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 MGMs, and AF bacilli per granuloma. Furthermore, Mm microbiological results were also considered. Investigated parameters were statistically analyzed. Mm microbiological isolation was negatively influenced by granulomas maturation and positively affected by AF bacilli's presence within the section. Granuloma maturation was positively influenced by granuloma size and negatively affected by the number of granulomas in the section and the number of MGMs within the granuloma. The obtained results indicate that granulomas' maturation ensured an efficient containment of Mm infection in the WB, making the intraspecific transmission of the disease an unlikely event. Finally, an outbreak of paratuberculosis in a group of scimitar-horned oryxes (SHOs) kept in a zoological park, gave us the chance to describe various aspects of this disease in an endangered animal species subject to an international conservation and reintroduction plan encompassing several countries. In particular, after the death of six of the 10 SHOs, serial investigations of dead and alive animals were performed. Necropsy, carried out on five out of six animals, identified intestinal thickening and mesenteric lymphadenomegaly in one of the animals. Histopathology (5/6) revealed lepromatous (2/5) and tuberculoid (2/5) intestinal forms or lack of lesions (1/5). ZN and immunohistochemistry stains identified two multibacillary, two paucibacillary forms, and one negative case. M. avium subsp. paratuberculosis (Map) was identified by quantitative PCR (qPCR) in tissue samples in five out of five SHOs and was microbiologically isolated from two of the three animals whose fresh tissue samples were available. Fecal samples were collected in four of the six dead animals: all four resulted positive to qPCR and Map was isolated in three. ELISA identified Map-specific antibodies in three of the five dead animals whose serum was available. qPCR identified Map in the freshly deposited feces of two out of the four alive animals. From the feces of these two animals, Map was microbiologically isolated in one case. All isolates were classified as Map type C and profiled as INMV2 and MVS27 by molecular analysis. Genomic analysis of a field isolate revealed clusterization with a European clade but was more similar to Italian than East European isolates. Our findings highlight again that paratuberculosis should always be considered in zoological parks where endangered species are hosted. Infection can be subclinical, and multiple combined testing techniques may be necessary. The studies included in this Thesis made the examination of certain aspects of significant animal mycobacterial diseases possible, and the results obtained demonstrate that a multidisciplinary approach is the best option when studying infectious diseases, and this is especially true in case of infections deserving to be managed in a One Health perspective.

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.

MYCOBACTERIAL DISEASES IN VETERINARY MEDICINE: MORPHOPATHOLOGY AND MYCOBACTERIAL PHENOTYPES

C. Pigoli
2021

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 MGMs, and AF bacilli per granuloma. Furthermore, Mm microbiological results were also considered. Investigated parameters were statistically analyzed. Mm microbiological isolation was negatively influenced by granulomas maturation and positively affected by AF bacilli's presence within the section. Granuloma maturation was positively influenced by granuloma size and negatively affected by the number of granulomas in the section and the number of MGMs within the granuloma. The obtained results indicate that granulomas' maturation ensured an efficient containment of Mm infection in the WB, making the intraspecific transmission of the disease an unlikely event. Finally, an outbreak of paratuberculosis in a group of scimitar-horned oryxes (SHOs) kept in a zoological park, gave us the chance to describe various aspects of this disease in an endangered animal species subject to an international conservation and reintroduction plan encompassing several countries. In particular, after the death of six of the 10 SHOs, serial investigations of dead and alive animals were performed. Necropsy, carried out on five out of six animals, identified intestinal thickening and mesenteric lymphadenomegaly in one of the animals. Histopathology (5/6) revealed lepromatous (2/5) and tuberculoid (2/5) intestinal forms or lack of lesions (1/5). ZN and immunohistochemistry stains identified two multibacillary, two paucibacillary forms, and one negative case. M. avium subsp. paratuberculosis (Map) was identified by quantitative PCR (qPCR) in tissue samples in five out of five SHOs and was microbiologically isolated from two of the three animals whose fresh tissue samples were available. Fecal samples were collected in four of the six dead animals: all four resulted positive to qPCR and Map was isolated in three. ELISA identified Map-specific antibodies in three of the five dead animals whose serum was available. qPCR identified Map in the freshly deposited feces of two out of the four alive animals. From the feces of these two animals, Map was microbiologically isolated in one case. All isolates were classified as Map type C and profiled as INMV2 and MVS27 by molecular analysis. Genomic analysis of a field isolate revealed clusterization with a European clade but was more similar to Italian than East European isolates. Our findings highlight again that paratuberculosis should always be considered in zoological parks where endangered species are hosted. Infection can be subclinical, and multiple combined testing techniques may be necessary. The studies included in this Thesis made the examination of certain aspects of significant animal mycobacterial diseases possible, and the results obtained demonstrate that a multidisciplinary approach is the best option when studying infectious diseases, and this is especially true in case of infections deserving to be managed in a One Health perspective.
GRIECO, VALERIA
GRIECO, VALERIA
17-mag-2021
Tuberculosis; Paratuberculosis; Granuloma; Histopathology; Anatomic Pathology; Mycobacteria
Settore VET/03 - Patologia Generale e Anatomia Patologica Veterinaria
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.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/843675
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