Estrategias de control de enfermedades micobacterianas

Abstract

Tuberculosis (TB), a zoonotic disease par excellence, and paratuberculosis (PTB), whose causal agent has zoonotic potential, are Mycobacterial diseases that, due to their importance from the economic and public health perspective, are the subject of this PhD Thesis. Overall, five strategies are used for the disease control which include: treatment, genetic improvement, vaccination, the adoption of hygienic-sanitary, biosecurity and management measures, animal health campaigns, and test and culling. Three of these strategies are treated in this PhD Thesis through two Chapters; the first (Chapter I) focuses on the strategies for taking hygienic-sanitary, management and biosecurity measures, and vaccination; and Chapter II, in the development of new diagnostic tests for application to animal health campaigns, the latter understood as the diagnosis of the disease and the reactors removal. Chapter I, in turn, consists of two subchapters. Chapter I.1. investigates whether European badger (Meles meles) is relevant as a maintenance host or reservoir of TB in Atlantic environments, in order to assess the effectiveness of possible disease control measures (vaccination) in Chapter I.2. The results obtained indicate that there is a spatio-temporal association of the disease between badgers and cattle, in addition to interspecific transmission, revealed by bacteriological and molecular techniques. Therefore, the badger must be considered a relevant species under certain circumstances in terms of TB maintenance, as is the case with wild ungulates such as wild boar or deer in Mediterranean environments. In this regard, badger health monitoring should be included in TB surveillance plans in wildlife, where new diagnostic techniques, such as the enzyme-linked immunosorbent assay (ELISA) based on the multi-protein complex p22, have shown be very useful and more sensitive and faster than bacteriological culture. In turn, and as part of the actions to be carried out on wildlife in terms of TB, the vaccination strategy must be considered. In Chapter I.2. the immune response induced at the local level by two vaccine candidates has been studied: live-attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine and the heat-Inactivated M. bovis (HIMB) vaccine. A relevant finding is that HIMB induces a greater humoral immune response that would predominate over the cellular response. Meanwhile, Chapter II of this Thesis consists of three subchapters whose objective consists on the search for new sensitive and specific diagnostic tools, capable of diagnosing cattle in different stages of PTB. These three studies are based on the work of Alonso-Hearn et al. (2019) in which numerous differential gene expression (ED) were identified by RNA-Seq analysis (massive sequencing of the complete transcriptome) of whole blood and ileocecal valve (VIC) samples of bovines with different types of histological lesions at the intestinal level and control animals without lesions. The proteins encoded by these ED are candidates for diagnostic biomarkers of the disease, with special interest in biomarkers capable of detecting subclinical infections, whose detection often escapes conventional diagnostic methods, as they are often characterized by the absence of clinical signs, the presence of focal histological lesions and a low bacterial load and antibody titer. Based on the previous transcriptomic analysis, the Chapter II.1. investigates the potential of ELISAs based on the detection of five candidate proteins, for diagnostic biomarkers. Among these proteins, the ATP-binding cassette subfamily A member 13 (ABCA13) and the matrix metallopeptidase 8 (MMP8), due to their biological function and high diagnostic precision to detect bovines with PTB lesions of focal and diffuse type, respectively, seems to have potential to be considered diagnostic biomarkers of PTB. As a continuation of this study, Chapter II.2. seeks to confirm these results with a large-scale validation of ELISAs based on the detection of both proteins, showing that the ELISA based on the detection of the ABCA13 protein constitutes a new diagnostic technique that is more sensitive than conventional diagnostic methods to detect subclinical infections, while the use of MMP8 as a biomarker for the diagnosis of PTB was ruled out. In addition, the diagnostic performance of the ELISA based on the detection of ABCA13 and other diagnostic methods conventionally used, was compared, applying the 20-year dynamic simulation model developed by Juste and Casal (1993), verifying that the use of the ELISA based on ABCA13 would allow the eradication of PTB in less than half the time needed to do it with the IDEXX ELISA and bacteriological isolation and real-time PCR in fecal samples. Although the benefit/cost ratio of using the ABCA13-based ELISA calculated at 20 years decreased between 8% and 54% compared to the ratio calculated for the conventional IDEXX ELISA, this technique offers a new alternative to farmers and animal health managers to help control any outbreak of PTB promptly. Similarly, Chapter II.3. analyzes the potential use of intelectin 2 (ITLN2), as a possible post mortem biomarker of infection. The ITLN2 precursor gene is overexpressed in VIC samples from bovines with focal and diffuse lesions compared to control animals without lesions, and it has been seen that it plays a very important role in the innate immune response to different infections. Characterization of ITLN2 expression in VIC samples by immunohistochemistry (IHQ), from animals with different types of histological lesions and controls without lesions, revealed a significantly higher number of immunolabeled Paneth and goblet cells in cattle with focal lesions and an inversely proportional relationship between the number of positive cells to the technique and the severity of the histological lesions. This finding suggests that ITLN2 may play an important role in the pathogenesis and progression of the disease, and that its quantification by IHQ constitutes a good post mortem diagnostic tool complementary to histology. In short, the work carried out over these four years is gathered in five scientific publications that make up the chapters on "Methodology and Results" presented in this PhD Thesis and, which constitute a unique document on advances in control strategies of TB and PTB, mycobacterial diseases that, given their serious repercussions worldwide, require a correct and complete approach for their adequate control.