Macroscopic and Histological Examination
After removal of fat and connective tissue, qualified staff inspected the lymph nodes macroscopically and recorded pathological changes. A subset of samples was additionally submitted for histology. Selection criteria were focusing on infections with the seven most prevalentMycobacterium species and presenting macroscopic lesions. In addition, all lymph nodes that tested positive for MTBC by real-time PCR were submitted for histological examination. Samples were fixed in 10% buffered formalin and embedded in paraffin. Two to three-micron-thick tissue sections were obtained and stained with hematoxylin and eosin (HE). Additionally, in cases where lesions consistent with mycobacterial infections were observed after HE staining, an additional ZN staining was performed.
Geographical Distribution and Statistical Analysis. The geographical distribution of the lymph nodes showing growth of mycobacteria and the circulation of the different NTM on the territory were investigated using the free software QGIS Desktop 3.6.1. Based on the collected data regarding sex and age of the animals, statistical analysis was performed using GraphPad Prism 8.2.1 (GraphPad Software, San Diego, USA). Fisher’s exact test was used to evaluate different age groups and the presence of viable NTM isolated from their lymph nodes. Moreover a possible association between the isolation of Mah , the most prevalent isolated species, and the three age groups was investigated with the same test. Statistical significance was set toP -value < 0.05.