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.

Differentiation among types I, II, and III is the primary step in typing Mycobacterium avium subsp. paratuberculosis. We propose an innovative approach based on detection of gyrase B ( gyrB ) gene polymorphisms by suspension array technology, with high discriminatory power and high-throughput potential.

Background: The presence of carbapenemase-producing bacteria (CPB) in animal hosts and along the food chain may result in the development of reservoirs for human infections. Several CPB strains isolated from animals have been reported, suggesting that transmission and dissemination of the corresponding genes between humans and animals may occur. Animal and food samples have complex backgrounds that hinder the detection of CPB present in low concentrations by standard detection procedures. Methods: We evaluated the possibility of detecting bla_KPC, bla_VIM, and bla_OXA-48-like carbapenemases in 286 animal and food samples (faeces from farm and companion animals, raw meat, bivalve molluscs) by culture-based and standard molecular methods and by ddPCR. Results: The proposed ddPCR managed to detect the target genes, also in samples resulting negative to standard methods. While the presence of bla_KPC and bla_VIM was detected in few samples (~3%), one third of the samples (n = 94/283) carried different variants of bla_OXA-48-like genes. Conclusion: A specific and sensitive method such as ddPCR could be suitable to evaluate the current veterinarian and environmental situation and to assess the dynamic transmission and persistence of CPB between animals and humans and vice versa.