Heat stress has divergent effects on the milk microbiota of Holstein and Brown Swiss cows.

Heat stress (HS) is one of the pivotal causes of economic losses in dairy industries and affects welfare and performance, but its effect on milk microbiota remains elusive. It is also unclear if and how different breeds may cope with HS in sustaining productive performance. The objectives of this study were to compare a) the performance of 2 dairy breeds, namely Holstein and Brown Swiss, subjected to HS and b) the different effects of HS on the milk microbiota of the 2 breeds in thermal comfort conditions and HS. The study was carried out on 36 dairy cows, 18 per breed. The HS was induced by switching off the cooling system during a natural heat wave for 4 d. Besides the Temperature Humidity Index (THI), the animal stress was confirmed by measuring respiratory frequency and rectal temperature twice daily at 4 a.m. and 3 p.m. The HS differently impacted the 2 breeds. Rectal temperatures were higher in Holstein cows, while no changes in rectal temperature were found in Brown Swiss. Milk yield recording and sampling were performed during the morning milking of d 1 (at 4.00 a.m.) and afternoon milking of d 4 (at 5.00 p.m.). Productive parameters were also different: milk yield, fat-corrected milk, energy-corrected milk, protein and casein content, and renneting parameters were decreased in Holstein but remained unaffected in Brown Swiss. The HS also modified the milk microbiota of the 2 breeds differently. During HS, the Brown Swiss milk microbiota was richer (α diversity) than the Holstein one. Comparing the time points before and during HS within breeds showed that Brown Swiss milk microbiota was less affected by HS than Holstein's. Under the same thermal comfort condition, milk microbiota did not discriminate between Brown Swiss and Holstein. Consistently with α and ß diversity, the number of operational taxonomic units (OTUs) at the genus level that changed their abundance during HS was higher in Holstein (74 OTUs) than in Brown Swiss (only 20 OTUs). The most significant changes in abundance affected Acinetobacter, Chryseobacterium, Cutibacterium, Enterococcus, Lactococcus, Prevotella-9, Serratia, and Streptococcus. In conclusion, the present report confirms and extends previous studies by demonstrating that Brown Swiss cows regulate their body temperature better than the Holstein breed. The relative thermal tolerance to HS compared with Holstein is also confirmed by changes in milk uncultured microbiota, which were more evident in Holstein than in Brown Swiss.

Methods in isolation and characterization of bovine monocytes and macrophages.

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 untargeted lipidomic profile of quarter milk from dairy cows with subclinical intramammary infection by non-aureus staphylococci.

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.

In vitro effects of conjugated linoleic acid (CLA) on inflammatory functions of bovine monocytes.

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.

Proteomics in veterinary medicine: applications and trends in disease pathogenesis and diagnostics.

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).

Citrus pectin modulates chicken peripheral blood mononuclear cell proteome in vitro.

Citrus pectin (CP) is a dietary fiber used in animal nutrition with anti-inflammatory properties. CP downregulates chicken immunoregulatory monocytes' functions, like chemotaxis and phagocytosis, in vitro. The molecular underlying background is still unknown. This study investigated the activity of CP on chicken peripheral blood mononuclear cells (PBMC) proteome. An overall number of 1503 proteins were identified and quantified. The supervised sparse variant partial least squares-discriminant analysis (sPLS-DA) for paired data highlighted 373 discriminant proteins between CP-treated and the control group, of which 50 proteins with the highest abundance in CP and 137 in the control group were selected for Gene Ontology (GO) analyses using ProteINSIDE. Discriminant Protein highly abundant in CP-treated cells were involved in actin cytoskeleton organization and negative regulation of cell migration. Interestingly, MARCKSL1, a chemotaxis inhibitor, was upregulated in CP-treated cells. On the contrary, CP incubation downregulated MARCKS, LGALS3, and LGALS8, which are involved in cytoskeleton rearrangements, cell migration, and phagocytosis. In conclusion, these results provide a proteomics background to the anti-inflammatory activity of CP, demonstrating that the in vitro downregulation of phagocytosis and chemotaxis is related to changes in proteins related to the cytoskeleton.

Conjugated linoleic acid (CLA) modulates bovine peripheral blood mononuclear cells (PBMC) proteome in vitro.

Conjugated linoleic acid (CLA) is a group of natural isomers of the n-6 polyunsaturated fatty acid (PUFA) linoleic acid, exerting biological effects on cow physiology. This study assessed the impact of the mixture 50:50 (vol:vol) of CLA isomers (cis-9, trans-11 and trans-10, cis-12) on bovine peripheral blood mononuclear cells (PBMC) proteome, identifying 1608 quantifiable proteins. A supervised multivariate statistical analysis, sparse variant partial least squares - discriminant analysis (sPLS-DA) for paired data identified 407 discriminant proteins (DP), allowing the clustering between the CLA and controls. The ProteINSIDE workflow found that DP with higher abundance in the CLA group included proteins related to innate immune defenses (PLIN2, CD36, C3, C4, and AGP), with antiapoptotic (SERPINF2 and ITIH4) and antioxidant effects (HMOX1). These results demonstrated that CLA modulates the bovine PBMC proteome, supports the antiapoptotic and immunomodulatory effects observed in previous in vitro studies on bovine PBMC, and suggests a cytoprotective role against oxidative stress. SIGNIFICANCE: In this study, we report for the first time that the mixture 50:50 (vol:vol) of cis-9, trans-11, and trans-10, cis-12-CLA isomers modulates the bovine PBMC proteome. Our results support the immunomodulatory and antiapoptotic effects observed in bovine PBMC in vitro. In addition, the present study proposes a cytoprotective role of CLA mixture against oxidative stress. We suggest a molecular signature of CLA treatment based on combining a multivariate sparse discriminant analysis and a clustering method. This demonstrates the great value of sPLS-DA as an alternative option to identify discriminant proteins with relevant biological significance.

Anti-inflammatory activity of citrus pectin on chicken monocytes' immune response.

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 effects of superoxide dismutase-rich melon pulp concentrate on inflammation, antioxidant status and growth performance of challenged post-weaning piglets.

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 1ß, 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.

Circulating miR-23b-3p, miR-145-5p and miR-200b-3p are potential biomarkers to monitor acute pain associated with laminitis in horses.

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.

Application of post-genomic techniques in dog cancer research.

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.

Circulating extracellular miR-22, miR-155, and miR-365 as candidate biomarkers to assess transport-related stress in turkeys.

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.

UCP1 and UCP2 expression in different subcutaneous and visceral adipose tissue deposits in 30 days old goat kids and effect of fatty acid enriched diets.

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.

Immunological role of the endosymbionts of Dirofilaria immitis: the Wolbachia surface protein activates canine neutrophils with production of IL-8.

Filarial nematodes, including Dirofilaria immitis and D. repens, harbour intracellular bacteria belonging to the genus Wolbachia. These bacteria have been implicated in the pathogenesis of filarial diseases, possibly through their endotoxins. Recent studies have shown that a major surface protein of Wolbachia (WSP) induces a specific IgG response in hosts infected by D. immitis. WSP from the Wolbachia of D. immitis was produced in recombinant form. The purified protein was used in stimulation assays on canine neutrophils. The assays performed using a modified Boyden chamber showed that WSP stimulates neutrophil chemokinesis. In addition, RT-PCR revealed increased production of chemokine IL-8 by cells incubated with this protein. Neutrophils have been shown to play a major role in the pathogenesis of river blindness, and to accumulate in the nodules of onchocerciasis patients. In dogs infected by D. immitis, neutrophils accumulate in kidneys and in the wall of pulmonary arteries. As shown by our studies, Wolbachia could contribute to these inflammatory phenomena through its surface protein WSP, independently from its endotoxin component.

Sequencing of the complete gene coding for the GroEL of the Wolbachia of Dirofilaria immitis and expression and purification of the recombinant protein.

Wolbachia are intracellular bacteria that infect arthropods and filarial nematodes. These bacteria play an important role in the immunology and pathogenesis of filarial diseases through their proteins and, possibly, other molecules. GroEL is a constitutively expressed bacterial protein; it is highly conserved among bacteria and is involved in the correct folding of newly synthesized proteins. Here we report the production of recombinant GroEL from the Wolbachia of Dirofilaria immitis. Our goal is to test the hypothesis that GroEL is involved in the immunopathology of filariases. The complete groel gene was PCR-amplified, sequenced and cloned into an expression vector. The recombinant GroEL was purified by affinity chromatography by using high-performance liquid chromatography (HPLC).

TIR8 receptor expression in bovine tissues.

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.