Advertisement

Cows selected for divergent mastitis susceptibility display a differential liver transcriptome profile after experimental Staphylococcus aureus mammary gland inoculation

Open AccessPublished:April 16, 2020DOI:https://doi.org/10.3168/jds.2019-17612

      ABSTRACT

      Infection and inflammation of the mammary gland, and especially prevention of mastitis, are still major challenges for the dairy industry. Different approaches have been tried to reduce the incidence of mastitis. Genetic selection of cows with lower susceptibility to mastitis promises sustainable success in this regard. Bos taurus autosome (BTA) 18, particularly the region between 43 and 59 Mb, harbors quantitative trait loci (QTL) for somatic cell score, a surrogate trait for mastitis susceptibility. Scrutinizing the molecular bases hereof, we challenged udders from half-sib heifers having inherited either favorable paternal haplotypes for somatic cell score (Q) or unfavorable haplotypes (q) with the Staphylococcus aureus pathogen. RNA sequencing was used for an in-depth analysis of challenge-related alterations in the hepatic transcriptome. Liver exerts highly relevant immune functions aside from being the key metabolic organ. Hence, a holistic approach focusing on the liver enabled us to identify challenge-related and genotype-dependent differentially expressed genes and underlying regulatory networks. In response to the S. aureus challenge, we found that heifers with Q haplotypes displayed more activated immune genes and pathways after S. aureus challenge compared with their q half-sibs. Furthermore, we found a significant enrichment of differentially expressed loci in the genomic target region on BTA18, suggesting the existence of a regionally acting regulatory element with effects on a variety of genes in this region.

      Key words

      INTRODUCTION

      Rapid progress in molecular biology and genetics has made it possible to include information on genomic variance in modern animal breeding alternatively or complementary to the classical pedigree-based approach (
      • Meuwissen T.H.
      • Hayes B.J.
      • Goddard M.E.
      Prediction of total genetic value using genome-wide dense marker maps.
      ;
      • Matthews D.
      • Kearney J.F.
      • Cromie A.R.
      • Hely F.S.
      • Amer P.R.
      Genetic benefits of genomic selection breeding programmes considering foreign sire contributions.
      ). Facing the challenges of rising antimicrobial resistances and an increasing awareness of animal welfare issues, this approach is particularly interesting for improving animal health (e.g., in dairy cattle). Genomic selection for reduced mastitis susceptibility is a sustainable option to reduce disease incidence, but it requires profound knowledge on the underlying genetic loci modulating the trait. In a previous study,
      • Brand B.
      • Baes C.
      • Mayer M.
      • Reinsch N.
      • Kuhn C.
      Identification of a two-marker-haplotype on Bos taurus autosome 18 associated with somatic cell score in German Holstein cattle.
      identified QTL on BTA18 associated with SCS (calculated from the SCC) in the German Holstein population. The SCS strongly correlates with mastitis (
      • Weller J.I.
      • Saran A.
      • Zeliger Y.
      Genetic and environmental relationships among somatic cell count, bacterial infection, and clinical mastitis.
      ;
      • Rupp R.
      • Boichard D.
      Genetics of resistance to mastitis in dairy cattle.
      ), the infection and inflammation of the mammary gland, one of the most common infectious diseases in dairy cows (
      • Halasa T.
      • Huijps K.
      • Osteras O.
      • Hogeveen H.
      Economic effects of bovine mastitis and mastitis management: a review.
      ;
      • De Vliegher S.
      • Fox L.K.
      • Piepers S.
      • McDougall S.
      • Barkema H.W.
      Invited review: Mastitis in dairy heifers: Nature of the disease, potential impact, prevention, and control.
      ).
      • Kühn C.
      • Reinhardt F.
      • Schwerin M.
      Marker assisted selection of heifers improved milk somatic cell count compared to selection on conventional pedigree breeding values.
      described that including genetic marker haplotype information associated with the SCS QTL on BTA18 improved selection for a favorable SCS compared with selection restricted to solely conventional pedigree information. Recently, differences between alternative paternally inherited BTA18 haplotypes in clinical performance were confirmed for first lactating heifers before and after challenge with mastitis pathogens at the early lactation stage (
      • Heimes A.
      • Brodhagen J.
      • Weikard R.
      • Hammon H.M.
      • Meyerholz M.M.
      • Petzl W.
      • Zerbe H.
      • Engelmann S.
      • Schmicke M.
      • Hoedemaker M.
      • Schuberth H.J.
      • Kuhn C.
      Characterization of functional traits with focus on udder health in heifers with divergent paternally inherited haplotypes on BTA18.
      ;
      • Meyerholz M.M.
      • Rohmeier L.
      • Eickhoff T.
      • Hülsebusch A.
      • Jander S.
      • Linden M.
      • Macias L.
      • Koy M.
      • Heimes A.
      • Gorríz-Martín L.
      • Segelke D.
      • Engelmann S.
      • Schmicke M.
      • Hoedemaker M.
      • Petzl W.
      • Zerbe H.
      • Schuberth H.J.
      • Kühn C.
      Genetic selection for bovine chromosome 18 haplotypes associated with divergent somatic cell score affects postpartum reproductive and metabolic performance.
      ;
      • Rohmeier L.
      • Petzl W.
      • Koy M.
      • Eickhoff T.
      • Hülsebusch A.
      • Jander S.
      • Macias L.
      • Heimes A.
      • Engelmann S.
      • Hoedemaker M.
      • Seyfert H.M.
      • Kühn C.
      • Schuberth H.J.
      • Zerbe H.
      • Meyerholz M.M.
      In vivo model to study the impact of genetic variation on clinical outcome of mastitis in dairy heifers.
      ). These results are in line with a large number of studies reporting a major QTL for functional traits (SCS, mastitis, longevity) in the respective genomic region on BTA18 (
      • Brand B.
      • Baes C.
      • Mayer M.
      • Reinsch N.
      • Kuhn C.
      Identification of a two-marker-haplotype on Bos taurus autosome 18 associated with somatic cell score in German Holstein cattle.
      ,
      • Brand B.
      • Baes C.
      • Mayer M.
      • Reinsch N.
      • Seidenspinner T.
      • Thaller G.
      • Kuhn C.
      Quantitative trait loci mapping of calving and conformation traits on Bos taurus autosome 18 in the German Holstein population.
      ;
      • Mao X.
      • Kadri N.K.
      • Thomasen J.R.
      • De Koning D.J.
      • Sahana G.
      • Guldbrandtsen B.
      Fine mapping of a calving QTL on Bos taurus autosome 18 in Holstein cattle.
      ;
      • Müller M.P.
      • Rothammer S.
      • Seichter D.
      • Russ I.
      • Hinrichs D.
      • Tetens J.
      • Thaller G.
      • Medugorac I.
      Genome-wide mapping of 10 calving and fertility traits in Holstein dairy cattle with special regard to chromosome 18.
      ;
      • Wu X.P.
      • Guldbrandtsen B.
      • Nielsen U.S.
      • Lund M.S.
      • Sahana G.
      Association analysis for young stock survival index with imputed whole-genome sequence variants in Nordic Holstein cattle.
      ;
      • Fang L.
      • Jiang J.
      • Li B.
      • Zhou Y.
      • Freebern E.
      • Vanraden P.M.
      • Cole J.B.
      • Liu G.E.
      • Ma L.
      Genetic and epigenetic architecture of paternal origin contribute to gestation length in cattle.
      ). Hence, information on BTA18 haplotypes in Holstein cattle breeding could contribute to improved animal health and particularly to reduced susceptibility to mastitis in the German Holstein cattle population. However, haplotype effects need to be first characterized more precisely to exclude potential detrimental side effects. Furthermore, a deep phenotypic evaluation of potential BTA18 haplotype effects could also shed light on the precise causal molecular background of the QTL localized in this chromosomal region. The genetic variation underlying this major QTL is still unclear in spite of very powerful analyses and promising candidate genes (
      • Fang L.
      • Jiang J.
      • Li B.
      • Zhou Y.
      • Freebern E.
      • Vanraden P.M.
      • Cole J.B.
      • Liu G.E.
      • Ma L.
      Genetic and epigenetic architecture of paternal origin contribute to gestation length in cattle.
      ;
      • Jiang J.
      • Cole J.B.
      • Freebern E.
      • Da Y.
      • VanRaden P.M.
      • Ma L.
      Functional annotation and Bayesian fine-mapping reveals candidate genes for important agronomic traits in Holstein bulls.
      ). The liver is at the center of the metabolic and immunological physiology of the dairy cow (
      • Moyes K.M.
      • Sørensen P.
      • Bionaz M.
      The impact of intramammary Escherichia coli challenge on liver and mammary transcriptome and cross-talk in dairy cows during early lactation using RNAseq.
      ). Hence, we focused in this study on the hepatic transcriptome using RNA sequencing (RNAseq) to gain comprehensive information about immunological as well as metabolic differences between divergent haplotypes at the transcriptomic level. Therefore, we have looked at the effects of an intramammary challenge with a mastitis pathogen on the hepatic transcriptome of half-sib cows, which had inherited either favorable or unfavorable paternal haplotypes. The challenge was performed with Staphylococcus aureus strain 1027, commonly known as a causative pathogen for subclinical mastitis (
      • Schukken Y.H.
      • Gunther J.
      • Fitzpatrick J.
      • Fontaine M.C.
      • Goetze L.
      • Holst O.
      • Leigh J.
      • Petzl W.
      • Schuberth H.J.
      • Sipka A.
      • Smith D.G.
      • Quesnell R.
      • Watts J.
      • Yancey R.
      • Zerbe H.
      • Gurjar A.
      • Zadoks R.N.
      • Seyfert H.M.
      Host-response patterns of intramammary infections in dairy cows.
      ;
      • Jensen K.
      • Günther J.
      • Talbot R.
      • Petzl W.
      • Zerbe H.
      • Schuberth H.-J.
      • Seyfert H.-M.
      • Glass E.J.
      Escherichia coli- and Staphylococcus aureus-induced mastitis differentially modulate transcriptional responses in neighbouring uninfected bovine mammary gland quarters.
      ).

      MATERIALS AND METHODS

      Selection Process to Establish Experimental Animal Cohorts

      The selection process was described in detail by
      • Heimes A.
      • Brodhagen J.
      • Weikard R.
      • Hammon H.M.
      • Meyerholz M.M.
      • Petzl W.
      • Zerbe H.
      • Engelmann S.
      • Schmicke M.
      • Hoedemaker M.
      • Schuberth H.J.
      • Kuhn C.
      Characterization of functional traits with focus on udder health in heifers with divergent paternally inherited haplotypes on BTA18.
      and
      • Meyerholz M.M.
      • Rohmeier L.
      • Eickhoff T.
      • Hülsebusch A.
      • Jander S.
      • Linden M.
      • Macias L.
      • Koy M.
      • Heimes A.
      • Gorríz-Martín L.
      • Segelke D.
      • Engelmann S.
      • Schmicke M.
      • Hoedemaker M.
      • Petzl W.
      • Zerbe H.
      • Schuberth H.J.
      • Kühn C.
      Genetic selection for bovine chromosome 18 haplotypes associated with divergent somatic cell score affects postpartum reproductive and metabolic performance.
      . Based on previous studies (
      • Kühn C.
      • Reinhardt F.
      • Schwerin M.
      Marker assisted selection of heifers improved milk somatic cell count compared to selection on conventional pedigree breeding values.
      ;
      • Brand B.
      • Baes C.
      • Mayer M.
      • Reinsch N.
      • Kuhn C.
      Identification of a two-marker-haplotype on Bos taurus autosome 18 associated with somatic cell score in German Holstein cattle.
      ), the targeted haplotypes were allocated to 2 BTA18 sub-regions (43–48 and 53–59 Mb). As presented earlier (
      • Meyerholz M.M.
      • Rohmeier L.
      • Eickhoff T.
      • Hülsebusch A.
      • Jander S.
      • Linden M.
      • Macias L.
      • Koy M.
      • Heimes A.
      • Gorríz-Martín L.
      • Segelke D.
      • Engelmann S.
      • Schmicke M.
      • Hoedemaker M.
      • Petzl W.
      • Zerbe H.
      • Schuberth H.J.
      • Kühn C.
      Genetic selection for bovine chromosome 18 haplotypes associated with divergent somatic cell score affects postpartum reproductive and metabolic performance.
      ) for all 11,503 German Holstein AI sires born between 1999 and 2012 and recorded in the VIT genome database, we calculated SNP effects for divergent paternal haplotypes in the respective chromosomal regions (Q, meaning favorable for SCS, and q, meaning unfavorable for SCS). We selected those sires with differences of summarized SNP effects of at least 2 standard deviations larger than the mean with the assumption that those sires should be segregating for a Q or q haplotype. We excluded sires with extreme breeding values for SCS and milk performance traits to achieve similar performance levels within half-sib groups. The maternal grandsires were selected for their breeding values for SCS (Relativzuchtwert Somatischer Zellgehalt, above 112 for the Q cohort and below 100 for the q cohort). Moreover, we searched for cohorts with at least 3 potential Q and q half-sib sisters within one sire, respectively, and a maximal calving age of 36 mo. After all these filtering steps, a total of 282 heifers were genotyped with the 50k Illumina SNP chip (Illumina Inc., San Diego, CA) and haplotyped for their inherited paternal haplotypes (Q or q). Finally, 24 healthy, pregnant heifers, which originated from 6 sires, were selected. Within sire, each of the heifers was allocated to the Q or q group according to the inherited SNP haplotypes, which enabled monitoring of alternative paternally inherited haplotypes in the analysis. The selected heifers were purchased from conventional dairy farms across Germany and brought to the Clinic for Cattle at the University of Veterinary Medicine Hannover (
      • Meyerholz M.M.
      • Rohmeier L.
      • Eickhoff T.
      • Hülsebusch A.
      • Jander S.
      • Linden M.
      • Macias L.
      • Koy M.
      • Heimes A.
      • Gorríz-Martín L.
      • Segelke D.
      • Engelmann S.
      • Schmicke M.
      • Hoedemaker M.
      • Petzl W.
      • Zerbe H.
      • Schuberth H.J.
      • Kühn C.
      Genetic selection for bovine chromosome 18 haplotypes associated with divergent somatic cell score affects postpartum reproductive and metabolic performance.
      ).

      Challenge Experiment

      Twenty-four animals (12 Q, 12 q) were challenged in an infection model in the Clinic for Cattle at the University of Veterinary Medicine Hannover essentially as described in detail in
      • Rohmeier L.
      • Petzl W.
      • Koy M.
      • Eickhoff T.
      • Hülsebusch A.
      • Jander S.
      • Macias L.
      • Heimes A.
      • Engelmann S.
      • Hoedemaker M.
      • Seyfert H.M.
      • Kühn C.
      • Schuberth H.J.
      • Zerbe H.
      • Meyerholz M.M.
      In vivo model to study the impact of genetic variation on clinical outcome of mastitis in dairy heifers.
      . The husbandry of these cows was previously described by
      • Meyerholz M.M.
      • Rohmeier L.
      • Eickhoff T.
      • Hülsebusch A.
      • Jander S.
      • Linden M.
      • Macias L.
      • Koy M.
      • Heimes A.
      • Gorríz-Martín L.
      • Segelke D.
      • Engelmann S.
      • Schmicke M.
      • Hoedemaker M.
      • Petzl W.
      • Zerbe H.
      • Schuberth H.J.
      • Kühn C.
      Genetic selection for bovine chromosome 18 haplotypes associated with divergent somatic cell score affects postpartum reproductive and metabolic performance.
      . Briefly, during the challenge experiment, the animals were kept in individual loose stall pens and received a component diet based on grass silage, corn silage, rapeseed extraction meal, soy extraction meal, concentrates, and minerals adjusted to milk performance. In the pre-challenge period, the animals were closely monitored for general health status and specific indicators of mastitis as described in
      • Rohmeier L.
      • Petzl W.
      • Koy M.
      • Eickhoff T.
      • Hülsebusch A.
      • Jander S.
      • Macias L.
      • Heimes A.
      • Engelmann S.
      • Hoedemaker M.
      • Seyfert H.M.
      • Kühn C.
      • Schuberth H.J.
      • Zerbe H.
      • Meyerholz M.M.
      In vivo model to study the impact of genetic variation on clinical outcome of mastitis in dairy heifers.
      . The experiment was performed under the reference number 33.12-42502-04-15/2024 with approval by the Lower Saxony Federal State Office for Consumer Protection and Food Safety. Furthermore, this study was approved by the ethics committee of the University of Veterinary Medicine Hannover. All ethical evaluations were performed as required by the German Animal Care law ().
      The intramammary challenge experiment was based on previous work by our research group regarding selection of challenge dose and sampling time (
      • Petzl W.
      • Zerbe H.
      • Gunther J.
      • Yang W.
      • Seyfert H.M.
      • Nurnberg G.
      • Schuberth H.J.
      Escherichia coli, but not Staphylococcus aureus triggers an early increased expression of factors contributing to the innate immune defense in the udder of the cow.
      ,
      • Petzl W.
      • Gunther J.
      • Pfister T.
      • Sauter-Louis C.
      • Goetze L.
      • von Aulock S.
      • Hafner-Marx A.
      • Schuberth H.J.
      • Seyfert H.M.
      • Zerbe H.
      Lipopolysaccharide pretreatment of the udder protects against experimental Escherichia coli mastitis.
      ). Thirty-six ± 3 d after parturition, 24 healthy animals (12 Q, 12 q) were challenged with 10,000 cfu of S. aureus1027 each in both hind quarters of the mammary gland and killed 96 h later. A control udder quarter was infused with sterile sodium chloride solution. Heifers were closely monitored for clinical signs of mastitis [e.g., declining milk yield, local (pain, swelling, redness) or systemic (fever) signs of inflammation] during the postchallenge period as described by
      • Rohmeier L.
      • Petzl W.
      • Koy M.
      • Eickhoff T.
      • Hülsebusch A.
      • Jander S.
      • Macias L.
      • Heimes A.
      • Engelmann S.
      • Hoedemaker M.
      • Seyfert H.M.
      • Kühn C.
      • Schuberth H.J.
      • Zerbe H.
      • Meyerholz M.M.
      In vivo model to study the impact of genetic variation on clinical outcome of mastitis in dairy heifers.
      . The animals were stunned with a penetrating captive bolt pistol, immediately followed by exsanguination via longitudinal section of the jugular veins and carotid arteries 96 h after the start of the challenge (
      • Meyerholz M.M.
      • Rohmeier L.
      • Eickhoff T.
      • Hülsebusch A.
      • Jander S.
      • Linden M.
      • Macias L.
      • Koy M.
      • Heimes A.
      • Gorríz-Martín L.
      • Segelke D.
      • Engelmann S.
      • Schmicke M.
      • Hoedemaker M.
      • Petzl W.
      • Zerbe H.
      • Schuberth H.J.
      • Kühn C.
      Genetic selection for bovine chromosome 18 haplotypes associated with divergent somatic cell score affects postpartum reproductive and metabolic performance.
      ). The time point was selected because it was predicted to be the zenith of mammary gland inflammation according to experience from previous experiments (
      • Petzl W.
      • Zerbe H.
      • Gunther J.
      • Yang W.
      • Seyfert H.M.
      • Nurnberg G.
      • Schuberth H.J.
      Escherichia coli, but not Staphylococcus aureus triggers an early increased expression of factors contributing to the innate immune defense in the udder of the cow.
      ,
      • Petzl W.
      • Gunther J.
      • Pfister T.
      • Sauter-Louis C.
      • Goetze L.
      • von Aulock S.
      • Hafner-Marx A.
      • Schuberth H.J.
      • Seyfert H.M.
      • Zerbe H.
      Lipopolysaccharide pretreatment of the udder protects against experimental Escherichia coli mastitis.
      ). During dissection of the animals, liver tissue was collected from the lobus caudatus, immediately shock frozen in liquid nitrogen, and subsequently stored at −80°C. About 6 mo after sampling, the tissue was used for transcriptome analysis as described below.

      Transcriptome Analysis by RNA Sequencing

      Frozen liver tissue samples (approximately 30 mg) were ground using the Precellys 24 tissue homogenizer with a lysing kit containing 1.4-mm ceramic beads (peQLab, Erlangen, Germany). Total RNA was extracted via an on-column purification following the protocol of the NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany), with an adapted DNase digestion step as described by
      • Weikard R.
      • Goldammer T.
      • Brunner R.M.
      • Kuehn C.
      Tissue-specific mRNA expression patterns reveal a coordinated metabolic response associated with genetic selection for milk production in cows.
      . The total RNA was controlled for presence of genomic DNA by PCR (
      • Weikard R.
      • Goldammer T.
      • Eberlein A.
      • Kuehn C.
      Novel transcripts discovered by mining genomic DNA from defined regions of bovine chromosome 6.
      ), and a second DNase digestion step was added if required. The RNA concentration and purity were quantified on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and a Qubit 2.0 fluorometer (Thermo Fisher Scientific), and RNA integrity was evaluated on the Bioanalyzer 2100 (Agilent Technologies, Böblingen, Germany). Subsequently, a stranded library preparation protocol for RNA sequencing was applied (TruSeq Stranded mRNA LP, Illumina) with application of indices for multiplexing during cluster generation and polyA-selection to focus on polyadenylated RNA (in the majority mRNA). The RNAseq libraries were checked for quality on the Bioanalyzer 2100. Using the Illumina HiSeq 2500 system (Illumina), we performed paired-end sequencing (2 × 90 bp).

      Bioinformatic Analysis

      The CASAVA v1.8 (Illumina) software was used for demultiplexing of reads. Scripts written in SAMtools (
      • Li H.
      • Handsaker B.
      • Wysoker A.
      • Fennell T.
      • Ruan J.
      • Homer N.
      • Marth G.
      • Abecasis G.
      • Durbin R.
      The Sequence Alignment/Map format and SAMtools.
      ), Linux, and R (
      • R
      ) were applied for data processing. We checked the quality of the raw reads with FastQC version 0.11.5 () and MultiQC version 1.4 (
      • MultiQC
      ). Adapters were removed using Cutadapt version 1.12 (
      • Martin M.
      Cutadapt removes adapter sequences from high-throughput sequencing reads.
      ) and low-quality bases were removed using QualityTrim (
      • Qualitytrim
      ). The reads were aligned to the bovine reference genome UMD 3.1 with Ensembl 87 reference annotation (
      UMD3.1.
      ) using Hisat2 version 2.1.0 (
      • Pertea M.
      • Kim D.
      • Pertea G.M.
      • Leek J.T.
      • Salzberg S.L.
      Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown.
      ). A guided transcript assembly was generated with StringTie version 1.3.2.d (
      • Pertea M.
      • Kim D.
      • Pertea G.M.
      • Leek J.T.
      • Salzberg S.L.
      Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown.
      ). The advantage of this approach is that a guided transcript assembly refers to the reference genome annotation and also enables the identification of transcripts, which have not yet been annotated. Using the StringTie (
      • Pertea M.
      • Kim D.
      • Pertea G.M.
      • Leek J.T.
      • Salzberg S.L.
      Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown.
      ) merge function, an annotation across samples was generated and read counting was carried out with FeatureCounts version 1.5.2 (
      • FeatureCounts
      ). Differential expression analysis was performed with DESeq2 version 1.18.1 (
      • Love M.I.
      • Huber W.
      • Anders S.
      Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
      ) with a threshold for significance of adjusted P (Padj) < 0.05. Ingenuity Pathway Analysis (Qiagen, Hilden, Germany) was used to identify enriched biological pathways and predicted upstream regulators in response to a pathogen challenge.
      During the selection process for Q and q heifers, the cholesterol deficiency (CD) defect was detected in the German Holstein population (
      • Kipp S.
      • Segelke D.
      • Schierenbeck S.
      • Reinhardt F.
      • Reents R.
      • Wurmser C.
      • Pausch H.
      • Fries R.
      • Thaller G.
      • Tetens J.
      • Pott J.
      • Haas D.
      • Raddatz B.B.
      • Hewicker-Trautwein M.
      • Proios I.
      • Schmicke M.
      • Grunberg W.
      Identification of a haplotype associated with cholesterol deficiency and increased juvenile mortality in Holstein cattle.
      ;
      • Menzi F.
      • Besuchet-Schmutz N.
      • Fragniere M.
      • Hofstetter S.
      • Jagannathan V.
      • Mock T.
      • Raemy A.
      • Studer E.
      • Mehinagic K.
      • Regenscheit N.
      • Meylan M.
      • Schmitz-Hsu F.
      • Drogemuller C.
      A transposable element insertion in APOB causes cholesterol deficiency in Holstein cattle.
      ). Hence, before final heifer selection, their CD carrier status was determined by haplotype analysis (
      • Kipp S.
      • Segelke D.
      • Schierenbeck S.
      • Reinhardt F.
      • Reents R.
      • Wurmser C.
      • Pausch H.
      • Fries R.
      • Thaller G.
      • Tetens J.
      • Pott J.
      • Haas D.
      • Raddatz B.B.
      • Hewicker-Trautwein M.
      • Proios I.
      • Schmicke M.
      • Grunberg W.
      Identification of a haplotype associated with cholesterol deficiency and increased juvenile mortality in Holstein cattle.
      ). In the end, 4 heterozygous CD carriers (CDC according to the World Holstein Friesian Federation, http://www.whff.info/documentation/genetictraits.php#go1) were included in the design, which were offspring of a single sire. The CDC heifers were distributed evenly across the Q and q groups. The CD carrier status was included as fixed effect in the differential expression data analysis.

      RESULTS

      RNA Sequencing Statistics

      The transcriptome analysis by RNAseq generated 2.6 billion reads (on average 107 million reads per sample). A total of 98% of reads mapped at least once to the reference genome UMD3.1 (
      UMD3.1.
      ). The data analysis revealed a total of 20,723 loci, which showed an expression level of at least 10 reads in at least 4 samples.

      Differential Hepatic Transcriptome Expression in Animals Infected with S. aureus

      In the expression analysis comparing S. aureus challenged animals that had either inherited the paternal haplotypes Q (n = 12) or q (n = 12), we found a total of 23 significantly (Padj < 0.05) differentially expressed (DE) loci (see Table 1 and Supplemental File S1; https://doi.org/10.3168/jds.2019-17612). Of these 23 significantly DE loci, 17 were annotated genes (74%), with 12 and 5 genes expressed at a higher and lower level, respectively, in Q compared with q animals.
      Table 1All significantly (adjusted P-value, Padj < 0.05) differentially expressed annotated genes in the differential expression analysis between paternally inherited haplotypes Q (favorable for SCS) versus q (unfavorable for SCS) of animals challenged with Staphylococcus aureus
      Gene symbolEntrez gene nameFunctionLog2 fold changePadj
      DYSFDysferlinRegulates cell adhesion in monocytes (
      • de Morrée A.
      • Flix B.
      • Bagaric I.
      • Wang J.
      • van den Boogaard M.
      • Grand Moursel L.
      • Frants R.R.
      • Illa I.
      • Gallardo E.
      • Toes R.
      • van der Maarel S.M.
      Dysferlin regulates cell adhesion in human monocytes.
      )
      0.360.015
      MS4A3Membrane spanning 4-domains A3Involved in cell cycle control (
      • Kutok J.L.
      • Yang X.
      • Folkerth R.
      • Adra C.N.
      Characterization of the expression of HTm4 (MS4A3), a cell cycle regulator, in human peripheral blood cells and normal and malignant tissues.
      )
      4.430.017
      GMIPGEM interacting proteinInvolved in vesicular transport and exocytosis (
      • Johnson J.L.
      • Monfregola J.
      • Napolitano G.
      • Kiosses W.B.
      • Catz S.D.
      Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GTPase-activating protein Gem-interacting protein.
      )
      0.460.017
      HMOX1Heme oxygenase 1Immunomodulatory and anti-inflammatory functions (
      • Naito Y.
      • Takagi T.
      • Higashimura Y.
      Heme oxygenase-1 and anti-inflammatory M2 macrophages.
      )
      0.970.019
      GPBAR1G protein-coupled bile acid receptor 1Involved in bile acid homeostasis and liver immunity (
      • Biagioli M.
      • Carino A.
      • Fiorucci C.
      • Marchianò S.
      • Di Giorgio C.
      • Roselli R.
      • Magro M.
      • Distrutti E.
      • Bereshchenko O.
      • Scarpelli P.
      • Zampella A.
      • Fiorucci S.
      GPBAR1 functions as gatekeeper for liver NKT cells and provides counterregulatory signals in mouse models of immune-mediated hepatitis.
      )
      0.580.019
      MARCH5Membrane associated ring-CH-type finger 5Involved in the regulation of mitochondrial morphology (
      • Tang H.
      • Peng S.
      • Dong Y.
      • Yang X.
      • Yang P.
      • Yang L.
      • Yang B.
      • Bao G.
      MARCH5 overexpression contributes to tumor growth and metastasis and associates with poor survival in breast cancer.
      )
      −0.260.019
      IGSF11Immunoglobulin superfamily member 11Inhibits T cell function (
      • Wang J.
      • Wu G.
      • Manick B.
      • Hernandez V.
      • Renelt M.
      • Erickson C.
      • Guan J.
      • Singh R.
      • Rollins S.
      • Solorz A.
      • Bi M.
      • Li J.
      • Grabowski D.
      • Dirkx J.
      • Tracy C.
      • Stuart T.
      • Ellinghuysen C.
      • Desmond D.
      • Foster C.
      • Kalabokis V.
      VSIG-3 as a ligand of VISTA inhibits human T-cell function.
      )
      −0.410.019
      FERMT2Fermitin family member 2Involved in TGFB1-, integrin-, Erk/MAPK-signaling pathway (
      • Wan C.
      • Borgeson B.
      • Phanse S.
      • Tu F.
      • Drew K.
      • Clark G.
      • Xiong X.
      • Kagan O.
      • Kwan J.
      • Bezginov A.
      • Chessman K.
      • Pal S.
      • Cromar G.
      • Papoulas O.
      • Ni Z.
      • Boutz D.R.
      • Stoilova S.
      • Havugimana P.C.
      • Guo X.
      • Malty R.H.
      • Sarov M.
      • Greenblatt J.
      • Babu M.
      • Derry W.B.
      • Tillier E.R.
      • Wallingford J.B.
      • Parkinson J.
      • Marcotte E.M.
      • Emili A.
      Panorama of ancient metazoan macromolecular complexes.
      ;
      • Rognoni E.
      • Ruppert R.
      • Fässler R.
      The kindlin family: Functions, signaling properties and implications for human disease.
      )
      −0.290.020
      ICAM3Intercellular adhesion molecule 3Chemotactic for macrophages, involved in phagocytosis (
      • Torr E.E.
      • Gardner D.H.
      • Thomas L.
      • Goodall D.M.
      • Bielemeier A.
      • Willetts R.
      • Griffiths H.R.
      • Marshall L.J.
      • Devitt A.
      Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells.
      )
      0.530.022
      PLCB2Phospholipase C β 2Inhibits expression of pro-inflammatory cytokines, regulates macrophage function (
      • Grinberg S.
      • Hasko G.
      • Wu D.
      • Leibovich S.J.
      Suppression of PLCbeta2 by endotoxin plays a role in the adenosine A(2A) receptor-mediated switch of macrophages from an inflammatory to an angiogenic phenotype.
      ;
      • Wang L.
      • Zhou Y.
      • Chen Z.
      • Sun L.
      • Wu J.
      • Li H.
      • Liu F.
      • Wang F.
      • Yang C.
      • Yang J.
      • Leng Q.
      • Zhang Q.
      • Xu A.
      • Shen L.
      • Sun J.
      • Wu D.
      • Fang C.
      • Lu H.
      • Yan D.
      • Ge B.
      PLCbeta2 negatively regulates the inflammatory response to virus infection by inhibiting phosphoinositide-mediated activation of TAK1.
      )
      0.500.022
      TNFAIP8L2TNF α-induced protein 8 like 2Negative immune regulator (
      • Lin Z.
      • Liu W.
      • Xiao C.
      • Fan Y.
      • Zhuang G.
      • Qi Z.
      TIPE2 inhibits GC via regulation of cell proliferation, apoptosis and inflammation.
      )
      0.470.031
      DNASE2Deoxyribonuclease 2, lysosomalClears the cell of damaged DNA (
      • Hacohen N.
      • Lan Y.Y.
      Damaged DNA marching out of aging nucleus.
      )
      0.430.037
      RNASE6Ribonuclease A family member k6Expressed by monocytes and neutrophils, antimicrobial activity (
      • Pulido D.
      • Arranz-Trullen J.
      • Prats-Ejarque G.
      • Velazquez D.
      • Torrent M.
      • Moussaoui M.
      • Boix E.
      Insights into the antimicrobial mechanism of action of human RNase6: Structural determinants for bacterial cell agglutination and membrane permeation.
      )
      0.530.046
      ARRDC3Arrestin domain containing 3Involved in placental development and pathogenesis of cancer (
      • Lei D.
      • Deng N.
      • Wang S.
      • Huang J.
      • Fan C.
      Upregulated ARRDC3 limits trophoblast cell invasion and tube formation and is associated with preeclampsia.
      )
      −0.630.046
      TNCTenascin CActivator of innate immunity, upregulated as response to inflammation (
      • Goh F.G.
      • Piccinini A.M.
      • Krausgruber T.
      • Udalova I.A.
      • Midwood K.S.
      Transcriptional regulation of the endogenous danger signal tenascin-C: A novel autocrine loop in inflammation.
      )
      2.460.048
      ZNF227Zinc finger protein 227Function unknown0.290.048
      PPP2R3AProtein phosphatase 2 regulatory subunit B''αMajor cellular Ser/Thr protein phosphatase, known for involvement in liver cancer (
      • Chen H.
      • Xu J.
      • Wang P.
      • Shu Q.
      • Huang L.
      • Guo J.
      • Zhang X.
      • Zhang H.
      • Wang Y.
      • Shen Z.
      • Chen X.
      • Zhang Q.
      Protein phosphatase 2 regulatory subunit B′'Alpha silencing inhibits tumor cell proliferation in liver cancer.
      )
      −0.420.048
      Within the 17 annotated DE genes between paternally inherited haplotypes Q versus q of S. aureus challenged animals 10 genes are listed, which are involved in immune response [e.g., IGSF11 with known effects on human T cells (
      • Wang J.
      • Wu G.
      • Manick B.
      • Hernandez V.
      • Renelt M.
      • Erickson C.
      • Guan J.
      • Singh R.
      • Rollins S.
      • Solorz A.
      • Bi M.
      • Li J.
      • Grabowski D.
      • Dirkx J.
      • Tracy C.
      • Stuart T.
      • Ellinghuysen C.
      • Desmond D.
      • Foster C.
      • Kalabokis V.
      VSIG-3 as a ligand of VISTA inhibits human T-cell function.
      ) and ICAM3, which is involved in apoptotic cell clearance (
      • Torr E.E.
      • Gardner D.H.
      • Thomas L.
      • Goodall D.M.
      • Bielemeier A.
      • Willetts R.
      • Griffiths H.R.
      • Marshall L.J.
      • Devitt A.
      Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells.
      )]. However, the top scorer of DE loci is a yet unannotated transcript on BTA18 located 2 Mb outside the genomic target region for our haplotype selection (see Supplemental File S1). Another locus localized on BTA18 at 48.9 Mb (according the UMD3.1) and still unannotated at the respective position in the current genome annotation ARS-UCD1.2 Ensembl 97 (https://www.ensembl.org/Bos_taurus/Info/Index) showed significant differential expression (see Supplemental File S1). The respective transcript does also not show any sequence homology to annotated genes in human or mouse genomes. Three DE (Padj < 0.05) loci are localized in the genomic region spanning 43 to 59 Mb containing the targeted chromosomal subregions for haplotype selection on BTA18. Within this region, a total of 525 loci are expressed in our data set. Thus, compared with the total number of 23 DE loci across all 20,724 expressed genomic loci in the entire genome there is a significant enrichment (P < 0.002) of DE loci in the BTA18 genomic region 43 to 59 Mb.

      Pathway Analysis of DE Genes in Animals Infected with S. aureus

      Ingenuity Pathway Analysis of those 17 DE loci, which are annotated in the bovine reference genome assembly (UMD3.1), provided 24 significantly (P < 0.05) enriched canonical pathways when comparing S. aureus challenged animals that had either inherited the paternal haplotypes Q or q (see Table 2 and Supplemental File S2; https://doi.org/10.3168/jds.2019-17612). Among them are the pathways integrin-linked protein kinase (ILK) signaling, IL-8 signaling, phospholipases, and phospholipase C signaling that are known to be directly related to immune response. The GPCR-mediated nutrient sensing in enteroendocrine cells, which was also significantly enriched between the paternal haplotypes Q or q, provides a link between the immune response and the dairy cow's metabolism (
      • Husted A.S.
      • Trauelsen M.
      • Rudenko O.
      • Hjorth S.A.
      • Schwartz T.W.
      GPCR-mediated signaling of metabolites.
      ;
      • Latorraca N.R.
      • Venkatakrishnan A.J.
      • Dror R.O.
      GPCR dynamics: Structures in motion.
      ).
      Table 2All significantly (P < 0.05) canonical pathways enriched in the hepatic transcriptome of animals challenged with Staphylococcus aureus with haplotypes Q (favorable for SCS) versus q (unfavorable for SCS) using Ingenuity Pathway Analysis (Qiagen, Hilden, Germany)
      Ingenuity canonical pathway−log10 (P-value)
      Phospholipases2.94E00
      Antioxidant action of vitamin C2.55E00
      GPCR-mediated nutrient sensing in enteroendocrine cells2.52E00
      p70S6K signaling2.41E00
      D-myo-inositol-5-phosphate metabolism2.25E00
      Gαq signaling2.24E00
      Dopamine-DARPP32 feedback in cAMP signaling2.18E00
      Heme degradation2.12E00
      ILK signaling2.11E00
      Synaptic long-term depression2.09E00
      Endothelin-1 signaling2.07E00
      IL-8 signaling2.06E00
      Breast cancer regulation by Stathmin12.05E00
      mTOR signaling2.01E00
      Superpathway of inositol phosphate compounds1.99E00
      Phospholipase C signaling1.84E00
      Choline biosynthesis III1.78E00
      Xenobiotic metabolism signaling1.77E00
      G Protein signaling mediated by tubby1.63E00
      D-myo-inositol (1,4,5)-trisphosphate biosynthesis1.62E00
      Cell cycle regulation by BTG family proteins1.60E00
      Role of CHK proteins in cell cycle checkpoint control1.41E00
      Wnt/Ca+ pathway1.35E00
      Mitotic roles of polo-like kinase1.35E00
      The analysis of the predicted upstream regulators (see Table 3) confirmed modulation of the hepatic immune system in Q heifers compared with the q half-sibs within S. aureus challenged animals. In the list of the top 10 significantly enriched upstream regulators (Table 3), 2 important immune receptors (CXCR3, C-X-C chemokine receptor type 3, and TLR3, toll-like receptor 3) as well as mitogen-activated protein kinase kinase kinase 1 (MAP3K1), a kinase involved in the TNF-α and NFκB signal cascades (
      • Ishizuka T.
      • Terada N.
      • Gerwins P.
      • Hamelmann E.
      • Oshiba A.
      • Fanger G.R.
      • Johnson G.L.
      • Gelfand E.W.
      Mast cell tumor necrosis factor alpha production is regulated by MEK kinases.
      ), were found.
      Table 3Top 10 significantly (P < 0.05) enriched upstream regulators in Ingenuity Pathway Analysis (Qiagen, Hilden, Germany) of haplotype Q (favorable for SCS) versus q (unfavorable for SCS) within animals challenged with Staphylococcus aureus (exogenous chemical excluded)
      Upstream regulatorP-value of overlap
      GCH12.79E-05
      SRF1.03E-04
      CXCR31.51E-04
      KDM3A4.10E-04
      GABPA4.51E-04
      FBXL177.35E-04
      BRIP17.35E-04
      MAP3K17.66E-04
      TLR38.89E-04
      CAT9.68E-04

      DISCUSSION

      The heifers investigated in this study had been deeply phenotyped before and after the intramammary pathogen challenge as reported by
      • Heimes A.
      • Brodhagen J.
      • Weikard R.
      • Hammon H.M.
      • Meyerholz M.M.
      • Petzl W.
      • Zerbe H.
      • Engelmann S.
      • Schmicke M.
      • Hoedemaker M.
      • Schuberth H.J.
      • Kuhn C.
      Characterization of functional traits with focus on udder health in heifers with divergent paternally inherited haplotypes on BTA18.
      ,
      • Meyerholz M.M.
      • Rohmeier L.
      • Eickhoff T.
      • Hülsebusch A.
      • Jander S.
      • Linden M.
      • Macias L.
      • Koy M.
      • Heimes A.
      • Gorríz-Martín L.
      • Segelke D.
      • Engelmann S.
      • Schmicke M.
      • Hoedemaker M.
      • Petzl W.
      • Zerbe H.
      • Schuberth H.J.
      • Kühn C.
      Genetic selection for bovine chromosome 18 haplotypes associated with divergent somatic cell score affects postpartum reproductive and metabolic performance.
      , and
      • Rohmeier L.
      • Petzl W.
      • Koy M.
      • Eickhoff T.
      • Hülsebusch A.
      • Jander S.
      • Macias L.
      • Heimes A.
      • Engelmann S.
      • Hoedemaker M.
      • Seyfert H.M.
      • Kühn C.
      • Schuberth H.J.
      • Zerbe H.
      • Meyerholz M.M.
      In vivo model to study the impact of genetic variation on clinical outcome of mastitis in dairy heifers.
      . The comparison of heifers with Q and q haplotypes revealed that the q animals were more susceptible to early-lactation diseases. They had a higher number of udder quarters with very low cell count (<10,000 cells/mL) in the first weeks of lactation, but in the later course of lactation the weekly SCS was significantly lower for Q compared with q animals. The q animals had a higher SCS at 24 and 36 h after S. aureus challenge and a higher shedding of bacteria 12 h after challenge as well as a lower decline in milk yield after pathogen challenge (
      • Heimes A.
      • Brodhagen J.
      • Weikard R.
      • Hammon H.M.
      • Meyerholz M.M.
      • Petzl W.
      • Zerbe H.
      • Engelmann S.
      • Schmicke M.
      • Hoedemaker M.
      • Schuberth H.J.
      • Kuhn C.
      Characterization of functional traits with focus on udder health in heifers with divergent paternally inherited haplotypes on BTA18.
      ;
      • Meyerholz M.M.
      • Rohmeier L.
      • Eickhoff T.
      • Hülsebusch A.
      • Jander S.
      • Linden M.
      • Macias L.
      • Koy M.
      • Heimes A.
      • Gorríz-Martín L.
      • Segelke D.
      • Engelmann S.
      • Schmicke M.
      • Hoedemaker M.
      • Petzl W.
      • Zerbe H.
      • Schuberth H.J.
      • Kühn C.
      Genetic selection for bovine chromosome 18 haplotypes associated with divergent somatic cell score affects postpartum reproductive and metabolic performance.
      ;
      • Rohmeier L.
      • Petzl W.
      • Koy M.
      • Eickhoff T.
      • Hülsebusch A.
      • Jander S.
      • Macias L.
      • Heimes A.
      • Engelmann S.
      • Hoedemaker M.
      • Seyfert H.M.
      • Kühn C.
      • Schuberth H.J.
      • Zerbe H.
      • Meyerholz M.M.
      In vivo model to study the impact of genetic variation on clinical outcome of mastitis in dairy heifers.
      ). Most interestingly, in addition to differences in clinical parameters there were also significant differences in the endocrine and metabolic profiles of the animals postpartum: those cows with Q haplotypes displayed higher IGF-1, lower growth hormone, and BHB levels in plasma and serum, respectively, compared with q half-sibs (
      • Meyerholz M.M.
      • Rohmeier L.
      • Eickhoff T.
      • Hülsebusch A.
      • Jander S.
      • Linden M.
      • Macias L.
      • Koy M.
      • Heimes A.
      • Gorríz-Martín L.
      • Segelke D.
      • Engelmann S.
      • Schmicke M.
      • Hoedemaker M.
      • Petzl W.
      • Zerbe H.
      • Schuberth H.J.
      • Kühn C.
      Genetic selection for bovine chromosome 18 haplotypes associated with divergent somatic cell score affects postpartum reproductive and metabolic performance.
      ).
      The hepatic transcriptome is suitable to monitor potential metabolic as well as immunological effects of the BTA18 haplotypes, as the liver is at the center of the metabolic and immunological physiology of the dairy cow (
      • Moyes K.M.
      • Sørensen P.
      • Bionaz M.
      The impact of intramammary Escherichia coli challenge on liver and mammary transcriptome and cross-talk in dairy cows during early lactation using RNAseq.
      ). To follow up the differences in IGF-1, growth hormone, and BHB plasma/serum levels between Q and q cows we had observed before the challenge, we looked at potential related hepatic transcriptomic signatures in Q versus q after intramammary S. aureus challenge. However, we did not find significant differences in gene expression for IGF1 or other key genes directly involved in (short chain) fatty acid metabolism (Supplemental File S1; https://doi.org/10.3168/jds.2019-17612). Interestingly, the canonical pathway GPCR-mediated nutrient sensing in enteroendocrine cells was significantly enriched in the hepatic transcriptome of S. aureus challenged animals with the Q haplotype compared with animals with the q haplotype. G-protein-coupled receptors (GPCR) are membrane proteins, which detect their ligands (e.g., hormones, neurotransmitters, chemokines) in the extracellular matrix and can subsequently initiate the respective intracellular signaling cascades (
      • Latorraca N.R.
      • Venkatakrishnan A.J.
      • Dror R.O.
      GPCR dynamics: Structures in motion.
      ). In the gastrointestinal tract, nutrient metabolites activate GPCR, resulting in the secretion of gut and pancreatic hormones (
      • Husted A.S.
      • Trauelsen M.
      • Rudenko O.
      • Hjorth S.A.
      • Schwartz T.W.
      GPCR-mediated signaling of metabolites.
      ). However, GPCR can also act as pro- and anti-inflammatory regulators of immune cells (
      • Husted A.S.
      • Trauelsen M.
      • Rudenko O.
      • Hjorth S.A.
      • Schwartz T.W.
      GPCR-mediated signaling of metabolites.
      ). Thus, they have a broad spectrum of influence, and the enrichment of the respective pathway in the hepatic transcriptome of S. aureus challenged animals with Q haplotypes compared with the half-sibs carrying a q haplotype indicates haplotype-specific differences regarding their metabolic and immune profiles.
      After the intramammary S. aureus challenge, the livers of Q or q cows showed a different hepatic transcriptomic profile with respect to immune genes (see Table 1, Table 2).
      The enrichment of the canonical pathways ILK signaling and IL-8 signaling indicates that immune pathways play a dominant role in the response of the divergent hepatic transcriptome response to intramammary S. aureus challenge. This is also indicated by the nominally significant enrichment of the Protein Information Resource category “Innate immunity” (P = 1.1 × 10−3) and also by the significantly DE genes themselves (see Table 1). DYSF, for example, which was originally observed for its role in muscle function, is involved in regulating cellular interactions, cell adhesion mechanisms, and has a function in inflammatory cells (
      • de Morrée A.
      • Flix B.
      • Bagaric I.
      • Wang J.
      • van den Boogaard M.
      • Grand Moursel L.
      • Frants R.R.
      • Illa I.
      • Gallardo E.
      • Toes R.
      • van der Maarel S.M.
      Dysferlin regulates cell adhesion in human monocytes.
      ). The receptor encoded by GPBAR1 is implicated in the suppression of macrophage functions and regulation of energy homeostasis by bile acids and is also essential for the regulation of liver immunity (
      • Biagioli M.
      • Carino A.
      • Fiorucci C.
      • Marchianò S.
      • Di Giorgio C.
      • Roselli R.
      • Magro M.
      • Distrutti E.
      • Bereshchenko O.
      • Scarpelli P.
      • Zampella A.
      • Fiorucci S.
      GPBAR1 functions as gatekeeper for liver NKT cells and provides counterregulatory signals in mouse models of immune-mediated hepatitis.
      ). HMOX1 has been recognized as having major immunomodulatory and anti-inflammatory properties and has been regarded as an adaptive cellular response against inflammatory response and oxidative stress (reviewed by
      • Naito Y.
      • Takagi T.
      • Higashimura Y.
      Heme oxygenase-1 and anti-inflammatory M2 macrophages.
      ). FERMT2 plays a role in the TGFB1 and integrin signaling pathways (reviewed by
      • Rognoni E.
      • Ruppert R.
      • Fässler R.
      The kindlin family: Functions, signaling properties and implications for human disease.
      ) and is related to the Erk/MAPK signaling pathway (
      • Wan C.
      • Borgeson B.
      • Phanse S.
      • Tu F.
      • Drew K.
      • Clark G.
      • Xiong X.
      • Kagan O.
      • Kwan J.
      • Bezginov A.
      • Chessman K.
      • Pal S.
      • Cromar G.
      • Papoulas O.
      • Ni Z.
      • Boutz D.R.
      • Stoilova S.
      • Havugimana P.C.
      • Guo X.
      • Malty R.H.
      • Sarov M.
      • Greenblatt J.
      • Babu M.
      • Derry W.B.
      • Tillier E.R.
      • Wallingford J.B.
      • Parkinson J.
      • Marcotte E.M.
      • Emili A.
      Panorama of ancient metazoan macromolecular complexes.
      ). Furthermore, ICAM3 and IGSF11 are both members of the immunoglobulin super-family. ICAM3 has been described as chemotactic for macrophages and is therefore implicated in the phagocytosis of apoptotic cells (
      • Torr E.E.
      • Gardner D.H.
      • Thomas L.
      • Goodall D.M.
      • Bielemeier A.
      • Willetts R.
      • Griffiths H.R.
      • Marshall L.J.
      • Devitt A.
      Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells.
      ), whereas IGSF11 inhibited T cell function in human blood cell culture (
      • Wang J.
      • Wu G.
      • Manick B.
      • Hernandez V.
      • Renelt M.
      • Erickson C.
      • Guan J.
      • Singh R.
      • Rollins S.
      • Solorz A.
      • Bi M.
      • Li J.
      • Grabowski D.
      • Dirkx J.
      • Tracy C.
      • Stuart T.
      • Ellinghuysen C.
      • Desmond D.
      • Foster C.
      • Kalabokis V.
      VSIG-3 as a ligand of VISTA inhibits human T-cell function.
      ). TNC, described to be upregulated in inflamed tissues, is an activator of innate immunity, which can stimulate the synthesis of inflammatory cytokines (
      • Goh F.G.
      • Piccinini A.M.
      • Krausgruber T.
      • Udalova I.A.
      • Midwood K.S.
      Transcriptional regulation of the endogenous danger signal tenascin-C: A novel autocrine loop in inflammation.
      ). The PLCB2 gene, also significantly differentially expressed, is part of the significantly enriched canonical pathways phospholipases and phospholipase C signaling. As a key regulator for macrophage function, PLCB2 is involved in the switch from an inflammatory (M1) to an angiogenic (M2-like) macrophage phenotype (
      • Grinberg S.
      • Hasko G.
      • Wu D.
      • Leibovich S.J.
      Suppression of PLCbeta2 by endotoxin plays a role in the adenosine A(2A) receptor-mediated switch of macrophages from an inflammatory to an angiogenic phenotype.
      ). Moreover, it can inhibit the expression of pro-inflammatory cytokines (
      • Wang L.
      • Zhou Y.
      • Chen Z.
      • Sun L.
      • Wu J.
      • Li H.
      • Liu F.
      • Wang F.
      • Yang C.
      • Yang J.
      • Leng Q.
      • Zhang Q.
      • Xu A.
      • Shen L.
      • Sun J.
      • Wu D.
      • Fang C.
      • Lu H.
      • Yan D.
      • Ge B.
      PLCbeta2 negatively regulates the inflammatory response to virus infection by inhibiting phosphoinositide-mediated activation of TAK1.
      ). As presented in Figure 1, the Ingenuity network analysis showed that DYSF, GPBAR1, HMOX1, FERMT2, ICAM3, TNC, and PLCB2 are interacting with each other in a network related to immune response, indicating an interrelationship between them.
      Figure thumbnail gr1
      Figure 1Highly enriched network in the differentially expressed gene analysis of Q (favorable paternal haplotypes for SCS) versus q (unfavorable haplotypes for SCS) in cows challenged with Staphylococcus aureus. Green = lower expression in Q cows compared with q cows, red = higher expression in Q compared with q cows (modified according to Ingenuity Pathway Analysis, Qiagen, Hilden, Germany).
      As potential transcriptional regulators CXCR3, TLR3, and MAP3K1 could be predicted (Table 3). The CXCR3 is expressed by monocytes, T cells, natural killer cells, dendritic cells, and cancer cells (
      • Tokunaga R.
      • Zhang W.
      • Naseem M.
      • Puccini A.
      • Berger M.D.
      • Soni S.
      • McSkane M.
      • Baba H.
      • Lenz H.J.
      CXCL9, CXCL10, CXCL11/CXCR3 axis for immune activation - A target for novel cancer therapy.
      ). After activation by its selective ligands CXCL9, CXCL10, and CXCL11, it is involved in the recruitment and clustering of T cells and natural killer cells (
      • Tokunaga R.
      • Zhang W.
      • Naseem M.
      • Puccini A.
      • Berger M.D.
      • Soni S.
      • McSkane M.
      • Baba H.
      • Lenz H.J.
      CXCL9, CXCL10, CXCL11/CXCR3 axis for immune activation - A target for novel cancer therapy.
      ;
      • Maurice N.J.
      • McElrath M.J.
      • Andersen-Nissen E.
      • Frahm N.
      • Prlic M.
      CXCR3 enables recruitment and site-specific bystander activation of memory CD8(+) T cells.
      ;
      • Read S.A.
      • Wijaya R.
      • Ramezani-Moghadam M.
      • Tay E.
      • Schibeci S.
      • Liddle C.
      • Lam V.W.T.
      • Yuen L.
      • Douglas M.W.
      • Booth D.
      • George J.
      • Ahlenstiel G.
      Macrophage coordination of the Interferon Lambda immune response.
      ). Toll-like receptor 3 is widely known for its crucial role in pathogen recognition and subsequent activation of innate immunity (
      • Chen N.
      • Xia P.
      • Li S.
      • Zhang T.
      • Wang T.T.
      • Zhu J.
      RNA sensors of the innate immune system and their detection of pathogens.
      ). Mitogen-activated protein kinase kinase kinase 1 takes part in the TNF-α and NFκB signal cascades (
      • Ishizuka T.
      • Terada N.
      • Gerwins P.
      • Hamelmann E.
      • Oshiba A.
      • Fanger G.R.
      • Johnson G.L.
      • Gelfand E.W.
      Mast cell tumor necrosis factor alpha production is regulated by MEK kinases.
      ;
      • Sanchez-Perez I.
      • Benitah S.A.
      • Martinez-Gomariz M.
      • Lacal J.C.
      • Perona R.
      Cell stress and MEKK1-mediated c-Jun activation modulate NFkappaB activity and cell viability.
      ) and is therefore eminent for the appropriate response of the immune system to invasive pathogens. Thus, the upstream analysis confirmed alteration of the hepatic immune system in Q heifers compared with their q half-sibs within S. aureus-challenged animals.
      Regarding a potential molecular background of the QTL for functional traits and udder health in particular, we found a significant enrichment of DE loci in the genomic target area of the different haplotypes and their close vicinity on BTA18. This suggests that a regionally acting regulatory element might be involved, which could modulate the expression of an array of genes located there. Regulatory variation in noncoding regions as background of the BTA18 telomeric functional QTL might also explain the lack of causal candidate variant evidence for this QTL. No causal mechanism for the QTL could be pinpointed despite genetic variants with predicted effects in coding and noncoding regions, as described in recent GWAS at the whole genome sequence level with powerful data sets (
      • Wang T.
      • Chen Y.P.
      • MacLeod I.M.
      • Pryce J.E.
      • Goddard M.E.
      • Hayes B.J.
      Application of a Bayesian non-linear model hybrid scheme to sequence data for genomic prediction and QTL mapping.
      ;
      • Fang L.
      • Jiang J.
      • Li B.
      • Zhou Y.
      • Freebern E.
      • Vanraden P.M.
      • Cole J.B.
      • Liu G.E.
      • Ma L.
      Genetic and epigenetic architecture of paternal origin contribute to gestation length in cattle.
      ;
      • Jiang J.
      • Cole J.B.
      • Freebern E.
      • Da Y.
      • VanRaden P.M.
      • Ma L.
      Functional annotation and Bayesian fine-mapping reveals candidate genes for important agronomic traits in Holstein bulls.
      ). This also underlines the need to improve the current annotation of the bovine genome in terms of functional regulatory elements as addressed by the global Functional Annotation of Animal Genomes action (https://www.animalgenome.org/community/FAANG/;
      • Andersson L.
      • Archibald A.L.
      • Bottema C.D.
      • Brauning R.
      • Burgess S.C.
      • Burt D.W.
      • Casas E.
      • Cheng H.H.
      • Clarke L.
      • Couldrey C.
      • Dalrymple B.P.
      • Elsik C.G.
      • Foissac S.
      • Giuffra E.
      • Groenen M.A.
      • Hayes B.J.
      • Huang L.S.
      • Khatib H.
      • Kijas J.W.
      • Kim H.
      • Lunney J.K.
      • McCarthy F.M.
      • McEwan J.C.
      • Moore S.
      • Nanduri B.
      • Notredame C.
      • Palti Y.
      • Plastow G.S.
      • Reecy J.M.
      • Rohrer G.A.
      • Sarropoulou E.
      • Schmidt C.J.
      • Silverstein J.
      • Tellam R.L.
      • Tixier-Boichard M.
      • Tosser-Klopp G.
      • Tuggle C.K.
      • Vilkki J.
      • White S.N.
      • Zhao S.
      • Zhou H.
      Coordinated international action to accelerate genome-to-phenome with FAANG, the Functional Annotation of Animal Genomes project.
      ).

      CONCLUSIONS

      Heifers with alternative paternal BTA18 haplotypes display a significantly different hepatic transcriptome upon intramammary S. aureus challenge. Animals with the favorable Q haplotypes showed a more activated immune system (as demonstrated by the higher activation of key pathways and divergent expression of relevant immune genes), which might be better able to defend the host during a bacterial challenge. Driver of these differences in the immune system might be different adaptations of metabolic pathways of the Q and q animals. These findings are in line with results from clinical data of the haplotypes, which showed Q cows to be more resistant to diseases, especially S. aureus mastitis (SCC, SCS, shedding of bacteria, milk yield) than their q half-sibs.

      ACKNOWLEDGMENTS

      The authors thank the laboratory staff at the FBN Dummerstorf as well as the PhD students of the ChronMast project at the University of Veterinary Medicine Hannover. We thankfully acknowledge Hans-Martin Seyfert (Dummerstorf, Germany) for very fruitful discussion. We also thank the Förderverein Bioökonomieforschung (Bonn, Germany) for continuous support. This work was supported by funds of the German Government's Special Purpose Fund held at Landwirtschaftliche Rentenbank (Frankfurt/Main, Germany). The authors have not stated any conflicts of interest.

      Supplementary Material

      REFERENCES

        • Andersson L.
        • Archibald A.L.
        • Bottema C.D.
        • Brauning R.
        • Burgess S.C.
        • Burt D.W.
        • Casas E.
        • Cheng H.H.
        • Clarke L.
        • Couldrey C.
        • Dalrymple B.P.
        • Elsik C.G.
        • Foissac S.
        • Giuffra E.
        • Groenen M.A.
        • Hayes B.J.
        • Huang L.S.
        • Khatib H.
        • Kijas J.W.
        • Kim H.
        • Lunney J.K.
        • McCarthy F.M.
        • McEwan J.C.
        • Moore S.
        • Nanduri B.
        • Notredame C.
        • Palti Y.
        • Plastow G.S.
        • Reecy J.M.
        • Rohrer G.A.
        • Sarropoulou E.
        • Schmidt C.J.
        • Silverstein J.
        • Tellam R.L.
        • Tixier-Boichard M.
        • Tosser-Klopp G.
        • Tuggle C.K.
        • Vilkki J.
        • White S.N.
        • Zhao S.
        • Zhou H.
        Coordinated international action to accelerate genome-to-phenome with FAANG, the Functional Annotation of Animal Genomes project.
        Genome Biol. 2015; 16 (25854118): 57
        • Biagioli M.
        • Carino A.
        • Fiorucci C.
        • Marchianò S.
        • Di Giorgio C.
        • Roselli R.
        • Magro M.
        • Distrutti E.
        • Bereshchenko O.
        • Scarpelli P.
        • Zampella A.
        • Fiorucci S.
        GPBAR1 functions as gatekeeper for liver NKT cells and provides counterregulatory signals in mouse models of immune-mediated hepatitis.
        Cell. Mol. Gastroenterol. Hepatol. 2019; 8 (31226434): 447-473
        • Brand B.
        • Baes C.
        • Mayer M.
        • Reinsch N.
        • Kuhn C.
        Identification of a two-marker-haplotype on Bos taurus autosome 18 associated with somatic cell score in German Holstein cattle.
        BMC Genet. 2009; 10 (19725965): 50
        • Brand B.
        • Baes C.
        • Mayer M.
        • Reinsch N.
        • Seidenspinner T.
        • Thaller G.
        • Kuhn C.
        Quantitative trait loci mapping of calving and conformation traits on Bos taurus autosome 18 in the German Holstein population.
        J. Dairy Sci. 2010; 93 (20172241): 1205-1215
        • Chen H.
        • Xu J.
        • Wang P.
        • Shu Q.
        • Huang L.
        • Guo J.
        • Zhang X.
        • Zhang H.
        • Wang Y.
        • Shen Z.
        • Chen X.
        • Zhang Q.
        Protein phosphatase 2 regulatory subunit B′'Alpha silencing inhibits tumor cell proliferation in liver cancer.
        Cancer Med. 2019; 8 (31647192): 7741-7753
        • Chen N.
        • Xia P.
        • Li S.
        • Zhang T.
        • Wang T.T.
        • Zhu J.
        RNA sensors of the innate immune system and their detection of pathogens.
        IUBMB Life. 2017; 69 (28374903): 297-304
        • de Morrée A.
        • Flix B.
        • Bagaric I.
        • Wang J.
        • van den Boogaard M.
        • Grand Moursel L.
        • Frants R.R.
        • Illa I.
        • Gallardo E.
        • Toes R.
        • van der Maarel S.M.
        Dysferlin regulates cell adhesion in human monocytes.
        J. Biol. Chem. 2013; 288 (23558685): 14147-14157
        • De Vliegher S.
        • Fox L.K.
        • Piepers S.
        • McDougall S.
        • Barkema H.W.
        Invited review: Mastitis in dairy heifers: Nature of the disease, potential impact, prevention, and control.
        J. Dairy Sci. 2012; 95 (22365187): 1025-1040
        • Fang L.
        • Jiang J.
        • Li B.
        • Zhou Y.
        • Freebern E.
        • Vanraden P.M.
        • Cole J.B.
        • Liu G.E.
        • Ma L.
        Genetic and epigenetic architecture of paternal origin contribute to gestation length in cattle.
        Commun. Biol. 2019; 2 (30886909): 100
        • FastQC
        https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
        Date: 2016
        Date accessed: December 15, 2016
        • FeatureCounts
        http://subread.sourceforge.net/
        Date: 2017
        Date accessed: November 29, 2017
        • Goh F.G.
        • Piccinini A.M.
        • Krausgruber T.
        • Udalova I.A.
        • Midwood K.S.
        Transcriptional regulation of the endogenous danger signal tenascin-C: A novel autocrine loop in inflammation.
        J. Immunol. 2010; 184 (20107185): 2655-2662
        • Grinberg S.
        • Hasko G.
        • Wu D.
        • Leibovich S.J.
        Suppression of PLCbeta2 by endotoxin plays a role in the adenosine A(2A) receptor-mediated switch of macrophages from an inflammatory to an angiogenic phenotype.
        Am. J. Pathol. 2009; 175 (19850892): 2439-2453
        • Hacohen N.
        • Lan Y.Y.
        Damaged DNA marching out of aging nucleus.
        Aging (Albany NY). 2019; 11 (31581135): 8039-8040
        • Halasa T.
        • Huijps K.
        • Osteras O.
        • Hogeveen H.
        Economic effects of bovine mastitis and mastitis management: a review.
        Vet. Q. 2007; 29 (17471788): 18-31
        • Heimes A.
        • Brodhagen J.
        • Weikard R.
        • Hammon H.M.
        • Meyerholz M.M.
        • Petzl W.
        • Zerbe H.
        • Engelmann S.
        • Schmicke M.
        • Hoedemaker M.
        • Schuberth H.J.
        • Kuhn C.
        Characterization of functional traits with focus on udder health in heifers with divergent paternally inherited haplotypes on BTA18.
        BMC Vet. Res. 2019; 15 (31296208): 241
        • Husted A.S.
        • Trauelsen M.
        • Rudenko O.
        • Hjorth S.A.
        • Schwartz T.W.
        GPCR-mediated signaling of metabolites.
        Cell Metab. 2017; 25 (28380372): 777-796
        • Ishizuka T.
        • Terada N.
        • Gerwins P.
        • Hamelmann E.
        • Oshiba A.
        • Fanger G.R.
        • Johnson G.L.
        • Gelfand E.W.
        Mast cell tumor necrosis factor alpha production is regulated by MEK kinases.
        Proc. Natl. Acad. Sci. USA. 1997; 94 (9177222): 6358-6363
        • Jensen K.
        • Günther J.
        • Talbot R.
        • Petzl W.
        • Zerbe H.
        • Schuberth H.-J.
        • Seyfert H.-M.
        • Glass E.J.
        Escherichia coli- and Staphylococcus aureus-induced mastitis differentially modulate transcriptional responses in neighbouring uninfected bovine mammary gland quarters.
        BMC Genomics. 2013; 14 (23324411): 36
        • Jiang J.
        • Cole J.B.
        • Freebern E.
        • Da Y.
        • VanRaden P.M.
        • Ma L.
        Functional annotation and Bayesian fine-mapping reveals candidate genes for important agronomic traits in Holstein bulls.
        Commun. Biol. 2019; 2 (31240250): 212
        • Johnson J.L.
        • Monfregola J.
        • Napolitano G.
        • Kiosses W.B.
        • Catz S.D.
        Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GTPase-activating protein Gem-interacting protein.
        Mol. Biol. Cell. 2012; 23 (22438581): 1902-1916
        • Kipp S.
        • Segelke D.
        • Schierenbeck S.
        • Reinhardt F.
        • Reents R.
        • Wurmser C.
        • Pausch H.
        • Fries R.
        • Thaller G.
        • Tetens J.
        • Pott J.
        • Haas D.
        • Raddatz B.B.
        • Hewicker-Trautwein M.
        • Proios I.
        • Schmicke M.
        • Grunberg W.
        Identification of a haplotype associated with cholesterol deficiency and increased juvenile mortality in Holstein cattle.
        J. Dairy Sci. 2016; 99 (27614835): 8915-8931
        • Kühn C.
        • Reinhardt F.
        • Schwerin M.
        Marker assisted selection of heifers improved milk somatic cell count compared to selection on conventional pedigree breeding values.
        Arch. Tierzucht. 2008; 51: 23-32
        • Kutok J.L.
        • Yang X.
        • Folkerth R.
        • Adra C.N.
        Characterization of the expression of HTm4 (MS4A3), a cell cycle regulator, in human peripheral blood cells and normal and malignant tissues.
        J. Cell. Mol. Med. 2011; 15 (19818099): 86-93
        • Latorraca N.R.
        • Venkatakrishnan A.J.
        • Dror R.O.
        GPCR dynamics: Structures in motion.
        Chem. Rev. 2017; 117 (27622975): 139-155
        • Lei D.
        • Deng N.
        • Wang S.
        • Huang J.
        • Fan C.
        Upregulated ARRDC3 limits trophoblast cell invasion and tube formation and is associated with preeclampsia.
        Placenta. 2020; 89 (31665660): 10-19
        • Li H.
        • Handsaker B.
        • Wysoker A.
        • Fennell T.
        • Ruan J.
        • Homer N.
        • Marth G.
        • Abecasis G.
        • Durbin R.
        The Sequence Alignment/Map format and SAMtools.
        Bioinformatics. 2009; 25 (19505943): 2078-2079
        • Lin Z.
        • Liu W.
        • Xiao C.
        • Fan Y.
        • Zhuang G.
        • Qi Z.
        TIPE2 inhibits GC via regulation of cell proliferation, apoptosis and inflammation.
        Oncol. Rep. 2018; 40 (30015980): 1307-1316
        • Love M.I.
        • Huber W.
        • Anders S.
        Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
        Genome Biol. 2014; 15 (25516281): 550
        • Mao X.
        • Kadri N.K.
        • Thomasen J.R.
        • De Koning D.J.
        • Sahana G.
        • Guldbrandtsen B.
        Fine mapping of a calving QTL on Bos taurus autosome 18 in Holstein cattle.
        J. Anim. Breed. Genet. 2016; 133 (26486911): 207-218
        • Martin M.
        Cutadapt removes adapter sequences from high-throughput sequencing reads.
        EMBnet.journal. 2011; 17
        • Matthews D.
        • Kearney J.F.
        • Cromie A.R.
        • Hely F.S.
        • Amer P.R.
        Genetic benefits of genomic selection breeding programmes considering foreign sire contributions.
        Genet. Sel. Evol. 2019; 51 (31311493): 40
        • Maurice N.J.
        • McElrath M.J.
        • Andersen-Nissen E.
        • Frahm N.
        • Prlic M.
        CXCR3 enables recruitment and site-specific bystander activation of memory CD8(+) T cells.
        Nat. Commun. 2019; 10 (31676770)4987
        • Menzi F.
        • Besuchet-Schmutz N.
        • Fragniere M.
        • Hofstetter S.
        • Jagannathan V.
        • Mock T.
        • Raemy A.
        • Studer E.
        • Mehinagic K.
        • Regenscheit N.
        • Meylan M.
        • Schmitz-Hsu F.
        • Drogemuller C.
        A transposable element insertion in APOB causes cholesterol deficiency in Holstein cattle.
        Anim. Genet. 2016; 47 (26763170): 253-257
        • Meuwissen T.H.
        • Hayes B.J.
        • Goddard M.E.
        Prediction of total genetic value using genome-wide dense marker maps.
        Genetics. 2001; 157 (11290733): 1819-1829
        • Meyerholz M.M.
        • Rohmeier L.
        • Eickhoff T.
        • Hülsebusch A.
        • Jander S.
        • Linden M.
        • Macias L.
        • Koy M.
        • Heimes A.
        • Gorríz-Martín L.
        • Segelke D.
        • Engelmann S.
        • Schmicke M.
        • Hoedemaker M.
        • Petzl W.
        • Zerbe H.
        • Schuberth H.J.
        • Kühn C.
        Genetic selection for bovine chromosome 18 haplotypes associated with divergent somatic cell score affects postpartum reproductive and metabolic performance.
        J. Dairy Sci. 2019; 102 (31521359): 9983-9994
        • Moyes K.M.
        • Sørensen P.
        • Bionaz M.
        The impact of intramammary Escherichia coli challenge on liver and mammary transcriptome and cross-talk in dairy cows during early lactation using RNAseq.
        PLoS One. 2016; 11 (27336699)e0157480
        • Müller M.P.
        • Rothammer S.
        • Seichter D.
        • Russ I.
        • Hinrichs D.
        • Tetens J.
        • Thaller G.
        • Medugorac I.
        Genome-wide mapping of 10 calving and fertility traits in Holstein dairy cattle with special regard to chromosome 18.
        J. Dairy Sci. 2017; 100 (28109604): 1987-2006
        • MultiQC
        http://multiqc.info/
        Date: 2017
        Date accessed: September 11, 2017
        • Naito Y.
        • Takagi T.
        • Higashimura Y.
        Heme oxygenase-1 and anti-inflammatory M2 macrophages.
        Arch. Biochem. Biophys. 2014; 564 (25241054): 83-88
        • Pertea M.
        • Kim D.
        • Pertea G.M.
        • Leek J.T.
        • Salzberg S.L.
        Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown.
        Nat. Protoc. 2016; 11 (27560171): 1650-1667
        • Petzl W.
        • Gunther J.
        • Pfister T.
        • Sauter-Louis C.
        • Goetze L.
        • von Aulock S.
        • Hafner-Marx A.
        • Schuberth H.J.
        • Seyfert H.M.
        • Zerbe H.
        Lipopolysaccharide pretreatment of the udder protects against experimental Escherichia coli mastitis.
        Innate Immun. 2012; 18 (21990573): 467-477
        • Petzl W.
        • Zerbe H.
        • Gunther J.
        • Yang W.
        • Seyfert H.M.
        • Nurnberg G.
        • Schuberth H.J.
        Escherichia coli, but not Staphylococcus aureus triggers an early increased expression of factors contributing to the innate immune defense in the udder of the cow.
        Vet. Res. 2008; 39 (18258172): 18
        • Pulido D.
        • Arranz-Trullen J.
        • Prats-Ejarque G.
        • Velazquez D.
        • Torrent M.
        • Moussaoui M.
        • Boix E.
        Insights into the antimicrobial mechanism of action of human RNase6: Structural determinants for bacterial cell agglutination and membrane permeation.
        Int. J. Mol. Sci. 2016; 17 (27089320): 552
        • Qualitytrim
        https://bitbucket.org/arobinson/qualitytrim
        Date: 2017
        Date accessed: September 11, 2017
        • R
        https://www.r-project.org/
        Date: 2016
        Date accessed: August 29, 2016
        • Read S.A.
        • Wijaya R.
        • Ramezani-Moghadam M.
        • Tay E.
        • Schibeci S.
        • Liddle C.
        • Lam V.W.T.
        • Yuen L.
        • Douglas M.W.
        • Booth D.
        • George J.
        • Ahlenstiel G.
        Macrophage coordination of the Interferon Lambda immune response.
        Front. Immunol. 2019; 10 (31798594)2674
        • Rognoni E.
        • Ruppert R.
        • Fässler R.
        The kindlin family: Functions, signaling properties and implications for human disease.
        J. Cell Sci. 2016; 129 (26729028): 17-27
        • Rohmeier L.
        • Petzl W.
        • Koy M.
        • Eickhoff T.
        • Hülsebusch A.
        • Jander S.
        • Macias L.
        • Heimes A.
        • Engelmann S.
        • Hoedemaker M.
        • Seyfert H.M.
        • Kühn C.
        • Schuberth H.J.
        • Zerbe H.
        • Meyerholz M.M.
        In vivo model to study the impact of genetic variation on clinical outcome of mastitis in dairy heifers.
        BMC Vet Res. 2020; 16: 33
        • Rupp R.
        • Boichard D.
        Genetics of resistance to mastitis in dairy cattle.
        Vet. Res. 2003; 34 (14556700): 671-688
        • Sanchez-Perez I.
        • Benitah S.A.
        • Martinez-Gomariz M.
        • Lacal J.C.
        • Perona R.
        Cell stress and MEKK1-mediated c-Jun activation modulate NFkappaB activity and cell viability.
        Mol. Biol. Cell. 2002; 13 (12181357): 2933-2945
        • Schukken Y.H.
        • Gunther J.
        • Fitzpatrick J.
        • Fontaine M.C.
        • Goetze L.
        • Holst O.
        • Leigh J.
        • Petzl W.
        • Schuberth H.J.
        • Sipka A.
        • Smith D.G.
        • Quesnell R.
        • Watts J.
        • Yancey R.
        • Zerbe H.
        • Gurjar A.
        • Zadoks R.N.
        • Seyfert H.M.
        Host-response patterns of intramammary infections in dairy cows.
        Vet. Immunol. Immunopathol. 2011; 144 (21955443): 270-289
        • Tang H.
        • Peng S.
        • Dong Y.
        • Yang X.
        • Yang P.
        • Yang L.
        • Yang B.
        • Bao G.
        MARCH5 overexpression contributes to tumor growth and metastasis and associates with poor survival in breast cancer.
        Cancer Manag. Res. 2018; 11 (30636894): 201-215
        • Tierschutzgesetz
        • Tokunaga R.
        • Zhang W.
        • Naseem M.
        • Puccini A.
        • Berger M.D.
        • Soni S.
        • McSkane M.
        • Baba H.
        • Lenz H.J.
        CXCL9, CXCL10, CXCL11/CXCR3 axis for immune activation - A target for novel cancer therapy.
        Cancer Treat. Rev. 2018; 63 (29207310): 40-47
        • Torr E.E.
        • Gardner D.H.
        • Thomas L.
        • Goodall D.M.
        • Bielemeier A.
        • Willetts R.
        • Griffiths H.R.
        • Marshall L.J.
        • Devitt A.
        Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells.
        Cell Death Differ. 2012; 19 (22117198): 671-679
      1. UMD3.1.
        ftp://ftp.ensembl.org/././pub/release-87/fasta/bos_taurus/dna/
        Date: 2016
        Date accessed: March 21, 2016
        • Wan C.
        • Borgeson B.
        • Phanse S.
        • Tu F.
        • Drew K.
        • Clark G.
        • Xiong X.
        • Kagan O.
        • Kwan J.
        • Bezginov A.
        • Chessman K.
        • Pal S.
        • Cromar G.
        • Papoulas O.
        • Ni Z.
        • Boutz D.R.
        • Stoilova S.
        • Havugimana P.C.
        • Guo X.
        • Malty R.H.
        • Sarov M.
        • Greenblatt J.
        • Babu M.
        • Derry W.B.
        • Tillier E.R.
        • Wallingford J.B.
        • Parkinson J.
        • Marcotte E.M.
        • Emili A.
        Panorama of ancient metazoan macromolecular complexes.
        Nature. 2015; 525 (26344197): 339
        • Wang J.
        • Wu G.
        • Manick B.
        • Hernandez V.
        • Renelt M.
        • Erickson C.
        • Guan J.
        • Singh R.
        • Rollins S.
        • Solorz A.
        • Bi M.
        • Li J.
        • Grabowski D.
        • Dirkx J.
        • Tracy C.
        • Stuart T.
        • Ellinghuysen C.
        • Desmond D.
        • Foster C.
        • Kalabokis V.
        VSIG-3 as a ligand of VISTA inhibits human T-cell function.
        Immunology. 2019; 156 (30220083): 74-85
        • Wang L.
        • Zhou Y.
        • Chen Z.
        • Sun L.
        • Wu J.
        • Li H.
        • Liu F.
        • Wang F.
        • Yang C.
        • Yang J.
        • Leng Q.
        • Zhang Q.
        • Xu A.
        • Shen L.
        • Sun J.
        • Wu D.
        • Fang C.
        • Lu H.
        • Yan D.
        • Ge B.
        PLCbeta2 negatively regulates the inflammatory response to virus infection by inhibiting phosphoinositide-mediated activation of TAK1.
        Nat. Commun. 2019; 10 (30765691): 746
        • Wang T.
        • Chen Y.P.
        • MacLeod I.M.
        • Pryce J.E.
        • Goddard M.E.
        • Hayes B.J.
        Application of a Bayesian non-linear model hybrid scheme to sequence data for genomic prediction and QTL mapping.
        BMC Genomics. 2017; 18 (28810831): 618
        • Weikard R.
        • Goldammer T.
        • Brunner R.M.
        • Kuehn C.
        Tissue-specific mRNA expression patterns reveal a coordinated metabolic response associated with genetic selection for milk production in cows.
        Physiol. Genomics. 2012; 44 (22669841): 728-739
        • Weikard R.
        • Goldammer T.
        • Eberlein A.
        • Kuehn C.
        Novel transcripts discovered by mining genomic DNA from defined regions of bovine chromosome 6.
        BMC Genomics. 2009; 10 (19393061): 186
        • Weller J.I.
        • Saran A.
        • Zeliger Y.
        Genetic and environmental relationships among somatic cell count, bacterial infection, and clinical mastitis.
        J. Dairy Sci. 1992; 75 (1452857): 2532-2540
        • Wu X.P.
        • Guldbrandtsen B.
        • Nielsen U.S.
        • Lund M.S.
        • Sahana G.
        Association analysis for young stock survival index with imputed whole-genome sequence variants in Nordic Holstein cattle.
        J. Dairy Sci. 2017; 100 (28551195): 6356-6370