WHOLE BLOOD BIOMARKER DISCOVERY IN HIGH-RISK STOCKER CATTLE AT-ARRIVAL: DIFFERENTIATING RESPIRATORY HEALTH AND DISEASE RISK

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1022778
• Grant No.: 2020-67016-31469
• Cumulative Award Amt.: $200,000.00
• Proposal No.: 2019-06015
• Project Start Date: Jul 1, 2020
• Project End Date: Jun 30, 2023
• Grant Year: 2020
• Program Code: [A1221]- Animal Health and Production and Animal Products: Animal Health and Disease

Recipient Organization
MISSISSIPPI STATE UNIV
(N/A)
MISSISSIPPI STATE,MS 39762

Performing Department
CVM Pathobiology/Population Me

Non Technical Summary
Bovine respiratory disease (BRD) is the leading cause of disease in beef cattle across North America and therefore, BRD is one of the main economic and welfare concerns in beef cattle production. BRD is also the leading cause of antimicrobial use in beef cattle; thus, discoveries that decrease BRD will also decrease antimicrobial use and decrease antimicrobial resistance. The diagnosis and treatment of BRD is often made from clinical signs, but this misses animals with subclinical disease. This frequently leads to misdiagnosing animals that have subclinical BRD as healthy. If veterinarians and cattle producers could accurately predict which cattle are likely to develop or resist BRD before it occurs, they could focus attention on the cattle likely to develop disease, which should improve accuracy and efficiency of detection and treatment. Our hypothesis is that beef cattle that remain healthy can be identified, at feeding facility arrival, to have increased levels of activity of specific genes involved in processes for BRD resistance. We will focus on two main objectives with this project. First, using blood testing, we will compare the activity of genes in cattle that never develop BRD versus those that develop BRD within 28 days of their arrival. Second, we will develop a more rapid and simple test to identify the genes that we learned are different in cattle that develop BRD versus cattle that remain healthy. We are expecting to find genes at arrival that accurately predict whether cattle develop BRD or remain healthy over time. This work will lead to a better understanding of why cattle develop or resist BRD, and will create tools that will help scientists, veterinarians, and cattle producers determine cattle that are likely to stay healthy or develop BRD, supporting the development of better protocols to identify, treat, and prevent BRD in cattle.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3310	1090	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1090 - Immunology;

Keywords

bovine

immune response

immunity

inflammation

pneumonia

Goals / Objectives
1. To evaluate and compare differential gene expression in at-arrival whole blood transcriptomes of high-risk stocker cattle that develop BRD within 28 days after arrival versus stocker cattle that do not.2. To evaluate expression of mRNA for genes associated with specialized proresolving mediators (SPM) by real time (quantitative) RT-PCR (RT-qPCR) of whole blood samples inmultiple independent populations of high-risk stocker cattle at arrival, and to confirm whether SPM expression is related to BRD resistance.

Project Methods
We have collected and properly stored several hundred at-arrival Tempus® blood tube (RNA preservation) samples from five independent populations of high-risk stocker cattle. We will also collect at-arrival samples from approximately 320 stocker calves over the next two years. Each sample has accompanying production (weight gain), BRD treatment, diagnostic, and management data (metadata). One of the strengths of this study is the number of RNA preserved samples stored from a wide assortment of cattle, in terms of geography, season, and year. We will select samples from this bank that represent the range of characteristics represented and including both cattle that develop bRD and cattle that do not for use in Objectives 1 and 2.Objective 1:Our experimental objective is to use on-arrival whole blood samples collected andstored from well-characterized stocker cattle populations in 2017 and 2019 to compare whole blood transcriptomic profiles of cattle that developed BRD and cattle that did not. Wewill select cattle that were managed similarly in terms of vaccination and other treatments atarrival, but that either developed BRD within 28 days or did not develop BRD. The rationale is that genes that are repeatably found to be differentially expressed at arrival between cattle that develop or resist BRD in temporally independent populations are candidate biomarkers for predicting BRD susceptibility.Approach: Briefly, RNA extraction, preparation, and sequencing will be performed at the UCLA Technology Center for Genomics and Bioinformatics. To avoid technical sequencing bias, the read processing and differential gene expression analysis will be performed in the identical manner as the samples that were used to generate our past preliminary data. Differentially expressed genes identified at a Benjamini-Hochberg adjusted p-value (FDR) of < 0.10 will be used for downstream enrichment analysis. Gene ontology (GO) enrichment and functional pathway enrichment analysis of the differentially expressed genes will be performed in WebGestalt online database (http://www.webgestalt.org/). Functional pathway and protein interaction analysis will be performed using WebGestalt, Reactome, and STRING. Pathways that possess an FDR cutoff of < 0.10 will be considered significantly enriched. Genes and pathways identified from this project will be compared to data analysis from the population used to generate our preliminary data. Euclidean metric modelling between the three populations will be applied through multi-dimensional scaling (MDS) in R to assess dissimilarities between healthy and diseased groups. Hierarchical clustering will be employed in R to identify genes that are differentially expressed between each population of cattle, and heatmap visualization utilized with the program pheatmap. Enriched functional pathways and biological processes between populations will be evaluated between healthy and diseased groups, both within and across each population of high-risk stocker cattle. DEGs will be confirmed by real time RT-qPCR using methods as described for Objective 2. The set of genes that best predict BRD or resistance will be determined by linear discriminant analysis (LDA). Whole blood samples collected prospectively for Objective 2 will be used for the testing population for confirmation.We expect to identify gene products with conserved differential expression amongindividuals from the 2017 and 2019 groups that developed BRD or remained healthy. We alsoanticipate identifying other biological pathways from these differentially expressed genes that segregate with disease and health. These data will be used to identify genes and pathophysiologic mechanisms that predict BRD outcome from cattle at arrival. This data will be made publicly available in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/sra/docs/submitgeo/). Because of the limited published information regarding stocker cattle host transcriptomics, we anticipate this analysis will provide novel comparative bioinformatic approaches and a wealth of host transcriptomic data for use by ourselves and others in ongoing and future research in beef cattle production and disease modelling.Objective 2:Our experimental objective is to quantify mRNA by RT-qPCR for specific pro-resolving mediators (SPM) and genes associated with angiotensinogen metabolism (AM) in at-arrival whole blood samples from calves that develop BRD and calves that remain healthy in multiple populations of stocker cattle from different geographical locations or years. The rationale for this objective is that our preliminary work identified an increase in transcripts specific to SPM and AM pathways in healthy calves. These pathways may support resistance to BRD through inflammatory resolution and metabolic regulation. An RT-qPCR test for genes that predict health or disease will be more efficient and inexpensive for longterm and widespread use by veterinarians and cattle producers.Approach: Primer construction: Primers will be developed for differentially expressed genes we previously identified (ALOX15, HPGD, CMA1, and CPB2) and other transcripts associated with SPM and AM. Primers for other DEG confirmed in Objective 1 will also be developed. Endogenous reference gene selection will be based on computation normalization of the RNA-Seq gene count data present from our preliminary data, via NormFinder. Primers will be designed to span exon-exon borders and melting curve analysis will be applied to identify any false-positive amplification of genomic DNA using. Principle design is based on MIQE guidelines and published considerations of developing assays. To determine specific whole blood mRNA abundance, RNA extraction and cDNA synthesis via commercially available testing kits will be utilized for each sample, and RT-qPCR will be performed for the detection of the genes of interest. Relative quantification analysis of RT-qPCR data will be analyzed using the 2 to the negative delta-delta Ct calculation. All experimentation will be carried out in triplicate. Statistical analysis will be performed with Student's T testing, ANOVA F-testing, and other proper analysis methods. The set of genes that best predict BRD or resistance will be determined by linear discriminant analysis (LDA).We expect to validate the association of SPM with disease outcome in multiple populations of high-risk stocker calves. By testing for several key regulatory genes related to SPM, as well as downstream gene products in these pathways, we aim to minimize differences due to biological variation and differences in time of sample collection relative to disease progressionEfforts to cause a change in knowledge and practice, and to evaluate impact:We anticipate the overall outcome of this research to be the identification of novel biomarkers and pathways related to BRD susceptibility and resistance, and an RT-qPCR test to detect these biomarkers in cattel populations. This work will also establish methodology in comparative bioinformatics for use by other cattle health researchers. We will report these results at meetings of geneticists, microbiologists, and immunologists, veterinarians and cattle producers, in order to disseminate the knowledge and lead to change in practice by which these groups identify cattle likely to develop or resist BRD.We will evaluate the impact of this by periodic review of the published peer-reviewed scientific literature and also lay publications to determine whether other groups are using our approach or data, or similar approaches, to assess on arrival the likelihood that cattle will develop BRD or stay healthy, and the degree to which these approaches are used for targeted management to improve cattle health while decreasing use of antimicrobials.

Progress 07/01/20 to 06/30/23

Outputs
Target Audience:The target audiencefor this project was scientist and veterinarians engaged in research related to bovine immunity and bovine respiratory diseases. Also, veterinarians in cattle practice could benefit from this project. Changes/Problems:The original plan for Objective 2 was to use quantitative real time reverse transcriptase PCR (qRT-PCR) to confirm in larger numbers of cattle from more sources the differential expression of genes identified in Objective 1. However, when it became possible to use NanoString analysis we decided instead to use that approach. As compared to qRT-PCR, NanoString has several benefits, including lack of bias introduced by amplification of cDNA. What opportunities for training and professional development has the project provided?This research was the subject of the PhD thesis of Matthew Scott, DVM PhD, who completed his PhD at Mississippi State University in 2021, and joined the faculty of Texas A&M University shortly thereafter. Since joining the faculty of Texas A&M University Dr. Scott has embarked on a productive research program building on the skills and knowledge he developed working on this project, already publishing new work and mentoring graduate students. Therefore this project was directly responsible for supporting the development of a new researcher who is using cutting edge techniques in transcriptomics and bioinformatics to identify new information to help veterinarians and farmers better predict BRD. How have the results been disseminated to communities of interest?Five scientific papers published in peer-reviewed journals, and 11 research abstracts/conference papers, resulted from this research. Scientific papers were presented at the Conference for Research Workers in Animal Disease in multiple years, the 2022 World Buiatrics Congress in Madrid, Spain, the American Association of Bovine Practitioners Annual Convention, the American College of Veterinary Internal Medicine Annual Forum, the 2019 Bovine Respiratory Disease Symposium, and the 2019 International Veterinary Immunology Symposium (IVIS). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Bovine respiratory disease is the leading cause of sickness and death in U.S. beef cattle, despite decades of research to determine improved methods to prevent and treat the disease. Little research has evaluated how the immune response of the cattle is related to outcomes. Using a new approach, transcriptomics, we evaluated how host immune responses on the day the cattle are first purchased (received) were related to whether or not the cattle developed BRD within the next month, in order to develop biomarkers of BRD risk. We discovered evidence that, on the day they are first received, cattle that will never be treated for BRD produce greater amounts of immune mediators not previously linked to BRD, specific pro-resolving mediators (SPM). In contrast, we found evidence that, on the day they are received, cattle that will eventually be treated for BRD are producing greater amounts of interferons. Because SPM have been linked with resistance to respiratory disease in other animals and humans, because they decrease inflammation, it makes sense that SPM are related to BRD resistance. Moreover, interferons are known to be produced by animals following virus infection, but interferon production on the day they are first received has not previously been associated with increased risk of BRD. These findings provide strong support for investigating SPM and interferons further to see if their measurement can repeatedly be used to accurately predict, on the day they are received, whether or not cattle will develop BRD at a later date. This will support their use as biomarkers of BRD risk. Our research also provided new information to scientists who conduct research on BRD, by demonstrating evidence that mediators previously little explored, SPMs, may play an important role in BRD resistance. These findings should guide future research that could help us better understand how the immune response of the cow determines whether or not the animal will develop BRD. If reliable biomarkers of BRD risk are confirmed, it would provide farmers and veterinarians with a novel tool to use to more accurately target antibiotic use to the cattle who actually need antibiotics, decreasing the need for mass administration of antimicrobials to entire groups of cattle at risk for BRD--the current practice used to decrease BRD.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Scott MA, Woolums AR, Swiderski CE, Finley A, Perkins AD, Nanduri B, Karisch BB. Hematological and gene co-expression network analyses of high-risk beef cattle defines immunological mechanisms and biological complexes involved in bovine respiratory disease and weight gain. 2022. PLoS One. 17:e0277033. doi: 10.1371/journal.pone.0277033
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Scott MA, Woolums AR, Swiderski CE, Thompson AC, Perkins AD, Nanduri B, Karisch BB, Epperson WB. Use of nCounter mRNA profiling to identify at-arrival gene expression patterns for predicting bovine respiratory disease in beef cattle. BMC Vet Res. 2022. Feb 23;18(1):77. doi: 10.1186/s12917-022-03178-8.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Scott MA, Woolums AR, Swiderski CE, Perkins AD, Nanduri B, Smith DR, Karisch BB, Epperson WB, Blanton JR Jr. Multipopulational transcriptome analysis of post-weaned beef cattle at arrival further validates candidate biomarkers for predicting clinical bovine respiratory disease. Sci Rep. 2021. Dec 13;11(1):23877. doi: 10.1038/s41598-021-03355-z.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Scott MA, Woolums AR, Swiderski CE, Perkins AD, Nanduri B, Smith DR, Karisch BB, Epperson WB, Blanton Jr JR. Comprehensive at-arrival transcriptomic analysis of post-weaned beef cattle uncovers type I interferon and antiviral mechanisms associated with bovine respiratory disease mortality. PLOS One. 2021. Apr 26;16(4):e0250758. doi: 10.1371/journal.pone.0250758. eCollection 2021. PMID: 33901263.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Scott MA, Woolums AR, Swiderski CE, Perkins AD, Nanduri B, Smith DR, Karisch BB, Epperson WB, Perkins A, Blanton Jr., Jr. Whole blood transcriptomic analysis of beef cattle at arrival identifies potential predictive molecules and mechanisms that indicate animals that naturally resist bovine respiratory disease. PLOS ONE 2020. DOI: https://doi.org/10.1371/journal.pone.0227507.

Progress 07/01/22 to 06/30/23

Outputs
Target Audience:Scientists and veterinarians engaged in research related to bovine immunity and bovine respiratory disease; veterinarians in cattle practice. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research was the subject of the PhD thesis of Matthew Scott, DVM PhD, who completed his PhD at Mississippi State University in 2021, and joined the faculty of Texas A&M University shortly thereafter. Since joining the faculty of Texas A&M University Dr. Scott has embarked on a productive research program building on the skills and knowledge he developed working on this project, already publishing new work and mentoring graduate students. Therefore this project was directly responsible for supporting the development of a new researcher who is using cutting edge techniques in transcriptomics and bioinformatics to identify new information to help veterinarians and farmers better predict BRD. How have the results been disseminated to communities of interest?As described above, the results were published in PLOS ONE, also, other data from previously reported work were presented in an abstract at the World Buiatrics Congress in Madrid, Spain, in September 2022. What do you plan to do during the next reporting period to accomplish the goals?This is the end of the project. We are currently working on a NIFA proposal for a Standard Research Grant for the August 2023 deadline that will build upon the results discovered in this project.

Impacts
What was accomplished under these goals? Major activities completed: To provide additional new knowledge regarding the relationship between gene expression pathways and other measures that can provide information about the health of cattle with bovine respiratory disease, we leveraged complete blood count (CBC) data collected with other funding to evaluate relationships between blood gene expression patterns measured for this proposal, and findings on the CBC. We demonstrated that some measurements that can be made on a CBC, which is easy for a practitioner to run in the local clinic, were associated with gene expression patterns that were related to BRD. This provides evidence that simple test that can easily be run in a veterinary clinic could be used to predict host immune gene expression patterns that are related to BRD resistance. This new information provides a foundation for development of a novel test to identify cattle at risk for BRD, which could enable more targeted management of cattle to decrease disease with less use of antimicrobials. Specific objectives met: All objectives for this project have been met. Significant results achieved: We demonstrated that a gene co-expression modules related to T cell responses were significantly correlated with increased red blood cells, platelets, and BRD development. Another gene co-expression module related to mitochondrial metabolism and rRNA maturation was significantly correlated with markers of parasitism, including blood eosinophil count, fecal egg count, and weight gain. Fifty-two interacting genes were related to BRD development in ways not previously described in the literature. Key outcomes: This work demonstrated new relationships between outcomes on the CBC, a test that can easily be run in a veterinary clinic, and host gene expression pathways related to BRD resistance. These findings could support the development of methods to use simple in-house tests to predict BRD risk in cattle. Such methods could support the development of more targeted approaches to treatment, which could keep more cattle healthy while decreasing antimicrobial use.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Scott MA, Woolums AR, Swiderski CE, Finley A, Perkins AD, Nanduri B, Karisch BB. Hematological and gene co-expression network analyses of high-risk beef cattle defines immunological mechanisms and biological complexes involved in bovine respiratory disease and weight gain. 2022. PLoS One. 17:e0277033. doi: 10.1371/journal.pone.0277033.
• Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Scott M, Woolums A, Swiderski C, Perkins A, Nanduri B, Smith DR, Karisch B, Epperson W, Blanton Jr, JR. Identification of potential predictive biomarkers and molecular mechanisms contributing to BRD-associated mortality in post-weaned beef cattle. 31st World Buiatrics Congress. Madrid, Spain. September 4-8, 2022. #AH-16.

Progress 07/01/21 to 06/30/22

Outputs
Target Audience:Scientists and veterinarians engaged in research related to bovine immunity and bovine respiratory disease; veterinarians in cattle practice. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research was the subject of the PhD thesis of Matthew Scott, DVM, who graduated in August 2021 with his PhD. Dr. Scott is now a faculty member in the Veterinary Education, Research, and Outreach (VERO) Program at Texas A&M University. How have the results been disseminated to communities of interest?As described above, they were presented in 4 manuscripts published in 2021 - 2022, and in 3 abstracts presented at national meetings in 2021. What do you plan to do during the next reporting period to accomplish the goals?We will investigate the relationship between expression of genes related to production of SPMs, which appear to protect cattle from BRD, and past history of treatment with antimicrobials, especially macrolide antimicrobials, which have been shown in vitro to induce expression of SPM by bovine cells. It may be that drugs given to cattle to kill bacteria are actually helping cattle resist disease by quelling information. If this is true, it may be possible to develop drugs that do not induce antimicrobial resistance, but still limit inflammation, improving cattle health while decreasing the dissemination of antimicrobial resistance.

Impacts
What was accomplished under these goals? Major activities completed: We completed the work for objective 2, confirming that gene expression shown in our earlier work to be associated with resistance to BRD, or development of BRD, were again differentially expressed in cattle that became sick or stayed healthy. Importantly, increased expression of mRNAs involved in specialized pro-resolving mediator synthesis (ALOX15, HPGD) were again upregulated in cattle that stayed healthy, while cattle that developed BRD cattle again demonstrated increased expression of CFB, and mRNA related to granulocyticprocesses (DSG1, LRG1, MCF2L) and type-I interferon activity (HERC6, IFI6, ISG15, MX1). Healthy and Treated_1 cattle were similar in terms of gene expression, while Treated_2+ cattle were the most distinct. ROC cutoffs were used to generate an at-arrival treatment decision tree, which classified 90% of Treated_2+ individuals. Specific objectives met: We have complete Objective 2. We also recognized that expression of specific pro-resolving mediators (SPM), which we have repeatedly found to be upregulated at arrival in cattle that go on to resist BRD, have also been shown to be upregulated by certain antimicrobial drugs, such as macrolides. Macrolides are the drugs most commonly given at arrival to prevent BRD, but it may be that the drugs have benefit from induction of SPM. This has not been investigated in live cattle; the work to date has entirely been in vitro. We have requested a no-cost extension for 2022-2023 in order to investigate the relationship between past antimicrobial therapy and gene expression in the cattle we have sampled, in order to test the hypothesis that SPM expression is related to administration of macrolide antimicrobials in cattle at risk for BRD. Investigation of this relationship could reveal new pathways for limiting the impact of BRD on cattle health. Significant results achieved: Assessment of gene expression with the NanoString nCounter FLEX system was effective to identify differentially expressed genes, leading to development of a an at-arrival treatment decision tree which classified 90% of Treated_2+ individuals. This decision tree could be used to determine at arrival which cattle are most likely to benefit from antimicrobial therapy, and which cattle are likely to thrive without antimicrobial therapy. In future research we will need to prospectively test this decision tree using a rapid test, such as PCR, to evaluate expression of the relevant genes, to confirm the feasibility of this decision tree tool to refine our management of cattle. Key outcomes: We have shown in repeated studies across multiple groups of cattle sampled at different times and in different location that expression of genes related to specific proresolving mediator (SPM) synthesis is increased on the day cattle arrive in those that go on to resist BRD, and expression of genes related to interferon production, and production of complement factor B, are increased on the day cattle arrive in those that go on to develop disease. These findings provide the basis for development of methods that allow precision management of cattle, keeping them more healthy with less reliance on antimicrobials.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Scott MA, Woolums AR, Swiderski CE, Thompson AC, Perkins AD, Nanduri B, Karisch BB, Epperson WB. Use of nCounter mRNA profiling to identify at-arrival gene expression patterns for predicting bovine respiratory disease in beef cattle. BMC Vet Res. 2022. Feb 23;18(1):77. doi: 10.1186/s12917-022-03178-8.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Scott MA, Woolums AR, Swiderski CE, Perkins AD, Nanduri B, Smith DR, Karisch BB, Epperson WB, Blanton Jr JR. Comprehensive at-arrival transcriptomic analysis of post-weaned beef cattle uncovers type I interferon and antiviral mechanisms associated with bovine respiratory disease mortality. PLOS One. 2021. Apr 26;16(4):e0250758. doi: 10.1371/journal.pone.0250758. eCollection 2021. PMID: 33901263.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Scott MA, Woolums AR, Swiderski CE, Perkins AD, Nanduri B, Smith DR, Karisch BB, Epperson WB, Perkins A, Blanton Jr., Jr. Whole blood transcriptomic analysis of beef cattle at arrival identifies potential predictive molecules and mechanisms that indicate animals that naturally resist bovine respiratory disease. PLOS ONE 2020. DOI: https://doi.org/10.1371/journal.pone.0227507.
• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Scott MA, Woolums AR, Swiderski CE, Perkins A, Nanduri B, Smith DR, Karisch BB, Epperson WB, Blanton JR. Blood biomarker discovery in high-risk stocker cattle at-arrival: differentiating respiratory health and disease. Conference for Research Workers in Animal Disease (CRWAD). Chicago IL. December 5-7, 2021.
• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Scott MA, Woolums AR, Swiderski CE, Thompson AC, Perkins AD, Nanduri B, Karisch BB, Goehl DR. Digital mRNA profiling of high-risk beef cattle at arrival substantiates markers of bovine respiratory disease. Conference for Research Workers in Animal Disease (CRWAD). Chicago IL. December 5-7, 2021.
• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Scott MA, Woolums AR, Swiderski CE, Perkins A, Nanduri B, Smith DR, Karisch BB, Epperson WB. Multipopulational transcriptomic analysis of high-risk beef cattle on arrival reveals genes and mechanisms which may predict bovine respiratory disease. Conference of the American Association of Bovine Practitioners (AABP). Salt Lake City, UT. October 7-9, 2021.

Progress 07/01/20 to 06/30/21

Outputs
Target Audience:Scientists and veterinarians engaged in research related to bovine immunity and bovine respiratory disease; veterinarians in cattle practice. Changes/Problems:The original plan for Objective 2 was to use quantitative real time reverse transcriptase PCR (qRT-PCR) to confirm in larger numbers of cattle from more sources the differential expression of genes identified in Objective 1. However, when it became possible to use NanoString analysis we decided instead to use that approach. As compared to qRT-PCR, NanoString has several benefits, including lack of bias introduced by amplification of cDNA. What opportunities for training and professional development has the project provided?This research was the subject of the PhD thesis of Matthew Scott, DVM, who has completed his thesis defense and submitted his thesis, Dr. Scott will graduate in August 2021 with his PhD. For his abstract presentation at the 2020 Annual Convention of the American Association of Bovine Practitioners, Dr. Scott won first place in the graduate student presentation competition. In June 2021 Dr. Scott began a new position as an Assistant Professor at Texas A&M University. How have the results been disseminated to communities of interest?As described above, they were presented as an abstract at CRWAD 2020, and have been submitted in manuscript form to Scientific Reports. What do you plan to do during the next reporting period to accomplish the goals?We will finish the analysis of the data collected for Objective 2, submit at least one manuscript describing this research, and we will also submit abstracts for presentation at CRWAD and other scientific meetings.

Impacts
What was accomplished under these goals? For Objective 2 we aimed to test whether genes found in previous investigations (including 48 cattle evaluated for Objective 1 of this proposal) to be differentially expressed at arrival in high risk stocker cattle treated 0, 1, or 2 or more times for BRD were consistently differentially expressed in larger numbers of cattle from diverse populations separated geographically and over time. Blood samples previously collected from 240 cattle into tubes of RNA preservation buffer on the first day they were at risk for naturally occurring respiratory disease were analyzed. Sampled cattle included those that were ultimately treated multiple times for disease (n = 34), treated once (n =91), or never treated (n = 115) over the subsequent 28 days were sampled. These cattle were in one of 7 different populations sampled between 2015 and 2020. Differential expression of genes, including many genes previously shown by us and/or others to be differentially expressed in cattle are treated for BRD versus cattle that do not go on to be treated for BRD, was assessed by NanoString analysis. Specific objectives met: We have complete Objective 1 and the results are described in the submitted manuscript described in the section "Products". Data collection for Objective 2 is complete and analysis is ongoing. These are the only 2 objectives of this seed grant. ?Significant results achieved: Data from 234 of the 240 samples were suitable for analysis. Gene expression was profiled with NanoString nCounter FLEX system, selecting 56 genes plus four internal controls. Differentially expressed genes (DEGs) were identified through nSolver v4.0 (p<0.05). Functional analysis of DEGs was performed with WebGestalt API. Statistical and visual analyses were performed within R. 17 DEGs were identified between healthy and BRD; 30 unique DEGs were identified between the three cohorts. Healthy cattle possessed increased specialized proresolvin mediator and defense response gene expression. BRD cattle possessed increased alternative complement and cell-cell adhesion gene expression. Type I interferon-related gene expression was increased in treated_2+ cattle. Average daily weight gain at time of sale (ADGend) significantly decreased with increased treatment frequency (p<0.0001). These data from multiple populations of cattle corroborate findings from previous transcriptome experiments. Key outcomes: Although preliminary pending the results of completed analysis, these results suggest that expression of certain genes, or expression of genes in certain host response pathways, is repeatedly associated with resistance or susceptibility to subsequent BRD in high-risk stocker cattle.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Scott M, Woolums A, Swiderski C, Perkins A, Nanduri B, Smith D, Karisch B, Epperson W, Blanton J. Multipopulational transcriptome analysis of post-weaned beef cattle at arrival further validates candidate biomarkers for predicting clinical bovine respiratory disease.
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Scott MA, Woolums AR, Swiderski CE, Perkins AD, Nanduri B, Smith DR, Karisch BB, Epperson WB, Blanton Jr JR. Transcriptomic profiling of BRD-attributed mortality in stocker cattle identifies active inflammatory and antiviral pathways at arrival. American Association of Bovine Practitioners, Annual Convention. Virtual (online only) meeting. Sept 24 - 26, 2020
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Woolums A, Scott MA, Swiderski C, Perkins AD, Nanduri B, Smith D, Karisch B, Epperson W, Blanton Jr JR. Blood biomarker discovery in high-risk stocker cattle at-arrival: differentiating respiratory health and disease. Conference of Research Workers in Animal Diseases (CRWAD). Virtual (online only) meeting. December 5-8, 2020. Abstract #253.