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.
Highlights
Known as Johne’s disease (JD), is a chronic infectious disease of ruminants resulting in diarrhea, wasting, weight loss, emaciation, and eventual death [1,2,3]
Holstein cattle were selected from four dairy farms with known paratuberculosis initial seroprevalences of >10% and were divided into age-cohorts by reproduction cycle: heifers, primiparous, and pluriparous cows
A dual mode DART-HRMS analysis was performed on sera extracted using polar and non-polar solvents with the aim of finding the changes in metabolites’ level in Mycobacterium avium subsp. paratuberculosis (MAP)-infected and infectious cattle as compared to those negative
Summary
Known as Johne’s disease (JD), is a chronic infectious disease of ruminants resulting in diarrhea, wasting, weight loss, emaciation, and eventual death [1,2,3]. In JD, animals can be classified into three groups: infected when MAP is present intracellularly in the animal, infectious when the animal is shedding MAP via feces and affected when clinical signs are visible [8]. Common diagnostic tests, such as fecal Polymerase Chain Reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) lack of sensitivity in earlier stages of infection, characterized by intermittent MAP shedding and absence of specific Th-2 response [9]. The biology of MAP infection, the long incubation period, the pathogenesis, and the difficulties in detecting infected animals in the absence of accurate diagnostic tests entail delayed diagnosis of JD, so disease control in infected herds is a challenge [10]
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