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no abstract provided by author / Master of Science

The cis and trans signals important in BPV replication were identified using a transient replication assay, the mobility shift assay, and a comparison between the BPV and LPV genomes. Replication of deleted BPV genomic clones, which contain the natural left (3’ OH end of the viral minus strand) and right (5’ PO, end of the viral minus strand) BPV termini, defined the minimum size of the BPV origin of replication (ori) to be the terminal 171 nucleotides of each terminus. Clones containing duplicate termini or altered left ends were also shown to replicate. The BPV ori was determined to have two domains identified by a computer analysis of homologus regions between these termini. Three proteins were identified that bind to the left terminal 171 nucleotides in the hairpin conformation. Inhibition of the formation of the DNA-protein complexes with competitor DNA localized two potential binding sites that correspond to the domains mentioned above. Two of the DNA-protein complexes were formed by BPV-coded proteins as determined by inhibition of the complex by anti-BPV antibodies. The third complex resulted from binding of a host cell S-phase protein that is a likely candidate for the S-phase factor required for autonomous parvovirus replication. The BPV ori thus appears to function by binding both cellular and viral proteins for the initiation of DNA synthesis from the hairpinned termini. The comparison of the BPV and LPV genome sequence suggest that the genomic organization of LPV may be more like BPV than that of the rodent parvovirus minute virus of mice; and therefore, LPV may contain similar cis signals. / Ph. D.

Cattle diseases caused by the protozoan parasite <i>Theileria parva</i> and transmitted by the brown ear tick <i>Rhipicephalus appendiculatus</i> are among the most costly diseases in eastern, central and southern Africa. The control of these diseases in areas where they occur, and the prevention of their spread to areas suitable for the survival and development of the vector, should be based on a strong understanding of their epidemiology. Therefore, methods to assist in transforming epidemiological information into a format that will assist animal health planners are needed. Computerized geographic information systems (GIS) may offer such valuable methodology. The purposes of this study were to define, characterize and display on a geographic basis, factors governing the epidemiology of cattle diseases caused by I parva. Factors studied included the distributions of the vector ticks (R appendiculatus and related species), major hosts (cattle and addition, continental representation of climatic parameters and satellite derived vegetation data were included. The c1imatedatabaseswereproducedusingmathematicalinterpolations of data from meteorological stations and further improved by the incorporation of an altitude data set. Interpolated climate databases were then used to run a climate matching model (CLIMEX)l to define areas of climatic suitability for tick distribution and abundance. A computerized geographic information system was used to store, manipulate, analyze buffalo) and display the data. / Ph. D.

The genome of bovine parvovirus (BPV) has been cloned by blunt end ligation of double-stranded virion DNA into the plasmid pUC8. The resulting genomic clones were infectious after transfection into bovine fetal lung (BFL) cells. Sequencing of the plasmids demonstrated that deletions were common at both ends of the cloned BPV genome. Deletions of up to 34 bases at the 3’ end lowered but did not abolish infectivity, while a deletion of 52 bases eliminated infectivity, End label analysis demonstrated the repair of deletions of up to 34 bases at the 3’ end or 35 bases at the 5’ end to the wild type length. Mutually inverted sequence orientations of the palindromic termini, known as the flip and flop forms, can occur during replication of parvovirus DNA. Cloning of BPV terminal sequences permitted the identification of the 3’ flop sequence inversion as a natural component of BPV DNA. This is the first report of sequence inversions within the 3’ end of an autonomous parvovirus. Clones with the 3’ flop or flip conformations were equally infectious. Wild type virion DNA was shown to have predominantly the 3’ flip conformation but a significant amount of 3’ flop was also detected. At the 5’ end, both the flip and flop sequence conformations were identified in nearly equal amounts. The progeny virion DNA from transfection of genomic clones had the same ratio of flip to flop as did wild type at both the 3’ and 5’ ends, regardless of the starting terminal conformations of the genomic clone. These data suggest that, while sequence inversion occurs at both termini during BPV DNA replication, some mechanism exists for the preferential replication of the 3’ flip conformation. Replicative form DNA from BPV infected cells had the same ratio of flip and flop at each end and the same termini as virion DNA. A set of deletion and frameshift mutants affecting each of the coding regions of BPV was constructed using one of the genomic clones. None of these mutants was infectious when transfected into BFL cells, which demonstrates that all three of the major open reading frames are essential for the production of infectious virus. / Ph. D. / incomplete_metadata

Typescript (photocopy). / Digitized by Kansas Correctional Industries

This study investigated the hypothesis that the particulate content of milk, as monitored with particle counters, is correlated to the health status of lactating dairy cows, in particular the condition mastitis. Twenty Holstein cows were monitored from the very first day of clinical mastitis outbreak until complete recovery from the disease. During the experiment, the changes in particle behaviour in all four quarters and mixture of milk from all of them were measured. For each sample the following parameters were measured: somatic cell count, fat content, lactose and protein concentration, number and size distribution of milk particles, electric conductivity and diameter of milk fat globules. In total over thirty mastitis outbreaks were observed and monitored throughout, including the first phase of this study when over three thousand samples of foremilk were collected and examined. An operational protocol and particle monitoring device were designed with the help of a commercial company Facility Monitoring Systems Ltd (FMS), Malvern. A particle counter and Peak Height Analyser (PHA) were used to monitor particulate content of milk and a compound phase contrast microscope was used to identify milk particles by photographic visualisation and to establish their diameter. It was observed that the number of particles, milk fat globule diameter and somatic cell counts were stable during periods without udder inflammation. Mastitis caused great changes in these parameters. Both milk particulate size and number were significantly affected by clinical and subclinical form of inflammation (change to the particulate behaviour). It was observed that the changes to the volume median diameter (VMD) of fat globules became evident a few days before clinical signs were present. Results obtained from a particle counter and the PHA were in agreement with data obtained by microscopy. Major changes were recorded in the number of total particles in milk before and during the outbreak of mastitis. Further research showed that changes took place in the pattern of particulate behaviour without visible signs of disease; additional data established that subclinical mastitis can be also identified through the monitoring of particles in milk. In summary monitoring of the behaviour (changes to size and number) of milk fag globules (MFG) can be used as an early indicator of the onset of mastitis. In addition data collected during study produced strong evidence supporting the theory of the interdependence of the quarters within the udder. It was found that the coefficient of correlation for size and number of particles for all four quarters within the udder was statistically significant. Particle counts and the VMD values behaviour were similar for the four quarters. This relationship was observed for all monitored animals. Moreover, the same relationship was also observed during both clinical and subclinical outbreaks of mastitis. Somatic cell count was affected only in an infected quarter while particulate content of milk ―responded‖ to disease in all four quarters within the udder (even if only one was infected). These results were the most surprising and unexpected outcome, suggesting that four quarters within the udder work together as one organ not four separate units. It was observed that the mean MFG cannot be used as a baseline to test individual animal deviations due to the unique particle profile of each observed animal. In all monitored animals particle counts obtained from PHA was found to be in the range of 1011 to 1013 with an average of 1012 particles per ml. The number of particles recorded in mastitis for one animal was at the healthy level for another. The particle pattern became a finger print for each animal and therefore MFG behaviour cannot be compared between animals. Following the first phase of the study the monitoring period was set at 15 to 20 days. This protocol allowed for minimising the influence of any other parameters on particles, which may influence the outcome of an experiment e.g. the number of particles in single samples detected by the particle counter. It was also essential to understand how the age, nutrition and stage of lactation might influence the particles and affect the results. Therefore two animals were chosen to be examined during their lactation. The analysed data did not present enough evidence to establish the relationship between nutrition and milk fat globules size and number. However to better understand this association additional studies should be carried out. Further work is required to optimise the monitoring device and build a fully automated system which will allow collecting and analysing data from the whole herd. This study proposed that particle pattern is unique for each animal – like a finger print. More research is needed to better understand the mechanism behind milk fat globules synthesis during inflammation. The results obtained during this study provide new evidence with regard to physiological changes within the udder before and during mastitis outbreak and supported the theory of interdependence between quarters within udder. The particle count in milk can be used as an indicator of the health status of the single animal. Combined the PHA and microscope can be used as a new tool to determine and monitor the particle count in milk. The understanding of the particulate behaviour will help to minimise the chance of mastitis outbreak by early detection and also to reduce the chance of the cross-contamination between animals during the milking process. Milk fat globule size and number can be used as an efficient indicator of the onset of mastitis.

Thesis (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The effect of the bacteriocin-like peptide AS-48, produced by Enterococcus faecalis FAIRE 92, was tested against a mastitis isolate of Staphylococcus aureus in an in vivo and in vitro study. During initial tests peptide AS-48 showed no significant activity towards S. aureus, even with a ten-fold concentrated cell-free supernatant. Activity was obtained only after purification with Triton X-114 phase partitioning, followed by cation exchange chromatography. Titers for the purified peptide varied between 3200 and 12800 AU/ml. The purified peptide also exhibited activity towards Streptococcus agalactiae and Streptococcus dysgalactiae, but not against Escherichia coli. The size of peptide AS-48 was determined at 7150 Da, based on electronspray mass spectrometry and SDS-PAGE. Complete inhibition of cell growth was obtained by adding 1ml of the purified peptide (3200 AU/ml) to 100 ml of cells of S. aureus in the lag growth phase. When the same concentration of peptide AS-48 was added to a culture of S. aureus in mid-exponential growth, a slight decrease in viable cell numbers was recorded, which lasted for only 30 min. Cell growth commenced thereafter. In situ experiments in cows were done with purified peptide AS-48, encapsulated in liposomes. These in vivo studies were conducted by administering peptide AS-48 (6400 AU/ml) to different udder quarters. In a prevention trial, i.e. where quarters were pretreated with peptide AS-48, a reduction close to 90% in the viable cell numbers of S. aureus was recorded relative to the control quarters, which were not treated with the peptide. A 50% reduction in somatic cell count (SCC) was recorded. In the treatment trial, i.e. infected quarters treated with peptide AS-48, a reduction of up to 94% in viable cell numbers of S. aureus was recorded. In the same quarters, a reduction in SCC amounted to almost 80%. A recombinant strain was constructed by conjugating plasmid 92 (p92), encoding peptide AS-48, from Enterococcus faecalis FAIRE 92 to E. faecalis FA2/Ent, which produces enterocins 1071A and 1071B. Southern blot hybridization experiments revealed thepresence of plasmid p92 in the recipient strain without the loss of plasmid pEF1071, which encodes enterocins 1071A and 1071B. All three antimicrobial peptides, i.e. enterocin 1071A, enterocin 1071B and peptide AS-48, were produced in transconjugant FA2/Ent/AS-48. The spectrum of antimicrobial activity of the transconjugant was greater than that recorded for strains FA2/Ent and FAIRE 92, respectively and included E. faecalis, Bacillus cereus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus sakei, Leuconostoc cremoris, Leuconostoc pentosaceus, Staphylococcus carnosus and S. aureus. These organisms are not inhibited by strain FA2/Ent. However, low levels of peptide AS-48 was produced by strain FA2/Ent/AS-48. Further research in fermentation and gene expression will be needed before the transconjugant E. faecalis FA2/Ent/AS-48 may be used in the treatment of mastitis. / AFRIKAANSE OPSOMMING: Die effek van die bakteriosien-agtige, peptied AS-48, geproduseer deur Enterococcus faecalis FAIRE 92, is gedurende ‘n in vivo en in vitro studie teen ‘n mastitiese Staphylococcus aureus-isolaat getoets. Aanvanklike toetse met peptied AS-48, selfs tienvoudig gekonsentreerde selvrye supernatant, het geen beduidende aktiwiteit teen S. aureus getoon nie. Aktiwiteit is eers verkry na suiwering met Triton X-114 fase-skeiding gevolg deur katioon uitruilingschromatografie. Titers vir die gesuiwerde peptied het tussen 3200 en 12800 AE/ml gewissel. Die gesuiwerde peptied het ook aktiwiteit teen Streptococcus agalactiae en Streptococcus dysgalctiae getoon, maar nie teen Escherichia coli nie. Peptied AS-48 het ‘n molekulêre massa van 7150 Da, soos bepaal met elektronsproeimassa spektrometrie en SDS-PAGE. Totale inhibisie van selgroei is verkry deur 1 ml gesuiwerde peptied AS-48 (3200 AE/ml) by ‘n 100 ml kultuur van S. aureus in die sloerfase te voeg. Dieselfe konsentrasie peptied AS-48, toegevoeg tydens die mideksponensiële groeifase, het egter slegs ‘n klein vermindering in die aantal lewende selle teweeg gebring en het ook vir slegs ‘n 30 min geduur. Selgroei het hierna weer normaal voort gegaan. In situ eksperimente op koeie is uitgevoer met gesuiwerde peptied AS-48, geenkapsuleerd in liposome. Hierdie In vivo studies is onderneem deur peptied AS-48 (6400 AE/ml) in verskillende kwarte van die uier, kunsmatig of reeds geïnfekteerd met S. aureus, toe te dien. In ‘n voorkomings-eksperiment waar kwarte vooraf met peptied AS- 48 behandel is, is ‘n verlaging van byna 90% in die lewende seltelling van S. aureus relatief tot die kontrole kwarte, sonder behandeling met peptied AS-48, verkry. ‘n 50% verlaging in die somatiese seltelling (SST) is verkry. In die behandelings-eksperiment, waar geïnfekteerde kwarte met peptied AS-48 behandel is, is ‘n verlaging van byna 90% in lewende S. aureus selle gevind. In dieselfde kwarte is ‘n verlaging van byna 80% in die SST genoteer.‘n Rekombinante ras is gekonstrueer deur plasmied 92 (p92), wat kodeer vir peptied AS- 48, vanaf Enterococcus faecalis FAIRE 92 na E. faecalis FA2/Ent, wat enterosien 1071A en 1071B produseer, te konjugeer. Southern-klad hibridisasie het die teenwoordigheid van plasmied p92 in die ontvanger ras, sonder die verlies van plasmied pEF1071 wat enterosien 1071A en 1071B kodeer, getoon. Al drie antimikrobiese peptiede, nl. enterosien 1071A, enterosien 1071B en peptied AS-48, is deur die transkonjugant FA2/Ent/AS-48 geproduseer. Die spektum van antimikrobiese aktiwiteit van die transkonjugant vand die transkonjugant is breër as dié van rasse FA2/Ent en FAIRE 92, onderskeidelik en het ook E. faecalis, Bacillus cereus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus sakei, Leuconostoc cremoris, Leuconostoc pentosaceus, Staphylococcus carnosus en S. aureus ingesluit. Hierdie organismes word nie deur ras FA2/Ent geïnhibeer nie. Lae vlakke van peptied AS-48 is egter deur ras FA2/Ent/AS-48 geproduseer. Verdere navorsing in fermentasie en geenuitdrukking is nodig voordat E. faecalis FA2/Ent/AS-48 in die behandeling van mastitis gebruik kan word.