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Porcine xenografts lacking swine leukocyte antigen (SLA) class I are thought to be protected from human T cell responses. We have previously shown that SLA class I deficiency can be achieved in pigs by CRISPR/Cas9-mediated deletion of β2 -microglobulin (B2M). Here, we characterized another line of genetically modified pigs in which targeting of the B2M locus did not result in complete absence of B2M and SLA class I but rather in significantly reduced expression levels of both molecules. Residual SLA class I was functionally inert, because no proper differentiation of the CD8+ T cell subset was observed in B2Mlow pigs. Cells from B2Mlow pigs were less capable in triggering proliferation of human peripheral blood mononuclear cells in vitro, which was mainly due to the nonresponsiveness of CD8+ T cells. Nevertheless, cytotoxic effector cells developing from unaffected cell populations (eg, CD4+ T cells, natural killer cells) lysed targets from both SLA class I+ wildtype and SLA class Ilow pigs with similar efficiency. These data indicate that the absence of SLA class I is an effective approach to prevent the activation of human CD8+ T cells during the induction phase of an anti-xenograft response. However, cytotoxic activity of cells during the effector phase cannot be controlled by this approach.

Between 2015 and 2017, a marked increase of anaphylactic-like reactions after intravenous administration of gentamicin was observed first in horses and, later, also in humans. This worldwide issue led to safety measures including product recalls and safety warnings. Here, a German Marketing Authorization Holder (MAH) of an early and intensely affected veterinary product containing gentamicin describes the clinical approach of the company to analyze the root cause and identify the causative agent in the active pharmaceutical ingredient (API). The pharmacovigilance data of the MAH are presented, along with pharmacovigilance phenomena observed during the affected period. An overview is given on further investigations of the API manufacturer and measures taken by all parties involved, including competent authorities to reestablish a safe use of gentamicin products. The histamine contamination of gentamicin was an exceptional incident of global extent, affecting not only veterinary but also human drug safety. The reactions in horses transpired to also be an indicator of a human health threat, which ultimately contributed to an improvement in the safety of human and veterinary medicinal products containing fermentative APIs. The extreme dimensions of this issue emphasise the important role that veterinary clinicians and practitioners play in spontaneous reporting based pharmacovigilance systems and, by this, in drug safety.

Mastitis has a high incidence in dairy cows. Experimental infection with Escherichia coli increased the number of leukocytes in milk and the gene expression of the chemokine receptor CXCR4 in mammary gland tissues. A link between CXCR4 expression and lipopolysaccharide sensing was demonstrated in other species using in vitro models. The receptor that binds the chemokine stomal cell-derived factor 1 might be associated with the inflammatory response in bovine mammary glands. However, studies in cows are rare, and data on the localization of CXCR4 in bovine mammary glands and its distribution in bovine leukocytes are lacking. Fatty acids (FA) affect the inflammatory response. In human peripheral blood monocytes, exposure to conjugated linoleic acids (CLA) decreases the expression of CXCR4, leading to a decreased inflammatory response in these cells. In this study, we analyzed the expression of CXCR4 in the mammary glands of dairy cows by immunohistochemistry (n = 5) and laser capture microdissection followed by qualitative PCR (n = 3). We characterized the surface expression of CXCR4 on bovine leukocytes, including monocyte subpopulations, first by flow cytometry (n = 5) and then confirmed these results by Western blotting (n = 3). Rumen fistulated dairy cows (n = 4; 126 ± 4 d in milk) were fitted with abomasal infusion tubes, arranged in a 4 × 4 Latin square design, and supplemented for 6 wk twice daily with rising doses of FA followed by a 3-wk washout period. Then, CXCR4 expression on leukocytes was analyzed. The cows received a corn-based diet and were supplemented with coconut oil delivering medium-chain FA (38 g/d), linseed-safflower oil mix delivering n-3 FA (EFA, 39 g of linseed oil and 2 g of safflower oil per day), Lutalin (cis-9,trans-11 and trans-10,cis-12 CLA, 5 g/d; BASF), and EFA + CLA. In the bovine mammary gland, the epithelial cells of the lactiferous duct, but not alveolar epithelial cells, showed clear CXCR4 protein and mRNA signals. Among the leukocyte subsets, monocytes displayed the highest percentage of CXCR4-positive cells (87%), whereas circulating neutrophils showed almost no CXCR4 surface expression (3%) but stored the receptor intracellularly. The percentage of CXCR4-positive leukocytes was not affected by the different FA supplements, but FA supplementation reduced the receptor abundance per cell (40% on average). In conclusion, CXCR4 was clearly detected in the lactiferous duct cells of the mammary gland but not in the alveolar epithelial cells. Compared with other leukocytes, bovine monocytes showed the highest signal intensity of CXCR4 on their surface, whereas granulocytes stored CXCR4 intracellularly. Supplementation with all the FA reduced the surface expression of CXCR4 per leukocyte and could therefore potentially affect the inflammatory status associated with the surface expression of CXCR4. The importance of our observations should be verified in cows with mastitis in the future.

Uterine epithelial cells (UEC) and migrated polymorphonuclear cells (PMN) play important roles in the uterine defence against microbial infection. The aims of the present study were to investigate i) whether undiluted uterine secretions modulate the expression of genes associated with the innate immune response in UEC and PMN in vitro, ii) whether these changes differ between the two cell populations and iii) whether uterine secretions from cows with subclinical endometritis produce a different response to those from unaffected cows. Therefore, undiluted uterine secretions, cytobrush and biopsy samples were collected from bovine uteri at a local abattoir. All cows had calved at least 3 months prior to sample collection. Subclinical endometritis was diagnosed by cytology (≥5% polymorphonuclear neutrophils) and histology. The uteri were thereby retrospectively categorised as endometritis-positive (E-pos; n = 14), if either the cytology or the histology results were positive, or endometritis-negative (E-neg; n = 17), if both diagnostics were negative. Cultured UEC responded to secretions from E-pos and E-neg cows with an increased gene expression of CXC ligand (CXCL) 8 and interleukin (IL) 6 compared to incubation with control medium alone. PMN expressed significantly higher mRNA levels of CXCL5, CXCL8 and IL1B in response to supernatant from UEC incubated with secretions from both groups (E-pos and E-neg) compared to those incubated with control medium alone. Gene expression of IL10 in uterine epithelial cells remained comparable to the control in cells exposed to E-pos secretions and was 3.6 times lower in those exposed to E-neg secretions. These results demonstrate that the expression of genes associated with the innate immune response in UEC and indirectly also PMN is affected by uterine secretions in vitro. Depending on the target gene, these changes differ between the two cell populations. UEC exposed to uterine secretions from cows without subclinical endometritis produce lower levels of IL10 compared to those exposed to secretions from affected cows or control medium alone. Therefore, the model established in this study can be used as a valuable tool to further understand the contributions of the two cell populations to the coordinated immune response in the uterus.

Background: In dairy herds, mastitis causes detrimental economic losses. Genetic selection offers a sustainable tool to select animals with reduced susceptibility towards postpartum diseases. Studying underlying mechanisms is important to assess the physiological processes that cause differences between selected haplotypes. Therefore, the objective of this study was to establish an in vivo infection model to study the impact of selecting for alternative paternal haplotypes in a particular genomic region on cattle chromosome 18 for mastitis susceptibility under defined conditions in dairy uniparous cows. Results: At the start of pathogen challenge, no significant differences between the favorable (Q) and unfavorable (q) haplotypes were detected. Intramammary infection (IMI) with Staphylococcus aureus 1027 (S. aureus, n = 24, 96 h) or Escherichia coli 1303 (E. coli, n = 12, 24 h) was successfully induced in all uniparous cows. This finding was confirmed by clinical signs of mastitis and repeated recovery of the respective pathogen from milk samples of challenged quarters in each animal. After S. aureus challenge, Q-uniparous cows showed lower somatic cell counts 24 h and 36 h after challenge (P < 0.05), lower bacterial shedding in milk 12 h after challenge (P < 0.01) and a minor decrease in total milk yield 12 h and 24 h after challenge (P < 0.01) compared to q-uniparous cows. Conclusion: An in vivo infection model to study the impact of genetic selection for mastitis susceptibility under defined conditions in dairy uniparous cows was successfully established and revealed significant differences between the two genetically selected haplotype groups. This result might explain their differences in susceptibility towards IMI. These clinical findings form the basis for further in-depth molecular analysis to clarify the underlying genetic mechanisms for mastitis resistance.

Bovine mastitis is a disease of major economic effects on the dairy industry worldwide. Experimental in vivo infection models have been widely proven as an effective tool for the investigation of pathogen-specific host immune responses. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are two common mastitis pathogens with an opposite clinical outcome of the disease. E. coli and S. aureus have proven to be valid surrogates to model clinical and subclinical mastitis respectively. Contemporary transcriptome profiling studies demonstrated that the transcriptomic response in the teat reflects the course of pathogen-specific mastitis, being ultimately determined by the immune response of the mammary epithelial cells. After an experimental in vivo challenge, E. coli induces a vigorous early transcriptional response in udder tissue being quantitatively and - notably - qualitatively distinct from the much weaker response against an S. aureus infection. E. coli mastitis models proved that the local response in the infected udder quarters is accompanied by a response in non-infected neighbouring udder quarters modulating systemically their immune responsiveness. Immunomodulation of the udder was investigated in animal models. Pathophysiological consequences were studied after intramammary administration of cytokines, chemokines, growth factors, steroidal anti-inflammatory drugs, or priming of tissue resident cells with pathogen-derived molecules. The latter approaches resulted only in a temporal protection of the udder, reducing transiently the risk of infection but sustained lowering of the severity of an eventually occurring mastitis. They offer an alternative to vaccination trials, which over decades also did not yield protection against new infections.

Lactation is characterized by an increase in energy demand and glucose consumption, which may also be linked to the concurrent compromise in immune function during early lactation. Magnesium (Mg) could be involved in this link based on its role for more than 600 metabolic reactions associated with energy metabolism, in addition to its essential role in glucose metabolism. Therefore, the current study aimed to investigate the influence of Mg supplementation on the expression of glucose and Mg transport-associated genes in blood cells (predominantly leukocytes) of lactating ewes and to identify possible correlations between the expression of these genes and leukocyte subpopulations. Pregnant ewes were divided into a control group (Control, n = 8) and a Mg group (Mg, n = 8), the latter supplemented with magnesium oxide at a daily Mg intake of approximately 0.30% and 0.38% of dry matter during the last four weeks of gestation and the first four weeks after lambing, respectively. At the end of the supplementation period, blood samples were collected in PAXgene tubes in order to determine the mRNA expression of glucose transporters (GLUT1, GLUT3 and GLUT4) and genes linked to Mg transport (SLC41A1, CNNM2, TRPM6, TRPM7 and MagT1) in blood cells. Mg supplementation tended to increase the expression of GLUT1 in blood cells (P = 0.087). Expression values of GLUT3, SLC41A1, CNNM2, TRPM6, TRPM7, and MagT1 were not significantly changed (P > 0.1). Interestingly, GLUT4 was not detected in either group. Counts of circulating neutrophils, lymphocytes and monocytes showed no significant differences between the two groups. Furthermore, no correlations were observed between the expression of the investigated genes and specific leukocyte subpopulation percentages/frequenciies. In conclusion, the expression of a variety of glucose and Mg transport-associated genes did not respond to dietary Mg supplementation. Nonetheless, a tendency towards higher expression of GLUT1 in the Mg group might support an involvement of Mg in the regulation of glucose metabolism of ovine blood cells.

Title: In vivo model to study the impact of genetic variation on clinical outcome of mastitis in dairy uniparous cows Background: In dairy herds, mastitis causes detrimental economic losses. Genetic selection offers a sustainable tool to select animals with reduced susceptibility towards postpartum diseases. Studying underlying mechanisms is important to assess the physiological processes that cause differences between selected haplotypes. Therefore, the objective of this study was to establish an in vivo infection model to study the impact of selecting for alternative paternal haplotypes in a particular genomic region on cattle chromosome 18 for mastitis susceptibility under defined conditions in dairy uniparous cows. Results: At the start of pathogen challenge, no significant differences between the favorable (Q) and unfavorable (q) haplotypes were detected. Intramammary infection (IMI) with Staphylococcus aureus 1027 (S. aureus, n = 24, 96 h) or Escherichia coli 1303 (E. coli, n = 12, 24 h) was successfully induced in all uniparous cows. This finding was confirmed by clinical signs of mastitis and repeated recovery of the respective pathogen from milk samples of challenged quarters in each animal. After S. aureus challenge, Q-uniparous cows showed lower somatic cell counts 24 h and 36 h after challenge (P < 0.05), lower bacterial shedding in milk 12 h after challenge (P < 0.01) and a minor decrease in total milk yield 12 h and 24 h after challenge (P < 0.01) compared to q-uniparous cows. Conclusion: An in vivo infection model to study the impact of genetic selection for mastitis susceptibility under defined conditions in dairy uniparous cows was successfully established and revealed significant differences between the two genetically selected haplotype groups. This result might explain their differences in susceptibility towards IMI. These clinical findings form the basis for further in-depth molecular analysis to clarify the underlying genetic mechanisms for mastitis resistance.

Background In human and different animal species, blood monocytes are classified based on their expression pattern of different monocytic markers into phenotypically and functionally different subsets. In the current study, we used flow cytometry and monoclonal antibodies to CD172a, CD14, CD163 and MHCII to identify monocyte subsets in peripheral blood of dromedary camels. Results Based on CD14, CD163 and MHCII expression, camel CD172a + monocytes were divided into three subsets: The major subpopulation of camel monocytes (mo-I) showed high expression of CD14 and CD163, but low expression of MHCII. A second subset of monocytes (mo-II) expressed highly all three markers, CD14, CD163 and MHCII. A third monocyte subset (mo-III) displayed low expression of CD14 and CD163 with high MHCII expression. While the two MHCIIhigh subsets (mo-II and mo-III) showed higher expression of CD11a in comparison to the MHCIIlow subset (mo-I), CD18 and CD11b were highest expressed on the two CD14high subsets (mo-I and mo-II). Bacterial stimulation of camel leukocytes identified mo-II cells as an antimicrobial monocyte subset with the highest phagocytic and ROS production capacity. The comparison of monocyte counts and phenotype between newborn calves and adult camels revealed significantly reduced numbers of mo-II cells in newborn animals. Monocytes of newborns expressed significantly more CD172a and CD163 molecules but less CD14 and MHCII molecules than monocytes of adult camels. Conclusions Camel monocyte subsets, mo-I, mo-II and mo-III are counterparts of bovine classical, intermediate and non-classical monocytes respectively. The distribution of camel monocyte subsets is influenced by age.

Background In human and different animal species, blood monocytes are classified based on their expression pattern of different monocytic markers into phenotypically and functionally different subsets. In the current study, we used flow cytometry and monoclonal antibodies to CD172a, CD14, CD163 and MHCII to identify monocyte subsets in peripheral blood of dromedary camels. Results Based on CD14, CD163 and MHCII expression, camel CD172a + monocytes were divided into three subsets: The major subpopulation of camel monocytes (mo-I) showed high expression of CD14 and CD163, but low expression of MHCII. A second subset of monocytes (mo-II) expressed highly all three markers, CD14, CD163 and MHCII. A third monocyte subset (mo-III) displayed low expression of CD14 and CD163 with high MHCII expression. While the two MHCIIhigh subsets (mo-II and mo-III) showed higher expression of CD11a in comparison to the MHCIIlow subset (mo-I), CD18 and CD11b were highest expressed on the two CD14high subsets (mo-I and mo-II). Bacterial stimulation of camel leukocytes identified mo-II cells as an antimicrobial monocyte subset with the highest phagocytic and ROS production capacity. The comparison of monocyte counts and phenotype between newborn calves and adult camels revealed significantly reduced numbers of mo-II cells in newborn animals. Monocytes of newborns expressed significantly more CD172a and CD163 molecules but less CD14 and MHCII molecules than monocytes of adult camels. Conclusions Camel monocyte subsets, mo-I, mo-II and mo-III are counterparts of bovine classical, intermediate and non-classical monocytes respectively. The distribution of camel monocyte subsets is influenced by age.

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. © 2020 American Dairy Science Association 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. © 2020 American Dairy Science Association Landwirtschaftliche Rentenbank

Background A major challenge in modern medicine and animal husbandry is the issue of antimicrobial resistance. One approach to solving this potential medical hazard is the selection of farm animals with less susceptibility to infectious diseases. Recent advances in functional genome analysis and quantitative genetics have opened the horizon to apply genetic marker information for efficiently identifying animals with preferential predisposition regarding health traits. The current study characterizes functional traits with a focus on udder health in dairy heifers. The animals were selected for having inherited alternative paternal haplotypes for a genomic region on Bos taurus chromosome (BTA) 18 genetically associated with divergent susceptibility to longevity and animal health, particularly mastitis. Results In the first weeks of lactation, the q heifers which had inherited the unfavorable (q) paternal haplotype displayed a significantly higher number of udder quarters with very low somatic cell count (

Porcine xenografts lacking swine leukocyte antigen (SLA) class I are thought to be protected from human T cell responses. We have previously shown that SLA class I deficiency can be achieved in pigs by CRISPR/Cas9-mediated deletion of β

Title: In vivo model to study the impact of genetic variation on clinical outcome of mastitis in dairy heifers Background: In dairy herds, mastitis causes detrimental economic losses. Genetic selection offers a sustainable tool to select animals with reduced susceptibility towards postpartum diseases. Studying underlying mechanisms is important to assess the physiological processes that cause differences between selected haplotypes. Therefore, the objective of this study was to establish an in vivo infection model to study the impact of selecting for alternative paternal haplotypes in a particular genomic region on cattle chromosome 18 for mastitis susceptibility under defined conditions in dairy heifers. Results: At the start of pathogen challenge, no significant differences between the favorable (Q) and unfavorable (q) haplotypes were detected. Intramammary infection (IMI) with Staphylococcus aureus 1027 (S. aureus, n = 24, 96 h) or Escherichia coli 1303 (E. coli, n = 12, 24 h) was successfully induced in all heifers. This finding was confirmed by clinical signs of mastitis and repeated recovery of the respective pathogen from milk samples of challenged quarters in each heifer. After S. aureus challenge, Q-heifers showed lower somatic cell counts 24 h and 36 h after challenge (P < 0.05), lower bacterial shedding in milk 12 h after challenge (P < 0.01) and a minor decrease in total milk yield 12 h and 24 h after challenge (P < 0.01) compared to q-heifers. Conclusion: An in vivo infection model to study the impact of genetic selection for mastitis susceptibility under defined conditions in dairy heifers was successfully established and revealed significant differences between the two genetically selected haplotype groups. This result might explain their differences in susceptibility towards IMI. These clinical findings form the basis for further in-depth molecular analysis to clarify the underlying genetic mechanisms for mastitis resistance. Keywords: Staphylococcus aureus, Escherichia coli, BTA18, genetic selection, haplotype, intramammary infection model, somatic cell count, mastitis

Diets of dairy cows are often based on maize silage (MS), delivering lower amounts of n-3 fatty acids (FA) compared to grass silage-based diets. The fatty acid composition of the cell membrane can affect the cell function. We evaluated the effects of an MS-based diet on bovine red blood cell (RBC) membrane FA composition and dietary effects on controlled ATP release of RBC. In trial 1, German Holstein cows were fed an MS-based total mixed ration for 24 weeks. The FA composition of RBC membranes from repeatedly taken blood samples was analysed in addition to the abundance of the RBC membrane protein flotillin-1, which is involved in, for example, cell signalling. In trial 2, four rumen fistulated MS-fed cows were abomasally infused in a 4 &times, 4 Latin square model with three successively increasing lipid dosages (coconut oil, linseed&ndash, safflower oil mix (EFA, rich in n-3 FA), Lutalin&reg, providing conjugated linoleic acids (CLA) or the combination of the supplements, EFA + CLA) for six weeks, followed by a three-week washout period. In trial 2, we analysed RBC ATP release, flotillin-1, and the membrane protein abundance of pannexin-1, which is involved in ATP release as the last part of a signalling cascade. In trial 1, the total amount of n-3 FA in RBC membranes decreased and the flotillin-1 abundance increased over time. In trial 2, the RBC n-3 FA amount was higher after the six-week infusion period of EFA or EFA + CLA. Furthermore, depending on the dosage of FA, the ATP release from RBC increased. The abundance of flotillin-1 and pannexin-1 was not affected in trial 2. It is concluded that changes of the membrane FA composition influence the RBC function, leading to altered ATP release from intact bovine RBC.

Cattle with subclinical endometritis (SCE) are sub-fertile and diagnosing subclinical uterine disease remains a challenge. The hypothesis for this study was that endometrial inflammation is reflected in mRNA expression patterns of peripheral blood leucocytes. Transcriptome profiles were evaluated in healthy cows and in cows with SCE using circulating white blood cells (WBC) and endometrial biopsy samples collected from the same animals at 45-55 days postpartum. Bioinformatic analyses of microarray-based transcriptional data identified gene profiles associated with distinct biological functions in circulating WBC and endometrium. In circulating WBC, SCE promotes a pro-inflammatory environment, whereas functions related to tissue remodeling are also affected in the endometrium. Nineteen differentially expressed genes associated with SCE were common to both circulating WBC and the endometrium. Among these genes, transcript abundance of immune factors C3, C2, LTF, PF4 and TRAPPC13 were up-regulated in SCE cows at 45-55 days postpartum. Moreover, mRNA expression of C3, CXCL8, LTF, TLR2 and TRAPPC13 was temporally regulated during the postpartum period in circulating WBC of healthy cows compared with SCE cows. This observation might indicate an advantageous modulation of the immune system in healthy animals. The transcript abundance of these genes represents a potential source of indicators for postpartum uterine health.

The susceptibility of animals to periparturient diseases has a great effect on the economic efficiency of dairy industries, on the frequency of antibiotic treatment, and on animal welfare. The use of selection for breeding cows with reduced susceptibility to diseases offers a sustainable tool to improve dairy cattle farming. Several studies have focused on the association of distinct bovine chromosome 18 genotypes or haplotypes with performance traits. The aim of this study was to test whether selection of Holstein Friesian heifers via SNP genotyping for alternative paternal chromosome 18 haplotypes associated with favorable (Q) or unfavorable (q) somatic cell scores influences postpartum reproductive and metabolic diseases. Thirty-six heifers (18 Q and 18 q) were monitored from 3 wk before calving until necropsy on d 39 (± 4 d) after calving. Health status and rectal temperature were measured daily, and body condition score and body weight were assessed once per week. Blood samples were drawn twice weekly, and levels of insulin, nonesterified fatty acids, insulin-like growth factor-I, growth hormone, and β-hydroxybutyrate were measured. Comparisons between the groups were performed using Fisher's exact test, chi-squared test, and the GLIMMIX procedure in SAS. Results showed that Q-heifers had reduced incidence of metritis compared with q-heifers and were less likely to develop fever. Serum concentrations of β-hydroxybutyrate were lower and insulin-like growth factor-I plasma concentrations were higher in Q- compared with q-heifers. However, the body condition score and withers height were comparable between haplotypes, but weight loss tended to be lower in Q-heifers compared with q-heifers. No differences between the groups were detected concerning retained fetal membranes, uterine involution, or onset of cyclicity. In conclusion, selection of chromosome 18 haplotypes associated with a reduced somatic cell score resulted in a decreased incidence of postpartum reproductive and metabolic diseases in this study. The presented data add to the existing knowledge aimed at avoiding negative consequences of genetic selection strategies in dairy cattle farming. The underlying causal mechanisms modulated by haplotypes in the targeted genomic region and immune competence necessitate further investigation.

Lists of IPA canonical pathways significantly enriched for differentially expressed genes in non-depleted (0) and depleted RNA samples (protocol variants A and B) from pathogen-challenged and non-challenged udder quarters. Pathways are classified for being observed across all protocols (0, A or B) or shared between two or all three protocols. (PDF 214 kb)

Distribution of gene expression level (FPKM) for the depleted and non-depleted RNA samples from non-challenged udder samples. 0, A, and B represent the non-depleted or the depleted RNA samples (protocol variant A and B), respectively. (PDF 283 kb)

IGV screen shot of the read distribution across the PAEP gene for the E. coli challenged udder sample from cow 2. O, A and B represent the non-depleted or the depleted RNA samples (protocol variant A and B), respectively. Exon 6 is zoomed in for better demonstration of the position of the antisense capture oligonucleotide (see red line). (PDF 235 kb)

Excessive stimulation of the TLR4 axis through LPS reduces the expression of some cytokine genes in immune cells, while stimulating the expression of immune defense genes during a subsequent bacterial infection. This endotoxin tolerance (ET) is mediated via epigenetic mechanisms. Priming the udder of cows with LPS was shown to induce ET in mammary epithelial cells (MEC), thereby protecting the udder against reinfection for some time. Seeking alternatives to LPS priming we tried to elicit ET by priming MEC with either lipopeptide (Pam2CSK4) via the TLR2/6 axis or inhibitors of histone-modifying enzymes. Pre-incubation of MEC with Pam2CSK4 enhanced baseline and induced expression of bactericidal (β-defensin; SLPI) and membrane protecting factors ( SAA3, TGM3), while reducing the expression of cytokine- and chemokine-encoding genes ( TNF, IL1β) after a subsequent pathogen challenge, the latter, however, not as efficiently as after LPS priming. Pre-treating MEC with various inhibitors of histone H3 modifiers (for demethylation, acetylation or deacetylation) all failed to induce any of the protective factors and only resulted in some dampening of cytokine gene expression after the re-challenge. Hence, triggering immune functions via the TLR axis, but not through those histone modifiers, induced the beneficial phenomenon of ET in MEC.

Intra-mammary bacterial infections can result in harmful clinical mastitis or subclinical mastitis with persistent infections. Research during the last decades closely examined the pathophysiology of inflamed udders. Initial events after pathogen perception but before the onset of mastitis have not been examined in vivo. The objective of this study was to develop a mastitis model in cows by monitoring initial transcriptional pathogen-specific host response before clinical signs occur. We applied a short-term infection model to analyse transcripts encoding chemokines, cytokines and antimicrobial molecules in the teat cistern (TC) and lobulo-alveolar parenchyma (LP) up to 3 h after challenge with E. and Staphylococcus aureus. Both pathogens elicited an immune reaction by 1 h after challenge. Escherichia coli induced all analysed factors ( CCL20, CXCL8, TNF, IL6, IL12B, IL10, LAP, S100A9); however, S. aureus failed to induce IL12B, IL10, LAP and S100A9 expression. The E. coli-induced up-regulation was 25–105 times greater than that after S. aureus challenge. Almost all the responses were restricted to the TC. The short-term mastitis model demonstrates that a divergent pathogen-specific response is generated during the first h. It confirms that the first transcripts are generated in the TC prior to a response in the LP.

Endometritis represents the main cause of reproductive failure in dromedary camels. In dromedary camels, associations between endometritis-causing pathogen-species, disease severity, and systemic changes in the immune system have not been evaluated. In the current study, there was use of flow cytometry and immunofluorescence of membrane proteins for the evaluation of leukocyte subsets and the cellular phenotype in blood of camels with clinical endometritis and evaluations of associations with disease severity and endometritis-causing pathogens. Animals with endometritis had markedly larger numbers of total leukocytes and neutrophils. Although total lymphocyte and monocyte counts did not differ between camels with and without clinical endometritis, there were lesser numbers of total and effector CD4-positive T cells in camels with endometritis. Among monocytes, number of camel inflammatory monocytes (Mo-II) was markedly greater, whereas Mo-III numbers were less in the blood of camels with clinical endometritis. Number of inflammatory monocytes was also indicative of endometritis severity grade. Among camels with clinical endometritis, E. coli- and S. aureus-infected animals had similar endometritis grades and comparable phenotype and composition patterns of leukocytes. Neutrophils and monocytes of camels with clinical endometritis had fewer cell adhesion molecules (i.e., CD11a and CD18). Collectively, the results from the current study allowed for identification of associations between endometritis severity grade and larger numbers of inflammatory monocytes. The results also indicate there is no association between endometritis pathogen-species and changes in phenotype or composition of blood leukocytes.

The blood cell counting methods used for diagnostic and research purposes in turkeys are, up to date, inferior to the techniques established for mammals and chickens. While microscopic counts are time consuming, previous flow cytometric approaches did not cover all blood cell types of interest due to the lack of turkey-specific markers for the different cell populations. Moreover, it is unknown to what extent the different leukocyte populations are affected by host and environmental factors including age, breed of the bird, and housing environment, respectively. In this study we established a whole blood based flow cytometric analysis method for turkeys. This method was used to determine baseline values depending on the age of the birds as well as under consideration of variations between trials and animal room effects. During three trials whole blood samples of B.U.T. 6 female turkeys were collected to analyze different leukocyte concentrations (cells/μl whole blood). In the first trial one group and in the second and third trial two groups with 22 birds/group were housed. Blood samples were collected at days one, 23, 43, 60, and 88 post hatch and concentrations of monocytes, MHC class II-positive, CD4+, and CD8+ lymphocytes, as well as thrombocytes and granulocytes were determined by flow cytometric analysis. Concentrations of all identified populations were not only influenced by the bird's age (p < 0.05), they varied also among trials (p < 0.05) and even for some of the populations between animal rooms within the same trial despite comparable housing and management conditions. Therefore, for the establishment of baseline values for leucocyte concentrations in whole blood effects of age and housing have to be considered. In addition, our data emphasize the importance of the establishment of baseline values for different age groups, as age had the strongest effect on the blood cell numbers in this study.