Abstract:
Reliable molecular identification of vertebrate species from morphologically unidentifiable tissue is critical to the prosecution of illegally traded wildlife products to limit their trade, as well us for surveillance to inform conservation policies and identification of blood-meal hosts. Currently, this is mainly dependent on sequencing of the cytochrome c oxidase subunit I (COI) ‘barcode’ genes, which remains costly for purposes of screening large numbers of unknown samples and routine surveillance. High-resolution melting (HRM) analysis was optimized as a cheaper supplement to conventional sequencing. Here, we adopted HRM analysis of COI, cytochrome b and 16S ribosomal RNA mini-barcode genes Polymerase chain reaction products. The process uses DNA intercalating dye to report the unique melting pattern of duplex DNA. We analysed 107 samples using the optimised approach and robustly differentiate 10 domestic species from 24 wildlife species that are common in the East African illegal wildlife trade. To validate the tool, we assessed whether bushmeat was sold in township butcheries by covert field surveillance sampling in Naivasha, Kenya. We identified one out of 90 samples as bushmeat (giraffe). This approach is being adopted for high-throughput pre-screening of potential bushmeat samples for exclusion of non-bushmeat samples from downstream processing in forensic species identification pipelines in Kenya and Tanzania. It is also useful as a sustainable surveillance and monitoring tool for illegal bushmeat trade, as well as for studies on hematophagous invertebrate vector-host interactions at wildlife-human interfaces in disease epidemiology
TABLE OF CONTENT
DECLARATION..............................................................................................................................ii DEDICATION.................................................................................................................................iii ACKNOWLEDGEMENT...............................................................................................................iv
TABLE OF CONTENT....................................................................................................................v
LIST OF TABLES..........................................................................................................................vii
LIST OF FIGURES.......................................................................................................................viii
LIST OF ACRONYMS....................................................................................................................x ABSTRACT..................................................................................................................................xiii
CHAPTER ONE.............................................................................................................................14
1.0 INTRODUCTION...............................................................................................................14
1.1 Background Information.................................................................................................14
1.2 Problem Statement..........................................................................................................16 1.3 Justification.....................................................................................................................17
1.4 Hypothesis/research questions........................................................................................18 1.5 Objectives........................................................................................................................18
1.5.1 General objective.....................................................................................................18
1.5.2 Specific Objectives..................................................................................................19
CHAPTER TWO............................................................................................................................20
2.0 LITERATURE REVIEW.....................................................................................................20
2.1 Techniques used in the identification of bushmeat.........................................................20
2.2 Mitochondrial DNA (mtDNA) for species identification...............................................21 ...............................................................................................................................................23
2.3 International Wildlife Trade (IWT).................................................................................23
2.4 Bushmeat in Africa..........................................................................................................24
2.5 Bushmeat in Kenya.........................................................................................................26
CHAPTER THREE........................................................................................................................29
3.0 METHODOLOGY..............................................................................................................29
3.1 Samples...........................................................................................................................29
3.1.1 Samples for optimization.........................................................................................29
3.1.2 Samples for validation.............................................................................................31
3.2 Genomic DNA extraction from tissue and blood............................................................31
3.3 Polymerase Chain Reaction with High Resolution Melting Analysis.............................32 v 3.4 Conventional Polymerase chain reaction........................................................................33
3.5 Sequence Analysis...........................................................................................................34
3.6 Blind validation...............................................................................................................34
3.7 Data Analysis...................................................................................................................35
CHAPTER FOUR..........................................................................................................................36
4.0 RESULTS.............................................................................................................................36
4.1 Species differentiation.....................................................................................................36
4.2 Marker discrimination comparison.................................................................................44
4.3 HRM identification of covertly sampled meat from butcheries in Naivasha, Kenya.....45
4.4 Covert samples sequence analysis...................................................................................48
CHAPTER FIVE............................................................................................................................50 5.0 DISCUSSION......................................................................................................................50 5.2 CONCLUSIONS..................................................................................................................55 5.3 RECOMMENDATIONS.....................................................................................................55 5.4 REFERENCES....................................................................................................................56