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    Assessment of the sensitivity of current standard procedures for the isolation of Yersinia enterocolitica from pork mince : a dissertation presented in partial fulfilment (25%) of the requirements for the degree of Master of Veterinary Studies in Veterinary Public Health at Massey University
    (Massey University, 1999) Subramaniam, Durai
    Y. enterocolitica and related species have been isolated from many types of food. The majority of isolates differ in biochemical and serological characteristics from typical pathogenic strains and are termed non-pathogenic or environmental strains. Usually the number of Y. enterocolitica organisms present in food products is low compared with the dominant background flora. The ability of current enrichment procedures to recover pathogenic strains of Y. enterocolitica from different foods is often inadequate probably because different strains require different conditions for optimum growth (De Boer 1992). An efficient enrichment procedure should confer some selective advantage to the desired type of microorganism by promoting its growth relative to the competing microflora. At present, there is no single ideal isolation procedure available for the recovery of pathogenic strains of Y. enterocolitica from foods. The aim of this study was to determine the recovery rate of Y. enterocolitica biotype 4/serotype 0:3 from samples of pork mince inoculated with known numbers of the microorganism using different enrichment parameters (Time, temperature and pH) and Cefsulodin-Irgasan-Novobiocin (CIN) agar as the selective medium. The experiment was conducted in two trials using different bacterial dilutions. Three pork mince samples in duplicate were inoculated with known quantities of Y. enterocolitica biotype 4/serotype 0:3 organisms and subjected to cold enrichment in phosphate buffered saline (PBS) with a pH of 7.6, 6.6 and 5.5 at 25°C for 2 days, 10°C for 7 days and 4°C for 21 days. CIN agar was used as the selective medium. Pre-inoculation control samples were selected and plated in CIN on day O and on day 21 after PBS enrichment at 4°C. In Trial one Y. enterocolitica organisms were recovered from all 3 samples incubated at 25°C for 2 days and from 1 out of 3 inoculated samples incubated at 4°C for 21 days. There were no organisms recovered from other inoculated samples. The control sample did not show any environmental contamination with Yersinia species. In Trial two, Y. enterocolitica was recovered from 1 out of 3 duplicate samples enriched in PBS with pH 6.6 and incubated at 25°C for two days. Y. enterocolitica was not recovered from other inoculated samples. Y. intermedia was isolated from all pH, temperature and time combinations and also from control samples. The following conclusions can be drawn from this experiment. Incubation at high temperature (25°C) and short duration (48 hours) can be used as an efficient method for isolating Y. enterocolitica from pork samples. The standard incubation period of 21 days required for cold enrichment at 4°C is too long for the isolation of pathogenic strains, because of possible growth of environmental microorganisms. A pH of 6.6 is less efficient than 7.6 for enrichment although occasional isolation can be made using this pH. Enrichment in PBS with a pH of 5.5 with any time as well as temperature combinations and incubation at 10°C for 7 days are not ideal for isolation of pathogenic Yersinia enterocolitica strains. Of the three enrichments (PBS 7.6, 6.6, 5.5) used in this experiment, PBS with pH 7.6 was found to be most efficient to others.
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    Neuropathology of ovine ceroid-lipofuscinosis : a thesis presented in partial fulfilment (30%) of the requirements for the degree of Master of Philosophy in Veterinary Pathology at Massey University
    (Massey University, 1987) Shimada, Akinori
    The purposes of this study were to describe the neuropathology of ovine ceroid-lipofuscinosis, to compare findings with those of the related entities in humans and other domestic animals, and to provide morphological information that might help elucidate the pathogenesis of these diseases. An established flock of South Hampshire sheep carrying the ceroid-lipofuscinosis gene have made it possible to perform a longitudinal study on the central nervous system of affected sheep of various ages including foetuses. The most striking gross pathological change of affected sheep was brain atropy. At terminal disease, the brain weights of affected sheep were 55% of those of normal sheep. Atrophy affected mainly the cerebrum. Sudan black and luxol fast blue positive autofluorescent neuronal pigment granules were detected by lightmicroscopy as early as the mid stage of foetal development, the earliest stage examined. Postnatally there were topographical differences in the quantity of accumulated lipopigments in neurones of various areas. Similarly, there were age related topographical differences in secondary degenerative changes. Neuronal loss was most severe in the parietal lobe cortex showing an initial laminar distribution. This pattern was well demonstrated by a concomitant astrocytosis. In addition to the complex electron dense cytosomes similar to those reported in the human syndromes, there were less complex cytosomes of smaller size in affected foetal brains. The latter were clearly bounded by a trilaminar membrane and contained whorls or loose stacks of trilaminar membranes resembling those of the limiting membranes. In some electronmicrographs there was a suggestion of continuity between the surrounding membrane and the internal membranes, but this was not definitely demonstrated. This is provisionally interpreted as being due to an internalization of surrounding limiting membrane rather than a recycling of membrane. Some of these small cytosomes also showed complex multilamellar profiles similar to those of large complex cytosomes. These latter appeared to be formed by coalescence of smaller complex ones. There thus appeared to be a sequence of changes in the development of storage cytosomes. This study revealed that the ovine disease has not only many neuropathological findings in common with analogous human diseases, but also some pathological features which have not been reported in affected humans or animals. Ovine ceroid-lipofuscinosis is thus a useful animal model for the study of the human ceroid-lipofuscinoses.
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    Thermophilic Campylobacter in animals and man : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Pathology and Public Health at Massey University
    (Massey University, 1984) Kakoyiannis, Charalambos Kleanthous
    During the course of the work reported in this thesis, more than 2,000 samples and isolates from animals and from humans, were examined for the presence of intestinal thermophilic Campylobacter. This figure does not include samples and isolates examined during the course of experimental studies. Culture of either rectal swabs from pigs or cloacal swabs from poultry usually provides an accurate assessment of the prevalence of distal gastro-intestinal (G.I.) tract infection by C. coli and C. jejuni, provided that the swabs are kept at 4°C and cultured within 6 hours. Cultures of isolates of C. jejuni can be preserved for at least 21 months if stored in FBP broth or PBS at -70°C. The major site of colonisation by C. coli in pigs, is the distal part of the G.I. tract and the proximal G.I tract in the case of C. jejuni. In poultry, the major sites of colonisation by both C. jejuni and C. coli are the distal ileum and the large intestine, especially the caeca. Surveys of pigs showed that 60 - 100% of post weaning pigs are infected with C. coli. In neonates the rates of infection increase with age, and infected sows are the major source of infection for piglets. C. jejuni is rarely isolated from pigs. Twenty eight percent (28%) of all poultry flocks examined were infected with intestinal thermophilic Campylobacter. In infected broiler flocks the rate of infection is almost 100%, while in older birds lower rates were found. Of all the isolates examined, 86% were C. jejuni and 14% C. coli. All poultry carcases and edible viscera, derived from infected broiler flocks, are contaminated with Campylobacter. The levels of contamination of these products by both C. jejuni and C. coli are approximately 106 and 104 cfu per whole carcase and packet of edible viscera respectively. Poultry chilled products for sale in supermarkets were also heavily contaminated by these two species Campylobacter which remained viable for the 'shelf life' of the products (ten days). The prevalence of infection by intestinal thermophilic Campylobacter in gulls was 59%, in ducks 29%, and in rats 60%. Infected sparrows were not detected. With the exception of an isolate from one pig, C. laridis is a species apparently host specific for sea birds, and isolates from gulls of what, on conventional taxonomic criteria, would be classified as C. coli are shown on more detailed examination to be C. laridis. The level of excretion in faeces of Campylobacter in most infected pigs is between 102 - 105 cfu/g, in poultry approximately 108 cfu/g, in rats 105 cfu/g, and in gulls 103 cfu/g. The infectivity of C. jejuni isolated from poultry for experimental chickens is in the region of 5 x 102 cfu and that of C. coli approximately 5 x 105. Both species of Campylobacter were a 100 to 1000-fold less infective for rats. Infection in both chickens and rats was self-limiting and attempts to reinfect chickens with the same species or organism, were usually unsuccessful. C. laridis was not infective for either chickens or rats at dose rates of up to 109 organisms. Campylobacter infections are the most common notified human enteric infection in New Zealand with an annual incidence rate of 31/100,000. Approximately 94% of the human cases of campylobacteriosis are due to C. jejuni and 6% to C. coli. People between 1 to 4, and 15 to 35 years of age are the most commonly affected, and patients who no longer have clinical signs of infection may shed up to 108 cfu of C. jejuni/g of faeces. By use of a bacterial restriction endonuclease DNA technique (BRENDA), up to 80 different types C. jejuni and C. coli were identified from 338 isolates from humans. One hundred and ninety four (61%) of the 316 isolates of C. jejuni from humans had similar BRENDA patterns to isolates of C. jejuni from animals. Poultry appear to be the major source of C. jejuni, and possibly C. coli, for humans, while pigs apparently are an insignificant source of C. coli for humans. Rats can be infected with strains of C. jejuni which can infect poultry, humans and other animals. BRENDA types recovered from gulls and ducks were not similar to any of the isolates from humans or other animals examined. Some strains of C. jejuni and C. coli developed an in-vitro resistance to nalidixic acid. This is an important finding in relation to conventional taxonomic criteria for differentiating C. laridis from other intestinal thermophilic Campylobacter. Isolates of 'C. coli' from gulls are phenotypic variants of C. laridis which may be either resistant, or non resistant, to nalidixic acid. Only by determining whether or not, anaerobic growth in the presence of TMAO occurs, can C. coli be differentiated from C. laridis.