Technical note: Assessment of neutrophil endocytosis and proteolytic degradation and its relationship with phagocytosis and oxidative burst in dairy cows.

Conventional assays of polymorphonuclear cell (PMN, neutrophil) function such as oxidative burst (OB) and phagocytosis (PC) are widely used to evaluate innate immunity in the transition period of dairy cows. Oxidative burst is commonly evaluated by measuring PMN median fluorescence intensity (MFI) involving the release of reactive oxygen species after in vitro stimulation. Phagocytosis can be measured by engulfment of fluorescent beads by PMN. DQ-ovalbumin (DQ-OVA) is a molecule suitable for the assessment of intracellular proteolytic degradation, so it might be informative about an additional pathway of pathogen handling by PMN. In this study, we evaluated the use of the DQ-OVA assay for the assessment of PMN function and the relationships among OB, PC, and DQ-OVA results in PMN isolated from blood of dairy cows between 5 and 21 d post partum. Results of the DQ-OVA validation assay were assessed with mixed linear regression models. Pearson correlation tests and kappa values for agreement were used to associate the MFI between each PMN function assay (OB, PC, and DQ-OVA). For the validation assay (9 cows in 3 replicates), PMN incubated with DQ-OVA were stimulated with IFN-γ or inhibited with cytochalasin D, and fluorescence was compared with untreated PMN. Stimulated and inhibited PMN had greater (970 ± 160 units) and lesser (593 ± 55 units) MFI relative to untreated PMN (791 ± 154 units), respectively, indicating that DQ-OVA fluorescence reflected enhanced or reduced endocytic and proteolytic function. To associate the MFI outcomes among OB, PC, and DQ-OVA, 153 samples from 40 cows were analyzed. Results showed significant, although weak association between DQ-OVA and PC MFI (Pearson r = 0.16). When values of MFI were categorized according to the first ("high" PMN functionality), second and third ("moderate" PMN functionality), or fourth ("low" PMN functionality) quartiles, agreement beyond chance (κ) was moderate: κ = 0.38 for DQ-OVA and OB, κ = 0.43 for DQ-OVA and PC, and κ = 0.43 for OB and PC. The DQ-OVA assay may complement traditional PMN functional assays because it provides additional information regarding the combination of endocytosis and proteolytic degradation, but it is not a substitute for assessment of OB or PC.

Feed restriction to induce and meloxicam to mitigate potential systemic inflammation in dairy cows before calving.

Most dairy cows experience a transient decrease in feed intake in the 1 to 2 wk before calving, which has been associated with systemic inflammation (SI), indicated by increased blood haptoglobin (Hp) concentration. We aimed to characterize the association between prepartum decrease in feed intake and the onset of SI and, if present, the ability of meloxicam (MEL), a non-steroidal anti-inflammatory drug, to mitigate SI. Holstein cows (n = 45) were assigned to control (n = 13), feed restriction (FR) untreated (FR-U; n = 15), and FR treated with MEL (FR-T; n = 17) groups. Daily feed intake was measured from -22 d from expected parturition until 35 d postpartum. Control cows were fed ad libitum, whereas FR-U and FR-T cows were reduced to 60% of their average intake for 4 consecutive days (-15 to -12 d from expected calving). The FR-T cows received MEL (0.5 mg/kg of body weight) once daily for 4 consecutive days (-13 to -10 d from expected calving). Blood samples were collected -22, -15, -14, -13, -12, -10, -7, -5, -3, 0, 1, 3, 5, 7, 15, 22, and 35 d relative to calving to measure serum concentrations of total calcium, total protein, albumin, globulin, cholesterol, urea, glucose, gamma-glutamyl transferase, aspartate aminotransferase, glutamate dehydrogenase, ß-hydroxybutyrate, nonesterified fatty acids, Hp, and insulin-like growth factor-1. Serum concentrations of lipopolysaccharide-binding protein were measured -22, -15, -14, -13, -12, and -10 d from expected calving. Simplified glucose tolerance tests were performed on -15, -12, -5, 1, and 5 d relative to calving. Mixed linear regression models were used to assess the effects of FR and MEL on each metabolite. The interaction between treatment group and blood sampling day was forced into each model. All models accounted for body condition score, parity, and the cow as a random effect. Nonesterified fatty acids concentrations in both the FR-U and FR-T groups significantly increased from the second until the last day of FR. Feed restriction increased urea concentrations compared with the control group on -14 d but decreased urea concentrations on -10 d from expected calving. Control cows had greater ß-hydroxybutyrate concentrations compared with FR cows on 15, 21, and 35 d postpartum. For all other metabolites, no differences were found. This model of FR produced substantial fat mobilization but based on serum Hp and lipopolysaccharide-binding protein concentrations did not generate measurable SI; therefore, we were unable to evaluate the ability of MEL to mitigate SI.

Effects of parity, blood progesterone, and non-steroidal anti-inflammatory treatment on the dynamics of the uterine microbiota of healthy postpartum dairy cows.

This study evaluated the effects of treatment with meloxicam (a non-steroidal anti-inflammatory drug), parity, and blood progesterone concentration on the dynamics of the uterine microbiota of 16 clinically healthy postpartum dairy cows. Seven primiparous and 9 multiparous postpartum Holstein cows either received meloxicam (0.5 mg/kg SC, n = 7 cows) once daily for 4 days (10 to 13 days in milk (DIM)) or were untreated (n = 9 cows). Endometrial cytology samples were collected by cytobrush at 10, 21, and 35 DIM, from which the microbiota analysis was conducted using 16S rRNA gene sequence analysis. A radioimmunoassay was used to measure progesterone concentration in blood serum samples at 35 DIM and cows were classified as ˃ 1 ng/mL (n = 10) or ≤ 1 ng/mL (n = 6). Alpha diversity for bacterial genera (Chao1, Shannon-Weiner, and Camargo's evenness indices) were not affected by DIM, meloxicam treatment, parity, or progesterone category. For beta diversity (genera level), principal coordinate analysis (Bray-Curtis) showed differences in microbiota between parity groups. At the phylum level, the relative abundance of Actinobacteria was greater in primiparous than multiparous cows. At the genus level, there was lesser relative abundance of Bifidobacterium, Lactobacillus, Neisseriaceae, Paracoccus, Staphylococcus, and Streptococcus and greater relative abundance of Bacillus and Fusobacterium in primiparous than multiparous cows. Bray-Curtis dissimilarity did not differ by DIM at sampling, meloxicam treatment, or progesterone category at 35 DIM. In conclusion, uterine bacterial composition was not different at 10, 21, or 35 DIM, and meloxicam treatment or progesterone category did not affect the uterine microbiota in clinically healthy postpartum dairy cows. Primiparous cows presented a different composition of uterine bacteria than multiparous cows. The differences in microbiota associated with parity might be attributable to changes that occur consequent to the first calving, but this hypothesis should be investigated further.

Dynamics of uterine microbiota in postpartum dairy cows with clinical or subclinical endometritis.

Our objectives were to describe and compare the uterine bacterial composition of postpartum Holstein cows diagnosed as healthy (n = 8), subclinical endometritis (SCE; n = 8), or clinical endometritis (CE; n = 5) in the fifth week postpartum. We did metagenomic analyses of 16S rRNA gene sequences from endometrial cytobrush samples at 10, 21, and 35 days in milk (DIM), and endometrial bacterial culture at 35 DIM. Uterine bacterial composition in healthy, SCE, and CE was stable at 10, 21, and 35 DIM. Alpha and beta diversities showed a different uterine microbiome from CE compared to healthy or SCE, but no differences were found between healthy and SCE cows. At the phylum level, the relative abundance of Bacteroidetes and Fusobacteria, and at genera level, of Trueperella was greater in CE than healthy or SCE cows. Trueperella pyogenes was the predominant bacteria cultured in cows with CE, and a wide variety of bacterial growth was found in healthy and SCE cows. Bacteria that grew in culture were represented within the most abundant bacterial genera based on metagenomic sequencing. The uterine microbiota was similar between SCE and healthy, but the microbiome in cows with CE had a loss of bacterial diversity.

Effect of anti-inflammatory treatment on systemic inflammation, immune function, and endometrial health in postpartum dairy cows.

Systemic inflammation (SI) is increasingly studied in several species because it may be central in many metabolic disturbances and be a risk factor for clinical disease. This proof-of-concept study evaluated the effects of the anti-inflammatory drug meloxicam on markers of SI and energy metabolism, polymorphonuclear neutrophil (PMN) function, and endometritis in clinically healthy postpartum dairy cows. Cows received meloxicam (0.5 mg/kg of body weight; n = 20) once daily for 4 days (10-13 days postpartum) or were untreated (n = 22). Blood samples were collected -7, 1, 3, 5, 7, 10, 11, 12, 13, 14, 18, 21, 28, and 35 days relative to calving to measure serum concentrations of metabolic and inflammatory markers. Function of peripheral blood PMN were evaluated at 5, 10, 14, and 21, and proportion of PMN in endometrial cytology were performed at 5, 10, 14, 21, 28 and 35 days postpartum. Meloxicam decreased serum haptoglobin from the second until the last day of treatment, and improved indicators of energy metabolism (lesser ß-hydroxybutyrate and greater insulin-like growth factor-1 during treatment, and greater glucose at the end of treatment than control cows). This improved PMN function at 14 days postpartum, but the endometrial inflammatory status was not affected.

Comparison of cow-side diagnostic techniques for subclinical endometritis in dairy cows.

A limited number of cow-side diagnostic techniques exist for the diagnosis of subclinical endometritis (SCE) in dairy cattle. The objectives of this study were to compare results of endometrial cytology from samples collected by cytobrush (CB) and low-volume lavage (LVL) and to assess leukocyte esterase (LE) test strips and Brix refractometry as surrogate cow-side tests for SCE. Two samples were consecutively collected from 248 Holstein cows between 29 and 35 days postpartum, using CB and LVL techniques. Each sample was analyzed using cytology with a cut-point of ≥5% polymorphonuclear (PMN) cells, LE strips using cut-points of ≥1 and ≥ 2, and a Brix refractometer. Each diagnostic technique was compared intra-sample using the respective cytology as a gold standard and inter-sample using CB samples as the referent. The concordance correlation coefficient (CCC, ρc) for PMN% between CB and LVL was ρc = 0.59 [95% confidence interval (CI): 0.50 to 0.67] and the Kappa (κ) for agreement was κ = 0.35 [sensitivity (Se) = 0.88, specificity (Sp) = 0.45]. The optimal cut-point of LE ≥ 2 resulted in moderate agreement between CB and LVL samples, κ = 0.56 (Se = 0.89, Sp = 0.65). Agreement between LE and cytology using CB (κ = 0.49; Se = 0.89, Sp = 0.57) and LVL (κ = 0.44; Se = 0.77, Sp = 0.67) were similar. The correlation between Brix values from CB and LVL was ρc = 0.12 (CI -0.01 to 0.26). The correlation between CB cytology and Brix was ρc = 0.33 (CI 0.20 to 0.45) but ρc = -0.07 (CI -0.21 to 0.06) between LVL cytology and Brix. While LE strips with a cut-point of LE ≥ 2 had moderate agreement with cytology, Brix refractometry had poor performance for the diagnosis of SCE. Samples taken by CB and LVL produced comparable cow-side diagnostic results and either is a viable method for the diagnosis of SCE.