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To enhance outbreak investigations of Shiga toxin-producing Escherichia coli O157:H7, PulseNet Canada has recently applied Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) as a supplemental subtyping tool in combination with the gold standard subtyping method Pulsed-field Gel Electrophoresis (PFGE) for enhanced resolution of isolates exhibiting indistinguishable/highly similar PFGE patterns. The objective was to assess the discriminatory power and level of specificity MLVA offers for outbreak detection and response. Results demonstrate that MLVA provides a statistically significant increase in discriminatory power for outbreak investigations (0.998) compared to PFGE alone (0.993). MLVA was able to provide additional resolution over PFGE analysis and generally agreed with PFGE when isolates were identical and epidemiologically linked. MLVA shows great promise as a molecular epidemiological tool to complement PFGE, as it improves case categorization during outbreak investigations, and the greatest benefits of MLVA may be realized during routine surveillance, when epidemiological information is not available.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2006. / Title from title screen (site viewed May 23, 2007). PDF text: viii, 161 p. : ill. (some col.) ; 1.95Mb UMI publication number: AAT 3237485. Includes bibliographical references. Also available in microfilm and microfiche formats.

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Single crystal X-ray work was carried out for four heterocyclic compounds: prior to this, definitive structures could not be written for any of these. The heterocyclic compounds reported are: 5,6-dihydro-1-thiobenzoylmethylene-1H-thiazolo[2,3-c][1,2,4] thiadiazole, (I); 4-N-(4'-bromophenylamino)-3-butylfuroxan, (II); 4,4'-methylenebis(1,3,5-trimethyl-4-imidazolin-2-one), (III), and 4-phenyl-3-phenylamino-1,2,4-thiadiazoline-5-one, (IV). These are then discussed, along with work which has been carried out for similar compounds. Trithiapentalenes and analogues having linear multisulphur systems are mentioned as criteria for deciding whether the NSS skeleton in (I) is best considered as N-S...S or N-S-S. Following this, the X-ray structure determinations of bis(4-methylpyridine)hydrogen(I) tetraphenylborate, (V), and bis(triphenylarsineoxide)hydrogen(I) tetrafluoroborate, (VI), arereported. Both (V) and (VI) are bis-cations which have strong hydrogen bonding. Other compounds with short 0...H...0 and N...H...N distances are mentioned, stabilities of such bis-cations compared to those of the monocations, and (amine-halogen-amine)+ complexes discussed. The final two X-ray structure determinations are for the compounds 1,1,1,3,3,3-hexaphenyldisilazane and μ-nitrido-bis (triphenylphosphorus)((I) thiocyanate. MNDO was used to calculate force constants for bending of linear molecular pseudohalides. The aim of such work was to rationalise 'unusual' geometries in some silicon-containing compounds via Second Order Jahn-Teller distortions, postulating that such compounds are easily distorted along a "soft' normal vibration coordinate.

Formation of L-cysteine, from L-serine and sulphide, represents the principal route of Sulphur incorporation into organic compounds in living organisms. Cysteine biosynthesis in plants is achieved by two enzymes, serine acetyltransferase (SAT) and 0-acetylserine (thiol) lyase (OASTL), which form a cysteine synthase complex. Three novel cDNA species, Sat-52, Sat-53 and Sat-106, encoding SAT isoforms from A. thaliana were isolated from a collection of cDNAs previously cloned by functional complementation of the E. coli cysE mutant strain JM15, which is defective in serine acetyltransferase. Deduced amino acid sequence analysis suggests that Sat-52 encodes a putatively mitochondrial isoform whilst Sat-53 and Sat-106 encode proteins with cytoplasmic locations. Sequence information derived from the Arabidopsis Genome Initiative allows mapping of Sat-52, Sat-53 and Sat-106 genes to locations on chromosomes V, I and II respectively. A fourth SAT cDNA from A. thaliana, Sat-1, was cloned prior to the work detailed here and encodes a putatively plastidic isoform of the enzyme. Southern hybridisation against digested genomic DNA suggests that each SAT gene is represented by a single copy in the A. thaliana genome. DNA probes specific to the SAT gene-family members were designed and used in various studies to examine expression of SAT genes. Northern blotting and hybridisation was used to determine transcript distribution between root, leaf, stem, flower and silique tissues and to study the expression of the gene-family in response to sulphate and nitrate nutrition. Spatial distribution of Sat-52 and Sat- 53 transcript in root, leaf and stem tissue was also examined by in situ hybridisation using specific riboprobes. Both genes are highly expressed in leaf trichomes. The Sat-52 transcript was also localised to the vascular- bundles of root, leaf and stem tissue. The isoforms encoded by Sat-52 and Sat-53 are hypothesised to have specific roles in these cell-types.

The technique of in situ hybridisation was optimised for the detection of viral RNA using radioactively-labelled single-stranded DNA and RNA probes, and applied to three areas of interest. Optimum hybridisation conditions were determined in vitro using cells infected with the single-stranded negative sense RNA paramyxoviruses. Transcription of RNA probes was the most rapid and efficient method of probe labelling, since electrophoretic purification was not required and large amounts of RNA were produced. However, their use for in situ hybridisation was problematic due to RNase contamination and low sensitivity. In contrast, DNA probes produced from M13 clones and oligonucleotide probes gave consistent hybridisation results and were preferred in subsequent studies for their ease of use, stability and sensitivity. The effect of virus-host interactions on the clearance of the paramyxovirus, SV5, in a mouse model was investigated by detection of viral RNA and protein in lung sections. Immunisation with purified SV5 proteins prior to infection provided protection against infection, indicated by a reduction in the level of viral RNA and protein, due to enhanced clearance of virus by primed T cells. X-irradiation of the host prior to infection resulted in prolonged or persistent infection in which RNA was detected up to 19 days post-infection. The potential of in situ hybridisation for detection of aetiological agents was demonstrated by investigation of the presence of measles virus in two chronic human diseases. Thus, measles virus RNA was detected in brain sections from a patient with subacute sclerosing panencephalitis and in the osteoclasts of bone sections from a patient with Paget's disease of bone. In situ hybridisation was used to analyse expression of the two immediate-early genes of herpesvirus saimiri, the 52K gene and the hinG gene. Differential expression was detected by hybridisation to mRNA using oligonucleotide probes, in productively-infected cells. The 52K gene was expressed asynchronously throughout the population in agreement with immunocytochemical detection of the 52K protein. In contrast, the hinG gene was expressed synchronously, with all cells showing similar levels of hybridisation, indicating a specific control mechanism for expression of the 52K gene, which differs from that of the hinG gene in requiring or being inhibited by additional factors. This may have relevance to the mechanism of establishment of latency in this virus.

Twenty years after chromosome doubling an unselected population of tetraploid rye appeared to have reached an "equilibrium state" in chromosome pairing behaviour. Cytogenetically the population was highly heterogeneous. Compared to early C-generations, meiotic behaviour in the population improved by an increase in quadrivalent frequency, mainly at the expense of trivalents and univalents. Quadrivalent frequency, however, failed to correlate with other meiotic features; instead bivalent frequency had significant positive correlations with the features of meiotic regularity including chiasma frequency. Furthermore, the average quadrivalent frequency in the population was considerably less than that of inbred lines (Hazarika & Rees, 1967). These facts led to the conclusion that disomic pairing dominated the chromosome behariour in this random mating population. No meiotic features ner morphological characters were correlated with seed-set in the population. A simultaneous selection for seed-set and regular tetrads was effective in increasing the bivalent frequency and reducing the frequencies of quadrivalents and aneuploids. A comparative study of three populations ("high", "low" and unselected) indicated the variable relationship between chiasma frequency and the frequencies of quadrivalents and bivalents. This and the higher frequency of qudrivalents in inbred materials was explained on the basis of "free" and "restricted" pairing of the four homologous chromosomes of an autotetraploid. It was concluded that the pairing pattern in inbred materials is predominantly tetrasomic whereas in outbred materials this may vary from tetrasomic to disomic depending on the chromosomal differentiation within the homologous sets. In the unselected population the lack of correlation of seed-set with meictic features and morphological characters proved to be due to a supplementary interaction between the cytological and the so-called physiclogical factors in determining the fertility of a plant. It was demonstrated that the genetical control of the cytological factors is independent from that of the so-called physiological factors. Once the interaction due to the latter was reduced by selection pressure, the effects of the cytological factors on seed-set became evident. But depending on whether the pairing pattern is predominantly tetrasomic or disomic, seed-set is correlated with quadrivalent or bivalent frequency. Quadrivalents were found to be sensitive to environmental changes whereas bivalents remained relatively stable. Furthermore, quadrivalent formations are restricted with 2/3 of the chromosomes. It is, therefore, concluded that the only way of ensuring meiotic stability in an autotetraploid is to induce disomic pairing. The possible ways of achieving this are outlined. Several chromosomal aberrations detected during the investigation are illustrated and discussed.

This study was conducted to determine if a relationship exists between temperament, stress response, and the shedding of Escerhichia coli O157:H7. Cattle (n = 150) were evaluated for disposition and stress response before shipping to the feeding operation, upon arrival at the feedlot, at approximately 70d on feed, and prior to transport to the harvesting facility. Chute and pen scores, as well as serum cortisol concentrations, were measured in order to assess individual temperament and stress response. A temperament index was created to classify cattle as Excitable, Intermediate, or Calm. The presence of E. coli O157:H7 was determined by rectal swabs on the live cattle and swabs of colons collected postmortem at the processing facility. As expected, variables for pre-shipment temperament index, exit velocity, pen score, arrival and midpoint exit velocity, and mid-point cortisol concentrations differed (P < 0.05) greatly between temperament groups. However, pre-shipment chute scores and cortisol concentration, as well as arrival and final cortisol concentrations differed (P < 0.05) only for Excitable cattle compared to both Calm and Intermediate groups. The percentage of cattle shedding the pathogen at arrival was approximately equal between temperament groups. When sampled before shipment to the processing facility, a higher proportion (P = 0.03) of cattle displaying Calm temperaments shed E. coli O157:H7 than the other groups. Results from postmortem colon samples exhibited a similar trend. When the results from all four sampling periods were pooled, the Calm cattle had a greater numerical percentage test positive for E. coli O157:H7. However, the pooled frequency distribution is largely dictated by the results of the final sampling time. Based on these results, it appears that Excitable cattle are not more likely to shed E. coli O157:H7. In fact, it seems that Calm cattle may be equally or more susceptible to shed at later points in the feeding period. However, it is important to note that a relatively small number of the samples tested positive for E. coli O157:H7, thus, potentially causing dramatic changes in the distributions.

a b s t r a c t Meat and meat products have been implicated in outbreaks of Escherichia coli O157:H7 in most parts of the world. In the Amathole District Municipality of the Eastern Cape Province of South Africa, a large number of households consume meat and meat products daily, although the microbiological quality of these types of food is questionable. The present study investigated the prevalence of E. coli O157:H7 isolated from selected meat and meat products (45 samples each of biltong, cold meat, mincemeat, and polony) sold in this area. Strains of E. coli O157:H7 were isolated by enrichment culture and confirmed by polymerase chain reaction (PCR). Also investigated were the antibiogram profiles of the E. coli O157:H7 isolates. Five (2.8%) out of 180 meat and meat products examined were positive for E. coli O157:H7 that carried the fliCH7, rfbEO157, and eaeA genes. Two of the E. coli O157:H7 isolates were resistant against all the eight antibiotics tested. To prevent E. coli O157:H7 infections, meat and meat products such as biltong, cold meat, mincemeat and polony should be properly handled, and packed in sterile polyvinyl wrappers.