Despite Africa accounting for ~20% of the global cattle population, prevalence estimates and related risk factors of bovine tuberculosis (bTB), caused by Mycobacterium bovis, are still poorly quantified in many countries across the continent. Control of bTB in Africa is difficult due to poor monitoring of cattle movements and limited abattoir surveillance. Also M. bovis is zoonotic and risk factors for transmission include living in close contact with cattle and consumption of unpasteurised milk. Cattle keeping is integral to some rural populations in Cameroon and understanding the epidemiology of bTB in cattle populations is important both to bovine and public health. Detection of bTB in cattle is difficult due to variability of immune responses to M. bovis infection. The interferon-γ (IFN-γ) assay maybe useful to estimate bTB prevalence and identify bTB risk factors in Cameroon. However its performance can vary at different stages of bTB pathogenesis and in different cattle populations. Recently Fasciola hepatica co-infections have been reported to suppress IFN-γ responses in M. bovis infected cattle but the potential effect with F. gigantica co-infections on bTB prevalence estimates in Cameroon is unknown. An abattoir study was conducted in Cameroon to assess the performance of the IFN-γ assay. In 2012-13; 2064 slaughtered cattle were sampled from Bamenda abattoir (North West Region; NWR) and Ngaoundere abattoir (Vina Division; VD). Individual animal data was collected from routine meat inspection including identification of bTB and Fasciola pathology. Cattle were also tested for bTB using the IFN-γ assay and an M. bovis antibody ELISA. In the absence of a gold-standard diagnostic, the IFN-γ assay was compared to other diagnostic tests to assess agreement and identify factors that affected performance of the assay. Agreement between IFN-γ assay, TB lesion identification and an M. bovis antibody ELISA was poor-moderate, probably partly related to differences in immune response detected. A presence of Fasciola gigantica also increased the odds of false negative IFN-γ assay results. On further investigation co-infected cattle had increased odds of TB lesions and reduced IFN-γ responses that potentially could lead to ~20% reduction in test sensitivity. In an attempt to take into account the potential impact of F. gigantica, when estimating bTB prevalence, an antibody ELISA was developed to detect the exposure in live cattle. To highlight the awareness of disease in cattle-rearing communities, estimate prevalence and identify risk factors of bTB in cattle populations; two cross-sectional studies were conducted in 2013. A stratified clustered cross-sectional study of pastoral cattle herds, in the NWR and the VD, sampled 1448 pastoral cattle reared by 100 pastoralists. A smaller cross-sectional study sampled 60 dairy cattle from 46 small-holder co-operative dairy farmers. Individual animal data and herd-level data were collected and animals were screened by both the single comparative intradermal skin test (SCITT) and IFN-γ assay. Awareness of zoonotic TB was low yet consumption of raw milk was high in cattle-keeping communities highlighting the need for accurate bTB prevalence estimates. Despite the high awareness of the clinical presentation of bTB, clinical signs identified by pastoral herdsmen were not associated with cattle being bTB positive. The SCITT was used to compare two manufacturers cut offs for the IFN-γ assay, ≥0.05 and ≥0.1, and highlighted that these two diagnostics may detect different populations of bTB positive cattle. Using the IFN-γ assay at ≥0.1, bTB prevalence was highest in dairy cattle (21.67%) and was also present in pastoral cattle in the NWR and VD (11.33% and 6.55% respectively). Importantly, as F. gigantica is endemic in Cameroon and its influence could mean the true prevalence of bTB could be higher. Female pastoral cattle were at lower odds of being IFN-γ assay positive potentially due to immunosuppressive factors had lower odds of disease. Husbandry practices also decreased the odds of being IFN-γ assay positive such as drinking from streams, antelope and contact with herds at grazing. Age increased the odds of pastoral cattle being IFN- assay positive potentially being a confounder to chronicity of bTB and other co-infections may influence IFN-γ responses. Dairy cattle herds had different risk factors for being IFN- positive likely due to differences in husbandry practices. Considering the potential risk to public health of M. bovis this thesis highlights the extent of bTB across two major cattle keeping regions in Cameroon and the public health risk in cattle-rearing communities. Furthermore the relationship between Fasciola co-infection and IFN- responses to M. bovis described has potential implications for bTB diagnosis in cattle populations where the parasite is present across the globe.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 4 uppsatser.

Genetic variation was examined in two endangered mussel species, Epioblasma brevidens and E. capsaeformis, and a common species Lampsilis fasciola, in the Clinch River, TN, by screening mitochondrial DNA (mtDNA) sequences and nuclear DNA microsatellites. These species use fish hosts with varying dispersal capabilities, ranging from low, moderate, and high, respectively. Patterns of mtDNA polymorphism exhibited different trends for long-term population sizes for each species during the Holocene (~10,000 ya to present); namely, E. brevidens has declined over time, E. capsaeformis has remained stable, and L. fasciola has expanded. Long-term effective population size (Ne) was smallest in E. brevidens, intermediate in E. capsaeformis, and highest in L. fasciola. Moderately diverged mtDNA lineages, perhaps indicative of secondary contact, were observed in E. brevidens and E. capsaeformis. High levels of gene flow (Nm) were estimated among demes of L. fasciola using traditional F-statistics and likelihood estimates of Nm, whereas such metrics were lower in E. brevidens and E. capsaeformis. Data are consistent with population dynamics and life history traits of each species and their fish hosts. Age, shell growth, and population demography of Epioblasma brevidens, E. capsaeformis, and Lampsilis fasciola were studied from 2004-2007 in a 32-km reach of the Clinch River, TN. Observed maximum age and length of E. brevidens was 28 y and 71.5 mm for males and 11 y and 56.6 mm for females; of E. capsaeformis, 12 y and 54.6 mm for males and 9 y and 48.6 mm for females; and of L. fasciola, 45 y and 91.3 mm for males and 13 y and 62.6 mm for females. For all three species, observed maximum age and length was greater among males than females. Estimated population size in this river reach was approximately 43,000 individuals for E. brevidens, 579,000 individuals for E. capsaeformis, and 30,000 individuals for L. fasciola. Mean recruitment y-1 of 1 y-old E. brevidens ranged from 7.1% to 20%, of E. capsaeformis from 4.0% to 32.4%, and of L. fasciola from 5.8% to 25.6%. Population growth rate y-1 was 24.9% for E. brevidens, 34.6% for E. capsaeformis, and -22.4% for L. fasciola. Mortality rates of females were higher than for males of E. capsaeformis and L. fasciola, but not E. brevidens. Juvenile mussels were collected but temporally and spatially variable in occurrence, and a significant component of the age-class structure of all three species. Recruitment was very high during 2006-2007 for E. capsaeformis and other species, likely due to low river discharges in the spring-summer of 2005-2007. Surplus individuals of E. brevidens and E. capsaeformis are currently available to conduct translocations for restoration purposes. Population modeling of Epioblasma brevidens and E. capsaeformis in the Clinch River was conducted to determine suitable harvest levels for translocation of sub-adults and adults, and to determine quantitative criteria for evaluating performance and recovery of extant and reintroduced populations. For both species, the recommended annual harvest was <1% of local population size to minimize risk of decline. Reintroduction modeling indicated that size of the initial population created during a 5 y build-up phase greatly affected final population size at 25 y, being similar to size at the end of the build-up phase, especially when expected growth rate was low, (e.g., 1-2%). Excluding age-0 individuals, age-1 juveniles or recruits on average comprised approximately 11% and 15% of a stable population of each species, respectively. The age-class distribution of a stable or growing population was characterized by multiple cohorts, to include juvenile recruits, sub-adults, and adults. Molecular genetic and demographic data indicated that the ratio of Ne/Nc was ~5% for both species. Based on this ratio and predicted declines of genetic variation at different population sizes, target sizes for reintroduced or recovered populations of each species should be ≥5,000 individuals (Ne=250) and ≥10,000 individuals (Ne=500), respectively, and should be comprised of multiple smaller demes spread throughout a river. Populations of both species are currently large enough to sustain harvest for translocation and reintroduction purposes, offering an effective species recovery strategy. / Ph. D.

Fascioloides magna (the giant liver fluke) originated from North America, is known in the Czech Republic since 1930s. This pathogenic fluke invades mostly cervids, but livestock too. Excretory-secretory products (ES products) contain number of esential biomolecules which are produced by excretory and secretory system of the fluke. These molecules play key role in many biological process during the life cycle not only of fascioloid flukes (e.g. migration in the host tissues, immune evasion and digestion). Due to their antigenic properties they could be also used in immunodiagnostics. Excretory-secretory proteins from adult Fascioloides magna and comparative related species Fasciola hepatica were purified and separated by the basic biochemical methods (1D, 2D electrophoresis, ion-exchange chromatography) and their activity was confirmed by specific (fluorogenic peptide) and nonspecific (gelatine) substrates. By using the mass spectrometry methods (MALDI TOF/TOF), the most abundant peptidolytically active proteins from ES products of F. magna were identified as cathepsin L (FmCL). Recombinant analog of FmCL was expressed in Pichia pastoris expression system. The peptidolytic activity was again confirmed using the synthetic fluorogenic substrates; the specifity of recombinant FmCL active site was...