http://hdl.handle.net/1842/1516 2013-05-25T23:18:51Z 2013-05-25T23:18:51Z Postigo, Milagros Postigo Mercades, Maria Milagros http://hdl.handle.net/1842/6576 2013-02-27T12:29:28Z 2007-06-22T00:00:00Z

Title: Molecular and antigenic characterisation of Ehrlichia ruminantium in Amblyomma variegatum ticks and in vitro cultures Authors: Postigo, Milagros; Postigo Mercades, Maria Milagros Abstract: The rickettsial pathogen Ehrlichia ruminantium, transmitted by ticks of the genus Amblyomma, causes heartwater, an economically important, often fatal disease of domestic and wild ruminants in sub-Saharan Africa and in the Caribbean. The studies described in this thesis have contributed to understanding several aspects of heartwater. First, a real-time PCR method was developed in order to study the kinetics of infection with E. ruminantium in the mammalian host. The assay was validated for specificity and sensitivity and was used to estimate numbers of the organisms in the blood of infected sheep. However, organisms were only detected during the clinical phase of infection, indicating that the way in which it was applied did not provide sufficient sensitivity to follow the early stages of infection. This PCR assay was then used, together with transcription and proteomic analyses, to investigate differential gene expression of E. ruminantium in the arthropod and mammalian hosts, in order to identify genes that may allow the organisms to successfully adapt to different environments. These studies used in vitro tick and mammalian cell culture systems, as well as tissues from infected A. variegatum ticks, and initially focused on the map1 multigene family. Although transcripts for most of the map1 paralogs were detected in organisms grown in vitro, in both mammalian and tick cells, only transcripts from map1 and map1-1 were detected in infected ticks. Moreover, map1-1 transcripts were more abundant in midguts than in salivary glands whereas map1 transcripts were most abundant in salivary glands and were expressed at higher levels following several days of tick feeding on a mammalian host. Because of the quantities of material required, proteomic analysis was only possible using in vitro-cultured organisms. Comparison of proteins encoded by the map1 cluster in E. ruminantium grown in tick or bovine endothelial cell cultures, using 2D gels and MALDI-TOF analysis, revealed that different proteins predominated in the corresponding spots in 2D gels from the different cultures; products of the map1-1 gene were abundant in tick cells, while products of map1 were abundant in endothelial cells. The detection of higher levels of map1 transcripts in salivary glands than in midguts of infected ticks, together with the presence of abundant MAP1 protein in organisms grown in mammalian but not in tick cell lines, suggest that expression of this protein may be associated with infectivity for mammalian cells. In contrast, map1-1 transcripts were abundant both in midguts of infected ticks and in tick cell lines, and the protein was expressed at high levels in infected tick cell cultures. Since both of these stages have low infectivity for sheep, these results suggest that the MAP1-1 protein may play an important role within the vector, possibly associated with colonisation and replication of E. ruminantium in the tick midgut. Collectively these findings suggest that this multigene family is involved in functions of biological relevance in different stages of the life cycle of E. ruminantium. Lastly the suppression subtractive hybridisation (SSH) technique was applied to RNA extracted from E. ruminantium-infected endothelial and tick cell cultures in an attempt to sample a large portion of the E. ruminantium genome for differentially expressed genes; although not resulting in identification of any differentially transcribed genes in the present study, this method was shown to work in principle.

2007-06-22T00:00:00Z Barrier, Alice Cécile Madeleine http://hdl.handle.net/1842/6527 2013-04-11T13:25:16Z 2012-06-30T00:00:00Z

Title: Effects of a difficult calving on the subsequent health and welfare of the dairy cows and calves Authors: Barrier, Alice Cécile Madeleine Abstract: Yearly calvings are essential to the sustainability of modern dairy farming. Currently, calving difficulty (or dystocia) affects one in six calvings among UK dairy herds but vary from 2 to 50% internationally. In dairy cows, despite reports of impaired performance, the extent and threshold of the effect of dystocia on health and performance remains unclear. Over the past years, there has also been increasing concerns about the levels of pain experienced by the dystocial cows. Better understanding of their parturition progress and behaviours is needed so that informed decisions on pain mitigation can be taken. Additionally, the impact of dystocia (besides stillbirth) should also be addressed in dairy calves. The objective of this study was to address the effects of a difficult calving on the health and welfare of both dairy cows and calves. Retrospective analyses of an experimental farm’s detailed records were used to relate calving difficulty with health and performance of the dairy cow. The results showed that after any difficulty at calving, dairy producers incur long-lasting shortfalls in milk sales. Dystocial cows also have impaired fertility, are more likely to leave the herd early and have a higher risk of dystocia at the following calving, thus there is a long-term detrimental impact on dystocial cows. Video monitoring of calvings allowed detailed investigation of the parturition progress and behaviours of dystocial Holstein cows giving birth to singleton liveborn calves. The study of calving behaviours and parturition progress indicated longer later stages of parturition, increased restlessness and tail raising in the six hours preceding expulsion of the calf, for dystocial cows receiving farm assistance compared with cows calving unaided. This may relate to the expression of higher levels of pain when dystocia occurs. The onset of maternal behaviour was not delayed following calving difficulty, and firm conclusions could not be drawn from investigation of some behavioural indicators of pain in the first three hours postpartum. Experimental work allowed the monitoring of a cohort of 496 calves born with various degrees of birth difficulty over two years. All but one vet assisted calves were born dead, and farmer assisted calves were more likely to be stillborn than calves born without assistance. Stillborn dystocial calves displayed larger internal damage, than stillborn eutocial calves, but they did not have a different body shape at birth than dystocial calves that survived. Dystocial dairy calves that survived the birth process had lower vigour at birth, had higher salivary cortisol, acquired lower passive immunity and received more health treatments in the neonatal period. Dystocial heifers also had higher mortality rates by weaning but had similar growth to first service. Historical records from the farm also showed that dystocial heifer calves were three times more likely to have died by weaning and by first service than calves born without assistance. For those who survived, there was, however, no indication of altered growth to weaning or subsequent impaired fertility. This may be explained by the early mortality of the most badly affected calves or by farm management. However, their high mortality rates still raise welfare concerns. Altogether, results suggest that dairy calves born with any difficulty have poorer welfare in the neonatal period and possibly beyond. The experience of any calving difficulty in dairy cattle therefore not only impairs the welfare of the cow, but also the welfare from their resulting calf. Any strategy implemented to lower the occurrence and mitigate the effects of dystocia will therefore improve the welfare of the cows, their calves and enhance the farm’s economic sustainability.

2012-06-30T00:00:00Z Baker, Rachael Helen http://hdl.handle.net/1842/6526 2012-11-15T14:47:47Z 2012-06-30T00:00:00Z

Title: GTP-Cyclohydrolase function in parasitic nematode development Authors: Baker, Rachael Helen Abstract: Parasitic nematodes of grazing livestock represent an increasing economic and welfare problem for British agriculture. By investigating specific life-cycle stages of these parasites, it may be possible to identify key molecules or pathways that are required for the survival of the worms, and thus exploit these for future control strategies. It has been shown previously that the third larval stages (L3) of the ovine parasitic nematode Teladorsagia circumcincta produce high levels of transcript for the enzyme GTP-Cyclohydrolase relative to later developmental stages. As the ratelimiting factor in the production of tetrahydrobiopterin, GTP-Cyclohydrolase is required for a number of different biochemical pathways, including those involved in the production of serotonin and melanin. As the L3 do not feed, it can be hypothesised that, if finite resources are being used in the production of transcript encoding this enzyme, then it may be important for survival. In this thesis, a number of approaches were taken to explore the function of GTPCyclohydrolase in the life-cycle development of T. circumcincta. The closely related parasite, Dictyocaulus viviparus, was used as a model organism to explore the role of GTP-Cyclohydrolase and serotonin production with regards to larval arrest, or hypobiosis. This process occurs readily under experimental conditions in D. viviparus, which is not possible with T. circumcincta. Quantitative PCR was used to examine GTP-Cyclohydrolase transcript levels in two different strains of D. viviparus, one that enters larval arrest when exposed to cold conditions and one that does not. No differences were observed between the two strains suggesting that GTP-Cyclohydrolase was unlikely to be involved in hypobiosis. The model nematode, Caenorhabditis elegans, was used to perform functional complementation experiments to assess the role of GTP-Cyclohydrolase in the cuticle, as it has been shown previously that C. elegans GTP-Cyclohydrolase mutants have a ‘leaky cuticle’ and are killed by lower doses of anthelmintics and bleach than the wild-type worms. The T. circumcincta gene for GTP-Cyclohydrolase was able to restore cuticular integrity of C. elegans GTP-Cyclohydrolase-deletion mutants, suggesting that the role played by the protein in both species is similar. In vitro inhibition experiments using a chemical inhibitor of GTP-Cyclohydrolase showed that T. circumcincta larval development was disrupted in the presence of the inhibitor. It was also shown that T. circumcincta L3 that were exposed to sunlight produced melanin, suggesting that the levels of GTP-Cyclohydrolase observed in the preparasitic stages of T. circumcincta may be required for the synthesis of melanin. Together, these data suggest that GTP-Cyclohydrolase is required by the preparasitic stages to survive on pasture. Ultraviolet radiation has been shown previously to be harmful to T. circumcincta L3, so if the melanin production provides protection from this, then it would be crucial for the survival of the pre-parasitic stages.

2012-06-30T00:00:00Z Buckley, Louise Anne http://hdl.handle.net/1842/6522 2012-11-15T14:27:31Z 2012-06-30T00:00:00Z

Title: Food choices for hungry broiler breeders: do they prefer quantitative or qualitative dietary restriction? Authors: Buckley, Louise Anne Abstract: This programme of research uses choice test methodologies to quantify hungry broiler breeder chickens’ preferences for qualitative or quantitative dietary restriction. It begins with an outline of quantitative dietary restriction, its severity and welfare implications before discussing methods of qualitative feed restriction and the difficulties ascertaining whether it represents a welfare improvement. Chapter two reviews the factors affecting diet preferences and discusses implications for feed restricted broiler breeder diet preferences. Chapter three outlines the use of a closed economy T-maze task to quantity the diet preferences of feed restricted broiler breeders. It concludes that broiler breeders can learn a food versus no food task but find it very difficult to learn a task in which both of the options are rewarded with food and this impeded diet preference quantification. Chapter four demonstrates that severity of feed restriction underlies these difficulties in learning. In Chapter five, a conditioned place preference task to identify the effects of diets on affective state (hunger versus satiety) is reported. A method validation group demonstrated that broilers show a state dependent preference for an environment associated with ad libitum access to food. However, birds failed to show a preference between an environment associated with quantitative dietary restriction and one associated with qualitative dietary restriction. Chapter six applies state- dependent learning (SDL) to quantifying the satiating effects of quantitative and qualitative dietary restriction. However, a validation group suggested that SDL preferences were probably an artefact of the test rather than a genuine state-led preference. Finally, the overall conclusion that no evidence was found that broiler breeders want, or that their welfare is improved by, qualitative feed restriction was drawn. However, the conditions under which a preference was reliably observed and the presence of hunger – state dependent effects on learning and expression of learnt preferences complicates the interpretation of any findings. Recommendations for further research are highlighted.

2012-06-30T00:00:00Z