Source: TEXAS A&M UNIVERSITY submitted to NRP
PARTNERSHIP: NATURAL EXPERIMENTS OF SARS-COV-2 TRANSMISSION IN CAPTIVE CERVID AGRO-ECOSYSTEMS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030294
Grant No.
2023-70432-39559
Cumulative Award Amt.
$799,999.00
Proposal No.
2022-11246
Multistate No.
(N/A)
Project Start Date
May 1, 2023
Project End Date
Apr 30, 2026
Grant Year
2023
Program Code
[A1181]- Tactical Sciences for Agricultural Biosecurity
Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
(N/A)
Non Technical Summary
The captive cervid industry is a robust agricultural sector with nearly 10,000 for-profit deer breeding operations across North America, with especially high concentrations in Texas. Unexpectedly, white-tailed deer (Odocoileus virginianus) have emerged as a reservoir for SARS-CoV-2, the agent of COVID-19. Several studies across the US and Canada have documented deer to be infected with many different lineages of the virus circulating in humans. In fall 2021, we reported the first findings of infection among farmed deer, in which over 94% of sampled animals on one farm were positive. Many questions remain about the viral transmission networks at deer farms, which may include spillover from humans or other animals and threats of onward transmission amongst other animals. Further, the impacts of SARS-CoV-2 infections on the health of agricultural deer is largely unknown. We will build on our multidisciplinary team's experience studying COVID-19 pandemic to understand the threat that SARS-CoV-2 poses to agricultural biosecurity so that plans can be made to mitigate the risks. Using the large deer farm network in Texas, we aim to quantify 1) the frequency of captive cervid infection; 2) the degree of horizontal transmission among captive deer and sympatric livestock, wildlife, and farm workers; 3) viral genome variants among deer and directionality of transmission and 4) the consequence of SARS-CoV-2 infection on animal health. Our ongoing partnerships with deer farms, established capacity for molecular and serological assays, and integrated team of epidemiologists and veterinarians will facilitate the project. This project will provide critical information for management decisions for improving agricultural biosecurity and protecting animal health.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31140301170100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4030 - Viruses;

Field Of Science
1170 - Epidemiology;
Goals / Objectives
Using the large deer farm network in Texas, we aim toquantify 1) the frequency of captive cervid infection; 2) the degree of horizontal transmissionamong captive deer and sympatric livestock, wildlife, and farm workers; 3) viral genomevariants among deer and directionality of transmission and 4) the consequence of SARS-CoV-2infection on animal health. Our ongoing partnerships with deer farms, established capacity formolecular and serological assays, and integrated team of epidemiologists and veterinarians willfacilitate the project. This project will provide critical information for management decisions toimproving agricultural biosecurity and protect animal health.
Project Methods
MethodsAim 1 - Estimate the infection prevalence of SARS-CoV-2 among captive cervids acrossTexas.Enrollment of deer farms. Building on our preliminary surveillance at three captive cervidfacilities in Texas, we will actively sample captive deer from agricultural facilities within years1-2 using a cross-sectional study design to estimate the infection prevalence. Using a cross-sectionalstudy design, our goal is to sample at least 10 captive cervids at each of 30 facilities within years1-2, for an overall sample size of 300 animals. Additionally, we can collect samples from captive deer that are harvested by legal hunting inhigh-fence operations when such opportunities arise.In order to facilitate enrollment and partnership with the Texas deer producers, we will offercomplementary enhanced diagnostic testing of the herd to deer breeders as an incentive. Basedon preliminary discussions, deer producers are interested in diagnostics for epizootichemorrhagic disease virus (EHDV) and bluetongue virus (BTV) in their farmed deer. The EHDVand BTV are vector-borne viral diseases caused by closely related orbiviruses (FamilyReoviridae) that affect domestic and wild ruminants and are transmitted by insect vectors of thegenus Culicoides [43]. Sampling. When deer are not physically restrained for standard agricultural operations, deer willbe chemically immobilized using protocols our team has optimized, including drug reversal uponcompletion of sampling (Cook, unpublished data based on [44]). Two swabs will becollected from each deer using polyester-tipped applicators with polystyrene handles (PuritanMedical Products, Guilford, ME, USA) and stored in viral transport media (VTM; madefollowing CDC SOP#: DSR-052-02): (i) respiratory (to consist of one nasal swab and one oralswab placed together into the vial) and (ii) rectal. Blood samples (10mL volume) will becollected using jugular venipuncture into clot activator tubes.Laboratory methods. Aliquots of VTM supernatant will be subjected to RNA/DNA extraction byMagMAX CORE Nucleic acid purification kit (Thermo Fisher Scientific, Waltham, MA). Analiquot of purified nucleic acid will be tested for SARS-CoV-2 RNA by specific real time RTqPCRto amplify the RdRp gene [45]; a control plasmid containing a portion of the RdRp genewill serve as a positive control (Integrated DNA Technologies, Coralville, IA, USA).Serum samples will be tested by plaque reduction neutralization tests (PRNT) to quantifyantibodies able to neutralize the formation of SARS-CoV-2 plaques on Vero CCL-81 cellcultures following standard protocols [47] in a Biosafety Level 3 laboratory at the Global HealthResearch Complex (see letter of support).Deer. Four of the farms in which positive deer are detected (Aim 1) will be enrolled into thelongitudinal aim of the study in which deer will be sampled (swabs and blood; same methods asdetailed in Aim 1) quarterly over a 18-month period (6 visits per farm). This will allow us todetect the duration of PCR-positivity and the duration of neutralizing antibodies in captive deer,which may exceed one year in some circumstances [4]. When possible, exotic cervids owned bydeer ranchers that interact with the more abundant captive white-tailed deer will also be sampledto best represent the hosts available in SARS-CoV-2 transmission networks.Farm laborers. Based on experience at deer farms, we anticipate identifying approximately 5individuals per farm that have close, regular occupational interactions with deer, including themanager, foreman, and other farm laborers. Up to 5 farm laborers with deer contact at eachinfected farm will be enrolled into the study, and each will provide data on the number of hoursspends in the cervid premises each week and nature of the work (e.g., direct deer contact;feeding/watering). After providing informed consent, human participants will allow provide selfcollectednasal swabs quarterly over a 18-month period following the finding of infected animalson the farm. Additionally, human participants will complete a log of respiratory disease/COVIDdiagnosis with instructions to self-swab during times of clinical disease so the sample can beprovided to the research team.Other agricultural animals and wildlife. Other agricultural and wild mammals may regularlyhave direct contact with farmed deer and be involved in infectious disease transmission pathwaysthat threaten agricultural biosecurity. For this reason, we will sample sympatric livestock andwildlife on four of the farms with infected deer at the same frequency used for the longitudinalsampling of deer in this study (quarterly over an 18-month period). Livestock sampling willfocus on domestic bovids given their abundance at captive cervid farms. Domestic livestock willbe sampled in farms at times of annual vaccination or other veterinary care, when possible, withswabs (respiratory and rectal) and blood collected in the same manner as described for the deerfrom 10 cattle per farm per visit that are likely to have the most interaction with the captive deer.This sampling effort will result in 240 total cattle samples. This sample size is required to be95% confident that SARS-CoV-2 infections are present at or below a prevalence of 1%, if noinfected cattle are detected.Wildlife sampling will focus on key species of locally abundant wildlife including rodents andmesomammals (raccoon, Procyon lotor; opossum, Didelphis virginianus; skunk, Mephitis spp.).Wild mammals will be sampled in and around farms positive for the presence of SARS-CoV-2.For this, we will use Sherman live traps (rodents) and Tomahawk live traps (mesomammals),with traps placed around deer feed troughs and the perimeter of the deer pens. We will deploy100 Sherman traps and 20 Tomahawk traps for three nights quarterly at the four farms that wedetected the highest SARS-CoV-2 seroprevalence in captive deer. Swab and blood collections will occur as explained in Aim 1.?In the laboratory, the same molecular and serologic diagnostic approaches will be used asdescribed in Aim 1. Both the qPCR and PRNT approaches ae not species-specific and aresuitable for use across a range of species; further, our laboratories are approved for working withbiological samples from diverse animal species and humans. Any qPCR-positive samples will besubjected to viral isolation efforts in the BSL 3 laboratory as we have previously done [42].For whole genome SARS-CoV-2 sequencing, RNA will be extracted from positive samples andsequencing will be performed using one of two platforms: library preparation with thePerkinElmer Nextflex variant-seq SARS-CoV-2 kit, with sequencing using Illumina NovaSeq SPPE 2 Å~ 150 flowcell v1.5 to generate an average of 8 million reads per sample, mapped andassembled using the Illumina DRAGEN-covid-pipeline-RUO-1.0.0; or, library preparation withthe Swift SNAP SARS-CoV-2 amplicon panel, with sequencing using Illumina NextSeq togenerate approximately 1 million reads per sample [54]. Mutations in the spike protein gene andother genes will be identified and variants will be identified based on comparisons to SARSCoV-2 genomes available in open repositories (GISAID; Genbank). All SARS-CoV-2 sequencesgenerated in this study will be deposited to GISAID.We will use longitudinal sampling of deer to make associations between SARS-CoV-2 infectionsand signs of respiratory disease, body condition, ingesta-free body fat, antler size, and fecundity.

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:We have conducted field sampling at multiple private captive cervid ranches, with deer producers as a target audience for this portion of the work. We also have presented at state-level and national conferences, with veterinarians and other animalhealth officials, public health officials, and biologists as the target audience. Changes/Problems:It is challenging to recruitdeer farms to the study, as the deer producers are already facing problems due to chronic wasting disease testing requirements and in some cases are not interested in volunteering for this infectious disease research project. Additionally, SARS_CoV-2 is a polarizing topic (politically) and one producer was not interested in having our team back for additional sampling, when he learned some of his animals were infected. Although we can maintain anonymity of the facility and report the facility status only to the county-level, there has been some resistance in enrolling. Nonetheless, many producers love to allow 'Aggies' to see their farms/animals and wish to contribute to the research mission and we are well on track to meeting sample size goals. What opportunities for training and professional development has the project provided?We held a training session set up at a captive cervid herd in Kingsville, TX, on Sept 22, 2023, during which time multiple trainees (postdoctoralassociates, graduate students) received training on deer immobilization, biological sample collection, and use of ultrasound to measure rump fat as a metric of body condition. Each visit to a deer facility has provided training opportunities to students at Texas A&M University and TAMU-Kingsville, including multiple undergraduatesfrom diverse programs; MS student in Veterinary Public Health Epidemiology program; MPH student and more. Additionally, we gave a Continuing Education seminarto the Texas Veterinary Medical Association in March 2024 to provide training and professional development on animals, deer and SARS-CoV-2 to members of the veterinarycommunity. How have the results been disseminated to communities of interest?We have shared individual herd findings with each participating producer.. What do you plan to do during the next reporting period to accomplish the goals?We plan to present our work at the Texas Deer Association meeting to share findings with stakeholders while recruiting additional participation. We also plan to present at the American Veterinary Medical Association for additional dissemination of results. We will involve more students in field and laboratory research. We will enroll additional facilities, expand the wildlife surveillance on the farms, and assess body condition parameters in infected and uninfected deer. Further, we will fully analyze the viral genome sequences and post the sequences to GISAID open access server. We will submit at least two publications for peer review in the coming year.

Impacts
What was accomplished under these goals? We have sampled animals from 14deer breeding ranches in 13 different Texas counties; (290deer total);some of these facilitiesthat have been visited multipletimes for the longitudinalaspects of the study. After both serologicand molecular diagnostics of the biological samples (blood, swabs) from the deer at these facilities, we have thus far identified 3 facilities with SARS-CoV-2 positive deer. Infected facility #1: We sampled 15 female white-tailed deer does (female) in November 2023 in Milam Co, TX,and found 11 to test positive by respiratory swab PCR. The SARS-CoV-2 viral genome was obtained from all 11 of these animals; all sequences group with the Omicron XBB clade. The closest matches to these deer sequences in GISAID sequence repository were from humans of southern Oklahoma and west Texas from early 2024. All 15 of the deer had neutralizing antibodies to SARS-CoV-2 with endpoint titers between 1:10 and 1:640. The producer did not authorize our team to return for additional deer, domestic animal or wildlifesampling. Infected facility #2: We sampled 73 deer in November 2023 in Dimmit Co, TX, and found 60% were seropositive with neutralizing antibodies to SARS-CoV-2; none were qPCR-positive. When we returned to this facility in March 2024, we sampled127 deer (different animals) and found 37.8% were seropositive with 4 deer also positive by qPCR. At this visit, we also deployed wildlife traps and sampled 31 rodents, 3 raccoons, and 1 cottontail. There were some raccoons with some neutralizing antibodies,but they did not meet the threshold of 50% or higher neutralization and thus are considered seronegative at this time. Additionally, one of the rodents tested PCR-positive but with a high CT value (low copy of viral RNA) and attempts to sequence the virus were not successful. As this would represent a new species to be infected, we sent an aliquot of the Viral Transport Media to the National Veterinary Services Laboratory (NVSL), and the sample tested negative at the lab. In April 2024, we returned to the facility to sample 30 cows, 42 rodents and 1 raccoon; these samples had no evidence of positivity. However, we realized that it was hard to obtain the minimum serum values needed to run the plaque reduction neutralization tests (PRNT) and so we obtained approval from the IACUC and Texas Parks and Wildlife Department to euthanize rodents to obtain larger blood volumes postmortem. Infected facilty #3: We sampled 47 white-tailed deer bucks in March 2024 in Hamilton Co., TX, and found 17% were seropositive with neutralizing antibodies to SARS-CoV-2; none were PCR-positive. We returned later in the spring to sample wildlife including rodents and opossums, and all have tested negative. We will return to this facility throughout the fall to sample harvested deer and wildlife. As we continue to expand the surveillance, we will be collecting data to understand how features of the facilities(deer density, pen structure) relate to positivity, and we will measure body condition and other health parameters of infected and uninfected deer.

Publications