Progress report for GNE22-279
Project Information
After June 2023, all US livestock producers will need a veterinarian-client-patient-relationship (VCPR) to be able to get a prescription to purchase any antimicrobial to treat and manage disease. The combination of limited livestock veterinarians and the lack of veterinary use by small ruminant producers creates urgency in identifying alternative strategies to combat disease. The utilization of estimated breeding values within the Katahdin breed has enabled sustainable improvement in resistance to parasites. Recent data indicates that selection for post-weaning worm egg count (PWEC) estimated breeding value (EBV) improves lamb survivability to weaning. Additionally, antibody response to vaccination was greater in Katahdin lambs with lower PWEC values. Taken together, these data provide evidence to indicate that Katahdin sheep with lower PWEC values are more immune competent. To test this hypothesis outside the context of parasitic infection we would need to model a bacterial infection, which can be achieved by using bacterial cell wall products like lipopolysaccharide (LPS). Thus, the objective of this project is to determine effects of PWEC genotype on immune response to LPS. To fully characterize this response, in vitro methodologies will be utilized to evaluate transcriptome-wide differences between PWEC genotypes in response to LPS. These data will be used to support implementation of selection strategies that incorporate PWEC and by doing so would potentially reduce the need for and use of antimicrobials. Reducing disease, the need to treat disease, and preserving the efficacy of medically important antimicrobials is fundamental to the sustainability of small ruminant production.
The approach of this project can be clarified by the objectives below:
- To characterize differential immune response between LoPWEC and HiPWEC sheep to LPS stimulation in-vitro
- To determine transcriptome-wide gene expression differences between LoPWEC and HiPWEC-derived cells stimulated with LPS
Funds requested from this proposal will principally be used to characterize immune response and perform transcriptome wide analysis in effort to elucidate patterns in Katahdin sheep with divergent genotypes for PWEC EBV. We intend to use this project as a starting point to allow for a comprehensive genetic analysis of the Katahdin breed in the context of bacterial infections. By using a transcriptome-wide approach, we have the capability to gain new insights into the differences in gene regulation in sheep bred for PWEC which has strong preliminary evidence to regulate immune function. This will give us potential avenues of exploration into the immunoregulation of individuals during a disease state. Fundamentally, we aim to substantiate the claim that selection for PWEC selects for improve immunocompetency outside of just parasitic infections. This work will be presented in the context of improved selection for animal health to producer groups both regionally and nationwide. Additionally, we plan to present findings to scientific community in efforts to build future collaborations that will study this phenomenon on other US sheep breeds.
The purpose of this project is to evaluate the effect of selection for parasite resistance on improved generalized immune responsiveness in sheep as a method to reduce the need for antimicrobial use. The use of antimicrobials (AM) in livestock production has been significantly reduced since implementing the veterinary feed directive, which made the use of AM in feed and water prescription based. To further reduce AM use by livestock producers, the FDA will regulate sale of over-the-counter AM to be by-prescription-only by June of 2023, in efforts to mitigate development of AM-resistance. Antimicrobial resistance is at the forefront of the FDA’s Center for Veterinary Medicine (CVM) challenge to national and worldwide public health (Patel et al., 2020). As part of its regulatory mission, CVM is responsible for ensuring the safety and effectiveness of animal drugs, including AM. The CVM has taken important steps to update the approved use conditions of medically important antimicrobials (i.e., antimicrobials important for treating human disease) to support their judicious use in food-producing animals (Patel et al., 2020). While most antibiotic use in livestock requires a prescription or a veterinary feed directive, individual decisions on administration are often made by farmers via guidelines provided by a veterinarian. Currently, surveillance data do not indicate dosages, or whether antibiotics were administered to prevent, control, or treat a disease (Patel et al., 2020). According to the National Resources Defense Council's (NRDC) analysis of the most recent data, some of the pitfalls surrounding antibiotic stewardship include animal husbandry practices on farms and the level of farm antibiotic-utilization rates (Wallinga and Kar, 2019). This indicates that livestock producers may inadvertently be misusing antibiotics and contributing to the emergence of resistant bacteria. Therefore, reducing AM use in animal agriculture is paramount to the sustainability of the livestock industry.
Antimicrobial stewardship at the producer level relies on implementation of non-AM disease control strategies. Currently, producers have limited options of non-antimicrobial strategies to utilize within their flock. The research proposed here intends to identify genetic underpinnings which account for differences in immune response in sheep bred to be parasite resistant. Parasite resistance is reported as an estimated breeding value (EBV) for post-weaning worm egg count (PWEC). Previous data indicate that Katahdin sheep with low PWEC (parasite resistant) have greater survivability to weaning. Since significant variability for PWEC exists within the Katahdin breed (-100 and +867) (NSIP Database, 2022), the opportunity for selection would allow producers to capitalize on genetic tools to implement quick progress. By investigating resistance mechanisms involved in differential PWEC genotypes, we will be better positioned to provide producers with an effective means to implement non-antimicrobial disease prevention within their flocks. Selection for LoPWEC will have sustainable outcomes on sheep production by improving animal health and reducing the need for AM use.
Research
General Approach: Katahdin ewes will be sourced from the Southwest Virginia Agricultural Research Extension Center (AREC) located in Glade Spring, Virginia. All individuals will have NSIP data and index values will be used to ensure ewes are relatively similar in genetic merit beyond PWEC. To achieve this goal, we lambs must have a minimum Katahdin Index of +104. Once that threshold has been met, then 10 sheep of each genotype will be randomly selected after they have met the following conditions. Low PWEC group (LoPWEC) will be required to have a PWEC EBV of less than -50 and high PWEC sheep (HiPWEC) will be required to have a PWEC EBV of greater than +200. In both instances, accuracy of PWEC estimates must be equal to or greater than 75%. Once identified, those lambs will be relocated to WVU Agronomy farm.
Objective 1: These sheep will then be used to collect peripheral blood mononuclear cells (PBMCs) for in vitro lipopolysaccharide (LPS) stimulation. To determine immune cell response to LPS, peripheral blood mononuclear cells (PBMC) will be isolated and quantified using a TC-20 automated cell counter (Bio-Rad, Hercules, CA). PBMC will be diluted in RPMI-1640 supplemented with fetal bovine serum and antibiotic. PBMC will be added to wells in triplicate by individual for both control and LPS treatments. Cells will be treated with 100µg/ml of LPS derived from E. coli O111:B4 (L4391, Sigma Aldrich, St. Louis, MO).
Objective 1a: Cell proliferation will be quantified using an Alamar blue assay measuring absorbance values of control versus LPS stimulated cells between the two groups. Cells from these divergent PWEC Katahdin ewes will be stimulated with LPS for 24 hrs in a humidified incubator at 37oC and 5% CO2. After which, Alamar Blue (Sigma Aldrich) will be added to each well and plate will be returned to incubator for 48 hrs, then absorbance of each well will be read using a plate spectrophotometer (BioTek, Winooski, VT) at 570nm. These data will be compared to control samples and relative proliferation will be calculated. These data will be analyzed by individual within genotype with the main effect of genotype. These data will be analyzed by one-way anova using Sigma Plot 14.5 software (San Jose, CA) and significance is determined when P < 0.05.
Objective 1b: To further determine differences in response to LPS activation, we will measure typical cellular response to LPS stimulation. PBMC (1x105) collected from each animal will be plated in triplicate and treated with LPS (100ug/ml). PBMC with and without stimulation will be incubated for 3, 6, 9, 12, 24 hrs. At each time point cells will be collected, and RNA extracted and converted to cDNA for Real-time PCR gene expression analysis. Our previous in vivo work has shown that LoPWEC sheep had greater serum levels of Tumor Necrosis Factor alpha (TNFα) than HiPWEC sheep for 8 hrs after LPS administration. Gene expression analysis of TNFα over a time course will allow us to determine the time point where the greatest difference exists between LoPWEC and will provide critical information to accomplish the next objective. Data from this study will be analyzed using proc mixed procedure of SAS 9.4 (Cary, NC) with time, treatment, and genotype as main effects. Interactions will be analyzed using appropriate post hoc procedures and significance is accepted when P < 0.05.
Objective 2: Once this optimum timepoint is determined PBMCs will once again be collected and stimulated (Control vs. LPS) for this amount of time and RNA will be isolated from these cells. For this analysis, 5 sheep from each genotype will be randomly selected so that a total of 20 RNA-seq analyses will be performed (10 LoPWEC; 5 control and 5 LPS-treated and 10 HiPWEC; 5 control and 5 LPS-treated). This RNA will be submitted to the WVU Genomics Core for cDNA library build and RNA sequencing. Good quality reads will then be aligned against the ovine reference genome (Oar_v4.0) using STAR, and unique reads will be quantified. Alignment mapping analysis will be conducted to determine the differences between the reference genome and the sequences provided by our experiment. This data will be normalized, and statistical analyses performed using DESeq2 (negative binomial distribution, outlier detection, and correction). The Benjamini-Hochberg procedure will be performed to obtain an adjusted P value (Benjamini-Hochberg adjusted P value) with a significance cut-off of P = 0.05. Following this we can compare gene upregulation in the LoPWEC versus HiPWEC resulting from LPS stimulation. This will give us the potential to evaluate the entire transcriptome and discover genes previously not known to be involved within the phenotypic differences we observe through divergent PWEC selection.
Education & Outreach Activities and Participation Summary
Participation Summary:
Outcomes from this project will be presented to sheep producers nationwide through a variety of media and events, and will specifically target Katahdin producers. Research results will be presented to producers at several events. Regionally, the Virginia Tech Southwest Virginia AREC Sheep Field Day and Ram Test Sale is an ideal venue for sharing our results. Annually, over 150 producers attend this educational day and presentations are recorded and available online. To continue our regional coverage, we will present our data at the Eastern Alliance for Production Katahdin annual meeting. This is a group of progressive producers (50-75) that are research-minded and interested in the latest information to improve their flocks. From a national perspective, we will present our finding at the annual meeting of the national Katahdin breed association. Typically, there are 150-200 producers at this meeting and presentations are recorded and posted on their website. Due to the applicability of these data, we will lobby the American Sheep Industry to present our data through both the ASI webinar series and to be a featured talk at their annual meeting. We will also aim to present our data to the scientific community though an abstract presentation at the Southern Section of the American Society for Animal Science meeting. Additionally, I would plan to submit a manuscript(s) to Translational Animal Science or The Journal of Animal Science for publication. These journals are read by small ruminant researchers across the country. Outside of my fellow researchers I also plan to write a short publication in a newsletter type format that is easily digestible for producers. This newsletter published in The Katahdin Hairald Magazine will serve as direct communication with small ruminant producers about the benefits of selection for PWEC EBV.
Project Outcomes
No research has been completed yet. Lambs will be born by mid February.