Location: Zoonotic and Emerging Disease Research
2023 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
Substantial progress was made in addressing the objectives of this research project through various collaborative projects. In diagnostics, molecular assay for the S and M segments were developed, validated, and transitioned to the field. These assays will detect the presence of the viral ribonucleic acids and allow investigators to determine if Crimean-Congo Hemorrhagic Fever (CCHF) virus is replicating. The laboratory developed and validated field deployable sequencing protocols and algorithms that will reduce errors and allow the detection of recombination events. Combinatorial arrayed reactions for multiplexed evaluation of nucleic acids (CARMEN) assays have been developed and evaluated for CCHF virus. Efforts were initiated to investigate the performance of existing serologic assays to evaluate their specificity and overall performance.
Efforts to use a proteomics pipeline to identify tick species and tick-borne pathogens using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) were initiated. If successful, the proteomics platform will identify the tick species and CCHF virus in ticks in a high throughput setting. Current efforts focus on collecting necessary materials, supplies, and equipment. This system will assist in efforts to monitor the changing geographic ranges or distributions of tick populations, data essential for accurate risk assessments and model development.
In collaboration with partners, work was performed to establish Amblyomma variegatum and Hyalomma spp. tick colonies (specifically H. asiaticum, H. anatolicum, H. dendriticum, H. marginatum, H. excavatum, H. rufipes). Animal feeding will commence in September. Efforts to investigate the impact of climate change on the host-seeking behaviors of key ticks using a behavioral assessment tool have been initiated. Of note, this is the first time such a system has been used in biocontainment.
Tick and animal sera samples are being conducted in several locations, including Tanzania, Sierra Leone, the Democratic Republic of Congo (DRC), Uganda, Ethiopia, and Kenya. Regional instabilities have impacted the collection of materials in some locations. In Tanzania and Sierra Leone, 54 cattle herds were sampled, 7,204 ticks and 89 milk samples were collected. Collected tick species included: Amblyomma, Hyalomma, Haemaphysalis, Boophilus and Rhipicephalus. Testing samples for antibodies to Crimean Congo Hemorrhagic Fever virus demonstrated a higher than anticipated seroprevalence rate, with all herds having at least one positive animal and some herds at 100% positive. The seroprevalence was generally higher in the North and east of Sierra Leone. Testing of the ticks collected failed to detect CCHF viral RNA. Additional work is required to confirm the serological and molecular results. Still, results generally indicate widespread CCHF virus exposure and infection at study sites with a trend towards age-related acquisition of anti-CCHFV antibodies.
In Kenya and Uganda, in-country partnerships were established with the Uganda Virus Research Institute (UVRI) and the Kenya Medical Research Institute (KEMRI) to collect ticks and detect CCHFV in and outside the cattle corridor. The distribution of potential vectors for Crimean-Congo Hemorrhagic Fever Virus and CCHFV prevalence in the area will be determined. So far, around 180 homesteads across 6 Ugandan districts across the cattle corridor have been sampled. Tick species identification is ongoing. No Hyalomma adults have been identified/collected yet but will likely be collected during the next sampling season. Most ticks from cattle identified so far belong to Rhipicephalus decoloratus. DNA and RNA isolation protocols for pooling and testing ticks for CCHF virus have been tested, optimized, and established. Molecular detection of Crimean-Congo Hemorrhagic Fever Virus in the ticks collected will start in the next few months once the ticks are shipped to Texas Tech (expected for mid-August).
In the DRC, multi-institutional partnerships were established, including with the local National Research and National Veterinary programs. A preliminary landscape analysis revealed little surveillance on infectious diseases among livestock in DRC and very limited reporting structures for adverse events. Cattle and pig density were mapped, preliminary study sites were identified, and collections were initiated.
New partnerships were established to facilitate monitoring tick populations in multiple regions and potential importation to the United States. Other partnerships established in 2023 include developing and using artificial intelligence for tick identification and improved low-complexity molecular diagnostics.
Accomplishments
1. Completion of serosurveys. ARS researchers at the National Bio Agro-Defense Facility in Manhattan, Kansas worked with partners in Sierra Leone and Tanzania to complete an expanded serosurvey. Sample collection was initiated in Uganda, Kenya, and the Democratic Republic of the Congo. This serosurvey provided the first data characterizing the presence of Crimean Congo hemorrhagic fever virus in the region. Critical is the finding that the virus may be much more prevalent than previously suspected. This data is essential for local ministries of health and agriculture. The high seroprevelance indicates that persons working closely with livestock in this region are at risk for exposure to this potentially deadly disease and Crimean Congo hemorrhagic fever should be included in the differential diagnosis in patients with unexplained illness in the region.
2. Training of in country teams. International field teams at the Sokoine University of Agriculture (Tanzania) and the University of Makeni (Sierra Leone) worked with ARS researchers from the National Bio Agro-Defense Facility in Manhattan, Kansas, and received training in tickborne disease surveillance and Crimean Congo hemorrhagic fever virus, molecular and serologic testing. The training was also completed in the Democratic Republic of the Congo using Research Electronic Data Capture (RedCap), sample collection and processing, and health assessment of animals. These capacity-building efforts are critical to ensure the rapid and accurate diagnosis of Crimean Congo hemorrhagic fever virus and other zoonotic threats in the region. Training staff to operate safely in the laboratory is another essential aspect of capacity building. Enhancing laboratory safety and capacity in the region will impact far-reaching improvements across public and animal health. The introduction of improved electronic data capture will improve the research quality in the region. The research electronic data capture is a widely used program that, once learned, can be applied to other studies. The use of this program ensures that metadata is readily linked to samples. This protects the data quality and integrity and aids in building a repository of well-characterized samples that may be of future use.
3. Validation and field testing of molecular assays. An antisense reverse transcription-polymerase chain reaction test (RTPCR) assays for the M and S segments of Crimean Congo hemorrhagic fever virus was validated in the labratory and transferred to the field for additional validation. This work was completed between ARS researchers at the National Bio and Agro-Defense Facility in Manhattan, Kansas, and its partners. These tests could improve the diagnosis of Crimean Congo hemorrhagic fever virus. Currently, there are limited assays available and of uncertain quality. The lack of diagnostics limits the ability to test for Crimean Congo hemorrhagic fever virus. If validated, these tests will have wide utility in human and animal health.
4. Development of proteomics and next-generation sequencing protocols. New protocols will reduce the error rate and allow the identification of recombinants. This data will be essential for characterizing the viral diversity and viral evolution of Crimean Congo hemorrhagic fever virus. Viral diversity is a challenge for molecular diagnostics. Even minor changes in sequences can significantly impact the performance of assays, potentially compromising their utility. Monitoring of diversity will also be essential for countermeasure development and risk assessment. Developing accurate field deployable strategies will be done between ARS researchers at the National Bio and Agro-Defense Facility in Manhattan, Kansas, and its partners, which will facilitate monitoring in even the most remote regions. The data generated from these efforts will be critical for modeling, transmission, and countermeasure efforts.
