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    Leptospirosis in humans and pastoral livestock in New Zealand : a thesis presented in partial fulfilment of the requirements for the doctoral degree of Doctor of Philosophy at Massey University
    (Massey University, 2013) Dreyfus, Anou
    This PhD investigated leptospirosis in humans and pastoral livestock in New Zealand (NZ). A longitudinal ‘abattoir study’, in which blood from workers (n=592) from sheep (n=4), deer (n=2) and beef (n=2) slaughtering abattoirs was tested by the microscopic agglutination test (MAT), revealed that 10-31%, 17-19% and 5% of workers respectively, had antibodies against Leptospira interrogans sv Pomona (Pomona) and/or L. borgpetersenii sv Hardjobovis (Hardjobovis). While the annual infection risk for meat workers of sheep plants was 11.1%, it was 0% in workers processing deer and 1.2% in those processing beef cattle. Sixty workers had a history of probable leptospirosis while working in abattoirs between 1962 and 2010 and three sheep abattoir workers within the one year study period. In sheep abattoirs, new infection with Hardjobovis or Pomona measured by serology was associated with a two-fold higher risk of ‘flu-like’ illness, and an average of four days absence from work. The average annual risk of experiencing flu-like symptoms due to infection with Leptospira measured by serology was 2.7%. The under-ascertainment of officially notified cases with leptospirosis in the last five years was estimated at between 16 and 56 times. Work position was the strongest risk factor for sero-positivity with Pomona and/or Hardjobovis in sheep and deer abattoir workers. The prevalence and new infection risk was highest in workers at the beginning of the slaughter board and the use of personal protective equipment (PPE) appeared not to reduce the risk of sero-positivity or new infection. The risk factor analysis revealed that the infection risk prevailed in the abattoirs and was not evident for non-work related risk factors, such as hunting, home slaughtering and farming. In a multi-species cross-sectional ‘farm study’ (n=238), 97% of sheep and beef and 76% of deer farms had at least one in 20 animals MAT sero-positive against Hardjobovis and/or Pomona. Overall, 50% of adult sheep, 58% of adult beef and 34% of yearling/adult deer were positive against either serovar. Hardjobovis was more prevalent in all three livestock species than Pomona. The regional prevalence distribution in sheep was different for Hardjobovis and Pomona. Grazing beef with deer reduced the likelihood of positivity against Pomona in beef. Co-grazing with another species did not increase the odds of the within-herd prevalence for deer and sheep of Pomona or Hardjobovis and for beef the within-herd prevalence of Hardjobovis controlling for other farm-level risk factors. The incidence of probable leptospirosis in cattle herds in 2009 was 2.6%, in sheep flocks 0% and in deer herds 1%. Tailing rates of sheep farms were positively correlated with prevalence of Hardjobovis: a 1% increase in prevalence was equivalent to a 0.11 increase in tailing percentages, which is unlikely to be causative since this association lacks biological plausibility. All other reproduction and culling rates of any species were not significantly associated with prevalence.
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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.
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    The spatio-temporal epidemiology of Bovine spongiform encephalopathy and Foot-and-mouth disease in Great Britain : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University
    (Massey University, 2003) Stevenson, Mark Anthony
    Great Britain suffered two of the most globally notable animal disease epidemics of recent decades — the bovine spongiform encephalopathy (BSE) epidemic which began in November 1986, and the foot-and-mouth disease (FMD) epidemic which lasted from February to September 2001. This thesis applies various analytical techniques to these two quite different epidemics: a rapidly spreading highly contagious disease for which urgent decisions are essential (FMD), and a feed-borne non-contagious disease with an exceptionally long incubation period (BSE). The BSE epidemic, in particular, presented major investigational challenges because its recent emergence meant that its epidemiological features were not yet fully clear. The studies of BSE reported here showed that the control measures recommended as a consequence of the first epidemiological study of the new disease were remarkably effective. The July 1988 meat and bone meal ban resulted in a 60% reduction in BSE risk for cattle born in the first 12 months after its introduction. Descriptive spatial analyses, using kernel density and regression techniques showed a marked concentration of BSE risk in the south of Great Britain. Following the July 1988 meat and bone meal ban BSE risk shifted to the east of the country, an effect partly explained by cross contamination of cattle feed with high-protein concentrates destined for the pig and poultry industry. Detailed investigation of the earliest BSE-exposed farm holdings identified the south of England as an area of excess exposure density. While interpretation of these findings is complicated by the fact that the disease must have been present for some years before it was first diagnosed, the evidence suggests initial amplification in the south provided risk material which progressively distributed the disease to the rest of the country. In contrast to BSE, FMD presents different challenges, in that affected farms can be diagnosed rapidly, but it is difficult to accurately evaluate the relative importance of the different mechanisms of transmission, and hence determine how best to apply control efforts. Foci of FMD infection of matched size in the English counties of Cumbria and Devon were compared to dissect out factors contributing to the two quite different epidemic patterns in these areas. This analysis showed evidence of strong spatio-temporal interaction of infection risk in Cumbria, due initially to cattle herds as the dominant influence, with a later growth in the role of sheep as a source of infection. During the FMD epidemic a stochastic spatial simulation model was used extensively as an aid for decision making. After the epidemic was over the predictive accuracy of earlier real-time modelling was assessed for the whole of Britain and the most concentrated focus of disease in Cumbria. The model predicted the temporal epidemic curve closely at both levels, and predicted the national spatial pattern of infection with high specificities (over 99%) and useful sensitivities (37% to 71%). It was concluded that the model predicted FMD-infected locations within 0 to 14 days after simulation start date with sufficient accuracy to guide surveillance activities and to provide estimates of resources required for contingency planning. The spatial accuracy of predictions might be further improved through the use of a series of sub-regional models, better-capturing the characteristics of individual outbreak foci that typically emerge during extended large scale, multicentred epidemics. The studies presented in this thesis demonstrate that the application of temporal, spatial and spatio-temporal analytical methods can enhance the understanding of the epidermiological features of diseases in animal populations. The value in applying these methods of analysis comes from the ability to identify high and low disease-risk time frames and locations, allowing more focused allocation of investigative resources.