Charles Hefer for their input and mentorship throughout the course of the study and for ensuring that this project was completed. Special thanks to all my colleagues and staff at ARC-BTP for your support, love and encouragement throughout this project, SLUTE. This study initiated the discovery of bacterial genera of medical and veterinary importance by metagenomics in the abomasum content of Dohne Merino sheep and adult male and female H.
Krona plot for taxonomic abundance of the abomasum content microbiota at the phylum level………..68 Figure 21. Krona plot for taxonomic abundance of the abomasum content microbiota at the genus level………..74 Figure 27. Krona plot depicting unique bacterial OTUs of the abomasum-content microbiota at the genus level……….78 Figure 31.
Introduction
In addition, this symbiosis between GIN parasites and microbiota affects the way food resources are used by the host, the maintenance of digestive tract homeostasis and the health of the host, as well as GIN parasites and microbiota (El-Ashram & Suo, 2017; Fisher et al., 2017). The GIN parasites cause health and welfare problems in the small agricultural ruminants because they survive on food sources they take from their hosts, as well as from the surrounding host environment (Roeber et al., 2013; Zvinorovaa et al., 2016). These GIN parasites colonize the GIT of animals and humans, but they can migrate to and invade other organs, although they prefer the intestinal wall (Duarte et al., 2016).
During these developmental stages, GIN parasites interact with diverse environmental and gastrointestinal microbiota, both pathogenic and beneficial (Lacharme-Lora et al., 2009). Previous studies by Lacharme-Lora et al. 2009) and El-Ashram & Suo, (2017) have reported that GIN parasites harbor diverse microbial communities and can act as vectors for transmission pathogenic bacteria. The underlying parasitic mechanisms of these losses result in major socioeconomic implications worldwide (Regassa et al., 2006; Roeber et al., 2013).
Bacteria
This burgeoning field has led to the discovery that the gastrointestinal microbiota provides crucial host services such as immune system development, protection of the host against pathogenic microbes, and prevention of autoimmune disease (Kreisinger et al., 2015; El-Ashram & Suo, 2017). Furthermore, NGS technologies, including high-throughput shotgun sequencing, have been used for various purposes, including the characterization of microbiota in complex ecosystems, such as soil and seawater (Venter et al., 2004) and the study of microbiota of veterinary and medical importance (Wittekindt et al., 2010). However, some bacteria are beneficial, including aiding in feed digestion, host health, and the production of medical drugs.
For example, bacteria Actinomycetes that produce antibiotics such as streptomycin and nocardicin (Clardy et al., 2009; Begum et al., 2017). Most bacteria live symbiotically with other microbes such as fungi, archaea and GIN parasites in the GIT, plant surfaces and roots while aiding in the breakdown of organic matter and digestion of food (Thursby & Juge, 2017).
Symbiosis
Symbiotic bacteria
Recently, an increasing number of GIN parasite microbiota studies have been conducted, focusing on characterizing microbial communities that are purely symbionts of GIN parasites and those that may pose a threat to GIN parasites and their host, with long-term goals of manipulating these microbes to control GIN parasites. El-Ashram & Suo., 2017; Sinnathamby et al., 2018).
The gastrointestinal microbiota (gut symbionts)
Gut bacterial symbionts of gastrointestinal nematode parasites
- The microbiota-gastrointestinal nematode parasites symbiosis complexes
Symbionts of mammalian gastrointestinal nematode parasites
- Filaria
- Ascaris suum
- Trichuris muris
- Haemonchus contortus
Composition of the gastrointestinal nematode microbiota
Functions of gastrointestinal nematode microbiota
- Nutrient metabolism
- Immunomodulation
- Antimicrobial protection
Molecular and microbiological based approach to studying the gastrointestinal
- Culture-dependent techniques
- Culture-independent techniques
However, culture-dependent techniques can only culture a fraction of the bacteria in the samples as 40 - 90% of gastrointestinal bacteria are uncultivable under artificial laboratory conditions (Gong & Yang, 2012). The main reasons for non-cultivation of most gastrointestinal bacteria include the inability to mimic the exact GI environment, unknown growth requirements of each of the intestinal bacteria and these techniques cannot detect the associations of bacteria with other microbes such as fungi, helminths and archaea in not simulating. the host GI environment (Zoetendal et al., 2004). Culture-independent techniques are commonly used methods to study microbiota without requiring cultivation of the bacteria.
These techniques are molecular biological methods based on the detection of common molecular markers found in the genome of microorganisms (Petrosino et al., 2009). Technique used in molecular ecology to multiply short pieces of DNA/RNA, creating thousands to millions of copies of a given DNA/RNA sequence in the sample. Quantitative PCR technique that uses DNA amplification to determine the exact/absolute or relative concentration of the target gene, such as 16S rRNA gene sequences, in the sample.
Colonization of gastrointestinal nematode parasites by microbiota
Modulation of host intestinal microbiota by gastrointestinal parasites infection21
Gastrointestinal nematode parasites
Biological control of gastrointestinal parasites
Genetic resistance to gastrointestinal nematode parasites in sheep
- Genetic control of gastrointestinal nematode parasites
- Gastrointestinal nematode parasites antigens and host antibodies
- Immune response cells associated with resistance
Gastrointestinal nematode parasites of veterinary importance in Dohne Merino
- Haemonchus contortus
- Morphology
- Life cycle
- Clinical signs, treatment and control of haemonchosis
- Diagnosis of haemonchosis in sheep
History of domestic sheep and Dohne merino breed
- Domestic sheep origin
- Dohne merino breed
The 16s metabarcoding
These studies have been used to study microbiota structure, function and dynamics (Thomas et al., 2012) as well as allowing us to gain more understanding of microbiota diversity in different environments; the complex structure of the microbiota they form and the impact they have on the surrounding environment (Huttenhower et al., 2013). Previous metagenomics-based methods such as shotgun metagenomics used to sequence many cultured microbial communities contained in environmental samples by randomly cutting DNA sequences into many short sequences and then reconstructing them into a consensus sequence (Tyson et al., 2004). It is essential for identifying the microbiota present in an environment, highlighting the differences in microbiota and determining the effect of external variables on the microbiota, i.e. food, drugs, age, geography and other microbial species through sequencing the 16S rRNA gene (Nicola) et al., 2013).
These metagenomic possibilities are especially important when studying the association of the microbiota disease and microbiota GIN parasites within a given environment (Wang et al., 2015). A study by Wang et al. 2015) reported that the abundance and proportions of the human gastrointestinal microbiota vary among individuals in the United States due to long-term dietary habits. In addition, metagenomics can be applied to solve various practical challenges in agriculture, medicine, technical sustainability and ecology (Nicola et al., 2013).
Next-generation sequencing (NGS)
- Roche 454 sequencing technology
- Ion semiconductor sequencing
- Illumina (solexa) sequencing technology
- Solid™ sequencing technology
- Oxford nanopore minion sequencing technology
This method was optimized and commercialized by Applied Biosystems (ABI), the European Molecular Biology Laboratory (EMBL) (Ansorge et al., 1986). For this reason, a series of new Massively Parallel Sequencing (MPS) technologies have been developed and called next generation technology (NGS) (Arsenic et al., 2015). However, the higher throughput of NGS reactions comes with limitations, including shorter sequences, as most sequencing platforms (Illumina, Roche, SOLiD) offer shorter read lengths (30–400 bp) than the conventional Sanger method (Ulahannan et al., 2013). ).
This technology refers to sequencing based on the reversible dye terminator method developed by Shankar Balasubramanian and David Klenerman in 1998 (Bentley et al., 2008). This technology was used to successfully read the genome of bacteriophage lambda, which is approximately 48,500 base pairs long, twice in one passage (Castro-Wallace et al., 2017). MinION technology is growing rapidly, being able to read 100,000 base pairs during a single capture of DNA, which has never been possible with traditional sequencing techniques (Kerkhof et al., 2017).
Bioinformatics
- Bioinformatics analysis of 16s metabarcoding data
Targeted amplicon-based analysis using 16S rRNA gene sequences is widely used for large-scale studies to explore complex microbiota from different sampling sites such as gastrointestinal tract, skin, soil or water (Turnbaugh et al., 2007). Microbial communities analysis requires the use of bioinformatics tools to process the large complex amount of data generated from whole genome and amplicon-based sequencing for taxonomic assignments (Kuczynski et al., 2012). The bioinformatics tools available for 16S rRNA gene sequence data analysis include Mothur, QIIME (Quantitative Insights Into Microbial Ecology), the RDPipeline (Ribosomal Database Project Pipeline), MG-RAST (Metagenomics Rapid Annotation using Subsystems Technology), MEGAN , METAGEN assist, VAMPSGEN assist. , Snowman, Genboree, EzTaxon, Pheonix2, Vegan, ade4, and monkey and CloVR-16S (Plummer et al., 2015; Oulas et al., 2015).
Common reference databases include Ribosomal Database Project, Greengenes, and Silva (DeSantis et al., 2006; Cole et al., 2009; Quast et al., 2013). The resulting output of the analysis can be visualized by statistical software packages such as R statistical package, Primer-E package and Metastats (Team, 2008; Clarke & Gorley, 2006; McMurdie & Holmes, 2013; Pavlopoulos et al., 2013). The current study was therefore formulated to initiate gastrointestinal parasites and microbiota association research in South Africa.
Problem statement
In Africa, there is a paucity of documented studies on the microbiota of GIN parasites and their association with the microbiota of their host in the literature. With this in mind, the current study focused on the characterization of adult male and female H. Here, morphological identification and molecular techniques were used to differentiate between adult male and female H.
Finally, metagenomic diagnosis was used to determine the microbiota of sampled adult male and female H . The data obtained from this study provide new insights into the bacterial community naturally associated with adult male and female H.
Aim, objectives and hypothesis
- Aim
- Objectives
- Research hypothesis
Outline of dissertation
Study site
Study approach
Experimental animals
Procedure for collection of abomasum content and adult H. contortus recovery
Morphological identification of the adult male and female H. contortus worms
- Sterilisation of adult H. contortus surface
- Ruling out microbial contamination on the adult H. contortus surfaces
- Microbial DNA extraction
- DNA extraction from abomasum content
- DNA extraction from adult male and female H. contortus worms
- DNA amplification and amplicon library preparation
- Amplicon PCR
- PCR clean-up
- Index PCR
- High-throughput sequencing
- Library quantification, normalization, and pooling
- Library denaturing and miseq sample loading
Bioinformatics analysis
Statistical analysis
Ethical approval
Morphometrics of H. contortus
Molecular analysis of the adult male and female H. contortus
Microbial DNA amplification by Polymerase Chain Reaction
Diversity index analysis of bacterial community
- Alpha diversity estimate of microbiota associated with abomasum content,
- Beta diversity estimate of microbiota associated with the abomasum content,
The microbiota composition and abundance associated with abomasum content
Unique and shared microbiota associated with the abomasum content, adult male
The relationship between H. contortus microbiota and its predilection site, the
Morphological and molecular identification of H. contortus
Investigation of parasite surfaces for abomasal microbial contamination and
As a result, in the present study, a number of measures were used to ensure that there is no cross-contamination of the abomasal microbial community in H. Previous studies by Berg et al. 2016) have shown that several washing steps with gentamicin or media containing Triton X-100 can be used to remove microbial contaminants on parasite surfaces. However, this protocol is not sufficient to completely remove all microbial contaminants on the surface of the parasite. Therefore, in the present study, after recovery of the parasite from the abomasum, we used a more rigorous treatment consisting of antibiotic solution (1mg/ml ampicillin/1mg/ml gentamicin) and 4% sodium hypochlorite to ensure removal of all microbial contamination. on the surface of the parasite.
The PCR test results with 16S rRNA gene primers showed that there is no contamination on the parasite originating from the abomasal microbial community. These results correlate with the previously published results by White, (2016) and El-Ashram & Suo, (2017) who reported that sterilization with antibiotic solution (1mg/ml ampicillin/1mg/ml gentamicin) and 4%. The final washes with (PBS, pH 7.4) ensure that all antibiotic solution and sodium hypochlorite residues that may be left on the surface of the parasites are removed.
These results are consistent with previously published studies that reported that PCR amplification with V3-V4 primer pairs can amplify microbial DNA from metagenomic samples. As a result, it enables sequencing and classification of digestive tract and environmental microbiota (Klindworth et al., 2013; . Fischer et al., 2016; Thijs et al., 2017). MICROBIOTA ASSOCIATED WITH THE ABOMASUM CONTENT OF THE DOHNE MERINO SHEEP NATURALLY POSSESSED WITH HAEMONCHUS CONTORTUS FIELD.
Microbiota associated with the abomasum content of the dohne merino sheep
Microbiota of H. contortus adult worms have a diverse and core microbiota
- The relationship between adult H. contortus microbiota and its predilection site,
- conclusion
- Major implications and limitations of the study
- Recommendations
