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Abstract 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

The quality of equine colostrum is typically defined by refractometry or the concentration of maternal antibodies. However, the activity of other equine colostral bioactive molecules has not yet been investigated. This study analyzed whether the administration of a Saccharomyces cerevisiae fermentation product (SCFP) influences the biological activity of mare colostrum and whether the biological activity of colostrum has a lasting immunomodulating effect for foals. A total of fourteen pregnant mares received 20 g/day of a SCFP for a period of twelve weeks prior to the calculated date of birth (SCFP-group). Twelve pregnant mares without supplementation served as controls (CON). Colostral Brix values were determined within three hours after parturition. The concentration of IgG in blood sera and colostrum samples was determined with an ELISA. The biological activity was determined in a cell growth assay with a porcine epithelial cell line (IPEC-J2). Mares (at three weeks before the calculated date of birth) and foals (at the age of 5–8 months) received a parenteral active vaccination against influenza and tetanus. The administration of SCFP did not alter the mare’s serum and colostrum IgG concentrations and did not exert a significant influence on the mares’ early response to the vaccination. Growth and diarrhea episodes were comparable between foals of supplemented mares (SCFP) and foals of mares without supplementation (CON). Colostrum samples from SCFP-supplemented mares exhibited heightened biological activity. While SFCP and CON foals did not differ in their early response to vaccination, the vaccination-induced alterations in circulating neutrophilic granulocyte numbers were significantly correlated with the biological colostrum activity. These findings suggest that the supplementation of mares in late gestation with SCFP can enhance the biological activity of colostrum, which subsequently influences the innate immune responses of their offspring in later life.

Feed supplements supporting animal welfare and performance are becoming increasingly important. Immunomodulatory effects of such products have been observed in many species. The aim of this study was to analyze whether food supplementation with a Saccharomyces cerevisiae fermentation product (SCFP) affects the occurrence of foal diarrhea in early life, and whether the SCFP feeding has an impact on the immediate response to a parenteral vaccination at the age of 6–9 months. Eleven foals received the SCFP (OLI) and eleven foals were fed a placebo (PLA) for 29 days. Growth, diarrhea, and diarrhea severity were observed until day 30. After weaning, at the age of 6–9 months, foals were vaccinated parenterally against influenza and tetanus. The supplementation had no statistically significant effect on diarrhea duration and severity. On the day of vaccination, PLA and OLI foals did not differ significantly regarding numbers of circulating blood leukocyte subsets. However, the response to vaccination differed significantly between OLI and PLA foals. In OLI foals, the numbers of the major leukocyte fractions (granulocytes, lymphocytes, monocytes, CD4+ T cells, CD8+ T cells, CD21+ B cells, and MHC-II+/CD21− cells) increased significantly 24 h after vaccination but remained unchanged in PLA foals. The observed results suggest that early life supplementation with an SCFP may affect the early immune response to an initial vaccination.

Abstract Background Respiratory diseases are among the most common and expensive to treat diseases in camels with a great economic impact on camel health, welfare, and production. Bronchoalveolar lavage fluid (BALF) has been proven as a valuable sample for investigating the leukocyte populations in the respiratory tract of several species. In the present study, fluorescent antibody labeling and flow cytometry were used to study the immune cell composition of BALF in dromedary camels. Animals with clinical respiratory diseases (n = seven) were compared with apparently healthy animals (n = 10). In addition, blood leukocytes from the same animals were stained in parallel with the same antibodies and analyzed by flow cytometry. Results Camel BALF macrophages, granulocytes, monocytes, and lymphocytes were identified based on their forward and side scatter properties. The expression pattern of the cell markers CD172a, CD14, CD163, and MHCII molecules on BALF cells indicates a similar phenotype for camel, bovine, and porcine BALF myeloid cells. The comparison between camels with respiratory disease and healthy camels regarding cellular composition in their BALF revealed a higher total cell count, a higher fraction of granulocytes, and a lower fraction of macrophages in diseased than healthy camels. Within the lymphocyte population, the percentages of helper T cells and B cells were also higher in diseased than healthy camels. The elevated expression of the activation marker CD11a on helper T cells of diseased camels is an indication of the expansion of helper T cells population due to infection and exposure to respiratory pathogens. The higher abundance of MHCII molecules on BALF macrophages from diseased camels indicates a polarization toward an inflammatory macrophage phenotype (M1) in respiratory diseased camels. No significant differences were observed in the systemic leukogram between healthy and diseased animals. Conclusions Collectively, the current study represents the first report on flow cytometric analysis of immune cell composition of bronchoalveolar lavage fluid (BALF) in dromedary camels.

Abstract Dairy cattle experience a profound nutrient deficit postpartum that is associated with immune dysfunction characterized by heightened inflammation and reduced pathogen clearance. The activation of the central nutrient-sensing mTOR pathway is comparatively reduced in leukocytes of early postpartum dairy cows during this time of most pronounced nutrient deficit. We assessed the effect of pharmacological mTOR inhibition (Torin-1, rapamycin) on differentiation of monocyte derived classically (M1) and alternatively (M2) activated macrophages (MPh) and dendritic cells (moDC) from 12 adult dairy cows. Treatment with mTOR inhibitors generated M1 MPh with increased oxidative burst and expression of IL12 subunits but decreased phagocytosis and expression of IL1B, IL6, and IL10. In M2 MPh, treatment inhibited expression of regulatory features (CD163, ARG2, IL10) skewing the cells toward an M1-like phenotype. In moDC, mTOR inhibition increased expression of pro-inflammatory cytokines (IL12A, IL12B, IL1B, IL6) and surface CD80. In co-culture with mixed lymphocytes, mTOR-inhibited moDC exhibited a cytokine profile favoring a Th1 response with increased TNF and IFNG production and decreased IL10 concentrations. We conclude that mTOR inhibition in vitro promoted differentiation of inflammatory macrophages with reduced regulatory features and generation of Th1-favoring dendritic cells. These mechanisms could contribute to immune dysregulation in postpartum dairy cows.

Abstract 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.

ABSTRACT: 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.

Innate immune training is defined as a property of innate immune cells to react stronger to a secondary contact with pathogens. Induction of innate immune training has been reported for a variety of pathogens and selected pattern recognition receptor-ligands, such as β-glucans (βG). We examined whether Saccharomyces cerevisiae cell wall component βG induces training in bovine monocytes in vitro based on a heightened TNF secretion after stimulation by trained monocyte-derived macrophages with Escherichia coli LPS. Sorted CD14-expressing monocytes (classical and intermediate monocytes), as well as single populations of sorted classical, intermediate and non-classical monocytes could not be trained by βG, whereas macrophages derived from plastic-adherent mononuclear cell preparations displayed features of a trained function. The hypothesis, that non-classical monocytes need to be present in a mixed monocyte population in order to be trained by βG could be verified by a successful training of positively sorted whole monocyte populations (CD14CD16/M) containing all three monocyte subpopulations. The trainability depended on conditions favoring M1 polarization of macrophages. Altogether, innate immune training of bovine monocytes seems to depend on the presence of non-classical monocytes. This adds new information to the role of this monocyte subpopulation in the bovine immune system.

Abstract Background Transition period (TP) is characterised by physiological and metabolic changes contributing to immunodysregulation. Since knowledge about this period in sheep is scarce, we analysed changes in selected immune variables during the TP in ewes and whether dietary magnesium (Mg) supplementation could modulate these immune variables. Pregnant ewes (2nd and 3rd lactation) were divided into a control group (CONT, n = 9) and a Mg group (MAG, n = 10) supplemented with Mg oxide resulting in a daily Mg intake of approximately 0.30 and 0.38% (MAG) of dry matter during ante- (a.p.) and post-partum (p.p.) periods, respectively. Blood samples were collected between days (d) 30 a.p. and d 30 p.p.. Whole blood neutrophil phagocytic activity, monocyte subset (classical cM, intermediate intM, non-classical ncM) composition and the proliferative capacity of lymphocytes were determined flow cytometrically. At d 14 a.p., all ewes were vaccinated against Mycobacterium avium subsp. paratuberculosis (MAP). Results Both groups showed a sharp increase in the total leukocyte counts (TLC) and neutrophil counts (P < 0.0001), at d 1 p.p., while, monocytes and their subpopulations displayed the highest values at d 30 p.p. (P ≤ 0.05). At d 1 p.p. the neutrophil phagocytic activity was higher (P < 0.05) in MAG ewes. Throughout the TP, the proliferative response of CD4+ cells was significantly higher in the MAG group (P < 0.05). Ewes in both groups responded with an increase in the TLC, neutrophil numbers (P ≤ 0.05) and ncM (P

Abstract 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.

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.

Abstract 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.

Feed supplements such as Saccharomyces cerevisiae fermentation products (SCFP) alter immune responses in horses. The purpose of this study was to analyze whether a prebiotic activity of the SCFP alters the gut microbiome in horses. Racehorses were fed either SCFP (Olimond BB, OLI, n = 6) or placebo pellets (PLA, n = 5) for 43 days. Fecal microbiota analysis was performed using 16S rRNA gene sequencing. The numbers and function of circulating immune cell subpopulations were analyzed by flow cytometry. SCFP supplementation resulted in non-consistent differences in fecal microbiota between the PLA and OLI during the feeding period. Rather, the individual animal had the highest impact on fecal microbiota composition. OLI and PLA horses displayed the same changes in numbers of blood leukocyte subpopulations over time. One day after a booster vaccination against equine influenza during the feeding period, the alpha diversity of fecal microbiota of PLA horses was significantly higher compared to OLI horses. This suggests that SCFP feeding altered the vaccination-induced spectrum of released mediators, potentially affecting gut microbiota. The overall non-consistent findings argue against a strong prebiotic effect of Olimond BB on the microbiota in racehorses. Fecal microbiota differences between the groups were also noticed outside the feeding period and, hence, are most likely not caused by the SCFP additive.

Camels are domesticated animals that are highly adapted to the extreme desert ecosystem with relatively higher resistance to a wide range of pathogens compared to many other species from the same geographical region. Recently, there has been increased interest in the field of camel immunology. As the progress in the analysis of camel immunoglobulins has previously been covered in many recent reviews, this review intends to summarize published findings related to camel cellular immunology with a focus on the phenotype and functionality of camel leukocyte subpopulations. The review also describes the impact of different physiological (age and pregnancy) and pathological (e.g. infection) conditions on camel immune cells. Despite the progress achieved in the field of camel immunology, there are gaps in our complete understanding of the camel immune system. Questions remain regarding innate recognition mechanisms, the functional characterization of antigen-presenting cells, and the characterization of camel NK and cytotoxic T cells.

Mastitis is one of the major risks for public health and animal welfare in the dairy industry. Two of the most important pathogens to cause mastitis in dairy cattle are Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). While S. aureus generally induces a chronic and subclinical mastitis, E. coli is an important etiological pathogen resulting in an acute and clinical mastitis. The liver plays a central role in both, the metabolic and inflammatory physiology of the dairy cow, which is particularly challenged in the early lactation due to high metabolic and immunological demands. In the current study, we challenged the mammary glands of Holstein cows with S. aureus or E. coli, respectively, mimicking an early lactation infection. We compared the animals' liver transcriptomes with those of untreated controls to investigate the hepatic response of the individuals. Both, S. aureus and E. coli elicited systemic effects on the host after intramammary challenge and seemed to use pathogen-specific targeting strategies to bypass the innate immune system. The most striking result of our study is that we demonstrate for the first time that S. aureus intramammary challenge causes an immune response beyond the original local site of the mastitis. We found that in the peripheral liver tissue defined biological pathways are switched on in a coordinated manner to balance the immune response in the entire organism. TGFB1 signaling plays a crucial role in this context. Important pathways involving actin and integrin, key components of the cytoskeleton, were downregulated in the liver of S. aureus infected cows. In the hepatic transcriptome of E. coli infected cows, important components of the complement system were significantly lower expressed compared to the control cows. Notably, while S. aureus inhibits the cell signaling by Rho GTPases in the liver, E. coli switches the complement system off. Also, metabolic hepatic pathways (e.g., lipid metabolism) are affected after mammary gland challenge, demonstrating that the liver restricts metabolic tasks in favor of the predominant immune response after infection. Our results provide new insights for the infection-induced modifications of the dairy cow's hepatic transcriptome following mastitis.

Saccharomyces cerevisiae (S. cerevisiae) fermentation products (SCFP) are used in animal husbandry as pre- and postbiotic feed supplements. A variety of immunomodulatory effects are noted in many species. The purpose of this study was to test the hypothesis that horses fed with SCFP containing feed additive Olimond BB display a modulated early immune response after influenza vaccination. Six horses received Olimond BB pellets (OLI) and five horses were fed placebo pellets (PLA) for 56 days. On day 40 all horses were vaccinated with a recombinant influenza A/equi-2 vaccine. At the day of vaccination, the groups did not differ in the composition of leukocyte subpopulations and reticulocytes. Twenty-four hours after vaccination total leukocyte counts and numbers of CD4+ T-cells significantly increased in both groups. In PLA horses, the numbers of neutrophil granulocytes significantly increased and numbers of CD8+ T-cells decreased, whereas the numbers of these cell types remained unchanged in OLI horses. Only OLI horses displayed a significant increase in reticulocyte percentages after vaccination. The numbers of lymphocytes, monocytes, CD21+ B-cells, and serum amyloid A levels remained unaffected in both groups after vaccination. Sixteen days after vaccination, PLA and OLI horses differed significantly in their enhanced ELISA IgG titres against Newmarket and Florida Clade 1 influenza strains. The observed differences after vaccination suggest that feed supplementation with Olimond BB leads to modulated early immune responses after influenza vaccination, which may also affect the memory responses after booster vaccination.

Abstract The etiology determines quality and extent of the immune response after udder infection (mastitis). Infections with Gram negative bacteria (e.g. Escherichia coli) will quickly elicit strong inflammation of the udder, fully activate its immune defence via pathogen receptor driven activation of IκB/NF-κB signaling. This often eradicates the pathogen. In contrast, Gram-positive bacteria (e.g. Staphylococcus aureus) will slowly elicit a much weaker inflammation and immune response, frequently resulting in chronic infections. However, it was unclear which immune regulatory pathways are specifically triggered by S. aureus causing this partial immune subversion. We therefore compared in first lactating cows the earliest (1–3 h) udder responses against infection with mastitis causing pathogens of either species. Global transcriptome profiling, bioinformatics analysis and experimental validation of key aspects revealed as S. aureus infection specific features the (i) failure to activating IκB/NF-κB signaling; (ii) activation of the wnt/β-catenin cascade resulting in active suppression of NF-κB signaling and (iii) rearrangement of the actin-cytoskeleton through modulating Rho GTPase regulated pathways. This facilitates invasion of pathogens into host cells. Hence, S. aureus mastitis is characterized by eliciting unbalanced immune suppression rather than inflammation and invasion of S. aureus into the epithelial cells of the host causing sustained infection.

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.

Evidence is emerging that the interaction between male seminal fluid and female tissues promotes fertility, pregnancy, and health of offspring. This includes the acceleration of ovulation in a species known as a spontaneous ovulator, the domestic pig. Earlier studies revealed that seminal plasma acts by a local mechanism in the female pig. The aim of the present study was to examine local short-term and mid-term effects of seminal plasma (SP) on mRNA expression of immunoregulatory genes and transcripts associated with follicle- and oocyte maturation. In the porcine animal model, effects on mRNA expression in the female tract and preovulatory follicles were examined. SP suppressed mRNA expression of Prostaglandin-Endoperoxide Synthase 2 (PTGS2) ipsilateral to the infused uterine horn which was associated with a lower presence of immune cells in the uterine epithelium and lower PTGS2 immunoreaction. Depending on the sampling time (2 h vs. 17 h) and hormonal status, SP altered significant correlative relations of mRNA expression between PTGS2 and the transcripts Tumor Necrosis Factor Alpha, Tumor Necrosis Factor Alpha-Induced Protein 6 and Pentraxin 3 in uterus, granulosa and cumulus cells. A modulatory effect of SP on the oocyte gene network comprising eight oocyte transcripts was observed: uterine exposure to SP induced positive correlations (r >0.08, p

Peripheral blood monocytes of several species can be divided into different subpopulations with distinct phenotypic and functional properties. Herein, we aim at reviewing published work regarding the heterogeneity of the recently characterized bovine monocyte subsets. As the heterogeneity of human blood monocytes was widely studied and reviewed, this work focuses on comparing bovine monocyte subsets with their human counterparts regarding their phenotype, adhesion and migration properties, inflammatory and antimicrobial functions, and their ability to interact with neutrophilic granulocytes. In addition, the differentiation of monocyte subsets into functionally polarized macrophages is discussed. Regarding phenotype and distribution in blood, bovine monocyte subsets share similarities with their human counterparts. However, many functional differences exist between monocyte subsets from the two species. In contrast to their pro-inflammatory functions in human, bovine non-classical monocytes show the lowest phagocytosis and reactive oxygen species generation capacity, an absent ability to produce the pro-inflammatory cytokine IL-1β after inflammasome activation, and do not have a role in the early recruitment of neutrophils into inflamed tissues. Classical and intermediate monocytes of both species also differ in their response toward major monocyte-attracting chemokines (CCL2 and CCL5) and neutrophil degranulation products (DGP) in vitro. Such differences between homologous monocyte subsets also extend to the development of monocyte-derived macrophages under the influence of chemokines like CCL5 and neutrophil DGP. Whereas the latter induce the differentiation of M1-polarized macrophages in human, bovine monocyte-derived macrophages develop a mixed M1/M2 macrophage phenotype. Although only a few bovine clinical trials analyzed the correlation between changes in monocyte composition and disease, they suggest that functional differences between human and bovine monocyte subsets are also reflected in their different clinical relevance for distinct diseases. In opposite to the human system, where higher blood cell number of non-classical monocytes was widely correlated with several human infectious and non-infectious diseases, higher counts of bovine intermediate monocytes are suggested as a potential biomarker for inflammatory responses postpartum.

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 × 4 Latin square model with three successively increasing lipid dosages (coconut oil, linseed−safflower oil mix (EFA; rich in n-3 FA), Lutalin®, 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.

Fatty acids may have an impact on immune functions, which is important in times of increased mobilization of body fat, e.g., around parturition. The aim of the present study was to investigate the effects of the CLA isomers cis-9,trans-11 and trans-10,cis-12, phytanic acid (PA), linoleic acid (LA) and a fatty acid (FA) mixture (containing 29.8% palmitic acid, 6.7% palmitoleic acid, 17.4% stearic acid and 46.1% oleic acid) on the proliferation of bovine blood mononuclear cells (PBMC) in vitro using alamar blue (AB) and 5-bromo-2′-deoxyuridine (BrdU) assay. Quantitative real time polymerase chain reaction analyses were performed to evaluate the expression of interleukin (IL)-4, IL-10, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and peroxisome proliferator-activated receptor (PPAR)-γ in response to cis-9,trans-11 and LA. The IC50 values did not differ between the investigated FA, but there were differences within the proliferation in the response of these FA in a concentration range between 20 and 148 µM (e.g., increased proliferation after treatment with lower concentrations of LA). No differences occurred when different FA combinations were tested. ConA stimulation increased the expression of TNF-α and IFN-γ, whereas IL-10 decreased. In general, neither the baseline expression nor the ConA-stimulated mRNA expression of cytokines and PPAR-γ were affected by the FA. In conclusion, all FA inhibit the proliferation of PBMC dose dependently without significantly altering the induced cytokine spectrum of activated bovine PBMC.

Murine and human peripheral blood monocytes are heterogeneous in size, granularity, nuclear morphology, phenotype and function. Whether and how bovine blood monocytes follow this pattern was analyzed in this study. Flow cytometrically, classical monocytes (cM) CD14⁺CD16⁻, intermediate monocytes (intM) CD14⁺ CD16⁺ and nonclassical monocytes (ncM) CD14⁺ CD16⁺ were identified, with cM being the predominant subset (89%). cM showed a significant lower expression of CD172a, intM expressed the highest level of MHC class II molecules, and ncM were low positive for CD163. Compared to cM and intM, ncM showed a significantly reduced phagocytosis capacity, a significantly reduced generation of reactive oxygen species, and reduced mRNA expression of CXCL8, CXCL1 and IL-1β after LPS stimulation. Based on IL-1β secretion after LPS/ATP stimulation, the inflammasome could be activated in cM and intM, but not in ncM. IFNγ increased the expression of CD16 selectively on cM and induced a shift from cM into intM in vitro. In summary, bovine CD172a-positive mononuclear cells define three monocyte subsets with distinct phenotypic and functional differences. Bovine cM and intM share homologies with their human counterparts, whereas bovine ncM are not inflammatory monocytes.

Prostaglandin F(2α) (PGF(2α)) induces luteolysis within a few days in cows, and immune cells increase in number in the regressing corpus luteum (CL), implying that luteolysis is an inflammatory-like immune response. We investigated the rapid change in polymorphonuclear neutrophil (PMN) numbers in response to PGF(2α) administration as the first cells recruited to inflammatory sites, together with mRNA of interleukin-8 (IL-8: neutrophil chemoattractant) and P-selectin (leukocyte adhesion molecule) in the bovine CL. CLs were collected by ovariectomy at various times after PGF(2α) injection. The number of PMNs was increased at 5 min after PGF(2α) administration, whereas IL-8 and P-selectin mRNA increased at 30 min and 2 h, respectively. PGF(2α) directly stimulated P-selectin protein expression at 5-30 min in luteal endothelial cells (LECs). Moreover, PGF(2α) enhanced PMN adhesion to LECs, and this enhancement by PGF(2α) was inhibited by anti-P-selectin antibody, suggesting that P-selectin expression by PGF(2α) is crucial in PMN migration. In conclusion, PGF(2α) rapidly induces the accumulation of PMNs into the bovine CL at 5 min and enhances PMN adhesion via P-selectin expression in LECs. It is suggested that luteolytic cascade by PGF(2α) may involve an acute inflammatory-like response due to rapidly infiltrated PMNs.

Experimental leishmaniasis is an excellent model system for analyzing Th1/Th2 differentiation. Resistance to Leishmania (L.) major depends on the development of a L. major specific Th1 response, while Th2 differentiation results in susceptibility. There is growing evidence that the microenvironment of the early affected tissue delivers the initial triggers for Th-cell differentiation. To analyze this we studied differential gene expression in infected skin of resistant and susceptible mice 16h after parasite inoculation. Employing microarray technology, bioinformatics, laser-microdissection and in-situ-hybridization we found that the epidermis was the major source of immunomodulatory mediators. This epidermal gene induction was significantly stronger in resistant mice especially for several genes known to promote Th1 differentiation (IL-12, IL-1beta, osteopontin, IL-4) and for IL-6. Expression of these cytokines was temporally restricted to the crucial time of Th1/2 differentiation. Moreover, we revealed a stronger epidermal up-regulation of IL-6 in the epidermis of resistant mice. Accordingly, early local neutralization of IL-4 in resistant mice resulted in a Th2 switch and mice with a selective IL-6 deficiency in non-hematopoietic cells showed a Th2 switch and dramatic deterioration of disease. Thus, our data indicate for the first time that epidermal cytokine expression is a decisive factor in the generation of protective Th1 immunity and contributes to the outcome of infection with this important human pathogen.