MYCOBACTERIAL DISEASES IN VETERINARY MEDICINE: MORPHOPATHOLOGY AND MYCOBACTERIAL PHENOTYPES
Tesi di Dottorato
Data di Pubblicazione:
2021
Citazione:
MYCOBACTERIAL DISEASES IN VETERINARY MEDICINE: MORPHOPATHOLOGY AND MYCOBACTERIAL PHENOTYPES / C. Pigoli ; tutor: V. Grieco ; coordinatore: V. Grieco. Universita' degli Studi di MILANO, 2021 May 17. 33. ciclo, Anno Accademico 2020.
Abstract:
Mycobacteria are an ancient bacterial taxon that has, over time, had a profound impact on mankind and domesticated animals. Mycobacteria are, in fact, responsible for significant human and animal diseases whose severe economic and public health repercussions make them still highly topical today.
The first topic of research in this Thesis is a widely characterized phenomenon in the context of human tuberculosis, but one which has, to date, been little considered in animal tubercular infections, namely mycobacterial dormancy. As bovine tuberculosis (bTB) is undoubtedly the most significant of the animal tubercular infections, these aspects have been investigated precisely in the context of this pathology. In particular, this study aimed to verify the intralesional existence of non-acid-fast (non-AF) mycobacteria phenotypes that in human medicine have been associated to the phenomenon of mycobacterial dormancy. Another important objective of this study was to develop a histological method capable of detecting in the same section the mycobacterial cells by combining AF stains and techniques capable of highlighting the mycobacteria regardless of their AF features. In this retrospective study, 250 bovine lymph nodes (LNs) in which Mycobacterium bovis (Mb) has been identified by the diagnostic algorithm routinely applied by the Italian National Reference Center for bTB, were randomly enrolled. Ziehl-Neelsen (ZN) stained sections were evaluated for each sample. Only LNs with at least one granuloma containing more than 20 ZN-positive bacilli were selected, and the corresponding formalin-fixed and paraffin-embedded blocks were retrieved. For each block, a 4-µm thick section was obtained and, after reducing the autofluorescence through a photobleaching protocol and unmasking the Mb antigens with enzymatic digestion, the sections were submitted to a protocol combining Auramine O stain and an indirect immunofluorescence assay targeting Mb antigens; a DAPI-containing mounting medium was used. Processed slides were viewed with a Leica DM6 B upright microscope, and the obtained images were elaborated with a deconvolution algorithm. Of the 250 enrolled LNs, 24 contained at least one granuloma with more than 20 ZN-positive bacilli, and in all of them, AF and non-AF bacilli were identified. In all (24/24) selected LNs, the non-AF bacillary load resulted greater than the AF one, especially in the central parts of the granuloma; non-AF bacilli were also identified within the cytoplasm of multinucleated giant macrophages (MGMs). This study revealed for the first-time non-AF Mb phenotypes within bovine lymph nodal granulomatous lesions. It also demonstrated that a widely studied pathology such as bTB still poses new issues regarding pathogenesis and host-pathogen interaction and suggested that bTB can be considered an effective animal model for studying mycobacterial dormancy.
The second study carried out was also focused on a tubercular infection but, in this latter case, both the mycobacterial species involved and the affected host were different. Indeed, in the second study, wild boar (WB) M. microti (Mm) infection, whose epidemiological and pathogenic dynamics are still largely unknown, was enquired into. Specifically, the natural WB Mm infection was investigated by evaluating the granulomatous lesions' histological features and Mm microbiological isolation. For this purpose, 103 WB retropharyngeal and submandibular LNs in which Mm was identified by gyrB restriction fragment length polymorphism PCR were retrospectively selected and histologically assessed. For each sample, Hematoxylin-eosin and ZN-stained slides were evaluated. Considered histological variables were the number of granulomas, size and maturational stage of granulomas, number of M
Tipologia IRIS:
Tesi di dottorato
Keywords:
Tuberculosis; Paratuberculosis; Granuloma; Histopathology; Anatomic Pathology; Mycobacteria
Elenco autori:
C. Pigoli
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