set up rapid screening approaches for Legionella pneumophila serotyping that aim to guide the selection of conventional methods, with subsequent reduction of the microbiology laboratory costs and times. 9 Our activities as a test laboratory According to EU regulation, manufacturers of processed foods are required to provide a nutrition fact label for each product that reports the energy value (kcal/kJ), and amounts of fat, saturated fatty acid, carbohydrate, sugar, protein, and salt.…”

“…In the field of clinical chemistry, we have defined the characteristic profiles of sera from cows affected by paratuberculosis to accelerate the early diagnosis of this bovine pulmonary disease. 8 In regards to routine microbial diagnostics, we have set up rapid screening approaches for Legionella pneumophila serotyping that aim to guide the selection of conventional methods, with subsequent reduction of the microbiology laboratory costs and times. 9…”