Unlocking environmental contamination of animal tuberculosis hotspots with viable mycobacteria at the intersection of flow cytometry, PCR, and ecological modelling

Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTBC), circulates in multi-host mammal communities. While interactions between different host species are mainly indirect, current knowledge postulates interspecific transmission is favored by animal contact with natural substrates contaminated with droplets and fluids from infected animals. However, methodological constraints have severely hampered monitoring of MTBC outside its hosts and the subsequent validation of this hypothesis. In this work, we aimed to evaluate the extent to which environmental contamination with M. bovis occurs in an endemic animal TB setting, taking advantage of a new real-time monitoring tool we recently developed to quantify the proportion of viable and dormant MTBC cell fractions in environmental matrices. Sixty-five natural substrates were collected nearby the International Tagus Natural Park region, in the epidemiological TB risk area in Portugal. These included sediments, sludge, water, and food deployed at unfenced feeding stations. The tripartite workflow included detection, quantification, and sorting of different M. bovis cell populations: total, viable, and dormant. Real-time PCR targeting IS6110 to detect MTBC DNA was performed in parallel. The majority of samples (54 %) contained metabolically active or dormant MTBC cells. Sludge samples had a higher burden of total MTBC cells and a high concentration of viable cells (2.3 x 104 cells/g). Ecological modelling informed by climate, land use, livestock and human disturbance data suggested eucalyptus forest and pasture cover as potential major factors affecting the occurrence of viable MTBC cells in natural matrices.

Automated summary

The presence of viable or dormant cells of Mycobacterium bovis, the causative agent of bovine tuberculosis, was found in the environment of an endemic TB area in Portugal. Ecological factors, such as eucalyptus forest and pasture cover, were identified as major factors influencing the occurrence of viable M. bovis cells in natural matrices.