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Infections caused by the Mycobacterium avium complex (MAC) are on the rise in both human and veterinary medicine. A means of effectively discriminating among closely related yet pathogenetically diverse members of the MAC would enable better diagnosis and treatment as well as further our understanding of the epidemiology of these pathogens. In this study, a five-target multiplex PCR designed to discriminate MAC organisms isolated from liquid culture media was developed. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all Mycobacterium species, a chromosomal target called DT1 that is unique to M. avium subsp. avium serotypes 2 and 3, to M. avium subsp. silvaticum, and to M. intracellulare, and three insertion sequences, IS900, IS901, and IS1311. The pattern of amplification results allowed determination of whether isolates were mycobacteria, whether they were members of the MAC, and whether they belonged to one of three major MAC subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. hominissuis. Analytical sensitivity was 10 fg of M. avium subsp. paratuberculosis genomic DNA, 5 to 10 fg of M. avium subsp. avium genomic DNA, and 2 to 5 fg of DNA from other mycobacterial species. Identification accuracy of the MAC multiplex was evaluated by testing 53 bacterial reference strains consisting of 28 different mycobacterial species and 12 nonmycobacterial species. Identification accuracy in a clinical setting was evaluated for 223 clinical MAC isolates independently identified by other methods. Isolate identification agreement between the MAC multiplex and these comparison assays was 100%. The novel MAC multiplex is a rapid, reliable, and simple assay for discrimination of MAC species and subspecies in liquid culture media.Since the early 1980s, there has been an increase in disease caused by organisms broadly categorized as nontuberculous mycobacteria (NTM), a generic term for mycobacteria not in the Mycobacterium tuberculosis complex and other than M. leprae (32). Of these NTM, Mycobacterium avium complex (MAC) species are the most common cause of human and animal disease globally (6,14,16,24). The clinical relevance of the MAC in humans has been amplified in recent decades with the increasing population of immunocompromised individuals resulting from longer life expectancy, immunosuppressive chemotherapy, and the AIDS pandemic (27 Members of the family Mycobacteriaceae, comprising the MAC, differ in virulence and ecology. Those designated M. avium subsp. hominissuis are genomically diverse, low-virulence, opportunistic pathogens for both animals and humans.

To analyze the co-occurrence of virulence genes among bovine and human commensal E. coli strains and visualize it in the network interface, we constructed a pairwise co-occurrence matrix for each gene (Supplemental Table S8). Only one co-occurrence

The objective of this longitudinal study was to evaluate the immune response against Treponema spp. infection in dairy heifers affected with digital dermatitis (DD). In addition, the accuracy of an indirect ELISA detecting anti-Treponema IgG antibodies in identifying clinical DD status has been assessed. A cohort of 688 pregnant Holstein heifers was evaluated at least 3 times before calving during a period of 6 mo. Complete clinical assessment of DD presence on the back feet of each heifer and blood extraction were performed in a stand-up chute. Digital dermatitis cases were characterized by the M-stage classification system and size and level of skin proliferation. An ELISA was performed on blood serum samples obtained from a subcohort of 130 heifers. For description purposes, the animals were classified by the number of clinical cases experienced during the study period as type I (no clinical cases were observed), type II (only 1 acute clinical case diagnosed), and type III (at least 2 acute clinical cases diagnosed). Multivariable repeated-measures models were used to evaluate the immune response against Treponema spp. infection. A binormal Bayesian model for the ELISA data without cut-point values was used to assess the accuracy of the ELISA as a diagnostic tool. Animals that never experienced a DD event throughout the study kept a constant low level of antibody titer. A 56% increase in mean ELISA titer was observed in heifers upon a first clinical DD case diagnosis. After topical treatment of an acute DD case with oxytetracycline, the antibody titer decreased progressively in type II heifers, achieving mean levels of those observed in healthy cows after a mean of 223 d. Surprisingly, antibody titer was not increased in the presence of M1 (DD lesion <20mm in diameter surrounded by healthy skin) and M4.1 (DD lesion <20mm in diameter embedded in a circumscribed dyskeratotic or proliferative skin alteration) DD stages. Type III cows showed a slight increase in antibody levels. The presence of skin proliferation at first DD diagnosis was found to be associated with an odds ratio of 2.04 of becoming a type III heifer in relation to heifers presenting first lesions without skin proliferation. The ELISA validity was estimated by an area under the curve of 0.88. Predicted probabilities of infection are provided for a range of ELISA values and prevalence of infection. Early detection and treatment is essential to control DD and the ELISA can be used in understanding the immunopathology of DD and shows great promise for prescreening purposes during DD management programs in combination with traditional clinical inspection.

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