Biodiversity and antibiotic resistance profile provide new evidence for a different origin of enterococci in bovine raw milk and feces.

Enterococci are widely distributed in dairy sector. They are commensals of the gastrointestinal tract of animals, thus, via fecal contamination, could reach raw milk and dairy products. The aims of this study were: 1) to investigate the enterococcal diversity in cow feces and milk samples and 2) to evaluate the antibiotic resistance (AR) of dairy-related enterococci and their ability to transfer resistance genes. E. faecalis (59.9%), E. faecium (18.6%) and E. lactis (12.4%) were prevalent in milk, while E. faecium (84.2%) and E. hirae (15.0%) were dominant in bovine feces. RAPD-PCR highlighted a high number of Enterococcus biotypes (45 from milk and 37 from feces) and none of the milk strains exhibited genetic profiles similar to those of feces biotypes. A high percentage of enterococci isolated from milk (71%) were identified as multidrug resistant and resistance against streptomycin and tetracycline were widespread among milk strains while enterococci from feces were commonly resistant to linezolid and quinupristin/dalfopristin. Only E. faecalis strains were able to transfer horizontally the tetM gene to Lb. delbrueckii subsp. lactis. Our results indicated that Enterococcus biotypes from milk and bovine feces belong to different community and the ability of these microorganisms to transfer AR genes is strain-dependent.

Effects of Live Saccharomyces cerevisiae Yeast Administration in Periparturient Dairy Cows.

Dairy cows face several challenges during the transition period, and the administration of live yeast might be useful to mitigate this stressful condition. In the current study, the effects of live yeast administration on milk production, feed intake, and metabolic and inflammatory conditions were evaluated. Multiparous Holstein cows were enrolled in this randomized controlled trial and received either a control diet (CTR, n = 14) or the control diet plus 4 g/d of live Saccharomyces cerevisiae yeast (LSC, n = 14) from -21 to 56 days relative to calving. Dry matter intake, milk yield and composition, and rumination time were monitored daily. Blood samples were collected at -21, -7, 3, 14, 28, 42, and 56 days relative to calving to evaluate the metabolic profile. Fecal samples were collected at 56 days relative to calving to measure volatile fatty acids and feed digestibility. No differences between groups were observed in dry matter intake. Compared with CTR, rumination time was lower in LSC in after calving. Although there were no differences in milk components between groups, LSC had greater milk yield in the last three weeks of the study than CTR. No differences were observed in inflammatory markers or other plasma metabolites, except for ß-hydroxybutyrate, which was higher in LSC, and reactive oxygen metabolites (ROMs), which were lower in LSC. Overall, these outcomes suggest that live yeast supplementation had some positive effects on milk yield and oxidative status.

Drying-off dairy cows without antibiotic therapy and orally supplemented with lyophilized Aloe arborescens: effects on rumen activity, immunometabolic profile, and milk yield.

The drying-off is a stressful stage of the lactation cycle of dairy cows that deeply affects cows' metabolism, inflammatory status, and immune system. The promising effects observed during the transition period resulting from supplementation with Aloe arborescens Mill. suggest its potential utility during this phase. A group of 23 Holstein dairy cows with somatic cell count (SCC) less than 200 × 103 cells/ml and without intramammary infections were enroled in the study. Cows were divided into two groups: one orally receiving 10 g/day of A. arborescens Mill. lyophilized powder (AL; 11 cows) between -7 and 7 days from dry-off (DFD), and a control group (CTR; 12 cows). From -14 to 7 DFD and 7 and 28 days from calving, the body condition score and rectal temperature were determined, and rumen fluid, feces, milk, and blood samples were collected. Daily rumination times and milk yield were recorded. Data were analyzed through repeated measures mixed models. Compared to the CTR group, AL cows tended to show reduced production of volatile fatty acids in the rumen with acetate proportion that tended to be higher and valerate proportion that was lower. Moreover, Aloe supplementation caused a reduction in fecal dry matter. At the end of drying-off, AL cows presented better liver function, as suggested by higher paraoxonase plasma concentrations at 7 DFD, higher glucose, and lower urea, but showed increased reactive oxygen metabolites. Aloe supplementation at dry-off ameliorated inflammatory status after calving (lower haptoglobin and ceruloplasmin levels), and improved milk yield in the first weeks of subsequent lactation, without influencing milk composition, SCC, and incidence of intramammary infections. These results confirmed the positive effects of Aloe administration on liver function in dairy cows but indicate the need for further studies investigating the effects of Aloe on rumen fermentation profile and oxidative status.

Effects of an Intravenous Infusion of Emulsified Fish Oil Rich in Long-Chained Omega-3 Fatty Acids on Plasma Total Fatty Acids Profile, Metabolic Conditions, and Performances of Postpartum Dairy Cows During the Early Lactation.

A group of 10 multiparous Italian Holstein cows were housed in individual tied stalls and infused with 150 ml of saline (CTR; 5 cows), or of 10% solution rich in long-chained omega-3 fatty acids (n3FA; 5 cows) at 12, 24, and 48 h after calving. From -7 to 21 days from calving (DFC), the body condition score, body weight, dry matter intake (DMI), and milk yield were measured, blood samples were collected to assess the plasma fatty acids (FA) and metabolic profiles, and milk samples were collected to assess the milk composition. Data underwent a mixed model for repeated measurements, including the treatment and time and their interactions as fixed effects. Plasma FA profile from n3FA cows had lower myristic and higher myristoleic proportions, higher cis-11,14-eicosadienoic acid and monounsaturated FA proportions at 3 DFC, and lower cis-10-pentadecanoic proportion at 10 DFC. Besides these, n3FA cows had higher eicosapentaenoic (EPA) and docosahexaenoic (DHA) proportions (1.09 vs. 0.71 and 0.33 vs. 0.08 g/100 g), confirming the effectiveness of the infusion in elevating plasma availability of these FA. The plasma metabolic profile from n3FA cows revealed a tendency toward a lower concentration of reactive oxygen metabolites at 1 DFC and lower haptoglobin at 2 and 3 DFC, reflecting a mitigated inflammatory state. Furthermore, n3FA cows had a higher DMI during the first week of lactation. Higher DMI of n3FA could account for the changes detected on their plasma FAs, the higher milk yield they had at 1 and 2 DFC, the reduced lactose and urea nitrogen content in their milk. Higher DMI could also account for the lower plasma urea that n3FA cows had at 1 and 2 DFC, suggesting a lower amount of endogenous amino acids deserved to gluconeogenic fate. Milk from n3FA cows had lower rennet clotting time and higher curd firmness, which is probably driven by a higher EPA and DHA inclusion in the milk fat. Together, these outcomes suggest that the infusion exerts a short-term anti-inflammatory action on dairy cows at the onset of lactation.

Gene network expression of whole blood leukocytes in dairy cows with different milk yield at dry-off.

Dairy cows at dry-off undergo several management and physiological changes, resulting in alterations in plasma biomarkers of inflammation, oxidative stress, and immune system. High milk yield at the end of lactation exacerbates these responses. The underlying mechanism of these changes has yet to be elucidated. We hypothesized altered leukocyte gene expression after dry-off and different responses in cows with different milk yield. Thirteen Holstein dairy cows were sampled at the turn of dry-off to investigated whole blood leukocyte gene expression and were grouped according to the average milk yield during the last week of lactation: low (< 15 kg/d) and high milk yield (> 15 kg/d). Blood samples were collected in PAXgene tubes (Preanalytix, Hombrechtikon, Switzerland) at -7, 7, and 34 days from dry-off (DFD) to measure mRNA abundance of 37 genes. Normalized gene abundance data were subjected to MIXED model ANOVA (SAS Institute Inc., Cary, NC). Compared with -7 DFD, at 7 DFD RNA abundance of lipoxygenase genes (ALOX5, ALOX15) and myeloperoxidase (MPO) increased, and that of the antioxidant gene (SOD2) decreased. Meanwhile, genes related to recognition and immune mediation (CD16, MYD88, TLR2), migration and cell adhesion (CX3CR1, ITGAL, ITGB2, TLN1), and the antimicrobial gene MMP9 were downregulated at 7 or 34 DFD, whereas the antimicrobial IDO1 gene was upregulated. Compared with low-producing cows, cows with high milk yield at dry-off cows had upregulated expression of the pro-inflammatory cytokines IL8 and IL18 and a greater reduction in transcript abundance of the toll-like receptor (TLR) recognition-related gene TLR2. Overall, the dry-off confirmed to be a phase of intense changes, triggering an inflammatory response and somewhat suppressing leukocyte immune function. In cows with high milk yield during the week before dry-off, the inflammatory response was exacerbated.

Associations between ultrasound measurements and hematochemical parameters for the assessment of liver metabolic status in Holstein-Friesian cows.

Metabolic disorders, including hepatic lipidosis and ketosis, severely affect animal health status and welfare with a large economic burden in dairy herds. The gold standard for diagnosing hepatic lipidosis is the liver biopsy, which is impractical and invasive for the screening at farm level. Ultrasound (US) imaging is a promising technique for identifying liver dysfunction, but standardized specifications in physiological conditions are needed. Herein, we described the features of four US measurements, namely the liver predicted triacylglycerol (pTAG) content, liver depth (LD), and portal vein area (PVA) and depth (PVD) and we investigated their associations with a set of hematochemical (HC) indicators in 342 clinically healthy Holstein Friesian dairy cows. Liver pTAG content was negatively associated with hematocrit and positively with globulin, whereas PVA was negatively associated with thiol group levels, and LD positively with ceruloplasmin. We found significant interactions between some HC parameters and parity: in particular, creatinine, thiol groups and globulin for PVA, and aspartate aminotransferase, paraoxonase and ceruloplasmin for PVD. This study offers new insights on variations in liver function occurring after calving and pave the way for the potential use of minimally invasive techniques for prompt detection of metabolic disorders in dairy herds.

Changes of Plasma Analytes Reflecting Metabolic Adaptation to the Different Stages of the Lactation Cycle in Healthy Multiparous Holstein Dairy Cows Raised in High-Welfare Conditions.

Here, we tested the changes occurring in several plasma analytes during different stages of the lactation cycle of high welfare raised multiparous Holstein cows, and provided reference intervals (RI) for plasma analytes concentrations. Eleven high-welfare farms (HWF) located in Northern Italy were selected and their herds used to recruit 361 clinically healthy cows undergoing the dry (from -30 to -10 days from real calving; DFC), the postpartum (from 3 to 7 DFC), the early lactation (from 28 to 45 DFC) and the late lactation phases (from 160 to 305 DFC). Cows affected by subclinical diseases (SCD) were retrospectively excluded, and a subset of 285 cows was selected. Data of plasma analytes underwent ANOVA testing using physiological phases as predictors. The individual effect of each phase was assessed using a pairwise t-test assuming p ≤ 0.05 as a significance limit. A bootstrap approach was used to define the reference interval (RI) for each blood analyte within physiological phases having a pairwise t-test p ≤ 0.05. The concentration of nonesterified fatty acids, albumin, cholesterol, retinol, paraoxonase and tocopherol changed throughout all the physiological phases, whereas the concentration of K, alkaline phosphatase and thiol groups remained stable. Triglycerides, Zn, and ferric ion reducing antioxidant power in the dry phase and BHB, Ca, myeloperoxidase, haptoglobin, reactive oxygen metabolites and advanced oxidation of protein product in postpartum differed compared with other physiological phases. During the dry phase, Packed cell volume, Cl, and urea concentrations were similar to during the postpartum phase. Similarly, Na, γ-glutamyl transferase and ß-carotene concentrations were similar to during the early lactation phase; fructosamine and bilirubin concentrations were similar to during the late lactation phase. During the postpartum phase, fructosamine and P concentrations were similar to during the early lactation phase, and the aspartate transaminase concentration was similar to during the late lactation phase. During the early lactation phase, Mg, creatinine, total protein, globulin and ceruloplasmin concentrations were similar to during the postpartum phase, while the urea concentration was similar to during the late lactation phase. All these plasma analytes differed among the other phases. This study identifies physiological trends affecting plasma analytes concentrations during the different stages of the lactation cycle and provides a guideline for the duration and magnitude of their changes when animals are healthy and raised in optimal welfare conditions.

Age-related metabolic changes of pre-weaned Simmental calves fed whole bulk milk and ad libitum calf starter.

The present study aimed to investigate blood biomarkers encompassing the metabolic status of Simmental calves, with emphasis on weekly differences in the pre-weaning period. Ten Italian Simmental calves were used for the experiment and were offered 6 L of bulk whole milk/d from an open-bucket starting at day 2 from birth and through 5 weeks. Blood samples were collected at birth and weekly to assess biochemical indicators related to energy metabolism and liver function, minerals, markers of inflammation, and oxidative stress. Body weight (BW), heart girth (HG), and solid feed intake were positively affected by aging. Calves increased solid feed intake consumption after 2 week and reached a BW gain of 28.16 kg at 5 weeks. Most of blood biomarkers were affected by age, mostly concerned the period after birth and day 1, whereas no changes were observed for haptoglobin, urea, ß-hydroxybutyrate, Na, and K. The amount of milk fed to calves throughout this study contributes to the maintenance of glucose concentration over time, coupled also with a considerable amount of starter intake. Glucose levels associated to the high ADG (0.89 ±â€¯0.09 kg/d, mean ±â€¯SEM) suggest that calves experienced a positive energy balance throughout the entire pre-weaning period. Similar levels of haptoglobin and ceruloplasmin, together with a gradual increase of albumin, reflect good liver functionality and a stable condition regarding the inflammatory status. These results suggest that blood biomarkers of Simmental calves in the pre-weaning phase vary with growing stages.

Effects of Aloe arborescens Whole Plant Homogenate on Lipid Metabolism, Inflammatory Conditions and Liver Function of Dairy Cows during the Transition Period.

The anti-hyperlipidemic and anti-inflammatory effects exerted by Aloe on monogastric mammals suggest it as a potential strategy to address the tremendous metabolic alterations that affect dairy cows during their transition to calving. A group of 20 multiparous Italian Holstein dairy cows were housed in freestalls and allocated into two homogeneous groups to receive either 200 g/d of water (CTR) or 200 g/day of Aloe arborescens Mill. whole plant homogenate through a rumen tube (AAM) between -14 and 14 days from calving (DFC). From -14 to 35 DFC, the BCS, and milk yield were measured, and blood samples were collected to assess the hematochemical profile. Data underwent ANOVA testing using a mixed model for repeated measurements, including the treatment and time and their interactions as fixed effects. Compared to CTR cows, AAM cows had a less pronounced BCS loss in early lactation (p < 0.01), indicating less mobilization of body reserves. Compared to CTR cows, AAM cows had a lower plasma concentration of nonesterified fatty acids and beta hydroxybutyrate (p < 0.01 and = 0.01 respectively) that, paired with the lower butterfat content and fat/protein ratio in their milk (p = 0.03 and < 0.01 respectively), indicates that Aloe reduced the mobilization of body fats. AAM cows had a reduced concentration of myeloperoxidase in plasma and a lower SCC in milk compared to CTR cows (p = 0.02 for both), indicating an anti-inflammatory effect of Aloe. Furthermore, AAM cows had a lower plasma concentration of ceruloplasmin (p < 0.05) and higher plasma concentration of cholesterol, retinol, and paraoxonase compared to CTR cows (p < 0.01, < 0.01 and < 0.05 respectively), indicating Aloe was effective in mitigating the acute phase response in early lactation. Finally, AAM cows had lower plasma creatinine concentrations around calving (p < 0.05), a lower concentration of plasma bilirubin, and a higher concentration of plasma tocopherol compared to CTR cows (p = 0.01 for both). These data suggest Aloe has anti-hyperlipidemic and anti-inflammatory effects on transition dairy cows that could have ameliorated liver and kidney function disruption and increased the availability of body antioxidants in early lactation.

Evaluation of circulating leukocyte transcriptome and its relationship with immune function and blood markers in dairy cows during the transition period.

Dairy cows during the transition period are faced with important physiological changes which include a dysfunctional immune system and an increased inflammatory state. New data are necessary to understand the key factors involved in the immune system regulation. Six dairy cows were sampled during transition period to investigate the leukocyte transcriptome changes and its relationship with blood biomarkers. Blood samples were collected at - 20 ± 2, - 3 ± 1, 3, and 7 days from parturition (DFP). Leukocyte transcriptome was analyzed by deep sequencing technology (Hiseq1000 Illumina, USA). Plasma was analyzed for metabolic biomarkers. Differentially expressed genes (DEG) were used to run an enrichment analysis through the Dynamic Impact Approach (DIA). Considering - 20 DFP as references time, the main KEGG impacted pathways were activated before calving (- 3 DFP) and were connected to lipid metabolism, lipid transport in plasma, and phagosome. The greatest differences were found after parturition with 281 DEG (179 upregulated and 102 downregulated). The activated pathways were mainly related to immunity and endocrine aspects, while metabolic pathways related to lipid and amino acid metabolism were inhibited. Plasma BHBA had a substantial inhibitory impact on KEGG pathways related to DNA replication and cell cycle, while plasma IL-1ß had an inhibitory impact on fatty acid elongation in mitochondria and an activated impact in several pathways related to cellular energy metabolism. Overall, this study confirmed that many changes in lipid metabolism and immune competence of the circulating leukocytes occurred in dairy cow around calving. Interestingly, BHBA and IL-1ß connected with the transcriptome.

Effect of Pegbovigrastim on Hematological Profile of Simmental Dairy Cows during the Transition Period.

Pegbovigrastim is a long-acting analog of recombinant bovine granulocyte colony-stimulating factor, that promotes and increases the count and functionality of polymorphonuclear cells in dairy cows. The present study aimed to explore, for the first time in Simmental cows, the clinical and hematological effect of pegbovigrastim during the transition period (TP). Cows were randomly assigned into two groups: treated group (PEG; n = 16) received pegbovigrastim at approximately 7 days before expected parturition and within 6 h after calving, and control group (CTR; n = 16) received saline solution. Blood samples were obtained at -7, 0, 1, 3, 7, 14, 21, and 30 days relative to calving. PEG group showed white blood cells (WBC) count consistently higher compared with CTR group (p < 0.001) until to 3 weeks after calving. Neutrophils remained higher in PEG group (p < 0.001) up to three weeks after calving, compared with CTR group, with slight increment of band cells. Moreover, PEG group displayed a lower index of myeloperoxidase at 1, 3, and 7 days after calving (p < 0.01) compared with CTR. Basophils and lymphocytes showed a similar trend to those observed for neutrophils at 1 day after calving in PEG group. Finally, monocytes remained markedly elevated until 3 days after calving in PEG compared to CTR group (p < 0.001), whereas in PEG group, eosinophils population showed lower percentage values at 1 and 3 days after calving but higher values at 30 days compared with CTR group. PEG group was characterized by lower red blood cells (RBCs) count compared with CTR group (p < 0.05) and higher % of red cell volume distribution width (RDW) from week 2 and mean corpuscular volume (MCV) at 30 days after calving. In addition, the mean platelet volume (MPV) was significantly higher in PEG group at calving, 1, 3, and 7 days after calving compared with CTR group (p < 0.05). For the first time, we described the effect of pegbovigrastim in a breed not specialized exclusively in milk production as Holstein, but with dual purpose (meat and milk), evaluating the complete hematological profile in cows during the transition period. These results provide evidence on the proliferative effect of pegbovigrastim on WBC in Simmental breed highlighting its possible side effect on RBCs.

Administration of an Immune Stimulant during the Transition Period Improved Lipid Metabolism and Rumination without Affecting Inflammatory Status.

Omnigen-AF (OAF) increases leukocyte functions in immunosuppressed animal models and reduces incidence of infectious diseases in early lactating dairy cows, although its mode of action is still unclear. This study aims to provide a wider perspective of the metabolic effect of OAF to test its potential as a strategy to address metabolic disorders of the transition period. A group of 10 Holstein dairy cows were divided into 2 groups: The treated group (IMS; 5 cows) received 32.5 g of OAF twice a day (65 g d-1) as top-dress in the morning and afternoon feeds from -55 to 42 days from calving (DFC), whereas the control group (CTR; 5 cows) received no supplementation. From -62 to 42 DFC, body condition score, body weight, dry matter intake, rumination time and milk yield were measured; blood samples were collected weekly to assess a wide hematochemical profile and to test white blood cell functions by ex-vivo challenge assays. At 30 DFC, rumen fluid was collected and analyzed for pH, volatile fatty acids composition, urea nitrogen, and lactate contents. Data were submitted to ANOVA using a mixed model for repeated measures, including treatment, time, and their interaction as fixed effects. OAF decreased blood nonesterified fatty acids and beta hydroxybutyrate concentrations and increased rumination time in early lactation. Leukocytes from IMS cows had lower lactate production and lower glucose consumption after ex-vivo stimulation. OAF did not reduce the acute phase response indicators and reduced the blood concentrations of albumin and antioxidants after calving, suggesting impairment of hepatic functions related to protein synthesis and antioxidant management. Nevertheless, the lack of effect on bilirubin and liver enzymes refutes the possibility of severe liver damage occurring with OAF supplementation. Positive effects in reducing mobilization of body fats and ketogenesis and in increasing rumination time after calving suggest OAF effectiveness in preventing metabolic disorders of the transition period.

Maternal consumption of organic trace minerals alters calf systemic and neutrophil mRNA and microRNA indicators of inflammation and oxidative stress.

Organic trace mineral (ORG) supplementation to dairy cows in substitution of sulfate (INO) sources has been associated with improvement in immune function during stressful states such as the peripartal period. However, the effect of supplemental ORG during pregnancy on the neonatal calf is unknown. Therefore, our aim was to investigate the effects of ORG supplementation during late pregnancy on the immune system and growth of the neonatal calf. Of specific interest was the evaluation of inflammation-related microRNA (miRNA) and target gene expression in blood neutrophils as indicators of possible nutritional programming. Forty multiparous cows were supplemented for 30d prepartum with 40 mg/kg of Zn, 20 mg/kg of Mn, 5 mg/kg of Cu, and 1mg/kg of Co from either organic (ORG) or sulfate (INO) sources (total diet contained supplemental 75 mg/kg of Zn, 65 mg/kg of Mn, 11 mg/kg of Cu, and 1 mg/kg of Co, and additional Zn, Mn, and Co provided by sulfates), and a subset of calves (n=8/treatment) was used for blood immunometabolic marker and polymorphonuclear leukocyte (PMNL) gene and miRNA expression analyses. Samples were collected at birth (before colostrum feeding), 1d (24 h after colostrum intake), and 7 and 21d of age. Data were analyzed as a factorial design with the PROC MIXED procedure of SAS. No differences were detected in BW, but maternal ORG tended to increase calf withers height. Calves from INO-fed cows had greater concentrations of blood glucose, GOT, paraoxonase, myeloperoxidase, and reactive oxygen metabolites. Antioxidant capacity also was greater in INO calves. The PMNL expression of toll-like receptor pathway genes indicated a pro-inflammatory state in INO calves, with greater expression of the inflammatory mediators MYD88, IRAK1, TRAF6, NFKB, and NFKBIA. The lower expression of miR-155 and miR-125b in ORG calves indicated the potential for maternal organic trace minerals in regulating the PMNL inflammatory response at least via alterations in mRNA and miRNA expression. Overall, these results indicate that maternal nutrition with organic trace minerals could alter the neonatal innate immune response at least in part via changes in gene and miRNA expression. Further studies involving inflammatory challenges during the neonatal period should be performed to determine the functional benefit of maternal organic trace minerals on the neonatal immune response.