The diagnostic performance of four serological assays for bovine brucellosis in African buffaloes, namely Rose-Bengal test (RBT), complement fixation test (CFT), indirect enzyme-linked immunosorbant assay (iELISA) and fluorescence polarisation assay (FPA) were evaluated and compared in a case-control study. The study followed the OIE assay validation pathway for validation of diagnostic tests applicable to wildlife species where there is a validated test available in a taxonomically closely related species. Two uninfected and four infected herds were recruited and an uninfected composite reference panel of 107 sera and infected composite reference panel of 93 were selected using composite reference standards. Diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were calculated for individual tests and for different combinations of two tests in series and in parallel. Cut-off points were adjusted using receiver operating characteristics (ROC) analysis. Using these cut-off values, the index tests performed as follows: RBT DSe of 98.9% (95% CI 96.83% - 100%) and DSp of 98.1% (95% CI 95.6% - 100%), iELISA (cut-off >40.5%) DSe 98.9% (95% CI 94.2% - 100%) and DSp 100% (95% CI 96.6% - 100%), CFT (cut-off >0 iU/ml) DSe 74.2% (95% CI 64.1% - 82.7%) and DSp 100% (95% CI 96.6% - 100%) and FPA (cut-off >16 mP) DSe 97.9% (95% CI 94.2% - 99.7%) and DSp 100% (95% CI 96.6% - 100%). Based on performance index and area under the ROC curve, the iELISA performed best (198.9% and 1.0), followed closely by the FPA (197.9% and 0.989) and the RBT (197.0%). The CFT s lower performance (174.2%, and 0.871) was due to low DSe. Kappa values for test agreement between the index tests was above 0 for all combinations, and varied from unweighted Kappa of 0.685 (95% CI 0.608 0.762) between FPA and iELISA to 0.26 (0.136-0.383b) between CFT and RBT. Consideration of the indices for positive and negative test agreement between the index tests supported the differential specificity of tests for different immunoglobulin classes and higher in line with the findings in cattle. Positive predictive value in herd C and E were 100% for the iELISA, CFT and FPA, 97.3% in herd C and 98.4% in herd E for the RBT. Negative predictive values in herd C ranged from 89% for the CFT to 99.2% for the RBT and in herd E 73.1% for the CFT to 98.7% for the RBT. Overall repeatability was satisfactory, except for the FPA, which was considered the result of sample quality related to prolonged storage in a freezer. The index tests were all found fit for use to detect or confirm brucellosis in populations and individual animals. The values for DSe and DSp that were estimated will be of use in the interpretation of serological results and determination of diagnostic strategies in different circumstances. Different combinations of tests in series and parallel increased the DSp and DSe. Using the RBT in combination with the CFT/FPA/iELISA interpreted in series or in parallel in relation to the epidemiological setting and objective of testing is recommended. / Mini-dissertation (MSc)--University of Pretoria, 2015. / tm2016 / Veterinary Tropical Diseases / MSc / Unrestricted

No description available.

Ph. D.

The polymerase activities involved in bovine parvoviral (BPV) DNA replication in vivo and in vitro have been described. In the in vitro system, purified and partially purified enzymes were used and the replication products were analyzed by gel electrophoresis and restriction enzyme digestion. DNA pol ૪ purified from fetal bovine liver, replicated BPV ssDNA to a unit-length covalently-linked dsDNA hairpin molecule but was unable to utilize purified BPV dsDNA as a template. Partially purified calf thymus DNA pol α replicated BPV DNA to a product 1 kbp smaller than unit-length dsDNA (11 kbp). Mapping of this product showed that the middle of the genome was under-represented. Purified pol a from bovine fetal lung (BFL) cells was capable of only end-labeling BPV ssDNA. If HeLa cell DNA pol α, which consisted of the core enzyme plus cofactors C₁ C₂, was used, the products consisted of both noncovalently- and covalently-linked unit-length dsDNA hairpin molecules. Hence, purification of pol a removed factor(s) necessary for the activity of the enzyme on B PV DNA. The polymerase activities involved in vivo in BPV DNA replication were analyzed using aphidicolin, a specific inhibitor of DNA pol α, and/or L-canavanine, an inhibitor of protein synthesis. DNA present in infected cells was visualized by autoradiography of Southern blots after probing with nick-translated BPV DNA. Aphidicolin, added at any time after-infection, reversibly inhibited each step of BPV DNA synthesis. Conversely, L-canavanine slowed the replication process, inhibited the synthesis of the viral-coded proteins NP-1 and VP3, and inhibited the production of replicative intermediates (RI) and progeny ssDNA. After removal of L-canavanine, both protein and DNA synthesis resumed. These results demonstrate that 1) pol α is involved in every stage of the replication process including the production of parental replicative form (RF), daughter RF, RI, and progeny genomes, 2) taken in conjunction with the in vitro data, that a pol α holoenzyme complex is required for BPV DNA replication, and 3) viral proteins are required for RI and progeny DNA synthesis, / Ph. D.

The mitochondrial utilization of a-ketoglutaric and pyruvic acids have been found to be depressed in guinea pig and bovine ketosis. Addition of sodium acetoacetate to the reaction medium resulted in depressed oxidation of pyruvate and 1-ketoglutarate of isolated normal rat liver mitochondria. / Ph. D.

Bovine parvovirus (BPV) is a small icosahedral virus containing single stranded DNA and belongs to the group of nondefective parvoviruses. Research on this virus group has revealed that viral replication occurs in cycling cells only, and evidence exists that the required factor provided by cycling cells is produced during early or mid S phase. Due to this S phase dependence, parvoviruses replicate synchronously only in synchronized cells. This study was initiated in order to determine the kinetics of replication of BPV in highly synchronized cells, the effects of viral replication on host macromolecular synthesis, and the properties of the double stranded DNA produced in infected cells. Bovine fetal spleen (BFS) cells, in which BPV replicates optimally, were synchronized at the G₁/S border by exposure of cells to 2 mM hydroxyurea (HU) for 32 hr. Immediately after release of cells from the HU block by washing, DNA synthesis began. Autoradiographic analysis revealed that within 2 hr, 80 to 85% of the cells were synthesizing DNA. The latent period for progeny BPV production was 8 hr in HU-synchronized cells infected at the beginning of S phase compared to a 16 hr latent period in asynchronous cells. In addition virus titers and the percentage of cells containing viral specific antigens increased more synchronously in HU-synchronized cells than in asynchronous cultures. Synthesis of BPV DNA always preceded the initial increase in virus titers and appeared to govern the rate of virus maturation. BPV DNA synthesis was always initiated during late S phase in cells infected at the beginning of S. Cellular S phase was not affected by BPV replication, but RNA and protein synthesis declined rapidly after the onset of BPV DNA synthesis in infected cells. The double stranded BPV DNA produced during infection was isolated in the supernatant after selective precipitation of cellular DNA with NaCl and sodium dodecyl sulfate (Hirt procedure) followed by hydroxylapatite chromatography. Some of the molecules of DNA isolated in this way existed in a covalently closed circular configuration as demonstrated by rapid reannealing rates following thermal denaturation, In addition, agarose gel electrophoresis revealed bands of DNA in this preparation comparable to ØX 174 closed and open circular replicative forms. No double stranded DNA isolated from cells infected with other animal parvoviruses has been shown to contain covalently closed circular forms. Although BPV is replicated to only a minor extent in stationary BFS cells, BPV was shown to replicate in cells infected 32 hr after the cells were exposed to 2 mM HU but not released. Throughout replication, cells remained in HU and no cellular DNA synthesis was detected. However, BPV DNA and progeny BPV were produced beginning at 16 hr postinfection. This synthesis was not due to viral production in a few cells only, since approximately 75% of the cells were involved in BPV replication as demonstrated by immunofluorescent staining. Therefore, it appears that a factor associated with S phase of the cell cycle and required for optimum BPV replication is produced even in the absence of cellular DNA synthesis per se. / Ph. D.

Call number: LD2668 .T4 1985 S336 / Master of Science

Bovine mammary glands respond to infection by foreign pathogens such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) through changes in gene expression. Monitoring the gene expression profiles will contribute to better understanding of the pathology of mastitis, and provide important selective markers for future animal breeding programs. Using cultured bovine mammary duct epithelial cells and somatic cells from infected bovine mammary glands, this study first examined the existence of Toll Like Receptors in these two systems. In cultured duct epithelial cells stimulated with E. coli LPS, both TLR 4 and 2 mRNA up regulation was detected at 2h-72h and 12h-48h respectively. For S. aureus LTA TLR 2 mRNA was up regulated at 48 and 72h whereas for TLR 4 mRNA expression up regulation was detected at 24, 48, and 72h in comparison to the Oh (p<0.05). In the case of PGN, an abundant structural component of S. aureus, the expression of TLR 2 mRNA was significant (p<0.05) at 72h whereas TLR 4 mRNA expression increased at 24, 48, and 72h. The expression of these receptors was also monitored in milk cells from cows infected with either E. coli or S. aureus. However, results obtained from the milk cells were inconclusive due to the high individual variability. Afterwards, differential gene expression profiles were monitored by the Differential Display Polymerase Chain Reaction technique in the cultured duct epithelial cells in response to E. coli and S. aureus structural components. A total of 6 candidate fragments were identified for E. coli LPS induction, whereas only one fragment was identified for S. aureus LTA induction. After LTA induction, a specific band was found to be up regulated and confirmed to be GCP-2, a chemokine involved in neutrophil recruitment. In contrast, PGN induction resulted in no change in GCP-2 levels. In different preparations of cultured duct epithelial cells both GCP-2 and IL-8 were confirmed by real time PCR to be up regulated by LTA with a significance of (p<0.01) when compared to the control cells. In the case of the E. coli identified bands, a different approach is necessary to potentially confirm the origin of these fragments. Further large scale screening of the GCP-2 and IL-8 genes in dairy cattle is necessary to test for their potential use as targets to differentiate the mastitis resistant from the mastitis prone cows.

No description available.

No description available.