Location: Zoonotic and Emerging Disease Research
2024 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 through various collaborative projects. Ways to detect the presence of the Crimean Congo Hemorrhagic Fever virus (CCHFV) and evidence of exposure to the virus are in development or have been developed, including assays that can be used on the farm or with little equipment or infrastructure. A gap analysis of CCHFV with experts from over a dozen countries raised concerns about the availability and quality of assays to assess if an animal or person had previously been exposed to CCHFV. Working with two partners, assays were developed to detect the presence of Immunoglobulin G (IgG) as a marker for last exposure. The assays detect the presence of IgG in a suspension and use technology that is reported to be more specific and more sensitive than the technology used by current assays. These assays are also designed to detect exposure to other viruses that may be confused with CCHFV. To mitigate risk a second approach using arrays of peptides has been started. Peptides that cover different CCHFV isolates, and other closely related viruses are coated onto arrays. Samples from CCHFV-exposed animals and people, as well as samples from uninfected animals and animals infected with other viruses, are placed on the arrays to identify specific parts of the virus the immune system responds to. Once identified, the most reactive peptides are targeted for assay development.
Two separate efforts are underway to aid in the identification of ticks. The first uses a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) to identify a unique signature or fingerprint based on the protein content of the tick. This approach may also identify if a tick is carrying CCHFV. Ticks collected as part of the Centers for Disease Control Center for the Surveillance of tick-borne diseases in the Southern States have been utilized to develop tick species databases. Thirteen different species of ticks have been collected from field sites in the United States, and permits have been put in place to obtain ticks from international locations. 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. colonies at partner locations. Efforts to investigate the impact of climate change on key ticks' host-seeking behavior 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 collections are being conducted in several locations, including Tanzania, Sierra Leone, the Democratic Republic of Congo (DRC), Uganda, Ethiopia, and Kenya. In Tanzania and Sierra Leone, a total of 54 cattle herds were sampled, 7,204 ticks were collected, and 89 milk samples were collected. Testing samples for previous exposure to Crimean Congo Hemorrhagic Fever virus (CCHFV) 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 the virus itself. Additional work is required but in general, results indicate widespread CCHFV exposure and infection at study sites with a trend towards age-related acquisition of anti-CCHFV antibodies. This finding reveals (at least for this limited sample set and preliminary laboratory testing) an apparent lack of association between herd-level evidence of CCHFV exposure and the presence of Hyalomma spp. ticks carrying CCHFV.
In Kenya and Uganda, in-country partnerships were established with the Uganda Virus Research Institute and the Kenya Medical Research Institute to collect ticks and detect CCHFV in and outside the cattle corridor. Approximately 180 homesteads across 6 Ugandan districts across the cattle corridor have been sampled to date. Uganda tick populations were shipped to Texas Tech University and screened for CCHFV. Two pools of Ugandan Rhipicephalus appendiculatus tick samples tested positive for CCHFV suggesting infection. Collection efforts suggest Hyalomma species are not prevalent in central Uganda, where previous outbreaks of CCHF have been reported. In Kenya, Hyalomma species were observed to be the predominant species collected in Kenyan pastoralism. Microsatellite genetic markers for Rhipicephalus appendiculatus have been optimized, and tick genotyping is in progress. Metatranscriptome and microbiome data processing of select Ugandan tick populations have been completed and data analysis is ongoing.
The U.S. National Tick Collection at Georgia Southern University in Statesboro, Georgia (U.S.A.), cooperated with facilities in Salford (United Kingdom), and Makerere University, Kampala, (Uganda) to collect ticks to test for CCHFV and other pathogens. The main vector, Hyalomma ticks, is found only sporadically in the west. However, desertification from human use and global climate change may be changing the habitats and allowing for vector expansion to new areas. Ticks of different genera collected from domestic/wild animals and by dragging the vegetation among the rural communities inside and around Queen Elizabeth National Park were evaluated to survey for tick-borne pathogens and to assess the importance of the progression CCHFV, other vectors, and pathogens.
In the DRC, multi-institutional partnerships were established, including with the local National Research and National Veterinary programs. Between July 2023 and June 2024, the ARS research team visited and completed sample collections in 9 provinces. 125 individual farms, slaughterhouses, and community farms were visited, and samples from over 1,000 cattle and 1,000 pigs were collected. As part of the work in the DRC, data was collected to inform agriculture husbandry practices. Approximately 60% of sites with cattle and sites with swine reported using any veterinary pharmaceuticals or dietary supplements. Only 22% of sites with cattle and 24% of sites with swine reported vaccinating their animals against any disease. Veterinary pharmaceuticals used in the DRC are commonly contributing to increased antimicrobial resistance regionally. Tetracycline antibiotics (e.g., oxytetracycline) are noted as the most frequently used therapeutic and prophylactic pharmaceuticals across the study area. The most common antibiotics used were oxytetracycline and a combination procaine penicillin G-dihydrostreptomycin sulfate drug. The most common antiparasitic agents reported were ivermectin and levamisole. Investing in proactive veterinary medicine by increasing vaccination rates has the potential for long-term national cost savings. Of note is that about half of the sites visited report notifying health officials about disease outbreaks. Approximately 50% of sites with cattle and 40% of sites with swine responded that they have reported an animal illness or death due to disease to health zones or province officials at least once.
Work was initiated in Nigeria with partners at Redeemers University. Nigeria is a hub with cattle migrations from Cameroon, Niger, Burkina Faso and Chad. The border is considered porous with an estimated 1500 unofficial border crossings. In Nigeria, evidence suggests that large animals such as cattle, donkeys, and camels are mainly brought in through Borno, Sokoto, Katsina, Adamawa, and Taraba states from Eastern and central African countries. The movement and trade of animals along these borders provide a route by which numerous pathogens can come together. Coupled with the inefficient quarantine and screening of incoming animals, people and animals can be exposed to zoonotic and transboundary pathogens, where novel pathogens can quickly amplify and spread internationally, posing health risks. Samples were collected from ill or deceased animals from 3 study sites in Northern Nigeria: Kano, Sokoto, and Adamawa states. Additionally, 300 tick vectors will be collected from sampled.
Partners at the Navy Medical Entomology unit initiated tick surveillance efforts in the Republic of Georgia. Next-generation metagenomic sequencing of 389 ticks was performed, and a bioinformatics pipeline was constructed to analyze the generated sequence data.
Partners at the Broad Institute developed a pan-viral open reading frame (ORFs) discovery platform using Massively Parallel Ribosome Profiling (MPRP), which identified 5,381 ORFs in approximately 700 human viruses, including 4,208 noncanonical ORFs. Currently we are working on experiments to identify these ORFs and evaluate their transition in the context of viral infection. These previously unidentified ORF may shed light on how these agents cause disease and may inform vaccine development.
Partners at the Broad Institute identified potential vaccine candidates. The team identified parts of CCHFV that we believe are most important to the immune system for preventing infection or disease. Expression of these targets and delivery systems were optimized. Immunogenicity studies in mice were initiated.
A mini-genome system was established to aid in the study of different CCHFV isolates and parts of the virus. A reporter gene, firefly luciferase, was incorporated into CCHFV genotypes 2 and 3. The inclusion of a reporter gene will facilitate in vitro work. Based on these results, experiments to rescue CCHFV from full-length infectious cDNA clones have been initiated. To aid in this, we have identified and tested several reagents (anti-CCHFV glycoprotein antibodies), that can be used to confirm replicating virus following infection.
Accomplishments
1. Deployment and refinement of AI tool for identification. An AI based instrument for the identification of ticks was deployed and 1600 ticks in the Democratic Republic of the Congo have been evaluated in partnership with ARS researchers in Manhattan, Kansas. The algorithm is under refinement and the instrument will be deployed to Liberia and Turkey.
2. Collection of samples from livestock and suspect animals. In partnership with ARS researchers in Manhattan Kansas samples from livestock have been collected from the Democratic Republic of the Congo, Sierra Leone, Tanzania, Uganda and Nigeria. Samples from animals with obvious signs of disease have been collected from the Democratic Republic of the Congo and Nigeria. These samples will inform the distribution of CCHFV to all improved risk assessment. Sequencing will provide critical information to all the development of diagnostics and countermeasures with the broadest specificity.
3. Development of bioinformatics pipeline. A bioinformatics pipeline for evaluation of sequencing data for the detection of Crimean-Congo hemorrhagic fever virus (CCHFV) has been developed in partnership with ARS researchers in Manhattan, Kansas. This platform will be made publicly available and will facilitate the analysis of genetic diversity. This information will facilitate the development of diagnostics and countermeasures with the broadest specificity.
4. Initiation of tick collections in Kenya, Uganda, Sierra Leone, Liberia, Republic of Georgia, Tanzania. Ticks have been collected from Kenya, Uganda, Sierra Leone, Liberia, Republic of Georgia, Tanzania and the Democratic Republic of the Congo in partnership with ARS researchers in Manhattan, Kansas. Ticks from Uganda and Sierra Leone have been tested for Crimean-Congo hemorrhagic fever virus. Positive samples have been identified from Uganda. These samples will inform the distribution of CCHFV to all improved risk assessment. Sequencing will provide critical information to all the development of diagnostics and countermeasures with the broadest
5. Development and down selection of lead mRNA vaccine candidates for immunogenicity testing. Multiple mRNA vaccine candidates were developed and lead candidates are undergoing immunogenicity testing Congo in partnership with ARS researchers in Manhattan, Kansas. If successful the vaccine may be able to interrupt transmission and spread of Crimean Congo Hemorrhagic Fever virus.