Extensive differential DNA methylation between tuberculosis skin test positive and skin test negative cattle.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), represents a significant problem for the agriculture industry as well as posing a risk for human health. Current diagnostic tests for bTB target the cell-mediated immune (CMI) response to infection with M. bovis, primarily through screening of animals with the tuberculin skin test. Epigenetic modifications have been shown to alter the course of the immune response and differentially methylated regions (DMRs) might also influence the outcome of the skin test in cattle. Whole Genome Bisulphite Sequencing (WGBS) was used to profile DNA methylation levels from peripheral blood of a group of cattle identified as test positive for M. bovis (positive for the single intradermal comparative tuberculin test (SICTT) and/or the interferon-γ release assay compared to a test negative control group [n = 8/group, total of 16 WGBS libraries]. Although global methylation profiles were similar for both groups across the genome, 223 DMRs and 159 Differentially Promoter Methylated Genes (DPMGs) were identified between groups with an excess of hypermethylated sites in SICTT positive cattle (threshold > 15% differential methylation). Genes located within these DMRs included the Interleukin 1 receptor (IL1R1) and MHC related genes (BOLA and BOLA-DQB). KEGG pathway analysis identified enrichment of genes involved in Calcium and MAPK signalling, as well as metabolism pathways. Analysis of DMRs in a subset of SICTT negative cattle that were IFN-γ positive showed differential methylation of genes including Interleukin 10 Receptor, alpha (IL10RA), Interleukin 17 F (IL17F) and host defence peptides (DEFB and BDEF109). This study has identified a number of immune gene loci at which differential methylation is associated with SICTT test results and the degree of methylation could influence effective host immune responses.

The transcriptomic response of bovine uterine tissue is altered in response to sperm from high- and low-fertility bulls†.

Despite stringent quality control checks, some bulls with apparently normal semen quality yield lower than expected pregnancy rates. This study profiled the transcriptome and performed histological analysis of the bovine uterus in response to sperm from high-fertility (HF) and low-fertility (LF) bulls. Postmortem uterine biopsies and uterine explants were collected from heifers 12 h after a fixed time artificial insemination (AI) to a synchronized estrus with frozen-thawed semen from five HF (fertility rate 4.01% ± 0.25) and five LF (fertility rate - 11.29% ± 1.11; mean ± SEM) bulls. Uterine biopsies were also collected from control (CTRL) heifers, which were not inseminated. RNA-sequencing and histological analysis were performed for differential gene expression and neutrophil quantification. In the HF treatment relative to CTRL heifers, there were 376 genes significantly differentially expressed in the endometrium with just one gene differentially expressed in the LF treatment relative to CTRL heifers. Comparing the HF and LF treatments directly, there were 40 significantly differentially expressed genes (P < 0.05). Transcriptomic analysis shows a predominant role for the inflammatory marker Interleukin-1 alpha, which was further confirmed by immunohistochemistry. Quantification of neutrophils in the endometrium showed a significant effect of sperm; however, there was no difference in neutrophil numbers between HF and LF groups. In conclusion, this novel study clearly shows a distinct inflammatory response to sperm in the endometrium and a divergent transcriptomic response to semen from HF and LF bulls.

Ewe breed differences in the cervical transcriptome at the follicular phase of a synchronised oestrous cycle.

BACKGROUND: Cervical artificial insemination (AI) with frozen-thawed semen results in unacceptably low pregnancy rates internationally. The exception is in Norway, where vaginal deposition of frozen-thawed semen to a natural oestrous routinely yields pregnancy rates in excess of 70%. Previous studies by our group has demonstrated that this is due to differences in cervical sperm transport. However, a potentially important contributory factor is that ewes are inseminated to a natural oestrous in Norway but to a synchronised oestrous across most of the rest of the world. In this study, we interrogated the gene expression of the sheep cervix of four ewe breeds with known differences in pregnancy rates following cervical AI using frozen-thawed semen under the effect of exogenous hormones to synchronise the oestrous cycle. These four ewe breeds (n = 8 to 11 ewes per breed) are from two countries: Ireland (Belclare and Suffolk; medium and low fertility, respectively) and Norway (Norwegian White Sheep (NWS) and Fur; both with high fertility compared to the Irish ewe breeds). RESULTS: RNA extracted from cervical biopsies collected from these breeds was analysed by RNA-sequencing and differential gene expression analysis. Using the low-fertility Suffolk breed as a reference level; 27, 1827 and 2641 genes were differentially expressed in Belclare, Fur and NWS ewes, respectively (P <  0.05 and FC > 1.5). Gene ontology (GO) analysis revealed that Fur and NWS had an up-regulation of enriched pathways involved in muscle contraction and development compared to Suffolk. However, there was a down-regulation of the immune response pathway in NWS compared to Suffolk. In addition, GO analysis showed similar expression patterns involved in muscle contraction, extracellular matrix (ECM) development and cell-cell junction in both Norwegian ewe breeds, which differed to the Irish ewe breeds. CONCLUSIONS: This novel study has identified a number of conserved and breed-specific biological processes under the effect of oestrous synchronisation that may impact cervical sperm transport during the follicular phase of the reproductive cycle.

Cervical immune activation during the luteal phase may compromise subsequent trans-cervical ram sperm transport†.

Worldwide, cervical artificial insemination using frozen-thawed semen yields low pregnancy rates. The only exception to this is in Norway, where vaginal insemination with frozen-thawed semen yields pregnancy rates in excess of 60% and which has been attributed to the specific ewe breed used. Our previous work demonstrated differences in cervical gene expression at the follicular phase of the estrous cycle in ewe breeds with known differences in pregnancy rates. In this study, we characterized the cervical transcriptome of the same ewe breeds [Suffolk, Belclare, Fur, and Norwegian White Sheep (NWS)] during the luteal phase, as an optimal environment at the luteal phase could better prepare the cervix for sperm migration through the cervix at the subsequent follicular phase. High-quality RNA extracted from postmortem cervical tissue was analyzed by RNA sequencing. After stringent filtering, 1051, 1924, and 611 differentially expressed genes (DEGs) were detected in the low-fertility Suffolk breed compared with Belclare, Fur, and NWS, respectively. Gene ontology analysis identified increased humoral adaptive immune response pathways in Suffolk. Increased expression of multiple immune genes supports the presence of an active immune response in the cervix of Suffolk ewes, which differentiates them significantly from the other three ewe breeds. Inflammatory pathways were upregulated in the Suffolk, resulting in higher expression of the potent pro-inflammatory cytokines. Therefore, higher levels of pro-inflammatory cytokines indicate unresolved inflammation in the cervix of the low-fertility Suffolk breed that could contribute to reduced cervical sperm transport in the next follicular phase.

Conserved and breed-specific differences in the cervical transcriptome of sheep with divergent fertility at the follicular phase of a natural oestrus cycle.

BACKGROUND: The outcome of cervical artificial insemination (AI) with frozen-thawed semen in sheep is limited by the inability of sperm to traverse the cervix of some ewe breeds. Previous research has demonstrated that cervical sperm transport is dependent on ewe breed, as sperm can traverse the cervix in greater numbers in some higher fertility ewe breeds. However, the molecular mechanisms underlying ewe breed differences in sperm transport through the cervix remain unknown. In this study, we aimed to characterise the cervical transcriptome of four European ewe breeds with known differences in pregnancy rates following cervical AI using frozen-thawed semen at the follicular phase of a natural oestrous cycle. Cervical post mortem tissue samples were collected from two Irish ewe breeds (Belclare and Suffolk; medium and low fertility, respectively) and from two Norwegian ewe breeds (Norwegian White Sheep (NWS) and Fur; high fertility compared to both Irish breeds) at the follicular phase of a natural oestrous cycle (n = 8 to 10 ewes per breed). RESULTS: High-quality RNA extracted from biopsies of the mid-region of the cervix was analysed by RNA-sequencing and Gene Ontology (GO). After stringent filtering (P <  0.05 and FC > 1.5), a total of 11, 1539 and 748 differentially expressed genes (DEGs) were identified in Belclare, Fur and NWS compared to the low fertility Suffolk breed, respectively. Gene ontology analysis identified significantly enriched biological processes involved in muscle contraction, extracellular matrix (ECM) development and the immune response. Gene co-expression analysis revealed similar patterns in muscle contraction and ECM development modules in both Norwegian ewe breeds, which differed to the Irish ewe breeds. CONCLUSIONS: These breed-specific biological processes may account for impaired cervical sperm transport through the cervix in sheep during the follicular phase of the reproductive cycle. This novel and comprehensive dataset provides a rich foundation for future targeted initiatives to improve cervical AI in sheep.

Functional analysis of bovine interleukin-10 receptor alpha in response to Mycobacterium avium subsp. paratuberculosis lysate using CRISPR/Cas9.

BACKGROUND: The interleukin-10 receptor alpha (IL10RA) gene codes for the alpha chain of the IL-10 receptor which binds the cytokine IL-10. IL-10 is an anti-inflammatory cytokine with immunoregulatory function during the pathogenesis of many inflammatory disorders in livestock, including Johne's disease (JD). JD is a chronic enteritis in cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is responsible for significant economic losses to the dairy industry. Several candidate genes including IL10RA have been found to be associated with JD. The aim of this study was to better understand the functional significance of IL10RA in the context of immune stimulation with MAP cell wall lysate. RESULTS: An IL10RA knock out (KO) bovine mammary epithelial cell (MAC-T) line was generated using the CRISPR/cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) gene editing system. These IL10RA KO cells were stimulated with the immune stimulant MAP lysate +/- IL-10, or with LPS as a positive control. In comparison to unedited cells, relative quantification of immune-related genes after stimulation revealed that knocking out IL10RA resulted in upregulation of pro-inflammatory cytokine gene expression (TNFA, IL1A, IL1B and IL6) and downregulation of suppressor of cytokine signaling 3 (SOCS3), a negative regulator of pro-inflammatory cytokine signaling. At the protein level knocking out IL10RA also resulted in upregulation of inflammatory cytokines - TNF-α and IL-6 and chemokines - IL-8, CCL2 and CCL4, relative to unedited cells. CONCLUSIONS: The findings of this study illustrate the broad and significant effects of knocking out the IL10RA gene in enhancing pro-inflammatory cytokine expression and further support the immunoregulatory role of IL10RA in eliciting an anti-inflammatory response as well as its potential functional involvement during the immune response associated with JD.

Association of genetic polymorphisms related to Johne's disease with estimated breeding values of Holstein sires for milk ELISA test scores.

BACKGROUND: Johne's disease (JD) is a chronic intestinal inflammatory disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) infection in ruminants. Since there are currently no effective vaccine or treatment options available to control JD, genetic selection may be an alternative strategy to enhance JD resistance. Numerous Single Nucleotide Polymorphisms (SNPs) have been reported to be associated with MAP infection status based on published genome-wide association and candidate gene studies. The main objective of this study was to validate these SNPs that were previously identified to be associated with JD by testing their effect on Holstein bulls' estimated breeding values (EBVs) for milk ELISA test scores, an indirect indicator of MAP infection status in cattle. RESULTS: Three SNPs, rs41810662, rs41617133 and rs110225854, located on Bos taurus autosomes (BTA) 16, 23 and 26, respectively, were confirmed as significantly associated with Holstein bulls' EBVs for milk ELISA test score (FDR < 0.01) based on General Quasi Likelihood Scoring analysis (GQLS) analysis. Single-SNP regression analysis identified four SNPs that were associated with sire EBVs (FDR < 0.05). This includes two SNPs that were common with GQLS (rs41810662 and rs41617133), with the other two SNPs being rs110494981 and rs136182707, located on BTA9 and BTA16, respectively. CONCLUSIONS: The findings of this study validate the association of SNPs with JD MAP infection status and highlight the need to further investigate the genomic regions harboring these SNPs.

Purulent vaginal discharge diagnosed in pasture-based Holstein-Friesian cows at 21 days postpartum is influenced by previous lactation milk yield and results in diminished fertility.

In a subset of dairy cows, prolonged pathological uterine inflammation results in purulent vaginal discharge (PVD), which can have negative consequences for both fertility and milk production. However, unlike for intensive systems, analysis of the effects of PVD in predominantly pasture-based herds is limited. The objective of this study was to assess the effect of PVD in spring-calving, pasture-based dairy cows on production and reproduction indices, stratified according to previous full-lactation milk yield. We assessed clinical disease as defined by vaginal mucus score (VMS) in 440 Holstein-Friesian cows from 5 farms. Cows were categorized as healthy (VMS 0) or having PVD (VMS 1-3) at 21 d postpartum. We recorded 305-d milk, milk protein, and milk fat yields (kg) before and after disease diagnosis, as well as fertility data, such as services per conception and the calving-conception period (CCP). Using SAS 9.4 (SAS Institute Inc., Cary, NC), we analyzed data using PROC MIXED, PROC PHREG, and PROC LOGISTIC to determine the least squares means differences and hazard and odds ratios between the groups, respectively. Overall, a 60% prevalence of PVD was recorded at 21 d postpartum. Milk yield and milk constituents were similar between all VMS categories and between healthy cows and cows with PVD. Although cows in the 4 VMS categories had statistically similar CCP, cows with PVD had a significantly longer CCP than healthy cows on average (9 d). The hazard ratio for cows with PVD was 0.66, indicating a 34% higher risk of a prolonged CCP than healthy cows. Odds ratio analysis determined that cows with PVD were 3 times more likely not to conceive at all, twice as likely not to conceive at first service, twice as likely not to conceive by 100 d postpartum, and 3 times more likely to fail to conceive before 150 d postpartum compared with healthy cows. Cows were retrospectively categorized as having low or high milk yield, based on whether they were above or below the median 305-d milk yield of the study population (6,571 kg) in the lactation before vaginal mucus scoring. Based on a univariate odds ratio, high-yield cows were 1.6 times more likely to present with PVD in the subsequent lactation. The number of services per conception did not differ between healthy and PVD cows in the low- and high-yield groups. In the high-yield group, cows with PVD were 4.9 times more likely not to conceive, 2.7 times more likely to require multiple services to conceive, 2.1 times more likely to remain not pregnant by 100 d postpartum, and 4.4 times more likely to remain not pregnant by 150 d postpartum. The CCP was also significantly longer in cows with PVD than their healthy counterparts (115.9 ± 4.9 and 104 ± 7.4 d, respectively). In conclusion, PVD significantly increased the CCP in all cows, but to a greater extent in cows with a high milk yield in the lactation before disease diagnosis.

Characterization of the bovine salivary gland transcriptome associated with Mycobacterium avium subsp. paratuberculosis experimental challenge.

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of Johne's disease is spread between cattle via the fecal-oral route, yet the functional changes in the salivary gland associated with infection remain uncharacterized. In this study, we hypothesized that experimental challenge with MAP would induce stable changes in gene expression patterns in the salivary gland that may shed light on the mucosal immune response as well as the regional variation in immune capacity of this extensive gland. Holstein-Friesian cattle were euthanized 33 months' post oral challenge with MAP strain CIT003 and both the parotid and mandibular salivary glands were collected from healthy control (n = 5) and MAP exposed cattle (n = 5) for histopathological and transcriptomic analysis. RESULTS: A total of 205, 21, 61, and 135 genes were significantly differentially expressed between control and MAP exposed cattle in dorsal mandibular (M1), ventral mandibular (M2), dorsal parotid (P1) and ventral parotid salivary glands (P2), respectively. Expression profiles varied between the structurally divergent parotid and mandibular gland sections which was also reflected in the enriched biological pathways identified. Changes in gene expression associated with MAP exposure were detected with significantly elevated expression of BoLA DR-ALPHA, BOLA-DRB3 and complement factors in MAP exposed cattle. In contrast, reduced expression of genes such as polymeric immunoglobin receptor (PIGR), TNFSF13, and the antimicrobial genes lactoferrin (LF) and lactoperoxidase (LPO) was detected in MAP exposed animals. CONCLUSIONS: This first analysis of the transcriptomic profile of salivary glands in cattle adds an important layer to our understanding of salivary gland immune function. Transcriptomic changes associated with MAP exposure have been identified including reduced LF and LPO. These critical antimicrobial and immunoregulatory proteins are known to be secreted into saliva and their downregulation may contribute to disease susceptibility. Future work will focus on the validation of their expression levels in saliva from additional cattle of known infection status as a potential strategy to augment disease diagnosis.

Improved detection of biomarkers in cervico-vaginal mucus (CVM) from postpartum cattle.

BACKGROUND: In the postpartum cow, early diagnosis of uterine disease is currently problematic due to the lack of reliable, non-invasive diagnostic methods. Cervico-vaginal mucus (CVM) is an easy to collect potentially informative source of biomarkers for the diagnosis and prognosis of uterine disease in cows. Here, we report an improved method for processing CVM from postpartum dairy cows for the measurement of immune biomarkers. CVM samples were collected from the vagina using gloved hand during the first two weeks postpartum and processed with buffer alone or buffer containing different concentrations of the reducing agents recommended in standard protocols: Dithiothriotol (DTT) or N-Acetyl-L-Cysteine (NAC). Total protein was measured using the bicinchoninic acid (BCA) assay; interleukin 6 (IL-6), IL-8 and α1-acid glycoprotein (AGP) were measured by ELISA. RESULTS: We found that use of reducing agents to liquefy CVM affects protein yield and the accuracy of biomarker detection. Our improved protocol results in lower protein yields but improved detection of cytokines and chemokines. Using our modified method to measure AGP in CVM we found raised levels of AGP at seven days postpartum in CVM from cows that went on to develop endometritis. CONCLUSION: We conclude that processing CVM without reducing agents improves detection of biomarkers that reflect uterine health in cattle. We propose that measurement of AGP in CVM during the first week postpartum may identify cows at risk of developing clinical endometritis.