The work presented in this thesis was undertaken as part of a larger research initiative in the field of ruminant paratuberculosis (Johne's disease). One of the group's main research objectives was the identification and characterisation of subspecies-specific genes and proteins of the two members of the <I>Mycobacterium avium </I>complex (MAC) known to have a causal role in ruminant paratuberculosis, namely <I>Mycobacterium avium</I> spp. <I>paratuberculosis (M. a. paratuberculosis</I>) and IS901/902-positive strains of <I>Mycobacterium avium</I> (<I>M. avium</I>). A protein antigen of 40kDa (p40) was identified in an IS901/902-positive strain of <I>M. avium</I>, but could not be detected in <I>M. a. paratuberculosis</I>, IS901/902-negative <I>M avium</I>, or in any of 13 other species of <I>Mycobacterium </I>tested. Examination of 19 further MAC field isolates confirmed the absolute association between p40 and IS901/902, suggesting that p40 is a novel, subspecies-specific protein which may potentially be of value as a diagnostic antigen. Soluble p40 antigen was precipitated from cleared cell lysates and purified to homogeneity using a series of chromatographic separations. Chemical cleavage and Edman degradation of the purified p40 antigen provided both amino-terminal and internal amino acid sequence data which showed no sequence identity with any protein sequence currently in the OWL database. Translations of IS901/902 in all six reading frames revealed amino acid sequences which confirmed that the genomic insertion sequence does not encode the p40 antigen. A second protein of 30kDa (p30) was purified simultaneously, and analysis of the first 10 amino-terminal amino acid residues revealed up to 90% sequence identity with the mature secretory antigens of the "antigen 85 complex" of 8 other species of <I>Mycobacterium.</I> Experiments were designed to ascertain whether the p40 antigen was expressed <I>in vivo</I> and to assess whether any immune response to the p40 could be exploited in the development of an immune-based assay to differentiate between animals infected with <I>M. a. paratuberculosis</I>, and others infected with IS901/902-positive status of <I>M. avium. </I>

No description available.

No description available.

The scavenging village chicken is important to millions of smallholders in Ethiopia, as in other less-economically developed countries, for its contribution to the economic, nutritional and social well-being of farmers, especially women and children. Infectious diseases are frequently cited as the greatest constraint to village chicken production, and in Ethiopia, most mortality is attributed to seasonal outbreaks of Newcastle disease (ND). This study conducted four cross-sectional surveys over an 18-month period in two geographically distinct regions of Ethiopia, to examine a range of bacterial, viral and parasitic infections in randomly-selected village chickens, and to look at their 6-month survival rate. The two chicken populations of the different regions were found to be different in terms of their population dynamics and phenotypic characteristics, and these may be driven by farmer demands, which are dictated by the local economic and cultural value placed on specific qualities in the chickens. Over the course of the study, no large outbreaks were observed in the eight villages which took part in the study, and only 9 out of 1280 birds (0.7%) were found to be serologically positive for ND. However, even in the absence of large outbreaks, around 20% of the birds in the study were reported to have died of disease within the 6-month follow-up period, and a further 13% lost to predation. Both location and seasonal variation influenced a bird’s fate, as did farmer decisions, such as choosing birds with specific characteristics to sell or eat. Rather than large outbreaks, the rainy season appeared to be associated with increased small-scale losses, and a variety of signs were described, suggesting several pathogens may be involved. No single infection measured at the time of sampling was a good predictor of subsequent death from disease; instead different pathogens appeared to be important in each region, and reduced the probability of survival through a variety of mechanisms. Positive correlations between Pasteurella and Salmonella, and between Marek’s disease and parasitic diseases were identified, but fewer birds than expected were identified with pathogens from both these groups, perhaps suggesting a decreased chance of survival for co-infected birds. Strong seasonal variation in prevalence was not observed for any of the infections in the study, suggesting that seasonal rises in disease mortality are unlikely to be attributable to a single infection, but other factors may play a role, including an increased probability of co-infection. This makes it difficult to prioritise control strategies for individual diseases; instead development programmes may find broad-based strategies, such as improving hygiene and chick management may be more beneficial to minimise the small-scale losses. Programmes also need to be tailored to local needs rather than assuming a blanket strategy will work equally well for all farmers or regions. Any development strategies to control single diseases should consider potential impacts on non-target infections, due to the existence of multiple interactions between pathogens in this system.

Neospora caninum is an obligate intracellular protozoan parasite, which is the most frequently diagnosed abortifacient in dairy cattle in the UK and is a leading cause of abortion worldwide. Neospora caninum infection in early gestation is associated with foetal death whereas in late gestation, infection can result in the birth of asymptomatic, but persistently infected animals. How the parasite kills the foetus is not fully understood, but it has been suggested that more mature foetuses are better able to mount a stronger immune response to control parasite multiplication and dissemination. The ability of the bovine foetus to respond to various antigens develops in a sequential fashion during the gestation period and foetal immunocompetence starts to develop at approximately 100 days gestation age (dg), but can only fully recognise antigens during mid-gestation at around 150 dg. Chapter 2 assessed the pathological effects of N. caninum on bovine foetuses in early and late gestation (70 and 210 days gestation, respectively) and also in foetuses from naturally infected dams after recrudescence of N. caninum in mid to late gestation. Based on results of an initial histological screen of 35 bovine foetuses and 2 new-born calves, a total of 12 foetuses and calves were selected and subjected to more detailed histological examination. Both haemolymphatic and non-haemolymphatic tissues were used. The distribution of N. caninum antigen, CD3-positive T cells, PAX5-positive B cells, monocytes/macrophages and neutrophils (myeloid/histiocyte antigen/calprotectin-positive), antigen presenting cells (MHCII), interferon gamma (IFN-γ) expressing cells, PCNA-positive proliferating cells and apoptotic cells (cleaved caspase 3-positive) was analysed by immunohistology. In uninfected, control foetuses in early gestation (90 days gestation), haemolymphatic tissues were moderately developed and exhibited normal morphological features with low lymphocyte turn over and no evidence of IFN-γ production. Uninfected foetuses in late gestation had fully developed haemolymphatic tissues with high lymphocyte turnover, indicative of a mature immune system. In the infected foetuses in early gestation, extensive apoptosis of lymphocytes was observed in the thymus and spleen compared to controls (p<0.001, student’s t-test). No histological changes were observed in the haemolymphatic tissues of infected foetuses in late gestation. In non-haemolymphatic tissues, infected foetuses in early gestation exhibited extensive hepatocellular necrosis and apoptosis, glial cell necrosis and apoptosis in the CNS and high parasite loads in the liver, CNS and myocardium. There was no evidence of cell death in the heart despite the high parasite loads. In late gestation, histological lesions were restricted mainly to the CNS where non-suppurative inflammation and low parasite loads were observed. Other non-haemolymphatic tissues exhibited only mild mononuclear inflammatory infiltrates. The results suggest that in early gestation, tachyzoites replicate preferentially in foetal liver, brain and myocardium in the absence of an inflammatory response and cause extensive necrosis in the liver and brain. Unlike foetuses in early gestation, those in late gestation exhibited a mild to moderate mononuclear inflammatory infiltrate in various tissues dominated mainly by lymphocytes, plasma cells and smaller numbers of macrophages. In Chapter 3, the observation that N. caninum appeared to induce cellular degeneration in hepatocytes but not in the myocardium was investigated in more depth. An in vitro tissue culture system using the human HepG2 hepatoma cell line and the murine HL-1 cardiomyocyte cell line was used to establish the mechanism of cell death following N. caninum infection. The activation of the initiator and effector caspases (caspases 3, 8 and 9) was measured and the mitochondrial organisation in cells following N. caninum infection evaluated. Quantitative (caspase 3) and semi-quantitative (caspase 8 and 9) analyses were used to assess differences in N. caninum-infected and uninfected HepG2 and HL-1 cells. A significant difference was observed in the numbers of cleaved caspase 3-positive HepG2 cells at 20-36 hours post infection (p=0.029, Mann-Whitney U test) in infected cultures compared to controls. No significant difference was observed for caspase 8 and 9 expression. In HL-1 cultures, no significant difference was observed in the number of caspase 3, 8 and 9-positive cells between infected and control cultures. This suggests that N. caninum infection is not associated with activation of the caspase cascade in cardiomyocytes. Neospora caninum tachyzoites were detected within intact HepG2 and HL-1 cells with normal cellular morphology and which were not labelled with the caspase antibodies; whereas uninfected surrounding cells were caspase 3, 8 and 9-positive, indicating that the parasites are involved in the inhibition of the caspase pathways (intrinsic and extrinsic). The mitochondrial organisation in N. caninum-infected and uninfected cells was assessed in both cell lines using double immunofluorescence, which involved staining with a N. caninum specific polyclonal antibody and COX 1 mitochondrial marker. In the control cultures of both HepG2 and HL-1 cells, mitochondrial clumping with large aggregates of mitochondria exhibiting a punctate pattern was observed in high numbers of cells, mainly in the perinuclear region and this is suggestive of mitochondrial fragmentation, which is associated with apoptosis. Other cells within the control cultures revealed an unaltered reticular pattern of mitochondria that is consistent with the normal cellular morphology. In the infected cultures, there was mitochondrial clumping with aggregates of mitochondria detected surrounding parasitophorous vacuoles; while in neighbouring uninfected cells, large aggregates of mitochondria, exhibiting a punctate pattern were present, suggesting mitochondrial clumping and fragmentation associated with cytochrome c release and apoptosis. Other uninfected HepG2 and HL-1 cells exhibited a diffuse, homogenous distribution of mitochondria, often with an unaltered reticular pattern as was observed in the control cultures and is consistent with the normal cellular morphology. The results indicate that N. caninum inhibits apoptosis in infected cells and is associated with increased apoptosis in infected HepG2 cultures, while not having any effects on HL-1 cardiomyocytes. Chapter 4 investigated the seroprevalence of N. caninum infection in Jamaican dairy herds. Serum samples were analysed from 499 Holstein-Friesian and Holstein Friesian crossbreed dairy cattle from three different farms in Jamaica. A seroprevalence of approximately 26% was found with the majority of seropositive animals aged 0-2 years old (25%), while the lowest seroprevalence was recorded in animals over 13 years old (13.3%). Pregnancy status was shown to influence the seroprevalence of cows, but no significant relation of seropositivity to age was found, suggesting that vertical transmission is the principal route of transmission in Jamaica.

The effect of the teratogenic agents, Insulin, Janus Green B, Lrthyroxine and 3-Acetylpyridine, as well as hypoxic and hyperoxic conditions, on neural tube development in the early chick embryo are investigated in relation to the extension of the normal necrotic zone at the neural fold region. Several substances involved in NAD+ metabolism are also considered as possible anti- teratogenic agents to Insulin and 3-Acetylpyridine. The teratogens and anti-teratogens were administered to embryos explanted by the New technique at St.7 of development and in the case of insulin, also in ovo. Due to the view that cell necrosis is an important consideration in teratology, the embryos were examined both at. the light- and electron-microscope levels for evidence of an increase in the physiological zone of necrosis at the neural fold region responsible for defects in neural tube development, including spina bifida. The result of biochemical estimations of NAD+ NADH, cyclic AMP and cyclic GMP carried out on insulin- and 3-acetylpyridine- treated neural tubes when considered along with previous reports on the effect of insulin and 3-acetylpyridine on NAD+metabolism, now lead to new proposals to explain the mechanism of action of the teratogens on the neural folds. Also discussed is the suggestion that a gradient of nutrients and essential molecules is present and responsible for the appearance of the normal necrotic zone at the neural folds. This view receives support from recent reports that a gradient of essential molecules is responsible for the commitment of mesodermal cells to a particular during chick limb development.

The literature relating to the production, absorption and utilisation of volatile fatty acids by growing and fattening ruminants was reviewed. It was concluded that the hypothesis which moat satisfactorily explained the poor utilisation of high fibre diets by fattening ruminants was that of Armstrong, Blaxter, Wainman and Graham (1958) which attributed the poor utilisation of such diets to an inefficient utilisation of acetate. However, subsequent work on the utilisation of the VP A, and of acetate in particular did not confirm this hypothesis, and it was suggested that apparently conflicting results reported in the literature might be reconciled if the efficiency with which acetate was utilised declined as its contribution to the energy available for lipogenesis increased. 2) In the work reported here the utilisation of acetate by growing lambs was investigated in two cooperative slaughter experiments and one calorimetry experiment. The utilization of propionate and butyrate was also investigated in the second of the comparative slaughter experiments. In all experiments the VFA were given as a mixture of sodium and calcium salts which were substituted for the ME provided by the carbohydrate of a barley based control diet. The ME value of the salts was assumed to be equal to the heat of combustion of their acid equivalents. The rumen pH, VFA and cation levels produced by some of the diets used were measured in too further experiments. 3) It was concluded that differences in the efficiency with which the diets were utilised for energy retention was mainly due to the utilisation of the VPA per sc. And was not due to differences in the metabolisability of the basal constituents of the diets, or to a cation effect. 4) Differences between diets in the efficiency with which they were utilised were caused by differences in fat deposition. There were no differences between VPA in their ability to promote protein deposition. 5) In the second comparative slaughter experiment, acetate provided 0, 7, 15 or 22% of ME and was utilised with a partial efficiency for the three acetate diets respectively. The difference between the Acetate-7 % diet and the Acetate-22 % diet was significant. When the overall efficiency with which the complete diet was utilised for energy retention (kf) was plotted against the proportion of ME provided by the acetate e. its there was a significant quadratic relationship. In the first comparative slaughter experiment (Experiment one), acetate provided 0, 14 or 19% of ME and was utilised with a partial efficiency of 2 or 10--13% for the two acetate diets respectively. The overall efficiency with which the two acetate diets were utilised to promote energy retention was significantly lower than that of the Control. In the calorimetry experiment (Experiment Five) acetate provided 4 or 16% of ME, end the partial efficiency with which the additional acetate of the Acetate-16% diet was utilised was 72%. The overall efficiency of the complete diets was practically identical. 6) The second comparative slaughter experiment (Experiment Three) diets which contained 22% of MS as propionate or butyrate were utilised less efficiently than the Control, but not significantly differently from the Acetate-22% diet described above. The partial efficiency with which propionate and butyrate were utilised was 35 and 17--15% respectively. 7) It was concluded that the efficiency with which acetate was utilised for energy retention was not constant but decreased as its contribution to the MS available for lipogenesis was increased, which is consistant with the concept that efficient utilisation of acetate for lipogenesis is dependent upon a sufficient supply of glucose or glucone precursor. The poor utilisation of butyrete sea consistent with this model if butyrate is utilised for lipo-genesis as a source of two-carbon units. The poor utilisation of propionate may have been due to the ability of the animals to synthetise or utilise glucoses being limited, and the fact that when acetate was substituted for 7% of barley ME, the diet was utilised alightly more efficiently than the control may imply that there is an optimum relationship for the efficient utilisation of both these metabolites. 8) The depot fat of lambs given propionate contained enhanded proportions of odd-chain and branched-chain fatty acids 9) The rumen VFA of cheap given diets containing VFA salts showed considerable diurnal variation which reflected the composition of the diet given. The rumen pH of sheep given diets containing VFA salts fluctuated less than when given the barley based control. The concentration of calcium in the digests of sheep given diets containing VFA salts was significantly increased whereas the concentration of sodium and potassium was similar to that from sheep when given the control diet.

The literature on the physiological processes involved in the establishment of pregnancy in the ewe was reviewed with particular reference to the prolific and nursing ewe. Experiments were designed to provide information on the early growth of foetuses in prolific ewes and on conception and embryo survival in the nursing ewe. Experiment 1 provided data on foetal growth during early pregnancy in Finnish Landrace x Dorset Horn ewes. In experiment 2, the consequence on the within-litter variability in foetal size of standardizing the number of embryos in each uterine horn was examined. Two embryos were transplanted into each uterine horn of 15 recipient ewes. The ewes were slaughtered on the 60th day of gestation and their gravid uteri dissected to ascertain litter size and the distribution of the foetuses within the uterus. Foetal weights and placental sizes were recorded. The implications of the findings of experiments 1 and 2 that within-litter differences in foetal weight are established in early pregnancy and are associated with within-litter differences in placental size are discussed in relation to the viability of the newborn lamb. Experiment 3 provided information on the endocrine changes in the ewes in experiment 1 following progestagen withdrawal. The timing and magnitude of the preovulatory surges of oestradiol-17 and LH and subsequent luteal secretion of progesterone were considered in relation to the ovulation rate of the ewes. In experiment 4, the effects of introducing a restricted-suckling regime after 5 weeks of lactation on the conception and lambing rates following matings to an induced oestrus in the ninth week of lactation were investigated, both outwith and during the breeding season. Pregnancy failure in free-suckling ewes was associated with both premature and delayed luteal regression. In experiment 5, detailed observations were made on the endocrine status of ewes subjected to different suckling frequencies after 4 weeks of lactation both outwith and during the breeding season. The resumption of ovarian activity in the post-partum period was delayed in nursing ewes when basal prolactin concentrations were low, but compared to the early-weaned ewe the nursing ewe remained anovular for an extended period post-partum when basal prolactin concentrations were high. Differences in the interval from parturition to the resumption of ovarian activity were not associated with differences in tonic LH secretion. Prolactin release at a suckling episode was greater during anoestrus than in the breeding season. During the breeding season basal prolactin concentrations in lactating ewes at the time of remating had reached the same low level observed in ewes weaned at 4 weeks. The transition from anoestrus to cyclic activity was marked by an increase in the frequency of the pulsatile release of LH and a decrease in the amplitude of each LH pulse. Following progestagen withdrawal and PMSG injection the pre-ovulatory rises in oestradiol-17, LH and prolactin were not influenced by lactation or the frequency of suckling but showed marked variations with season. At the induced oestrus in December, oestradiol-17 concentrations rose higher and reached maximum concentrations earlier than in May. The interval from progestagen withdrawal to the LH surge was shorter and the magnitude of the surge lower in December than in May. In December when basal concentrations of prolactin were very low, the pre-ovulatory surge of prolactin was more pronounced than in May.

No description available.

To establish the prevalence of diseases in sheep flocks in North-east Scotland responsive to the application of preventative measures which may be incorporated in flock management plans. A survey of a sample of commercial sheep flocks was carried out to ascertain management and marketing practices. A critical assessment was made of the 'Flock Diary' 'Detailed' and 'Daily' Lambing Diaries produced by the Meat and Livestock Commission and their use. A mastitis survey showed an overall incidence for acute severe and chronic mild mastitis of 1.5% and 4.2% respectively. Orf was a particularly important predisposing factor. Antibiotic therapy at weaning resulted in a reduction in the number of ewes culled for chronic mastitis but growth rate of subsequent lambs was unaffected. A survey of trace element status of flocks showed that deficiencies were not always recognised especially selenium. On-farm trials were set up to examine the implications of copper, cobalt and selenium deficiencies and possible methods of control and monitoring. Trials of these procedures in commercial flocks included measurements of blood composition, ewe body condition score, lambing data, lamb birth weight and early growth rate. In lamb based trials blood composition and growth rate were the only parameters measured. Trials over a 4 week period were carried out to test the effectiveness of selenium-cobalt supplemented anthelminthics. The cobalt content of such products was inadequate to maintain pre-existing cobalt/vitamin B12 status in weaned lambs, however, selenium content induced a sustained rise in selenium/GSH-Px. Treatment with oxytetracycline reduced the number of chlamydial abortions in vaccinated gimmers while selenium treatment reduced the stillbirth rate. There was a suggestion that a compliment fixation titre of 1/16 was indicative of infection. Factors limiting implementation of preventive measures in flocks were the absence of performance and health records, accurate diagnosis, effective communication between farmers and external agencies and sources of integrated advice.