published_or_final_version / Microbiology / Master / Master of Philosophy

published_or_final_version / Microbiology / Master / Master of Philosophy

A rapid detection method combining immunomagnetic separation (IMS), PCR and slot blot detection was developed for the detection of Salmonella and Listeria monocytogenes in milk. Bacteria were first isolated and concentrated from phosphate-buffered saline (PBS) or milk by IMS. After extraction from diluted bacteria culture with the extraction buffer, bacterial DNA was subjected to PCR. Slot blot assay was optimized and used to measure PCR products. The lowest level of detection by this method was 40 cfu/ml in PBS or milk for both pathogens. The whole detection procedure could be completed within 7 h. Moreover, this detection method is simple and easy to handle for a large number of samples. Using multiplex PCR (amplification of two different bacterial DNA in the same PCR tube) and slot blot, simultaneous detection of both bacteria was also assessed. The detection sensitivities of 103 cfu/ml for both bacteria were the same as when PCR and slot blot were used for each bacterium separately. The combination of IMS, PCR and slot blot seems to give a highly sensitive and time-efficient procedure, which could be used for routine detection of Salmonella and L. monocytogenes in milk.

The epidemiological study of pathogens largely depends on three technologies, serology, microscopy and the polymerase chain reaction (PCR). Serological methods are unable to differentiate between current and past infections. Microscopy has historically been the mainstay of epidemiological study. In recent times the use of microscopy has been in decline, as it has been shown to have an inherent lack of sensitivity and specificity and produces many false negative results. PCR is now the method of choice for screening samples for the presence or absence of pathogens. Although PCR is widely regarded as an extremely sensitive technique, the fact that it assays a very small volume of sample is often overlooked. If the target pathogen is not present in the tiny aliquot of sample from an infected host, then a false negative results will occur. In endemic situations were the pathogen is present at low infection intensities, then the potential for false negatives results of this type is high. This intensity related false negative effect can lead to serious underestimation of diagnosed prevalence and incidence with consequent misinterpretation of the resulting data. This phenomenon has been reported in the literature for a range of pathogens and especially for epidemiological study of schistosomiasis. The extensive occurrence of false negatives during study of schistosomiasis samples was such an obstacle to epidemiological study it prompted the world health organisation to repeatedly call for quantitative methods to be employed to combat the problem. The main objectives of this thesis are to rationalise and simplify the methods of diagnosing African trypanosomes in epidemiological studies and to investigate the consequences of, and methods of dealing with infection intensity related false negative results that occur as a result of widespread sub-patent infections in the study population A new PCR assay was developed that was capable of analysing whole blood placed onto treated filter paper. The PCR assay was capable of differentiating between all the important African trypanosome species, producing a unique size of amplicon for each species of trypanosome. Initial results from repeated screening of human and cattle samples known to be parasitologically positive indicated that many false negative results occur. A more extensive analysis of thirty five bovine blood samples randomly chosen from a collection of field samples revealed that false negative results occurred regularly. The prevalence of infection after a single screening was 14.3% whereas the cumulative prevalence after over 100 repeated screenings rose to 85.7%. This showed that a severe underestimation of prevalence occurs from a single screening of the samples. In order to investigate the consequences of, and develop methods of dealing with this problem, computer based simulations were used to model the dynamics of screening samples with sub-patent infections. In order to construct the model the data obtained from repeat screening of the thirty-five bovine blood samples was fitted to a number of mathematical distributions. A negative binomial distribution best described the distribution of trypanosomes across the hosts. Exploration of the phenomenon with the resulting model showed the extensive underestimation of true prevalence that is possible. The simulations also showed that it is possible for populations with very different patterns of infection and true prevalence to all have the same diagnosed prevalence from a single screening per sample. Statistical comparison of these very different populations by diagnosed prevalence alone would conclude there was no significant difference between the populations. It was therefore concluded that the diagnosed prevalence from a single (or even multiple) screenings is an inadequate and potentially misleading measure of both infected hosts and parasite numbers. In order to deal with these problems new methods were evaluated for use in epidemiological studies. A simple method of producing quantitative measures of infection was advocated. The insensitivity of existing screening methods in detecting significant difference between populations was highlighted and a greatly improved methodology was shown. Finally, a method for inferring the true population prevalence from the data obtained from repeat screening of samples was suggested. Although some of these new methodologies have limitations, they represent a great improvement on the use of a single diagnostic test for each host. The work presented in this thesis highlights a serious potential limitation to our understanding of the epidemiology of pathogens that exist at sub-patent levels, and develops some possible methods of overcoming these limitations.

Odontoglossum ringspot virus (ORSV) is one of the most prevalent orchid viruses that infects greenhouse-grown orchids worldwide. In order to prevent the spread of viruses in greenhouses and to cultivate clones from virus-free mother plants, it is necessary to develop a more sensitive technique for the detection of viruses in orchids. SYBR green real-time RT-PCR is a highly sensitive technique that can specifically detect ORSV in orchid tissue. By harvesting tissue at the inoculation site and at specific distances from the inoculation site at different times past inoculation, this technique can also be used to study the rate of spread of ORSV in orchids. Orchid clones were inoculated with ORSV and other clones were mock-inoculated with molecular grade water. Leaf tissue was harvested from the ORSV-inoculated and mock-inoculated clones at the site of inoculation and at specific distances from this site at 16 h, 24 h, and 72 h past inoculation. Total RNA was extracted from the harvested tissue. Competitive RTPCR was going to be used for the quantification and detection of ORSV in the samples, but attempts at cloning an ORSV fragment into a vector in order to form a competitive standard were unsuccessful. Instead, a highly sensitive qualitative approach called SYBR green real-time RT-PCR was used for the detection of ORSV. ORSV was detected in all virus-inoculated orchids, except for one. Therefore, all of the ORSV inoculated plants except for one were infected with the virus. Unexpectedly, ORSV was also detected in all of the mock-inoculated orchids. Most likely the orchids were previously infected with ORSV, but the viral titer was too low to be detected by commercial techniques. However, there is a small possibility that the orchids were contaminated during experimentation, despite careful technique. The rate of spread of the virus could not be studied because the mock-inoculated samples also contained the virus. Although viral amplification was demonstrated in the mock-inoculated plants, SYBR green real-time RT-PCR is still a sensitive and consistent method for ORSV detection in orchids. With additional controls, this method could prove to be the ideal method for reliable detection of ORSV in commercially-grown orchids. / Department of Biology

Several powdered food products were screened using repetitive element palindrome PCR (rep-PCR) for the presence of enterotoxin producing species of Bacillus. Samples from these products were screened by being placed into a tryptone-peptoneglucose-yeast enrichment medium (TPGY), heat-treated, and shake-incubated. DNA was extracted using a modification of established protocol, leading to the development of an optimized method for each food system. Purified DNA was amplified through rep-PCR using extragenic sequence-targeting primers and optimized for each food product. Amplified PCR products were analyzed electrophoretically and viewed using an ultraviolet photodocumentation system. Bacillus cereus positive control DNA fingerprints were compared to banding patterns from enriched food samples, revealing the presence of the typical diagnostic 1,230 bp band in non-fat dry milk (NFDM). Restriction Fragment Length Polymorphism (RFLP) with Alu I restriction enzyme was performed on the 1230 bp diagnostic band from NFDM and displayed a profile consistent with Bacillus cereus positive control. RPLA (Reverse Passive Latex Agglutination) and BDE ELISA (Bacillus Diarrhoeal Enterotoxin Enzyme Linked Immunosorbent Assay - Tecra Diagnostics) confirmed the presence of HBL and NHE enterotoxin production in NFDM, Coffee creamer, infant milk formula, and two lecithin samples. / Department of Biology

The regulatory enzyme 6-phosphofructo-l-kinase (PFK) controls the key, rate-limiting step in glycolysis. There are 3 known mammalian isoforms termed PFK-muscle (PFK-A), PFK-liver (PFK-B), and PFK-brain (PFK-C) that randomly aggregate to form active homo- and heterotetrameric isozymes with their respective frequencies and kinetic properties contingent upon the presence and concentration of individual subunits. This study utilized RT-PCR and densitometry analyses to characterize the expression patterns of the mRNA for each isoform during mouse preimplantation development. PFK-B is increasingly expressed across these stages with a significant increase in PFK-B transcript between 8-cell (0.425 ± 0.158) and morula (0.579 ± 0.197) stages (p < 0.0005). Neither PFK-A nor PFK-C mRNA was detected at any of the preimplantation stages tested. The statistically significant increase in PFK-B corresponded with the known juncture of the switch from the oxidation of maternally supplied pyruvate to a predominant glycolyticmetabolism. Such timing suggested the direct involvement of elevated PFK-B transcription with an increase in glycolysis. / Department of Biology

To improve the existing methods based on phenotype and/or genotype a new genotyping method was investigated to identify Lactobacillus species. / The method used the polymerase chain reaction (PCR) to amplify specific sequences of aminopeptidase (pepN) genes. The primers for the PCR reactions derived from a pepN sequence of Lactobacillus rhamnosus S93. PepN amplification products of 387 bp were obtained from forty three Lactobacillus strains and from some strains of Lactococcus (3), Streptococcus (2) and Bifidobactertium (5). / Restriction fragment length polymorphisms (RFLPs) and single-strand conformation polymorphisms (SSCP) methods were used to detect polymorphisms among amplified aminopeptidase DNA fragments from the different Lactobacillus strains. / The results of RFLPs after digestions with Sau3A I, Rsa I and Tru9 I confirmed that the PCR products were specific. According to the fingerprints generated, Lactobacillus species tested could be grouped in four. / SSCP allowed a good discrimination between different pepN PCR products of the same size. Some Lactobacillus strains, Lb. plantarum and Lb. rhamnosus showed the different ssDNA patterns. Though for many strains of Lactobacillus the SSCP patterns were similar, no general comparison can be made because all the samples were not loaded on the same SSCP polyacrylamide gel. The SSCP, PCR-based method can be easily modified to increase the rate of polymorphism detection. / This new genetic identification method is different from others because it uses specific pepN DNA sequences for each strain tested and it uses SSCP to detect the presence of polymorphisms. The method is also applicable to other genera of lactic acid bacteria.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 120-131). / Microfiche. / xiii, 131 leaves, bound ill. 29 cm

Thesis (PhD)--University of Stellenbosch, 2005. / Bibliography. Also available via the Internet.