Cows with diverging haplotypes show differences in differential milk cell count, milk parameters and vaginal temperature after S. aureus challenge but not after E. coli challenge.

BACKGROUND: In dairy cattle, mastitis causes high financial losses and impairs animal well-being. Genetic selection is used to breed cows with reduced mastitis susceptibility. Techniques such as milk cell flow cytometry may improve early mastitis diagnosis. In a highly standardized in vivo infection model, 36 half-sib cows were selected for divergent paternal Bos taurus chromosome 18 haplotypes (Q vs. q) and challenged with Escherichia coli for 24 h or Staphylococcus aureus for 96 h, after which the samples were analyzed at 12 h intervals. Vaginal temperature (VT) was recorded every three minutes. The objective of this study was to compare the differential milk cell count (DMCC), milk parameters (fat %, protein %, lactose %, pH) and VT between favorable (Q) and unfavorable (q) haplotype cows using Bayesian models to evaluate their potential as improved early indicators of differential susceptibility to mastitis. RESULTS: After S. aureus challenge, compared to the Q half-sibship cows, the milk of the q cows exhibited higher PMN levels according to the DMCC (24 h, p < 0.001), a higher SCC (24 h, p < 0.01 and 36 h, p < 0.05), large cells (24 h, p < 0.05) and more dead (36 h, p < 0.001) and live cells (24 h, p < 0.01). The protein % was greater in Q milk than in q milk at 0 h (p = 0.025). In the S. aureus group, Q cows had a greater protein % (60 h, p = 0.048) and fat % (84 h, p = 0.022) than q cows. Initially, the greater VT of S. aureus-challenged q cows (0 and 12-24 h, p < 0.05) reversed to a lower VT in q cows than in Q cows (48-60 h, p < 0.05). Additionally, the following findings emphasized the validity of the model: in the S. aureus group all DMCC subpopulations (24 h-96 h, p < 0.001) and in the E. coli group nearly all DMCC subpopulations (12 h-24 h, p < 0.001) were higher in challenged quarters than in unchallenged quarters. The lactose % was lower in the milk samples of E. coli-challenged quarters than in those of S. aureus-challenged quarters (24 h, p < 0.001). Between 12 and 18 h, the VT was greater in cows challenged with E. coli than in those challenged with S. aureus (3-h interval approach, p < 0.001). CONCLUSION: This in vivo infection model confirmed specific differences between Q and q cows with respect to the DMCC, milk component analysis results and VT results after S. aureus inoculation but not after E. coli challenge. However, compared with conventional milk cell analysis monitoring, e.g., the global SCC, the DMCC analysis did not provide refined phenotyping of the pathogen response.

Macrophages in bovine term placenta: An ultrastructural and molecular study.

Retention of foetal membranes (RFM) is a major reproductive disorder in dairy cows. An appropriate immune response is important for a physiological expulsion of the foetal membranes at parturition. Our study aims to provide a deeper insight into characteristics of foetal and maternal macrophages in bovine term placenta. We used transmission electron microscopy (TEM), immunohistochemistry and semi-quantitative RT-PCR to provide a deeper insight into characteristics of foetal and maternal macrophages in bovine term placenta. Semi-quantitative RT-PCR was used to define macrophage polarization in foetal and maternal compartments of normal term placenta. Gene expression of factors involved in M1 polarization [interferon regulatory factor-5 (IRF5), interleukin (IL)-12A, IL12B] and in M2 polarization (IL10) were studied. Ultrastructurally, foetal macrophages showed an irregular shape and large vacuoles, whereas the maternal macrophages were spindle shaped. By immunohistochemistry, macrophages were identified by a strong staining with the lysosomal marker Lysosome-associated membrane glycoprotein 1 (LAMP-1), while myofibroblast in the maternal stroma was positive for alpha-smooth muscle actin. We used the LAMP-1 marker to compare the density of foetal stromal macrophages in placentas of cows with RFM and in controls, but no statistically significant difference was observed. RT-PCR showed a higher expression of all studied genes in the maternal compartment of the placenta and generally a higher expression of M1-, compared to M2-associated genes. Our results indicated that at parturition placental macrophages predominantly show the pro-inflammatory M1 polarization. The higher expression of all the target genes in the maternal compartment may denote that maternal macrophages in bovine term placenta are more frequent than foetal macrophages.

Factors associated with ewe and lamb survival, and subsequent reproductive performance of sheep undergoing emergency caesarean section.

Clinical records of all 212 ewes undergoing emergency caesarean surgery at a veterinary teaching hospital between January 2008 and December 2019 were evaluated retrospectively. Their age ranged from 1 to 10 years (median = 4 years), with German merino the predominant breed (48.1% of cases). The most frequently diagnosed indications were insufficient cervical dilatation (n = 94, 44.3%), uterine torsion (n = 50, 23.6%), foetopelvic disproportion (n = 31, 14.6%) and vaginal prolapse intra partum (n = 11, 5.2%). Fifty-four (25.5%) of the 212 ewes additionally suffered from one or more concurrent, pre-existing conditions. Overall ewe mortality until hospital discharge was 10.8% (23/212), and 3.8% (n = 6) for the 158 ewes without a history of concurrent disorders. Mortality during hospitalization increased to 31.5% (17/54) for those with pre-existing conditions. Total lamb mortality was 49.1% (173/352) until hospital discharge. Pre-existing conditions (p = .001) and the presence of post-surgical complications (p = .025) were identified as significant factors influencing dam mortality, while delayed presentation for veterinary attention with an observed duration of labour of >12 hr was identified as the most influential factor on total lamb mortality (p = .010). The presence of dead or emphysematous foetuses was not significant for ewe mortality. Follow-up information on further outcomes was available for 156 (82.5%) of the 189 discharged ewes. Eighty-nine animals (57.1%) were re-bred in the following season and achieved a 93.3% (83/89) pregnancy rate, while the remainder had either been slaughtered (n = 56, 35.9%), sold (n = 5, 3.2%) or had died of unknown causes (n = 3, 1.9%). The subsequent incidence of dystocia was 15.6% (n = 12) in the 77 ewes with available information on lambing ease. Adequate management of underlying conditions and timely intervention are important factors for best possible short-term outcomes. In the long term, the subsequent pregnancy rate was good and the incidence of subsequent dystocia was within the normal range.

Development and evaluation of a milk protein transcript depletion method for differential transcriptome analysis in mammary gland tissue.

BACKGROUND: In the mammary gland transcriptome of lactating dairy cows genes encoding milk proteins are highly abundant, which can impair the detection of lowly expressed transcripts and can bias the outcome in global transcriptome analyses. Therefore, the aim of this study was to develop and evaluate a method to deplete extremely highly expressed transcripts in mRNA from lactating mammary gland tissue. RESULTS: Selective RNA depletion was performed by hybridization of antisense oligonucleotides targeting genes encoding the caseins (CSN1S1, CSN1S2, CSN2 and CSN3) and whey proteins (LALBA and PAEP) within total RNA followed by RNase H-mediated elimination of the respective transcripts. The effect of the RNA depletion procedure was monitored by RNA sequencing analysis comparing depleted and non-depleted RNA samples from Escherichia coli (E. coli) challenged and non-challenged udder tissue of lactating cows in a proof of principle experiment. Using RNase H-mediated RNA depletion, the ratio of highly abundant milk protein gene transcripts was reduced in all depleted samples by an average of more than 50% compared to the non-depleted samples. Furthermore, the sensitivity for discovering transcripts with marginal expression levels and transcripts not yet annotated was improved. Finally, the sensitivity to detect significantly differentially expressed transcripts between non-challenged and challenged udder tissue was increased without leading to an inadvertent bias in the pathogen challenge-associated biological signaling pathway patterns. CONCLUSIONS: The implementation of selective RNase H-mediated RNA depletion of milk protein gene transcripts from the mammary gland transcriptome of lactating cows will be highly beneficial to establish comprehensive transcript catalogues of the tissue that better reflects its transcriptome complexity.

Evaluation of a bovine visual pregnancy test for the detection of pregnancy-associated glycoproteins in sheep.

As sheep produce similar pregnancy-associated glycoproteins (PAGs) to cattle, a commercially available bovine visual pregnancy test based on the detection of PAGs (visual-PAG-test) was evaluated in sheep. The test was performed with whole blood (WhB), plasma (P) and serum (S) of 163 pregnant and 153 non-pregnant ewes. Additionally, 11 pregnant ewes were tested weekly from day 14 to 49 of gestation and monthly from day 60 to day 149. Ten ewes were sampled weekly from the date of lambing until day 63 post-partum (p.p.). The sensitivity in mid-pregnancy (n = 163) was 98.16% (WhB), 99.39% (P) and 99.39% (S) compared to transabdominal ultrasonography as the gold standard while the specificity (n = 153) was 94.12% (WhB), 76.47% (P) and 93.46% (S), respectively. During early pregnancy, all 11 ewes were correctly identified as pregnant on day 42 (100%); however, the test sensitivity decreased to 54.6% (WhB) and 63.6% (S and P, respectively) at day 49. The ewes were again consistently identified as pregnant on day 63 (P) or on day 119 (S, WhB). The test was consistently negative from day 42 p.p. onwards in eight out of ten ewes. Two ewes remained consistently positive until the last sample on day 63 p.p. In conclusion, the test could be used to accurately select pregnant ewes at day 42 with a drop in sensitivity at day 49. The sensitivity of the visual-PAG-test was good in mid to late pregnancy, and early detection of pregnancy was possible in individual animals. In some ewes, the PAGs were however detectable for more than 63 days p.p.-the previous breeding history should therefore always be taken into account for correct interpretation of the test results.

Staphylococcus aureus Phenol-Soluble Modulins Impair Interleukin Expression in Bovine Mammary Epithelial Cells.

The role of the recently described interleukin-32 (IL-32) in Staphylococcus aureus-induced mastitis, an inflammation of the mammary gland, is unclear. We determined expression of IL-32, IL-6, and IL-8 in S. aureus- and Escherichia coli-infected bovine mammary gland epithelial cells. Using live bacteria, we found that in S. aureus-infected cells, induction of IL-6 and IL-8 expression was less pronounced than in E. coli-infected cells. Notably, IL-32 expression was decreased in S. aureus-infected cells, while it was increased in E. coli-infected cells. We identified the staphylococcal phenol-soluble modulin (PSM) peptides as key contributors to these effects, as IL-32, IL-6, and IL-8 expression by epithelial cells exposed to psm mutant strains was significantly increased compared to that in cells exposed to the isogenic S. aureus wild-type strain, indicating that PSMs inhibit the production of these interleukins. The use of genetically complemented strains confirmed this observation. Inasmuch as the decreased expression of IL-32, which is involved in dendritic cell maturation, impairs immune responses, our results support a PSM-dependent mechanism that allows for the development of chronic S. aureus-related mastitis.

Investigating the contribution of IL-17A and IL-17F to the host response during Escherichia coli mastitis.

Mastitis remains a major disease of cattle with a strong impact on the dairy industry. There is a growing interest in understanding how cell mediated immunity contributes to the defence of the mammary gland against invading mastitis causing bacteria. Cytokines belonging to the IL-17 family, and the cells that produce them, have been described as important modulators of the innate immunity, in particular that of epithelial cells. We report here that expression of IL-17A and IL-17F genes, encoding two members of the IL-17 family, are induced in udder tissues of cows experimentally infected with Escherichia coli. The impact of IL-17A on the innate response of bovine mammary epithelial cells was investigated using a newly isolated cell line, the PS cell line. We first showed that PS cells, similar to primary bovine mammary epithelial cells, were able to respond to agonists of TLR2 and to LPS, provided CD14 was added to the culture medium. We then showed that secretion of CXCL8 and transcription of innate immunity related-genes by PS cells were increased by IL-17A, in particular when these cells were stimulated with live E. coli bacteria. Together with data from the literature, these results support the hypothesis that IL-17A and IL-17 F could play an important role in mediating of host-pathogen interactions during mastitis.

Effects of Elective Caesarean Sections in Healthy Near-Term Ewes on Subsequent Reproductive Performance.

Post-surgical reproductive performance following ovine caesarean section has not been well studied. To assess any direct effects of surgical delivery in the absence of confounders such as dystocia or underlying diseases, we studied elective surgery performed in healthy animals for teaching purposes. Four hundred and eleven paired breeding records following vaginal delivery (n = 233), elective caesarean section (n = 122), and subsequent further vaginal deliveries in animals with a history of one prior elective caesarean operation (n = 56) were evaluated retrospectively. The overall subsequent pregnancy rate was 95%. Multivariable statistical analyses did not reveal any significant influence of planned caesarean surgery on subsequent conception, stillbirth, perinatal lamb mortality, lamb birth weights, or the incidence of premature foetal death (mummification and abortion). A significantly higher number of mating attempts was, however, necessary. Also, a significant reduction in litter size was seen in the first pregnancy immediately following a surgical delivery in comparison to animals which had previously only delivered vaginally (p = 0.001), but litter size returned to pre-caesarean levels in further follow-up pregnancies in animals with a history of one elective caesarean section (p = 0.436). Subsequent long-term reproductive performance of sheep following elective caesarean section is thus excellent, and the results encourage retention for breeding.

Escherichia coli- and Staphylococcus aureus-induced mastitis differentially modulate transcriptional responses in neighbouring uninfected bovine mammary gland quarters.

BACKGROUND: The most important disease of dairy cattle is mastitis, caused by the infection of the mammary gland by various micro-organisms. Although the transcriptional response of bovine mammary gland cells to in vitro infection has been studied, the interplay and consequences of these responses in the in vivo environment of the mammary gland are less clear. Previously mammary gland quarters were considered to be unaffected by events occurring in neighbouring quarters. More recently infection of individual quarters with mastitis causing pathogens, especially Escherichia coli, has been shown to influence the physiology of neighbouring uninfected quarters. Therefore, the transcriptional responses of uninfected mammary gland quarters adjacent to quarters infected with two major mastitis causing pathogens, E. coli and Staphylococcus aureus, were compared. RESULTS: The bacteriologically sterile, within-animal control quarters exhibited a transcriptional response to the infection of neighbouring quarters. The greatest response was associated with E. coli infection, while a weaker, yet significant, response occurred during S. aureus infection. The transcriptional responses of these uninfected quarters included the enhanced expression of many genes previously associated with mammary gland infections. Comparison of the transcriptional response of uninfected quarters to S. aureus and E. coli infection identified 187 differentially expressed genes, which were particularly associated with cellular responses, e.g. response to stress. The most affected network identified by Ingenuity Pathway analysis has the immunosuppressor transforming growth factor beta 1 (TGFB1) at its hub and largely consists of genes more highly expressed in control quarters from S. aureus infected cows. CONCLUSIONS: Uninfected mammary gland quarters reacted to the infection of neighbouring quarters and the responses were dependent on pathogen type. Therefore, bovine udder quarters exhibit interdependence and should not be considered as separate functional entities. This suggests that mastitis pathogens not only interact directly with host mammary cells, but also influence discrete sites some distance away, which will affect their response to the subsequent spread of the infection. Understanding the underlying mechanisms may provide further clues for ways to control mammary gland infections. These results also have implications for the design of experimental studies investigating immune regulatory mechanisms in the bovine mammary gland.

Lipopolysaccharide priming enhances expression of effectors of immune defence while decreasing expression of pro-inflammatory cytokines in mammary epithelia cells from cows.

BACKGROUND: Udder infections with environmental pathogens like Escherichia coli are a serious problem for the dairy industry. Reduction of incidence and severity of mastitis is desirable and mild priming of the immune system either through vaccination or with low doses of immune stimulants such as lipopolysaccharide LPS was previously found to dampen detrimental effects of a subsequent infection. Monocytes/macrophages are known to develop tolerance towards the endotoxin LPS (endotoxin tolerance, ET) as adaptation strategy to prevent exuberant inflammation.We have recently observed that infusion of 1 µg of LPS into the quarter of an udder effectively protected for several days against an experimentally elicited mastitis. We have modelled this process in primary cultures of mammary epithelial cells (MEC) from the cow. MEC are by far the most abundant cells in the healthy udder coming into contact with invading pathogens and little is known about their role in establishing ET. RESULTS: We primed primary MEC cultures for 12 h with LPS (100 ng/ml) and stimulated three cultures either 12 h or 42 h later with 107/ml particles of heat inactivated E. coli bacteria for six hours. Priming-related alterations in the global transcriptome of those cells were quantified with Affymetrix microarrays. LPS priming alone caused differential expression of 40 genes and mediated significantly different response to a subsequent E. coli challenge of 226 genes. Expression of 38 genes was enhanced while that of 188 was decreased. Higher expressed were anti-microbial factors (ß-defensin LAP, SLPI), cell and tissue protecting factors (DAF, MUC1, TGM1, TGM3) as well as mediators of the sentinel function of MEC (CCL5, CXCL8). Dampened was the expression of potentially harmful pro-inflammatory master cytokines (IL1B, IL6, TNF-α) and immune effectors (NOS2, matrix metalloproteases). Functional network analysis highlighted the reduced expression of IL1B and of IRF7 as key to this modulation. CONCLUSION: LPS-primed MEC are fitter to repel pathogens and better protected against misguided attacks of the immune response. Attenuated is the exuberant expression of factors potentially promoting immunopathological processes. MEC therefore recapitulate many aspects of ET known so far from professional immune cells.

Gene expression profiling of bovine peripartal placentomes: detection of molecular pathways potentially involved in the release of foetal membranes.

The mechanisms underlying detachment of foetal membranes after birth in cows are still unclear. To address this problem in a systematic manner, we performed the first holistic transcriptome study of bovine placentomes antepartum (AP; n=4 cows) and intrapartum (IP; n=4 cows) using Affymetrix GeneChip Bovine Genome Arrays. Three placentomes were extracted from each cow, and tissue samples from the contact zones of the placentomes (foeto-maternal units) were recovered by systematic random sampling and processed for RNA extraction and for stereological quantification of cellular composition. Statistical analysis of microarray data (false discovery rate 1%) revealed 759 mRNAs with at least twofold higher levels in the samples of the AP group, whereas 514 mRNAs showed higher levels in the IP group. The differentially expressed genes were classified according to biological processes and molecular functions using the Functional Annotation Clustering tool of the DAVID Bioinformatics Resources. Genes with higher mRNA levels in the AP group were nearly completely related to mitotic cell cycle and tissue differentiation. During parturition, a complete shift occurred because the genes with higher mRNA levels in IP were nearly all related to three different physiological processes/complexes: i) apoptosis, ii) degradation of extra cellular matrix and iii) innate immune response, which play a fundamental role in placental detachment. These results are an excellent basis for future studies investigating the molecular basis of retained foetal membranes.

Retrospective analysis of 302 ovine dystocia cases presented to a veterinary hospital with particular attention to uterine torsion.

BACKGROUND: Dystocia is common in sheep, and foetal causes are predominant. Among maternal causes, insufficient cervical dilatation is the most frequent problem. Uterine torsion has been considered rare by many authors. OBJECTIVES: This study was conducted to investigate causes of dystocia in sheep presented for veterinary attention, and particular focus was set on the description of uterine torsion and analysis of potentially predisposing factors for this condition. METHODS: Clinical records of 302 sheep treated for dystocia were evaluated retrospectively. Known and proposed risk factors for uterine torsion in cattle were analysed regarding their potential importance in sheep. These included lamb birth weights, ewe age, parity, season, nutrition, breed type, litter size and husbandry. RESULTS: Maternal causes of dystocia accounted for 67.2% (203/302) of the presented cases. Of these, insufficient cervical dilatation (121/203, 59.6%) was the most frequent diagnosis. Another substantial proportion of maternal causes (60/203, 29.6%) was identified as uterine torsion. Husbandry, breed type and litter size showed significance in univariate analyses, with lower odds for meat breeds (OR 0.22; p < 0.001), twin- (OR 0.49; p = 0.020) or multiple-bearing ewes (OR 0.19; p = 0.013) and higher odds for fully housed animals (OR 17.87; p < 0.001). Year-round housing was identified as the most influential factor in a subsequent multivariate analysis. CONCLUSIONS: Uterine torsion was identified as a relevant cause of dystocia in our case load. The condition is likely to be underdiagnosed in sheep, and increased farmer and veterinary awareness is necessary to ensure adequate treatment of affected animals and to prevent unnecessary suffering.

Vitrification and in-straw warming do not affect pregnancy rates of biopsied bovine embryos.

In the cattle industry, in vivo or in vitro embryo production combined with genotyping and cryopreservation technologies allows the selection and conservation of embryos carrying genes for desirable traits. This study aimed to assess the efficiency of a vitrification method suitable for in-straw warming of biopsied in vivo derived (IVD) bovine embryos. Three experiments were carried out using two methodologies: the Cryotop®, the gold standard vitrification and 3-step warming methodology, or the VitTrans, a vitrification/in-straw 1-step warming method that enables direct embryo transfer to the uterus. In experiment 1, intact and biopsied in vitro produced (IVP) day 7 expanded blastocysts were vitrified using the Cryotop® and warmed in 1- or 3-steps. No differences in survival rates were recorded at 24 h after warming for intact or biopsied IVP blastocysts irrespective of the warming procedure. In experiment 2, the effect of the time from trophectoderm (TE) biopsy to vitrification/in-straw warming on post-warming survival rate was assessed. No significant differences in survival were observed when blastocysts were vitrified/in-straw warmed immediately after biopsy or after 3 h in culture when compared to intact blastocysts. In experiment 3, IVD embryos were vitrified 3 h after biopsy using the Cryotop® or the VitTrans method and pregnancy rates were assessed at day 60 after transfer. Fresh, biopsied embryos served as control. Similar pregnancy rates were observed when IVD biopsied embryos were transferred fresh or vitrified/warmed by the Cryotop® or VitTrans method. No significant effect of the embryo quality or developmental stage was detected on the percentage of pregnant recipients when IVD biopsied embryos were transferred fresh or after vitrification. While fresh female IVD embryos produced significantly higher pregnancy rates than male embryos, there were no differences in pregnancy rates when male or female vitrified/warmed embryos were transferred. About 81% from the biopsies analyzed successfully determined the embryo sex, confirming that DNA was there, and it was efficiently amplified. To conclude, our findings indicate that both vitrification methodologies produced similar post-warming outcomes for both intact and biopsied IVP embryos. Besides, vitrification/in-straw warming of biopsied IVD bovine embryos did not affect the viability to originate pregnancy, being a useful option for their direct transfer in field conditions.

Is the IL1RA/IL1B Ratio a Suitable Biomarker for Subclinical Endometritis in Dairy Cows?

The adequate expression of cytokines is essential for the prevention and healing of bovine endometrial inflammation. This study investigated the intra-uterine concentration of the proinflammatory cytokine interleukin (IL)1B and its antagonist IL1RA in cows with and without subclinical endometritis (SE). Samples were taken from 37 uteri at the abattoir and 26 uteri in vivo. Uterine secretion samples were classified as showing no signs of SE (SEneg; polymorphonuclear neutrophil granulocyte (PMN) < 5%) or showing signs of SE (SEpos; PMN ≥ 5%). Concentrations and ratios for IL1B and IL1RA were measured using a commercial and a newly established AlphaLISA kit, respectively. In both groups, a higher concentration of IL1B was detected in the SEpos group compared with the SEneg group (abattoir: p = 0.027; in vivo p < 0.001). No significant differences were observed in the concentration of IL1RA (p > 0.05). In uterine secretion samples retrieved in vivo, a lower IL1RA/IL1B ratio was detected in the SEpos group compared with the SEneg group (p = 0.002). The results of this study highlight the important role of IL1B and IL1RA during endometritis and the potential of the IL1RA/IL1B ratio as a possible biomarker for SE.

Allele-biased expression of the bovine APOB gene associated with the cholesterol deficiency defect suggests cis-regulatory enhancer effects of the LTR retrotransposon insertion.

The insertion of an endogenous retroviral long terminal repeat (LTR) sequence into the bovine apolipoprotein B (APOB) gene is causal to the inherited genetic defect cholesterol deficiency (CD) observed in neonatal and young calves. Affected calves suffer from developmental abnormalities, symptoms of incurable diarrhoea and often die within weeks to a few months after birth. Neither the detailed effects of the LTR insertion on APOB expression profile nor the specific mode of inheritance nor detailed phenotypic consequences of the mutation are undisputed. In our study, we analysed German Holstein dairy heifers at the peak of hepatic metabolic load and exposed to an additional pathogen challenge for clinical, metabolic and hepatic transcriptome differences between wild type (CDF) and heterozygote carriers of the mutation (CDC). Our data revealed that a divergent allele-biased expression pattern of the APOB gene in heterozygous CDC animals leads to a tenfold higher expression of exons upstream and a decreased expression of exons downstream of the LTR insertion compared to expression levels of CDF animals. This expression pattern could be a result of enhancer activity induced by the LTR insertion, in addition to a previously reported artificial polyadenylation signal. Thus, our data support a regulatory potential of mobile element insertions. With regard to the phenotype generated by the LTR insertion, heterozygote CDC carriers display significantly differential hepatic expression of genes involved in cholesterol biosynthesis and lipid metabolism. Phenotypically, CDC carriers show a significantly affected lipomobilization compared to wild type animals. These results reject a completely recessive mode of inheritance for the CD defect, which should be considered for selection decisions in the affected population. Exemplarily, our results illustrate the regulatory impact of mobile element insertions not only on specific host target gene expression but also on global transcriptome profiles with subsequent biological, functional and phenotypic consequences in a natural in-vivo model of a non-model mammalian organism.

Comparative kinetics of Escherichia coli- and Staphylococcus aureus-specific activation of key immune pathways in mammary epithelial cells demonstrates that S. aureus elicits a delayed response dominated by interleukin-6 (IL-6) but not by IL-1A or tumor necrosis factor alpha.

Infections of the udder by Escherichia coli very often elicit acute inflammation, while Staphylococcus aureus infections tend to cause mild, subclinical inflammation and persistent infections. The molecular causes underlying the different disease patterns are poorly understood. We therefore profiled the kinetics and extents of global changes in the transcriptome of primary bovine mammary epithelial cells (MEC) after challenging them with heat-inactivated preparations of E. coli or S. aureus pathogens. E. coli swiftly and strongly induced an expression of cytokines and bactericidal factors. S. aureus elicited a retarded response and failed to quickly induce an expression of bactericidal factors. Both pathogens induced similar patterns of chemokines for cell recruitment into the udder, but E. coli stimulated their synthesis much faster and stronger. The genes that are exclusively and most strongly upregulated by E. coli may be clustered into a regulatory network with tumor necrosis factor alpha (TNF-α) and interleukin-1 (IL-1) in a central position. In contrast, the expression of these master cytokines is barely regulated by S. aureus. Both pathogens quickly trigger an enhanced expression of IL-6. This is still possible after completely abrogating MyD88-dependent Toll-like receptor (TLR) signaling in MEC. The E. coli-specific strong induction of TNF-α and IL-1 expression may be causative for the severe inflammatory symptoms of animals suffering from E. coli mastitis, while the avoidance to quickly induce the synthesis of bactericidal factors may support the persistent survival of S. aureus within the udder. We suggest that S. aureus subverts the MyD88-dependent activation of immune gene expression in MEC.

Concentration-Dependent Type 1 Interferon-Induced Regulation of MX1 and FABP3 in Bovine Endometrial Explants.

The inadequate maternal recognition of embryonic interferon τ (IFNτ) might explain subfertility in cattle. This study aimed at modeling the inducibility of type 1 interferon receptor subunits 1/2 (IFNAR1/2), mimicking competition between IFNτ and infection-associated interferon α (IFNα), and simulating type 1 interferon pathways in vitro. Endometrial explants (n = 728 from n = 26 healthy uteri) were collected at the abattoir, challenged with IFNτ and/or IFNα in different concentrations, and incubated for 24 h. Gene expression analysis confirmed the inducibility of IFNAR1/2 within this model, it being most prominent in IFNAR2 with 10 ng/mL IFNα (p = 0.001). The upregulation of interferon-induced GTP-binding protein (MX1, classical pathway) was higher in explants treated with 300 ng/mL compared to 10 ng/mL IFNτ (p < 0.0001), whereas the non­classical candidate fatty acid binding protein 3 (FABP3) exhibited significant downregulation comparing 300 ng/mL to 10 ng/mL IFNτ. The comparison of explants challenged with IFNτ + IFNα indicated the competition of IFNτ and IFNα downstream of the regulatory factors. In conclusion, using this well-defined explant model, interactions between infection-associated signals and IFNτ were indicated. This model can be applied to verify these findings and to mimic and explore the embryo-maternal contact zone in more detail.

Escherichia coli infection induces distinct local and systemic transcriptome responses in the mammary gland.

BACKGROUND: Coliform bacteria are the most common etiologic agents in severe mastitis of cows. Escherichia coli infections are mostly restricted to a single udder quarter whereas neighboring quarters stay clinically inapparent, implicating the presence of a systemic defense reaction. To address its underlying mechanism, we performed a transcriptome study of mammary tissue from udder quarters inoculated with E. coli (6 h and 24 h post infection), from neighboring quarters of the same animals, and from untreated control animals. RESULTS: After 6 h 13 probe sets of differentially expressed genes (DEG) were detected in infected quarters versus control animals. Eighteen hours later 2154 and 476 DEG were found in infected and in neighboring quarters vs. control animals. Cluster analysis revealed DEG found only in infected quarters (local response) and DEG detected in both infected and neighboring quarters (systemic response). The first group includes genes mainly involved in immune response and inflammation, while the systemic reaction comprises antigen processing and presentation, cytokines, protein degradation and apoptosis. Enhanced expression of antimicrobial genes (S100A8, S100A9, S100A12, CXCL2, GNLY), acute phase genes (LBP, SAA3, CP, BF, C6, C4BPA, IF), and indicators of oxidative stress (GPX3, MT1A, MT2A, SOD2) point to an active defense reaction in infected and neighboring healthy quarters. Its early onset is indicated by increased transcription of NFIL3 at 6 h. NFIL3 is a predicted regulator of many genes of the systemic response at 24 h. The significance of our transcriptome study was evidenced by some recent findings with candidate gene based approaches. CONCLUSIONS: The discovery and holistic analysis of an extensive systemic reaction in the mammary gland significantly expands the knowledge of host-pathogen interactions in mastitis which may be relevant for the development of novel therapies and for genetic selection towards mastitis resistance.

Germ-line transmission of lentiviral PGK-EGFP integrants in transgenic cattle: new perspectives for experimental embryology.

Lentiviral vectors are a powerful tool for the genetic modification of livestock species. We previously generated transgenic founder cattle with lentiviral integrants carrying enhanced green fluorescent protein (EGFP) under the control of the phosphoglycerate kinase (PGK) promoter. In this study, we investigated the transmission of LV-PGK-EGFP integrants through the female and male germ line in cattle. A transgenic founder heifer (#562, Kiki) was subjected to superovulation treatment and inseminated with semen from a non-transgenic bull. Embryos were recovered and transferred to synchronized recipient heifers, resulting in the birth of a healthy male transgenic calf expressing EGFP as detected by in vivo imaging. Semen from a transgenic founder bull (#561, Jojo) was used for in vitro fertilization (IVF) of in vitro matured (IVM) oocytes from non-transgenic cows. The rates of cleavage and development to blastocyst in vitro corresponded to 52.0 +/- 4.1 and 24.5 +/- 4.4%, respectively. Expression of EGFP was observed at blastocyst stage (day 7 after IVF) and was seen in 93.0% (281/302) of the embryos. 24 EGFP-expressing embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos, flushed from the uterus on day 15, two fetuses recovered on day 45, and a healthy male transgenic calf revealed consistent high-level expression of EGFP in all tissues investigated. Our study shows for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. The pattern of inheritance was consistent with Mendelian rules. Importantly, high fidelity expression of EGFP in embryos, fetuses, and offspring of founder #561 provides interesting tools for developmental studies in cattle, including interactions of gametes, embryos and fetuses with their maternal environment.

Investigation of mammary blood flow changes by transrectal colour Doppler sonography in an Escherichia coli mastitis model.

The objectives of this preliminary investigation were to evaluate the feasibility of transrectal colour Doppler sonography (TCDS) for determining blood flow of the pudendoepigastric trunk in cows with experimentally induced Escherichia coli Mastitis. Five primiparous Holstein dairy cows, 4-6 months after calving, were examined in two trials. All monitored udder quarters were initially clinically healthy, somatic cell count (SCC) was <50 000 cells/ml and bacteriologically negative. The blood flow of the left and the right pudendoepigastric trunk was described by the blood flow volume (BFV). In the methodological part of the study, the intra-observer precision of the method was evaluated. The coefficients of variation of the BFV were 7.1% for the left and 9.4% for the right pudendoepigastric trunk. The intraclass correlation coefficients of the BFV were 0.99 (P<0.001) for the left and 0.75 (P=0.004) for the right vessel. BFV did not differ significantly between the left and the right side nor between pre- and post-milking nor between oestrus and dioestrus. In the experimental part of the study, significant differences of increasing BFV between 0 and 12 h p.i. (post infectionem) (P=0.043) and decreasing BFV between 12 and 24 h p.i. (P=0.043) were discovered for the pudendoepigastric trunk of the infected right side. In the left-right (control-infection) comparison a significant increase of the right BFV was observed at 12 h p.i. (P=0.043). The difference of an increasing SCC correlated positively with the difference of an increasing BFV between 0 and 12 h p.i. (Spearman's rho=1.00; P=0.043) for the right infected side. It was shown that TCDS is a reproducible technique for investigating pathological mammary blood flow changes at an early stage of acute mastitis.