US-UK COLLAB: ECOLOGY AND EVOLUTION OF PATHOGEN- MICROBIOME-HOST INTERACTIONS DURING POPULATION-LEVEL INTERMINGLING

Project Methods
For the first field trial, we propose to sample weaned calves from a variety of sources, which will be selected based on a pre-trial survey of the BCV variants circulating within target sources. Specifically, we will obtain fecal samples from ~10 randomly selected calves on ~20 large source farms, and subject these samples to BCV variant typing. Calves will be sourced from up to 8 pedigree Holstein dairy farms with different circulating variants of BCV and transported to the research facility where they will be quarantined for two weeks. The calves will then be assigned to pens in a manner that generates groups with varying source-farm – and thus variant – diversity. Variant diversities (H) will be varied by high or low source-farm richness (RS; number of unique sources, and thus BCV variants), and high or low evenness (E; relative abundance of calves from each source). Calves in each pen will be sampled five times: before and after quarantine, and then 24 hours, 96 hours, and 3 weeks after placement. At each sampling time point and from each animal, we will collect deep nasopharyngeal samples (DNP, one per nostril), and per-rectum fecal samples. Pre-entry and three-week samples will be subjected to BCV antigen and antibody testing in order to establish the following commingling dynamics: pen-level incidence of any BCV; pen-level incidence of seroconversion; pen-level BCV variant richness and diversity at entry and at three-week interval; host-level change in BCV variant; and, pen-level incidence of variant-specific BCV during commingling. BCV will be detected using well-established, high-fidelity reverse transcription-PCR (RT-PCR) methods that target the highly-conserved nucleocapsid protein. Seroconversion will be measured using a validated BCV ELISA test kit (Svanovir® BCV-Ab). BCV variants on source farms and at week 3 will be characterized using nested RT-PCR for the hypervariable region of the spike (S) glycoprotein gene, followed by gel purification and sequencing. The pen-level diversity and BCV information will be included in the risk factor models of individual BCV status and clinical disease as part of SA7. To incorporate pen-level diversity into our SEIR compartmental model of pathogen transmission in SA8, we will utilize The Spatiotemporal Epidemiological Modeler's (STEM's) "mixing graph edge generator".To perform the second field trial, we will randomly enroll and allocate 99 calves at birth at asingle heifer raiser facility. The calves will be placed into individual hutches for 6 weeks and managed as per standard protocol. At 6 weeks of age and on the same day, all study calves will be subjected to one of three treatments, based on the random allocation: no commingling/no stressor (I-NS), commingling/no stressor (C-NS), commingling/stressor (C-S).To apply the physiologic stressor, calves in the relevant groups will be subjected to a 3-hour truck ride,which has been shown repeatedly to induce a robust stress response as measured using physiological and behavioral parameters.After the truck ride, calves will be returned to the heifer facility and placed into group housing (C-S group). The calves allocated to "NS" groups will not be transported (i.e., no physiologic stressor), either remaining in their original hutches (I-NS group) or being moved into new housing in groups of 6-9 animals with no prior contact (C-NS group). This commingling protocol and the related facilities present a unique opportunity to disentangle the impacts of commingling and host physiologic stress using a randomized controlled field trial. Calves will be sampled at arrival (S1) and just prior to the commingling and physiologic stressor events (S2) to establish a baseline microbiome and BCV status (S1). Then, the calves will be sampled three more times after the treatments are applied: 24 hours (S3), 72 hours (S4) and 168 hours (S5) after initiation of the treatment. Wewill measure several components of host immune and pathogen responses during commingling, at each of the timepoints listed. Intra-host pathogen replication will be measured using quantitative PCR for BCV on deep nasopharyngeal swabs (DNP). Host immune response will be measured via serum neutralizing antibodies and nasal IgA directed against BCV. Host inflammatory response will be measured via ELISA for proinflammatory cytokines TNF-aand IL-1b in serum. Serum cortisol will be measured by ELISA. Whole blood will be collected for transcriptome analysis. DNP swabs will be collected in duplicate to be used for BCV and metagenomic analyses. Total RNA will beused for quantitative (q)RT-PCR of BCV RNA. The spike and nucleocapsid genes will be used for viral detection. Sequence data will be generated for RNAseq analysis. RNASeq data will be subjected to standard transcriptome analysis, and integrated with immunological data using a stepwise approach. Metagenomic data will be analyzed for microbial composition and potential function, using dynamic multi-level network modeling approach. Signatures of microbial transmission will be detected using nucleotide-level analysis of the metagenomic data. All data generated from field trials 1 and 2 will be used to populate a spatiotemporal SEIR model for BCV transmission; as well as a Bayesian network model for clinical respiratory disease risk.