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Browsing Biosciences by Author "Feto, Naser Aliye, Dr."

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    Characterization and identification of microbial communities in pigeon droppings using Culture-Independent techniques
    (Vaal University of Technology, 2015-08) Leareng, Samuel Keeng; Feto, Naser Aliye, Dr.; Pillay, M., Prof.
    Pigeon droppings, found in abundance in most cities and towns where pigeons are found, are a source of potential yeast and molds into the environment. Invasive fungal infections are a cause of morbidity and often mortality in immunocompromised individuals. The objective of this study was to the identification of bacterial and mold agents from pigeon droppings. Pigeon droppings samples were collected from three locations during the winter and summer months and studied for the occurrence of bacteria, yeast and molds by utilising culture-independent techniques. Amplification of the 16S rDNA gene and the internal transcribed spacer (ITS) region, cloning and ARDRA and DGGE were used for the characterisation of the microbial populations followed by sequencing. Several mold and yeasts, as well as bacteria were found to be present in pigeon droppings, which can spread into the environment and be transmitted to immunocompromised individuals and children. DGGE analysis of the bacterial communities revealed banding patterns that clustered all but one winter samples and all summer samples, showing a high similarity among the microbial members in both seasons and sample locations. Fungal DGGE analysis revealed clusters that grouped summer and winter samples from Johannesburg and Pretoria while VUT samples were clustered on their own. From the identification of fungal and bacterial DNA, Cryptococcus species was the majority of fungi isolated from the dropping samples. Geotrichum, Kazachstania and Fusarium species were isolated from phylotypes obtained from ITS amplicons analysed by ARDRA. Lactobacillus and Enteroccoccus species, organisms usually found in the gastrointestinal tract were the common bacterial members identified. The results showed no difference in microbial communities across all sample locations, while seasonal changes also had no impact in microbial community patterns.
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    Investigation of seasonal prevalence of low pathogenic avian influenza (LPAI) in a heterogeneous wild waterfowl population in Pretoria
    (Vaal University of Technology, 2018-06) Phiri, Thandeka P.; Abolnik, Celia, Prof.; Feto, Naser Aliye, Dr.
    Influenza-A virus is a single stranded negative sense RNA virus that is a member of a Orthomyxoviridae group. The virus is diverse and consists of 16 haemagglutinin (H) and 9 neuraminidase (N) glycoproteins subtypes that form a serotype. Avian influenza virus (AIV) has been detected in more than 100 bird species from 26 different families, although Anseriformes and Charadriiformes are considered the natural hosts of the virus. A 12-month study was conducted at the African Pride Irene Country Club lodge in Pretoria where the prevalence of AIV was monitored in a community of wild birds. The African Pride Irene Country Club lodge houses a population of wild bird species such as Egyptian geese (Alopochen aegytptiaca), Yellow-billed duck (Anas undulata), Red knobbed coot (Fulica cristata), African sacred ibis (Threskiornis aethiopicus) and Hadeda ibis (Bostrycha hagedash). A total of 3674 faecal samples were collected over a period of 12 months and screened for AIV group using the Matrix gene (M-gene) real time reverse-transcriptase PCR (rRT-PCR). Positive samples were submitted for virus isolation in embryonated chicken eggs. In addition, the RNAs were screened using H5 and H7 subtype specific rRT-PCR and a conventional universal RCR assay that targets the HA gene was also used. Polymerase Chain Reaction (PCR) products were requenced using Sanger sequencing and the viral isolates were subjected to Next Generation sequencing (NGS). Twenty percent of the samples tested positive for the AIV group and four virus subtypes were identified. One virus isolate was identified through NGS as H3N6; two through conventional PCR and Sanger sequencing as H9Nx and H6Nx. Of the twenty percent samples that tested positive for AIV 98% were identified as H7Nx by subtype specific through rRT-PCR. The highest frequency of AIV positive samples was detected between the months of January and February 2017 (20%), with smaller peaks detected in february and March 2016 (0.3%). Lower peaks were also detected between the months July and November 2016 (0.1%), respectively. A high prevalence of AIV was detected in the late summer months with a frequency of 65% positive, although a low prevalence was also detected in the autumn (0.6%) winter (0.6%) and spring 0.08%). Thus, the study provides a valuable insight into the seasonal prevalence of AIV in a heterogeneous wild duck population in Gauteng Province.
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    Mining of genes encoding for DNA-manipulating enzymes from hot springs using metagenomic techniques
    (Vaal University of Technology, 2019-09) Mokoena, Morena India; Rashamuse, K., Dr.; Feto, Naser Aliye, Dr.
    The use of conventional culture-based approach results in vast majority of microbes (90 - 99%) unaccounted for. However, over the past years, the use of metagenomics, which is a culture-independent comprehensive approach has enabled researchers to access nearly 100% of the microbiome. In this study, three hot springs (44 – 70 oC) in Limpopo province of South Africa were investigated as potential sources of genes encoding for DNA-manipulating enzymes (DNA polymerase, DNA ligase and endonuclease), which are central in genetic engineering. They are usually grouped into four broad classes (nucleases, ligases, polymerases and modifying enzymes) depending on the type of the reaction they catalyze. Accordingly, hot spring metagenomic DNA was successfully extracted using modified SDS-CTAB method involving gel purification and electroelution. Consequently, a portion of the extracted metagenomic DNA was used for sequencing and another for fosmid library construction. Sequencing was done using Illumina MiSeq next generation sequencing platform and sequence data analyzed and de novo-assembled using CLC Genomic Workbench, which resulted in 5 681 662 reads and 7 338 contigs. A metagenome expression fosmid library of approximately 2.16 x 103 clones was also constructed using CopyControl™ HTP Fosmid Library Production Kit with pCC2FOS™ Vector. A BLAST algorithm in NCBI revealed 57 distinct genes for DNA polymerase, 29 genes for DNA ligase and more than 100 genes for endonuclease II enzymes. Hence, three genes related to thermophiles representing genes for DNA polymerase, DNA ligase and endonuclease II were selected. Accordingly, the three genes were codon-optimized, synthesized and successfully cloned into pET- 30a (+) and overexpressed in Escherichia coli BL21 (DE3) by inducing with 0.5 mM IPTG and incubating overnight at 16ºC. The cells were lysed using B-PER Reagent, protein extracted and purified using AKTA start protein purification system and purity of 85- 95 % was achieved. From this study, it can be concluded that metagenomics as an approach, can be used to mine for putative DNA-manipulating enzymes from hot spring metagenome. Besides, further study should be conducted to formulate the developed DNA-manipulating enzymes and study the practical application and chart way for commercialization. Moreover, the constructed fosmid library could also be screened for potentially novel thermo-stable biomolecules of industrial and therapeutic importance.
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    Sequence and function based screening of the goat rumen metagenome for novel amylases
    (Vaal University of Technology, 2019-09) Rabapane, Kgodiso Judith; Nelson, K. E., Prof.; Feto, Naser Aliye, Dr.
    During one of our preliminary studies in 2015, metagenomic DNA extracted from the goat rumen was sequenced and the in silico mining of the biorefining enzyme showed the presence of significant number of different biocatalysts, such as amylases (E.C 3.2.1.1), xylanases (E.C 3.2.1.8), pectinases (E.C 3.2.1.15) and cellulases (E.C 3.2.1.4). Hence, a subsequent study was conducted which is aimed at extracting metagenomic DNA from the goat rumen, constructing the metagenomic library using pCC2-FOS™ plasmid vector (Epicentre®), and eventually screening the constructed library for potential novel amylases using soluble starch as a substrate. Accordingly, rumen digesta was aseptically collected from four compartments of each goat and pulled before extraction of metagenomic DNA. The conventional CTAB protocol was modified to extract the metagenomic DNA from the rumen digesta. As a result, high molecular weight DNA was obtained and used to construct the metagenomic fosmid library. Since the host (Escherichia coli EPI 300-T1r) supplied with CopyControlHTP Fosmid Library Production Kit has background amylase expression we opted for a knockout E. coli strain with deleted starch hydrolysis (amylase expression) pathway. The library was subsequently screened for the presence of amylase isoforms using soluble starch as a substrate. Therefore, for the purpose of this study, four fosmids clones showing amylase activity were selected, recombinant vector isolated and MiSeq-sequenced. Out of four recombinant proteins, only one (pET30a(+)-amy-vut12) was successfully expressed. Subsequently, pET30a(+)-amyvut12 was further characterize physicochemically. Interestingly, the recombinant enzyme showed maximum activity in the pH and temperature ranges of 6.0 - 8.0 and 70 - 90oC, respectively. Hence, this implies that novel recombinant protein has sound activity from acidic to alkaline pH range and potently thermostable. Further work should be done to optimize and improve the solubility of three other recombinant proteins (pET30a(+)-amy-vut2, pET30a(+)-amy-vut9 and pET30a(+)-amy-vut14) studied, which might harbour important traits. Most importantly, immobilization as well as crystallographic studies of pET30a(+)-amy-vut12 and downstream applications should further be investigated.