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Essential fatty acids (EFA) and conjugated linoleic acids (CLA) are unsaturated fatty acids with immune-modulatory effects, yet their synergistic effect is poorly understood in dairy cows. This study aimed at identifying differentially abundant proteins (DAP) and their associated pathways in dairy cows supplied with a combination of EFA and CLA during the transition from antepartum (AP) to early postpartum (PP). Sixteen Holstein cows were abomasally infused with coconut oil as a control (CTRL) or a mixture of EFA (linseed + safflower oil) and CLA (Lutalin, BASF) (EFA + CLA) from − 63 to + 63 days relative to parturition. Label-free quantitative proteomics was performed on plasma samples collected at days − 21, + 1, + 28, and + 63. During the transition time, DAP, consisting of a cluster of apolipoproteins (APO), including APOE, APOH, and APOB, along with a cluster of immune-related proteins, were related to complement and coagulation cascades, inflammatory response, and cholesterol metabolism. In response to EFA + CLA, specific APO comprising APOC3, APOA1, APOA4, and APOC4 were increased in a time-dependent manner; they were linked to triglyceride-enriched lipoprotein metabolisms and immune function. Altogether, these results provide new insights into metabolic and immune adaptation and crosstalk between them in transition dairy cows divergent in EFA + CLA status.

Monocytes and macrophages belong to the mononuclear phagocyte system and play important roles in both physiological and pathological processes. The cells belonging to the monocyte/macrophage system are structurally and functionally heterogeneous. Several subsets of monocytes have been previously identified in mammalian blood, generating different subpopulations of macrophages in tissues. Although their distribution and phenotype are similar to their human counterpart, bovine monocytes and macrophages feature differences in both functions and purification procedures. The specific roles that monocytes and macrophages fulfil in several important diseases of bovine species, including among the others tuberculosis and paratuberculosis, brucellosis or the disease related to peripartum, remain still partially elusive. The purpose of this review is to discuss the current knowledge of bovine monocytes and macrophages. We will describe methods for their purification and characterization of their major functions, including chemotaxis, phagocytosis and killing, oxidative burst, apoptosis and necrosis. An overview of the flow cytometry and morphological procedures, including cytology, histology and immunohistochemistry, that are currently utilized to describe monocyte and macrophage main populations and functions is presented as well.

A growing body of evidence shows that microRNA (miRNA), play important roles in regulating adipose tissue (AT) physiology and function. The objective was to characterize the AT miRNA profile in over-conditioned (HBCS, n = 19) versus moderate-conditioned (MBCS, n = 19) periparturient dairy cows. Tail-head subcutaneous AT biopsied on d -49 and 21 relative to parturition were used for miRNA sequencing. The miR-486 was the most significant miRNA among the upregulated miRNA on d -49, which might be related to more pronounced changes in lipogenesis and altered insulin sensitivity in AT of HBCS cows at dry-off. Comparing HBCS to MBCS on d 21, 23 miRNA were downregulated and 20 were upregulated. The predicted targets of upregulated differentially expressed (DE)-miRNA on d 21 were enriched in different pathways, including pathways related to lysosomes and peroxisomes. The predicted targets of downregulated DE-miRNA on d 21 were enriched in various pathways, including epidermal growth factor receptor, insulin resistance, hypoxia-inducible factor 1 signaling pathway, and autophagy. The results showed that over-conditioning was associated with changes in SCAT miRNA profile mainly on d 21, of which most were downregulated. The enriched pathways may participate in over-conditioning-associated metabolic challenges during early lactation.

The objective of this study was to determine the circulating microRNA (miRNA) profile in over-conditioned (HBCS) versus normal-conditioned (NBCS) dairy cows in combination with pathway enrichment analyses during the transition period. Thirty-eight multiparous Holstein cows were selected 15 wk before anticipated calving date based on their current and previous body condition scores (BCS) for forming either a HBCS group (n = 19) or a NBCS group (n = 19). They were fed different diets during late lactation to reach the targeted differences in BCS and backfat thickness until dry-off. A subset of 15 animals per group was selected based on their circulating concentrations of nonesterified fatty acids (on d 14 postpartum) and β-hydroxybutyrate (on d 21 postpartum), representing the greater or the lower extreme values within their BCS group. Blood serum obtained at d -49 and 21 relative to parturition (3 pools with 5 cows per each group and time point) were used to identify miRNA that were differentially expressed (DE) between groups or time points using miRNA sequencing. No DE-miRNA were discovered between NBCS versus HBCS. Comparing pooled samples from d -49 and d 21 resulted in 7 DE-miRNA in the NBCS group, of which 5 miRNA were downregulated and 2 miRNA were overexpressed on d 21 versus -49. The abundance of 5 of these DE-miRNA was validated in all individual samples via quantitative PCR and extended to additional time points (d -7, 3, 84). Group differences were observed for miR-148a, miR-122 as well as miR-455-5p, and most DE-miRNA (miR-148a, miR-122, miR-30a, miR-450b, miR-455-5p) were downregulated directly after calving. Subsequently, the DE-miRNA was used for bioinformatics analysis to identify putative target genes and the most enriched biological pathways. The most significantly enriched pathways of DE-miRNA were associated with cell cycle and insulin signaling as well as glucose and lipid metabolism. Overall, we found little differences in circulating miRNA in HBCS versus NBCS cows around calving.

The conjugated linoleic acid (CLA) isomers, a group of naturally occurring isomers of the essential fatty acid (FA) linoleic acid, have received special attention in animal and human nutrition. Although they have long been used as dietary integrators in dairy cows, the effects of CLA isomers on bovine immune cells remain mostly undisclosed. The present study aimed to cover this gap and investigate the in vitro effects of CLA on inflammatory functions, including chemotaxis, phagocytosis, killing capability, and extracellular respiratory burst of purified bovine monocytes (CD14+). The apoptosis rate of monocytes was addressed as well. Once assessed, the effects of different concentrations (10, 50, 100, and 500 μM) of the 2 main CLA isomers, namely cis-9,trans-11 and trans-10,cis-12, the experiments were carried out using a concentration of 50 μM of the CLA isomers, both individually and in a mixture (50:50). The immunomodulatory activities of linoleic acid, an essential FA, and stearic acid, a saturated FA, were also investigated. Only the 50:50 CLA mixture was able to reduce monocyte apoptosis and to increase the extracellular respiratory burst during experimental proinflammatory conditions, as assessed by measuring production of reactive oxygen species. Linoleic acid and CLA had no effects on chemotaxis, phagocytosis, or killing capability. Remarkably, treatment of monocytes with stearic acid significantly reduced their chemotactic capability. The present results demonstrated that CLA isomers do have immunomodulatory effects on some functions of bovine monocytes, and that the mixture of the 2 CLA isomers is more effective than the CLA isomers individually.

International audience; This study aimed at investigating the synergistic effects of essential fatty acids (EFA) and conjugated linoleic acids (CLA) on the liver proteome profile of dairy cows during the transition to lactation. 16 Holstein cows were infused from 9 wk. antepartum to 9 wk. postpartum into the abomasum with either coconut oil (CTRL) or a mixture of EFA (linseed + safflower oil) and CLA (EFA + CLA). Label-free quantitative proteomics was performed in liver tissue biopsied at days -21, +1, +28, and + 63 relative to calving. Differentially abundant proteins (DAP) between treatment groups were identified at the intersection between a multivariate and a univariate analysis. In total, 1680 proteins were identified at each time point, of which between groups DAP were assigned to the metabolism of xenobiotics by cytochrome P450, drug metabolism - cytochrome P450, steroid hormone biosynthesis, glycolysis/gluconeogenesis, and glutathione metabolism. Cytochrome P450, as a central hub, enriched with specific CYP enzymes comprising: CYP51A1 (d - 21), CYP1A1 & CYP4F2 (d + 28), and CYP4V2 (d + 63). Collectively, supplementation of EFA + CLA in transition cows impacted hepatic lipid metabolism and enriched several common biological pathways at all time points that were mainly related to ω-oxidation of fatty acids through the Cytochrome p450 pathway. SIGNIFICANCE: In three aspects this manuscript is notable. First, this is among the first longitudinal proteomics studies in nutrition of dairy cows. The selected time points are critical periods around parturition with profound endocrine and metabolic adaptations. Second, our findings provided novel information on key drivers of biologically relevant pathways suggested according to previously reported performance, zootechnical, and metabolism data (already published elsewhere). Third, our results revealed the role of cytochrome P450 that is hardly investigated, and of ω-oxidation pathways in the metabolism of fatty acids with the involvement of specific enzymes.

International audience; Repeated measurements analysis of variance - simultaneous component analysis (ASCA) has been developed to handle complex longitudinal omics datasets and combine novel information with existing data. Herein, we aimed at applying ASCA to 64 liver proteomes collected at 4-time points (day -21, +1, +28, and + 63 relative to parturition) from 16 Holstein cows treated from 9 wk. antepartum to 9 wk. postpartum (PP) with coconut oil (CTRL) or a mixture of essential fatty acids (EFA) and conjugated linoleic acid (CLA) (EFA + CLA). The ASCA modeled 116, 43, and 97 differentially abundant proteins (DAP) during the transition to lactation, between CTRL and EFA + CLA, and their interaction, respectively. Time-dependent DAP were annotated to pathways related to the metabolism of carbohydrates, FA, and amino acid in the PP period. The DAP between FA and the interaction effect were annotated to the metabolism of xenobiotics by cytochrome P450, drug metabolism - cytochrome P450, retinol metabolism, and steroid hormone biosynthesis. Collectively, ASCA provided novel information on molecular markers of metabolic adaptations and their interactions with EFA + CLA supplementation. Bioinformatics analysis suggested that supplemental EFA + CLA amplified hepatic FA oxidation; cytochrome P450 was enriched to maintain metabolic homeostasis by oxidation/detoxification of endogenous compounds and xenobiotics.Significance: This report is among the first ones applying repeated measurement analysis of variance-simultaneous component analysis (ASCA) to deal with longitudinal proteomics results. ASCA separately identified differentially abundant proteins (DAP) in 'transition time', 'between fatty acid treatments', and 'their interaction'. We first identified the molecular signature of hepatic metabolic adaptations during postpartum negative energy balance; the enriched pathways were well-known pathways related to mobilizing fatty acids (FA) and amino acids to support continuous energy production through fatty acid oxidation, TCA cycle, and gluconeogenesis. Some of the DAP were not previously reported in transition dairy cows. Secondly, we provide novel information on the mechanisms by which supplemented essential FA and conjugated linoleic acids interact with hepatic metabolism. In this regard, FA amplified hepatic detoxifying and oxidation capacity through ligand activation of nuclear receptors. Finally, we briefly compared the strengths and weaknesses of the ASCA model with PLS-DA and outlined why these methods are complementary.

Mastitis by non-aureus staphylococci (NAS) is a significant issue in dairy buffalo farming. In a herd with subclinical NAS mastitis, we identified Staphylococcus microti as the predominant species. To assess milk protein integrity and investigate potential disease markers, we characterized 12 NAS-positive and 12 healthy quarter milk samples by shotgun peptidomics combining peptide enrichment and high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS). We observed significant changes in the milk peptidome. Out of 789 total peptides identified in each group, 49 and 44 were unique or increased in NAS-positive and healthy milk, respectively. In NAS-positive milk, the differential peptides belonged mainly to caseins, followed by milk fat globule membrane proteins (MFGMP) and by the immune defense/antimicrobial proteins osteopontin, lactoperoxidase, and serum amyloid A. In healthy milk, these belonged mainly to MFGMP, followed by caseins. In terms of abundance, peptides from MFGMP and immune defense protein were higher in NAS-positive milk, while peptides from caseins were higher in healthy milk. These findings highlight the impact of NAS on buffalo milk quality and mammary gland health, even when clinical signs are not evident, and underscore the need for clarifying the epidemiology and relevance of the different NAS species in this dairy ruminant.

Tuberculosis (TB) is a zoonotic disease primarily caused by pathogens belonging to the genus of Mycobacterium. Programs of control and eradication for bovine TB include a screening using single intradermal tuberculin (SIT) test with Mycobacterium bovis (M. bovis)-purified protein derivatives (PPD-B) single or concurrent with Mycobacterium avium (M. avium)-purified protein derivatives (PPD-A). This study aimed to determine the effects of intradermal PPD-B and PPD-A test on immune-related mRNA and microRNAs in dermal oedema exudates of water buffaloes (Bubalus bubalis). The investigation was carried out on RNA extracted from dermal oedema exudates of 36 animals, of which 24 were M. bovis positive (M. bovis+) and 12 M. avium positive (M. avium+). The lymphocyte polarization toward Th1, Th2, TReg, and Th17 lineages was addressed by measuring the abundance of the respective cytokines and transcription factors, namely TBET, STAT4, IFNγ, and IL1β for Th1; STAT5B, and IL4 for Th2; FOXP3 and IL10 for TReg; and RORC, STAT3, and IL17A for Th17. Due to the very low abundance of Th17-related genes, a digital PCR protocol was also applied. The abundance of microRNAs involved in the immune response against PPDs, including miR-122-5p, miR-148a-3p, miR30a, and miR-455-5p, was equally measured. Results showed that IFNγ (fold change = 2.54; p = 0.037) and miR-148a-3p (fold change = 2.54; p = 0.03) were upregulated in M. bovis+ as compared to M. avium+ samples. Our preliminary results supported the pivotal role of IFNγ in the local immune response related to PPD-B and highlighted the differential expression of miR-148a-3p, which downregulates the proinflammatory cytokines and the TLR4-mediated NF-κB activation, providing an anti-inflammation modulator in responses to mycobacterial infection.

This observational study determined the lipidome of cow milk during subclinical intramammary infection (IMI) by non-aureus staphylococci (NAS), also defined as coagulase-negative staphylococci, using an untargeted approach. Among the pathogens causing bovine IMI, NAS have become the most frequently isolated bacteria from milk samples. Although the application of system biology approaches to mastitis has provided pivotal information by investigating the transcriptome, proteome, peptidome, and metabolome, the milk lipidome during mammary gland inflammation remains undisclosed. To cover this gap, we determined the milk lipidome of 17 dairy cows with IMI caused by NAS (NAS-IMI), and we compared the results with those of healthy quarter milk from 11 cows. The lipidome was determined following a liquid chromatography-quadrupole time-of-flight mass spectrometry approach. Sixteen subclasses of lipids were identified in both groups of animals. From 2,556 measured lipids, the abundance of 597 changed more than 10-fold in quarter milk with NAS-IMI compared with healthy quarters. The results demonstrate the influence of NAS-IMI on the milk lipidome, implying significant changes in lipid species belonging to the family of triacylglycerols and sphingomyelins, and contribute to the understanding of inflammatory processes in the bovine udder, highlighting potential novel biomarkers for improving mastitis diagnostics.

Coagulase-negative staphylococci (CNS) are the most prevalent microorganisms isolated from cow milk and are associated with subclinical mastitis and persistent increases in the bulk milk somatic cell count (BMSCC) of low BMSCC herds. By combining peptide enrichment, LC-ESI-MS/MS, and statistical analysis, we investigated the influence of subclinical mastitis and CNS infection on the milk peptidome. Quarter milk samples from clinically healthy Holstein cows were subjected to bacteriological culture (BC) and somatic cell counting (SCC) for two consecutive samplings and 28 (including 11 negatives and 17 positives) were selected for peptidomic analysis. The study identified 1363 different endogenous peptides and highlighted a significant increase of peptides in CNS-positive milk, mainly represented by casein fragments. Milk peptidome changes increased with the SCC, as also demonstrated by protein electrophoresis and densitometry. Peptides significantly different in CNS or CONTROL samples were identified and characterized. Our results indicate that subclinical mastitis by CNS can induce significant changes in the milk peptidome, opening the way to future studies for the identification of a biomarker panel as well as for the understanding of their consequences for the technological and sensorial characteristics of cow milk and dairy products. SIGNIFICANCE: This is the first investigation on the impact of subclinical CNS mastitis on the bovine milk peptidome. The peptide enrichment strategy combined with a highly sensitive MS/MS analysis enabled the compilation of a very large peptide dataset for healthy and mastitic milk. The comparison of CNS and Control samples, also considering SCC classes, highlighted several peptides with potential for understanding milk protein and peptide dynamics in subclinical mastitis, with possible implications for its detection.

The transition from late pregnancy to early lactation is a critical period in a dairy cow's life due to the rapidly increasing drain of nutrients from the maternal organism towards the foetus and into colostrum and milk. In order to cope with the challenges of parturition and lactation, comprehensive adaptive reactions comprising the endocrine and the immune system need to be accomplished. There is high variation in this coping ability and both metabolic and infectious diseases, summarized as “production diseases”, such as hypocalcaemia (milk fever), fatty liver syndrome, laminitis and ketosis, may occur and impact welfare, productive lifespan and economic outcomes. Proteomics and metabolomics have emerged as valuable techniques to characterize proteins and metabolite assets from tissue and biological fluids, such as milk, blood and urine. In this review we provide an overview on metabolic status and physiological changes during the transition period and the related production diseases in dairy cows, and summarize the state of art on proteomics and metabolomics of biological fluids and tissues involved in metabolic stress during the peripartum period. We also provide a current and prospective view of the application of the recent achievements generated by omics for biomarker discovery and their potential in diagnosis. Biological significance For high-yielding dairy cows there are several “occupational diseases” that occur mainly during the metabolic challenges related to the transition from pregnancy to lactation. Such diseases and their sequelae form a major concern for dairy production, and often lead to early culling of animals. Beside the economical perspective, metabolic stress may severely influence animal welfare. There is a multitude of studies about the metabolic backgrounds of such so called production diseases like ketosis, fatty liver, or hypocalcaemia, although the investigations aiming to assess the complexity of the pathophysiological reactions are largely focused on gene expression, i.e. transcriptomics. For extending the knowledge towards the proteome and the metabolome, the respective technologies are of increasing importance and can provide an overall view of how dairy cows react to metabolic stress, which is needed for an in-depth understanding of the molecular mechanisms of the related diseases. We herein review the current findings from studies applying proteomics and metabolomics to transition-related diseases, including fatty liver, ketosis, endometritis, hypocalcaemia and laminitis. For each disease, a brief overview of the up to date knowledge about its pathogenesis is provided, followed by an insight into the most recent achievements on the proteome and metabolome of tissues and biological fluids, such as blood serum and urine, highlighting potential biomarkers. We believe that this review would help readers to be become more familiar with the recent progresses of molecular background of transition-related diseases thus encouraging research in this field.

Staphylococcus aureus biofilm-related infections are of clinical concern due to the capability of bacterial colonies to adapt to a hostile environment. The present study investigated the capability of the acute phase protein alpha

The concept that ruminant mammary gland quarters are anatomically and physiologically unrelated has been recently challenged by immunological evidence. How this interdependence reflects on individual quarter milk microbiota is unknown. The aim of the present study was to cover this gap by investigating the interdependence of quarters among the same mammary gland at the milk microbiota level using next-generation sequencing of the V4-16S rRNA gene. A total of 52 samples were included in this study and classified as healthy or affected by subclinical mastitis. Extraction of DNA, amplification of the V4-16S rRNA gene, and sequencing using Ion Torrent Personal Genome Machine (Thermo Fisher Scientific, Waltham, MA) were carried out. We found that the intra-individual variability was lower than the inter-individual one. The present findings further support at milk microbiota level the hypothesis of the interdependence of quarters, as previously demonstrated following immunological studies, suggesting that individual factors (e.g., immunity, genetics) may have a role in modulating milk microbiota.

Altres ajuts: PRC/IZS/ME/10/13 RC. AC Water buffalo mastitis represents a major issue in terms of animal health, cost of therapy, premature culling and decreased milk yeld. The emergence of antibiotic resistance has led to investigate strategies to avoid or reduce antibiotics' based therapies, in particular during subclinical mastitis. The use of Generally Regarded As Safe bacteria (GRAS) such as Lactobacillus rhamnosus to restore the unbalance in mammary gland microbiota could provide potential corrective measures. The aim of this study was to investigate the changes in milk microbiota after the intramammary treatment with inactivated cultures of Lactobacillus rhamnosus of mammary gland quarters naturally affected by subclinical mastitis as compared to antibiotic therapy.A number of 43 quarters affected by subclinical mastitis with no signs of clinical inflammation and aerobic culture positive for pathogens were included in the study. The experimental design was as follows: 11 quarters were treated with antibiotics, 15 with inactivated cultures of Lactobacillus rhmnosus and 17 with PBS as negative control, by means of intrammary injection. Samples were collected at eight time points, pre- (T-29, T-21, T-15, T-7, T0 days) and post- treatment (T1, T2, and T6 days). Microbiological culture and Somatic Cell Count (SCC) were perfomed on all the samples, and microbiota was determined on milk samples collected at T0 and T6 by amplifying the V4 region of 16S rRNA gene by PCR and sequencing using next generation sequencing technique. Treatment with Lactobacillus rhamnosus elicited a strong chemotactic response, as determined by a significant increase of leukocytes in milk, but did not change the microbiological culture results of the treated quarters. For what concerns the analysis of the microbiota, the treatment with Lactobacillus rhamnosus induced the modification in relative abundance of some genera such as Pseudomonas and 5-7N15. As expected, antibiotic treatment caused major changes in microbiota structure with an increase of Methylobacterium relative abundance. No changes were detected after PBS treatment. In conclusion, the present findings demonstrated that the in vivo intrammmary treatment with Lactobacillus rhamnosus has a transient pro-inflammatory activity by increasing SCC and is capable to modify the microbiota of milk after six days from inoculation, albeit slightly, even when the bacterial cultures were heat inactivated. Further studies are necessary to assess the potential use of this GRAS as supportive therapy against mastitis.

Heat stress (HS) has a negative impact on dairy cows' health, milk production, reproductive performance and immune defenses. Cellular and molecular responses to high temperatures in bovine polymorphonuclear cells and peripheral blood mononuclear cells (PBMCs) have been investigated so far. On the contrary, the effects of high temperatures on isolated monocytes remain almost undisclosed. The aim of this study was to unravel the in vitro effects of high temperatures, simulating a severe HS related body hyperthermia, on bovine lifespan and M1/M2 polarisation. The PBMCs were isolated from whole blood of 9 healthy dairy cattle. Monocytes were sorted by magnetic activated cell sorting and cultured over night at 39 °C (normothermia) or 41 °C (HS). Apoptotic rate and viability were assessed and mRNA abundance for heat shock proteins (HSPs), heat transcription factors (HSFs) and genes involved in monocyte/macrophage polarization (STAT1, STAT2, STAT3, STAT6, IL1β, TGF1β, IL-10, COX2) were quantified by qPCR. We found that apoptosis increased in monocytes exposed to 41 °C, as compared to control, while viability conversely decreased. HS increased the abundance of HSF1 and HSP70. The concomitant decrease of STAT1 and STAT2 and the increase of STAT6 genes abundance at 41 °C suggest, at transcriptional factors level, a polarization of monocytes from a classical activated M1 to a non-classically activated M2 monocytes. In conclusion, the exposure of bovine monocytes to high temperatures affects their lifespan as well as the abundance of genes involved in HS response and in monocyte/macrophages polarization phenotype, confirming that bovine immune response may be significantly affected by hyperthermia.

Bovine viral diarrhea virus (BVDV) has been detected in peripheral blood mononuclear cells (PBMCs) of immunocompetent animals, not being clear whether the development of a specific humoral immune response can prevent BVDV infection. The aim of this study was to evaluate the ability of non-cytopathic BVDV to replicate and produce infectious virus in PBMCs from calves pre-infected with BVDV and to elucidate the immunomodulatory effect of BVDV on these cells in an in vitro model. Quantification of virus was by quantitative PCR, while its replicative capacity and shedding into the extracellular environment was evaluated by viral titration. Apoptosis was assessed by flow cytometry analysis of annexin V and propidium iodide, and by expression of caspase-3/7. Flow cytometry was used to analyze the expression of CD14/CD11b/CD80, CD4/CD8/CD25, MHC-I/MHC-II and B-B2 markers. Our results showed that PBMCs from cattle naturally infected with BVDV were more susceptible to in vitro BVDV infection and showed a more severe apoptosis response than those from naïve animals. Non-cytopathic BVDV in vitro infection also resulted in a lack of effect in the expression of antigen presentation surface markers. All these findings could be related to the immunosuppressive capacity of BVDV and the susceptibility of cattle to this infection.

The serum amyloid A (SAA) and haptoglobin (Hp) are the most prominent acute phase proteins (APPs) in cow. Liver mainly produces APPs, but extra hepatic expression has also been demonstrated in some tissues. The major aim of the present study was to assess the constitutive SAA and Hp mRNA expression by quantitative PCR (qPCR) in a wide panel of 33 bovine tissues, including gastrointestinal tract, respiratory system, urogenital system, mammary gland, hematopoietic system, central nervous system, eye, thyroid and heart. Normalization of gene expression in different samples requires reference genes, which are stably expressed. Therefore, seven reference genes were investigated (ACTB, GAPDH, HMBS, SDHA, YWHAZ, SF3A1, EEF1A2) and three genes, namely SF3A1, HMBS and ACTB, were selected after assessing their stability with geNorm™ and NormFinder(©) softwares. The qPCR analysis confirmed liver as the principal source of SAA and Hp, but also identified both APPs' mRNA in almost all tissues. The highest expression rate of SAA was found in thyroid, followed by pancreas and submandibulary gland. Hp mRNA expression was detected at high concentration in pancreas and submandibulary gland. The present data indicated a widespread expression of SAA and Hp also in non pathological conditions, thus envisaging a possible role as immunomodulatory and protective molecules. To understand where SAA and Hp come from is the prerequisite to their utilization as Acute Phase Reaction biomarkers.

Iron is an essential mineral required for many functions in animal physiology. The iron redox couple reaction mediates the transfer of single electrons through the reversible oxidation/reduction of Fe2+ and Fe3+. This redox cycling is useful for many metabolic pathways essential to life, such as cell cycle progression and division, oxygen transport, mitochondrial activity and immune system function. As a consequence of iron redox reactions, the same metabolic pathways generate reactive oxygen species (ROS) that provide the background for oxidative stress (ie, an imbalance between the production of free radicals and the ability of the body to counteract their harmful effects). Iron homeostasis is finely tuned: if dysregulated, conditions of iron overload or iron deficiencies can occur. Iron deficiency effects go beyond the well-known importance of iron in the synthesis and function of haemoglobin and myoglobin (and subsequently oxygen transport), by also having further impact on the oxidative status of animals. Iron is a cofactor of enzymes involved in the scavenging of ROS, namely catalase, peroxidase and superoxide dismutase (SOD)1. Therefore, iron deficiency reduces organism protection against oxidative stress. On the other hand, excess of iron may have similar effects: iron can bind to the Mn-SODs, with similar affinities and geometries as manganese,2,3 resulting in the inactivation …

Combined quantitative (real-time) polymerase chain reaction and immunohistochemistry were used to evaluate the expression of the minor acute phase protein alpha 1-acid glycoprotein (AGP, orosomucoid) in bovine extrahepatic tissues. AGP was produced mainly in the salivary glands and spleen, whereas minor expression was detected in all other tissues sampled, including lung, lymph nodes, uterus, ovary, kidney and tongue. The findings were consistent with immunohistochemical results. In view of the immunomodulatory and direct antibacterial activity of AGP, its expression in the salivary glands may signal an involvement in the regulation of the local immunity, even in non-pathological conditions.

Heat stress exerts a direct negative effect on farm animal health, triggering physiological responses. Environmental high temperature induces immunosuppression in dairy cows, increasing the risk of mastitis and milk somatic cell counts. The influence of heat stress on leukocytes activities has not been fully elucidated. The present in vitro study was aimed at assessing whether the exposure to temperature simulating conditions of severe whole body hyperthermia affects defensive functions of bovine blood polymorphonuclear cells. Blood was collected from seven clinically healthy, multiparous, late lactating Holstein cows. After isolation, PMN were incubated at either 39 or 41°C. Phagocytosis, respiratory burst and apoptosis were then investigated. The selected temperatures of 39°C or 41°C mimicked conditions of normothermia or severe heat stress, respectively. Phagocytosis assay was carried out by measuring the fluorescence of phagocyted fluorescein-labelled E. coli bioparticles. The modulation of oxidative burst activity was studied by the cytochrome C reduction method. Apoptosis was determined by measuring the activities of two enzymes that play an effector role in the process, namely Caspase-3 and Caspase-7. Statistical analyses were performed using SPSS 22.0. A Student t-test for paired samples and a Generalised Estimating Equation were used based on data distribution. The phagocytosis rate was reduced (-37%, P

alpha(1)-Acid glycoprotein (AGP, orosomucoid) is a normal constituent of bovine blood. AGP is an immunocalin, a binding protein that can also exert several immunomodulatory functions. In this paper we investigated the effect of bovine alpha(1)-acid glycoprotein (boAGP) on spontaneous and staurosporine-induced apoptosis of blood derived monocytes purified using magnetic cell sorting techniques. Bovine AGP was purified from blood following a chromatographic protocol. The homogeneous protein was used to stimulate the cells as well to raise a polyclonal antibody, that was used throughout all the experiments. When monocytes were incubated with high concentrations of boAGP (0.9 mg/ml), similar to those found in bovine plasma during systemic reaction to inflammation, their spontaneous apoptosis rate was suppressed, as determined by caspase-3/7 enzymatic activity assay. Similar results were obtained when apoptosis was induced by adding staurosporine, a potent protein kinase inhibitor. The apoptosis-modulating activity of boAGP was dependent on its concentration, since physiological concentrations of boAGP (0.3 mg/ml) did not exhibit a statistically significative anti-apoptotic activity. We also investigated whether this apoptosis-modulating activity was dependent on the terminal sialic acid residues exposed on the surface of the protein. Enzymatic treatment with neuraminidase, that cleaves terminal sialic acid residues, completely abolished boAGP's anti-apoptotic activity. These results suggest that the protective effect of AGP is likely mediated by its sialic acid terminal groups.

International audience; $\alpha$1-acid glycoprotein (AGP) is an immunomodulatory protein expressed by hepatocytes in response to the systemic reaction that follows tissue damage caused by inflammation, infection or trauma. This paper presents the detection of bovine AGP (boAGP) in mammary secretions (colostrum and milk) and mammary gland tissue. Bovine AGP was detected by Western blotting in all the samples analysed, and could be quantified in colostrum at 162 (± 63.7) $\mu$g/mL and 114.5 (± 67.8) $\mu$g/mL during the first 12 h and 24 h respectively. In mature milk, the boAGP concentration clearly decreased and was no longer detectable using the Radial Immunodiffusion (RID) technique. The concentration of mature milk boAGP was therefore semi-quantified using an anion-exchange chromatographic procedure that allowed the concentration of the protein to be determined. The presence of AGP in bovine milk was confirmed by the internal sequence analysis performed following purification to homogeneity of the protein from milk. The concentration of AGP in bovine milk with low SCC (< 250 000) was very similar to that from bovine milk with high SCC (> 250 000). In order to investigate the origin of AGP in bovine milk, a search for mRNA was carried out in somatic cells and mammary gland tissue: mRNA expression of the boAGP gene was detected in mammary gland tissue, but not in somatic cells. Finally, the cDNA sequence of the boAGP was determined, and is hereby presented.

Lipopolysaccharide-binding protein (LBP) is an acute phase protein involved in host response to Gram-negative and Gram-positive pathogens. It is synthesized by hepatocytes and released as 60-65kDa glycoprotein in plasma. Little is known about the distribution of LBP in non-pathological bovine tissues. The aim of the present study was to investigate the extra hepatic expression of LBP in different bovine tissues by qualitative and quantitative real time (RT) PCR. The presence of the protein was also confirmed by immunohistochemistry using an anti-human LBP antibody preliminarily validated by cross-reactivity in bovine tissues. While a wide panel of organs and tissues was investigated, the attention was focused on the digestive tract and mammary gland. Moderate amount of mRNA was detected in most of the tissues involved in this study. Extra hepatic LBP mRNA expression was particularly high in parotid and submandibular salivary glands. Remarkably, LBP mRNA was found in rumen, reticulum and omasum. High expression was also found in the mammary gland. Intensity of protein staining paralleled mRNA expression in most tissues, with the exception of lung, ovary and thyroid gland. The presence of LBP throughout epithelial mucosal tissues is indicative of an important role of LBP in mucosal immunity at sites of bacterial exposure. These results suggest that ruminant forestomachs may mount a local acute phase reaction.

MicroRNAs (miRNAs) are a family of regulatory molecules involved in many physiological processes, including activation of cells of the immune system. This study investigated the effect of Escherichia coli lipopolysaccharide (LPS) and Staphylococcus aureus enterotoxin B (SEB) on the expression of five miRNAs involved in the inflammatory response, including miR-9, miR-125 b, miR-155, miR-146 a and miR-223, in bovine CD14(+) cells (monocytes). Incubation of monocytes with SEB induced down-regulation of miR-155, miR-223 and miR-125 b, but not the anti-inflammatory miRNA miR-146 a. Conversely, incubation with LPS upregulated both miR-155 and miR-146 a. In vitro incubation of isolated CD14(+) bovine monocytes with LPS and SEB elicited different and opposite expression of miRNAs reportedly involved in inflammatory reactions.

In this article the localization of the acute phase protein Serum Amyloid A (SAA) in different depots of bovine adipose tissue (AT) and liver is reported. Quantitative (Real Time) PCR was paired to immunohistochemistry after the production of a specific polyclonal antibody. SAA's mRNA was found in all analyzed AT depots included in the present study, the AT located in the withers being the major source of SAA mRNA. A polyclonal antibody was raised against bovine SAA and was used to validate gene expression analyses. Western Blotting confirmed that SAA is present in all the seven adipose tissue depots include in the present experiment. Anti-SAA polyclonal antibody also stained diffusely adipocytes. In liver, intracytoplasmic immunolabeling was observed in hepatocytes. Staining was generally mild and not diffuse: negative hepatocytes were intermixed with positive ones. A positive intracytoplasmic immunostaining was occasionally observed in endothelial cells lining small blood vessels within AT septa and liver parenchyma. Our data confirm that bovine AT may provide an important source of SAA in healthy subjects. It remains to be determined which is the contribution of AT in the serum concentration of SAA.

Adipose tissue is an endocrine compartment that plays an important role in immune defence by producing and releasing a wide range of proteins, including acute phase proteins (APPs). The liver is the main organ of APP synthesis, although extrahepatic production has also been reported. In the present study, expression of two APPs in dairy cattle, lipopolysaccharide binding protein (LBP) and α1-acid glycoprotein (AGP), was determined in four visceral (pericardial, mesenteric, omental and retroperitoneal) and three subcutaneous (withers, tail head and sternum) adipose tissue depots. mRNA expression was evaluated using qualitative and quantitative PCR, protein profiles were assessed by Western blot analysis and cellular localisation was determined by immunohistochemistry. The presence of LBP and AGP was demonstrated at mRNA and protein levels in all seven adipose tissue depots. Expression of AGP and LBP suggests that they may have roles as local and systemic inflammatory adipokines.

The flavoenzyme d -aspartate oxidase from beef kidney (DASPO, EC 1.4.3.1) has been overexpressed in Escherichia coli. A purification procedure, faster than the one used for the enzyme from the natural source (bDASPO), has been set up yielding about 2 mg of pure recombinant protein (rDASPO) per each gram of wet E. coli paste. rDASPO has been shown to possess the same general biochemical properties of bDASPO, except that the former contains only FAD, while the latter is a mixture of two forms, one active containing FAD and one inactive containing 6-OH-FAD (9–20% depending on the preparation). This results in a slightly higher specific activity (about 15%) for rDASPO compared to bDASPO and in facilitated procedures for apoprotein preparation and reconstitution. Redox potentials of −97 mV and −157 mV were determined for free and l -(+)-tartrate complexed DASPO, respectively, in 0.1 M KPi, pH 7.0, 25°C. The large positive shift in the redox potential of the coenzyme compared to free FAD (−207 mV) is in agreement with similar results obtained with other flavooxidases. rDASPO has been used to assess a possible oxidative activity of the enzyme towards a number of compounds used as agonists or antagonists of neurotransmitters, including d -aspartatic acid, d -glutamic acid, N-methyl- d -aspartic acid, d,l -cysteic acid, d -homocysteic acid, d,l -2-amino-3-phosphonopropanoic acid, d -α-aminoadipic acid, d -aspartic acid-β-hydroxamate, glycyl- d -aspartic acid and cis-2,3-piperidine dicarboxylic acid. Kinetic parameters for each substrate in 50 mM KPi, pH 7.4, 25°C are reported.

A two-dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins has been established. About 250 spots were detected after silver staining and polypeptides from 24 spots have been N-terminally sequenced. Major proteins already described in bull seminal plasma, like PDC-109 and aSFP, have been located on the map; proteins not yet reported in male reproductive tracts have been evidenced; for some polypeptides showing a previously unknown N-terminal sequence, structural similarities with proteins described in other organisms have been found. A reference map of seminal plasma proteins could be useful in relating protein pattern changes to physiopathological events influencing the reproductive sphere.

Adipose tissue is not only a tissue where energy is stored but is also involved in regulating several body functions such as appetite and energy expenditure via its endocrine activity. Moreover, it thereby modulates complex processes like reproduction, inflammation and immune response. The products secreted from adipose tissue comprise hormones and cytokines that are collectively termed as adipocytokines or "adipokines"; the discovery and characterization of new proteins secreted by adipose tissue is still ongoing and their number is thus increasing. Adipokines act in both endocrine manner as well as locally, as autocrine or paracrine effectors. Proteomics has emerged as a valuable technique to characterize both cellular and secreted proteomes from adipose tissues, including those of main cellular fractions, i.e. the adipocytes or the stromal vascular fraction containing mainly adipocyte precursors and immune cells. The scientific interest in adipose tissue is largely based on the worldwide increasing prevalence of obesity in humans; in contrast, obesity is hardly an issue for farmed animals that are fed according to their well-defined needs. Adipose tissue is nevertheless of major importance in these animals, as the adipose percentage of the bodyweight is a major determinant for the efficiency of transferring nutrients from feed into food products and thus for the economic value from meat producing animals. In dairy animals, the importance of adipose tissue is based on its function as stromal structure for the mammary gland and on its role in participating in and regulating of energy metabolism and other functions. Moreover, as pig has recently become an important model organism to study human diseases, the knowledge of adipose tissue metabolism in pig is relevant for the study of obesity and metabolic disorders. We herein provide a general overview of adipose tissue functions and its importance in farm animals. This review will summarize recent achievements in farm animal adipose tissue proteomics, mainly in cattle and pigs, but also in poultry, i.e. chicken and in farmed fish. Proteomics advancement in adipocyte cell lines, have also been included. Adipose tissue is not only a tissue where energy is stored but is also involved in regulating several body functions such as appetite and energy expenditure via its endocrine activity. Moreover, it thereby modulates complex processes like reproduction, inflammation and immune response. The products secreted from adipose tissue comprise hormones and cytokines that are collectively termed as adipocytokines or "adipokines"; the discovery and characterization of new proteins secreted by adipose tissue is still ongoing and their number is thus increasing. Adipokines act in both endocrine manner as well as locally, as autocrine or paracrine effectors. Proteomics has emerged as a valuable technique to characterize both cellular and secreted proteomes from adipose tissues, including those of main cellular fractions, i.e. the adipocytes or the stromal vascular fraction containing mainly adipocyte precursors and immune cells. The scientific interest in adipose tissue is largely based on the worldwide increasing prevalence of obesity in humans; in contrast, obesity is hardly an issue for farmed animals that are fed according to their well-defined needs. Adipose tissue is nevertheless of major importance in these animals, as the adipose percentage of the bodyweight is a major determinant for the efficiency of transferring nutrients from feed into food products and thus for the economic value from meat producing animals. In dairy animals, the importance of adipose tissue is based on its function as stromal structure for the mammary gland and on its role in participating in and regulating of energy metabolism and other functions. Moreover, as pig has recently become an important model organism to study human diseases, the knowledge of adipose tissue metabolism in pig is relevant for the study of obesity and metabolic disorders. We herein provide a general overview of adipose tissue functions and its importance in farm animals. This review will summarize recent achievements in farm animal adipose tissue proteomics, mainly in cattle and pigs, but also in poultry, i.e. chicken and in farmed fish. Proteomics advancement in adipocyte cell lines, have also been included.

Most of the acute intestinal diseases are caused by foodborne pathogens with infants and elderly people being at major risk. The aim of this study was to develop a procedure to simultaneously detect 20 foodborne pathogens in complex alimentary matrices such as milk, cheese and meat. The list of targets include, among the others, Listeria spp., Salmonella spp., Shigella spp., Escherichia coli spp., Campylobacter spp., Clostridium spp. and Staphylococcus aureus. The accuracy of detection was determined by using ATCC strains as positive and negative controls. The achieved sensitivity of each of assays was 1 pg of genomic DNA, which was equivalent to ∼1 cfu. The working ranges of the TaqMan(®) Real-time PCR assays, when used quantitatively on cheese and meat samples inoculated with serial dilution of Listeria spp., Listeria monocytogenes, S. aureus, Salmonella enterica, Shigella boydii, E. coli O157:H7, Bacillus cereus, Campylobacter coli, Yersinia enterocolitica, Enterobacter sakazakii and Pseudomonas aeruginosa was 10(8) cfu/g to 10(4) cfu/g. No matrix interferences were observed.

Advancement in electrophoresis and mass spectrometry techniques along with the recent progresses in genomics, culminating in bovine and pig genome sequencing, widened the potential application of proteomics in the field of veterinary medicine. The aim of the present review is to provide an in-depth perspective about the application of proteomics to animal disease pathogenesis, as well as its utilization in veterinary diagnostics. After an overview on the various proteomic techniques that are currently applied to veterinary sciences, the article focuses on proteomic approaches to animal disease pathogenesis. Included as well are recent achievements in immunoproteomics (ie, the identifications through proteomic techniques of antigen involved in immune response) and histoproteomics (ie, the application of proteomics in tissue processed for immunohistochemistry). Finally, the article focuses on clinical proteomics (ie, the application of proteomics to the identification of new biomarkers of animal diseases).

Serum amyloid A3 (SAA3) is the predominant SAA isoform secreted by mammary epithelial cells in dairy cows; it is also expressed in bovine adipose tissue (AT). The adipokine SAA3 is linked to obesity and insulin resistance of AT and the respective inflammatory response, at least in mice. Dietary treatment with conjugated linoleic acids (CLA) reportedly also affects insulin sensitivity and inflammatory status in monogastrics. Both SAA3 and CLA thus seem to alter similar functions. Based on changes in insulin sensitivity and the inflammatory status throughout lactation, we hypothesized that the mRNA abundance of SAA3 in various tissues might be regulated as well and that CLA could be a modulator of SAA3 mRNA expression. In 2 trials, 21 pluriparous and 25 primiparous Holstein cows were fed 100 g/d of a CLA or a control fat supplement from d 1 to 182 or 105 postpartum, respectively. Biopsies from liver and subcutaneous (s.c.) AT from pluriparous cows and samples from 3 different visceral AT and 3 s.c. AT, muscle, mammary gland, and liver tissue from slaughtered primiparous cows were obtained. In an adipocyte cell culture system, cell samples were collected during differentiation of bovine preadipocytes at d 0, 2, 6, 8, 10, 12, and 13 relative to the onset of differentiation. The SAA3 mRNA abundance in tissues and in differentiating bovine preadipocytes was measured by real-time PCR. The presence of the SAA protein was confirmed by Western blotting. Treatment with CLA yielded only few and inconsistent effects on SAA3 mRNA abundance. In both trials, SAA3 mRNA peaked at d 1 postpartum in all tissues except in mesenteric AT, in which the change was not significant. The highest SAA3 mRNA expression was observed in the mammary gland, followed by omental AT. The SAA protein was present in the visceral and s.c. AT depots investigated. Adipocytes as one source of SAA3 were confirmed by the SAA3 mRNA profile in differentiating adipocytes. The longitudinal changes observed point to SAA3 being involved in the inflammatory situation around parturition.

α1-Acid glycoprotein (AGP) is an acute phase protein that modulates innate immunity and increases in response to infection or injury. The effects of native (phosphorylated) and partially dephosphorylated AGP on the antimicrobial activities of bovine polymorphonuclear leucocytes (PMNs) and monocytes were evaluated. Native AGP inhibited phagocytosis and killing of Escherichia coli by PMNs and monocytes. Engulfment and killing of E. coli were reduced at the acute phase concentration of AGP (0.9 mg/mL) compared with a physiological concentration (0.3mg/mL). The ability of AGP to inhibit phagocytosis by monocytes and the killing of E. coli by PMNs was reduced following dephosphorylation. The findings indicate that the functions of PMNs and monocytes are differentially regulated by varying concentrations of AGP and its phosphorylation state.

The properties of D-aspartate oxidase from Octopus vulgaris (EC 1.4.3.1) have been investigated. The protein is a monomer of M(r) 37,000 containing one mol flavin/mol protein. The enzyme as isolated exists at least in two forms, one containing FAD and the other, which is catalytically inactive, probably containing 6-OH-FAD, as inferred from the absorption spectrum of the enzyme. An additional form of the enzyme, as far as the nature of the coenzyme is concerned, has been detected in the purified enzyme and shown to derive from the form originally containing FAD. The modulation of the coenzyme reactivity exerted by Octopus D-aspartate oxidase, as studied by spectrophotometric techniques, conforms to the one expected for an enzyme belonging to the oxidase class of flavoproteins. Structural investigations show similarities in both the amino-acid composition and the N-terminal amino-acid sequence to bovine D-aspartate oxidase and porcine D-amino-acid oxidase. In summary, the general properties of the enzyme from Octopus vulgaris closely resemble those of the enzyme from beef kidney. Moreover, kinetic analyses suggest that two active-site residues with pKa of 7.1 and 9.1 are critical for catalysis, and that the ionization of such residues has different effects on the catalytic activity depending whether mono- or dicarboxylic D-amino acids are used as substrate.

It is well known that the plasminogen-activating (PA) system plays a key role in the bovine mammary gland during tissue remodelling. However, the modulation of the PA cascade after bacterial infections needs to be elucidated. This study examined the effects of Escherichia coli lipopolysaccharide (LPS) on cell viability, the modulation of cell-associated u-PA activity, and the regulation of u-PA and u-PA receptor (u-PAR) RNA expression using the BME-UV1 bovine mammary epithelial cell line. LPS did not affect cell viability, but induced an increase in u-PA activity, with the maximum response after 6 h of incubation. Moreover, u-PA and u-PAR mRNA expression were both up-regulated in BME-UV1 cells after 3 h of incubation with LPS. These data indicated that E. coli LPS led to an increase in u-PA activity and RNA expression of u-PA and u-PAR in BME-UV1 cells, thus strengthening the role of the PA system during pathological processes.

Acute phase proteins (APPs) are produced mainly by the liver and their concentration is increased during the systemic inflammatory response. Expression of haptoglobin (Hp), serum amyloid A (SAA), lipopolysaccharide-binding protein (LBP) and α-1 acid glycoprotein (AGP) was determined in the mucosa of the normal bovine forestomachs and abomasum by qualitative and quantitative reverse transcriptase-PCR for mRNA and by Western blot analysis and immunohistochemistry for proteins. Although expression of SAA mRNA was evident in the forestomachs and abomasum, SAA protein was identified only in the abomasum. Expression of Hp protein was high in the forestomachs and abomasum, even though expression of Hp mRNA was negligible. The main site of expression of LBP mRNA was the omasum, whereas the highest protein expression was evident in the abomasum. AGP was expressed at low levels in the bovine forestomachs. Western blot analysis revealed a heterogeneous electrophoretic pattern for AGP, LBP and Hp, indicating that different stomach compartments produce isoforms that are different to those expressed by the liver. Expression of APPs by the bovine forestomachs and abomasum may contribute to regulation of the innate immune response against pathogens.

Although the mRNA that encodes progesterone receptor membrane component 1 (PGRMC1) is present in mammalian oocytes, nothing is known about either PGRMC1's expression pattern or function in oocytes during maturation, fertilization, and subsequent embryonic development. As PGRMC1 associates with the mitotic spindle in somatic cells, we hypothesized that PGRMC1 is involved in oocyte maturation (meiosis). Western blot analysis confirmed the presence of PGRMC1 in bovine oocytes. This study also shows that PGRMC1 is present at the germinal vesicle (GV)- and MII-stage oocytes and is associated with male and female pronucleus formation of the zygote and is highly expressed in blastocysts. A more detailed examination of PGRMC1 localization using confocal imaging demonstrated that in GV-stage oocytes, PGRMC1 was concentrated throughout the GV but did not localize to the chromatin. With the resumption of meiosis in vitro, PGRMC1 concentrated in the centromeric region of metaphase I chromosomes, while in the anaphase I/telophase I stages the majority of PGRMC1 concentrated between the separating chromosomes. At the metaphase II stage, PGRMC1 re-associated with the centromeric region of the chromosomes. A colocalization study demonstrated that PGRMC1 associated with the phosphorylated form of aurora kinase B, which localizes to the centromeres at metaphase. Finally, PGRMC1 antibody injection significantly lowered the percentage of oocytes that matured and reached the metaphase II stage after 24 h of culture. The majority of the PGRMC1 antibody-injected oocytes arrested in the prometaphase I stage of meiosis. Furthermore, in most of the PGRMC1 antibody-injected oocytes, the chromosomes were disorganized and scattered. Taken together, these data demonstrate that PGRMC1 is expressed in bovine oocytes and its localization changes at specific stages of oocyte maturation. These observations suggest an important role for PGRMC1 in oocyte maturation, which may be specifically related to the mechanism by which chromosomes segregate.

The TIR8 receptor (also called SIGIRR) is an orphan member of the TIR superfamily. Its function is still elusive, but it is believed to trigger a negative pathway of regulation of the Toll-like/IL-1 receptor system, crucial for modulating inflammation in gastrointestinal (GI) tract and in other tissues (lung and kidney). The expression pattern of TIR8 in bovine tissues is unknown. Given the importance of GI diseases in cattle, the aim of this investigation was to study the distribution of TIR8 in a wide panel of non-pathologic tissues and organs. TIR8 expression was assessed by Northern blot analysis and further confirmed and comparatively quantified by qualitative and quantitative (Real-Time) PCR. The possible presence of tissue-specific isoforms was determined by Western blot immunodetection, using an anti-human TIR8 polyclonal antibody previously validated in bovine tissues. Similarly to humans and mice, bovine TIR8 was found in the GI tract and kidney. Expression of TIR8 mRNA was also detected in lymph nodes, thymus and thyroid gland. Interestingly, several isoforms of bovine TIR8 were detected in the same organs, suggesting the occurrence of different post-translational processings.

In this paper, the possible involvement of the acute phase protein α 1 -acid glycoprotein (AGP) in the local immunomodulation of inflammation was investigated. The dose response of bovine neutrophils to AGP as to mobilization of primary and secondary granules was studied. It was found that AGP fulfils a protective role against spontaneous exocytosis of secondary, but not primary, granules. This downregulatory effect is much more evident when degranulation is challenged with Zymosan activated serum (ZAS). AGP activity is dose-dependent, the acute phase concentration being more active than the physiological one. Carbohydrate moiety of AGP was found to be critical, since experimentally desialylated protein does not maintain its exocytosis-modulatory activity. The fact that AGP may modulate the degranulation of neutrophils confirms the hypothesis that AGP is heavily involved in the fine tuning of neutrophil activity in the inflammatory environment.

The present study was designed to investigate the capability of bovine neutrophil granulocytes to produce the minor acute phase protein α 1 -acid glycoprotein (AGP, Orososmucoid). Bovine neutrophils contain a high MW (50–60 kDa) AGP isoform (PMN-AGP), as determined by Western blotting and confirmed by fluorescence microscopy. The presence of AGP in bovine neutrophils has been confirmed by fluorescence immunocytometry. In addition, bovine neutrophils contain also a 42–45 kDa isoform, which has the same MW as plasma-, liver-delivered, AGP. cDNA sequence of plasma- and PMN-AGP revealed that (i) the two proteins are products of the same gene; (ii) the differences in molecular weight are due do different post-translational modifications. This result was confirmed by deglycosylation of the two glycoforms. Exocytosis studies showed that isolated neutrophils exposed to several challengers, including Zymosan activated serum (ZAS) and phorbol 12-myristate 13-acetate (PMA), which mimic the inflammatory activation, released PMN-AGP as early as 15 min. AGP's mRNA is physiologically expressed by mature resting neutrophils. Real-time PCR on LPS, ZAS and PMA challenged cells revealed that the level of expression apparently does not increase after inflammatory activation. Collectively, the findings reported in this paper proved that PMN-AGP: (i) is a hyperglycosylated glycoform of plasma AGP, (ii) is stored in granules, and (iii) is released by neutrophils in response to activation. Due to its anti-inflammatory activity, PMN-AGP may work as a fine tuning of the neutrophils functions in the inflammatory focus, i.e. it can reduce the damages caused by an excess of inflammatory response.

Doppel (Dpl) protein is a recently identified prion-like protein. Although Dpl might be expressed in the brain after prion gene deletion, in both human and mice Dpl is normally expressed only in testis and spermatozoa, where it appears to be involved in male fertility. Little information is available so far about the expression pattern of Dpl in bovines, thus, hampering possible research on the role of this protein in bovine infertility. We have thus, designed, produced and validated through Western blotting a polyclonal antibody against bovine Dpl. With this antibody we then screened bovine tissues for Dpl expression by immunohistochemistry. Ejaculated spermatozoa were screened by flow cytometry and immunocytochemistry. Bovine Dpl was expressed in all the developing stages of germinal cells, from spermatogones to ejaculated spermatozoa, in Sertoli cells and in ovarian follicles (granulosa cells and follicular fluid). Dpl immunoreactivity was also found on other tissues, where endothelial cells, peripheral nerves and scattered lymphocytes stained positive. This distribution pattern suggests that Dpl might be involved in sperm maturation/capacitation in bovines, like it might be in mice. This hypothesis needs to be verified by widespread application of the flow cytometric protocol established in this paper on spermatozoa from animals with reduced fertility.

Doppel (Dpl) protein shares some structural features with prion protein (PrP), whose pathologic isoform (PrPsc) is considered to be the causative agent of transmissible spongiform encephalopathies. Dpl is mainly expressed in testes but, when ectopically expressed in the central nervous system, is neurotoxic. We have examined the expression pattern of Dpl and PrP on bovine lymphoid tissues and circulating leukocytes. A polyclonal anti-Dpl antibody along with a panel of monoclonal antibodies specific for leukocyte membrane antigens or PrP were used to examine frozen sections from spleen, lymph nodes, and bone marrow by immunohistochemistry. Blood was analyzed by flow cytometry. Double staining was used to study the possible coexpression of the two proteins and to characterize cells expressing Dpl and/or PrP. Dpl was expressed in B-cells, in dendritic cells within lymphoid follicles, bone marrow, circulating myeloid cells, and circulating B-cells. The distribution of Dpl was quite similar to that of PrP. The only differences in expression observed concerned the low number of Dpl + cells in lymph nodes and the strong Dpl positivity of circulating granulocytes. The two proteins were rarely co-expressed, suggesting an independent expression mechanism in resting cells. The role of Dpl+ leukocytes in the pathogenesis of Dpl- or PrP-induced diseases merits further investigation.

A protein trypsin inhibitor from seeds of snail medic (Medicago scutellata), MsTI, has been purified by ion-exchange chromatography, gel-filtration chromatography and reverse-phase HPLC. The protein inhibits the catalytic activity of bovine beta-trypsin, with an apparent Kd of 1.8 x 10(-9), but exhibits no activity towards bovine alpha-chymotrypsin. Moreover, MsTI inhibits the trypsin-like proteinase activity present in larvae of the crop pests Adoxophyes orana, Hyphantria cunea, Lobesia botrana and Ostrinia nubilalis. The complete amino acid sequence of MsTI consists of 62 residues corresponding to a M(r) of 6925. Sequence comparison shows that MsTI exhibits significant similarity to other proteins belonging to the Bowman-Birk trypsin inhibitor family, and the closest identity (81%) with the wound-induced trypsin inhibitor from Medicago sativa leaves.

The complete primary structure of the peroxisomal flavoenzyme D-aspartate oxidase from beef kidney has been determined by analyses of the peptides obtained through fragmentation of the carboxymethylated protein with trypsin, CNBr, heptafluorobutyric acid/CNBr and Staphylococcus aureus V8 protease. The protein consists of a single polypeptide of 338 residues, accounting for a M(r) of 37,305 for the apoprotein. A form of the enzyme lacking Lys-338 and therefore ending with Pro-337 has been detected. The N-terminal residue is blocked. Seven cysteines and no disulfide bridges are present. Residue 228 can be either Ile or Val. Thus, D-aspartate oxidase presents two types of heterogeneity in the polypeptide chain in addition to the one already described concerning the possible content of FAD or 6-hydroxyflavin adenine dinucleotide. Comparison of the primary structure of D-aspartate oxidase with other known sequences reveals that D-aspartate oxidase is homologous with D-amino acid oxidase (another flavo-oxidase) and does not present significant sequence similarities with any other protein, including flavoenzymes.

The acute phase protein 1-acid glycoprotein (AGP - Orosomucoid) is a lipocalin with immunomodulatory functions. The present study provides evidence that the plasma glycoforms of AGP inhibit the migration of bovine monocytes in response to classical chemoattractants. The inhibition is specific, since neutrophils are apparently not affected. To investigate the molecular basis of this finding, the expression of the molecules mostly involved in chemotaxis, including CD18, CD11b and CD47 was studied. It was found that the incubation of activated monocytes with acute phase concentration of AGP (0.9mg/mL) induces a down-regulation of CD18, and has no apparent influence on CD11b and CD47. RT-PCR expression studies on CD18, CD11b and CD47 mRNA revealed that AGP treatment does not modify the expression rate of these genes. Since AGP treatment is related to a down-regulation of CD18 on the surface of the monocytes, the authors suggest that one of its possible functions consists in specifically reducing the firm adhesion phase of bovine monocytes to the endothelium. alpha-1 acid glycoprotein / acute phase protein / CD18 / monocytes / chemotaxis

α1-Acid glycoprotein (AGP) is a lipocalin that is produced mainly by the liver and secreted into plasma in response to infections and injuries. In this study, we evaluated AGP isoforms that can be detected in bovine milk. We found that milk-AGP content is made up of at least two isoform groups, a low MW group (44 kDa) that is produced in the mammary gland (MG-AGP), and a higher MW group (55–70 kDa), that is produced by somatic cells (SC-AGP). Identical SC-AGP isoforms can be found both in milk and blood PMN cells. Analysis of the mammary tissue cDNA showed that the sequence of the MG-AGP isoform is identical to that of plasma AGP. Each group contains several proteins with different MWs and different isoelectric points, as shown by 2D-electrophoresis. The glycosylation patterns of these isoforms were analysed by means of specific lectin binding, to evaluate the degree of sialylation, fucosylation and branching. The MG-AGP glycan pattern was identical to plasma AGP produced by the liver. Several differences were detected, however, between plasma and SC-AGP isoforms, the most evident being the strong degree of fucosylation and the elevated number of di-antennary glycans in SC-AGP. Immunohistochemistry showed that AGP is found in all tissues that make up the mammary gland, but that it is most likely produced for the main part by the alveoli.