Masters Degrees (Genetics)

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Analysis of the gut microbiome sheds insights into breed resilience and the challenges of antimicrobial resistance in Dohne merino sheep.
(2024) Mgaga, Andiswa.; Dzomba, Edgar Farai.; Muchadeyi, Farai Catherine.; Pierneef, Riaan.
This study focused on analysing the gut microbiome of Dohne Merino, one of South Africa’s leading sheep breeds, that is also reared in Australia, New Zealand, and other European countries. Dohne Merino is of high economic importance in South Africa as it maintains livelihoods in many small rural communities. In South Africa, sheep and other livestock are exposed to multiple diseases and parasites. The efforts to manage these diseases and infections while keeping high productivity have led to an increased usage of antimicrobials in agriculture. This has resulted in high prevalence of antimicrobial resistance (AMR), that is a primary global concern demanding surveillance and action. Some sheep breeds and populations are known to be resilient to diseases, harsh production, and environmental conditions and have low AMR prevalence, which could be due to the defense provided by their gut microbiome. Studying the gut microbiome is essential because the gut microbiome contributes to animal nutrition and health. The study investigated the gut microbial environment of South African Dohne merino sheep. Metagenomic sequencing of the rumen, reticulum, omasum, and abomasum of six mature Dohne Merino ewes that were raised on open pasture was performed. Illumina HiSeq2500 was used to generate a total of 15million reads of 150bp illumina base pairs. The members of the microbial population were fully characterised, and the resistome of the gut was analysed. The microbial population was studied at phylum, class, order, genus, and species level, with bacteria being the most abundant and diverse domain. Other present domains included archaea and viruses. Majority of the microbial population was Bacteroides (53,9%) and Firmicutes (25,4%). Two illness-related bacterial phyla were also identified, Actinobacteria and Cyanobacteria. Identified archaea belonged to the phyla Candidatus Asgardarchaeota, Candidatus Thermoplasmatota, and Euryarchaeota. The observed viral population was very diverse with a total of 5 realms, 8 kingdoms,13 phyla, and 21 classes. Many of the viruses were dsDNA phages belonging to the Duplodnaviria realm. Disease-linked RNA viruses belonging to the Riboviria realm were also observed. Riboviria members have been associated with diseases such as influenzae, rabies and the corona virus. The relationships between microbiome composition and AMR prevalence across the four gut compartments were assessed. A total of 12 AMR genes were identified in the gut and were found to confer resistance to 15 antimicrobials. The observed antimicrobial resistance profiles were low compared to those reported for other breeds and species implying breed resilience of the Dohne Merino sheep. The analysis of the resistance profiles within the compartments revealed that, the higher the diversity, the lower the prevalence of AMR. The overall high diversity of the gut microbiome is the probable cause of breed resilience in Dohne Merino sheep. Additionally, the observed AMR high prevalence with no clinical symptoms of AMR in the animals is also indicative of breed resilience. More studies that will investigate the relationships between the gut microbiome and AMR prevalence are required and the knowledge generated can then be applied to overcome the challenges of AMR in livestock species.
Genomic analysis of Swakara sheep sub-populations.
(2024) Njilo, Andiswa.; Dzomba, Edgar Farai.; Muchadeyi, Farai Catherine.
The Swakara, originally derived from the Karakul breed of Uzbekistan, is a fat-tailed sheep breed first imported into Namibia in the 1900’s and then later spread throughout Southern Africa. With the aim of producing superior pelts, the breed was subjected to intense crossbreeding and selection with the white wooled indigenous Namaqua Afrikaner and Blackhead Persian breeds, with the aim of producing superior pelts possessing wavy hair with short, lustrous fibres and clear patterns. Swakara sheep can be found in four different coat colours (black, grey, brown, and white), with only the black being inherent to the original Karakul with the presence of coat colour variation thought to be the basis of some of the inbreeding and selection imposed on the breed. The sheep are prone to genetic disorders such as the subvital factor that causes the animals to die within 48 hours of birth and is prevalent in grey and white-wooled sheep. It is suggested that these genetic disorders are due to intensive selection imposed on a breed of limited population size. Little is known about the genomic architecture of the Swakara sheep and its divergence between the sub-populations and from the founding breeds. Such lack of information makes it difficult to understand the factors contributing to the appearance of genetic disorders. Furthermore, it imposes a challenge in the implementation of future breeding programs, trying to select for new traits as well as in the conservation of genetic resources. In this study, 244 sheep from 8 sub-populations representative of Swakara Sheep from Namibia (n = 171), and South Africa (n = 44) and founding Karakul sheep from Germany (n = 5) were sampled and genotyped using the OvineSNP50 beadchip. These sheep were of Brown (n = 25), Black (n = 51), Grey (n = 42), White Vital (n = 34), and White Subvital (n = 63) sheep. In addition, ancestral breeds of Namaqua Afrikaner (n = 10) and Blackhead Persian (n = 14) were obtained from previously published data. The first set of analysis investigated the genetic diversity and structure of Swakara sub-populations and its presumed founding breeds. Genetic diversity ranged from HO = 0.29±0.15 for the White Subvital to HO = 0.41±0.22 for the Karakul. The first principal component analysis (PCA) produced five clusters with PC1 explaining 27.35% of the total variation whilst PC2 accounted for 19.25% of the total variation. Cluster A consisted of only the Brown Swakara, Cluster B consisted of the Black, Grey, and White Swakara. Some of the White Vital and Black Vital also clustered together with the Karakul in Cluster C. The Namaqua Afrikaner and Blackhead Persian clustered separately in Clusters D and E, respectively. Per marker FST showed differentiated SNPs within QTLs associated with milk production, wool quality traits, and coat colour. Signatures of selection were identified utilising three methods of within population (iHS), between population (XP-EHH) and across the global population (HapFLK). A total of 73, 619 and 1931 selective sweeps were detected from each of the analysis, respectively. A large number of significant selective sweeps (|iHS|>3.0) were identified within the ancestral populations across 16 different chromosomes (OAR1, OAR2, OAR3, OAR4, OAR5, OAR6, OAR7, OAR8, OAR12, OAR15, OAR17, OAR18, OAR20, OAR21, OAR22, OAR23) and 28 of the signals were detected within the coat colour groups and ancestral groups. Overlapping genes were associated with QTLS of body weight (FBN2, CNTNAP5) and milk fat percentage (TMEM163, NDUFB3). The strongest signals were observed between the coat colour sub-populations with XP-EHH values ranging from 3.93 to 5.27. Further analysis detected signatures of selection related to 56 candidate genes, including PTGER3, ADAMTS3, TROAP, NEIL2, FBXO8, BTBD10, KLF13, and CCNT1 that are associated with hot carcass weight, testes weight and milk yield. HapFLK revealed strong selection signals on chromosome 2 and 3. Genes related to gastrointestinal parasites such as GPC6, KAZN, MCTP1, and HS3ST3A1 were detected. Runs of homozygosity (ROH) analysis revealed the Brown Vital and Blackhead Persian to have ROHs of the longest mean lengths. Furthermore, FROH based inbreeding estimates were low across populations with the highest inbreeding (FROH = 0.26) identified in the Namaqua Afrikaner. This study sought to provide insight into the genomic architecture of Swakara sheep and the presumed ancestors to understand the prevalence of genetic disorders, guide future breeding programs and facilitate preservation of genetics.
Multiple oxidation events increase signal specificity in the Tpx1-Pap1 pathway for hydrogen peroxide.
(2024) Parkies, Selloane Limpho Wandile.; Pillay, Che Sobashkar.
Oxidative stress can be split into two states: eustress is associated with beneficial physiological states and cell proliferation, and distress correlates with deleterious states, neurological disease, diabetes and disease. Many redox pathways in these physiological states across cellular life require multiple oxidation events for full activation. OxyR in E. coli needs four events, Nrf2-Keap1 in mammals requires three, Yap1 in baker’s yeast needs three to four and Pap1 in S. pombe requires two oxidation events. This study investigated the purpose of the multiple oxidation events in hydrogen peroxide signal transduction in redox pathways. To achieve this, we developed and analysed a single and double oxidation computational Pap1-Tpx1-Trr1 model to compare how they respond to different hydrogen peroxide concentrations. We also utilised in vitro recombinant proteins, Tpx1 and Pap1, to assemble the Tpx1-Pap1 system and assessed the signal output at different hydrogen peroxide concentrations. The computational analysis showed that adding an oxidative step increases the system’s capability to attenuate the signal at low hydrogen peroxide concentrations and amplify it at high concentrations. The in vitro system showed that a simplified Tpx1-Pap1 system can differentiate between different hydrogen peroxide concentrations. Multiple oxidation steps impart high pass filtering properties on the system, allowing systems to better differentiate between low and high signal inputs.
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.
Molecular phylogenetic structure between geographically distant marine fish Macrourus holotrachys and M. berglax.
(2021) Zuma, Nkululeko.; Miya, Tshoanelo Portia.
The taxonomic status of Macrourus species has been in disarray due to their morphological similarities and overlapping distribution. Given the number of research that have been conducted in an attempt to resolve the taxonomic confusion in this group, there are still some species with unconfirmed taxonomy. Macrourus holotrachys found in the Southern Ocean and M. berglax from the North Atlantic Ocean, are among the species that need taxonomic review. These species differ morphologically, and the minor differences have been used as a basis to separate them into different species. DNA molecular studies that have reviewed the taxonomy of these species using the cytochrome c oxidase subunit I (COI) gene found low sequence divergence between M. holotrachys and M. berglax. The findings of these studies provide motivation for population genetic studies to compare genetic structure between these distantly distributed species. The present study aimed to close this gap by assessing the genetic structure between M. holotrachys and M. berglax using the COI and displacement loop (D-loop) gene regions. Aligned sequences of these genes were 532 base pairs (bp) and 703 bp long, respectively. The COI gene revealed seven haplotypes from 26 sequences, while D-loop region had eight haplotypes from 55 sequences. The mismatch distribution curves were unimodal and the haplotype network trees had a star shape pattern for both datasets, which is consistent with populations undergoing demographic expansion. Although neutrality test values were not significant, negative values were observed for Tajima’s D, supporting the populations undergoing demographic expansion. The FST pairwise distance method revealed no substantial differentiation between the populations of M. holotrachys and M. berglax. These results suggest that a recent dispersal of populations may have occurred such that there was not enough time for the separated populations to develop different genetic traits. In conclusion, this study demonstrated no significant genetic differentiation between M. holotrachys and M. berglax and confirms that they are one species consisting of North and South Pole populations, respectively. Additionally, this study has added more literature on the population genetic structure of these species.
The use of DNA barcoding in the forensic identification of animal species in processed meat products from KwaZulu-Natal, South Africa.
(2021) Naicker, Annicia Elizè.; Zishiri, Oliver Tendayi.
The price of meat products in South Africa are exceptionally high, to the extent that these products have been categorized as a luxury for many South Africans. The estimated price of white meat per kg is R115,00 while the price of red meat can average at R190,00 per kg, this makes processed meat products an easy target for meat adulteration and substitution. This study aimed to use DNA barcoding to determine if samples collected from local meat markets around eThekwini, KwaZulu-Natal have been subject to food fraud by means of meat adulteration and mislabelling. Universal primers for the amplification of the mitochondrial gene, Cytochrome C oxidase subunit 1 (COI) coupled with bioinformatic analyses using Barcode Of Life Database was utilized in this study. This study has determined that 62% of the samples were indeed adulterated, lamb samples were reportedly substituted with mainly beef(64%), however species of duiker(12%), rat(1%), frog (1%) and even chimpanzee(2%) were also identified in the processed meat products. Chicken samples indicated substitution with fish, crab and even beef. Beef samples however reported the least substituted with only 1 samples showing substitution of beef to fish. Consumers have become increasingly aware of what they are consuming and for this reason this study serves to bring awareness to food fraud by means of meat adulteration and mislabelling. This study has provided valid information on the meat adulteration that is occurring in many local meat markets around Ethekwini, together with violations of the regulations pertaining to consumer protection and food labelling. The area of research is still in the novel stages in South Africa, for this reason it is recommended continued research is conducted not only looking at processed meat products but also fish products and even game meat available throughout South Africa.
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.
Genetic analyses of antimicrobial resistance and virulence genes in Enterococcus species isolated from livestock production systems in South Africa.
(2021) Mnguni, Anele Buhle.; Zishiri, Oliver.
Enterococcus species are widely dispersed in the environment this includes soil, water, plants, food and animals. Although Enterococcus constitute mostly as a commensal bacterium; over the past years the bacterium has evolved to cause nosocomial infections. The proliferation of this pathogen is attributed to its ability in successfully transferring antimicrobial and virulence genes using several channels such as mobile genetic elements. This study investigated the prevalence of Enterococcus spp. in small-scale commercial farms in rural South Africa. The dissemination of virulent E. faecium and E. faecalis isolates allied with livestock production in the Eastern Cape and KwaZulu-Natal provinces was investigated. A total of 276 samples randomly sampled from livestock and their associated environments (feed, soil and water) were screened for Enterococcus spp. using selective media and using DNA molecular methods. E. faecalis and E. faecium prevalence was confirmed by the amplification of the tuf and sodA genes. Sixty-one percent of total presumptive isolates were E. faecalis (n=61) and only 8% (n=8) were identified as E. faecium. The presence of virulence determining factors such as asa1, ccf, cylA, esp, gelE and hyl was screened in all samples that tested positive for Enterococcus species. Presumptive E.faecalis and E. faecium isolates were mostly recovered from Amandawe (KZN). E. faecalis isolates harboured the most virulence genes asa1 (25%; n=), ccf (84%; n=), esp(4%;n= ), gelE (69%; n=) and hyl (12%; n= ). Whilst E. faecium isolates only harboured of asa1(12.5%; n=1), ccf (100%; n=8), gelE (75%;n=6 ) and hyl (25%;n=2). The current study also evaluated the antibiotic resistance profiles and their associated genes in these two species. Antibiotic susceptibility profiles of E. faecium and E. faecalis were assessed using Kirby-Bauer disk-diffusion assay as per the CSLI guidelines. Erythromycin had the highest occurrence of resistant isolates in both species with 75% (n=6) and 54.1% (n=33) respectively. Isolates were least resistant to ampicillin, with 0.03% resistance in E. faecalis and 0% in E. faecium. E. faecalis had the highest prevalence of Multi Drug Resistance (MDR), exhibiting phenotypic resistance to macrolides, aminoglycoside, tetracyclines and fluoroquinolones. TET-CIP-ERY was the most observed antibiotic resistance pattern. Furthermore, the isolates were screened for vanA, vanB, vanC1, vanC2/3, aac(6”)-aph(2”) ,ermA and ermB. The resistance genes that amplified in E. faecalis included vanB (8%;n=5), vanC1 (37%;n=23), vanC2/3 (37%; n=23), ermB (96%;n=58), ermA (8%;n=5) and aac(6”)-aph(2”) (1.6%;n=1). The immense dissemination of E. faecalis that has potentially pathogenic virulent determinants is a cause for concern in livestock production systems. In addition, faecal contamination from livestock poses a threat to the dissemination of virulent strains. The study demonstrated that E. faecium and E. Faecalis isolated from livestock and their associated environment were predominantly resistant to macrolides, glycopeptides, tetracyclines and fluroquinolones. In addition to be the first study in South Africa to document the emergence of inducible vanC determinants in Vancomycin Resistant Enterococci isolates.
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.
Phylogenetic diversity, host specificity and geographic distribution of avian malaria in Africa.
(2022) Van Zweiten, Sam Falcon.; Willows-Munro, Sandi.
Abstract available in PDF.
The thioredoxin redox charge as a measure of cell redox homeostasis in Schizosaccharomyces pombe.
(2022) Bhagwandeen, Tejal.; Pillay, Che Sobashkar.
Thiol-based redox systems play essential roles in repairing oxidatively damaged proteins, deoxyribonucleotide synthesis, sulfur metabolism, protein folding, and oxidant detoxification and signaling. The principal thiol systems in most cells are the thioredoxin (Trx) and the glutathione/glutaredoxin (GSH/Grx) systems. In the thioredoxin system, reducing equivalents from NADPH are transferred by thioredoxin reductase to thioredoxin, resulting in reduced thioredoxin. Thioredoxin in the reduced form further reduces target proteins and is itself consequently oxidized. Given the system’s essential role in cellular physiology, inhibition of the thioredoxin system is an important drug target for communicable and non-communicable diseases. However, measuring the activity of the thioredoxin system in vivo is challenging. The thioredoxin redox charge (reduced thioredoxin/total thioredoxin) was proposed as a novel, surrogate measure of the thioredoxin system’s activity and could be used as a general measure of the cellular redox state. Indeed, published data showed that the thioredoxin redox charge and cell viability collapsed if a chemical inhibitor directly targeted thioredoxin reductase. To evaluate the utility of the thioredoxin redox charge as a generic indicator of redox stress, the fission yeast Schizosaccharomyces pombe, was subjected to various stressors including hydrogen peroxide, heat, cadmium sulfate and potassium ferricyanide and the thioredoxin redox charge and cell viability were measured over time. We found dynamic changes in the thioredoxin redox charge profiles, in response to these stressors, but only obtained weak, positive correlations between the thioredoxin redox charge and cell viability. Thus, and in contrast to our initial hypothesis, the thioredoxin redox charge appeared to be buffered in response to high-stress perturbations, even when cell viability was clearly inhibited. These results show that the redox poise of the thioredoxin system can presumably only be disrupted by direct inhibitors of the system. Future work should aim to elucidate the mechanisms underlying the preservation of the thioredoxin redox charge.
Genetic characterization and molecular detection of antimicrobial resistance genes and virulence genes present in campylobacter spp. isolated from broiler chickens and human clinical samples in Durban.
(2016) Reddy, Samantha.; Zishiri, Oliver Tendayi.
Campylobacter jejuni and Campylobacter coli have evolved as the most prevalent Campylobacter species which are responsible for gastroenteritis infections in humans. Successful infection and continued fitness of Campylobacter species depend on virulence determinants and antimicrobial resistance elements which differ amongst strains of different origin. There is a paucity of information regarding Campylobacter virulence genes and the antimicrobial resistance genes in developing countries such as South Africa. Therefore, research is essential to characterize pathogenic markers and to implement strategies for proper control and prevention of infection caused by this pathogen. This study aimed to detect the presence of virulence genes such as: cadF, hipO, asp, ciaB, dnaJ, pldA, cdtA, cdtB and cdtC as well as the detection of genes associated with antimicrobial resistance which included gyrA, blaOXA-61and tetO present in C. jejuni and C. coli. Following ethical approval, 100 commercial chicken fecal samples were collected and 100 human clinical isolates were selected from a collection of Campylobacter spp. which originated from a private pathology laboratory in South Africa. From the 100 chicken fecal samples 78% were positive for Campylobacter growth on mCCDA and from the collection of a 100 human clinical isolates 83% demonstrated positive Campylobacter spp. growth following culturing methods. The cadF gene was present in 100% of poultry and human clinical isolates. This could indicate that the presence of this gene is needed for successful infection in a host. C. jejuni was the main species detected in both poultry and human clinical isolates while C. coli were detected at a low incidence (chicken =13%, human=17%) and therefore, not statistically significant in either host (p>0.05). The resistance genes gyrA(235bp), gyrA(270bp), blaOXA-61 and tetO were also detected at a higher percentage (51%, 36%, 58% and 68% respectively) in chicken samples compared to human clinical samples (49%, 36%, 53% and 64% respectively). In conclusion, this study demonstrated high prevalence of virulence and antibiotic resistance genes in Campylobacter species from South Africa. The high prevalence rates demonstrated the importance of Campylobacter spp. as a food borne zoonotic pathogen capable of causing persistent infection due to acquisition of antimicrobial resistance genes and virulence genes via the food chain. The study finally recommended limited and prudent use of antimicrobial agents as a mitigating measure to combat the evolution of multiple drug resistance.
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.
The role of thioredoxin in the redox regulation of the Tpx1/Pap1 pathway in Schizosaccharomyces pombe.
(2022) Naidoo, Kelisa Cheyen.; Pillay, Che Sobashkar.
Reactive oxygen species (ROS) can damage cellular components leading to dysfunction and cell death. Paradoxically, ROS, such as hydrogen peroxide, are also essential for a range of metabolic and signalling functions within cells. Given these opposing functions, cells have developed several redox signalling mechanisms to manage ROS within specific homeostatic limits. In bacterial cells, thiol-peroxidases (peroxiredoxins) and other enzymes detoxify ROS, while the antioxidant transcriptional response is induced by transcription factors directly oxidized by ROS. In many eukaryotes, these functions are combined with peroxiredoxins detoxifying ROS as well as activating redox-sensitive transcription factors. The relative benefits and disadvantages of such sensor-mediated redox signalling systems are unknown, and we aimed to understand the logic underlying this signalling mechanism using the Schizosaccharomyces pombe Tpx1/Pap1 pathway. In this pathway, the peroxiredoxin Tpx1 reduces hydrogen peroxide and oxidizes the redox transcription factor Pap1. Following a hydrogen peroxide perturbation, the Pap1 signal profile revealed a biphasic profile with a rapid initial increase followed by a relatively prolonged decrease in Pap1 oxidation. These dynamics were suggestive of an incoherent feedforward loop, and we hypothesized that the Trx1 protein was responsible for the incoherence as it could both dampen and increase the signal by reducing Pap1 and Tpx1, respectively. To test this hypothesis, we analyzed the effect of several oxidants (hydrogen peroxide, tert-butyl hydroperoxide, and diamide) on Pap1 activation to determine if we could selectively modulate signal duration. However, we could not quantitatively delineate the effects of these oxidants on the signal profiles obtained. We, therefore, utilized computational modelling to analyze the Tpx1/Pap1 pathway and found that excess Trx1 reduced Tpx1 faster, preventing the association of Tpx1 and Pap1. On the other hand, insufficient Trx1 allowed for Pap1 to be oxidized over a longer interval which increased the signal duration. Thus, our analysis showed that, in contrast to our hypothesis, Trx1 limitation, rather than incoherence, was responsible for the Pap1 oxidation profile. These results indicate that in the presence of ROS, Trx1 plays a vital role in determining the signal profile of Pap1.
Genetic diversity of some Moringa oleifera Lam. cultivars available in South Africa.
(2020) Makgolane, Thandy Ramathetje.; Tsvuura, Zivanai.; Zishiri, Oliver Tendayi.; Ndhlala, Ashwell Rungano.
Moringa oleifera Lam. (Moringaceae) is a nutritious and high medicinal value tree. The species is native to India but grown globally due to its properties. The nutritional content of the species includes high levels of vitamins, minerals, calcium, magnesium and iron. It is now known that different cultivars of M. oleifera are grown throughout the world depending on the intended products. However, in South Africa, there is limited knowledge on the genetic variation of the cultivars most farmers and researchers are growing. In this study, a partial sequence of chloroplast atpB gene was used to determine genetic diversity of M. oleifera cultivars from different regions across the world but grown for research at the Agricultural Research Council, Vegetable and Ornamental Plants campus in Roodeplaat, Gauteng Province, South Africa. Thirteen cultivars were collected, of which three were domesticated cultivars from South Africa, eight were from Thailand, and one cultivar each was from Taiwan and the United States of America. Each cultivar consisted of six replicates. DNA extraction, PCR and gel electrophoresis were performed at the University of KwaZulu-Natal whereas DNA sequencing was done at Inqaba Biotec laboratory. DNA sequence analysis was done using BioEdit, MegaX, POPArT and DnaSP softwares. Based on the phylogenetic analysis, the average total length of the sequences was 404 nucleotides with the number of polymorphic segregating sites and mutation of 21 and 24, respectively. From the maximum likelihood tree and neighbour-joining tree, South African domesticated cultivars SH and Limpopo were more related to each other than to the domesticated cultivar, CHM (South African). The cluster which was well supported with 84% bootstrap value was between cultivars TOT 5077 (Thailand) and TOT 4100 (Taiwan). The polymorphic data indicated a nucleotide diversity of 0.01654 and average number of nucleotide difference (k) of 6.58095. The haplotype network also showed less genetic differences between the cultivars with some cultivars recorded as similar. The low genetic diversity observed in these cultivars suggests that the cultivars might have originated from a common ancestor. However, further study is necessary by collecting more cultivars of M. oleifera South Africa and other parts of the world to get a clear view on the genetic diversity. The results from this study will be an addition to the already existing knowledge of M. oleifera available in the world and new knowledge on genetic variation among South African cultivars, which may be relevant in cultivar development and conservation.
The glutaredoxin/glutathione post-stress recovery system is dependent on the availability of glutathione in the cell.
(2020) Blom, Erin Shelby.; Pillay, Che Sobashkar.
The cellular response to oxidative stress involves three interconnected processes: reactive oxygen species detoxification, adaptation and repair. Glutathionylation is an adaptive response in which glutathione binds to labile proteins protecting them from oxidative damage but also inactivating them. While it has been established that glutaredoxins play a crucial role in deglutathionylating these proteins, the kinetic regulation of this post-stress repair process is less clear. Intriguingly, aged cells have decreased glutathione levels, although the mechanistic significance of this decrease has not been well-understood. We hypothesized that in these cells, the lower glutathione levels reduced the efficiency of the glutaredoxin/glutathione system which impaired the recovery of the cell post-stress. To test this hypothesis, we used a validated computational model of the glutaredoxin/glutathione system to determine how perturbation of the glutaredoxin system affected the availability of active glutaredoxin as well as the rate of deglutathionylation. We separated the effects of the kinetic and thermodynamic components of glutaredoxin activity and found that the overall flux was primarily controlled by the kinetic effects and that the activity of the system was largely dependent on the availability of reduced glutathione. To test whether reduced deglutathionylation activity was a characteristic of aging, aging and glutathione determination experiments were undertaken in the fission yeast, Schizosaccharomyces pombe. In contrast to our hypothesis and data from other studies, fission yeast cells aged for five days were shown to have increased glutathione concentrations, from 36.62 μM to 43.09 μM in minimal media when compared with two-day old cells, except in the presence of additional glutathione or Lbuthionine sulfoximine, a glutathione synthesis inhibitor. Further, glutathionylation levels decreased or remained unchanged in the aged cultures which we speculate was due to an adaptive response by the glutathione synthesis pathway in these cells. Future experiments will need to measure both the glutaredoxin system and the metabolic pathways that provide reductive inputs into the system in order to understand the role of the glutathionylation cycle in post-stress recovery.
Quantification of time-dependent redox signalling in the Tpx1/Pap1 pathway in Schizosaccharomyces pombe.
(2019) Lind, Diane Justine.
Reactive oxygen species (ROS) can damage cellular components leading to cell death, but paradoxically, ROS also play essential roles in metabolism and signalling in eukaryotic cells. Dysregulation of this balance is associated with a range of host diseases and cells have consequently evolved sophisticated signalling networks to sense, detoxify and adapt to changes in ROS levels. Hydrogen peroxide, for example, is reduced by thiol-peroxidases which in turn, can trigger the oxidation of thiol-dependent redox transcription factors. However, the relationship between hydrogen peroxide stimuli and the level of redox transcription factor activation has largely been described in qualitative terms. Because quantitative measures of the redox signal have been lacking, we tested whether three signalling parameters viz. the signalling time, duration and amplitude could be used to quantify the hydrogen peroxide-dependent redox signal in the Tpx1/Pap1 pathway in Schizosaccharomyces pombe. We found significant differences in the signalling time and duration, but not signal amplitude as hydrogen peroxide concentrations were increased from 100 to 1000 μM in our assays. By way of comparison, we also found that the general oxidant, tert-butyl hydroperoxide at 200 μM, decreased signal time and duration in the Pap1 pathway when compared to an equivalent hydrogen peroxide concentration. This method was also used to compare the hydrogen peroxide signalling by OxyR in Escherichia coli and Yap1 in Saccharomyces cerevisiae showing that these measures could be used to characterize and compare redox signalling from different oxidants and in different species. Thus, quantification of time-dependent redox signalling revealed new insights into hydrogen peroxide signalling that could not be readily obtained by qualitative methods and, these measures are expected to facilitate a better understanding of the role of redox signalling in health and disease.
Methylation profiling and validation of candidate tDMRs for identification of human blood, saliva, semen and vaginal fluid and its application in forensics.
(2017) Naidoo, Natalie.; Ghai, Meenu.
Identification of body fluids and tissues is an essential step in forensic investigation because it can be used as strong evidence in identifying suspects and victims. Currently in forensic investigations, catalytic, enzymatic and immunological techniques are used to identify body fluids, however, are limited due to lack of sensitivity and specificity. Hence, researchers are always on the lookout for novel methods that can be used to identify and analyse body fluids. Recently, DNA methylation-based markers have proven to be more sensitive and specific than conventional methods for body fluid identification. Genome-wide methylation studies have demonstrated that tissue specific differentially methylated regions (tDMRs) vary in methylation profiles in various cell types and tissues. The differences in methylation profiles of tDMRs can be targeted to be used as biomarkers to differentiate between body fluids and tissues. To date, only a few DNA methylation-based markers have been reported to identify body fluids. To enhance the specificity and robustness of DNA methylation-based identification, novel markers are required. Additionally, methylation-based markers require further interrogation, to evaluate the stability of their methylation profiles under simulated forensics conditions such as UV light, temperature, rain and microbes, which could cause DNA degradation and affect DNA recovery as well as the methylation status of body fluids. In a previous study, based on differential gene expression in blood, saliva, semen and vaginal fluid, gene body CpG islands were selected, in genes Zinc finger protein 282 (ZNF282), Protein tyrosine phosphatase, receptor S (PTPRS) and Hippocalcin like 1 (HPCAL1), that have potential tDMRs to differentiate between, blood, saliva, semen and vaginal fluid. It was proposed that differential gene expression could be possibly due to differences in methylation patterns. The present study was undertaken to establish the methylation status of potential tDMRs in target body fluids by using methylation specific PCR (MSP) and bisulfite sequencing (BS). In both MSP and BS, the methylation status of 3 genes ZNF282, PTPRS and HPCAL1 were analysed in 10 samples of each body fluid. With MSP analysis the ZNF282 and PTPRS1 tDMR displayed semen-specific hypomethylation while HPCAL1 tDMR showed saliva-specific hypomethylation. The PTPRS 2 tDMR did not differentiate between any body fluids due to presence of methylation and unmethylation for all body fluids. With quantitative analysis by BS the ZNF282 tDMR showed statistically significant difference in overall methylation status between semen and all other body fluids as well as at individual CpG sites (p < 0.05). Therefore, ZNF282 tDMR has the potential to be used to be a semen-specific hypomethylated marker. However, no statistically significant difference in methylation profiles was observed for PTPRS 1 and PTPRS 2 tDMR between body fluids or at individual CpG sites (p > 0.05). The BS study showed that the tDMR for the HPCAL1 gene displayed non-specific amplification therefore was not further analysed. Furthermore, a sensitivity and forensic simulation study was conducted to determine the stability of methylation profiles. To determine the lowest DNA concentration that can be evaluated with MSP, a sensitivity study was conducted using five-fold serial dilution (25, 20, 15, 10, 5, 1 ng) of blood DNA samples. Each DNA dilution was subjected to bisulfite modification, followed by amplification with ZNF282, PTPRS 1, PTPRS 2, and HPCAL1 primers. The results showed that the detection limits were 10 ng for ZNF282 tDMR, 5 ng for PTPRS 1, 15 ng for PTPRS 2, and 5 ng for HPCAL1 tDMR. Thus, it was concluded that a DNA concentration greater than 10 ng would yield successful results with MSP analyses. To evaluate whether environmental conditions has an effect on the stability of methylation profiles of the ZNF282 tDMR, five samples of each body fluid were subjected to five different forensic simulated conditions (dry at room temperature, wet in an exsiccator, outside on the ground, sprayed with alcohol and sprayed with bleach) for 50 days. Following the 50 days, vaginal fluid showed highest DNA recovery under all conditions while semen had least DNA quantity. Under outside on the ground condition, all body fluids except semen showed decrease in methylation level, however, significant decrease in methylation level was observed for saliva. A statistical significant difference was observed for saliva and semen (p < 0.05) in the outside on the ground condition. No differences in methylation level were observed for the ZNF282 tDMR under all conditions for vaginal fluid samples. Thus, ZNF282 tDMR is stable under environmental insults and can be used as reliable semen-specific hypomethylated marker. The analysis of tDMRs represents a unique, efficient and reliable technique that can be used to differentiate between human body fluids. In the future, identification and validation of new tDMRs based markers as well as determining methylation differences in other forensically relevant body fluids will be beneficial for forensics applications.
Genetic characterization of resistance and virulence genes in Enterococcus species from animal isolates in Durban.
(2018) Eberechi, Phoebe Nnah.; Adeleke, Matthew Adekunle.; Zishiri, Oliver Tendayi.
Misuse of antimicrobials in animal agriculture has given rise to strains of bacteria that are resistant to multiple antibiotics. Enterococci bacteria have emerged among such antibiotic-resistant strains of bacteria and infections due to antibiotic-resistant bacteria is one of the world’s critical health challenge. Enterococci are gut commensal bacteria but are currently confirmed pathogenic bacteria responsible for so many hospital-acquired infections like urinary tract infections. The aim of this research was to detect the occurrence of Enterococcus species in chickens, cats, and dogs; their phenotypic and genotypic resistance to antibiotic drugs and virulence genes. Isolation of Enterococcus species was done using microbiological culture methods and confirmed using specific primers through Polymerase Chain Reaction (PCR). Presumptive Enterococcus growth on bile esculin agar was positive for 94% of all the isolates. Overall, 77.3% of the isolates were positive for Tuf gene (Enterococcus genus-specific gene). Enterococcus faecalis was detected at a higher frequency (40.4%; P <0.05) compared to Enterococcus faecium (8.5%). All the Enterococcus isolates were susceptible to High-Level Gentamicin on antimicrobial susceptibility test. Enterococcus species in chickens exhibited higher resistance to the antibiotics than the pets. Highest resistance was observed in Quinupristin/Dalfopristin (89.4%) followed by Vancomycin (87.9%), Rifampicin (85%), Ampicillin (76.6%), Erythromycin (72.3%), and Tetracycline (64.5%). Chloramphenicol (24.8%), High-Level Streptomycin Resistance (24.1%), and Ciprofloxacin (14.2%). Eighty-four percent (84%) of the Enterococcus isolates expressed multidrug resistance (MDR). Three of the four resistance genes screened were detected: 21.3%, 7.8% and 4.3% for Kanamycin, Streptomycin, and Vancomycin resistance genes respectively. Gentamicin resistance gene was absent in all the isolates. PCR detection of virulence gene showed highest prevalence in EfaA gene at 88.7% frequency followed by GelE (82.3%), ccf (81.6%), Esp (26.2%) and CylA (25.5%). All E. faecalis and E. faecium detected harbored multiple virulence genes. These findings show that chickens, cats, and dogs can be colonized by pathogenic Enterococci which harbor resistance and virulence genes and are multidrug resistant. It is therefore important that antibiotics are used prudently in animal husbandry to mitigate emergence and transfer of Enterococci pathogens to humans via food chain and direct contact of pets by their owners.
Prevalence of Staphylococcus aureus and its antimicrobial resistance and virulence genes in pet dogs of Durban: the risks of reservoir populations.
(2017) Brouckaert, Mary-Anne Frances.; Adeleke, Matthew Adekunle.; Zishiri, Oliver Tendayi.
Abstract available in pdf.

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