Dissertations / Theses on the topic 'Footrot'

Virulence, in relation to ovine footrot, was examined in a review which emphasised the primary role of Bacteroides nodosus, an anaerobic strict parasite of ungulates. The association of this parasite with other bacteria in the footrot lesion resulted in complex interactions of host. parasite and environment. However, experimentation showed that the severity of the footrot lesion was associated principally with two different properties of B. nodosus: protease stability and surface translocation, the latter being a probable function of the pilus. The relationship between virulence, extracellular protease and translocation was elucidated in terms of function rather than structure. For example, the severity of footrot lesions was not related specifically to the electrophoretic mobility of protease isoenzymes or outer membrane proteins of B. nodosus. Although there were only two levels of protease stability, surface translocation, measured as either colony size or degree of cellular twitching, varied continuously between isolates. It was suggested that surface translocation was the basis for a continuous spectrum of virulence observed in ovine footrot. Nevertheless, protease stability was associated specifically with microbial penetration of the epidermal matrix of the hoof , hence justifying a classification of B. nodosus isolates into virulent (stable protease) and benign (unstable protease) strains. Although this classification was considered realistic, the complexity of ovine footrot was emphasised by evidence that twitching motility mediated the effects of both ambient temperature and the footrot microbial flora on the severity of all forms of the disease.

Dichelobacter nodosus, an anaerobic bacterium, is the major transmissible agent of ovine footrot. The disease expresses as a virulent or benign lesion in the hoof. Virulence is related to the production of serine proteases, particularly a thermostable protease. Isolates of D. nodosus are characterised according to the type of protease produced (either heat-stable or heat-labile) and the electrophoretogram (zymogram) of the protease. This study reports on the use of the DNA-based typing techniques Pulsed-Field Gel Electrophoresis (PFGE) and Infrequent-Restriction-Site-PCR (IRS-PCR) to investigate the molecular epidemiology of D. nodosus, including a consideration of the relationship between genetic type, zymogram patterns and whole cell protein profiles. The aim of the project was to obtain a better understanding of D. nodosus strain diversity and dissemination in Australia and its relationship to virulence within the population. The overall intention was to use this information to assist in the long-term control of virulent footrot. Field isolates of D. nodosus from Western Australia (n = 735), New South Wales (n = 16), Victoria (n = 24) and South Australia (n = 21) were obtained and analysed. Both typing techniques that were used offered good differentiation between isolates for epidemiological purposes, and the results were in general agreement. PFGE provided slightly better discrimination between isolates, with 214 PFGE types (181 from Western Australia) compared to 94 IrsT types (77 from Western Australia). Within this diverse range of molecular types clonality was observed - with clones being defined as clusters of isolates having closely related PFGE types. The strains were categorised as genetically diverse, genetically similar or identified as the same strain. This diversity of genetic types was found overall, within flocks of sheep on a farm and within a single hoof where, on a number of occasions, multiple molecular types and zymogram types were found colonising a single hoof. One isolate that was experimentally inoculated into a flock of sheep produced six different genetic types when tested 12 months after the initial infection. This indicates that D. nodosus undergoes rapid genetic change, which means that follow-up epidemiological investigation of disease outbreaks and trace-backs need to be done as soon after infection as possible. The genetic differences appeared to be due to large insertions or deletions of DNA. Amongst sheep on some properties, isolates that had a different protease expression and virulence expression were found to have the same molecular type. Investigation of these isolates by SDS-PAGE showed that they also had the same whole cell protein profiles. Isolates from the same clonal groups also had the same protein profile, whereas genetically diverse isolates had different protein profiles. The lack of protein differences between isolates of the same molecular type, or within a clonal group, suggests that the differences in protease thermostability may be due to conformational changes in the protein, rather than to overall detectable genetic change and/or expression of different proteins. These results demonstrate that PFGE typing can be useful in predicting likely phenotypic expression of whole cell proteins. Further work is required to elucidate differences between virulent and benign strains of D. nodosus.

Dichelobacter nodosus, an anaerobic bacterium, is the major transmissible agent of ovine footrot. The disease expresses as a virulent or benign lesion in the hoof. Virulence is related to the production of serine proteases, particularly a thermostable protease. Isolates of D. nodosus are characterised according to the type of protease produced (either heat-stable or heat-labile) and the electrophoretogram (zymogram) of the protease. This study reports on the use of the DNA-based typing techniques Pulsed-Field Gel Electrophoresis (PFGE) and Infrequent-Restriction-Site-PCR (IRS-PCR) to investigate the molecular epidemiology of D. nodosus, including a consideration of the relationship between genetic type, zymogram patterns and whole cell protein profiles. The aim of the project was to obtain a better understanding of D. nodosus strain diversity and dissemination in Australia and its relationship to virulence within the population. The overall intention was to use this information to assist in the long-term control of virulent footrot. Field isolates of D. nodosus from Western Australia (n = 735), New South Wales (n = 16), Victoria (n = 24) and South Australia (n = 21) were obtained and analysed. Both typing techniques that were used offered good differentiation between isolates for epidemiological purposes, and the results were in general agreement. PFGE provided slightly better discrimination between isolates, with 214 PFGE types (181 from Western Australia) compared to 94 IrsT types (77 from Western Australia). Within this diverse range of molecular types clonality was observed - with clones being defined as clusters of isolates having closely related PFGE types. The strains were categorised as genetically diverse, genetically similar or identified as the same strain. This diversity of genetic types was found overall, within flocks of sheep on a farm and within a single hoof where, on a number of occasions, multiple molecular types and zymogram types were found colonising a single hoof. One isolate that was experimentally inoculated into a flock of sheep produced six different genetic types when tested 12 months after the initial infection. This indicates that D. nodosus undergoes rapid genetic change, which means that follow-up epidemiological investigation of disease outbreaks and trace-backs need to be done as soon after infection as possible. The genetic differences appeared to be due to large insertions or deletions of DNA. Amongst sheep on some properties, isolates that had a different protease expression and virulence expression were found to have the same molecular type. Investigation of these isolates by SDS-PAGE showed that they also had the same whole cell protein profiles. Isolates from the same clonal groups also had the same protein profile, whereas genetically diverse isolates had different protein profiles. The lack of protein differences between isolates of the same molecular type, or within a clonal group, suggests that the differences in protease thermostability may be due to conformational changes in the protein, rather than to overall detectable genetic change and/or expression of different proteins. These results demonstrate that PFGE typing can be useful in predicting likely phenotypic expression of whole cell proteins. Further work is required to elucidate differences between virulent and benign strains of D. nodosus.

Footrot is prevalent in most sheep-producing countries. Two forms are generally recognised: virulent and benign. Virulent footrot has major economic and animal welfare impacts. Footrot has a complex aetiology; the clinical manifestations of the disease result from interactions between the essential causative agent, Dichelobacter nodosus, and the bacterial community of the foot. The severity of these manifestations varies according to environmental conditions and host susceptibility, thus clinical diagnosis of virulent footrot can be challenging, and laboratory tests are used to assist diagnosis. A comparative evaluation of phenotypic and genotypic virulence tests was undertaken, including the elastase test and a qPCR test targeting the aprV2 protease gene, which is thought to be a key virulence marker. The qPCR had a low diagnostic specificity, with aprV2-positive D. nodosus strains detected flocks with clinically benign footrot. As such, aprV2 was deemed an unreliable virulence marker. In contrast, expression of elastase was closely associated with virulence. D. nodosus strains are divisible into ten immunologically distinct serogroups (A to I, M). A culture-independent testing procedure was developed that enhanced the speed and accuracy of serogrouping, and a cPCR test targeting serogroup M was developed. Such a test was previously unavailable. The foot microbiomes of healthy and footrot-affected Merino sheep were characterised and compared using next-generation sequencing and analysis of the bacterial 16S rRNA gene. Fifteen bacterial genera were found to be preferentially abundant on the feet of footrot-affected Merino sheep, several of which were not previously known to contribute to the disease process. A pasture-based experimental model was also developed that provides a more accurate representation of the conditions in which footrot is naturally transmitted and expressed.

Ovine footrot (FR) is an economically important disease that causes lameness and affects sheep flocks worldwide. It is characterized by interdigital skin inflammation (interdigital dermatitis [ID]) with, or without, separation of the hoof horn from the underlying tissue (severe footrot [SFR]). The primary causative agent is the gram-negative anaerobic bacterium Dichelobacter nodosus, which is present in diseased feet and thought to be transmitted via contaminated surfaces. Periods of apparent zero prevalence of FR in a flock can be followed by disease occurrence when the climate becomes favourable for pathogen transmission. This suggests that there are sites where D. nodosus persists in the absence of disease. These sites might include healthy feet, the gingival cavity and faeces of sheep and also the environment. The aim of this thesis was to investigate persistence of D. nodosus, by investigating possible sites of survival of D. nodosus over time. Prospective longitudinal studies were used to investigate persistence. Samples were collected from sheep and from the pasture in three studies (Studies 1 and 2: England, study 3: Spain). Quantitative PCR was used to detect and quantify D. nodosus and to investigate associations between D. nodosus presence in feet, in the gingival cavity and on pasture and a range of predictor variables including climate. A multiple locus variable number tandem repeat analysis (MLVA) suitable for use on mixed DNA and environmental samples was optimized and validated to investigate D. nodosus strains within and between sites. A novel approach to characterize individual strains in a sample was designed. D. nodosus was detected in all sample types in all studies but not on all occasions. The feet of sheep were the only site where D. nodosus was detected in loads exceeding 103 cells per swab. In study 1, D. nodosus was detected in amounts exceeding103 cells in samples collected from the pasture in week 1 only, when detection frequency of D. nodosus on feet was high and the weather was wet. A minimum of 14 strains of D. nodosus were detected on the feet of sheep by MLVA. A decline in detection of D. nodosus in the environment coincided with periods of dry weather, however, dry weather did not coincide with a decline in D. nodosus loads on feet or incidence of disease. D. nodosus was more likely to be detected in the gingival cavity when a sheep had FR. It was detected in 25 % of gingival cavity samples and strain types identified in the gingival cavity were the same as the dominant strain types on the feet of sheep. In study 2, disease prevalence and D. nodosus detection frequencies were lower than in study 1. When sheep from the study group were separated from the main flock in week 1 and moved onto pasture that had been unoccupied for 10 days, D. nodosus was transferred to the study group on healthy feet. One dominant strain of D. nodosus persisted throughout an episode of disease and this strain was present on the healthy feet of sheep until up to 5 weeks before the development of lesions in high bacterial loads. There was a reduction in lesion severity and reduced detection of D. nodosus in soil in a period of dry weather. Only 1 sample from the gingival cavity was positive for D. nodosus. Two faecal sample were positive for D. nodosus, indicating for the first time that faecal shedding is possible. In study 3, there were high loads of D. nodosus on healthy feet of a sheep that was classed as susceptible when there had been no cases of FR for at least 2 month. D. nodosus was still present in the flock during the long non-transmission period in the summer. We conclude that D. nodosus is more likely to persist on the feet of sheep, whereas long-term environmental reservoirs of D. nodosus are unlikely. Future research should focus on the feet of sheep and possibly faeces as possible sites of persistence of D. nodosus in the absence of disease.

The interaction between host genetics and epidemiological processes in ovine footrot was investigated using a combination of data analysis and simulation modelling. The study’s aims were to determine the potential for genetic selection to be used to reduce the prevalence of footrot in the UK and to assess different strategies for use of conventional epidemiological interventions. A stochastic simulation model was developed, incorporating host genetics for traits controlling footrot resistance, bacterial population dynamics, sheep population dynamics and epidemiological processes. Sensitivity analysis of the model showed survival time of Dichelobacter nodosus in the environment and infection rate were the key determinants of disease outcomes. Antibiotics were predicted to be the most effective conventional control method, reducing prevalence of footrot to 1-2% when administered promptly. Pasture rotation, selective culling and vaccination were all predicted to reduce prevalence but to a lower extent. Analysis of field data confirmed the likely role for some degree of host genetic control of footrot resistance, i.e. resistance appears to be lowly to moderately heritable. Using the simulation model it was then shown that genetic selection could be effective at reducing footrot prevalence. In combination with antibiotic treatment or pasture rotation elimination of footrot from an individual flock could be achieved. Genetic selection was predicted to be effective at reducing prevalence and improving resistance but the choice of selection criteria impacts the results seen. It is likely that progress would be slower in field situations because footrot traits would be diluted by simultaneous selection for other traits affecting profitability. Field studies are required to determine optimal combinations of interventions and genetic selection and to validate modelling outcomes. Combined data from longitudinal disease observations, genetic information and bacterial samples are necessary to address current knowledge gaps and to further advance understanding of host and disease processes in ovine footrot.

Ovine footrot is the major cause of lameness in sheep within the UK and an economic and welfare concern for sheep farmers globally. It is characterised by under-running of the hoof-horn and preceded by interdigital dermatitis (ID). Under-running footrot is attributed to the host immune response, which is provoked primarily by Dichelobacter nodosus in addition to other bacteria pathogens. However, the precise role of these other bacteria is yet unknown. Therefore, we hypothesise that bacterial invasion and colonisation of the ovine interdigital skin contributes to a strong host inflammatory response leading to the characteristic histopathology observed. In this context, this study examined host inflammatory response [inflammatory cell infiltration, pro-inflammatory cytokines (IL-1β)], histopathological lesions and virulent D. nodosus abundance in healthy, ID and footrot conditions in an attempt to gain further insights into the pathogenesis of this important disease. To investigate this hypothesis, two studies were designed: (i) to grade histological lesions in different clinical conditions and (ii) to determine bacterial localisation in post-slaughtered interdigital skin biopsies from the abattoir. Standardised histology lesion grading systems were developed and applied using histochemical techniques (haematoxylin and eosin (H&E), periodic acid Schiff PAS). Bacterial localisation was determined in serial horizontal sections across skin depths combining histology (transverse cryosections + H&E) and qPCR technique for the quantification of bacterial DNA. Furthermore, parallel data of IL-1β expression and virulent D. nodosus load obtained from a different study were compared to histology lesions. Key findings were as follows: (i) histological lesions (cell ballooning, parakeratosis, epidermal micro-abscesses and inflammatory cell infiltration) were similar in all clinical conditions, (ii) increased inflammatory cell infiltration score corresponded significantly with high levels of IL-1β expression (p < 0.05) in footrot, and virulent D. nodosus load (p < 0.001) across all clinical conditions, (iii) across different skin depths, eubacteria localisation was consistent, D. nodosus localisation was highly variable while F. necrophorum was localised in deeper sections of healthy feet. In addition, eubacteria load was significantly higher (p=0.0002) in the epidermis near the skin surface (≤200μm) of footrot disease samples when compared to healthy samples. Eubacteria components may play contributory roles in footrot pathogenesis based on their localisation in interdigital skin. In conclusion, contrary to previous notion that the severity of disease condition was dictated by progressive pathology, data in this study showed no appreciable difference in the levels of histological lesions and inflammatory response between healthy and diseased (ID, footrot) conditions. Histological lesions and the bacterial components of the skin including the virulent D. nodosus contribute to the local inflammatory response which probably drives the progression of footrot disease.

Ovine footrot is a contagious disease of sheep caused by Dichelobacter nodosus which results in production losses and compromises the welfare of animals. The severity of disease which develops is on a spectrum, with benign, intermediate and virulent forms of disease described. The pathogenicity of three aprV2-positive D. nodosus field isolates were tested in a pen trial. Foot lesions typically associated with benign or intermediate footrot developed in the animals inoculated with the arV2- positive isolates. The efficacy of a serogroup specific bi-valent vaccine against benign and intermediate footrot was tested in four sheep farms in NSW. The use of the serogroup-specific vaccine can be effective at controlling some intermediate strains of D. nodosus. The antibody concentrations for the serogroups included in the vaccine at each farm were generally above concentrations required for protective immunity. The utility of pooled sample testing was examined using 572-foot swabs samples collected from six sheep farms in Tasmania and one farm in NSW. The test sensitivity (Se) and specificity (Sp) of the three multiplex PCR assays differed between sample types, individual serogroups and each of the three multiplex PCR assays. The pooling of five DNA samples may improve the cost effectiveness of diagnostic testing. A questionnaire survey was developed and completed by 43 sheep farmers in NSW. Risk factors associated with footrot and other hoof diseases were identified and it was determined sheep farmers in NSW consider benign and intermediate footrot an important disease which negatively affects the health and welfare of affected animals. The association between the aprV2 gene and virulence has not been established in Australian strains of D. nodosus. A method was developed to examine gene expression levels and when incubated for 5 days, the level of gene expression of isolates classified as virulent was higher than isolates classified as benign.

The aim of this thesis is to quantify the benefits of selection for resistance to an important sheep disease in Britain. Specific aspects addressed are i) the choice of the specific disease based on economic costs and potential savings from selection, ii) genetic parameters for the disease, such as the heritability (h²) and the relation with production traits, iii) prediction of the response to selection on a trait that is measured in only two classes (healthy or diseased) and depending on environmental factors affecting exposure and prevalence, and iv) modelling of the combined effect of increased genetic resistance and reduced pathogen burden as a result of selection. It is concluded that footrot is a disease of economic importance, with additive genetic variability. Selection for resistance can be effective if based on simple binary scores, especially if animals are scored repeatedly. The response to such selection can be predicted with a newly developed theory for binary traits, which also covers situations when exposure to infection is variable. Selection for resistance is expected to result in animals that can better cope with the disease, in terms of reduced weight loss. A new epidemiological model predicts likely responses to selection, showing a considerable additional decrease in the prevalence of footrot compared to purely genetic predictions. It is concluded that selection for increased resistance to footrot can be expected to be successful in reducing costs of the disease to the British sheep industry.

Dichelobacter nodosus, an anaerobic bacterium, is the major transmissible agent of ovine footrot. The disease expresses as a virulent or benign lesion in the hoof. Virulence is related to the production of serine proteases, particularly a thermostable protease. Isolates of D. nodosus are characterised according to the type of protease produced (either heat-stable or heat-labile) and the electrophoretogram (zymogram) of the protease. This study reports on the use of the DNA-based typing techniques Pulsed-Field Gel Electrophoresis (PFGE) and Infrequent-Restriction-Site-PCR (IRS-PCR) to investigate the molecular epidemiology of D. nodosus, including a consideration of the relationship between genetic type, zymogram patterns and whole cell protein profiles. The aim of the project was to obtain a better understanding of D. nodosus strain diversity and dissemination in Australia and its relationship to virulence within the population. The overall intention was to use this information to assist in the long-term control of virulent footrot. Field isolates of D. nodosus from Western Australia (n = 735), New South Wales (n = 16), Victoria (n = 24) and South Australia (n = 21) were obtained and analysed. Both typing techniques that were used offered good differentiation between isolates for epidemiological purposes, and the results were in general agreement. PFGE provided slightly better discrimination between isolates, with 214 PFGE types (181 from Western Australia) compared to 94 IrsT types (77 from Western Australia). Within this diverse range of molecular types clonality was observed - with clones being defined as clusters of isolates having closely related PFGE types. The strains were categorised as genetically diverse, genetically similar or identified as the same strain. This diversity of genetic types was found overall, within flocks of sheep on a farm and within a single hoof where, on a number of occasions, multiple molecular types and zymogram types were found colonising a single hoof. One isolate that was experimentally inoculated into a flock of sheep produced six different genetic types when tested 12 months after the initial infection. This indicates that D. nodosus undergoes rapid genetic change, which means that follow-up epidemiological investigation of disease outbreaks and trace-backs need to be done as soon after infection as possible. The genetic differences appeared to be due to large insertions or deletions of DNA. Amongst sheep on some properties, isolates that had a different protease expression and virulence expression were found to have the same molecular type. Investigation of these isolates by SDS-PAGE showed that they also had the same whole cell protein profiles. Isolates from the same clonal groups also had the same protein profile, whereas genetically diverse isolates had different protein profiles. The lack of protein differences between isolates of the same molecular type, or within a clonal group, suggests that the differences in protease thermostability may be due to conformational changes in the protein, rather than to overall detectable genetic change and/or expression of different proteins. These results demonstrate that PFGE typing can be useful in predicting likely phenotypic expression of whole cell proteins. Further work is required to elucidate differences between virulent and benign strains of D. nodosus.

Footrot (FR) is a highly infectious and debilitating disease of sheep, which has a significant economic impact on the sheep farming industry, in the UK and worldwide and causes significant suffering of sheep. Despite some recent advances, FR remains a scientifically challenging disease to understand. To help improve our understanding of disease pathogenesis, two culture-independent techniques were developed to examine the microbial succession events between the causative agent, Dichelobacter nodosus and an accessory agent, Fusobacterium necrophorum, the latter also postulated to be involved in disease initiation. The two populations were monitored in relation to disease initiation and progression during a longitudinal study and disease presentation in tissue biopsies (in situ). Finally, the distribution of these two species of bacteria in the environment was examined to highlight possible sources of infection. The work in this thesis has demonstrated that FR is a disease where expression is related to D. nodosus load present in the ovine interdigital space. D. nodosus (rpoD) load increased from that on a healthy foot to one presenting with interdigital dermatitis (ID) and feet with a higher D. nodosus (rpoD) load were more likely to go on to develop FR one week later. FISH analysis of the D. nodosus population present within the epidermis also revealed similar findings; D. nodosus cell counts increased during stages of ID, but the organism was less frequently detected in biopsies from feet with FR. Suggesting that ID might be the most infectious stage of the disease process. A fact that needs to be highlighted to farmers to encourage treatment at this stage of disease. In contrast, F. necrophorum (rpoB) load did not correlate with ID presentation or prior to the development of FR, but increased the week of FR onset. FISH analysis also revealed that F. necrophorum cell counts were higher in feet with FR than those with ID. It is possible therefore that F. necrophorum may thrive in the altered environment of a foot presenting with FR, possibly contributing to disease persistence and severity. Finally, both pathogens were detected in a range of environmental samples from a farm with endemic FR, highlighting possible sources of infection and material, which once contaminated with D. nodosus and F. necrophorum may contribute to the spread of FR. This study has provided an improved understanding of the microbial population dynamics involved in the development of ID and FR in sheep, which may have implications for control and treatment practices not only in the UK, but world-wide.

Studies presented in this thesis looked at developing new methods for the diagnosis of virulent footrot (VFR) in sheep and identification of serogroups of Dichelobacter nodosus, the principal causative agent of footrot. Earlier studies had shown that immunological memory response in sheep recovered from VFR can be aroused by natural or recurrent infection or by injection of outer membrane protein (OMP) antigens to be used as a retrospective diagnostic test for VFR. But OMP antigen was found to be non-specific in older animals. To overcome this non-specificity of OMP antigen in anamnestic response, pilus antigen was evaluated in a trial at Camden. The results of this trial indicated that antibodies to pilus antigen can be detected over time in a manner similar to OMP antibodies so a retrospective assessment of VFR status can be made by anamnestic test with pilus antigens. The anamnestic response to pilus was similar in character to OMP antigen but unlike OMP was highly specific. The response to anamnestic challenge with pilus was determined by severity of the lesions they had expressed, with severe lesions triggering the greater responses. However, there was variation between individuals, with some (6 of 46 with severe lesions) failing to respond. This individual variation is probably mediated genetically as is response to vaccination. This anamnestic test was tested in flocks of sheep in Nepal that had a history of VFR which had apparently been eradicated. That assessment, based on clinical findings, was confirmed by the uniformly negative results in the pilus anamnestic test applied to a representative sample of the population. This allowed a conclusion that the virulent strains of D. nodosus involved had been eliminated from these flocks. As mentioned in the preceding study pilus antigen was found to be very specific and ideal for retrospective diagnosis of virulent footrot with an anamnestic challenge ELISA test. However, serogroup specificity was seen as a disadvantage of using pilus antigen for the anamnestic test. The possibility of using multivalent pilus antigens was tested in another trial. These animals had been involved in a clinical expression experiment conducted by another research group and had a clinical and bacteriological history extending over more than 12 months. After these initial trials all these animals were treated for footrot and managed for 5 months as a single flock at Camden. These were then challenged with multivalent pilus antigen (serogroup A - I) as a single injection. The results obtained indicate that multivalent pilus anamnestic ELISA is equally effective as monovalent pilus. This has the added advantage that prior knowledge of the serogroups present in the flock is not required. It has the possibility of being used as an indirect test to check the presence of serogroups in a flock without doing the bacterial cultures. This test can identify most animals with pre-existing underrunning lesions (Scores of 3 or higher). However, the sensitivity and specificity of this test need to be tested extensively in flocks of known clinical history before it could be adopted as a routine test. As a key component of a larger study to determine the role of fimbrial genes (fimA and fimB) of D. nodosus in the pathogenesis of footrot using allelic exchange to disrupt these genes of a strain (serogroup G), the study presented in this thesis contributed a detailed characterisation of the resultant mutant and the wild strains and tested these strains for virulence in sheep. The results presented provided the first definitive evidence that the fimA gene is essential for virulence of D.nodosus in sheep. In vivo virulence testing of two fimA mutants showed that they were not able to establish any footrot whereas the wild type of the same strain produced virulent footrot in the same trial conducted under similar conditions. These mutant bacteria were not re-isolated from interdigital skin after in vivo challenge. This indicated that fimA mutant strains could not colonise the ovine foot, and the simplest and most likely explanation for these results was that colonisation of the interdigital skin and subsequent penetration of the stratum corneum requires the adhesive activity of type IV fimbriae. However, since these mutants also had altered ability to secrete extracellular proteases, and perhaps other as yet unknown extracellular proteins, the possibility of the involvement of these factors as major determinants of host colonisation or invasion cannot be excluded. Current methods for the identification of the serogroup of D. nodosus present in the bacterial population requires isolation of the organism and after purification by subculture, antigenic analysis with agglutination tests. This usually takes at least 3 to 4 weeks. With the objective of developing a rapid serogroup specific PCR assay, the basis of serogroup variation in D. nodosus localised in the fimbrial gene region was exploited. A common forward primer and 9 serogroup specific reverse primers were selected from the fimbrial gene sequences of the prototype strains. Analytical sensitivity of the serogroup specific primers on chromosomal DNA was similar to PCR tests in other bacterial species reported before. Immuno-magnetic bead capture PCR method was able to detect 5 to 10 cells in cell lysates. Specificity within and between the serogroups of D. nodosus was tested with all the prototype strains. They were found to be very specific to each serogroup and specific only to D. nodosus when tested with 84 commonly found bacterial strains or strains related to D. nodosus. To overcome the time delay in conducting 9 different amplifications to find out the prevalence of all possible serogroups in a flock multiplex PCR reactions with common forward primer and groups of 3, 4 and 5 reverse primers were successful in reducing the number of reactions to 2 (with groups of 4 and 5) or 3 (with groups of 3) primers. A drawback of the multiplex reaction was that if a template was 1000 times less concentrated that the others in the mixture it was not amplified but the margin for difference is very high. The main aim of developing rapid serogroup specific PCR was to apply these tests directly on footrot lesion samples to make it a rapid diagnostic test for field samples. The sensitivity of the test on lesion samples was found to be very low. To try and improve the sensitivity an overnight or four days old pre-enrichment culture in broth was tested but was found to be no better than direct PCR. The immuno-magnetic capture method which improved the sensitivity of pure culture samples by 10 -100 fold also had very low sensitivity with lesion samples. However, this drawback can be overcome by picking up colonies from 4 days old lesion cultures on hoof agar (HA) plates for serogroup specific multiplex PCR. If the colonies are too small/ too few on the lesion cultures these can be sub cultured onto a quarter of 4 percent HA plates and then used for the PCR test which also reduces the time taken for serogrouping at least by 2 weeks. The other advantage is that individual colonies do not need to be isolated. A PCR test can be done on pooled colonies just as well and can be used to identify all serogroups present in the sample. Serogroup specific PCR is much faster and is more sensitive and accurate than slide agglutination tests which take 3 to 4 weeks to complete. Multiplex PCR makes it easier to detect different serogroups in a sample with a maximum of 3 PCR tests. Serogroup specific multiplex PCR will be a useful tool for footrot control based on specific vaccination. The difficulty in using the test on direct lesion swabs needs to be further looked into. There may be future advances in the application of PCR tests to clinical samples.

The bacterium, Bagteroides nogosu , is the principal causative organism of footrot in sheep. a disease which causes major economic losses in Australia. Current footrot vaccines contain strains from each of the eight serogroups present in Australia. However, they are expensive and difficult to produce because B.nodosus an anaerobe has complex growth requirements and expression of the pilus, a major protective antigen is variable in liquid culture. The pilus (plural, pili) is composed essentially of one subunit protein called pilin and is the antigen which forms the basis for serotyping the organism. Purified pilus vaccines protect sheep against footrot challenge. It is therefore important to clone and characterise the genes reponsible for piliation in §.godosus. so that its regulation may be understood. With this knowledge, it should be possible to produce a cheap, reliable footrot vaccine based on genetically engineered strains that express high levels of pilus antigen. This work describes the cloning of the pilin gene from §.nodosu§ strain 215, which belongs to serogroup B. During the course of this project, three distinct b. nodosus loci have been cloned and expressed in Escherichia coli, an easily grown and well characterised bacterium. The first locus is represented by two clones pDWl and pDW3. carrying DNA inserts of 7.2 and 4.5 kilobases (kb). respectively. Analysis in maxicells shows that they both encode a protein which co—migrates with b. nodosus pilin and reacts weakly with pilus antiserum in Western blotting. It appears not to be the same pilin locus as that isolated from b. nodosus strain 198 (serogroup A: Anderson et al., 1984; Elleman et al., 1984). because the DNA shows no restriction homology with the strain 198 pilin clones. It is suggested that pDWl and pDW3 may encode an alternative pilin locus. The second locus is represented by pDW4, a clone carrying an insert of 10.2 kb. Western blotting shows that pDW4 encodes three proteins that are present in the pilus preparation used to raise antisera in rabbits. It does not encode pilin. These first two loci may encode proteins associated with pilus production, as the clones were detected using antibodies that had been raised against purified pili. The third locus is represented by pDWS and pDW6, carrying inserts of 10.0 and 5.6 kb repectively. These clones show strong expression of pilin in Western blotting experiments, and the DNA restriction maps exhibit homology with DNA from strain 198 pilin clones. Suggestions for further work, to determine the genes and conditions necessary for full pilus expression. are presented.

Ovine footrot is an infectious cause of lameness in sheep that has significant economic impact for the UK sheep farming industry. It is also a major concern for animal health and welfare. The causal agent is Dichelobacter nodosus, and Fusobacterium necrophorum is an opportunistic secondary pathogen that increases disease severity. The primary reservoirs for F. necrophorum in sheep were believed to be sheep faeces and the environment, however, no studies had demonstrated the presence of F. necrophorum at either of these sites. Two longitudinal studies (Study A and Study B) were conducted to determine reservoir sites of F. necrophorum in ovine footrot. Study A included 10 sheep sampled on four occasions at two week intervals. Study B included 40 sheep sampled weekly for 20 weeks. Samples collected from sheep and their environment were foot swabs, mouth swabs, faeces, soil and grass. Quantitative PCR was used to detect and quantify F. necrophorum. A multiple locus variable number tandem repeat analysis (MLVA) community typing scheme for F. necrophorum was developed and validated, and used to analyse samples from Study A and Study B. Contrary to prior assumption, the environment was not a significant reservoir of F. necrophorum. F. necrophorum persisted in sheep, primarily on feet with footrot. MLVA indicated that the strains of F. necrophorum found on the feet of sheep were closely related, and they may therefore share characteristics that make them well adapted to feet and footrot. Mouths and faeces were an intermittent reservoir for the strains of F. necrophorum involved in footrot. Mouths and faeces may therefore facilitate persistence of F. necrophorum in the absence of footrot, or facilitate transmission of F. necrophorum between flocks. Mouths were a persistent reservoir for strains of F. necrophorum not involved in footrot.

In this research, PCR-based detection assays were developed to detect and quantify three N. haematococca pathogenicity genes (PDA, PEP3 and PEP5) both from isolates and DNA extracted from agricultural fields with footrot history. Denaturing gradient gel electrophoresis (DGGE), gene cloning and sequencing assays were used to explore the diversity of these genes in agricultural soils. The applicability of using quantitative (real-time) PCR to quantify these genes in infected soils was validated. Furthermore, biotic and abiotic factors that interact to cause pea footrot disease in soil were also studied. Results showed that the PDA^H allele of the PDA gene, responsible for rapid demethylation of the phytoalexin pisatin, together with PEP3 and PEP5 genes promotes maximal footrot disease in peas. DGGE results showed diversity amongst PDA and PEP5 partial gene sequences amplified from agricultural soil-DNA. Partial PEP3 gene sequences showed no diversity. There was a positive correlation between numbers of pathogenic N. haematococca spores and numbers of pea pathogenicity genes in soil. Pea pathogenicity gene numbers of up to 100 per gram of soil constituted a threshold number for pea footrot infection; potentially capable of causing footrot disease of economic proportion. A disease model that included total phosphate, carbon and ammonium-nitrogen was identified with stepwise regression analysis (R² = 0.42). The PCR-based techniques developed herein are viable and reliable alternatives to culture-based assays for accurate detection and quantification of pathogenic N. haematococca in soils. They offer the opportunity for quantitative prediction of pea footrot infections in agricultural soils prior to cultivation.

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Doenças podais são uma das principais injúrias em rebanhos de pequenos ruminantes em diversos países e a pododermatite infecciosa (Footrot) é relatada como a mais frequente em ovinos no Brasil. No Rio Grande do Sul, as doenças podais ainda são um grave problema para os criadores de ovinos e pouco tem sido feito para saná-las. O Footrot, mesmo se tratando de uma doença de notificação obrigatória e frequente na região, os registros oficiais sobre a situação da doença nos rebanhos são escassos. Esse trabalho teve como objetivo descrever as principais características das lesões podais observadas em ovinos da Mesorregião Sudoeste do Rio Grande do Sul, com ênfase nos aspectos epidemiológicos, macroscópicos, microscópicos e radiográficos das lesões de Footrot. O estudo foi realizado em duas etapas. Inicialmente foram avaliados ovinos em 27 propriedades rurais, das quais 21 registraram a ocorrência de lesões podais em ovinos com perdas econômicas significativas. Aproximadamente 1.700 ovinos, em média 10% dos animais do rebanho, apresentavam diferentes graus de claudicação decorrente de lesões podais que, macroscopicamente, variavam de brandas a severas. Posteriormente, foram avaliados os variados graus de lesões de Footrot nos ovinos. Em casos de abate e necropsia, os cascos dos ovinos com as lesões foram submetidos à avaliação macroscópica, radiográfica e microscópica. Dessa forma a doença foi classificada em cinco graus de severidade que variaram de 1 (lesões leves) a 5 (lesões graves). Verificou-se que diversos fatores como clima e manejo foram favoráveis para o desenvolvimento das lesões podais e essas estão associadas, na maioria dos casos, a Footrot em diferentes estágios de evolução. A partir dessa classificação em graus foi possível classificá-los macroscopicamente em duas síndromes clínicas propostas, a saber Footrot benigno e maligno. Essa classificação facilita o estabelecimento das medidas de controle com intuito de limitar a propagação da doença e evitar a evolução das lesões nos cascos acometidos.
Foot diseases are one of the main disorders in small ruminant flocks in several countries and infectious pododermatite (Footrot) is reported as the most frequent podal lesions in sheep in Brazil. In Rio Grande do Sul state, foot diseases still a serious problem for sheep farmers and little has been done to remedy them. Footrot is a notifiable disease and frequent in the region, the official records on the disease situation in herds are scarce. This study aimed to describe the main features of foot lesions observed in sheep from Mesoregion Southwest of Rio Grande do Sul, with emphasis on epidemiology, macroscopic, microscopic and radiographic changes of Footrot injuries. The study was conducted in two steps. Initially, sheep were evaluated on 27 farms, of which 21 showed records of the occurrence of foot lesions in sheep and significant economic losses. Approximately 1,700 sheep, about 10% of the flocks, showed varying degrees of lameness due to foot lesions, macroscopically characterized as mild to severe. Subsequently, they assessed the varying degrees of injuries Footrot in sheep. Hooves with injuries were submitted to macroscopic, radiographic and microscopic evaluation. Thus the disease was classified into five grades of severity ranging from 1 (mild injury) to 5 (severe damage). It has been found that several factors such as weather and handling were favorable for the development of foot injuries and these are associated in most Footrot cases in different stages of evolution. Based on the classification in degrees of infectious pododermatitis it was possible to classify them macroscopically in clinical syndromes proposed as benign and malignant Footrot. This classification facilitates the establishment of control measures with the intention of reduce spread of disease and prevent the development of lesions in affected hooves.

Dissertação de Mestrado em Engenharia Zootécnica / Produção Animal
Neste estudo foram utilizados registos produtivos de 6808 lactações de 2596 ovelhas da raça Assaf, mantidas num sistema com dieta à base de pastagem. Avaliaram-se vários caracteres produtivos e reprodutivos (produção de leite total, produção média diária, duração da lactação, prolificidade e intervalo entre partos) e a ocorrência de peeira, em função de vários fatores. Por exemplo para a produção do leite considerouse o efeito da prolificidade, do ano do parto, da estação do parto, do número de ordem de lactação e da ocorrência de peeira. As médias registadas foram de 1,11 para a prolificidade, 299 L para a produção de leite total, 1,50 L para a produção média diária, 195,40 dias para a duração da lactação, 17% para a ocorrência de peeira e 337 dias para o intervalo entre partos. Nem todos os fatores analisados apresentaram efeitos significativos sobre os carateres produtivos e reprodutivos. A peeira e a prolificidade foram as únicas que não apresentaram efeito significativo em todos os caracteres produtivos. Foram estimadas curvas de lactação médias com a forma linearizado do modelo de Wood utilizando regressões aleatórias, em função dos seguintes fatores: prolificidade, número de ordem de lactação, estação do parto e ocorrência de peeira. Verificaram-se diferenças importantes em relação à produção inicial, dia de pico, pico de produção e persistência de lactação entre as curvas dos diferentes fatores.
ABSTRACT - Milk production and Lactation curves of Assaf sheep - In this study we used information on 6808 lactations by 2596 Assaf ewes raised on a diet based on grass. Different production and reproductive traits (total milk yield, daily milk yield, lactation length, prolificacy and lambing interval) and the occurrency of footrot, were analyzed as a function of several factors. For example, for milk yield the factors considered in the analyses were prolificacy, year of lambing, season of lambing, lactation number and footrot occurrency. The obtained mean average were 1,11 for prolificacy, 299 L for total milk yield, 1,5 L for daily milk yield, 195 days for duration of lactation, 17% for foorot occurrency and 337 days for lambing interval. Not all factors analyzed showed a significant effect on productive and reproductive traits. Footrot occurrence and prolificacy did not present significant effects on all the productive traits assessed. Mean lactation curves were estimated with the linear form of Wood model using random regressions, considering prolificacy, lactation number, lambing season and footrot occurrence. Important differences were identified in initial milk yield, day of peak yield, peak yield and lactation persistency, among curves estimated for the different factors.
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O objetivo desta dissertação de mestrado foi avaliar a eficácia de uma vacina autógena no controle do footrot (FR) dos ovinos, para tanto foram desenvolvidos dois experimentos em duas propriedades rurais distintas, no Estado do Rio Grande do Sul (RS). O primeiro experimento foi conduzido em uma propriedade rural do município de Santiago, com um extenso histórico de surtos de footrot no rebanho ovino. Inicialmente foi colhido material infeccioso presente no rebanho para a produção de uma vacina autógena, posteriormente 347 ovelhas foram vacinadas (grupo V) com duas doses da vacina com 30 dias de intervalo, a dose foi 2 ml por via subcutânea. Desse grupo, 150 animais receberam a vacina na região axilar (grupo Va) e 197 animais receberam a vacina na região inguinal (grupo Vb). Um grupo de 75 ovelhas formou o grupo controle (grupo C) sem vacinação. Os dados mostraram que a prevalência do FR no grupo V que inicialmente era de 4%, sofreu uma redução para 2% na semana 23, chegando à zero na semana 30. No grupo C a prevalência de animais infectados foi de 6,7% no início do experimento, teve uma redução para 5,3% na semana 23 e ao final estava em 3,7%. Observou-se uma redução gradativa no número de ovinos infectados nos dois grupos, entretanto a eliminação seletiva ocorrida no grupo controle prejudicou a análise estatística dos dados. Amostras de sangue foram colhidas da jugular dos animais para verificar títulos de anticorpos aglutinantes contra o antígeno presente na propriedade em cinco ocasiões durante o experimento. Os resultados mostraram diferenças significativas (p<0,001) entre os títulos de anticorpos aglutinantes contra Dichelobacter nodosus no soro de ovinos vacinados e não vacinados durante o experimento. A análise das reações vacinais locais apontou a região inguinal como o melhor local para a aplicação da vacina com adjuvante oleoso por via subcutânea e também demonstrou uma relação direta entre a idade dos ovinos e o percentual de reações vacinais locais e a severidade dessas reações. Os resultados sugerem que a vacina autógena com adjuvante oleoso obteve sucesso no controle da doença. O segundo experimento foi conduzido em uma propriedade rural do município de Glorinha, com o objetivo de avaliar a resposta imunológica provocada por uma vacina monovalente e por uma vacina polivalente (7 sorogrupos) contra o FR. Trinta fêmeas ovinas, com idades variadas, foram divididas aleatoriamente em 3 grupos de 10 animais: grupo controle (C) que não foi vacinado, grupo vacinado com vacina monovalente (Vm) e grupo vacinado com vacina polivalente (Vp). Os ovinos vacinados receberam duas doses com quatro semanas de intervalo, a dose foi de 2 ml por via subcutânea na região inguinal. Amostras de sangue foram colhidas da jugular dos animais para verificar títulos de anticorpos aglutinantes contra o D. nodosus em quatro ocasiões durante o experimento. Os resultados mostraram diferenças significativas (p<0,001) entre os títulos médios geométricos (GMT) de anticorpos aglutinantes contra D. nodosus no soro de ovinos dos grupos Vm, Vp e C na quarta, sétima e 12ª semanas do experimento. Em relação aos títulos médios geométricos (GMT) entre os grupos Vm e Vp houve diferenças estatisticamente significativas na quarta e sétima semanas. A vacina monovalente induziu títulos de aglutininas superiores contra o D.nodosus em comparação com a vacina polivalente.
The objective of this work was to evaluate the efficacy of an autogenous vaccine in the control of footrot (FR) in sheep. Two field experimental works were carried on two different farms located in the State of Rio Grande do Sul (RS), Brazil. The first experiment was conducted on a farm in the municipality of Santiago, with a long history of FR outbreaks in their sheep flock. At beginning of the trail samples from FR infected sheep were collected for the production of an autogenous vaccine. Following 347 sheep were vaccinated (group V) with two doses of 2 ml subcutaneously vaccine 30 days apart. Of this group, 150 animals received the vaccine in the axillary region (group Va) and 197 animals received the vaccine in the inguinal region (group Vb). A group of 75 sheep formed the control group (group C) without vaccination. The data showed that the prevalence of FR in the group V initially 4%, was reduced to 2% at 23 weeks, reaching to zero at week 30. In the group C the prevalence of infected animals of 6.7% at the beginning of the experiment, was reduced to 5.3% at week 23, decreasing to 3.7%, at the end. There was a gradual reduction in the number of infected sheep in both groups, however the selective elimination occurred in the group C affected the statistical analysis. Blood samples were collected from the jugular vein of the animals to see evidence of agglutinating antibodies against the antigen present on the property on five occasions during the experiment. The results showed significant differences (p<0,001) between antibody titers against Dichelobacter nodosus in the serum of sheep vaccinated and not vaccinated during the experiment. The analysis of local vaccine reactions showed the inguinal region as the best place for the application subcutaneously oil-adjuvant vaccine and also demonstrated a direct relationship between the age of the sheep and the percentage of local vaccine reactions and the severity of these reactions. The results suggest that autogenous oil-adjuvant vaccine succeeded in controlling the disease. The second experiment was conducted on a farm in the municipality of Glorinha, in order to evaluate the immune response elicited by a monovalent and a polyvalent vaccine against FR, containing seven serogroups. Thirty ewes, of various ages were randomly divided into 3 groups of 10 animals each: control group (C) was not vaccinated, group vaccinated with monovalent vaccine (Vm) and the group vaccinated with polyvalent vaccine (Vp). The sheep were vaccinated with two doses of 2 ml subcutaneously in the inguinal region, four weeks apart. Blood samples were collected from the jugular vein of the animals to determine agglutination titers against D. nodosus in the beginning of the experiment (day zero) and in other three occasions, weeks 4, 7 and 12. The results showed significant differences (p<0,001) between the geometric mean titers (GMT) of antibodies against D. nodosus in the serum of sheep of groups Vm, Vp and group C in the fourth, seventh and 12th weeks of the experiment. For the geometric mean titers (GMT) between the groups Vm and Vp there was statistically significant differences in the fourth and the seventh weeks. The monovalent vaccine induced titers of higher against D. nodosus compared with the polyvalent vaccine.

Footrot is a contagious hoof disease of ruminants. It is endemic in New Zealand and throughout sheep and goat farming regions of the world. The disease results from a mixed bacterial infection, but the essential agent is Dichelobacter nodosus, a Gram-negative, anaerobic bacterium that possesses type-IV fimbriae on its surface. Genetic variation in the fimbriae of D. nodosus was investigated in this study. Using the polymerase chain reaction (PCR), the variable region of the gene encoding the fimbrial subunit (fimA) was amplified from bacterial DNA extracted from footrot lesions. Different fimA amplimers were differentiated by single-strand conformation polymorphism (SSCP) analysis. In conjunction with DNA sequencing, 15 unique sequences of D. nodosus fimA were obtained from 14 footrot samples taken from 6 farming regions throughout New Zealand. When these sequences were compared to fimA of known serogroups, it revealed that there were at least 15 D. nodosus strains, representing 8 serogroups, present on New Zealand farms. The predominant serogroup was B which contained 6 strains, followed by serogroups F, H and G. No strains from serogroups D and I were detected in this investigation. Twelve out of the 15 New Zealand D. nodosus strains had fimbriae different to those previously reported and the presence of multiple strains on a single hoof was common (86% of samples). The fimA sequences from the 12 D. nodosus strains incorporated into the footrot vaccine currently available in New Zealand were determined. A primer set targeting the relatively conserved fimA regions and based on the published sequence of serogroup M Nepalese isolates (designated M-Nep), failed to amplify fimA from the vaccine serotype M strain (designated as M-SPAHL). When the downstream primer was substituted with a primer that was specific for other serogroups of D. nodosus, the fimA gene was successfully amplified. Cloning followed by DNA sequencing, revealed that M-SPAHL fimA was different to M-Nep fimA. The predicted amino acid sequence of M-SPAHL fimA did not show homology to any known serogroups or serotypes. The most similar sequence was from serotype F1, and not M-Nep. The sequence difference between M-SPAHL and M-Nep was larger than that expected within a serogroup. The consequences of serological relatedness and sequence dissimilarity are discussed. Only eight of the 15 New Zealand field strains had fimbriae identical to those of the vaccine strains, while the remaining seven strains possessed different fimbriae. In addition, the vaccine contained two more D. nodosus strains, representing two sera groups, that were not found on the New Zealand farms investigated in this study. This may, to some extent, explain why the current footrot vaccine is at times less efficient in New Zealand. Another 17 footrot samples were screened for new or additional D. nodosus strains. Two PCR amplimers (designated X and Y) derived from footrot samples generated SSCP patterns different to those of previously identified strains. DNA sequencing revealed that these two fragments possessed novel sequences. The upstream of X (nt 1-183) was identical to serotype M1 while its downstream (nt 223-414) was identical to serotype F1; the upstream of Y (nt 1-116) was identical to serotype E1 whereas its downstream (nt 148-423) was identical to serotype F1. A 14-mer sequence consisting of two partially overlapping Chi-like sequences, 5'-GCTGGTGCTGGTGA-3', was also found in these fragments. Two primer sets with the downstream primer specific for serotype Fl and the upstream primer specific for serotype M1 or E1, produced PCR products of the expected sizes from the footrot samples from which fragments X and Y were isolated, respectively. These primer sets did not appear to amplify artificially mixed genomic DNA from serotypes M1 and F1 or E1 and F1. However, when the reactions were re-amplified, PCR recombination artefacts were observed, suggesting that PCR recombination does occur, but at a low frequency. It therefore seems more likely that fragments X and Y reflect genuine fimA sequences of D. nodosus which have resulted from in vivo DNA recombination, than from a PCR recombination artifact. The genetic capability for recombination at the fimbrial subunit locus may therefore endow D. nodosus with the ability to alter its antigenic appearance. D. nodosus strains present in footrot lesions can be genotyped using a PCR-SSCP/sequencing technique. However, this typing technique requires cloning and screening of D. nodosus fimA sequences, which is both laborious and costly. A rapid molecular typing system for D. nodosus was therefore developed in this study. A close examination of available D. nodosus fimA sequences revealed regions that appear to be specific for serogroups and serotypes. These regions were used to design a panel of sequence-specific oligonucleotide probes (SSOPs), and a rapid and accurate D. nodosus typing system using PCR and reverse dot-blot hybridisation (PCR/oligotyping) was subsequently developed. The variable region of D. nodosus fimA, amplified and labelled with digoxigenin (DIG) in a single multiplex PCR amplification, was hybridised to a panel of group- and type-specific, poly-dT tailed oligonucleotides that were immobilised on a nylon membrane strip. A mixture of positive control poly-dT tailed oligonucleotides was also included on the membrane. After hybridisation the membrane was washed to a defined specificity, and DIG-labelled fragments that had hybridised were detected. The specificity of the oligonucleotides was verified by the lack of cross-reactivity with D. nodosus fimA sequences that had a single base difference. DNA from 14 footrot samples previously genotyped by PCR-SSCP/sequencing, was assayed using the PCR/oligotyping technique. All types of D. nodosus which had been detected previously with a PCR-SSCP/sequencing method were detected by this procedure. However, for three of the 14 footrot samples, PCR/oligotyping detected additional types of D. nodosus. Further PCR amplification using type-specific primers, confirmed that these types were present in the original footrot samples. These results indicate that PCR/oligotyping is a specific, accurate, and useful tool for typing footrot samples. In combination with a rapid DNA extraction protocol, D. nodosus present in a footrot sample can be accurately genotyped in less than two days. Individual animals from the same farm, or the same paddock, were often infected by different strains of D. nodosus. This suggests a host role in mediating footrot infection, or that the interaction between the pathogen and the host is important. In order to better understand the interaction between the bacterium and the host, two polymorphic ovine class II MHC genes DQA1 and DQA2, which have been previously shown to be important in footrot infection, were also investigated in this study. PCR-SSCP/sequencing analysis of the DQA1 locus revealed ten unique ovine DQA1 sequences, with five of them being newly identified. This increases the number of known ovine DQA1 alleles from 8 to 13 (including a null allele), implying a high level of polymorphism at the ovine DQA1 locus. D. nodosus present on 20 footrot infected sheep from the same flock were genotyped, together with the ovine DQA1 and DQA2 genotypes of their hosts. Preliminary results showed that sheep with the same DQA1 and DQA2 genotypes tended to be infected by similar types of D. nodosus. Different types of D. nodosus were generally found on sheep with different genotypes at either the DQA1 or the DQA2 locus. This suggests the diversity in D. nodosus infection may be associated with the heterogeneity in the host MHC. However, as only a small number of animals from the same sire were analysed, further investigation is needed to gain a better understanding of the interaction between D. nodosus and the host MHC.

The judges of the Supreme Court of Louisiana issued a court order, in the year 1840, mandating a required reading course of legal materials for prospective bar applicants. Included in the list of materials was Sir William Blackstone's Commentaries on the Laws of England. Louisiana's legal system has, from its inception, been based on the civil law, a system tracing its roots back to Rome. The other organized system of law is the common law, which originated in the customs and judicial decisions of England, and is the legal system followed in the other forty-nine states. A question arises as to the reason the Commentaries was included in the "Course of Studies," since there exist fundamental differences between the two systems. This thesis explores the possible motive by examining the decisions of the Territorial Superior Court, and the Supreme Court of Louisiana between the years 1809 and 1875.

The diploma thesis is focused on draft of footrests for endure Honda Transalp XL700V of 2008. Attention is paid to the draft of the design of the footrest which is, which is designed for driving in light off-road. There is also described the issue if the choice of the material from aluminium alloy and the verification of the methods of the designed geometry using FEM and Rapid Prototyping. The experimental part deals with the design of appropriate tools and cutting conditions and with the creation of the NC program in ShopMill. There is enclosed the technical documentation and the NC program in the final part.

The Anglo-American interpretation of the Second World War has continuously overlooked the significance of the Scandinavian region to the outcome of the war. This thesis seeks to address some of the more glaring errors of omission that have dampened the Anglo-American understanding of the war. Attention will first be paid to Finland and how its war against the Soviet Union in 1939-1940, known as the Winter War, influenced Adolf Hitler and his decision to launch Operation ‘Barbarossa.’ In regards to Sweden, attention will be paid to how critical Swedish iron ore was to the Nazi war economy. Finally, the thesis will examine how the Anglo- American interpretation of the German invasion of Norway is flawed. The thesis seeks to change the way that the role Scandinavia played during the Second World War is understood amongst Anglo-American historians and begin a new conversation on the story of World War II.

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2020
A Peeira é uma doença que afecta ovinos e outros ruminantes, sendo atualmente uma das principais causas de claudicação em ovelhas. Tem como principal agente patogénico a bactéria gram-negativa anaeróbica Dichelobacter nodosus. Esta doença tem grandes impactos económicos e relevância em termos de bem-estar animal, tornando-se por isso essencial o seu controlo, tratamento e erradicação. Existem essencialmente duas manifestações clínicas desta doença. A manifestação ligeira consiste na inflamação da epiderme interdigital, sendo por essa razão denominada Dermatite Interdigital (ID – Interdigital Dermatitis). Por outro lado, na manifestação mais grave, esta inflamação pode progredir e causar a separação do casco e do tecido subjacente, sendo por essa razão chamada de Peeira Grave (SFR – Severe footrot). A severidade desta doença está principalmente dependente de três fatores: a virulência da estirpe infectante de D. nodosus, as condições ambientais e a resistência natural do hospedeiro. Alguns dos fatores ambientais que contribuem para a progressão e transmissão da doença são a temperatura, a humidade e o tipo de solo. Foi também demonstrado que a coinfecção com bactérias de outros géneros desempenha um papel deveras importante no desenvolvimento e progressão da doença. Algumas das bactérias oportunistas que foram identificadas na pele interdigital de ovinos com Peeira são Fusobacterium necrophorum e Treponema spp.. O papel exercido por estas bactérias oportunistas na progressão da doença ainda não é totalmente compreendido. Verificou-se, no entanto, que F. necrophorum, também uma bactéria gram-negativa anaeróbica, contribui para o desenvolvimento e progressão da peeira e que exerce uma relação sinergística com D. nodosus, tornando esta bactéria oportunista um foco central no estudo da doença. Em relação à virulência de D. nodosus, os dois factores principais reconhecidos são as fímbrias do tipo IV e as proteases extracelulares. A análise das fímbrias permite a discriminação de dez serogrupos (A-I e M) com base na sua diversidade estrutural que é causada por variações na região carboxi-terminal da subunidade fimbrial, também chamada proteína FimA e codificada pelo gene fimA. A protease acídica 2 (AprB2/V2), em particular, é um importante fator de virulência responsável pela atividade de elastase presente nas estirpes virulentas que, por sua vez, é essencial para o desenvolvimento das lesões características da Peeira. Os genes aprV2 e aprB2 diferem apenas numa substituição de dois pares de base (TA/CG) na posição 661/662, o que resulta na alteração de um aminoácido (Tyr92Arg) na proteína madura, sendo que esta alteração determina a atividade de elastase. O principal objetivo do presente estudo foi determinar a prevalência de D. nodosus e F. necrophorum em ovelhas da região do Alentejo com diferentes manifestações de Peeira e isolar e caracterizar D. nodosus quanto à sua virulência e serogrupo. Foram utilizadas amostras de tecido da epiderme interdigital provenientes de ovelhas saudáveis (grau 0) e com Peeira (graus 1-5) de 17 explorações de 13 concelhos da região do Alentejo. Amostras de DNA das biópsias recolhidas (n=261) foram utilizadas para a deteção e identificação de D. nodosus e F. necrophorum e para determinar a virulência e os serogrupos de D. nodosus. Estes testes foram realizados através de PCR em tempo real. Os fatores de virulência foram ainda caraterizados com recurso a sequenciação de Sanger. A determinação dos serogrupos foi realizada por PCR em formato multiplex. O isolamento de D. nodosus a partir de amostras de biópsia foi realizado por cultura bacteriológica em meios seletivos. Os resultados deste estudo mostraram que, na região do Alentejo, existe uma prevalência de D. nodosus (51%, n= 132) e F. necrophorum (46,4%, n=121) elevada em casos clínicos de Peeira. Verificou-se também uma associação entre a prevalência destes dois agentes e a severidade da lesão, ou seja, com a progressão da doença para fases mais avançadas. D. nodosus não foi detetado em quatro das 17 explorações em estudo enquanto F. necrophorum apenas estava ausente numa dessas explorações. Observou-se ainda uma tendência para explorações com um maior número de animais infetados por D. nodosus também apresentarem uma elevada prevalência de F. necrophorum. A coinfecção por ambas as espécies é a situação epidemiológica mais comum, estando associada a uma maior severidade da doença, quando comparada com as situações em que se registou infeção apenas com D. nodosus. Em relação à prevalência geográfica de D. nodosus, este foi detetado em 11 dos 13 concelhos, não se tendo detetado no Alandroal e Moura. As prevalências mais elevadas foram registadas em Almodôvar (83%), Portel (72,4%) e Évora (68%). A análise dos fatores de virulência nas amostras de DNA de biópsia onde D. nodosus foi detetado (n=132) revelou que a maioria dos isolados (n=127) era virulenta, com exceção de cinco cujo resultado foi indeterminado. Com base em critérios espaciais e qualitativos, das 132 amostras de DNA de biópsia positivas para a presença de D. nodosus foram selecionadas 53 para a determinação dos serogrupos, tendo-se confirmado o serogrupo em apenas 19 dos casos, distribuídos nas seguintes proporções: serogrupo B (90%), serogrupo C (5%) e serogrupo F (5%). Quanto à cultura bacteriológica, das 132 amostras processadas onde foi detetado D. nodosus foram obtidos 17 isolados identificados como D. nodosus por PCR em tempo real, tendo estes sido caraterizados quanto à virulência e serogrupo a partir de DNA extraído de culturas puras. Com a exceção de um isolado, os resultados dos testes de virulência foram concordantes com os resultados obtidos nas amostras de DNA de biópsia, confirmando os isolados como virulentos. O serogrupo de 15 dos 17 isolados foi identificado, tendo dois permanecido indeterminados. Os 15 isolados pertenciam a seis diferentes serogrupos, B, C, F, G, D e H, sendo que a maioria dos isolados pertencia ao serogrupo B (40%). O segundo serogrupo mais comum foi o serogrupo C (20%), seguindo-se, na mesma proporção, os serogrupos H e G (13%). Combinando os resultados provenientes da análise por PCR multiplex de DNA de biópsia total e de DNA de culturas puras, determinou-se os serogrupos de 29 (22%) das 132 amostras de ovelhas positivas para D. nodosus. Estes 29 serogrupos foram identificados em animais de explorações de oito dos 11 concelhos positivos para D. nodosus (73%), tendo o serogrupo B sido detetado em sete concelhos com a exceção de Odemira. Em contraste, os serogrupos D e H só foram encontrados num concelho cada (Portel e Ponte de Sôr, respectivamente). Em relação à localização geográfica, é importante mencionar a elevada prevalência em concelhos geograficamente próximos, incluindo o concelho de Évora (68%), Portel (72,4%) e Alvito (65,4%). Dois outros municípios com elevada prevalência são Almodôvar (83%) e Odemira (67%), os concelhos mais a sul incluídos neste estudo e também geograficamente próximos. Interessantemente, os três concelhos com menor prevalência aparente, Alandroal (0%), Moura (0%) e Serpa (20%), situam-se perto da fronteira com a Espanha. O presente estudo parece indicar que, na região do Alentejo, existe uma elevada prevalência de D. nodosus, assim como de F. necrophorum, em casos clínicos de peeira. Confirmou-se uma relação entre as duas espécies bacterianas, sendo que a infeção por D. nodosus é influenciada pela presença de F. necrophorum. Os resultados obtidos sugerem ainda que esta espécie bacteriana atua como agente patogénico secundário, aumentando a severidade das lesões e estando envolvida predominantemente nas fases mais tardias da Peeira. Conclui-se também que as estirpes de D. nodosus presentes nesta região de Portugal são maioritariamente estirpes virulentas e que os serogrupos mais comuns são os serogrupos B e C. Verificou-se ainda que poderão existir possíveis “hotspots” de infeção por D. nodosus nas zonas em torno do concelho de Évora e no sul do Alentejo, junto aos concelhos de Almodôvar e de Odemira. Esta investigação consiste no segundo estudo realizado em Portugal com foco na caracterização da Peeira e no primeiro com a utilização de métodos atualizados de diagnóstico e caracterização molecular de D. nodosus (PCR em tempo real e PCR multiplex). Os resultados desta dissertação salientam a importância do estudo da Peeira em Portugal, tendo em consideração a sua elevada prevalência e a virulência das estirpes de D. nodosus. A identificação dos serogrupos predominantes e a caraterização dos isolados neste estudo epidemiológico é um primeiro passo para um melhor controlo e possível erradicação da doença no país, nomeadamente através da implementação de medidasimunoprofiláticas e medidas de biossegurança nas explorações afetadas e noutras que, com estas, mantêm relações comerciais.

碩士
國立臺北科技大學
機電整合研究所
96
This research deals with the design of the mechanisms of a wheelchair footrest. According to the related references of this research, most of wheelchair footrest only have a fixed pivot when users want to stretch their legs, the axles of the knees and footrest are not in the different positions, and when footrest and ground are parallel, footrest didnt stretch, and the legs will get a bearing to make the knees a little bend and it will make the users feel uncomfortable and didnt accord with ergonomics. The design of the wheelchair footrest is four-bar linkage which has one degree of freedom mechanism. The mechanism is used motion generator for three-positon motion synthesis. The moves process of the footrest which make the user obtain comfortable extending and carry. In addition, by using the Solid Works 2007 SP0.0 to build up the 3D model and simulate the movements, and implement COSMOSWorks Designer to analyze stress, strain and displacement, so as to confirm the strength of the wheelchair footrest structure. So the automatic adjustable wheelchair footrest by the one degree of freedom, and its lightweight and easy to use. Its the ideal auxiliary apparatus of wheelchair, and suitable for geriatric or defective.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2009.
Title from title screen (site viewed October 15, 2009). PDF text: viii, 117 p. : ill. ; 1.43 Mb. UMI publication number: AAT 3355631. Includes bibliographical references. Also available in microfilm and microfiche formats.

There are Stories that we are told, stories that we tell, and Stories that are told through us. This text sets out to ask whether self-regulation is a tool to support the progressivist educator or something that undermines the goals of progressivism. But we cannot avoid the footnotes or philosophy in educational research. What is ‘progressivism?’ How does its theory connect or disconnect from its practice? Can it function or live up to its name if those who call themselves ‘progressive’ teachers or parents are distracted by The Question of ‘How do I get them to do what I want them to do?’ And, what about the follow-up fear of control or chaos or the belief that ‘Those kids can’t handle their freedom?’ Employing an ‘out-of-the-box’ narrative academic writing approach, weaving stories from personal parenting and teaching moments with case studies, the questions surrounding self-regulation reveal some surprising answers. Can the narrative surrounding Classroom Management co-exist with progressivist educational goals or the tool of self-regulation? Can democracy be promoted, taught, or lived without praxis?
Graduate

碩士
國立中正大學
資訊管理學系暨研究所
101
Extensible Business Reporting Language (XBRL) could be deemed to a common language of the global corporate financial reports. To catch up with the pace of advanced country, our country is actively promoting XBRL and International Financial Reporting Standards (IFRS) for applying to financial reporting. As our country fully prepared to adopt the IFRS as our financial reporting framework, we should note the difference with the Generally Accepted Accounting Principles (GAAP) and the IFRS, particularly the impact of fair value. While reading and analyzing the financial report, we also rely on the disclosure of footnote that provides additional insight into the companies of what the accounting policies and reasons they have chosen. For these depiction that describe the business operations and the value of the investment, this study’s aim is to establish a mechanism, which combine XBRL and the word-frequency analysis of Zipf’s law to assist users achieving the purpose of finding a specific company checklist group ,and the explanation of unusual word-frequency. It can help us to focus on anomalies word-frequency in order to increase the company's auxiliary reference by judgments on footnote. After the system is built, this study takes the footnotes disclosure of the investment property in significant accounting policies as a testing case. From the result and the data analysis of each module found that the contents and qualities of the footnotes varies due to accountancy firms’ effects. Besides, through this mechanism also be able to identify the degree of difference footnote disclosures with other companies.