Masters Degrees (Genetics)

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Antimicrobial resistance, plasmid profiles and sequence typing of enterotoxigenic escherichia coli isolates causing colibacillosis in neonatal and weaning piglets of South Africa.
(2016) Ranketse, Mary.; Dzomba, Edgar Farai.; Muchadeyi, Farai Catherine.; Madoroba, Evelyn.
Abstract available in PDF file.
Autolytic characterization of selected Enterococcal strains, (previously Streptococcal)
(2007) Sukkhu, Melisha.; Beukes, Mervyn.
Autolysins are enzymes that cleave specific structural components within the bacterial cell wall. They contribute to numerous cellular activities such as cell growth, cell division, peptidoglycan recycling and turnover. In this study, twelve Enterococcal isolates (previously from the genus Streptococcus) were examined for susceptibility to the antibiotics Penicillin G and Vancomycin, using a Disk Diffusion and a Microtitre plate assay. In both methods, all twelve strains were resistant to Vancomycin. Six of these strains were susceptible to Penicillin G. The minimum inhibitory concentration (MIC) values were twice that of the disk diffusion assay values. In the presence of antibiotic, the growth rates for the six strains were halved. Autolysins were extracted from the respective cell cultures using a 4% SDS precipitation method. The protein concentrations were calculated and estimated to be within the range of 5.47- to 6.35 μg/μl. Profiles of the SDS precipitate were analyzed on SDS-PAGE. Autolytic proteins were identified and partially analyzed by renaturing SDS-PAGE (zymograms) using gels containing cell wall substrate. Seven lytic bands of molecular weights 25, 30, 50, 63, 75 95 and 145 kDa (designated Autolysin A to G, respectively) were selected for further analysis. The temporal distribution of the enzymes ranged from the mid exponential phase to the early death phase. The seven proteins were blotted onto polyvinylidene difluoride (PVDF) membranes and excised for N-terminal sequencing. Blast analysis of the respective N-terminal sequences showed autolysins A, C, D, E and F to have 100% similarity to the muramidase, amidase and peptidase from S. cremoris, S. suis, S. pneumonia, S. pyogenes and E. faecium, respectively. Biochemical characterization confirmed autolysins A, B, E and F to exhibit muramidase activity, and autolysin C and G to exhibit peptidase activity. Autolysin D displayed 100% similarity to the protein LytA, a peptidoglycan hydrolase that is known to exhibit amidase activity. Blast analysis could not determine any significant similarities for autolysins B and G to previously identified autolysins, thus indicating they may perhaps be novel autolysins.
Behavioural, reproductive and growth studies on Oreochromis mossambicus (Peters 1852)
(2010) Weber, Raimund Michael.; Laing, Mark Delmege.
A major obstacle facing the successful creation of an African aquaculture industry, based upon Oreochromids, is the irregular supply of good quality fish seed. There are several causative biological processes behind its irregular supply. The aim of this research was therefore to determine the basic requirements for the establishment and maintenance of a small breeding facility, for O. mossambicus. The goal was to make a unit that was simple and which could be easily replicated in rural, satellite aquaculture seed stations. The results obtained illustrate that a small reproduction unit can produce large quantities of healthy 90-day fry. Asynchronous hatching of the eggs and spawning asynchrony in female Oreochromis mossambicus are two elements which negatively affect uniformity in the fry produced. Typical fish seed production uses large ponds partitioned into breeding allotments or a series of breeding pools. While the earthen ponds provide a substrate in which a nest can be excavated, its presence is not required for mating success in the closely related O. niloticus (Linneaus 1758). Female mate choice, as well as apparent fecundity, according to nest size has been clearly recorded in related cichlids but no investigations have been made as to nest size and spawning synchrony in O. mossambicus. The main focus of this investigation was to ascertain whether O. mossambicus would accept artificial nest substitutes in preference to their own constructed ones and secondly, whether different alternatives would elicit different levels of acceptance. The observed results indicate a ready acceptance for artificial nest alternatives, with nest lip height being prioritised by the fish . The implications thereof are discussed in relation to the potential for optimization of breeding arenas for O. mossambicus by the provision of artificial nests whose dimensions satisfy both male and female preferences. In established communities, Oreochromis mossambicus display various complex and ritualised behaviours during stable and disruptive events. The aim of this research was primarily to produce a glossary of behaviours defining these interactions, particularly with reference to male-male behaviours. Three males and six females were allowed to acclimatise over one month, with various social groupings being established within the first few days. Results from this study illustrated not only a dynamic social structure, signaled via various chemosensory and visual methods, but also supported recent findings in apparent male-male courtship and the underlying ii causes. Furthermore, the observed male-male activity of the nestholder malesfirmly corroborate the current practice in aquaculture whereby only one male is allocated per breeding arena. The use of artificial incubation of Oreochromis spp. eggs has become widespread in high intensity fish seed production. Various types of incubator exist, and their selection is dependent on the specific attributes of the egg to be incubated. Currently available incubators are typically of a funnel (up-flow) or round bottomed (down-flow) design. Neither permits easy access to the eggs, which is particularly important when dealing with poor quality water as is typically found in rural areas. The aim of this study was to devise and test an easy-to-use incubator, applicable to rural seed production projects, which offers advantages over currently available incubator types. The final design, WETNURSE Type II, offered improved hatching rates over Type I, with a mean hatching success of 75%. While falling short of the desired 80% success rate (Rana 1986), the various other benefits provided by the design justify further optimization and testing. Three distinct populations of O. mossambicus, representing populations of inbred, randomly mated and genetically unknown (wild-caught) pedigree were analysed according to their food conversion efficiency (FCE). The intra- and inter-sample crosses were done with single males in order to produce half-sib progeny batches which allowed for the assessment of sire influences on the FCE of the progeny batches. The results show that the population of unknown pedigree is comparable to that of the randomly mating population, indicating the presence of sufficient genetic variation to permit further selection; the genetic contribution of the males to their respective progeny was insignificant in relation to that made by the female.
Breed effects on the virulence gene profiles and genetic diversity at FUT1, MUC4, MUC13 and MUC20 candidate genes for controlling diarrhoea-causing Escherichia coli.
(2013) Mohlatlole, Ramadimetja Prescilla.; Dzomba, Edgar Farai.; Chimonyo, Michael.
Escherichia (E) coli infections result in diarrhoea and oedema in growing pigs. Enterotoxigenic (ETEC), shigatoxin producing (STEC) and enteroaggregative (EAEC) E. coli have been identified as the principal causes of colibacillosis in most pig production systems. These E. coli use fimbrial and non-fimbrial adhesins to adhere to the intestines and cause infection. Absence or presence of the receptors on the intestinal walls determines the resistance or susceptibility of the host to the E. coli. In other populations, candidate genes linked to the receptors have been found to be associated with resistance/susceptibility to infection and are used in marker-assisted selection programs. This study investigated the presence and prevalence of ETEC, STEC and EAEC and the associated virulence genes in 263 E. coli isolates sampled from Landrace, Large White, Duroc and Indigenous piglets from the Animal Production Institute of the Agricultural Research Council (ARC) in Irene and Middledrift farm in Eastern Cape Province. The study also investigated polymorphisms at six candidate genes associated with two E. coli receptors in the same pig populations. Over 39 % of the isolates tested positive for the E. coli virulent genes investigated. None of the samples had fimbrial adhesins. The mode of attachment of the investigated E. coli was through non-fimbrial adhesins which were found in 49.06% of the isolates. The 106 E. coli isolates were categorized into 25 pathotypes carrying definable and unique combinations of E. coli virulence factors. The resistant allele for Alfa (1) fucosyltransferase 1 (FUT1) M307, a candidate gene for FI8R, was present in less than 1 % of the population. Various mutations of mucin genes MUC4 g.8227, MUC20 c1600 and g.191 were found in the population. Their respective alleles for controlling F4ab/ac E. coli adhesion in pigs were predominant in both breeds. Three loci (FUT1, MUC20 g.191 and MUC20 c.1600) deviated from Hardy Weinberg equilibrium (HWE) in the Indigenous and the Large White breeds. Heterozygotes deficiency and high levels of within breed diversity was observed in these two breeds at the mentioned loci. Overall, the study observed a wide range of toxin and colonisation factors (CFs) giving rise to diverse pathotypes in South African pigs. The absence of fimbrial adhesins suggests a different colibacillosis control program from that previously used. The presence of the resistant alleles in most of the loci investigated was low, however their presence suggest it is possible to use them to generate a resistant population using marker assisted selection. This study serves as a foundation for future pig colibacillosis control and immunity studies in the South African pig herds.
Breeding of advanced generation of Eucalyptus macarthurii-growth parameters and development of a near infrared (NIR) calibration model to predict whole tree pulp yield using non destructive cores.
(2008) Ndlovu, Zama Thandekile Laureen.; Tongoona, Pangirayi.; Swain, Tammy-Lyn.
Eucalyptus macarthurii is one of the cold tolerant eucalypt species grown in South Africa for pulp and paper. However, little research has been done on this species’ growth performance. A study was therefore initiated to: i) analyse growth characteristics of Eucalyptus macarthurii at two sites and to calculate genetic parameters (genetic and phenotypic correlations, heritabilities and genetic gains), ii) develop a non-destructive near infrared calibration model to predict whole tree pulp yield of Eucalyptus macarthurii, and iii) screen a second generation Eucalyptus macarthurii breeding population, using the developed near infrared calibration model on core samples, to predict screened pulp yield and to rank and identify families with superior pulping properties. Eucalyptus macarthurii population growth data (diameter under bark, diameter over bark, bark thickness, bark stripping, height, basic wood density and stem form) were measured at Pinewoods and Vlakkloof sites and their respective genetic parameters calculated. Genotype by environment interaction was found in this population, indicating that different populations should perhaps be developed independently of each other for the two sites. Genetic and phenotypic correlations between diameter over bark and diameter under bark were, 0.96 and 0.98 for Pinewoods and 0.98 and 0.99 for Vlakkloof, respectively. These correlations indicated that selection of diameter over bark would lead to a positive indirect selection for diameter under bark. The heritability estimates also ranged from 0.03 to 0.23 at both sites, which indicated a reasonable response to selection. The predicted gains for all traits found at Pinewoods were higher than those at Vlakkloof for progeny trials E76/P1, except height for progeny trial E76/P2, which was 2.09m at Pinewoods site and 3.52m at Vlakkloof site which showed that, selection for taller trees will be more effective at Vlakkloof site. A preliminary study was undertaken from eleven second generation trees (2007 tree collection) to investigate if the radial strip core taken at breast height predicts the whole tree wood properties. Correlations found between laboratory Kraft pulping of whole tree wood discs and whole tree NIR spectra with that of the radial strip core NIR spectra were 0.9472 and 0.9506, respectively. These results confirmed that NIR spectra of the radial strip core at breast height predict the whole tree wood properties. A non-destructive near infrared calibration model using wood samples was obtained from Eucalyptus macarthurii felled trees. The wood samples were chipped into wood chips, pulped using Kraft pulping (reference method) and a sub-sample of wood chips of the same trees were ground into sawdust samples and analysed through near infrared spectroscopy for screened pulp yield. The screened pulp yield values obtained from both processes had a narrow screened pulp yield range of 40 to 48%. The Eucalyptus macarthurii screened pulp yield values obtained from both processes, as well as from values obtained from other eucalypt species, were subjected to Vision® Software for calibration and validation of the near infrared calibration model. The results indicated a strong calibration correlation coefficient of 94%, between Kraft pulping and near infrared spectroscopy with a validation coefficient of 89%. The strong correlation and validation coefficient indicated that a reliable non-destructive near infrared model to predict screened pulp yield was successfully developed. The successful development of the valid calibration model required a wider range of other eucalypts species, which improved the development of the model. The developed calibration model was applied to the second generation breeding population planted in KwaZulu-Natal and Mpumalanga provinces, using wood core samples obtained from standing trees for the prediction of screened pulp yield. The highest screened pulp yield achieved was 48%, which compared well to that found for Kraft pulping, which confirmed the success of the development of the calibration model. There was a wide scope of growth variation found amongst traits, which will be useful in selecting superior trees for the next generation. The development of the nondestructive near infrared calibration model was a success due to the strong correlation coefficients found between the screened pulp yields obtained from Kraft pulping and near infrared spectroscopy processes, which was achieved by the inclusion of other eucalypt species in the dataset. The calibration model can be used to select the top performing individual and family trees for the next generation based on screened pulp yield. Tree improvement trials can now be conserved for further breeding, without felling the trees for determination of pulping properties.
Characterisation of antibiotic resistance in Streptococcus, Enterococcus and Staphylococcus using a bioinformatics approach.
(2005) Ramsuran, Veron.; Beukes, Mervyn.
The rate at which bacterial pathogens are becoming resistant to antibiotics is quite alarming, and therefore much attention has been focussed on this area. The mechanism whereby the bacterial cells acquire resistance is studied in order to determine how this process works as well as to determine if any future resistance mechanisms can be circumvented. In this study three different genera and the antibiotics that are resistant to them were used, namely, penicillin resistant Streptococcus, vancomycin resistant Enterococcus and methicillin resistant Staphylococcus. The results prove that the active sites SXXK, SXN and KT(S) G in the penicillin resistance Streptococcus plays a major role in resistance. It is seen in this study that the SXXK active site is found in all the resistant and most of the intermediate strains, therefore proving to be an important component of the cell wall resistance. It was subsequently noticed the greater the number of mutations found in the sequences the higher the resistance. Three dimensional structures showed the actives sites and their binding pockets. The results also show the change in conformation with a mutation in the active site. The results also proved that the Penicillin Binding Protein (PBP) genes essential for resistance are PBP Ia, PBP 2b and PBP 2x. The results obtained, for the vancomycin resistance in Enterococcus study, proved that the VanC and VanE cluster are very much alike and VanE could have evolved from VanC. There is also close similarity between the different ligase genes. The VanX 3D structure shows the position of the critical amino acids responsible for the breakdown of the D-Ala-D-Ala precursors, and the VanA ligase 3D structure shows the amino acids responsible the ligation of the D-Ala-D-Lac precursors. The analysis performed on the methicillin resistance in Staphylococcus study showed that the genes used to confer resistance are very similar between different strains as well as different species.
Characterisation of the divergence of the Elsenburg Merino resource flock.
(2012) Naidoo, Pavarni.; Dzomba, Edgar Farai.; Cloete, Schalk Willem P.
The Elsenburg Merino flock has been divergently selected for the ability of ewes to rear multiple offspring since 1986. Updated genetic trends for reproduction are reported for the Elsenburg Merino resource flock. The objective was to determine whether genetic trends estimated previously for the Elsenburg Merino Resource flock changed significantly with the introduction of genetic material from the industry to the high (H) line. All analyses included the full pedigree file, consisting of 6547 individuals. Heritability estimates were 0.08 ± 0.02 for number of lambs weaned and 0.11 ± 0.02 for corrected weight of lamb weaned. The ewe permanent environment variance was estimated at 0.09 ± 0.02 and 0.11 ± 0.02 for number of lambs weaned and for corrected weight of lamb weaned, respectively. Genetic trends for the H and low (L) lines were divergent (P < 0.05) for all reproduction traits during the period prior to the observed breakpoints. Progress for number of lambs weaned in the H line stabilised after 1999 while a decline in response for weight of lamb weaned in the H line occurred after 2003. The change points may result from reduced selection intensity during the formation of reciprocal crossbred lines, or the introduction of unrelated industry sires in the H line. The pedigree was analysed and inbreeding trends computed for the H and L lines with the aim to test the significance of inbreeding within the lines. The software packages used for the statistical analyses were ENDOG v4.8 and POPREP web analysis software. The average inbreeding coefficients (F) were 1.47% and 0.73% for the divergently selected H and L lines. The rate of inbreeding (ΔF) per generation was 0.5% for the H line and 0.6% in the L line. The overall rates of inbreeding per generation were different in the H and L lines but within acceptable levels. The L line, however, showed an unwanted recent increase in inbreeding that will need to be considered in future. Since 2003, part of the Elsenburg Merino breeding flock was subjected to structured reciprocal within-breed crossing. Lamb survival traits and ewe reproductive performance of purebred (H and L) and reciprocal crosses (HxL and LxH) were evaluated using least squares analyses. Levels of heterosis were also assessed. The mean survival of the two crossbred lines was notably superior to the midparent value in absolute terms, although the contrast did not reach statistical significance (P = 0.098). Further research is required to establish whether this within breed heterosis for lamb survival can be exploited to decrease lamb losses. Reproduction, number of lambs born (NLB) and number of lambs weaned (NLW) in the H line was higher than in the L line (P < 0.05) while the two crossbred lines were intermediate and different from both the H line and the L line (P < 0.05) from the analyses of annual reproduction and overall “lifetime” reproduction across three lambing opportunities. Individual heterosis for annual reproduction was estimated at 2.2% for NLB, 13.8% for NLW and 8.5% for corrected weight of lamb weaned (TWW), with the estimate for NLW reaching significance (P < 0.05). Corresponding estimates for total production over three lambing opportunities were 8.7% for TNLB, 19.1% for TNLW and 13.8% for TTWW, with the estimate for NLW reaching significance (P < 0.05). Ten RAPD markers were used to study molecular divergence between the H and L lines. Phenotypic data on the lifetime reproduction of ewes born in 1999 and 2000 indicated that reproduction in the H line ewes was markedly higher than that of L line contemporaries (P < 0.01). The RAPD assay, conducted on 15 ewes from each line, used eight primers and produced 87% polymorphic loci. The mean coefficient of genetic differentiation between lines (Gst) was estimated to be 0.25. In conclusion, the H and L lines were shown to be divergent for genetic trends and levels of inbreeding. The derived estimates of heterosis may also be used to infer divergence between the lines and significant molecular divergence proven using RAPD assays.
Characterization of streptococcal infections in KwaZulu-Natal Durban by random amplified polymorphic DNA anaylsis and DNA macrorestriction analysis.
(2004) Madlala, Paradise Zamokuhle.; Beukes, Mervyn.
A collection of 29 clinical streptococcal isolates obtained from the University of KwaZulu-Natal, Medical School, Durban Metro area (South Africa) were studied to establish their penicillin G susceptibility patterns often refered to as minimal inhibitory concentration (MIC) and to determine the genetic diversity among them using two genotyping methods, randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) analysis. All isolates with MIC less than or equal to 0.12 µg/ml were considered susceptible, intermediate resistant if MIC was between 0.25 µg/ml and 4 µg/ml and resistant if greater than 4 µg/ml, The percentage of isolates with resistance was relatively high (75.9%), only 10.3% of isolates showed intermediate resistance and 13.8% of the isolates were completely susceptible to penicillin G. Some of the resistant isolates were highly resistant reaching penicillin G MIC levels of 5000 µ/ml. They were speculated to contain Path altered penicillin binding proteins and high level of crosslinking cell wall induced by the gene products of the MurMN operon. RAPD analysis was performed using three primers, MBPZ-1, MBPZ-2, and MBPZ-3, respectively. RAPD analysis allowed for the identification of 27 RAPD types with MBPZ-1 and MBPZ-3 and 26 RAPD types with MBPZ-2. Ninety-eight percent of these isolates were clustered into two groups, group I and group II, with 90% to 100% dissimilarity among them. Fifty two percent of the isolates of MBPZ-1 group I were in MBPZ-2 group I, 72% isolates of MBPZ-1 group I were in MBPZ-3 group I, and 72% of the isolates of MBPZ-2 group I were in MBPZ-3 group 1. This shows the discriminatory ability of the primers used in this study. Despite clustering of isolates, relatively high diversity was seen. PFGE analysis of macrorestriction fragments obtained after digestion of chromosomal DNA by restriction enzyme, SmaI showed 24 PFGE patterns. The 24 PFGE patterns were divided into three groups (I, II and III) of isolates, with an average of 85% dissimilarity (15% homology) among them. At 25% homology, four clusters, A (13 isolates), B (9 isolates), C (4 isolates), and D (4 isolates) were observed. Two pairs of isolates in group I, cluster A, showed 100% homology. This suggested that each represent the same strain. Four isolates of group I, cluster B, also exhibited 100% homology. This study showed that most of streptococcal isolates with the same penicillin G susceptibility patterns grouped together in a phylogenetic tree by both RAPD and PFGE analysis. There was also some similarity between the results obtained by RAPD analysis and PFGE analysis. Seventeen and nine of the 29 isolates grouped into group I and group II, respectively, two pairs of isolates were indistinguishable, and two pairs of islates were closely related by both RAPD (using MBPZ-3) and PFGE analysis. Although, RAPD analysis is sensitive, specific, faster and cost effective, the ease with which PFGE analysis can be performed, high discriminatory power, reproducibility of the results, and the polymorphism seen in the patterns, suggests that PFGE method has the potential to be very useful for epidemiological evaluations of nosocomial streptococcal infections in KwaZulu-Natal.
Characterization of the autolytic systems in selected streptococcal species.
(2005) Naidoo, Kershney.; Beukes, Mervyn.
Autolysins are endogenous enzymes responsible for the cleavage of specific bonds in the bacterial sacculus resulting in damage to the integrity and protective properties of the cell wall. The true biological functions of these enzymes are largely unknown. However, they have been implicated in various important biological synthesis processes making their characterization important. Antibiotic susceptibility testing showed these streptococcal strains to have broad spectrum inhibitory concentrations. The major autolysins of selected streptococcal strains were detected and partially characterized by renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (zymograms). The autolysins were isolated from the specific culture supematants using 4% SDS precipitation and were shown to have apparent molecular masses ranging from 60kDa to 20kDa. Four major autolysins named A, B, C, and D from the Streptococcus milleri 77 strain were characterized. Lytic enzymes were blotted onto polyvinylidene difluoride (PVDF) membrane and N-terminally sequenced. Sequences showed between 100% and 80% similarity to that of a muramidase, glucosaminidase and a peptidase from S. mutans, S. pyogenes and S. pneumonia respectively. Biochemical characterization confirmed autolysin A to exhibit muramidase activity with both autolysin Band C exhibiting endopeptidase activity. Autolysin D showed an 80% N-terminal sequence similarity to Millericin B, a peptidoglycan hydrolase that is known to exhibit peptidase activity. Autolysis was determined using different buffers at two optimal pHs. Assaying for autolytic activity at different growth stages showed autolysis to be moderate during the lag and early exponential phases of the growth cycle. The activities of autolysins were the highest in the late exponential phase and the stationary phase of growth. Zymogram analysis showed that the Streptococcal milleri strains had moderate autolytic expression during the early and late exponential phases of the growth cycle. Control regulatory mechanisms of autolysins were determined in the presence or absence of specific charged groups, such as teichoic acids. In each case the absence of these charged groups inhibited the rate of autolysis, suggesting that the absence of teichoic acids could play a role in the regulation of the autolysins. Two-dimensional-SDS and zymographic-electrophoresis was used to determine total protein profiles for each strain. This is the first report using twodimensional zymography. Specific proteins which were either up- or down-regulated were identified.
Cloning, expression and purification of the immunity factor associated with leucocin A production.
(2004) Pillay, Kovashni.; Beukes, Mervyn.
Leucocin A is a bacteriocin produced by Leucoconostoc gelidium UAL 187-22. Bacteriocin producer strains possess an immunity protein, which enables the strain to protect itself against its own bacteriocin. The immunity gene from Leucoconostoc gelidium was isolated via PCR from a recombinant clone pJF5.5. This fragment was cloned by amplifying the immunity gene from pJF5.5 and ligating it into pMALc2. The resulting recombinant plasmid pKP1 was then transformed into Escherichia coli strain JM103. The clone putative, was confirmed by DNA sequencing and southern blot hybridization using the primers EAL-2 and EAL-3. It was shown to contain an insert of 3.6 kb. Expression analysis showed the construct as an in frame malE fusion protein expressed within E. coli. The fusion construct was isolated by affinity chromatography. Leucocin A was purified to 90% purity, from the supernatant of Leucocnostoc gelidium UAL 187-22 by ion-exchange chromatography and HPLC. It was found to elute from a C18 reverse phase column at 55% actetonitrile, 0.1% TFA. Binding interaction and the stability of the immunity gene fusion protein were compared using a Biacore 2000. The supernatant and cytoplasmic extract isolated from Leucocnostoc gelidium UAL 187-22 were tested for interaction with the fusion construct. Surface Plasmon resonance studies indicated that there was no binding partner present in the supernatant which would influence the immunity process. However, a stable interaction was found between the immunity protein and an orphan ligand within the cytoplasm.
Cloning, expression and purification of the subunits of the Mannose PTS Permease of Listeria monocytogenes EGD.
(2010) Mia, Rizwana.; Beukes, Mervyn.; Watson, Gregory M. F.
The disease listeriosis is caused by Listeria monocytogenes. This common food-borne disease has been responsible for about 0.1 to 10 cases per million inhabitants per year. However, this disease is serious with its high fatality rates of 20% - 30%, and 40% of all cases reported have been in pregnant women suffered from a foetal abortion. Recently the organism has acquired resistance to antibiotic treatment and the development of an alternative treatment is necessary. Class IIa bacteriocins such as leucocin A have been shown to be active against L. monocytogenes. However, the leucocin A receptor molecule responsible for growth inhibition within L. monocytogenes remains unclear. Various studies have implicated the mannose PTS permease (EIIt Man) of L. monocytogenes as the putative receptor for class IIa bacteriocins. The results from studies reviewed indicate that the EIIt Man of L. monocytogenes could be the chiral receptor needed for bacteriocin interaction at the surface of targeted cells. Specifically, the membrane associated IIDMan and IICMan subunits were implicated in direct interaction with class IIa bacteriocins. Our study focused on cloning, expression and purification of the subunits of the mannose PTS permease of L. monocytogenes EGD. Primers were designed to amplify the subunit genes of the mptACD operon. The mptC, mptD and mptAB genes which were then successfully cloned into pET28a expression vector and transformed into E. coli JM109(DE3) host strain. Recombinant plasmids were screened using colony PCR. Subsequently recombinant pET28-C, pET28-D and pET28-AB was once again transformed and expressed in the E. coli BL21(DE3) pLysS expression host strain. After an induction at 30°C for 5 hours, IICMan and IIDMan were found to be expressed in the cell membrane, whilst IIABMan was expressed in the cytosol of the host expression strain. Membrane proteins His-IICMan, His- IIDMan, and cytosol associated His-IIABMan were purified using Ni2+-NTA affinity chromatography. Results for His-IICMan yielded a 28 kDa protein and a 55 kDa co-purified protein. Results for His-IIDMan yielded a 31 kDa protein and a 60 kDa co-purified protein. Results for His-IIABMan yielded a 35 kDa protein and a 68 kDa co-purified protein. A western blot analysis revealed that all proteins purified carried an attached His-tag as detected by an anti-mouse peroxidase conjugate anti-His-tag antibody.
Computer simulation of marker-assisted selection utilizing linkage disequilibrium.
(2006) Keildson, Sarah.; Hancock, Carolyn Elizabeth.
The face of animal breeding has changed significantly over the past few decades. Traditionally, the genetic improvement of both plant and animal species focussed on the selective breeding of individuals with superior phenotypes, with no precise knowledge of the genes controlling the traits under selection. Over the past few decades, however, advances in molecular genetics have led to the identification of genetic markers associated with genes controlling economically important traits, which has enabled breeders to enhance the genetic improvement of breeding stock through linkage disequilibrium marker-assisted selection. Since the integration of marker-assisted selection into breeding programmes has not been widely documented, it is important that breeders are able to evaluate the advantages and disadvantages of marker-assisted selection, in comparison to phenotypic selection, prior to the implementation of either selection strategy. Therefore, this investigation aimed to develop deterministic simulation models that could accurately demonstrate and compare the effects of phenotypic selection and marker-assisted selection, under the assumption of both additive gene action and complete dominance at the loci of interest. Six computer models were developed using Microsoft Excel, namely 'Random Mating,' 'Phenotypic Selection,' 'Marker-Assisted Selection,' 'Selection with Dominance,' 'Direct Selection' and 'Indirect selection.' The 'Random Mating' model was firstly used to determine the effects of linkage disequilibrium between two loci in a randomly mating population. The 'Phenotypic Selection' and 'Marker-Assisted Selection' models focused primarily on examining and comparing the response to these two selection strategies over five generations and their consequent effect on genetic variation in a population when the QTL of interest exhibited additive gene action. In contrast, the 'Selection with Dominance' model investigated the efficiency of phenotypic selection and marker-assisted selection under the assumption of complete dominance at the QTL under selection. Finally, the 'Direct Selection and 'Indirect Selection' models were developed in order to mimic the effects of marker assisted selection on two cattle populations utilizing both a direct and indirect marker respectively. The simulated results showed that, under the assumption of additive gene action, marker-assisted selection was more effective than phenotypic selection in increasing the population mean, when linkage disequilibrium was present between the marker locus and the QTL under selection and the QTL captured more than 80% of the trait variance. The response to both selection strategies was shown to decrease over five generations due to the decrease in genetic variation associated with selection. When the QTL under selection was assumed to display complete dominance, however, marker-assisted selection was markedly more effective than phenotypic selection, even when a minimal amount of linkage disequilibrium was present in the population and the QTL captured only 60% of the trait variance. The results obtained in this investigation were successful in simulating the theoretical expectations of markerassisted selection. The computer models developed in this investigation have potential applications in both the research and agricultural sectors. For example, the successful application of a model developed in this investigation to a practical situation that simulated markerassisted selection, was demonstrated using data from two Holstein cattle populations. Furthermore, the computer models that have been developed may be used in education for the enhancement of students understanding of abstract genetics concepts such as linkage disequilibrium and marker-assisted selection.
Conservation genetics of the Hooded vulture Necrosyrtes monachus.
(2023) Le Roux, Rynhardt.; Willows-Munro, Sandi.; Van Vuuren, Bettine.; Thompson, Lindy Jane.
African vulture species have experienced rapid population declines, due to many anthropogenic threats. Hooded vultures are no exception and have experienced dramatic declines and are now listed as Critically Endangered on the IUCN Red Data list. Two subspecies of Hooded vulture have been described : Necrosyrtes monachus monachus which occurs in West Africa and Necrosyrtes monachus pileatus which occurs in East and southern Africa. The two subspecies differ in their feeding behaviour and morphology supporting the validity of the subspecies status. However, the validity of this taxonomic grouping is still being questioned. Clarifying the taxonomic status of the subspecies is important as if the two subspecies are genetically distinct then they should not be managed as a single species and current conservation policies would need to be updated. In addition, there is limited information available on many aspects of Hooded vulture life history including the factors affecting reproduction in the wild. In Chapter 2 I use microsatellite data collected from across the distributions of the two subspecies and Approximate Bayesian Computation (ABC) to test the hypothesis that the two subspecies are genetically distinct and should be elevated to separate species. In Chapter 3 I examine the genetic variation present in the South African Hooded vulture population. This population only includes 100-200 individuals and is at the edge of the southern range of the species. The conservation value of peripheral populations is debatable as these populations are often isolated and smaller with genetic drift and inbreeding leading to reduced genetic variability. In contrast, studying the genetic diversity in range-edge populations is important for understanding range shifts and adaptive capacity under climate change. These edge populations could potentially also retain unique genetic diversity which helps with the adaptation of species to different environments. Vulture colonies act as “food finding information hubs” allowing for the exchange of information regarding potential food resources. This explains, in part, the high-levels of relatedness often found within colonies as close relatives are more likely to tolerate the cost of sharing food by increasing their inclusive fitness. Hooded vultures are tree nesters with a single breeding pair per tree. In Chapter 4 I use the genetic data to test if individuals nesting close to each other are closely related and if the same individuals use the same nest over multiple years. The analyses conducted in Chapter 2 did not support the existence of the two subspecies classification, due to different demographic events experienced between the two groups. The next factor indicating that there is no subspeciation is the contemporary gene flow that is still seen between the population (m = 0.188) and the little variance seen between the two subspecies (11.9%). Structure analysis also does not support the formation of two distinct subspecies. Thus, this study supports the claim made by Mundy 2021 that it is size cline and not speciation. In Chapter 3 the genetic data did not support the hypothesis that the small South African population was genetically depauperate, instead the results show that the South African population contained similar levels of genetic diversity (Ho = 0.495) to that recorded for the Ghanaian population (Ho = 0.315) where Hooded vultures are more abundant. Levels of heterozygosity were similar to those recorded for other species of Old World vultures such as Cape Vultures (Gyps coprotheres, Ho = 0.380), and Bearded vultures (Gypaetus barbatus Ho = 0.400 – 0.480), but differed from the Griffon Vulture (Gyps fulvus Ho = 0.530 – 0.600) found in Europe. Worryingly, both populations of Hooded vultures show elevated levels of inbreeding and relatedness. The bottleneck analysis for both populations show no sign of a recent bottleneck and a normal L shaped distribution for both populations. In Chapter 4 breeding pairs were not found to reuse the same nests over multiple years. A negative correlation was seen between genetic distance and geographical distance (R2 = 0.0117; p-value = 0.012) the closer related individuals thus tend to nest further away from each other. The spatial autocorrelation shows a positive correlation between genetic and geographical distance between distance classes 8 km – 16km, 32 km – 40km and then between 88 km – 112km, but no clear support for increased relatedness between closer nesting individuals. Thus no support is seen for the formation of loose colonies to function as food finding information sharing hubs. African vultures are facing a number of challenges and most species are considered of conservation concern. Despite this limited genetic data is available for many species. This study aimed to fill this knowledge gap by generating and analysing microsatellite data for the Critically Endangered Hooded vulture to answer a number of key hypotheses. As such this study makes an important contribution towards the conservation of Hooded vultures across Africa.
Designing T-cell epitope-based vaccine against Eimeria infection in chicken using immunoinformatics approach.
(2021) Madlala, Thabile.; Adeleke, Matthew Adekunle.; Okpeku, Moses.
Chicken coccidiosis is the most significant ubiquitous, intestinal parasitic disease known to infect chickens globally. It is recognised for incurring significant production loss to the poultry industry, caused by single or multiple Eimeria spp. infections which threaten chicken welfare and productivity. The emergence of drug resistance in parasites and pathogenicity reversion has put pressure on the poultry industry to reduce chemoprophylactic drugs and live vaccines as preventive measures against coccidiosis. Recombinant DNA vaccines have shown promising results as an alternative option, but complete protection has not been reported highlighting the need for the design of new vaccine against this disease. In this study, Eimeria antigens Immune Mapped Protein-1(IMP1) and Microneme Protein-2(MIC2) were explored using reverse vaccinology and immunoinformatics tool to predict and design potential multiepitope vaccine candidate against coccidiosis. A total of 28 and 19 antigenic T-cell epitopes were predicted and used to construct two multiepitope vaccines with 610 and 512 amino acids for IMP1 and MIC2, respectively. The produced vaccines exhibited favoured characteristics for an ideal vaccine candidate; they were antigenic (Vaxijen score of 0.5989 and 0.5103), immunogenic (scores: 10.15 and 9 419), thermostable (instability index <40 ), and non-allergic. The presence of IFN-gamma and IL-4 inducing epitopes in the constructed vaccine enables vaccine to trigger a cellular and humoral response within the host. Molecular docking of designed vaccines with toll-like receptors (TLR4 and TLR5) to determine vaccine interaction and stability was confirmed by molecular dynamics simulation root-mean square deviation (RMDS) and root-mean-square fluctuation (RMSF) analysis. The designed vaccines induced immune response through production of cytokines and antibodies associated with tertiary response. When exposed to online immune simulation C-ImmSim, both vaccines produced potent immune response through production of IgG, Tc and Th cell and memory Bcells. The constructed multiepitope vaccine in this present study is highly promising and as such further experimental work should be done to confirm its suitability against chicken coccidiosis.
Direct transformation of maize (Zea mays L.) tissue using electroporation and particle bombardment, and regeneration of plantlets.
(1996) Jenkins, Megan Joy.; Shanahan, Paul Edward.
Please open electronic version for Abstract.
Diversity of Eimeria tenella apical membrane antigen-1 from chickens in Mpumalanga province and its in silico epitope prediction as a vaccine candidate.
(2021) Tenza, Petronella Nokukhanya.; Adeleke, Matthew Adekunle.; Fatoba, Abiodun Joseph.
Coccidiosis has been a significant challenge in the poultry industry. There is a high request for the modification of a cost-effective immunizing agent to curtail this disease. Apical membrane antigen 1 (AMA1) has been reported as a protective antigen in sub-unit vaccine development against several apicomplexan parasites such as Plasmodium falciparum, Eimeria tenella and Eimeria maxima. However, knowledge of genetic diversity in this vaccine candidate is imperative. Also, to minimize the cost and time involved in producing a vaccine, computational vaccine design has received much attention through the immunoinformatics method. Therefore, screening for the potential vaccine epitopes in AMA1 that can induce cellular and humoral immune response through the immunoinformatics technique looks promising. This investigation aimed to detect the level of genetic diversity amid Eimeria tenella Apical Membrane Antigen 1(EtAMA1) in selected farms in Mpumalanga province and predict vaccine epitopes from this antigen. Four hundred fresh faecal samples were collected from 10 selected broiler chicken farms in Mpumalanga. The samples were screened for Eimeria oocyst using a compound microscope, and samples containing oocyst were further screened for E. tenella using molecular methods. AMA1 (n=103) was amplified from positive samples for E. tenella, and resulted amplicons were sent to Inqaba Biotec for sequencing and analyzed using MEGA6.06 and DnaSP programs. The results revealed low levels of genetic diversity among Mpumalanga EtAMA1 sequences which were measured by nucleotide diversity (0.0007) diversity, haplotype diversity (0.113) and haplotype number (3). Correspondingly, the haplotype network revealed 4 haplotypes, 3 of which consist of samples from Mpumalanga. Identification of immunogenic B- and T-cell epitopes from EtAMA1 sequences was further carried out and were used to construct a multiepitope vaccine (MEV) using immunoinformatics approaches. The constructed MEV is 311 amino acids long. It was constructed by linking 6 B-cell, 3 CD8+ epitopes and 6 CD4+ epitopes with appropriate adjuvant and linkers. Both adjuvant and linkers were used to increase the immunogenicity of the MEV. The designed MEV was highly antigenic and non-allergenic. The results showed a strong binding affinity of MEV with TLR4. These results suggest that the predicted vaccine could be a significant vaccine candidate against chicken coccidiosis through further experimental validation is still necessary.
Dynamics of the glutathione/glutaredoxin system.
(2014) Mashamaite, Lefentse Nelly.; Pillay, Che Sobashkar.; Rohwer, Johann M.
The glutathione/glutaredoxin system is made up of glutaredoxins, glutathione (GSH) and glutathione reductase (GLR). Glutaredoxins, which are involved in essential cellular functions such as DNA synthesis, iron metabolism and iron-sulfur cluster assembly, become oxidised during their catalytic cycle and are reduced by GSH and GLR. Glutaredoxins also play a critical role in regulating the glutathionylation/deglutathionylation cycle. Under oxidative stress conditions, protein thiols may be glutathionylated and glutaredoxin activity is important for restoring the functions of these proteins. While the individual components of this system have been studied extensively, the dynamics of the system as a whole has not been described despite its importance in the glutathionylation/deglutathionylation process. Computational systems biology approaches could be used to describe this type of regulation but the kinetic mechanism used by glutaredoxins for deglutathionylation is unclear as a monothiol and a dithiol mechanism have both been proposed for glutaredoxin activity. The in vitro data supporting these mechanisms have been contradictory with a number of discrepancies observed in the literature, including contrasting activities of mutant glutaredoxin Cxx(C→S) and wild-type glutaredoxins. Further, Lineweaver-Burk plots showed a curved line pattern in some studies, while other studies reported a linear pattern in response to GSH. Finally, analyses of the Lineweaver-Burk plots in two substrate kinetics experiments revealed both parallel line and intersecting initial velocity line patterns for deglutathionylation. Computational and mathematical models were used to resolve these discrepancies and we showed that the mono- and di- thiol mechanisms, are in fact identical. Mathematical models of mutant and wild-type glutaredoxin activities revealed that the GSH concentration and the rate constant for GSH oxidation significantly affected these relative activities which explained the contradictory data for wild-type and mutant glutaredoxins. The sigmoidal response to GSH was due to the kinetic order of this reaction and our results demonstrated that the resulting parallel and intersecting kinetic line patterns observed in some studies depended on the reversibility of the deglutathionylation reaction. Finally, fitting experiments showed that our models were able to accurately describe the in vitro data. Collectively, our results showed how deglutathionylation should be described in computational systems biology models and further revealed how the formation of oxidised glutaredoxin may play a vital role in the regulation of glutaredoxin activity.
The dynamics of thioredoxin-dependent reactions.
(2014) Photolo, Mampolelo M.; Pillay, Che Sobashkar.
The thioredoxin system, comprising thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, is present in most organisms from prokaryotes to eukaryotes. The system plays a central role in the redox regulation of several key cellular processes including DNA synthesis, apoptosis, glycolysis and redox signal transduction and changes in this system are associated with the progression of a number of diseases including certain cancers, malaria and HIV. Understanding the regulation of this network from a systems perspective is therefore essential. Our lab developed the first computational model of the Escherichia coli thioredoxin system and analyzed this system using mathematical and core models. In contrast to the commonly held assumption of the thioredoxin network as an electrical circuit with no cross-talk, our analysis showed this system displayed ultrasensitivity, adaptability and was interconnected via the thioredoxin redox cycle. In this study, a model of the Saccharomyces cerevisiae thioredoxin system was developed and computational modeling showed that an increase in concentration of one of the substrates in the thioredoxin system could decrease the flux of another thioredoxin oxidation reaction in a concentration-dependent manner. To complement these computational analyses, yeast thioredoxin reductase and thioredoxin were cloned, expressed and purified. An in vitro kinetic assay using insulin as a substrate and immunoglobin Y (IgY) as a competing substrate was subsequently undertaken. Our results showed that in some cases an increase in IgY concentration affected the rate of insulin reduction as measured by turbidity at 650 nm confirming the computational model's prediction. However, unexpectedly, with an increase in IgY concentration, there was a decrease in apparent absorbance at 650 nm at longer time points. These in silico and in vitro analyses shed light on how the thioredoxin system connects seemingly unrelated parts of metabolism into an integrated network, but additional experiments are required in order to improve the kinetic analyses in this study.
The effect of genotype on avian malaria infections in the Amur falcon (Falco amurensis)
(2022) Stoffberg, Rachel Caitlin.; Willows-Munro, Sandi.
Avian malaria is caused by haemosporidian parasites (Plasmodium, Haemoproteus and Leucocytozoon) that are transmitted by dipteran vectors. Passerines have been the focus of avian malaria research however raptors are generally keystone species in ecosystems making them important hosts to investigate. The Amur falcon (Falco amurensis) is a small raptor with the longest migration recorded in any raptor species. This host is particularly interesting to investigate as although it is a raptor it belongs to the order Falconiforms which ultimately is more closely related to parrots and passerines compared to other raptors. The falcons congregate in large flocks during migration which may impact the infection rate of the parasites. The Amur falcon has had a depletion in numbers due to mass harvesting in 2012 as well as two hailstorms that killed approximately 1000 falcons in Kwa-Zulu Natal, South Africa, making it a novel host to test for bottleneck events as well as genetic diversity and population structure. The main aim of this thesis was to determine the significance of age, sex and individual heterozygosity on avian malaria infections in the Amur falcon. The results of this study indicated that the Amur falcon had a high rate of haemosporidian parasite infection, particularly Haemoproteus. Phylogenetic analyses indicated that Haemoproteus was host specific while Leucocytozoon was found to be more generalist, infecting many different species of birds. The Amur falcon population had high genetic diversity and low levels of inbreeding indicating a healthy population. There was a lack of population structure. Generalized linear models were used to test whether sex (male or female), age (juvenile or adult) and individual heterozygosity were drivers of avian malaria infection in the Amur falcon. No significant associations were found except when the different lineages of Haemoproteus were considered independently. The data and results presented in this thesis provide a baseline for future studies on the Amur falcon, and also contributes towards a growing body of work examining haemosporidian parasite infections in migratory birds.
Elucidation of gene function using RNA interference in a cancer cell culture model.
(2011) Daniels, Aliscia Nicole.; Singh, Moganavelli.; Ariatti, Mario.
RNA interference (RNAi), mediated by small interfering RNA (siRNA), has emerged as a powerful tool for elucidating gene function and also holds great potential for the treatment of acquired and inherited diseases. The delivery of siRNAs still remains a major obstacle for their therapeutic use. Cationic liposomes, a group of positively charged nanovesicles, represent a class of non-viral vectors that have shown the ability to efficiently bind and deliver siRNA. In this study, six cationic liposome formulations containing either cationic cholesterol derivative [N-(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) or N,Ndimethylaminopropylaminylsuccinylcholesterylformyl- hydrazide (MSO9) were prepared with the neutral lipid dioleoylphosphatidylethanolamine (DOPE). Varying amounts of distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), (0, 2 and 5 mole percent) were also included in the liposomal formulations as polyethylene glycol is known to improve the lipoplex bioavailability in vivo. We present evidence that siRNA may be delivered to mammalian cells, in vitro, using a novel cationic liposome carrier system and that siRNA binding and transfection efficiency of the cationic liposomes are affected by the degree of pegylation. Cryoelectron microscopy revealed that the liposome vesicles were unilamellar and were in the 30 - 130 nm size range, while band shift assays confirmed the formation of complexes between the siRNA and the liposome preparations. These siRNA lipoplexes were shown to afford protection to their siRNA cargoes against serum nuclease degradation and were also shown to be relatively non-toxic to the HeLa tat luc cell line which stably expresses the firefly luciferase gene. Cryoelectron microscopy revealed that an inverse relationship exists between the lipoplex size and the degree of pegylation. To determine the transfection efficiency of the cationic liposome preparations in the HeLa tat luc cell line, complexes were prepared with anti-luciferase siRNA, which is specific for the firefly luciferase gene, and knockdown of the luciferase gene was monitored in transfected cells. The results show that liposomes containing the cytofectin Chol-T were particularly effective, achieving up to 93.4% gene knockdown at the 30 nM siRNA concentration. The non- pegylated and pegylated cationic liposomes that have been formulated and examined in this study therefore warrant further development to facilitate in vivo studies.

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