Mycobacteria Lecture Notes - Jayne Hope PDF

Lecture title: Mycobacteria Lecturer: Professor Jayne Hope Learning outcomes Gain knowledge of key Mycobacterial species and diseases they cause: bovine tuberculosis and Johne’s disease Understand the basis of diagnostic tests including tuberculin testing to identify cattle...

Lecture title: Mycobacteria Lecturer: Professor Jayne Hope Learning outcomes Gain knowledge of key Mycobacterial species and diseases they cause: bovine tuberculosis and Johne’s disease Understand the basis of diagnostic tests including tuberculin testing to identify cattle infected with M. bovis and be aware of barriers to vaccination against Mycobacterial diseases Understand the basic pathogenesis of M. bovis, including knowledge of the bacterial cell wall and granuloma formation The Mycobacterium genus contains some of the most significant human and animal pathogens. The bacteria are rod-shaped, aerobic, non motile, non-spore forming and characterised by large amounts of cell wall lipid including mycolic acids, which help retain the Ziehl-Neelsen stain after acid washing and are therefore known as acid-fast bacteria. They occasionally form easily broken filaments showing their similarity with the Actinomycete family of bacteria. The isolates can be characterised into fast- and slow-growing isolates, the virulent strains being those that generally take longer to culture. For example, it may take several weeks to develop visible growth of certain pathogenic mycobacterial species on selective media. Certain Mycobacterial species (for example M. leprae) can only be grown in association with host cells (obligate intracellular bacteria). Several species are opportunistic or obligate pathogens. The mycobacteria are reported to have derived from a single common ancestor more than 3 million years ago. Via a series of deletions and insertions, and adaptations for particular niches two major clades have evolved. This includes the Mycobacterium avium subspecies including M. avium and M. avium paratuberculosis: important pathogens of birds and ruminants respectively, and the M. tuberculosis complex (MTBC). Mycobacterium tuberculosis, M. bovis, M. microti, M. africanum and M. canetti are all part of the MTBC as is BCG, the vaccine strain derived from M. bovis. Mycobacteria of (veterinary) clinical importance Mycobacterium bovis: bovine tuberculosis [wide host range] Mycobacterium avium paratuberculosis: Johne’s disease in ruminants Mycobacterium avium complex: TB in birds, pigs Mycobacterium microti: TB in cats Johne’s disease is a chronic wasting disease primarily of ruminants caused by infection with M. avium paratuberculosis (MAP). It presents as severe diarrhoea with associated weight loss and is progressively fatal. The disease is characterised by infection in early life, typically within days of birth, followed by a long (2-4) years subclinical period. A trigger – which is not fully understood but often relates to calving or co-infection – leads in some animals to progression into clinical disease. This likely reflects alterations in immune status. The typical response is a chronic granulomatous inflammation of the small intestine, largely the ileum. A large number of animals are thought to be infected with estimates at up to 80% of dairy cattle herds positive for MAP. Importantly it is thought that MAP can infect humans and lead to Crohn’s disease – this has typical corrugation of the ileum lining like Johne’s and has been linked to drinking infected milk, although clearly this is not the whole picture as genetic differences play a role. Nevertheless, ‘supershedder’ cattle exist that are often subclinical Johne’s disease cases that excrete enormous numbers of MAP into the environment posing an animal health risk, and 1 potential risk to humans through infected milk and meat products. The disease is not well controlled, hence the high prevalence. In part this can be controlled by improved farm conditions but poor diagnostics and lack of effective/licenced vaccines are also an issue. Bovine Tuberculosis In cattle herds in the UK, parts of the USA, and New Zealand bovine TB, caused by infection with Mycobacterium bovis, is a disease of relatively high prevalence. Tuberculosis in cattle is very similar to that seen in humans in terms of pathology and immunology – this likely relates to the high similarity at the genetic level of M. tuberculosis (human) and M. bovis (cattle) [> 99%]. M. bovis is also important to human health as this is a zoonotic organism also capable of causing TB in humans. Prior to milk pasteurisation (and in countries where milk is not treated) many cases of human tuberculosis were caused by the bovine organism. In the UK, the incidence of TB in cattle continues to rise, despite control measures being introduced more than 60 years ago. This causes major economic losses to the farming industry (estimated at > £1billion over the next ten years if disease control cannot be improved). Control measures against bovine TB were introduced in the UK in the 1950s – and initially led to a greatly reduced prevalence. However, since the mid-1980s the incidence has been on the increase. For example, in 1986 the number of cattle testing positive for bovine TB was around 300 but has now reached over 30,000. The disease is still largely geographically isolated in the UK, but the disease areas are increasing in size and TB is now being found in areas that were typically TB free (North West England/Scotland). In order to control TB and Johne’s disease in cattke there is a need for improved diagnostic tests, and potentially vaccines. Development of sensitive, specific tests and effective vaccines requires knowledge of the immune response that is induced by infection and ways in which it can be measured (diagnosis). Understanding the type of immune response that is needed to induce protection (immunity versus disease) is needed for the development of effective vaccines. Pathogenesis Immune responses to Mycobacterial infections Mycobacteria are facultative intracellular bacteria, and a key feature of infections involving these bacteria is their ability to survive and grow inside professional phagocytes (macrophages and dendritic cells). As a consequence, cell-mediated immunity is critical to controlling infection, in particular macrophage activation by interferon-gamma (IFNγ) released by stimulated T-cells is central to immunity. Humoral immunity and secretion of antibodies is not commonly seen in TB but may only be observed at very late stage of infection and correlates with disease progression. Antibodies are also seen late following MAP infection, usually as animals progress to clinical Johne’s. Immune responses to mycobacteria are initiated through uptake of bacteria by macrophages or dendritic cells that line the respiratory tract or are present within the lung. Mycobacteria have evolved mechanisms to avoid intracellular destruction following uptake by these cells. Usually, bacteria are taken up into a phagosome which will then fuse with a lysosome inside the cell: the lysosome contains low pH and enzymes which contribute to destruction of the bacteria. Mycobacteria can block phago-lysosome fusion enabling them to survive inside the macrophage. They do not completely evade detection as even the early events associated with uptake allow the macrophage to signal to the immune system (via pattern recognition receptors, Immunology lecture 3). These signals then induce a cascade of events that lead to the formation of granulomas within the tissues (lungs and lymph nodes). Granulomas or ‘lesions’ are aggregates of cells produced by the host in response to infection and are the site of host- pathogen interactions. Within the granuloma mycobacteria may be free living, or contained within macrophages which they can manipulate for their survival based on metabolism and cell wall components. The replication of mycobacteria within macrophages induces signals for lymphocytes which traffic to the site of infection in an attempt to clear the bacteria. This results 2 in a ‘walling’ off of the bacteria. This can lead to a strong immune response where the bacteria are cleared. In most cases, the granuloma is a site where mycobacteria can survive but don’t replicate and granuloma formation will not result in anything other than a small lesion in the tissue. The bacteria can be reactivated later in life e.g. by other infections, or old age when immune system weakens. The granulomas contain large numbers of T cells. Both CD4+ (T helper) and CD8+ (cytotoxic) are present, along with other non-conventional populations (e.g. gamma delta T cells). The major role of T cells in the granuloma is to release factors such as IFNγ and lymphotoxin which influence the growth of mycobacteria within the lesions. IFNγ activates macrophages; these activated macrophages are generally better at killing mycobacteria via the actions of reactive nitrogen and oxygen intermediates. IFNγ also enhances phago-lysosome fusion and bacterial destruction. Cytotoxic actions of CD8+ T cells can induce macrophage death which may also kill mycobacteria. In some cases immune control breaks down completely and the lesion becomes caseous or liquefies which is followed by release of bacteria and can result in haematogenous spread. This is rare. Mycobacterial virulence factors The basis to mycobacterial virulence is their survival within host macrophages and surviving the hostile environment of the granuloma. The predominant virulence ‘factor’ is the protective capability of the glycolipid-containing cell wall. The mycobacterial cell wall has a plasma membrane containing proteins, phosphatidylinositol mannosides and lipoarabinomannan. The peptidoglycan layer has 10% of N-glycolymuramic acid residues covalently attached to arabinogalactan via phosphodiester bonds. In turn, the penultimate and final arabinogalactan moieties are attached to high molecular weight mycolic acids. Into these, on the bacterial surface, are interwoven lipids and glycolipids. This complex structure makes the bacteria very hydrophobic, resistant to common lysis products (lysozyme) and also helps resist the oxygen and nitrogen radicals released in phagosomes. The bacterial also produce enzymes such as superoxide dismutase to resist the impact of these intermediates. Cell wall components, as outlined below, also affect normal functioning and signalling of host cells. Cell wall components: Mycolic acids – resist phagocytic digestion. Sulfatides – prevent phagocyte activation and phagosome-lysosome fusion. Trehalose di-mycolate (cord factor) – Inhibits phagocyte chemotaxis, activation, phagosome- lysosome fusions and digesion. Lipoarabinomannan (LAM) – prevents phagocyte activation and digestion within the phagocyte. Mycosides – prevent intracellular killing and digestion. Detection and diagnosis For both Johne’s disease and bovine TB, there are a range of diagnostic techniques based on either detection of the pathogen or on measurement of immune responses to infection. For Johne’s disease MAP may be detected in faeces using PCR – however, there are a number of issues: intermittent nature of shedding and low shedding in the subclinical phase; difficulty of processing faecal material. Evidence for MAP infection/Johne’s disease is usually from blood based diagnostic which measures MAP specific antibody. This is an ELISA test – suffers from low sensitivity (although specific). Many animals are antibody negative despite being infected – this reflects late onset of humoral immunity in MAP infection (immune evasion, preferential stimulation of cell mediated immunity contribute to this). Improved diagnostics could target cell mediated immunity – ongoing research. For bovine TB statutory diagnostic testing takes place in the UK: this uses the tuberculin skin test; positive animals are culled. Alongside this, another indirect test measures IFNγ secretion. 3 These two tests measure the host immune response. Other direct methods of detection may also be used as confirmatory tests: these are usually carried out when TB is suspected based on the skin test and/or IFNγ test. Post-mortem tissues can be examined for the presence of lesions/granulomas – these are not always macroscopic and may be missed. Tissue samples (and respiratory discharges, milk), may be subjected to Ziehl-Neelsen staining to detect bacteria. Culture may be performed from tissues or fluids but the generation time of M. bovis is very slow – minimum 3-6 weeks for a positive result. As growth is so slow PCR may be used to detect the presence of the organism. The Tuberculin test is used to detect current or previous infection with mycobacteria and uses purified ‘protein’ derivatives (PPD) which contain a complex mixture of proteins, lipids, carbohydrates and DNA from the relevant Mycobacterium species. An intradermal inoculation is used to test for delayed-type hypersensitivity (swelling and reddening) after 72 hrs. Previous exposure to non-pathogenic Mycobacteria spp. can sometimes give a positive response so a comparative test is usually performed: The tuberculin test is carried out at 1, 2, 3, or 4 year intervals depending on the frequency of TB in the area. PPDs from M. bovis and M. avium are separately injected intradermally into the shoulder. Skin thickness is measured at the same time, and again 3 days later. DTH indicative of an ongoing immune response will be detected as an increase in the skin thickness. DEFRA definitions of TB reactor, inconclusive reactor and negative are used to determine individual animal status. If the reaction to M. bovis PPD is 4mm greater than to the M. avium PPD then the animal is defined a ‘reactor’ i.e. has TB and sent for slaughter. If 1-4 mm different this is ‘inconclusive’ and the animal is then retested within 40-60 days. Where reactors or inconclusive reactors are found, the herd then is placed under TB restrictions until the entire herd tests clear. This test has been in use for several decades but the incidence of TB is still increasing. There are many possible reasons including a lack of sensitivity or specificity of the skin test. In order to improve detection of TB in certain geographical areas where TB incidence is low [to maintain TB free status], additional control measures have been introduced. A blood based detection method where specific IFNγ gamma responses to bovine PPD are measured is used alongside the skin test. This may be more sensitive and specific and usually detects animals earlier in infection than the skin test. Other considerations include co-infections e.g. liver fluke associated with failure of tuberculin skin testing; wildlife reservoirs which make eradication difficult and a lack of understanding of disease spread between animals or wildlife. Vaccination No vaccines are currently licenced for use in cattle for Johne’s disease or bovine TB. Partially effective vaccines exist but, because these are either attenuated versions of the causative organism (BCG) or killed whole bacteria (e.g. Gudair, Silirum), they cannot be deployed as these interfere with current diagnostic tests. This means either 1) develop new vaccines that don’t interfere with current diagnostics or 2) develop new diagnostics that enable us to differentiate infected from vaccinated animals (so called DIVA diagnostics). A DIVA diagnostic is being developed for bovine TB to enable BCG vaccination to be deployed in cattle. BCG is the human TB vaccine which was first used in humans more than 100 years ago. BCG can protect cattle from bovine TB especially if it is given to them within first week of life. It is an attenuated (or weakened) form of the cattle pathogen M. bovis and was derived by continual growth on slices of potato over a number of years. This resulted in the loss of a number of regions of the M. bovis genome (regions of deletion or RD genes). By comparing the genomes of BCG and M. bovis it was possible to identify genes within these regions that could be used as diagnostic tools. Thus, these specific gene products can be used in either the skin test or the IFNγ blood test and only infected animals will respond. Vaccinated animals cannot respond as the vaccine does not have the gene expressed to stimulate the immune response. 4