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Item Development of methylation-specific PCR (MSP) and multiplex methylation SNaPshot assay for efficient identification of human body fluids.(2020) Haripersad, Sumina.; Ghai, Meenu.The information gathered from crime scenes have the unique ability to direct the course of forensic investigations. In terms of criminal cases, the correct categorisation of human body fluids can provide significant leads e.g., alleged sexual offences can be supported by the presence of vaginal fluid and semen, and signs of probable physical struggle can be inferred by blood at the crime scene. Methods for body fluid identification were initially developed using catalytic, enzymatic, chromatographic, and immunological procedures. Conventional methods, however, do not facilitate multiplexing and are not deoxyribonucleic acid (DNA) based. Tissue-specific differentially methylated regions/sites (tDMRs/tDMSs) are locations/sites on the human genome which display differential methylation patterns in tissues or cells, thus, enabling their use in body fluid identification. Techniques evaluating differential methylation in tDMRs have been particularly beneficial as only extracted DNA is processed, conserving additional physical evidence. Other benefits of DNA methylation-based methods include efficiency and multiplexing to evaluate multiple tissues in a single assay, therefore, is time-saving. The present study aimed to develop two DNA methylation-based assays: methylation-specific polymerase chain reaction (MSP) and methylation SNaPshot, in order to efficiently identify four different body fluids viz. blood, saliva, semen, and vaginal fluid, simultaneously and in a mixture. Methylation profiling of novel DNA methylation markers was carried out by a qualitative MSP assay. The in-house developed ZNF282 and HPCAL1 gene-based tDMRs were employed for analysis of semen and saliva, respectively. The novel MSP primers were designed to target CpG islands in genes ZNF282 and HPCAL1. Two previously reported tDMS markers for blood (cg08792630; Park et al., 2014) and vaginal fluid (cg09765089-231d; Lee et al., 2015) were modified, to design MSP primers which targeted CpG sites flanking the reported sites for blood and vaginal fluid. MSP analysis showed that saliva, semen, and vaginal fluid were correctly identified and differentiated from one another by HPCAL1, ZNF282 and cg09765089-231d markers, respectively. The complete unmethylation of HPCAL1 and ZNF282 in saliva and semen, respectively; and complete methylation in non-target body fluids demonstrated the immense potential both markers have for forensic application. Vaginal fluid marker revealed complete methylation in vaginal fluid. MSP analysis of the blood-specific MSP marker indicated that blood could neither be efficiently identified, nor differentiated from other body fluids of the study, due to the presence of both methylation and unmethylation. A total of four SNaPshot primers were designed based on MSP amplicons, to target a single site which showed differential methylation between the body fluids. All SNaPshot markers were able to identify and differentiate their respective body fluids from others in SNapShot simplex reactions, by generating green (unmethylated) or blue (methylated) peaks only in target body fluids. Excluding the saliva marker, all others displayed high levels of specificity in the multiplex SNaPshot reaction. The clear observation of either complete methylation or complete unmethylation, depicted the high specificity of the markers for criminal investigations. However, obtaining single-body fluid samples is a luxury that crime scenes do not afford. Most evidence is severely degraded, available in low quantities, and present in the form of mixtures. To assess the sensitivity of the designed multiplex MSP-SNaPshot assay, an assortment of body fluid mixtures in varying ratios were subjected to the MSP-SNaPshot assay. The high sensitivity of the semen and saliva marker was validated by the unambiguous identification of semen and saliva, even when target body fluids were minor constituents of the mixture. Vaginal fluid could not be identified when present in lower concentrations. The newly designed blood marker showed significant blood-methylation specificity and sensitivity by efficiently identifying the body fluid in low quantities. The present study reports novel MSP and SNaPshot markers for the identification of blood, saliva, semen, and vaginal fluid in single samples as well as mixtures. Future research would involve the use of the described markers, under different forensic conditions which will enhance their applicability in forensic analysis.Item 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.Item Identification of arthropods of forensic importance during cold and warm seasons in KwaZulu-Natal Province of South Africa.(2021) Tembe, Danisile.; Mukaratirwa, Samson.Forensic entomology is the study and use of insects and other arthropods in forensic investigations associated with death, abuse and neglect of both humans and animals. Although there has been an increased interest in forensic entomology and its application in predicting post-mortem interval (PMI) amongst other issues in many developed countries, the results cannot be extrapolated beyond the countries/regions of study since the arthropods species spectra may vary with region and geographical conditions. The present study aimed to determine the arthropod species of forensic importance found during different stages of decomposition of sheep (Ovis aries) and pig (Sus scrofa domesticus) carrion during the warm and cold season in KwaZulu-Natal province of South Africa. A scoping review was conducted to determine the state of knowledge of forensic entomology research and application in southern Africa. To determine the arthropod species associated with sheep and pig carcass during different stages of decomposition, two medium sized Large-White pigs and two medium sized Merino sheep were humanely killed and used for the cold and warm season. Adult arthropods found on and around the carcasses during different stages of decomposition were collected and identified using combined morphological identification keys and molecular technique based on the mitochondrial gene. The review showed that arthropod species that were found on a decomposing carcass could be useful in the estimation of PMI and provided clues in cases of criminal investigations. The review also confirmed the scarcity of forensic entomology research, and its application in southern Africa. Experimental results from this study showed that dipteran flies from the families Calliphoridae, Muscidae and Sarcophagidae were the first to colonize the sheep and pig carcasses during both warm and cold seasons. These include species of Chrysomya marginalis, Ch. putoria, Ch. albiceps, Ch. chloropyga, Lucilia cuprina, Musca domestica and Sarcophaga calcifera. On the sheep carcasses, Ch. marginalis, Ch. albiceps and M. domestica were the most dominant fly species, contributing 63.2 % of the collected flies in the warm season, and 68.9 % in the cold season. Colonization by coleopterans during the warm season started as early as the fresh stage with Dermestes maculatus, Thanatophilus micans and Onthophagus crassicollis. In the cold season these same beetle species were collected from the bloated stage of the sheep carcass. On the pig carcasses, Ch. marginalis (n = 111), Ch. albiceps (n = 99) and M. domestica (n = 131) were the most abundant species during the warm season. The same species were the most abundant species in the cold season (n = 55), (n = 34) and (n = 81) respectively, although in lower numbers than the warm season. Among the collected Coleoptera species, D. maculatus (n = 112) and N. rufipes (n=62) were the most abundant species found on the carcass during the warm season and the same species were the most abundant species in the cold season (n = 66) and (n = 48) respectively. In the warm season Dermestes maculatus was recorded on the pig carcass during the fresh stage and persisted on the carcass until the last of decomposition. However, in the cold season Dermestes maculatus was first recorded on the carcass during the active stage of decomposition. Molecular analyses confirmed the identification of twelve (12) arthropod taxa collected from both sheep and pig carcasses during the cold season. Results showed that 11/12 arthropod species were common in both sheep and pig carcasses, with exception to Onthophagus sp. and Atherigona soccata species which were unique to sheep and pig carcasses respectively. However, during the warm season, the sheep carcass attracted more (n=13) taxa as compared to the pig carcass. The variation in the arthropod was due to the presence of Onthophagus sp. which was also unique to the sheep carcass during this season. Furthermore, there was an addition of a beetle species Hycleus lunatus, which was collected from both sheep and pig carcasses but unique to the warm season. This study generated important information on the endemic arthropod species that are of forensic importance KwaZulu-Natal province. The arrival time and association of arthropods species with different stages of decomposition during the warm and cold season highlighted their value in estimating the PMI in forensic investigations in the locality of KwaZulu-Natal province. The studied arthropods can potentially be useful in the estimation of PMI and other cases of criminal investigations. The seasonal variations in abundance of both Diptera and Coleoptera in the two seasons seemed to indicate influence of seasons which subsequently influenced temperature. It is recommended that similar studies be conducted at other geographical locations of South Africa with a different ecological system to build a database of dipteran and coleopteran species of forensic importance which are endemic in these areas.