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Item Molecular epidemiology and whole genome sequencing analysis of clinical Mycobacterium bovis from Ghana.(PLOS, 2019-03-04) Otchere ID; van Tonder AJ; Asante-Poku A; Sánchez-Busó L; Coscollá M; Osei-Wusu S; Asare P; Aboagye SY; Ekuban SA; Yahayah AI; Forson A; Baddoo A; Laryea C; Parkhill J; Harris SR; Gagneux S; Yeboah-Manu D; Spigelman MBackground Bovine tuberculosis (bTB) caused by Mycobacterium bovis is a re-emerging problem in both livestock and humans. The association of some M. bovis strains with hyper-virulence, MDR-TB and disseminated disease makes it imperative to understand the biology of the pathogen. Methods Mycobacterium bovis (15) among 1755 M. tuberculosis complex (MTBC) isolated between 2012 and 2014 were characterized and analyzed for associated patient demography and other risk factors. Five of the M. bovis isolates were whole-genome sequenced and comparatively analyzed against a global collection of published M. bovis genomes. Results Mycobacterium bovis was isolated from 3/560(0.5%) females and 12/1195(1.0%) males with pulmonary TB. The average age of M. bovis infected cases was 46.8 years (7-72years). TB patients from the Northern region of Ghana (1.9%;4/212) had a higher rate of infection with M. bovis (OR = 2.7,p = 0.0968) compared to those from the Greater Accra region (0.7%;11/1543). Among TB patients with available HIV status, the odds of isolating M. bovis from HIV patients (2/119) was 3.3 higher relative to non-HIV patients (4/774). Direct contact with livestock or their unpasteurized products was significantly associated with bTB (p<0.0001, OR = 124.4,95% CI = 30.1–508.3). Two (13.3%) of the M. bovis isolates were INH resistant due to the S315T mutation in katG whereas one (6.7%) was RIF resistant with Q432P and I1491S mutations in rpoB. M. bovis from Ghana resolved as mono-phyletic branch among mostly M. bovis from Africa irrespective of the host and were closest to the root of the global M. bovis phylogeny. M. bovis-specific amino acid mutations were detected among MTBC core genes such as mce1A, mmpL1, pks6, phoT, pstB, glgP and Rv2955c. Additional mutations P6T in chaA, G187E in mgtC, T35A in Rv1979c, S387A in narK1, L400F in fas and A563T in eccA1 were restricted to the 5 clinical M. bovis from Ghana. Conclusion Our data indicate potential zoonotic transmission of bTB in Ghana and hence calls for intensified public education on bTB, especially among risk groups..Item A study of tuberculosis in hedgehogs so as to predict the location of tuberculous possums : a thesis submitted in partial fulfilment of the requirements for the degree of Masters of Veterinary Studies at Massey University, Palmerston North, New Zealand(Massey University, 1998) Gorton, Robyn JaneHedgehogs are spillover hosts for Mycobacterium bovis, which means the prevalence of disease in the hedgehog is directly related to the prevalence of disease in a local reservoir population such as the possum. Possums have home ranges similar to that of hedgehogs and on large farms, locating a tuberculous hedgehog coud substantially reduce the area where extensive control is required to eliminate tuberculosis from the wild animal population. Male animals usually have a larger home range than females and this is true of the hedgehog. In utilising the knowledge of a hedgehog’s home range, female hedgehogs could provide a specific local indicator of the presence of tuberculous possums and male hedgehogs could locate the general region on the farm with tuberculous possums. The hedgehog could also be considered a temporal indicator of tuberculosis in the wild animal population especially where there has been a history of tuberculosis. The longevity of the hedgehog is reasonably short (2-3 years in the wild) and should sufficient control of other tuberculous animals occur then the disease will also disappear from the hedgehog population. Hedgehogs from this study were noted to be carriers of Salmonella enteriditis, Sarcoptes scabiei. This is believed to be the first report of these pathogens associated with hedgehogs in New Zealand.Item Transmission of tuberculosis (Mycobacterium bovis) by possums : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University(Massey University, 1995) Jackson, R.Tuberculosis caused by Mycobacterium bovis was diagnosed in 59 of 632 possums (Trichosurus vulpecula) individually identified over a 52 month period, during a longitudinal study of the naturally occurring disease in possums at a 21 hectare bush pasture location on a farm at Castlepoint in the Wairarapa. The disease exhibited marked spatial and temporal clustering and was continuously present in the population for the whole period. The disease had a relatively long duration of up to 22 months and four distinct stages were demonstrated in cross-sectional studies. Among tuberculous possums, prevalences of up to 0.15 (±0.11) were recorded in the first stage prior to development of gross lesions. After dissemination started, the disease showed rapid generalisation to multiple sites by haematogenous and/or lymphatic spread to the next stage when gross lesions were evident, particularly in lung, axillary and inguinal lymphocentres. In the third stage, lesions were disseminated through almost all lung lobes, discharging fistulae were common and kidney, intestine and mammary gland were commonly affected by both gross and microscopic lesions. Behaviour and outward signs of health were unaffected prior to the terminally-ill stage, lasting for up to 2 months. In common with other marsupials studied to date and in contrast with most eutherians, there are no popliteal lymph nodes and efferent drainage from the inguinal lymphocentre passes directly to the deep axillary group of lymph nodes via an inguinoaxillary trunk. All subcutaneous lymph drainage passes through either the superficial cervical or the axillary lymphocentres before entering the venous system. Studies of survival of Mycobacterium bovis organisms in different natural habitats showed a relatively short period of survival of M. bovis outside hosts and support a conclusion that environmental contamination of pasture, particularly in summer months, may be relatively unimportant in the epidemiology of tuberculosis in cattle, deer and possums. The weight of evidence favours transmission of infection by the respiratory route and it would seem that transmission of tuberculosis between possums occurs through two major and one minor pathway. The first major pathway is pseudo-vertical transmission from mother to joey during the rearing process. The second major transmission mechanism is direct horizontal transmission among adult possums with available evidence suggesting that this takes place around the locality where a possum dens, probably during competition and threat/agonistic behaviour and during courting and mating activity. The third and probably least important pathway is indirect transmission among mature possums. None of three ELISA assays reliably detected possums infected with tuberculosis and poor test performance was exacerbated by inconsistency between results from serially collected samples from known tuberculous possums.Item A genetic approach to identify Mycobacterium bovis exported protein antigens : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Biology, Massey University(Massey University, 1997) Borich, Suzanne MarieA novel approach, combining phoA-fusion technology with T cell screening of a recombinant cosmid library, was used to detect Mycobacterium bovis exported T cell antigens. An M. bovis BCG library of phoA-fusions was constructed in Escherichia coli and Mycobacterium smegmatis using the plasmid vector pJEM11. The M. bovis BCG DNA inserts from ten PhoA+ clones were partially sequenced and used to search databases for similarities to known genes. These revealed similarities to a family of genes coding for high temperature-requirement serine proteases and a Mycobacterium leprae putative exported lipoprotein gene (pel). The DNA inserts from PhoA+ clones were used to probe an M. bovis cosmid library expressed in M. smegmatis 10 identify cosmids containing the full-length genes coding for these exported proteins. Culture filtrates (CFs) prepared from selected M. smegmatis recombinants (cosmids) were assayed for their ability to induce proliferation and IFN-γ-production from peripheral blood mononuclear cells (PBMCs) taken from M. bovis BCG-immunised and non-immunised control cattle. Culture filtrates from two recombinant M. smegmatis (cosmids 44 and 56) induced significant IFN-γ-production and proliferation by PBMCs from immunised animals. An exported protein gene, identified using the phoA-fusion technology, was subcloned from cosmid 56 and its sequence determined and analysed. Database searches using the deduced amino acid sequence of this gene revealed similarities to an M. leprae putative exported lipoprotein (Pel) and a family of MalE maltose-binding proteins. The M. bovis pel gene was shown to be expressed by recombinant M. smegmatis. Preliminary evidence from this study indicates that the M. bovis Pel protein is recognised by antigen-specific lymphocytes from M. bovis BCG-immunised animals. The PBMCs taken from M. bovis challenged and M. bovis BCG vaccinated / challenged cattle also recognised CF from recombinant M. smegmatis expressing the pel gene in in vitro immunoassays. The combined strategy of using phoA-gene fusions and T cell screening of CFs from a recombinant M. bovis cosmid library proved a sensitive and rapid method for the detection of potential M. bovis T cell antigens.