Prevalence and diversity of non-tuberculous mycobacteria isolated from slaughtered tuberculin-positive bovine samples: Epidemiological and Diagnostic implications

The isolation of non-tuberculous mycobacteria (NTM) is common in cattle positive for tuberculosis (TB) in official diagnostic tests, whereas data on specific NTM species in Spanish cattle remain limited. This study identifies the most frequently isolated NTM species from Single Intradermal Tuberculin Test (SITT)-positive cattle in Extremadura, western Spain. Among 1669 Mycobacterium Growth Indicator Tube (MGIT) positive cultures collected in 2018, 493 (29.54%) were identified as NTM, and 194 were randomly selected for further analysis. Polymerase Chain Reaction (PCR)-restriction analysis of the hsp65 gene and partial sequencing of 16S ribosomal DNA (rDNA) confirmed a diverse range of species. The most prevalent complex was Mycobacterium avium (40.12%), including M . senriense , M . intracellulare , and M. avium subsp. paratuberculosis . Other notable NTM species (23.35%) included M. bourgelatii, M. kansasii, M. gordonae, and M. shinjukuense . Less frequent complexes included M . simiae (11.38%), M. ulcerans (3.59%), M. parafortuitum (2.99%), and M. terrae (1.20%), along with M. holsaticum (1.20%), a species related to the M. tuberculosis complex . Phylogenetic analysis and geographic mapping revealed weak correlation between genetic and geographic distances (Mantel test: R xy = 0.015, P = 0.253), suggesting limited spatial structuration of genetic diversity. Alpha diversity metrics indicated moderate diversity (Shannon’s H = 2.641, Simpson’s D = 0.106), with some zones exhibiting greater species evenness. Diversity analyses showed moderate dissimilarity among clusters. These findings enhance understanding of Mycobacterium diversity and distribution while emphasizing the diagnostic challenges posed by NTM in TB detection and the importance of molecular tools in species identification and epidemiological surveillance. • NTM represent a significant diagnostic challenge that must be considered in TB eradication programs. • NTM were highly prevalent in SITT-positive cattle from western Spain. • Integrating genetic and epidemiological analyses enhances species identification and clarifies the ecological factors driving NTM distribution. • 16S rDNA sequencing and diversity indices revealed a complex, evenly distributed NTM community, with no dominant species and wide environmental dispersion.