Johne's disease, caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic wasting disease of ruminants. Hallmark symptoms of clinical Johne's disease include diarrhea, progressive weight loss, and premature death; symptoms due largely to chronic inflammation in the small intestine. MAP colonizes resident macrophages within the ileum of the small intestine, subsequently establishing a persistent infection in the host. It has been proposed that regulatory T cells may play a role in the progression of Johne's disease, either through promotion of tolerance to MAP or via a loss in homeostasis that subsequently allows widespread inflammation. In this report, we evaluated the presence of Tregs, as well as other immune parameters, in the ileum and draining lymph nodes of MAP associated lesions. A lesion classification scheme was developed to categorize severity of MAP-induced lesions within infected tissues and subsequently regulatory T cell presence and overall immune activity were assessed corresponding to lesions of varying severity, in comparison to tissues from healthy control animals. Our results revealed a relationship between animal health and overall lesion severity within the infected tissues, as well as a relationship between bacterial burden and severity of pathology. Regulatory T cell abundance was shown to decrease with increasing lesion severity. Within the ileum, the expression of many Th1, Th2, and Treg-associated genes increased in mild lesions and decreased in severe lesions, whereas in the lymph nodes the expression of these genes tended to increase with increasing lesion severity. Based on our results, we conclude that a local loss of T cell (including Treg) activity occurs within severe Heal lesions associated with MAP, resulting in a loss of homeostasis that ultimately leads to the progression of clinical Johne's disease. (C) 2016 Elsevier B.V. All rights reserved.