Location: Zoonotic and Emerging Disease Research
Project Number: 3022-32000-021-009-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2023
End Date: Aug 31, 2026
Objective:
The United States National Tick Collection (USNTC) at Georgia Southern University and the United States Department of Agriculture - Agricultural Research Service (USDA – ARS) desire to enter into this agreement for the supporting research to be carried out at the USNTC laboratory in Statesboro, GA (U.S.A.) and cooperator facilities in Salford (U.K.), and Makerere University, Kampala, (Uganda). The project will collect ticks to test for Crimean Congo Hemorrhagic Fever virus (CCHFV) and other pathogens. CCHFV has a fatality rate of up to 40%. This virus is prevalent in the Northeast corresponding to the Ugandan/Kenyan cattle corridor, but recently CCHFV has been reported outside the corridor, including in Western Uganda (Kasese region). The main vector, Hyalomma ticks are found only sporadically in the west, but desertification from human use and global climate change may be changing the habitats and allowing for vector expansion to new areas. Rhipicephalus ticks are the predominant tick in west Uganda and have been found infected with CCHFV, although they are not a proven viral vector. The first objective is to examine ticks of different genera collected from domestic/wild animals and by dragging the vegetation, among the rural communities around Queen Elizabeth National Park (QENP) and inside the park, to survey for tick-borne pathogens and, in particular, to assess the importance of the progression CCHFV, other vectors, and pathogens. The second objective is to enhance the local preparation and ability to respond to emerging tick-borne viral pathogens by training Ugandan staff and students and by providing sustainable resources. These resources have been adapted to on-site conditions and requirements that can work closely with other Centers seeking to provide CCHFV study and response capacity (all the proposed work will be carried out in Uganda and the Mweya laboratory). The third objective is to test the circulation of pathogens between wild and domestic animals, in order to better inform the local rural communities of the ticks (or lack thereof) associated with wild ungulates. The fourth objective is to collaborate on a study of the population genetic structure and dynamics of the progressive expansion of the geographic range of Hyalomma ticks based on microsatellite markers.
Approach:
Principle investigators and collaborators will collect ticks from livestock and wild animals among rural communities around and inside the Queen Elizabeth National Park. To detect and identify viruses and other tick-borne pathogens, traditional polymerase chain reaction and sequencing methods will be complemented using third generation Nanopore sequencing technology (portable technology, cheaper, capable of sequencing large DNA fragments from individual or pooled tick batches). Collaborators have established experience of Nanopore capacity building in Uganda, including Tropical Disease capacity and using similar tiled amplicon approaches to study COVID-19 in Northern Uganda in 2021-2022. If CCHFV is detected, samples of cattle blood will be preserved at -80°C at the Mweya Laboratory. Tick samples will be collected 4 times a year for 3 years from livestock in the rural communities (4 villages) around the QENP where cattle and wild ungulates, often intermingle. We expect approximately 2000-3000 ticks to be collected in 3 years. Tick cDNA will be synthesized by using conventional Reverse Transcriptase kits, and the presence of Nairoviridae detected by using well-established amplification protocols. Positive samples will be sequenced for further identification of the species/strain/variant involved. Nanopore sequencing of whole CCHFV viral genomes will be achieved using two approaches. The first approach is to use a tiled-amplicon approach similar to that developed for COVID-19 whole genome sequencing and the second approach is to use a Nanopore adaptive sampling native to the new instrumentation that can enrich (c)DNA samples for non-tick DNA (and therefore natively identify infecting viruses/bacteria/protozoa etc. For the first approach, multiplex PCR primer schemes will be designed using PrimalScheme software and tested at Georgia Southern University and Salford University prior to deployment in Mweya; For the second approach, as this is native to Nanopore technology, this will be testing in situ in Mweya once capacity has been established. On the basis of this, we will develop taxon-specific conventional (q)PCR assays for molecular diagnosis of relevant pathogens. Design microsatellite molecular markers through shotgun genomic sequencing of different Ugandan Hyalomma species and analyze the population structure of different populations along transects spanning from areas of established through areas of non-established populations. Training of local scientists and technicians has already started and will further be completed by organizing additional training sessions for them in the U.S. and, more importantly, at the local Mweya laboratory. One of the main scopes of this study is the transfer of knowledge and technologies to Ugandan facilities that will result in personnel and facilities to be independent, equipped, and prepared for facing future emerging outbreak. Therefore, it is essential to perform all steps of this study in Ugandan facilities.Based on obtained results, create educational outreach support to possibly improve relationships between QENP and local communities.
