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Integratie van muggenbarcodering, eDNA-metabarcodering, Wolbachia-profilering en CRISPR-Cas voor geavanceerde vector- en pathogeenbewaking en -bestrijding in Ethiopië

Year: 2025
Type: dissertation
Abstract: Arboviruses, including dengue virus (DENV), chikungunya virus (CHIKV), Zika virus (ZIKV), yellow fever virus (YFV), Rift Valley fever virus (RVFV), and West Nile virus (WNV), pose significant public health and economic challenges, particularly in tropical and subtropical parts of the world. Mosquitoes primarily transmit these diseases, and their burden is increasing due to climate change, urbanization, and global trade dynamics. Ethiopia's diverse ecology and growing urbanization create favorable conditions for arboviral transmission; however, its surveillance systems remain inadequate. The absence of an efficient vector and pathogen monitoring system hinders the implementation of timely interventions and effective disease control strategies. The present research integrates advanced molecular tools, including mosquito barcoding, mosquito blood meal analysis, Wolbachia profiling, environmental DNA (eDNA) metabarcoding, and CRISPR-Cas technology, to enhance vector surveillance and pathogen detection as well as to support vector control and management in Ethiopia. Thus, this study aims to address the limitations inherent in traditional vector surveillance and control methods. The outcomes will enhance the accuracy and efficiency of arboviral disease monitoring and management, ultimately improving public health response efforts in Ethiopia. The study was conducted in the Mid Rift Valley of Ethiopia, which features diverse habitats. Mosquito specimens were collected from various environments and identified using morphological characteristics and DNA barcoding techniques using the cytochrome c oxidase subunit I (COI) gene. Blood meal analysis utilized 12S rRNA molecular markers to determine mosquito feeding preferences. eDNA metabarcoding was performed on water and soil samples collected from mosquito breeding sites to detect the presence of mosquito DNA, offering a non-invasive approach to monitoring vector populations. Wolbachia profiling employed Wolbachia 16S rRNA-encoding gene, Wolbachia surface protein-encoding gene (wsp), and prophage WO capsid protein gene (orf7) genetic markers to detect and characterize Wolbachia and Prophage WO infections, providing potential insights into biological control strategies for vectors. Furthermore, a CRISPR-Cas12 assay was developed for the detection of RVFV, providing a rapid and highly specific diagnostic tool for early disease detection. DNA barcoding has effectively identified 16 mosquito species in Ethiopia, the dominant ones being Anopheles coustani, Culex pipiens complex, Cx. tritaeniorhynchus, Mansonia africana, and Ma. uniformis. These species are recognized as significant vectors of arboviruses and play a crucial role in transmitting numerous diseases. Blood meal analyses of engorged mosquitoes have provided evidence on their host preferences. Cx. tritaeniorhynchus and Ma. uniformis feed on a broad range of hosts, including humans, cattle, and other animals. This diversity in feeding behavior underscores their potential as bridge vectors, capable of transmitting pathogens between animals and humans. In contrast, Cx. pipiens complex exhibits pronounced anthropophilic behavior. Genetic diversity analyses have indicated notable population expansions among key mosquito species, suggesting that these populations may be adapting to environmental changes, such as shifts in climate or urbanization, which could impact their ability to spread diseases. eDNA metabarcoding has effectively identified species such as An. coustani, Cx. poicilipes, and Ma. uniformis, demonstrating its potential as a cost-effective and scalable solution for vector surveillance. Its ability to detect mosquito DNA from environmental samples without capturing live specimens offers a significant advantage, particularly in remote or resource-limited areas where collecting live mosquitoes is often challenging. This method can provide real-time data on mosquito population dynamics, enabling early detection of potential disease outbreaks and informing targeted interventions. The study also investigated the prevalence and genetic diversity of Wolbachia and prophage WO. Wolbachia was detected in 44.2% of mosquitoes using the 16S rRNA gene and 46.8% using the wsp gene. The highest prevalence was observed in the Cx. pipiens complex (100%) and Ma. uniformis (92.3%). Furthermore, prophage WO, a bacteriophage associated with Wolbachia, was detected in 46.8% of mosquitoes, with evidence of multiple-strain co-infections in some species. Phylogenetic analysis classified the Ethiopian Wolbachia isolates within Supergroup B, which is commonly associated with arthropods, including mosquitoes. Most Wolbachia strains remain uncharacterized; however, this study identified the wPip strain in Cx. neavei and Cx. tritaeniorhynchus, as well as the wNo strain in Cx. tritaeniorhynchus and Coquillettidia microannulata. This study developed a highly sensitive and specific diagnostic assay, termed CRISPR-RVFDx, by integrating CRISPR-Cas12a, Recombinase Polymerase Amplification (RPA), and lateral flow assay for the detection of Rift Valley Fever Virus (RVFV). CRISPR-RVFDx is a portable assay with high specificity and sensitivity, making it well-suited for point-of-care testing (POCT) in resource-limited settings. The ability to quickly and accurately identify RVFV field isolates significantly enhances outbreak response times, improving the overall capacity to manage RVFV outbreaks and reducing their impact on public health and livestock. The findings of this research have several important implications for the control of arboviral diseases in Ethiopia and beyond. Integrating molecular tools such as DNA barcoding, eDNA metabarcoding, and CRISPR-Cas diagnostics can significantly enhance the capacity for early detection and monitoring of arboviral vectors and the pathogens they transmit. The genetic diversity of various Wolbachia strains in mosquito populations can be leveraged for biological control strategies, such as releasing Wolbachia-infected mosquitoes to reduce arbovirus transmission. In conclusion, this thesis presents a comprehensive framework for enhancing arboviral disease surveillance and control in Ethiopia using advanced molecular and genomic technologies. By leveraging mosquito DNA barcoding, eDNA metabarcoding, Wolbachia profiling, and CRISPR-Cas diagnostics, the research contributes to global efforts to reduce the burden of arboviral diseases and improve public health preparedness. The findings underscore the importance of integrating innovative tools and strategies into existing vector control programs to address the growing threat of arboviral diseases in a rapidly changing world. Keywords: Arboviruses, Blood meal, CRISPR-Cas, DNA Barcoding, eDNA, Ethiopia, metabarcoding, mosquito, prophage WO, Rift Valley Fever, Vector Surveillance, Wolbachia
Source: Lirias
Author: Regassa, Samson Leta
Institution: KU Leuven
Language: English
Field: Medicine
Open Access status: green
OpenAlex ID: W7112644595
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