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Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are omega-3 long-chain polyunsaturated fatty acids (n-3 PUFA) found mostly in fish oil. They have been commonly used as dietary integrators in human and animal nutrition, modulating the immune system, mostly by exerting anti-inflammatory activities as demonstrated by in vivo and in vitro studies. The precise mechanisms of action at the background of EPA and DHA immunomodulatory activity are still not fully elucidated. Moreover, no information on their effects on porcine monocytes immune response is available yet. To cover this gap, the study aimed to evaluate DHA and EPA's in vitro impact on porcine monocytes (CD14 +) defensive functions. Briefly, monocytes were isolated from the blood of twenty-six healthy pigs, using a magnetic-activated cell sorting technique (MACS). Monocytes were first treated with increasing concentrations of DHA and EPA (25, 50, 100 and 200 µM) and apoptosis and viability were measured to assess potential cytotoxic effects. Once determined EPA and DHA subtoxic working concentrations (25, 50 and 100 µM), their effects on chemotaxis, phagocytosis and total, intracellular and extracellular reactive oxygen species (ROS) production were evaluated. DHA and EPA only decreased porcine monocytes viability at the highest concentration (200 µM), but their apoptosis was unaffected. DHA (100 µM) decreased the cells' chemotaxis, while EPA (25 µM) increased their intracellular ROS production after 60 min under non-inflammatory or resting conditions and at 90 min under pro-inflammatory conditions (PMA challenge). EPA (50 µM) decreased monocytes' intracellular ROS levels only under resting conditions at 30 min. No effects were observed on porcine monocytes phagocytic capacity. In conclusion, this study demonstrates that DHA and EPA can exert differential in vitro immunomodulatory effects in pigs, by dampening monocytes chemotaxis and potentiating their oxidative burst, respectively. Thus, our results suggest these n-3 PUFA might exert both anti-inflammatory and/or immune-enhancing effects in pigs.

Specific microRNAs expressions may accurately characterize different stages of canine myxomatous mitral valve disease. This preliminary pilot study aimed to (1) describe the clinical and echocardiographic parameters of Cavalier King Charles Spaniels affected by myxomatous mitral valve disease at different American College of Veterinary Internal Medicine (ACVIM) stages (B1, B2 and C) and healthy control group (ACVIM A), comparing the parameters collected during the first examination (T0) and the end of the follow-up (T1); (2) assess the association between the values of echocardiographic parameters at T1 and the expression profile of miR-30b-5p at T0. Thirty-five Cavalier King Charles Spaniels (median age 4.29 years and median weight 9 Kg) in different ACVIM stages were included (7 A, 19 B1, 6 B2 and 3 C). Inverse probability weighting analysis was performed to estimate the association of the exposure variable (miR-30b-5p) with the outcome variables (clinical and echocardiographic variables). Time was included as variable. The results pointed out that high levels of plasma miR-30b-5p corresponded to lower values of left ventricular end-diastolic diameter normalized for body weight, end-diastolic and end-systolic volumes indexed for body weight, and left atrium-to aortic root ratio. Hence, higher miR-30b-5p expressions were associated with milder forms of mitral valve disease in our study population. In contrast, the results obtained for the intensity of heart murmur, the mitral regurgitation severity, and the Mitral INsufficiency Echocardiographic score) were not statistically significant. A relationship between high abundance of miR-30b-5p and myxomatous mitral valve disease that appear echocardiographically more stable over time has been demonstrated. In conclusion, Cavalier King Charles Spaniels affected by myxomatous mitral valve disease that at the first cardiologic evaluation showed an upregulation of miR-30b-5p are expected to experience lesser variations on their echocardiographic examination between T0 and T1.

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.

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.

The identification of milk microbial communities in ruminants is relevant for understanding the association between milk microbiota and health status. The most common approach for studying the microbiota is amplifying and sequencing specific hypervariable regions of the 16S rRNA gene using massive sequencing techniques. However, the taxonomic resolution is limited to family and, in some cases, genus level. We aimed to improve taxonomic classification of the water buffalo milk microbiota by amplifying and sequencing the full-length 16S rRNA gene (1,500 bp) using Nanopore sequencing (single-molecule sequencing). When comparing with short-read results, we improved the taxonomic classification, reaching species level. We identified the main microbial agents of subclinical mastitis at the species level that were in accordance with the microbiological culture results. These results confirm the potential of single-molecule sequencing for in-depth analysis of microbial populations in dairy animals.

There is a growing interest in developing new molecular markers of heart disease in young dogs affected by myxomatous mitral valve disease. The study aimed to measure 3 circulating microRNAs and their application as potential biomarkers in the plasma of Cavalier King Charles Spaniels with early asymptomatic myxomatous mitral valve disease. The hypothesis is that healthy Cavalier King Charles Spaniels have different microRNA expression profiles than affected dogs in American College of Veterinary Internal Medicine (ACVIM) stage B1. The profiles can differ within the same class among subjects of different ages. This is a prospective cross-sectional study. Thirty-three Cavalier King Charles Spaniels in ACVIM stage B1 were divided into three groups (11 younger than 3 years, 11 older than 3 years and younger than 7 years, and 11 older than 7 years), and 11 healthy (ACVIM stage A) dogs of the same breed were included as the control group. Three circulating microRNAs (miR-1-3p, miR30b-5p, and miR-128-3p) were measured by quantitative real-time PCR using TaqMan® probes. Diagnostic performance was evaluated by calculating the area under the receiver operating curve (AUC). MiR-30b-5p was significantly higher in ACVIM B1 dogs than in ACVIM A subjects, and the area under the receiver operating curve was 0.79. According to the age of dogs, the amount of miR-30b-5p was statistically significantly higher in group B13y (2.3 folds, P = 0.034), B1 3-7y (2.2 folds, P = 0.028), and B17y (2.7 folds, P = 0.018) than in group A. The area under the receiver operating curves were fair in discriminating between group B13y and group A (AUC 0.780), between B1 3-7y and A (AUC 0.78), and good in discriminating between group B17y and A (AUC 0.822). Identifying dogs with early asymptomatic myxomatous mitral valve disease through the evaluation of miR-30b-5p represents an intriguing possibility that certainly merits further research. Studies enrolling a larger number of dogs with preclinical stages of myxomatous mitral valve disease are needed to expand further and validate conclusively the preliminary findings from this report.

Brucellosis is an infectious disease caused by bacteria from the Brucella genus that can be transmitted to humans through contact with infected animals or contaminated animal products. Brucellosis also causes financial losses in animal production. Ruminants are highly susceptible to brucellosis, and the causative agent water buffaloes (Bubalus bubalis) is Brucella abortus. Circulating microRNAs (miRNAs) are cropping up as promising biomarkers for several infectious diseases. The goals of this study were to characterize the serum miRNA signature associated with brucellosis in water buffaloes and investigate the miRNAs’ potential use as biomarkers in vaginal fluids. Next Generation Sequencing was used to assess miRNA expression profiles in Brucella-positive and Brucella-negative blood sera; dysregulated miRNAs in blood serum and vaginal fluids were validated using RT-qPCR. ROC curves were generated to evaluate the diagnostic value of miRNAs for Brucella. GO and KEGG pathway enrichment analyses were exploited to investigate the biological functions of dysregulated miRNAs. The results showed that 20 miRNAs were modulated, of which, 12 were upregulated and 8 were downregulated. These findings were corroborated by RT-qPCR, and ROC curves indicated that the miRNAs can serve as potential biomarkers for Brucella. GO and KEGG pathway analyses pointed out that some of these miRNAs are related to immune response and apoptosis. These results provided an overview of miRNA expression profiles and highlighted potential biomarkers for Brucella infection in water buffaloes. We also demonstrated the potential of vaginal fluids in studies involving microRNA detection. Further functional and mechanistic studies of these miRNAs may improve our understanding of the biological processes involved in Brucella infection and host immune response.

BACKGROUND Pain in horses is an emergent welfare concern, and its assessment represents a challenge for equine clinicians. This study aimed at improving pain assessment in horses through a convergent validation of existing tools: we investigated whether an effective analgesic treatment influences the horse grimace scale (HGS) and the concentration of specific circulating microRNAs (miRNAs). METHODS Eleven stallions underwent routine surgical castration under general anaesthesia. They were divided into two analgesic treatment groups: castration with the administration of preoperative flunixin and castration with preoperative flunixin plus a local injection of mepivacaine into the spermatic cords. HGS and levels of seven circulating miRNAs were evaluated pre-, 8 and 20 hours post-procedure. RESULTS Compared to pre-castration, HGS, miR-126-5p, miR-145 and miR-let7e increased significantly in horses receiving flunixin at 8 hours post-castration (Friedman test, p

Post-surgical management is an important issue in veterinary medicine, requiring biomarkers with high sensitivity and specificity for timely and effective treatment. Emerging evidence suggests that miRNAs are promising stress- and pain-related markers. The aims were to profile the circulating miRNA signature in plasma of turtles (Trachemys scripta) and point out potential candidate biomarkers to assess the status of the animal. The plasma of female turtles underwent surgical gonadectomy were collected 24 h pre-surgery, and 2.5 h and 36 h post-surgery. The expression of miRNAs was profiled by Next Generation Sequencing and the dysregulated miRNAs were validated using RT-qPCR. The diagnostic value of miRNAs was calculated by ROC curves. The results showed that 14 miRNAs were differentially expressed over time. RT-qPCR validation highlighted that 2-miR-499-3p and miR-203-5p-out of 8 miRNAs tested were effectively modulated. The Area Under the Curve (AUC) of miR-203-5p was fair (AUC 0.7934) in discriminating pre- and 36 h post-surgery samples and poor for other time points; the AUC of miR-499-3p was excellent (AUC 0.944) in discriminating pre-surgery and 2.5 h post-surgery samples, and fair in discriminating pre-surgery and 36 h post-surgery (AUC 0.7292) and 2.5 h and 36 h post-surgery (AUC 0.7569) samples. In conclusion, we demonstrated for the first time that miRNAs profile changes in plasma of turtles underwent surgical oophorectomy and identified miR-203-5p and miR-499-3p as potential candidate biomarkers to assess animals' status. Further studies are necessary to confirm their diagnostic value and to investigate functional and mechanistic networks to improve our understanding of the biological processes.

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.

Cutaneous mast cell tumours (MCTs) are common skin neoplasms in dogs. MicroRNAs (miRNAs) are post-transcriptional regulators involved in several cellular processes, and they can function as tumour promoters or suppressors. However, the role of miRNAs in canine MCTs has not yet been elucidated. Thus, the current study aimed to characterize miRNA profiles and to assess their value as biomarkers for MCTs. miRNA expression profiles were assessed in formalin-fixed, paraffin-embedded samples by next-generation sequencing. Ten samples were MCT tissues, and 7 were healthy adjacent tissues. Nine dysregulated miRNAs (DE-miRNAs) were then validated using RT-qPCR in a larger group of MCT samples, allowing the calculation of ROC curves and performance of multiple factor analysis (MFA). Pathway enrichment analysis was performed to investigate miRNA biological functions. The results showed that the expression of 63 miRNAs (18 up- and 45 downregulated) was significantly affected in MCTs. Five DE-miRNAs, namely, miR-21-5p, miR-92a-3p, miR-338, miR-379 and miR-885, were validated by RT-qPCR. The diagnostic accuracy of a panel of 3 DE-miRNAs—miR-21, miR-379 and miR-885—exhibited increased efficiency in discriminating animals with MCTs (AUC = 0.9854) and animals with lymph node metastasis (AUC = 0.8923). Multiple factor analysis revealed clusters based on nodal metastasis. Gene Ontology and KEGG analyses confirmed that the DE-miRNAs were involved in cell proliferation, survival and metastasis pathways. In conclusion, the present study demonstrated that the miRNA expression profile is changed in the MCT microenvironment, suggesting the involvement of the altered miRNAs in the epigenetic regulation of MCTs and identifying miR-21, miR-379 and miR-885 as promising biomarkers.

The present study aimed to investigate whether acute pain associated with castration and tail docking of male piglets may modulate the expression of salivary microRNAs (miRNAs) and to explore their potential use as biomarkers. Thirty-six healthy 4-d-old piglets (Hermitage × Duroc) were randomly assigned to three groups: the first group (12 piglets) has been pretreated with anesthetic and anti-inflammatory drugs (ANA) and then castrated and tail docked; the second one (12 piglets) has been castrated and tail docked without any drugs (CONV); the third one (12 piglets) has been only handled (SHAM). Saliva was collected 10 min before (control group) and 30 to 45 min after the procedures. Salivary cortisol has been quantified. The expression concentrations of seven miRNAs, namely miR-19b, miR-27b-3p, miR-215, miR-22-3p, miR-155-5p, hsa-miR-365-5p, and hsa-miR-204, were measured and assessed as potential biomarkers of pain by quantitative Polimerase Chain Reaction using TaqMan probes. The area under the receiver operating curve (AUC) was used to evaluate the diagnostic performance of miRNAs. The concentration of salivary cortisol increased after treatment in CONV and ANA, while no significant variation was observed in the SHAM group. The comparative analysis demonstrated that the concentrations of salivary miR-19b (P = 0.001), miR-27b (P = 0.042), and miR-365 (P < 0.0001) were significantly greater in CONV as compared with pretreatment. The AUC of pretreatment vs. CONV and CONV vs. ANA were excellent for miR-19b and miR-365 and fair for miR-27b. Combining two miRNAs, namely miR-19b and miR-365, in a panel increased the efficiency of distinguishing between pre- and post-treatment groups. No differences have been identified between SHAM and ANA groups. mRNA potential targets of differentially expressed-miRNA were investigated, and genes related to pain and inflammation were identified: miR-19b potentially modulates TGF-beta and focal adhesion pathways, miR-365 regulates cytokines expression (i.e., IL-1, Tumor Necross Factor-alpha, and IL-8 cytokine), and miR-27b regulates macrophage inflammatory protein pathways (i.e., MIP1-beta). In conclusion, we demonstrated that the abundance of miR-19b, miR-27b, and miR-365 increases in the saliva of piglets castrated and tail docked without the administration of pain-relieving drugs. Further studies are needed to assess their potential during routine husbandry procedures and to extend their assessment in other stressful events, such as weaning or chronic pain.

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.

Otitis externa is one of the most common diseases in dogs. It is associated with bacteria and yeast, which are regarded as secondary causes. Cerumen is a biological substance playing an important role in the protection of ear skin. The involvement of cerumen in immune defense is poorly understood. MicroRNAs can modulate the host immune response and can provide promising biomarkers for several inflammatory and infectious disorder diagnosis. The aims of this study were to profile the cerumen miRNA signature associated with otitis externa in dogs, integrate miRNAs to their target genes related to immune functions, and investigate their potential use as biomarkers. Cerumen was collected from healthy and otitis affected dogs and the expression of miRNAs was profiled by Next Generation Sequencing; the validation of the altered miRNAs was performed using RT-qPCR. The potential ability of miRNAs to modulate immune-related genes was investigated using bioinformatics tools. The results pointed out that 32 miRNAs, of which 14 were up- and 18 down-regulated, were differentially expressed in healthy vs. otitis-affected dogs. These results were verified by RT-qPCR. To assess the diagnostic value of miRNAs, ROC analysis was carried out, highlighting that 4 miRNAs are potential biomarkers to discriminate otitis-affected dogs. Bioinformatics showed that cerumen miRNAs may be involved in the modulation of host immune response. In conclusion, we have demonstrated for the first time that miRNAs can be efficiently extracted and quantified from cerumen, that their profile changes between healthy and otitis affected dogs, and that they may serve as potential biomarkers. Further studies are necessary to confirm their diagnostic value and to investigate their interaction with immune-related genes.

Otitis externa is a common multifactorial disease in dogs, characterized by broad and complex modifications of the ear microbiota. The goal of our study was to describe the ear cerumen microbiota of healthy dogs, within the same animal and between different animals, and to compare the cerumen microbiota of otitis affected dogs with that of healthy animals. The present study included 26 healthy dogs, 16 animals affected by bilateral otitis externa and 4 animals affected by monolateral otitis externa. For each animal cerumen samples from the right and left ear were separately collected with sterile swabs, and processed for DNA extraction and PCR amplification of the 16S rRNA gene. Amplicon libraries were sequenced using an Ion Torrent Personal Genome Machine (PGM), and taxonomical assignment and clustering were performed using QIIME 2 software. Our results indicate that the bacterial community of the cerumen in healthy dogs was characterized by extensive variability, with the most abundant phyla represented by Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes and Fusobacteria. The analysis of both alpha and beta diversity between pairs of left and right ear samples from the same dog within the group of affected animals displayed higher differences than between paired samples across healthy dogs. Moreover we observed reduced bacterial richness in the affected group as compared with controls and increased variability in population structure within otitis affected animals, often associated with the proliferation of a single bacterial taxon over the others. Moreover, Staphylococcus and Pseudomonas resulted to be the bacterial genera responsible for most distances between the two groups, in association with differences in the bacterial community structure. The cerumen microbiota in healthy dogs exhibits a complex bacterial population which undergoes significant modifications in otitis affected animals.

MicroRNAs (miRNAs) are a class of small, noncoding RNA that post-transcriptionally regulate protein expression. miRNAs are emerging as clinical biomarkers of many diseases, including tumors. The aim of this study was to investigate whether miRNA expression could vary in melanoma samples derived from formalin-fixed, paraffin-embedded (FFPE) tissues. The study included 4 groups: (1) 9 samples of oral canine malignant melanoma, (2) 10 samples of cutaneous malignant melanoma, (3) 5 samples of healthy oral mucosa, and (4) 7 samples of healthy skin. The expression levels of 6 miRNAs—miR-145, miR-146a, miR-425-5p, miR-223, miR-365, and miR-134—were detected and assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) using TaqMan probes. Cutaneous canine malignant melanoma showed a decrease of the expression level of miR-145 and miR-365 and an increase of miR-146a and miR-425-5p compared to control samples. MiR-145 was also downregulated in oral canine malignant melanoma. The miRNAs with decreased expression may regulate genes involved in RAS, Rap1, and transforming growth factor β (TGF-β) signaling pathways, as well as upregulated genes associated with phosphatidylinositol signaling system, adherens junction, and RAS signaling pathways. In conclusion, miR-145, miR-365, miR-146a, and miR-425-5p were differentially expressed in canine malignant melanoma and healthy FFPE samples, suggesting that they may play a role in canine malignant melanoma pathogenesis.

The aim of the present study was to investigate how maternal diet can influence the adipose tissue of goat kids. Omental adipose tissue proteomes of goat-kids from mothers fed with diet enriched with stearic acid (ST-kids), fish oil (FO-kids) and standard diets (CTRL) were determined by quantitative iTRAQ 2D-LC-MS/MS analysis. Twenty proteins were found to be differentially expressed in suckling kids' omental adipose tissue. Stearic acid induces changes in a higher number of proteins when compared to fish oil. Eleven proteins, namely AARS, ECl1, PMSC2, CP, HSPA8, GPD1, RPL7, OGDH, RPL24, FGA and RPL5 were decreased in ST-kids only. Four proteins, namely DLST, EEF1G, BCAP31 and RALA were decreased in FO-kids only, and one, NUCKS1, was increased. Four proteins, namely PMSC1, PPIB, TUB5 x 2 and EIF5A1, were be less abundant in both ST- and FO- kids. Most of the protein whose abundance was decreased in ST kids (10 out of 15) are involved in protein metabolism and catabolism pathways. Qualitative gene expression analysis confirmed that all the proteins identified by mass spectrometry, with the exception of FGA, were produced by adipose tissue. Quantitative gene expression analysis demonstrated that two proteins, namely CP, a minor acute phase protein, and ECl1, involved in fatty acid beta oxidation, were downregulated at mRNA level as well. ECl1 gene expression was downregulated in ST-kids AT as compared to Ctrl-kids and CP was downregulated in both ST- and FO-kids. The present results demonstrate that it is possible to influence adipose goat-kid proteome by modifying the maternal diet.

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

Haemorrhagic enteritis (HE) is a viral disease affecting intestinal integrity and barrier function in turkey (Meleagris gallopavo) and resulting in a significant economic loss. Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH-MS) was applied to identify crucial proteins involved in HE infection. A total of 938 proteins were identified and used to generate a reference library for SWATH-MS analysis. In total, 523 proteins were reliably quantified, and 64 proteins were found to be differentially expressed, including 49 up-regulated and 15 down-regulated proteins between healthy and HE-affected intestinal mucosa. Functional analysis suggested that these proteins were involved in the following categories of cellular pathways and metabolisms: 1) energy pathways; 2) intestine lipid and amino acid metabolism; 3) oxidative stress; 4) intestinal immune response. Major findings of this study demonstrated that natural HE infection is related to the changes in abundance of several proteins involved in cell-intrinsic immune defense against viral invasion, systemic inflammation, modulation of excessive inflammation, B and T cell development and function and antigen presentation. mRNA quantitative expression demonstrated that most of the proteins involved in innate immunity that were found to be differentially abundant were produced by intestinal mucosa, suggesting its direct involvement in immune defences against HE infection.

Pectin is a dietary fibre composed of galacturonic acid, primarily found in the citrus fruits' cell walls. Citrus pectin (CP) has demonstrated antioxidative, anticancer, and anti-inflammatory properties in humans and animals. In broilers, CP supplementation improves energy utilization and nutrient digestibility, but limited information on its effects on chicken immunity is available so far. This study aimed to assess the in vitro impact of CP on chicken monocytes' immune response. Cells were purified from whole blood of healthy chickens and incubated with increasing concentrations (0, 0.25, 0.5, 0.75, 1 mg/mL) of CP to determine CP working concentration. The effects of different CP concentrations on cells' apoptosis and viability were assessed by measuring caspase-3 and -7 and the cells' metabolic activity (MTT assay), respectively. CP had no dose-dependent effect on monocyte apoptosis and viability.Then, the effects of CP (0.5 mg/mL) on chicken monocytes' chemotaxis and phagocytosis were assessed by measuring transwell migration and fluorescein-labelled E. coli incorporation, respectively. CP inhibited both monocytes' chemotaxis and phagocytosis.These data demonstrate that CP exerts an immunomodulatory role in chicken monocytes, supporting its integration in nutrition strategies that might be beneficial for the animal's immunity and health.

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.

α1-acid glycoprotein (orosomucoid, AGP) is an Acute Phase Protein produced by liver and peripheral tissues in response to systemic reaction to inflammation. AGP functions have been studied mostly in human, cattle and fish, although the protein has been also found in many mammalian species and birds. AGP fulfils at least two set of functions, which are apparently different from each other but in fact intimately linked. On one hand, AGP is an immunomodulatory protein. On the other hand, AGP is one of the most important binding proteins in plasma and, beside modulating pharmacokinetics and pharmacodynamics of many drugs, it is also able to bind and transport several endogen ligands related to inflammation. The focus of this review is the immunomodulatory activity of AGP. This protein regulates every single event related to inflammation, including binding of pathogens and modulating white blood cells activity throughout the entire leukocyte attacking sequence. The regulation of AGP activity is complex: the inflammation induces not only an increase in AGP serum concentration, but also a qualitative change in its carbohydrate moiety, generating a multitude of glycoforms, each of them with different, and sometimes opposite and contradictory, activities. We also present the most recent findings about the relationship between AGP and adipose tissue: AGP interacts with leptin receptor and, given its immunomodulatory function, it may be included among the potential players in the field of immunometabolism.

Piglets can often suffer impaired antioxidant status and poor immune response during post-weaning, especially when chronic inflammation takes place, leading to lower growth rates than expected. Oral administration of dietary antioxidant compounds during this period could be a feasible way to balance oxidation processes and increase health and growth performance. The aim of the trial was to study the effects of an antioxidant feed supplement (melon pulp concentrate) that contains high concentration of the antioxidant superoxide dismutase (SOD) on inflammation, antioxidant status and growth performance of lipopolysaccharide (LPS) challenged weaned piglets. In total, 48 weaned piglets were individually allocated to four experimental groups in a 2×2 factorial design for 29 days. Two different dietary treatments were adopted: (a) control (CTR), fed a basal diet, (b) treatment (MPC), fed the basal diet plus 30 g/ton of melon pulp concentrate. On days 19, 21, 23 and 25 half of the animals within CTR and MPC groups were subjected to a challenge with intramuscular injections of an increasing dosage of LPS from Escherichia coli (serotype 0.55:B5) (+) or were injected with an equal amount of PBS solution (−). Blood samples were collected at the beginning of the trial and under the challenge period for interleukin 1β, interleukin 6, tumour necrosis factor α, haptoglobin, plasma SOD activity, total antioxidant capacity, reactive oxygen species, red blood cells and plasma resistance to haemolysis, and 8-oxo-7, 8-dihydro-2’-deoxyguanosine. Growth performance was evaluated weekly. A positive effect of melon pulp concentrate was evidenced on total antioxidant capacity, half-haemolysis time of red blood cells, average daily gain (ADG) and feed intake, while LPS challenge increased pro-inflammatory cytokines and haptoglobin serum concentrations, with a reduced feed intake and gain : feed (G : F). The obtained results show that oral SOD supplementation with melon pulp concentrate ameliorates the total antioxidant capacity and the half-haemolysis time in red blood cell of post-weaning piglets, with positive results on growing performance.

Omics techniques have been widely applied to veterinary science, although mostly on farm animal productions and infectious diseases. In canine oncology, on the contrary, the use of omics methodologies is still far behind. This review presents the most recent achievement in the application of postgenomic techniques, such as transcriptomics, proteomics, and metabolomics, to canine cancer research. The protocols to recover material suitable for omics analyses from formalin-fixed, paraffin-embedded tissues are presented, and omics applications for biomarker discovery and their potential for cancer diagnostics in veterinary medicine are highlighted.

MicroRNA (miRNA) have been identified in circulating blood and might have the potential to be used as biomarkers for several pathophysiological conditions. To identify miRNA that are altered following stress events, turkeys (Meleagris gallopavo) were subjected to 2 h of road transportation. The expression levels of five circulating miRNA, namely miR-22, miR-155-5p, miR-181a-3p, miR-204 and miR-365-3p, were detected and assessed by quantitative polymerase chain reaction using TaqMan® probes, as potential biomarkers of stress. The areas under the receiver operating characteristic curves were then used to evaluate the diagnostic performance of miRNA. A panel of three stress-responsive miRNA, miR-22, miR-155 and miR-365 were identified; their expression levels were significantly higher after road transportation and the area under the curve (AUC) were 0.763, 0.71 and 0.704, respectively. Combining the three miRNA a specificity similar to the one found for the three miRNA separately was found. The AUC of the weighted average of the three miRNA was 0.763. This preliminary study suggests that the expression levels of circulating miR-22, miR-155 and miR-365 are increased during transport-related stress and that they may have diagnostic value to discriminate between stressed- and unstressed animals.

Beside its importance in the first hours of life, brown adipose tissue has also significant roles in the following stages of growth and in adults by regulating energy metabolism, but its identification in adult ruminants is still controversial. Quantitative PCR, followed by histological confirmation, was used to investigate UCP expression and brown and white adipocytes' distribution in 30-day-old goat kids. The influence of maternal diet enriched with either fish oil or stearic acid was investigated as well. Results showed the differential expression of both UCP1 and UCP2 genes between subcutaneous and visceral adipose tissues, suggesting a different thermogenic activity between the two macro areas. The maternal diet influenced neither UCP1 nor UCP2 gene expression. The presence of multilocular adipocytes in 1-month goat kids is remarkable, as suggests thermogenic activity in non-newborn animals. Further insights into characteristics and functions of adipose tissue in young and adult goats are worth exploring.

Circulating microRNAs (miRNAs) are emerging as promising biomarkers for several disorders and related pain. In equine practice, acute laminitis is a common disease characterised by intense pain that severely compromises horse welfare. Recently, the Horse Grimace Scale (HGS), a facial expression-based pain coding system, was shown to be a valid welfare indicator to identify pain linked to acute laminitis. The present study aimed to: determine whether miRNAs can be used as biomarkers for acute pain in horses (Equus caballus) affected by laminitis; integrate miRNAs to their target genes and to categorise target genes for biological processes; gather additional evidence on concurrent validity of HGS by investigating how it correlates to miRNAs. Nine horses presenting acute laminitis with no prior treatment were recruited. As control group, nine healthy horses were further included in the experimental design. Samples were collected from horses with laminitis at admission before any treatment ('pre-treatment') and 7 days after routine laminitis treatment ('post-treatment'). The expression levels of nine circulating miRNAs, namely hsa-miR-532-3p, hsa-miR-219-5p, mmu-miR-134-5p, mmu-miR-124a-3p, hsa-miR-200b-3p, hsa-miR-146a-5p, hsa-miR-23b-3p, hsa-miR-145-5p and hsa-miR-181a-5p, were detected and assessed as potential biomarkers of pain by quantitative PCR using TaqMan® probes. The area under the receiver operating curve (AUC) was then used to evaluate the diagnostic performance of miRNAs. Molecular data were integrated with HGS scores assessed by one trained treatment and time point blind veterinarian. The comparative analysis demonstrated that the levels of miR-23b-3p (P=0.029), miR-145-5p (P=0.015) and miR-200b-3p (P=0.023) were significantly higher in pre-treatment and the AUCs were 0.854, 0.859 and 0.841, respectively. MiR-200b-3p decreased after routine laminitis treatment (P=0.043). Combining two miRNAs in a panel, namely miR-145-5p and miR-200b-3p, increased efficiency in distinguishing animals with acute pain from controls. In addition, deregulated miRNAs were positively correlated to HGS scores. Computational target prediction and functional enrichment identified common biological pathways between different miRNAs. In particular, the glutamatergic pathway was affected by all three miRNAs, suggesting a crucial role in the pathogenesis of pain. In conclusion, the dynamic expression of circulating miR-23b-3p, miR-145-5p and miR-200b-3p was detected in horses with acute laminitis and miRNAs can be considered potentially promising pain biomarkers. Further studies are needed in order to assess their relevancy in other painful conditions severely compromising horse welfare. An important implication would be the possibility to use them for the concurrent validation of non-invasive indicators of pain in horses.

The need to provide in vivo complex environments to understand human diseases strongly relies on the use of animal models, which traditionally include small rodents and rabbits. It is becoming increasingly evident that the few species utilised to date cannot be regarded as universal. There is a great need for new animal species that are naturally endowed with specific features relevant to human diseases. Farm animals, including pigs, cows, sheep and horses, represent a valid alternative to commonly utilised rodent models. There is an ample scope for the application of proteomic techniques in farm animals, and the establishment of several proteomic maps of plasma and tissue has clearly demonstrated that farm animals provide a disease environment that closely resembles that of human diseases. The present review offers a snapshot of how proteomic techniques have been applied to farm animals to improve their use as biomedical models. Focus will be on specific topics of biomedical research in which farm animal models have been characterised through the application of proteomic techniques.

The present study investigated the in vitro effects on caprine monocytes of two ω-3 PUFAs, namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on lipid droplet formation, an emerging process of fundamental importance in innate immunity regulation. The mRNA abundance of PAT protein family (PLIN1, PLIN2 and PLIN3), involved in the formation and trafficking of the droplets, was also assessed. The effects of EPA and DHA on monocyte apoptosis were studied as well. The number of lipid droplets per cell was found to be dependent on both type and concentration of fatty acid. ω-3 PUFAs upregulated PLIN3 and PLIN2 gene expression, as well as apoptosis rate. The present findings suggest that PUFA might modify innate immune functions of goat monocytes by interfering with the formation of lipid droplets and by upregulating proteins belonging to PAT protein family.

Acute phase proteins (APP) are plasma proteins that can modify their expression in response to inflammation caused by tissue injury, infections, immunological disorders or stress. Although APP are produced mainly in liver, extrahepatic production has also been described. As a prerequisite to get insight the expression of APP in chicken during diseases, this study investigated the presence of five APP, including alpha1-acid glycoprotein (AGP), Serum Amyloid A (SAA), PIT54, C-Reactive protein (CRP) and Ovotransferrin (OVT) in twenty tissues collected from healthy chicken (Gallus gallus) by quantitative Real Time PCR and immunohistochemistry. As expected, APP gene abundance was higher in liver compared with other tissues. The mRNA coding for CRP, OVT and SAA was detected in all analyzed tissues with a higher expression in gastrointestinal tract, respiratory and lymphatic samples. SAA expression was particularly high in cecal tonsil, lung, spleen and Meckel's diverticulum, whereas OVT in lung, bursa of Fabricius and pancreas. AGP and PIT54 mRNA expression were detected in all tissues but at negligible levels. Immunohistochemical expression of AGP and OVT was variably detected in different organs, being identified in endothelium of every tissue. Positive cells were present in the epithelium of the mucosal layer of gastrointestinal tract and kidney. Lung and central nervous system stained for both proteins. No positive staining was detected in lymphoid tissues and muscle. These results suggest that most tissues can express different amount of APP even in healthy conditions and are therefore capable to mount a local acute phase reaction.

Hemorrhagic enteritis (HE) is an acute viral disease that affects avian species, particularly turkeys, compromising their commercial production and having a negative effect on animal welfare. Turkey adenovirus 3 (TAdV-3), is the main causal agent of the disease. In this study, we considered 3 groups of turkeys to achieve 2 purposes: 1) A preliminary investigation on the microbiota content in the 4 parts of healthy turkey’s intestine (group A), namely duodenum, jejunum, ileum, and ceca was done; 2) an investigation on the relationship between natural infections with TAdV-3 and the intestinal microbiota in the jejunum, where HE mostly develops, comparing group A with animals with molecular positivity for the virus and with clinical signs of HE (group B) and animals with molecular positivity for the virus but without clinical signs (group C). Massive sequencing of the hypervariable V1–V2 regions of 16S rRNA gene and QIIME 1.9.1 software analysis was performed, and operation taxonomic units (OTUs) were classified into 4 abundant phyla: Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. The microbial population of small intestine was distributed almost homogeneously in the healthy turkeys, and Firmicutes was the prevalent phylum (79.85% in duodenum, 89.57% in jejunum and 99.28% in ileum). As compared with small intestine, ceca microbial community was much more heterogeneous: Firmicutes (48.03%), Bacteroidetes (33.60%) and Proteobacteria (12.32%). In the natural infections of HEV, the main bacterial families were Bacteroidaceae (Bacteroidetes) and Peptostreptococcaceae (Firmicutes), uniquely detected in group B and C. Also Clostridiaceae (Firmicutes) was detected, uniquely in group B.

The aim of this study was to define the microbiota of water buffalo milk during sub-clinical and clinical mastitis, as compared to healthy status, by using high-throughput sequencing of the 16S rRNA gene. A total of 137 quarter samples were included in the experimental design: 27 samples derived from healthy, culture negative quarters, with a Somatic Cell Count (SCC) of less than 200,000 cells/ml; 27 samples from quarters with clinical mastitis; 83 samples were collected from quarters with subclinical mastitis, with a SCC number greater of 200,000 cells/ml and/or culture positive for udder pathogens, without clinical signs of mastitis. Bacterial DNA was purified and the 16S rRNA genes were individually amplified and sequenced. Significant differences were found in milk samples from healthy quarters and those with sub-clinical and clinical mastitis. The microbiota diversity of milk from healthy quarters was richer as compared to samples with sub-clinical mastitis, whose microbiota diversity was in turn richer as compared to those from clinical mastitis. The core microbiota of water buffalo milk, defined as the asset of microorganisms shared by all healthy milk samples, includes 15 genera, namely Micrococcus, Propionibacterium, 5-7N15, Solibacillus, Staphylococcus, Aerococcus, Facklamia, Trichococcus, Turicibacter, 02d06, SMB53, Clostridium, Acinetobacter, Psychrobacter and Pseudomonas. Only two genera (Acinetobacter and Pseudomonas) were present in all the samples from sub-clinical mastitis, and no genus was shared across all in clinical mastitis milk samples. The presence of mastitis was found to be related to the change in the relative abundance of genera, such as Psychrobacter, whose relative abundance decreased from 16.26% in the milk samples from healthy quarters to 3.2% in clinical mastitis. Other genera, such as SMB53 and Solibacillus, were decreased as well. Discriminant analysis presents the evidence that the microbial community of healthy and clinical mastitis could be discriminated on the background of their microbiota profiles.

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.

The acute phase proteins (APP) are a group of serum proteins that change their concentration in animals following external or internal challenges, such as infection, inflammation or stress. The concentrations of four APPs, including serum amyloid A (SAA), haptoglobin (Hp), alpha(1)-acid glycoprotein (AGP) and ceruloplasmin (Cp) were determined in serum collected from healthy Alpine ibexes (Capra ibex) and ibexes with Sarcoptes scabiei mange. Primary structures of all four APPs were determined by cDNA sequencing. The concentrations of all four APPs were higher in serum of animals with clinical signs of sarcoptic mange when compared to healthy animals. Two of the APPs, including SAA and AGP, acted as major APPs, since their serum concentrations were increased more than 10-folds when compared to healthy animals (P0.001). The other two APPs, including Hp and Cp, acted as minor acute phase proteins, as their concentrations were increased from two to five folds (P0.001). These findings provide a remarkable potential as diagnostic markers for the early detection of sarcoptic mange in free ranging animals.

An in vitro study was carried out to examine the influence of two fish-oil-derived long chain omega-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on goat polymorphonuclear leukocytes (PMN). Twelve Saanen healthy goats were used as blood donors. Neutrophils were isolated from blood and incubated with increasing concentration of EPA and DHA (25, 50, 100, 200muM). Control samples were incubated in the absence of omega-3 PUFAs. Phagocytosis was evaluated by fluorescein-labeled Escherichia coli incorporation, while extracellular Reactive Oxygen Species (ROS) production was determined by cytochrome c reduction assay, which was selected among the others due to its specificity for extracellular superoxide anion release. Phagocytic activity was significantly increased by EPA (P

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.

The aim of this study was to investigate the effects of transport-related stress on the liver gene expression of four acute phase proteins (APP), namely α1-acid glycoprotein (AGP), C-Reactive Protein (CRP), Serum Amyloid A (SAA) and PIT54, in turkeys (Meleagris gallopavo). A group of seven BUT BIG 6 commercial hens was subjected to a two-hour long road transportation and the quantitative gene expression of APP in the liver was compared to that of a non transported control group. The expression of AGP and CRP mRNA was found to be increased in animals slaughtered after road transport. The presence of AGP protein was also confirmed by immunohistochemistry and Western blotting. The results of this study showed that road-transport may induce the mRNA expression of immune related proteins. The finding that AGP and CRP can be upregulated during transport could suggest their use as for the assessment of turkey welfare during transport.

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.

Adiponectin is an adipocyte-derived hormone, which circulates in the form of homo-multimers. The individual oligomers have a distinct profile of activity, playing crucial roles in several biological processes, including metabolism and inflammation. Adiponectin exerts many of its effects by interacting with the receptors, AdipoR1 and AdipoR2. In the present study, mRNA expression of adiponectin, AdipoR1 and AdipoR2 was evaluated by quantitative PCR in different areas of the mammary gland in healthy lactating cows. The adiponectin isoforms in milk and blood were investigated by Western blotting and 2D-electrophoresis, and the presence of adiponectin protein was determined by immunohistochemistry. Low level expression of adiponectin mRNA was found in all areas of bovine mammary gland tissues examined. AdipoR1 and AdipoR2 mRNAs were also detected in mammary tissues and their expression was particularly prominent in the parenchyma and cistern. Western blotting revealed a heterogeneous electrophoretic pattern, indicating that different adiponectin isoforms exist in milk, compared with blood. In particular, milk shows a low molecular weight isoform of adiponectin, corresponding to the globular domain. Adiponectin in milk is characterised by a more complex 2D electrophoretic pattern, compared with blood, as illustrated by the presence of proteins of different molecular weights and isoelectric points. Adiponectin protein was detected by immunohistochemistry in epithelial cells lining the secretory alveoli, in secretum within the alveolar lumen and in small peripheral nerves. The study findings support a role for adiponectin in regulating metabolism and immunity of the bovine mammary gland and potentially the calf intestine, following ingestion of milk.

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.

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).