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    Bovine tuberculosis in brushtail possums (Trichosurus vulpecula) : studies on vaccination, experimental infection, and disease transmission : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University
    (Massey University, 2001) Corner, L A L
    The objectives of the research program were to obtain a better understanding of BCG as a tuberculosis vaccine in possums, and assess its potential as a tool for controlling tuberculosis in wild possum populations. A series of vaccination and challenge experiments were conducted, as well as studies on alternative experimental infection procedures. The program included two field studies, one on the epidemiology of tuberculosis in a population of possums regenerating after localised possum eradication, and the other examined the efficacy of BCG vaccine in a wild population in which tuberculosis was endemic. The first experiments confirmed the earlier published findings that BCG delivered as an intranasal aerosol induced a protective response. The protective response was found to be present 12 months after vaccination and therefore of sufficient longevity to make vaccination a practical control tool. A second study demonstrated that revaccination of possums enhanced protection and a third showed that conjunctival vaccination was as effective as intranasal aerosol. These findings supported the development of a possum activated self-vaccinator that would deliver vaccine as an aerosol. In delivering the spray to both the external nares and the eyes a simple and cheap device could be designed to efficiently vaccinate wild possums. The intratracheal experimental infection procedure used in the vaccination and challenge experiments was not entirely suitable for our purposes. Although it provided an assured level of exposure and repeatable results, all infected possums developed fulminant, rapidly progressive disease, irrespective of the vaccination regime used. Two alternative methods of challenge were examined; the conjunctival route of infection, and natural transmission between experimentally infected possums and susceptible in-contact possums. Conjunctival infection was shown to be a reliable procedure for infecting possums, with the disease that resulted from infection having many of the cardinal features of natural tuberculosis in wild possums. Infection following conjunctival inoculation progressed slowly and may be suitable for studying pseudo-vertical transmission and the efficacy of post-infection vaccination. In studies with captive possums there was little or no transmission of infection between experimentally infected possums and susceptible in-contact possums in the same pen when the experimentally infected animals were selected at random. However, when possums with high levels of social interaction were experimentally infected there was a significant increase in transmission rates. In addition, the possums that became infected by transmission were more socially active than those that remained free of infection. Two aspects of the pathogenesis of tuberculosis in possums were clarified during the experimental infection and natural transmission studies. The duration of preclinical infection, impossible to determine accurately in longitudinal studies on wild possums, was found to range from 6 - 20 weeks. Secondly, the pre-eminence of the aerosol route in naturally transmitted tuberculosis was confirmed. After eradication of possums from a 36 ha site, tuberculosis reappeared within four months. Re-emergence of infection on the site was due to immigration of infected possums, not to the survival of M. bovis in the environment. Each of the four restriction endonuclease analysis (REA) types of M. bovis that caused disease in the possum population showed a different temporal and spatial pattern. BCG vaccine had high efficacy in a wild possum population. Over 2 years, 300 possums were recruited to a study of BCG vaccination. Approximately 50% of the possums were vaccinated, where each possum was vaccinated using both intranasal aerosol and conjunctival instillation. There were significantly more cases of tuberculosis in unvaccinated possums than in vaccinates, with a relative risk of tuberculosis in unvaccinated possums of 3.21. The vaccine efficacy was 69%. The most important question relating to BCG vaccine that remains to be addressed is the ability of vaccination to control tuberculosis in possum populations. This research has demonstrated that BCG vaccine provided protection against M.bovis infection in both captive and wild possums. Future research should be directed towards developing delivery systems for vaccinating wild possums and strategies for vaccine use in wild tuberculous possum populations.
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    Identification and characterisation of an exported immunogenic protein of Mycobacterium avium subspecies paratuberculosis : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand
    (Massey University, 2002) Dupont, Christine
    Exported proteins of mycobacteria are available to interact with the immune system at an early stage of infection and are potent inducers of immune responses. Potentially exported proteins of Mycobacterium avium subspecies paratuberculosis were identified using alkaline phosphatase gene fusion technology. A library of partial gene fusions from a New Zealand clinical isolate of M. a. paratuberculosis was constructed in the shuttle vector pJEM11 and expressed in the surrogate hosts E. coli and M. smegmatis. The DNA inserts from a portion of the resulting clones expressing alkaline phosphatase-positive fusion proteins were partially sequenced to identify the proteins. Eleven proteins not previously described for M. a. paratuberculosis were identified as containing signal sequences for export. One of these, a putative lipoprotein named P22 was selected for further study. The full nucleic acid sequence of the p22 gene was determined and the open reading frame was cloned into die mycobacterial expression vector pMIP12. This enabled P22 to be produced as a polyhistidine-tagged protein in M. smegmatis and facilitated purification by chromatography. N-terminal sequencing of the recombinant protein confirmed cleavage of an N-terminal signal sequence. Native P22 was detected in culture supernatants and cell sonicates of M. a. paratuberculosis strain 316F using rabbit antibody raised to P22. Investigation of the presence of genes similar to p22 in other mycobacterial species, revealed p22 was present in Mycobacterium avium subspecies avium and similar genes existed in M. intracellularae (88.5% identity) and M. scrofulaceum (87.7% identity). Database searches showed P22 belonged to the LppX/LprAFG family of mycobacterial lipoproteins also found in M. leprae and in members of the M. tuberculosis complex. P22 shared less than 75% identity to these proteins. Recombinant P22 was able to elicit significantly increased interferon-gamma secretion in blood from a group of eight sheep vaccinated with a live, attenuated strain of M. a. paratuberculosis (strain 316F) compared to a group of five unvaccinated sheep. Antibody to P22 was detected by Western blot analysis in 10 out of 11 vaccinated sheep, in two out of two clinically affected cows and in 11 out of 13 subclinically infected cows.