Expression network analysis of bovine skin infested with Rhipicephalus australis identifies pro-inflammatory genes contributing to tick susceptibility.

The skin is the primary feeding site of ticks that infest livestock animals such as cattle. The highly specialised functions of skin at the molecular level may be a factor contributing to variation in susceptibility to tick infestation; but these remain to be well defined. The aim of this study was to investigate the bovine skin transcriptomic profiles of tick-naïve and tick-infested cattle and to uncover the gene expression networks that influence contrasting phenotypes of host resistance to ticks. RNA-Seq data was obtained from skin of Brangus cattle with high (n = 5) and low (n = 6) host resistance at 0 and 12 weeks following artificial tick challenge with Rhipicephalus australis larvae. No differentially expressed genes were detected pre-infestation between high and low resistance groups, but at 12-weeks there were 229 differentially expressed genes (DEGs; FDR < 0.05), of which 212 were the target of at least 1866 transcription factors (TFs) expressed in skin. Regulatory impact factor (RIF) analysis identified 158 significant TFs (P < 0.05) of which GRHL3, and DTX1 were also DEGs in the experiment. Gene term enrichment showed the significant TFs and DEGs were enriched in processes related to immune response and biological pathways related to host response to infectious diseases. Interferon Type 1-stimulated genes, including MX2, ISG15, MX1, OAS2 were upregulated in low host resistance steers after repeated tick challenge, suggesting dysregulated wound healing and chronic inflammatory skin processes contributing to host susceptibility to ticks. The present study provides an assessment of the bovine skin transcriptome before and after repeated tick challenge and shows that the up-regulation of pro-inflammatory genes is a prominent feature in the skin of tick-susceptible animals. In addition, the identification of transcription factors with high regulatory impact provides insights into the potentially meaningful gene-gene interactions involved in the variation of phenotypes of bovine host resistance to ticks.

Application of quantitative proteomics to discover biomarkers for tick resistance in cattle.

Introduction: Breeding for tick resistance is a sustainable alternative to control cattle ticks due to widespread resistance to acaricidal drugs and the lack of a protective vaccine. The most accurate method used to characterise the phenotype for tick resistance in field studies is the standard tick count, but this is labour-intensive and can be hazardous to the operator. Efficient genetic selection requires reliable phenotyping or biomarker(s) for accurately identifying tick-resistant cattle. Although breed-specific genes associated with tick resistance have been identified, the mechanisms behind tick resistance have not yet been fully characterised. Methods: This study applied quantitative proteomics to examine the differential abundance of serum and skin proteins using samples from naïve tick-resistant and -susceptible Brangus cattle at two-time points following tick exposure. The proteins were digested into peptides, followed by identification and quantification using sequential window acquisition of all theoretical fragment ion mass spectrometry. Results: Resistant naïve cattle had a suite of proteins associated with immune response, blood coagulation and wound healing that were significantly (adjusted P < 10- 5) more abundant compared with susceptible naïve cattle. These proteins included complement factors (C3, C4, C4a), alpha-1-acid glycoprotein (AGP), beta-2-glycoprotein-1, keratins (KRT1 & KRT3) and fibrinogens (alpha & beta). The mass spectrometry findings were validated by identifying differences in the relative abundance of selected serum proteins with ELISA. The proteins showing a significantly different abundance in resistant cattle following early and prolonged tick exposures (compared to resistant naïve) were associated with immune response, blood coagulation, homeostasis, and wound healing. In contrast, susceptible cattle developed some of these responses only after prolonged tick exposure. Discussion: Resistant cattle were able to transmigrate immune-response related proteins towards the tick bite sites, which may prevent tick feeding. Significantly differentially abundant proteins identified in this research in resistant naïve cattle may provide a rapid and efficient protective response to tick infestation. Physical barrier (skin integrity and wound healing) mechanisms and systemic immune responses were key contributors to resistance. Immune response-related proteins such as C4, C4a, AGP and CGN1 (naïve samples), CD14, GC and AGP (post-infestation) should be further investigated as potential biomarkers for tick resistance.

Transcriptional changes in the peripheral blood leukocytes from Brangus cattle before and after tick challenge with Rhipicephalus australis.

BACKGROUND: Disease emergence and production loss caused by cattle tick infestations have focused attention on genetic selection strategies to breed beef cattle with increased tick resistance. However, the mechanisms behind host responses to tick infestation have not been fully characterised. Hence, this study examined gene expression profiles of peripheral blood leukocytes from tick-naive Brangus steers (Bos taurus x Bos indicus) at 0, 3, and 12 weeks following artificial tick challenge experiments with Rhipicephalus australis larvae. The aim of the study was to investigate the effect of tick infestation on host leukocyte response to explore genes associated with the expression of high and low host resistance to ticks. RESULTS: Animals with high (HR, n = 5) and low (LR, n = 5) host resistance were identified after repeated tick challenge. A total of 3644 unique differentially expressed genes (FDR < 0.05) were identified in the comparison of tick-exposed (both HR and LR) and tick-naive steers for the 3-week and 12-week infestation period. Enrichment analyses showed genes were involved in leukocyte chemotaxis, coagulation, and inflammatory response. The IL-17 signalling, and cytokine-cytokine interactions pathways appeared to be relevant in protection and immunopathology to tick challenge. Comparison of HR and LR phenotypes at timepoints of weeks 0, 3, and 12 showed there were 69, 8, and 4 differentially expressed genes, respectively. Most of these genes were related to immune, tissue remodelling, and angiogenesis functions, suggesting this is relevant in the development of resistance or susceptibility to tick challenge. CONCLUSIONS: This study showed the effect of tick infestation on Brangus cattle with variable phenotypes of host resistance to R. australis ticks. Steers responded to infestation by expressing leukocyte genes related to chemotaxis, cytokine secretion, and inflammatory response. The altered expression of genes from the bovine MHC complex in highly resistant animals at pre- and post- infestation stages also supports the relevance of this genomic region for disease resilience. Overall, this study offers a resource of leukocyte gene expression data on matched tick-naive and tick-infested steers relevant for the improvement of tick resistance in composite cattle.