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    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 Jane
    Hedgehogs 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.
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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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    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.
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    The contribution of wild mammals to the epidemiology of tuberculosis (Mycobacterium bovis) in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University
    (Massey University, 1997) Lugton, Ian William
    The objective of these studies was twofold. The primary aim was to gain a better understanding of the role of free-living mammalian species, other than possums, in the epidemiology of wildlife tuberculosis in New Zealand. The other objective was to continue the operation of the Castlepoint longitudinal so that hypotheses regarding the epidemiology of M. bovis infection in possums could be further refined and clarified. Of the wild carnivores found in New Zealand, the disease persists at high prevalence only in ferrets, and is probably maintained principally by ingestion of tuberculous carrion. Although a moderate number of ferrets excrete M. bovis orally, there appears to be only minor intraspecific transmission by bite wounding. Although cats and stoats can also become infected through scavenging, they appear to be less susceptible to oral infection than ferrets. There is no substantial evidence to suggest that any of New Zealand's free-living carnivores are likely to be reservoir hosts of M. bovis. Observational studies involving twelve domestic red deer suggested that cervids probably become infected through close inspection and investigation of moribund tuberculous possums, and that the likelihood of exposure to M. bovis was related to the curiosity and social ranking of the deer. Necropsies conducted principally on wild red deer and involving 152 animals provided evidence to suggest that significant bacillary excretion from infected deer was uncommon, and that only the few with advanced disease had the potential to be highly infectious. However, behavioural phenomena and disease characteristics preclude the ready transmission of disease amongst cohorts. There is now strong evidence to suggest that a high prevalence of tuberculosis infection in wild deer can only be maintained through contact with infected possums. However, deer may still be able to maintain the disease amongst themselves, albeit at a low prevalence, in the absence of infection in possums. This study also confirmed the importance of lymphoepithelial tissues, such as the oropharyngeal and nasopharyngeal tonsils, as primary sites for the establishment of M. bovis infection, and the subsequent excretion of organisms in deer. The gross and histopathological appearance of the lesions found in six infected hedgehogs are described. It is likely that infection arose from the scavenging behaviour of hedgehogs. The moderate prevalence (3.9%) of tuberculosis in these animals, combined with their small home ranges may allow them to be used successfully in wildlife surveys to pinpoint the locality in which tuberculous possums have died. To gain an understanding of the potential role of wild pigs, goats, sheep, rabbits, hares, rats and mice in the dynamics of Mycobacterium bovis infection in free-ranging animals, numbers of these species were examined for evidence of infection. Of these, only the pig appears to have sufficient potential for intraspecific transmission to be of concern in tuberculosis control programmes. Sheep and goats appear to be simply spillover hosts, which may have a limited role in disease amplification following possible, but limited, intraspecific transmission. Rodents and lagomorphs are most unlikely to play any substantial role in the epidemiology of tuberculosis in New Zealand, under current circumstances. A longitudinal study was established in 1989 to examine the disease behaviour in an infected possum population on a farm in the southern North Island of New Zealand, by trapping, using a fixed set of 295 traps for at least 3 days per month. Animals captured were examined at 2 monthly intervals for evidence of tuberculosis. During the first 5.5 years of this project over 900 individual possums were captured and tagged. Blood was collected from each possum examined, and the sera retained were stored frozen. Using these stored sera, three indirect ELISAs were evaluated as diagnostic tests for tuberculosis in possums. All ELISAs had low sensitivity when a cutoff selected to maximise the specificity was chosen. None of the ELISAs reliably detected possums infected with tuberculosis and they therefore have limited value for epidemiological studies. The lymphocyte transformation assays performed on blood taken from possums was estimated to have a sensitivity for detection of tuberculosis of approximately 80%, when the specificity was set at 99%. The lymphocyte transformation assay was the best of the in vivo tests evaluated, with the moderate sensitivity allowing it to be used with a degree of confidence to retrospectively diagnose disease, and aid the development of hypotheses regarding the epidemiology of tuberculosis in possums. The evaluated tests were applied retrospectively to sera and blood samples from possums from the Castlepoint longitudinal study. The additional data arising from these assays suggested that perhaps as few as one fifth of study site possums which had contact with M. bovis had been previously detected as infected by clinical examination. A proportion of these test positive/examination negative animals may have been exhibiting resistance to M. bovis infection, and/or had resolved lesions or cryptic infection. Such animals may have formed a pool of possums in which future reactivation of tuberculosis was possible. The time from earliest evidence of infection till death, in those possums which showed clinical disease, varied from months to several years. Cortisol assays performed on stored sera, and monitoring of trends in body weight, were used to investigate the role of stressful environmental phenomena in the epidemiology of tuberculosis in possums. Major stressful periods involving inadequate nutrition, heat, cold and moisture stress appear to precipitate severe tuberculosis outbreaks, which are believed to have their origins in the reactivation of subclinical/latent infection in the population. As the period of pre-clinical disease varies substantially, and can be as long as several years, this epidemic of tuberculosis takes several years to subside. Thereafter a small number of clinically diseased possums are likely to be restricted to "hot spots" conducive to transmission of M. bovis. Isolates of M. bovis recovered from a variety of species, both wild and domestic, in the Castlepoint environs, and in particular the Castlepoint study site, were subjected to restriction endonuclease analysis to DNA fingerprint the strains present, and hence gain a better understanding of the inter- and intraspecific epidemiology of tuberculosis. The results do not challenge the accepted view of possums being the major reservoir hosts of tuberculosis in the Wairarapa. There was also no evidence to suggest that host adaptation of M. bovis has occurred, except in the case of possums, where they appear to be able to maintain clusters of individuals infected with particular restriction types, in microhabitats for at least 5 year periods. The occurrence of newly introduced restriction types has made possible new observations on the epidemiology of infection, including the documentation of the occurrence of latent infections, duration of primary progressive disease in newly infected possums (7-8 months), and the likely occurrence of post-primary reactivation of tuberculosis.