Within the pig population of Northeast Scotland resistance to tetracycline and ampicillin was found to be widespread among commensal <I>Escherichia coli, </I>as also noted among anaerobic commensals of the genera <I>Bacteroides </I>and <I>Lactobacillus. E. coli </I>resistant to apramycin and nalidixic acid and enterococci resistant to vancomycin were less frequently isolated but remained common. Production system, piglet age and recent transportation were found to influence the antibiotic resistant proportion of the faecal <I>E. coli </I>population whilst dietary copper inclusion did not. Within the intestinal tract higher proportions of resistance to ampicillin were noted among the <I>E. coli </I>of the ileum and caecum than more distal sections and to apramycin and nalidixic acid among the ileal mucosal associated <I>E. coli </I>than those of the ileal lumen. Novel techniques for characterising and modelling antibiotic resistant bacterial populations were suggested and investigated. A spiral-plater system was used to investigate the phenotypic expression of antibiotic resistance among faecal <I>E. coli, </I>complemented by the development of a PCR technique to identify the genetic basis of tetracycline resistance. The effects of antimicrobial supplementation on the indigenous enteric flora were monitored through the validation and use of an <I>in vitro </I>porcine ileal simulation. Within the same model transmission of genetic material encoding antibiotic resistance was demonstrated between commensal and pathogenic representatives of the <I>Enterobacteriaceae </I>under ileal conditions. Variation in the mucosal adherence of <I>E. coli </I>expressing different antibiotic resistant attributes was investigated <I>in vitro </I>following the implementation of a modified cell culture technique.

The rationale of this thesis was to examine and extend knowledge of boar semen quality, its evaluation and factors which influence quality; with the objective of improving the effectiveness of pig AI. The literature review led to the conclusion that the development of a semen evaluation technique which describes the overall quality of the sperm, correlates well with most semen quality parameters, and most important, applicable at farm level would be a valuable advance. In addition knowledge is needed of the factors affecting semen quality and in particular better semen dilution techniques. Studies on the effect of various factors on semen quality showed: i. With two boar lines (purebred, PB) and (crossbred, CB), CB gave higher total sperm counts (P<0.05), but PB gave better farrowing rates (87% V 78%, P<0.05), ii. Semen collected in Autumn/Winter had higher total sperm counts than semen collected in Spring/Summer (66.4 V 47.8 x 10⁹, P<0.001), iii. General semen quality did not change significantly over 48 hours in the study, iv. Pooling of semen had no effect on semen quality. Semen dilution by a controlled 'Anti-shock' procedure gave a higher proportion of litters with 11 or more piglets born than semen diluted by the standard procedure (78% vs 62%, P<0.05). Sperm motility was affected when, in Kiev diluent, glucose (G) was replaced by fructose (F). Motility was F vs GF vs G: 84.8%; 84.1; 81.0, but by day-3 was 27.7, 60.2 and 62.0 (P<0.01). No significant differences were found between diluents in AI sow fecundity, probably due to the limited numbers of sows. It is concluded that although farrowing rate and litter size are the ultimate indices of semen quality, good correlations with <I>in vitro</I> techniques have yet to be established. The 'swim-up' test is suggested as the most suitable on farm <I>in vitro</I> semen assessment, and that the 'Anti-shock' dilution technique has commercial promise. Fructose should be the usual sugar used in the diluent.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

Leishmania aethiopica causes two distinct forms of cutaneous leishmaniasis in the highlands of Ethiopia and in northern Kenya: the generally self-healing localised cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL), which does not self-heal. Unlike other Leishmania parasites, the pathological and infectious processes associated with exposure to this parasite are not well studied in experimental systems, mainly due to the lack of a suitable in vivo model. This study was undertaken to develop an experimental model to examine the distribution and fate of parasites and pathological processes that take place after infection with L. aethiopica. It was also the aim of this work to develop a diagnostic system to directly identify parasites in potentially infected tissue with or without the presence of clinical signs of disease. Several laboratory animals including 12 strains of mice from a spectrum of genetic backgrounds, two strains of hamsters and other experimental animals such as rabbits, guinea pigs, gerbils and cotton rats were tested. All animals were inoculated with late stationary phase culture promastigotes of L. aethiopica and examined over a period of 18 months. Evaluation of the outcome of infection was monitored by observing the production of lesions and isolating cultivable parasites from sites of inoculation. The fate and distribution of parasites was investigated using culture, histology and PCR to identify L. aethiopica-specific DNA. The results revealed differences in susceptibility to L. aethiopica infection within the strains of mice used. Cultivable parasites were isolated from the footpads of BALB/c, BALB/c nude and CBA nude mice up to 16 days post infection whereas SCID mice retained live parasites for a longer period of 20 days. CBA and C57/BL mice cleared cultivable parasites from sites of inoculation within 8 and 12 days respectively. Overall, BALB/c mice retained detectable parasite DNA for the longest period (550 days) post-infection in the footpads, draining lymph nodes and the liver. Among mice with intact genetic backgrounds, only BALB/c mice showed cellular in vivo responses at the site of parasite inoculation that peaked at day 13. Hamsters were found to be the only animals that produced clinical lesions (46%). In addition, live parasites were isolated from the site of inoculation (55%) and from the draining lymph nodes (50%) after 390 days post-infection. L aethiopica kinetoplast DNA was detected up to 470 days post infection in 82% of noses and 91% of draining lymph nodes. Hamsters exhibited the ability to retain cultivable parasites late in the course of infection regardless of the presence or absence of lesions. These results indicate that hamsters are potentially suitable models to study the fate and distribution of L. aethiopica. The detection of parasites using the amplified DNA signatures was found to be superior to conventional methods such as histology and culture in that it was sensitive, specific and simple. The PCR method that was developed during this work involved extraction of nucleic acids from cultured parasites, tissues and smears, amplifying L. aethiopica-specific DNA using a universal primer for a conserved Leishmania kinetoplast sequence together with an L. aethiopicaspecific oligonucleotide. The amplified fragments were 864bp in length and were initially characterised by restriction digest analysis using Hae HI restriction enzyme digestion as a result of which several patterns were recognised. To confirm this diversity in k-DNA sequences, amplified fragments were cloned using pCRII® TA cloning kit and the recombinant clones were digested to confirm the patterns observed initially and were sequenced. The sequences that were generated were analysed using the BLAST alignment facility of the National Centre for Biotechnology Information (NCBI). All fragments were Leishmania kinetoplast in origin but were found to possess varying percentages of similarity ranging from 50% to 100% among themselves. Analysis of these results suggested that L. aethiopica isolates that originated from individual patients possess genetic differences in their k-DNA sequences. In summary, the results generated from this work indicate that the hamster is a suitable model for studying cutaneous leishmaniasis caused by L. aethiopica and that both hamsters and mice can be used to study parasite distribution in experimental leishmaniasis. It was also revealed that there are genetic differences within the kinetoplast of L. aethiopica isolates. These genetic differences within L. aethiopica DNA could be exploited to better understand the diversity within the causative agent of Ethiopian Cutaneous Leishmaniasis.

K99 was chosen as a model target for this study, to explore the application of recombinant antibody technology to livestock infection. Our aims were to isolate and characterise single-chain Fv antibodies against K99 using phage display. Escherichia coli B41, a clinical isolate that expresses K99 fimbriae, was grown and fimbriae were extracted by heat-shock treatment and then precipitated by ammonium sulphate. The major K99 subunit (Fan C) was purified from crude fimbriae extract by an ion-exchange chromatography method using SP-XL columns. The semi-synthetic Tomlinson I and J libraries (Center of Protein Engineering, Cambridge, UK) were used to select phage antibodies against K99 using immunotubes coated with the major fimbrial subunit. After three rounds of selection, phage were transfected into E. coli HB2151 for the expression of soluble scFvs. Fifteen scFv clones with high activity against K99 fimbriae were identified by ELISA and sequenced. Of these, six scFvs carried sequences that were reasonably diverse. These proteins were purified for further characterization. The recombinant antibodies were shown to react with fimbriae present at the surface of E. coli B41 using immunofluorescence microscopy and immunogold electron microscopy. Some of the purified scFv antibodies were also able to inhibit the agglutination of sheep erythrocytes by E. coli B41 grown at 37°C. To pursue this observation, attempts were made to use the scFvs in an in vitro model of bacterial colonisation in which bacteria were tested for attachment to isolated bovine intestinal villi. Although bacteria could be observed adhering to the brush border, the scFvs appeared unable to prevent this attachment. Further experiments with this in vitro model or a mouse model of ETEC infection, allied with epitope mapping studies should determine if anti-colonisation activity is attributable to binding of scFvs to the receptor-recognition site on the major subunit of the adhesin.