PARTNERSHIP: NATURAL EXPERIMENTS OF SARS-COV-2 TRANSMISSION IN CAPTIVE CERVID AGRO-ECOSYSTEMS

<b>Project Methods</b><br> 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immobilized using protocols our team has optimized, including drug reversal uponcompletion of sampling (Cook, unpublished data based on [44]).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.