Location: Zoonotic and Emerging Disease Research
2022 Annual Report
Objectives
Objective 1. Develop CCHF virus diagnostic tests for the early detection and surveillance of Crimean-Congo Hemorrhagic Fever Virus.
Develop viral detection methods for ticks.
Develop direct viral detection methods for cattle, sheep, and goats.
Develop antibody detection methods to determine CCHF exposure in cattle, sheep, goats, and relevant wildlife.
Objective 2. Determine mechanisms of CCHF transmission.
Develop CCHF tick and animal infection methods.
Develop CCHF tick-animal transmission models.
Objective 3. Investigate the epidemiology of CCHF and the role of ticks in maintaining reservoirs of infection in endemic settings.
Determine the prevalence of CCHF in vector and animal hosts in endemic areas.
Determine the competence of hard tick species in endemic areas.
Determining the risk for the establishment of tick host vector in the United States considering climatic and ecological conditions.
Approach
The research addresses the following research components in the 2022-2027 Animal Health National Program (NP 103) Action Plan: 1) Component 1: Biodefense, Problem Statement 1A, Control and eradicate foreign animal diseases. The research addresses ARS Strategic Plan Goal 4.3 and the following Performance Measure: Provide scientific information to protect animals, humans, and property from the negative effects of pests and infectious diseases, and develop and transfer tools to the agricultural community, commercial partners, and government agencies to control or eradicate domestic and exotic diseases and pests that affect animal and human health.
Progress Report
To address the objectives of this research project, collaborative projects were initiated with the University of Texas Medical Branch (UTMB) and the University of California Davis (UCD). To maximize funds at ARS in Manhattan, Kansas, UCD and UTMB teamed to capitalize on each teams unique expertise. UCD is leading the field collection of tick and sera samples from cattle, the local training, and capacity building for collection and serologic and molecular testing of samples. UTMB brings unique experience in the development of assays, next generation sequencing, as well as experience working in biosafety level-4. The partnership with another maximum containment laboratory is critical while location facilities are still coming online.
The joint project directly addresses Objective 3: Investigate the epidemiology of Crimean Congo Hemorrhagic Fever virus (CCHFV) and the role of ticks in maintaining reservoirs of infection in endemic settings and Objective 2. Determine mechanisms of CCHFV transmission and proteomics will be performed on vertebrate and tick cells infected with CCHFV, to compare the expression profiles and identify potential pathogenesis and maintenance pathways. In addition, the project will begin to address Objective 1: develop CCHFV diagnostic tests for the early detection and surveillance of CCHFV. Development of molecular assays including protocols for next generation sequencing will be performed as part of this effort. In addition, a commercially available assay will be compared to a gold standard in house research assay.
Efforts to develop and validate assays to be used for research and diagnostic purposes are well advanced. An antisense reverse transcription polymerase chain reaction (RTPCR) assay for the S segment of CCHF has been developed and validated. A second assay for the M segment equivalent has been developed and is currently undergoing validation. The development of the anti-sense assay will be critical to demonstrate CCHF infection and replication. Protocols for next generation sequencing (NGS) using a field deployable Min-Ion have been optimized and are undergoing validation. We expect these protocols to be field tested in early 2022. The necessary equipment, reagents and controls have been acquired for the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) platform, and protocol optimization has been completed. Lastly, efforts focused on tick host identification have down selected polymerase chain reaction (PCR) amplification combined with probe blotting as the most promising method. All reagents, equipment, and primers and probes needed were ordered.
Despite travel restrictions and prioritization of COVID-19 related activities, substantial progress has been accomplished. Critical staff members were hired and trained at UCD and UTMB. Remote trainings with partner countries were performed. International field teams at the Sokoine University of Agriculture (Tanzania) and University of Makeni (Sierra Leone) received remote training in tickborne disease surveillance,CCHFV biology and safe collection and processing of samples. In Sierra Leone, a total of 4 study staff members and 8 government district livestock officers were also trained. In Tanzania, 4 staff members and 2 government livestock officers were trained. This training was essential for future efforts.
A pilot seroprevalence survey of CCHFV in cattle was performed. Due to COVID-19 travel restrictions the work was primarily performed by in-country partners after receipt of remote virtual training. In total 754 cattle from 37 cattle herds were sampled (20 in Sierra Leone; 17 in Tanzania) including milk specimens from 89 animals.Teams have also collected 7,204 ticks from cattle.
Tanzania cattle serology results indicate widespread CCHFV exposure and infection at study sites with a trend towards age-related acquisition of anti-CCHFV antibodies. Further testing and evaluation of a commercial enzyme linked immunosorbent assay (c-ELISA) (ID-VET) with current gold-standard ELISAs will be conducted at both Sierra Leone and Tanzania partner sites during the latter part of 2022.
Other progress includes UCD obtaining the necessary USDA import permits. UCD is in in the process of establishing a Amblyomma variegatum as well as a Hyalomma marginatum and Hyalomma anatolicum colony.
COVID-19 related travel delays and interruptions greatly impacted the UCD and UTMB team’s ability to travel to Sierra Leone and Tanzania to accomplish laboratory testing and training. The UCD team plans to resume travel and conduct PCR testing of these tick specimens in September to October of 2022. Despite these limitations significant progress was achieved.
Accomplishments
1. Completion of initial serosurvey. Collection of ticks and a serosurvey study was initiated. Samples were analyzed to determine the seroprevelance of Crimean Congo Hemorrhagic Fever virus (CCHFV) in Sierra Leone and Tanzania. This study was essential for down selection of future study sites and provides some of the first insights into CCHFV prevalence in these regions. Work was performed by in-country partners with remote support from researchers from ARS in Manhattan, Kansas, University of California Davis, and University of Texas Medical Branch.
2. Training of in-country teams. International field teams at the Sokoine University of Agriculture (Tanzania) and University of Makeni (Sierra Leone) received remote training in tickborne disease surveillance and Crimean Congo Hemorrhagic Fever virus (CCHFV), establishing their competency with the necessary skills and knowledge accomplish project goals. A total of 4 staff members and 8 District Livestock officers in Sierra Leone, and 4 staff members and 2 Livestock Officers in Tanzania were trained. Trainings were performed by University of California Davis and University of Texas Medical Branch staff in partnership with ARS scientists in Manhattan, Kansas.
3. Validation of anti-sense assay. An antisense reverse transcription polymerase chain reaction (RTPCR) essay for the S segment of Crimean Congo Hemorrhagic Fever virus (CCHFV) has been developed and validated at University of Texas Medical Branch (UTMB). A second assay for the M segment equivalent has been developed and is currently undergoing validation at UTMB. The development of the anti-sense assay will be critical to distinguish CCHFV infection and replication. Work was performed by University of California Davis scientists in partnership with ARS researchers in Manhattan, Kansas.
4. Development of proteomics and next generation sequencing protocols. Protocols for next generation sequencing (NGS) using a field deployable Min-Ion have been optimized and are undergoing validation. The necessary equipment, reagents and controls have been acquired for the Matrix-assisted laser desorption/ionization-time of flight (MALDI-Tof) platform and protocol optimization has been performed. Work was performed by University of California Davis scientists in partnership with ARS researchers in Manhattan, Kansas.
