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Characterisation of anthelmintic resistance in a multiple drug resistant Teladorsagia circumcinta isolateStenhouse, Lindsay Joanne January 2007 (has links)
The purpose of this study was to undertake detailed molecular and phenotypic characterisation of a MDR isolate of T. circumcincta (MTci5) with particular focus upon the mechanisms underlying benzimidazole (BZ) resistance. MTci5 was isolated from a farm in Central Scotland, which employed a suppressive anthelmintic dosing regime and was closed in 2002 when control of the parasite population became unsustainable. Underpinning all of the experiments in this study was an anthelmintic selection process whereby the MTci5 isolate was pressured individually with three broad-spectrum anthelmintics (benzimidazole, ivermectin and levamisole). There are three main areas of investigation in this study, the first being an investigation of the population genetic structure or a MDR isolate. A central question was whether the MDR phenotype of MTci5 is conferred by the inheritance of genes present in a single interbreeding population or whether there is genetic sub-structuring, whereby discrete sub-populations of the isolate each show resistance to different anthelmintics. Microsatellite analysis was employed to investigate the population genetic structure of the MTci5 isolate. The results suggest that the MTci5 isolate is a single, freely interbreeding population with triple resistance, showing no evidence of genetic sub-structuring. The second area of investigation was the role of the F200Y isotype I ß-tubulin mutation in the determination of BZ resistance and the potential involvement of this mutation in resistance to ivermectin (IVM) and levamisole (LEV). There was no evidence of an effect of IVM or LEV selection upon the F200Y isotype I ß-tubulin mutation. The third area of investigation was the origin and diversity of BZ resistance alleles in the MTci5 isolate. Single strand conformation polymorphism (SSCP) analysis of small region extending through exons 1 and 2 and intron 1 of the isotype I ß-tubulin gene was used to assess the genetic diversity of this locus in the MTci5 isolate and of five other UK T. circumcincta populations. Results are consistent with the theory of multiple independent, spontaneous mutations at the P200 locus of the isotype I ß-tubulin gene.
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Epidemiology and control of ruminant helminths in the Kericho Highlands of KenyaRugutt, Moses Kipngeno Arap January 1999 (has links)
The studies reported in this thesis have been conducted in a high potential area of the country where no previous studies had been undertaken. The two major components of the research were an epidemiology study and an intervention study, both of which were conducted on farms in the peri-urban area of Kericho. An initial background study was conducted to collect data from the local Veterinary Investigation Laboratory, from extension staff in a participatory rapid appraisal (PRA) study and a cross-sectional socio-economic survey was undertaken with local farmers. These provided some understanding of the problems caused by helminths and the approaches taken to control them, together with socioeconomic data relating to the keeping of livestock in the area. Disease appeared to be a major constraint affecting ruminant productivity, with nematodoses appearing to be the most important helminth disease in the area particularly in small ruminants. In the cross-sectional survey, more than 60 % of farmers reported that they administered anthelmintics therapeutically and over 30 % gave routine treatments. On average, cattle were reported as being given up to 1.5 treatments per year, sheep just over 1.1 and goats 0.9 treatments per annum. In general, extension workers recommend that animals are wormed every 3 - 4 months, however this practice was not adopted by the farmers because of the expense involved. The epidemiology study conducted on 27 smallholder farms over a 22 month period obtained data on the prevalence of a) helminth ova in cattle, goat and sheep faeces b) helminth larvae on herbage and c) the different nematode species acquired by introduced Dorper tracer lambs which grazed on communal land. Grazing ruminants in Kericho appear to be exposed to infection with gastrointestinal nematodes throughout the year, there was no evidence of a marked seasonal influence on the availability of infective larvae on pasture. The two commonest genera Trichostrongylus and Haemonchus are both well adapted to the three ruminant species that are commonly grazed together.
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A cell biological approach to studying lameness in the dairy cowMaccallum, Amanda Jane January 1999 (has links)
This thesis describes a cell biological approach to studying lameness in the dairy cow. Lameness has been associated with altered keratinisation of the epidermis of the bovine hoof. Claw tissue was obtained by an in vivo claw biopsy technique. The biopsies were cultured in the presence of L-[35S]-methionine and [3H]-thymidine for measurement of protein synthesis and cell proliferation respectively. The influence of nutritional and environmental stressors on keratinisation in the claw was investigated. Protein synthesis was found to be significantly higher after challenge with these factors. It was not clear, however, if this was an effect of challenge or the biopsy procedure itself. Physiological and endocrinological changes associated with pregnancy and lactation also appeared to have an effect on claw keratinisation. In a long-term developmental study of first-calving heifers changes in cell proliferation and protein synthesis were related to reproductive state. A dramatic seasonal effect on claw cell biology was also demonstrated. The keratinocytes were actively proliferating and keratinising during the summer months but were quiescent during the winter. A final study investigated the cell biological changes which occur in the claw during the onset, development and recovery stages of weight-bearing challenge and the effects of concrete flooring. During challenge cell proliferation increased significantly in claw subjected to weight-bearing. The tissue may have been responding to challenge, however, the biopsy procedure itself could also have had an effect due to the short-time period between samplings. To conclude, altered keratinisation in the claw may be related to physiological and endocrinological changes associated with season and reproduction. However, changes in management may also be important and further investigation is required.
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Proteomic and metabolomic studies on milk during bovine mastitisMansor, Rozaihan January 2012 (has links)
The principal objectives of the study presented in this thesis were to study the changes of milk proteomes, peptidomes and metabolomes during the course of bovine mastitis in comparison with normal milk samples and to discover new bovine mastitis biomarkers using various modern and up-to-date methodologies such as proteomics, peptidomics and metabolomics. Bovine mastitis caused by bacterial infection of the mammary gland of dairy cows is often associated with loss of milk production due to a reduction in milk composition and quality which in turns, lead to negative economic impact on dairy industry. Two important acute phase proteins (APPs) which serve as valuable biomarkers in bovine mastitis were investigated in every chapter using developed and validated enzyme linked immunosorbent assay (ELISA) for bovine milk haptoglobin and commercially available ELISA for bovine milk serum amyloid A3 (M-SAA3). These APPs were quantified alongside somatic cell counts (SCC) and California Mastitis Test (CMT) to confirm the disease status of each animal used in this study. Proteomic methodologies were applied including 1D gel electrophoresis, 2D gel electrophoresis, MALDI-TOF analysis and difference gel electrophoresis to investigate the changes of milk proteome in both subclinical and clinical mastitic milk samples in comparison with healthy milk samples. However these investigations did not reveal novel biomarkers for mastitis. Next, peptidomic methodologies were used to study the changes in milk peptidome and to detect the presence of any significant disease biomarkers in the presence of bovine mastitis by using CE-MS and LC-MS/MS. A total of 31 and 14 polypeptides can be used to discriminate control from infected groups and E. coli from S. aureus infected groups respectively. Lastly, metabolomic methodology was applied with an intention to study the changes in milk metabolome and ultimately to detect the presence of novel biomarkers in bovine mastitis. Di- and tri-peptides were found higher in S. aureus than in E. coli infected groups and based on metabolic pathways, arachidonic, arginine and galactose metabolites were seen increased in mastitic milk samples in comparison to healthy milk samples. Overall, the findings detailed in this thesis indicate that the use of advanced proteomic and metabolomic methodologies could deliver on their promise of the discovery of potential significant bovine mastitis biomarkers. Further studies are needed for validation of these proposed biomarkers and it was hoped that better prevention and treatment methods for bovine mastitis can be achieved in the future.
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Analysis of the role of fimbriae in the virulence of Salmonella enterica in poultryClayton, Debra Jayne January 2008 (has links)
Salmonella is a Gram-negative bacterium that consists of two species; S. enterica and S. bongori. The species S. enterica can be further divided into 6 subspecies and subspecies I is predominantly associated with disease in warm blooded animals and contains over 2,500 antigenically distinct serovars. Each serovar is >90% identical at the DNA level but can infect a different range of hosts and cause different diseases. Poultry are an important reservoir of entry of Salmonella into the human food chain owing to the contamination of their eggs and meat. The molecular mechanisms underlying colonisation of food producing animals with Salmonella are unknown. Fimbrial genes encode proteinaceous surface exposed appendages which have been shown to mediate adhesion of bacterial cells but the precise role for fimbriae in the carriage and virulence of Salmonella is poorly defined. The purpose of this study was to annotate and characterise the fimbrial genes of the poultry-associated S. enterica serovars Enteritidis and Gallinarum and relate this role to host-specificity. The availability of the genome sequences of several strains of S. enterica allowed a comparison of the sequence, location and repertoire of fimbrial genes and although no unique fimbrial genes were identified all serovars possessed a unique repertoire. The host-specific serovars contain a higher number of pseudogenes within fimbrial operons than the ubiquitous serovars and the rate of attrition of fimbrial genes was 3-4 fold higher than the genomic mean. Such gene decay may partially explain the narrowing of host-range of the host-restricted and host-specific serovars. Polymorphisms that may alter transcription were identified along with targets that may be associated with phase variation of the fimbrial genes. Lambda red-mediated homologous recombination was used to construct a panel of S. Enteritidis P125109 and S. Gallinarum 287/91 strains lacking major fimbrial subunit genes which were examined in vitro and in vivo. Several fimbrial subunits played a role in the adherence to and invasion of different cell lines in different growth conditions and the role appeared to be serovar-specific. A mutation in the steA gene impaired interactions with different cell lines in vitro but this phenotype was found to be due to a polar effect on genes downstream of steA. The majority of fimbrial subunits played no significant role in the colonisation of the alimentary tract in an established chicken model. Mutation of the stcA gene resulted in the greatest degree of attenuation in vivo of all of the fimbrial mutants examined. This phenotype was trans-complemented and was not the result of a polar or second-site defect thereby fulfilling molecular Koch’s postulates. The stcA genes therefore play a significant role in the colonisation of the chicken caeca.
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A study of the anatomical variations of the carotid arterial tree in EquidaeHayah, Nurul January 2012 (has links)
The internal carotid artery in the horse is of significant veterinary importance due to its intimate relationship with the guttural pouch, and mycotic disease thereof. The relevance of recognising and identifying variations involving the internal carotid artery lies in the fact that surgical occlusion of the artery is the treatment of choice for guttural pouch mycosis. However, occlusion could be hampered when there is doubt about the anatomy of this vessel and its variation. Conventional angiography and automatic rotational angiographic techniques were used to study the anatomy of the carotid trifurcation and the internal carotid artery on cadavers of three species included in the genus Equus; 50 horses, 26 donkeys and one zebra. Following angiography, arterial latex casting was performed on the horse and donkey specimens with subsequent dissection to harvest the hardened arterial casts. Rotational angiography with 3-dimensional image reconstruction represent a major advantage in the angiographic diagnosis of carotid and cerebral vascular variation compared to conventional angiography. This technique generated superior angiographic images of the carotid and cerebral vascular system of horses, donkey and zebra. In the horse, five variations of the internal carotid artery were identified as follows: [1] the internal carotid artery and occipital artery arising as a common trunk, [2] an aberrant branch of the internal carotid artery that unites with the basilar artery, [3] an aberrant branch of the internal carotid artery that does not unite with the basilar artery, [4] aberrant branch of the internal carotid artery that gives rise to several satellite branches, [5] aberrant branch of the internal carotid artery that has a satellite branch connected to the caudal branch of the ipsilateral occipital artery. Variations of the carotid arterial tree in donkeys were identified as follow: [1] the internal carotid and occipital arteries shared a common trunk, [2] the linguofacial trunk originated from the common carotid artery causing the common carotid artery to terminate as four branches, [3] a short external carotid artery before giving rise to the linguofacial trunk, mimicking the appearance of the common carotid artery terminating into four branches, [4] the internal carotid artery originating far more caudal from the common carotid artery termination. The carotid arterial anatomy of the one zebra studied here showed no discrepancy to the accepted common anatomical pattern of this structure. Aneurysm formation was not identified in any of the specimens.
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Total intravenous anaesthesia in dogs : development of a target controlled infusion (TCI) scheme for propofolBeths, Thierry January 2008 (has links)
The aim of this work was to develop a propofol (PPF) based Total Intravenous Anaesthesia (TIVA) technique to be used in dogs by veterinary surgeons in practice. As PPF is a poor analgesic agent, this work also looked into the development of a co-infusion scheme for the potent alpha2-adrenoceptor agonist medetomidine (MED) and its active enantiomer dexmedetomidine (DEX). The study was divided into 4 phases. In phase one, canine PPF pharmacokinetic (PK) parameters, derived from previous studies reported in the literature, were incorporated to a Target Controlled Infusion (TCI) system. This approach, comprising computer software incorporated into a syringe driver, which will deliver PPF to a predicted blood concentration, is widely used in humans. The performance of the system was investigated in 6 greyhounds and 10 mixed-breed dogs undergoing routine dental work by comparing predicted with measured PPF concentrations in venous blood samples obtained during and following TCI PPF anaesthesia. The optimal induction target was 3 mgml-1, and an adequate depth and a satisfactory quality of anaesthesia were achieved with mean maintenance targets of between 3.4 and 4.5 mgml-1 of PPF. The performance of the TCI system was considered clinically acceptable as the Median Prediction Error (MDPE%), a measure of bias, and the Median Absolute Performance Error (MADPE%), a measure of the accuracy, were -12.47 and 28.47 respectively, in the greyhounds and 1.56 and 24.79 respectively, in the mixed-breed dogs. The system was easy to use and the quality of anaesthesia was judged to be adequate for dental work. Phase 2 illustrated the inhibitory effect of MED and DEX on PPF metabolism at the level of the cytochrome P450 in rat and canine hepatic tissue and highlighted, therefore, the possible effect on the metabolism dependant performance of the TCI system. Before designing an infusion scheme for MED and studying its possible effect on PPF PK in vivo (phase 4), the purpose of phase 3 was to characterise cardiovascular and respiratory effects of MED administered IV to dogs anaesthetised with a TCI of PPF, and to assess its suitability for use in a TIVA regime. Eighty dogs, ASA 1 or 2, aged 0.5 to 8 years, were randomly allocated into 8 groups of 10 dogs according to the dose of MED administered (Groups 1-8: 0 (saline, 0.9%, 1 ml), 0.01, 0.03, 0.1, 0.3, 1, 3, 10 mcgkg-1 MED, respectively). Following premedication, anaesthesia was induced with a PPF target blood concentration of 3 mcgml-1 and maintained with a target concentration of 3.5 mcgml-1. Cardiovascular and respiratory parameters were recorded for 15 min post induction and before saline (group 1) or MED (groups 2-8) was injected slowly over 1 min. Medetomidine induced a dose-dependent reduction in heart rate (HR) and increase in systolic arterial blood pressure (ABP). At the time of maximum observed effect (2 min post MED injection), the ED50 for ABP and HR were 2.05 and 0.187 mcgkg-1 respectively, while the ED95 (doses of MED inducing 95% of the maximum effect) values were estimated to be 18.1 mcgkg-1 and 3.1 mcgkg-1, respectively. The no effect doses for MED were 0.01 mcgkg-1 for HR, and 0.1 mcgkg-1 for ABP. Minimal respiratory effects were observed in all groups except the group receiving 10 mcgkg-1 of MED where, by the end of the recording period (20 min post MED injection), 8 of 9 spontaneously breathing dogs became apnoeic after MED administration. Phase 4 was designed to develop and assess a stepped infusion scheme for MED and DEX in the TCI PPF anesthetised dog using MED PK parameters from O. Vainio (V1 = 470 mlkg-1, K12 = 0.0954, K21 = 0.0438, K10 = 0.0489); to observe the possible PK and PD (pharmacodynamic) interactions between PPF and the 2 alpha2-adrenoceptor agonists during co-infusion; to determine the minimum blood PPF infusion target (MIT) necessary to prevent purposeful movement during supramaximal noxious stimulation (tetanic twitch for 5 sec at the level of the 4th and 5th coccygeal vertebrae) with and without a co-infusion of MED or DEX and to confirm the DEX minimum analgesic blood concentration of 0.85 ngml-1. Six female beagle dogs, 7.3 (± 2.3) years old, were anaesthetised on 4 occasions, following a randomised cross over design: PPF TCI with either co-stepped infusion of saline (PS), MED (blood target of 1.7 ngml-1, PM), low DEX (blood target of 0.85 ngml-1, PLD) or high DEX (blood target of 1.7 ngml-1, PHD). The co-infusion was started 25 min after the start of anaesthesia (instrumentation period), while the MIT determination was conducted 15 min after the last step of the co-infusion. Venous blood samples were taken at specific times for determination of the PPF, MED and DEX plasma concentrations. The performance of the TCI system for PPF in the dog was only clinically acceptable in the PS and PLD treatments with MDPE% values of 18.85 and 25.94 respectively, and MDAPE% values of 18.85 and 35.80 respectively. In this study the use of DEX 0.85 ngml-1 had a similar PPF sparing effect to the equivalent MED blood concentration of 1.7 ngml-1, but with less effects on ABP, as well as on the performance of the TCI for PPF in the dog. Therefore, it could be concluded that DEX was more advantageous than MED given by infusion in PPF anaesthetised dogs. The study also confirmed the validity of the PK of MED from the previous study. The study redefined specific PK parameters for DEX, although the MED PK parameters could also be used. The study indicated that DEX blood concentrations as low as 0.85 ngml-1 decreased the measured PPF blood concentrations necessary to maintain anaesthesia during noxious stimulation by about 38%. However, although this study supported the suitability of the co-infusion of DEX during PPF anaesthesia in the dog, and the analgesic/sedative effects of DEX were present at the lowest blood concentrations with well maintained respiratory parameters, the CV effects were marked with a decrease in HR and CO and an increase in systolic and mean ABP. Further studies are therefore necessary to establish if a lower blood concentration of DEX will provide analgesia while preserving the CV system.
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Equine sarcoids and bovine papillomavirus : unravelling the viral pathogenesisFinlay, Margaret January 2011 (has links)
The equine sarcoid is the most commonly detected skin tumour in equidae worldwide and has been reported in horses, donkeys, mules and zebra. Sarcoids can be defined as locally aggressive, fibroblastic, benign tumours of the equine skin and although they do not metastasise, they exhibit variable clinical presentations ranging from small alopecic areas to rapidly growing, ulcerated fleshy masses. Clinical behaviour may also vary, from aggressive infiltrative growth to spontaneous regression. Diverse treatment modalities have been reported, and these have been used separately, or have been combined, with variable efficacy. No single therapeutic approach has been found to be universally successful. Although rarely life-threatening, equine sarcoids can have important welfare and economic implications. There is a large body of evidence now supporting the hypothesis that bovine papilloma virus (BPV) is the aetiological agent of equine sarcoids and our understanding of the general pathophysiology of the disease continues to progress; however, several fundamental aspects of the disease remain unclear. In the first chapter, the clinical aspects of the disease are discussed and evidence to support a viral aetiology is presented, along with background information regarding papilloma virus infections in the natural host. An epidemiological overview of the disease that considers the most current theories and understanding of equine sarcoids is also given. Chapter II describes the materials and methods used in the course of carrying out the investigations detailed in Chapters III, IV and V. The aim of Chapter III was to investigate the potential role of flies as vectors in transmission of equine sarcoids between infected and susceptible animals. We found that BPV-1 DNA can be detected in flies trapped from areas where animals with BPV-1 infected sarcoids are housed. This study provides evidence to support the hypothesis that flies may therefore be significant in disease transmission. Such evidence will be of benefit in formulating management control strategies for fly control measures that will reduce the incidence and spread of equine sarcoids. The work described in Chapter IV was carried out to investigate the hypothesis that the development of the sarcoid tumour arises primarily through inhibition of apoptotic pathways by viral gene products. Assessment of DNA-damage-induced apoptosis in BPV transformed equine fibroblasts revealed that sarcoid derived cells and BPV-1 transfected fibroblasts are less resistant to apoptosis than normal, BPV-negative control equine fibroblasts, but are more likely to recover from DNA damage and continue to grow and divide. Further research was directed towards identifying the viral genes responsible for conferring resistance to apoptosis through siRNA knock-down and monitoring expression of endogenous cellular proteins known to be involved in apoptotic pathways. Using an siRNA targeted to a particular viral protein, we were able to reduce the ability of the BPV transformed cells to survive after DNA damage. Potentially, this information can now be used to develop novel therapeutic strategies. The final chapter describes the results of a study in to the expression of the p53 protein in sarcoids in vivo and in BPV transformed cells in vitro in tissue culture. In vivo, analysis of 51 equine sarcoid lesions showed that 48% of tumours are associated with nuclear p53 protein expression and that high levels of p53 were associated with clinically aggressive tumour types (fibroblastic). Interestingly, we observed high levels of cytoplasmic p53 staining with no nuclear staining in one tumour in vivo. In vitro, p53 mRNA levels were assessed in BPV transformed and BPV negative fibroblasts. The half-life of the p53 protein, the cellular location and functionality were also investigated in vitro. The results showed that BPV transformed equine fibroblast cells also exhibit increased nuclear p53 protein expression and one cell line (EqS04b) showed a cytoplasmic staining pattern similar to that observed in vitro. However, the abnormal level and location of the p53 does not appear to affect the transactivational functionality of p53 since p21 expression was induced by exposing the cells to UVB irradiation.
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Quantitative epidemiological studies on recurrent airway obstruction in the horse population of Great Britain using a risk-screening questionnaireHotchkiss, Joel W. January 2004 (has links)
The principal aim of this study was to investigate the epidemiology of recurrent airway obstruction (RAO) in horses in Great Britain using risk-screening questionnaires (RSQ). Three processes were used to aid construction of the RSQ for RAO, namely: a review of the scientific literature, a survey of equine practitioners in the UK and a modified Delphi consultation with experts in the field of RAO. The demographic information, generated by the questionnaire, enabled investigation of risk factors associated with the disease using multilevel, multivariable logistic regression. Two models were constructed. The first related to host and environmental risk factors and the second explored the effect of early life factors. The host and environmental model identified an increased risk of RAO in association with increasing age and the horse residing in an urban or semi-urban environment. There were also some associations that were contrary to what would be expected from knowledge of the aetiology of RAO. In particular, horses fed soaked (wet) hay had increased odds of having RAO, whilst horses fed dry hay had decreased odds. The early life model identified an increased risk of a horse having RAO if its owner had acquired them after the age of two years or that in early life it had been fed hay or had a respiratory infection. The final stage of the study was to develop and assess an educational package for horse owners regarding the disease. RAO appears to be worryingly prevalent in the horse population of Great Britain; a real concern in terms of welfare. Much can be done to alleviate this chronic disease by controlling a horse’s environment to reduce respiratory challenge. Greater emphasis could be placed on assisting horse owners in making this transition by providing detailed guidance.
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Assessing MHC class I diversity in dairy cattle populationsCodner, Gemma Frances January 2010 (has links)
The gene dense major histocompatibility complex (MHC) region, present in all jawed vertebrates, encodes molecules involved in self-non-self discrimination and the binding and presentation of antigenic peptides to T cells during the adaptive immune response. Variation in MHC genes is thought to be driven largely by pathogen-mediated selection, with diversity at MHC loci believed to benefit populations by allowing responses to rapidly evolving disease pathogens. However, in economically important dairy cattle, there are concerns that intensive selection for production and fitness traits may override natural selection. It had been hypothesised that these focussed dairy breeding practices may lead to a reduction in MHC diversity and leave cattle populations susceptible to new disease pathogens. The purpose of this study was to estimate current levels of MHC class I diversity in the UK Holstein-Friesian dairy cattle population, primarily through the assessment of diversity in bull populations with genetic input into the UK herd. In a sample of Canadian Holstein bulls, levels of class I allelic diversity were low given the size of the population sampled, but no significant loss of diversity over a twenty year period of selection was detected. Simulations of gene flow implicated trait selection as an influential force shaping diversity in the Canadian Holstein bull population. Haplotypes detected at high frequency were often negatively associated with selection traits indicating the action of heterozygote advantage. A SNP-based assay has been designed to facilitate rapid detection of common haplotypes and thus enable breeders to make more efficient selective breeding decisions whilst also maintaining MHC diversity in cattle populations. Investigations of class I diversity were expanded to incorporate the British Friesian bull population which were shown to have a markedly different pattern of class I diversity to that observed in the Canadian Holstein sample. A number of novel allele sequences and haplotypes were detected in the British Friesian bulls, the characterisation of which has contributed to our knowledge of the mechanisms driving diversity in the cattle class I region. MHC class I typing data from two bull populations and statistical analysis of trait associations with MHC haplotypes provides a comprehensive picture of MHC class I diversity in the wider UK herd and the selective forces integral to shaping diversity.
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