Comprehensive at-arrival transcriptomic analysis of post-weaned beef cattle uncovers type I interferon and antiviral mechanisms associated with bovine respiratory disease mortality

Abstract

Background Despite decades of extensive research, bovine respiratory disease (BRD) remains the most devastating disease in beef cattle production. Establishing a clinical diagnosis often relies upon visual detection of non-specific signs, leading to low diagnostic accuracy. Thus, post-weaned beef cattle are often metaphylactically administered antimicrobials at facility arrival, which poses concerns regarding antimicrobial stewardship and resistance. Additionally, there is a lack of high-quality research that addresses the gene-by-environment interactions that underlie why some cattle that develop BRD die while others survive. Therefore, it is necessary to decipher the underlying host genomic factors associated with BRD mortality versus survival to help determine BRD risk and severity. Using transcriptomic analysis of at-arrival whole blood samples from cattle that died of BRD, as compared to those that developed signs of BRD but lived (n = 3 DEAD, n = 3 ALIVE), we identified differentially expressed genes (DEGs) and associated pathways in cattle that died of BRD. Additionally, we evaluated unmapped reads, which are often overlooked within transcriptomic experiments. Results 69 DEGs (FDR<0.10) were identified between ALIVE and DEAD cohorts. Several DEGs possess immunological and proinflammatory function and associations with TLR4 and IL6. Biological processes, pathways, and disease phenotype associations related to type-I interferon production and antiviral defense were enriched in DEAD cattle at arrival. Unmapped reads aligned primarily to various ungulate assemblies, but failed to align to viral assemblies. Conclusion This study further revealed increased proinflammatory immunological mechanisms in cattle that develop BRD. DEGs upregulated in DEAD cattle were predominantly involved in innate immune pathways typically associated with antiviral defense, although no viral genes were identified within unmapped reads. Our findings provide genomic targets for further analysis in cattle at highest risk of BRD, suggesting that mechanisms related to type I interferons and antiviral defense may be indicative of viral respiratory disease at arrival and contribute to eventual BRD mortality.

Integrated Network Analysis to Identify Key Modules and Potential Hub Genes Involved in Bovine Respiratory Disease: A Systems Biology Approach

“…In agreement with similar BRD studies ( Tizioto et al, 2015 ; Behura et al, 2017 ; Johnston et al, 2021c ; Lebedev et al, 2021 ), the leukocyte transendothelial migration was identified as one of the important pathways of the immune system in the blue module. Additionally, some of the hub–hub genes identified in the blue module have also been reported in the previous BRD studies, including TLR4 ( Scott et al, 2021 ), ANXA5 ( Mitchell et al, 2008 ), C3 , PSEN1 ( Neupane et al, 2018 ), CTSB , CD59 , FTL ( Nilson et al, 2020 ), CAT ( Joshi et al, 2018 ), TLR7 , CD86 ( Palomares et al, 2014 ), ATG7 ( Lipkin et al, 2016 ), and IFNAR1 ( Amat et al, 2019 ). For example, stresses from weaning, transport, and commingling reduced the expression level of the ANXA5 (hub–hub gene) and, thus, lead to an increase in apoptotic cells in the lungs or epithelial lining fluid, which can increase the susceptibility of cattle to a primary infection ( Mitchell et al, 2008 ).…”

“…Several recent studies have shown that the type I interferon signaling pathway has been identified as a key pathway in cows with BRD atfeedlot entry, which indicates that animals show antiviral responses at the entry stage ( Sun et al, 2020 ; Johnston et al, 2021a ; Scott et al, 2021 ). Interestingly, some of the important hub–hub genes, which are involved in anti-viral interferon response were identified in the purple module, such as IFI6 , ISG15 , MX1 , OAS2 , IFIH1 , DDX58 , DHX58 , RSAD2 , IFI44 , IFI44L , EIF2AK2 , ISG20 , MX2 , IFIT5 , IFITM3 , OAS1Y , and HERC5 , have been suggested by these studies as potential biomarkers for diagnostic and prediction of subclinical BRD at early stage of infection ( Sun et al, 2020 ; Johnston et al, 2021a ; Scott et al, 2021 ). Additionally, some of the key hub–hub TFs that regulate the expression of coexpressed genes in this module, and play critical roles in interferon antiviral responses during BRD, were included IRF9 , STAT1 , STAT2 ( Sun et al, 2020 ), and IRF7 ( Johnston et al, 2019 ).…”

“…In agreement with the previous studies, functional terms including “VEGF signaling pathway,” “T-cell receptor signaling pathway” ( Tizioto et al, 2015 ), and “C-type lectin receptor signaling pathway” as well as hub–hub genes including LCK ( Smirnova et al, 2009 ), TLR3 ( Marin et al, 2016 ), TIA1 ( Johnston et al, 2021b ), TNF ( El-Deeb et al, 2020 ), and STAT5A hub–hub TF ( Lin et al, 2015 ) demonstrate the importance of the red module during BRD. Furthermore, the salmon module was mainly enriched in “cellular defense response,” “regulation of immune response,” “leukocyte cell–cell adhesion,” and “natural killer cell-mediated cytotoxicity.” Recent studies have demonstrated that some of the hub–hub genes in the salmon module, such as CCR5 ( Salem et al, 2019 ), CCR8 ( Amat et al, 2019 ; Lopez et al, 2020 ), CX3CR1 ( Scott et al, 2021 ), ITGAL , IL12RB2 ( Neupane et al, 2018 ), NCR1 ( Osman and Griebel, 2017 ), CCL5 ( N’jai et al, 2013 ), and PRF1 ( Johnston et al, 2021a ) tend to participate in BRD. Additionally, GZMB gene, which plays a major role in stimulating cytotoxic T-cell responses and limiting virus replication in the host ( Jiminez et al, 2021 ), was found among hub–hub genes in the salmon module.…”

“…Furthermore, the salmon module was mainly enriched in "cellular defense response," "regulation of immune response," "leukocyte cell-cell adhesion," and "natural killer cell-mediated cytotoxicity." Recent studies have demonstrated that some of the hub-hub genes in the salmon module, such as CCR5 (Salem et al, 2019), CCR8 (Amat et al, 2019;Lopez et al, 2020), CX3CR1 (Scott et al, 2021), ITGAL, IL12RB2 (Neupane et al, 2018), NCR1 (Osman andGriebel, 2017), CCL5 (N'jai et al, 2013), and PRF1 (Johnston et al, 2021a) tend to participate in BRD. Additionally, GZMB gene, which plays a major role in stimulating cytotoxic T-cell responses and limiting virus replication in the host (Jiminez et al, 2021), was found among hub-hub genes in the salmon module.…”

“…Functional genomic methods such as RNA-sequencing-based transcriptomics can provide a global gene expression profile, and their use in BRD studies can accelerate the understanding of disease mechanisms and the development of diagnosis ( Rai et al, 2015 ). Several transcriptome studies have been performed on BRD in various tissues, such as the lung, bronchial lymph nodes ( Behura et al, 2017 ; Johnston et al, 2019 ), and whole blood ( Jiminez et al, 2021 ; Scott et al, 2021 ). For example, Sun et al (2020) reported differentially expressed genes (DEGs) in the blood tissue as biomarkers to recognize BRD cattle at entry, such as MX1 , IFIT3 , ISG15 , OAS2 , and IFI6 involved in the interferon signaling pathway.…”