Sequencing the genomes of the environmental mycobacteria that are encountered by livestock may provide researchers the ability to identify bacterial proteins or nucleic acid targets that will not cross react with M. bovis; thus enabling the development of assays with increased specificity and sensitivity. Identifying the mycobacteria that infect livestock and wildlife may enable the rational prioritization of the mycobacterial genomes to sequence to aid diagnostic test development.
A number of mycobacteria reported here have been isolated from the environment associated with cattle feed or water. Many of these have been reported to elicit reactions to PPDb based skin tests including isolates belonging to Runyan Group III , M. flavescens, M. terrae complex, M. gordonae, M. intracellulare, M. fortuitum, M. smegmatis, M. senegalense, M. scrofulaceum, (reviewed in ) and M. kansasii[23, 24]. In addition to cattle, M. fortuitum has been reported to interfere with the skin and γ-interferon test in African Buffalo [13, 14]. Immunological cross-reaction between these mycobacteria and PPDa and PPDb suggest that immunologically similar proteins exist between mycobacteria. Cross-reaction reduces specificity and sensitivity of diagnostic tests.
M. bovis and M. avium complex species are the most common mycobacteria isolated from cervids in the United States. These results are consistent with those reported for New Zealand . In contrast to cattle, more M. terrae/terrae complex were isolated from cervids than M. fortuitum/fortuitum complex.
Cervids may be more susceptible to mycobacterial infections. In this study, mycobacteria were isolated at a rate of 107 per thousand cases compared to cattle with a rate of 49.9 per thousand cases. If a herd is infected with a greater range of environmental mycobacteria there is a greater probability that the immune response to the environmental mycobacteria may cross-react with PPDa and/or PPDb thus reducing the specificity and/or sensitivity of the diagnostic test.
There are two caveats to the current data. First, not all isolates were identified to the species level. These totals may be different if all samples were speciated as far as possible. This may be most prominent in the M. avium complex isolates. Hughes et al. report that when improve molecular techniques were applied members of the M. avium group resulted in identification of a larger number of mycobacterial species . We reason that the major isolates would be similar, but some of the minor species may play a larger role than currently indicated by the data. Not all isolates were speciated due to the priority of the diagnostic laboratory to identify and report M. bovis infections. Some isolates were not speciated because they could not be confidently assigned to the species level (Unable to Speciate). This inability to speciate each mycobacterial isolate was also reported by Hues et al.  in Northern Ireland, suggesting that not all mycobacterial species have been identified.
A second caveat is that not all mycobacteria are cultivatable. The possibility exists that the mycobacteria that cause significant interference with current diagnostic tests are unculturable. A prime example of this scenario is the infrequent yet persistent finding of CCT positive cattle with lesions only found in the skin. Histopathological examination reveals the presence of acid-fast bacteria, but culture attempts are always unsuccessful.
With the advent of next generation sequencing, the possibility exists that mycobacterial species could be sequenced with little capital investment. Regions or proteins unique to M. bovis could be derived by comparing the M. bovis genome with environmental mycobacteria. These unique regions could then be tested for use in protein or nucleic acid based tests. The problem with this approach has been the difficulty in selecting the environmental mycobacteria to sequence. Here we report the mycobacteria isolated from cattle and cervids in the United States. These data provide a rational for selection of mycobacteria for genome sequencing to aid in the development of improved diagnostic tests.