Thesis (M.S.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains viii, 57 p. Vita. Includes abstract. Includes bibliographical references (p. 44).

Thesis (doctoral)--Université de Paris, Faculté de médecine, année 1958, no. 41. / Description based on: Année 1958, no. 41; contains seven theses published between 1939 and 1958. Includes bibliographical references.

The aim of this study was to develop and evaluate an in vitro proliferative response of peripheral blood mononuclear cells (PBM) isolated from Holstein-Friesian cattle, induced by formalin-killed Staphylococcus aureus, a major mastitis pathogen, as a potential indicator of resistance or susceptibility to mastitis. Different strains of S. aureus, isolated from cows with subclinical mastitis, and identified by restriction enzyme fragmentation pattern analysis, were shown to induce different levels of proliferation of PBM in vitro. One particular strain (strain A), induced the strongest proliferation response compared to the other strains tested and differences in the magnitude of the proliferative response, induced by S. aureus strain A, was noted among cows. Phenotypic analysis of PBM of cows, by flow cytometry, showed that normal proportions of cell sub-populations were present at the start of culture and that the proliferating cell population consisted mainly of T cells expressing CD4, CD8 or markers. Proliferation of PBM of cows, induced by Staphylococcal Enterotoxin B (SEB), showed that the peak day of proliferation occurred 72 to 96 hours earlier in the presence of SEB compared to in the presence of S. aureus strain A antigen. A statistically significant difference in the proliferative response induced by S. aureus strain A was seen in two progeny groups sired by two different commercial bulls, suggesting that genetic control of this response may be important in protection of the mammary glands against infection. The proliferative assay was, therefore, subsequently performed in bulls.

A risk assessment approach was used to: a) define the risk of acquisition of meticillin-resistant Staphylococcus aureus (MRSA) in pet dogs over a defined time period, b) attempt to identify important priority data gaps for future research efforts in this area and c) comment on the usefulness of risk assessment in this regard, in a data sparse area and in a field in which it has not previously been used. A conceptual model was defined that identified the potential pathways for acquisition of MRSA in a pet dog over any given 24 hours. A qualitative risk assessment, using categorical qualitative estimators and combining estimates using a matrix approach was undertaken. It was found that this approach was unsatisfactory for the specification of this biological model that encompassed a non-modular, non-sequential form, characterised by non-mutually exclusive pathways of acquisition. Modification of the categorical descriptors enabled a relative, rather than absolute assessment of risk and resulted in the conclusion that both veterinary and non-veterinary routes were potentially important for the acquisition of MRSA in pet dogs and that family members and staff were likely to be the most important sources of the organism in the community and veterinary clinic environments respectively. Given the limitations encountered, a quantitative risk assessment was pursued. Data gaps that were defined within the qualitative risk assessment were addressed through dedicated data-collection studies and an expert opinion elicitation exercise. The studies found a lower veterinary environmental and staff prevalence than had previously been reported, corroborated prior estimates of dog-dog and dog-human interactions and justified the inclusion of dog foods as environmental sources of MRSA rather than independent and important sources in their own right. The expert opinion elicitation exercise used a modified technique to obtain numerous estimates relating to prevalence and transmission of MRSA. However, it was found that experts lacked confidence in estimation of transmission variables in particular, and the resulting distributions for these variables demonstrated divergence between experts and resulted in wide and poorly-informative combined distributions. These results were utilised, along with published and unpublished data, to parameterise a second order stochastic simulation-based quantitative risk assessment model. The model produced a biologically plausible outcome and allowed the application of sensitivity analyses with the intention of identifying areas of putative importance for future research efforts. The implementation of logistic regression analyses directly to the input/output relationship within the simulation model represented a novel application of a variance-based sensitivity analysis technique in the area of veterinary medicine, and was implemented with and without the consideration of interaction terms. In addition, one-at-a-time (OAT) and Plackett-Burman (P-B) analyses were also completed. The results of the sensitivity analyses were complicated and ambiguous. While family members and the environment were identified as potentially important independent and non-independent sources of MRSA, respectively, it was not possible to discount, or defensibly rank the importance of other sources. In conclusion, it was found that, despite the application of well researched and previously utilised methods, marked limitations were encountered in the use of risk assessment to address a biologically complex phenomenon that is characterised by sparse data such as this.

The condition known as tick-borne fever (TBF),which occurs in sheep and cattle, is due to a tick-transmitted rickettsia called Cytoecetes phagocytophila , and has frequently been suggested to have an immunosuppressive effect, predisposing animals to other disease conditions. The work in this thesis was directed towards examining c. phagocytophila as a pathogen per se and its role in other diseases of sheep such as tick pyaemia, louping-ill and pneumonic pasteurellosis. The mechanism whereby the immunosuppressive effect occurred was investigated and methods for the oontrol or prevention of 'lBF and tick pyaemia explored.

This study took place over 3 years during which the occurence of vulval discharge was monitored in a commercial sheep flock of 400 mule ewes. Vulval discharges were found to be physiological (Type 1), occuring immediatly after normal parturition and consisted of clear red coloured mucus which was frequently sufficiently profuse to drip from the vulva and stain the inside of the thighs. This discharge lasted for up to 7-8 days after lambing. Ewes were clinically normal with rectal temperatures between 39.1C adn 39.6C. White to brownish, watery discharge with a foetid smell was designated Type 2. Affected ewes had rectal temperatures of 39.9C ti 41.5C. The most severe discharge (Type 3) was yellow/white with large lumps of pus and affected ewes were inappetant, visibly depressed with rectal temperatures in excess of 41.5C. Ewes with Type 2 and 3 discharges were found to produce fewer lambs per ewe than the flock average in the season following the discharge (1.6 vs 1.95 in 2008 and 1.75 vs 1.92 to scanning in 2009). Bacteria present in the discharges included E.coli and other faecal bacteria, staphylococci, especially S. aureus, corynebacteria of a number of species, C. minutissimum, C. jeikeium, C. stratum, C. pseudodiptheriticum, C. afermentans, C. urealyticum, C. renale, C. cystitidis, C. propinquum and C. argentoratense and Arcanobacterium pyogenes. Some bacterial species isolated represent first records for the sheep, especially Aerococcus urinae. Others such as Globicatella sanguinis had not previously been recorded in the sheep reproductive tract. This study was affected by the occurence of Scrapie, which led to the culling of many of the ewes studied: Enzootic Abortion of Ewes (Chlamydophora abortus infection) which resulted in a whole flock treatment with parental oxytetracycline in two years and assistance at lambing when antimircrobial was administered. A. pyogenes was not found in vulval discharges from ewes which had received assistance and the assiciated antimicrobial treatment. The finding that severe discharges in ewes were not associated with major productivity problems was novel. The observation differs from the situation in cattle where post-partum discharges are recorded as being associated with poorer or delayed conception.

The work described in this thesis was initially directed towards the elucidation of the self-cure phenomenon as a flock phenomenon under natural grazing conditions. As a consequence of these studies it became apparent that several important aspects of the epidemiology and of the clinical syndromes associated with haemonchosis required further investigation. The thesis is therefore essentially a description of haemonchosis of sheep in an endemic area supported, where indicated, by observations obtained by experimental infections. Each of the separate aspects studied is prefaced by a review of the relevant literature and the details of many individual observations which are not included in the text may be found in the various appendices. Some results, for example the individual faecal egg counts and worm burdens at autopsy, were too voluminous to be included and these are available at The Veterinary School of the University of Glasgow.

The principal aims of the work presented in this thesis were to investigate the biochemical properties and protein compositions of nasal secretion (NS) from healthy cattle and to document changes in the protein expression in NS from diseased and vaccinated cattle using advanced proteomic methods. Bovine respiratory disease (BRD) is the principal source of economic loss for the cattle industry throughout the world. Even though it has been studied extensively, it still remains the number one cause of disease and death in cattle. Although the etiopathogenesis of BRD is multifactorial and complex, all of the pathogens have the common route of infection which is intranasal. Thus, NS is potentially a valuable source of biological sample in the detection of biomarkers for BRD. Therefore, a method for the collection of substantial volumes of NS from cattle was developed in this study to establish a reference range of analytes that are present in the NS of healthy cattle. Biochemical profiles of NS from a group of 38 healthy Holstein-Friesian cows revealed high alkaline phosphatase (AP) activity of up to 2392 IU/L which is 15.7 fold higher compared to AP activity in serum reference ranges. This study further investigated the source of the high AP activity in bovine NS. Histochemical analysis using AP specific staining confirmed the localization of the AP enzyme activity to epithelial cells and serous glands of the nasal mucosa. Advanced molecular methods were used to determine the characterization of nasal AP. The analysis at mRNA levels from nasal mucosa by endpoint RT-PCR and PCR product sequencing confirmed that the AP was locally produced and is identical at the nucleotide level to the non-specific AP splice variant gene (ALPL) found in bovine liver, bone and kidney. Investigation using isoelectric focussing (IEF) confirmed that AP was produced locally at a high level in nasal epithelium demonstrating that novel AP isoenzymes in NS had pIs in the same range as those of the nasal mucosa extracts (pH 4.8-6.2) but were clearly different from the extracts of other tissues. The differences in IEF migration of the AP extracts is likely to be due to post translational modifications (PTMs) such as glycosylation or phosphorylation which would be areas worthy of future research. Preliminary proteomic investigation of NS from healthy cattle using 1D gel electrophoresis, 2D gel electrophoresis and ESI-MS/MS analysis putatively identified 10 major proteins in NS consisting of 7 vascular proteins and other glandular and cellular proteins such as lactoferrin, an anti-bacterial protein commonly present in mucosal secretions, odorant binding protein known to have a role in scent recognition and glutathione-S-transferase, an enzyme capable of detoxifying noxious compounds. The final objective was therefore to compare the nasal proteins from healthy, disease and vaccinated group of animals using 2D difference gel electrophoresis (DiGE). The experimental model used for this investigation was an immunisation study against bovine malignant catarrhal fever (MCF). It was concluded that quantitative proteomics technology such as DiGE identified and measured changes in nasal protein expression in response to MCF and following vaccine protection. In conclusion, this research has contributed to the scientific knowledge regarding the biochemical properties and protein compositions that is present in bovine NS. In addition, the research also explored the use of proteomic technologies as a novel tool to analyse protein expression and identify possible biomarkers in NS. This thesis is an important step forward for a better understanding of bovine NS, and thus provides a basis for future studies involving bovine NS by way of providing reference data and alternative source of biological sample.

This study was an investigation of aspects of the biology of sheep blowflies in relation to their control by trapping. In order to determine the species composition of blowfly larvae in ovine analysis cases in Scotland, samples of dipteran larvae were collected from live sheep throughout Scotland, reared in the laboratory, and identified once adult flies emerged. Lucilia sericata was found in 77% of samples, and other species in 49%. The most common alternative species were L. caesar, which occurred in 31% of samples, and Protophormia terraenovae, which occurred in 18%. Three other calliphorid species, Calliphora vomitoria, C. vicina, and L. illustris, and the muscid Muscina pabulorum were also found. The proportion of samples containing alternative species was significantly lower in eastern Scotland than in western Scotland. Significantly higher proportions of samples containing alternative species were collected at altitudes of 200 metres and above; from sheep of hills breeds; from rough grazing conditions and moorland; in the absence of trees; and in the presence of bracken. The importance of Lucilia caesar in myiasis cases in Scotland having been confirmed, the capture of this species was investigated using four different trap designs, all baited with beef liver and sodium sulphide solution. A horizontal target coated with a polybutene-based adhesive performed significantly better than a similar vertical target. Both of these adhesive designs demonstrated significantly higher catches of both male and female flies than a water trap and a commercially-produced enclosed trap, Fly City (P < 0.05). Subsequent investigations showed that catches on adhesive targets were significantly greater at a height of 0.2m than at ground level (P < 0.05), 0.6m, or 0.8m (P < 0.01). Catches were also increased on larger targets (P < 0.05), but there was no significant increase in catch per unit area with target size.

A number of biochemical and haematological changes are known to occur in horses involved in long distance riding competitions of 40- 160 km, particularly changes associated with disturbances in fluid/ electrolyte balance, catabolism of body fuel stores and alterations in the integrity of the muscle cell membrane. This study investigated these changes in more detail in 50 horses involved in competitive rides and in four horses undergoing two 80 km rides under controlled conditions. In addition, experiments were carried out on horses and ponies exposed to a hot, humid environment (41°C, 33°C wet bulb) and during intravenous adrenaline infusion, in order to study further the fluid/ electrolyte alterations associated with sweating and in particular the composition of equine sweat. Changes in plasma and urine biochemistry were also studied over 24 hours in horses at rest for comparative purposes. Significant changes were shown in 13 of the 14 plasma parameters measured in the resting horses. Most of these could be related to feeding, in particular to hay feeding which caused alterations in fluid/ electrolyte balance associated with salivary secretion. Urine composition changed very markedly during the 24 hours. Urine flow rate and creatinine and urea excretion were higher during the day and increased following drinking, as in man. Urine potassium and chloride excretion were much higher than sodium excretion and all three electrolytes (and pH, which was alkaline) showed diurnal variations markedly different from those in man. The competing endurance horses demonstrated moderate haemoconcentration, but plasma electrolyte alterations, particularly an increase in sodium concentration, were not always consistent with the production of apparently hypertonic sweat. The pattern of fuel utilisation was one of exhaustion of liver glycogen after about 40 km with extensive fat mobilisation and the use of glycerol for gluconeogenesis. Breakdown of phosphocreatine was extensive and evidence of protein catabolism was observed. Large variable increases in plasma CK and AST activities unassociated with fatigue suggested a non-pathological alteration in muscle cell membrane integrity in a number of horses. During heat exposure changes in PCV and plasma proteins were poorly related to fluid losses but changes in electrolyte concentrations were consistent with the sweat tonicity. Hypertonic chloride and potassium, and isotonic sodium concentrations (relative to plasma) were maintained in the sweat for 4.5 hours. In contrast sweat magnesium and protein concentrations were initially high but decreased exponentially with time. There was a very close correlation between these two parameters. Most of the changes in plasma parameters seen during adrenaline infusion were attributable to the adrenaline per se, but the profuse sweating induced in the horses caused some haemoconcentration. Small ponies sweated much less in response to adrenaline than Thoroughbred horses. Hypertonic sweat concentrations of chloride, sodium and potassium were maintained for 3 hours and significant differences, particularly in Na/K ratio, were found between heat and adrenalineinduced sweat. Sweat urea concentration was related to plasma urea concentration and glucose appeared in the sweat when the plasma glucose concentration exceeded 10 - 12 mmol/l. The sweat magnesium was not protein-bound and the two main electrophoretic fractions of the sweat protein were not present in serum. The possible function of this protein as a wetting agent was discussed. During the controlled 80 km rides total fluid loss was 33.5% of the total ECF volume: 78% of this was sweat and 22% respiratory evaporation. Although sweat electrolyte concentrations were again hypertonic to plasma, theoretical concentrations in total body (sweat plus respiratory) water loss were much closer to plasma concentrations - slightly hypotonic for sodium, slightly hypertonic for chloride. As a result changes in plasma electrolyte concentrations were small, an increase of 9 mmol/1 (5.7%) in sodium and a decrease of 4 mmol/l (3.5%) in chloride. Potassium appeared to move out of the intracellular fluid at the start of exercise and back in immediately afterwards and this obscured any effect of sweat losses on plasma concentration. The only urinary constituent which was conserved in the exercising horses was chloride, and the absence of any decrease in urea excretion suggested that the increase in plasma urea concentration was due to increased protein catabolism, probably from the liver. Body fuel utilisation was similar to that seen in the competing horses and plasma CK and AST activities again suggested that a non-pathological disruption of muscle cell membrane integrity was occurring in some horses which continued intermittently for several months. It was concluded that in the horse, unlike man, the thermoregulatory fluid is approximately isotonic to plasma, which minimises electrolyte imbalances and allows prolonged exercise with less need for drinking. After exercise the large caecum and colon and the sodium contained in them appear to be important in the controlled replacement of the extensive water and electrolyte losses which result.