Intrauterine infusion of a pathogenic bacterial cocktail is associated with the development of clinical metritis in postpartum multiparous Holstein cows.

Metritis is a uterine disorder common in dairy cattle caused by bacterial infection, with greater incidence in the early postpartum period. The disease causes delayed uterine involution, with a fetid, watery, red-brown discharge, with animals presenting different clinical signs including fever, dullness, inappetence, decreased milk yield, and dehydration. We developed an in vivo model of clinical metritis in Holstein multiparous cows using a pathogenic cocktail of Escherichia coli, Fusobacterium necrophorum, and Trueperella pyogenes. A total of 36 multiparous cows were randomly allocated to 1 of 3 treatment groups of 12 animals each. Cows assigned to the control group received an intrauterine administration of sterile saline solution, those in the low-dose group received a bacterial inoculum containing 106 cfu of Escherichia coli, Trueperella pyogenes, and Fusobacterium necrophorum; and those in the high-dose group received 109 cfu of these same organisms, all within 24 h of parturition. Clinical signs; milk yield; dry matter intake; serum concentration of acute phase proteins, metabolites, and cytokines; and bacterial counts in vaginal discharge were measured from parturition until 14 d in milk. Based on clinical signs, cows in the low-dose group had a greater incidence of metritis (83.3%) compared with the control (9%) or high-dose (25%) group. The low-dose group also had lesser dry matter intake compared with controls and an acute phase protein profile that typified metritis. The low-dose group presented greater relative abundance of bacteria from the genus Fusobacterium in the vaginal discharge compared with control cows, and the high-dose group was similar to the control group. Unexpectedly, intrauterine infusion of cows with a lower dose of bacteria was more effective than a higher dose, indicating that the bacterial load may affect metritis incidence in a nonlinear manner. These findings demonstrate that clinical metritis can be experimentally induced in postpartum multiparous Holstein cows, creating a relevant model for the study of uterine diseases.

Recombinant protein subunit vaccine reduces puerperal metritis incidence and modulates the genital tract microbiome.

The objective of this study was to evaluate the efficacy of 3 vaccine formulations containing proteins (FimH, leukotoxin, and pyolysin), inactivated whole cells (Escherichia coli, Fusobacterium necrophorum, and Trueperella pyogenes), or both, in the prevention of postpartum uterine diseases. A randomized clinical trial was conducted at a commercial dairy farm; 800 heifers were assigned into 1 of 4 different treatment groups: control, vaccine 1 (bacterin and subunit proteins), vaccine 2 (bacterin), and vaccine 3 (recombinant subunit proteins), and each heifer received a subcutaneous injection of its respective treatment at 240 ± 3 and 270 ± 3 d of gestation. Vaccination significantly reduced the incidence of puerperal metritis when compared with control (9.1% vs. 14.9%, respectively; odds ratio 0.51). Additionally, vaccine 3 was found to reduce the incidence of puerperal metritis when compared with the control (8.0% vs. 14.9%, respectively; odds ratio 0.46). Reproduction was improved for metritic cows that were vaccinated, and the effect was stronger for cows that were treated with vaccine 3. In general, vaccination decreased the total vaginal bacterial load and decreased the vaginal load of F. necrophorum by 9 d in milk. Vaccination reduced the prevalence of puerperal metritis in the first lactation of dairy cows, leading to less metritic disease and improved reproduction.

Effects of recombinant bovine interleukin-8 (rbIL-8) treatment on health, metabolism, and lactation performance in Holstein cattle I: Production and functional characterization of rbIL-8 in vitro and in vivo.

In the present study, we standardized processes of cloning and purification of recombinant bovine interleukin-8 (rbIL-8) from bacterial culture and assessed its biological activity in Holstein cattle. Plasmid containing a subclone of bovine IL-8 was expressed using Escherichia coli BL21 and cell lysate was purified by chromatography. The presence of rbIL-8 was assessed by Western blot analyses and function was confirmed in vitro using a chemotaxis chamber. Based on optical density values, chemoattractant properties of rbIL-8 were 10-fold greater compared with control wells. Two in vivo studies were conducted to assess the biological activity of rbIL8. For study 1, one-year-old Holstein heifers (n = 20) were randomly allocated to receive a single intravaginal administration containing 1,125 µg of rbIL-8 diluted in 20 mL of saline solution (rbIL-8, n = 10) or a single intravaginal administration of 20 mL of saline solution (control, n = 10). For study 2, nonpregnant lactating Holstein cows (n = 31) were randomly allocated to receive an intrauterine administration with 1,125 µg of rbIL-8 diluted in 20 mL of saline solution (rbIL-8, n = 11), a positive control consisting of resin-purified lysate of E. coli BL21 not transfected with the plasmid coding for rbIL-8 diluted in 20 mL of saline solution (E. coli, n = 10), and a negative control administered with 20 mL of saline solution (control, n = 10). An increase in vaginal neutrophils was observed in heifers treated with rbIL-8 within 3 h of treatment, but not in control heifers. Additionally, intrauterine administration of rbIL-8 increased the proportion of PMN cells in uterine cytological samples from 3.5% before treatment to 75.8% 24 h later-an increase that was not observed in the negative control group and cows treated with resin-purified lysate of E. coli. To further evaluate the effect of local and systemic rbIL-8 stimulation on the dynamics of circulating white blood cells, a third study was conducted. In study 3, nonpregnant 8-mo-old Holstein heifers (n = 30) were randomly allocated into 1 of 3 treatment groups: intravenous rbIL-8 (1,125 µg of rbIL-8 diluted in 5 mL of saline solution, n = 10); intravaginal rbIL-8 (1,125 µg of rbIL-8 diluted in 20 mL of saline solution; n = 10); or intravaginal saline (20 mL of saline solution, n = 10). Intravenous injection of rbIL-8 resulted in a transient increase in rectal temperature, which was greater at 2 h after treatment compared with cows treated intravaginally with rbIL-8 or heifers treated with saline solution. Heifers treated with rbIL-8 intravenously displayed a marked reduction in neutrophils, basophils, lymphocytes, and monocytes within the first 4 h posttreatment compared with heifers treated intravaginally. However, at 6 h after treatment, heifers treated with rbIL-8 intravenously displayed a rebound in white blood cell counts caused by an increase in neutrophil counts. These results show that the presented purification method is effective and results in biologically active rbIL-8 that can be used safely to modulate immune responses in cattle.

Effects of recombinant bovine interleukin-8 (rbIL-8) treatment on health, metabolism, and lactation performance in Holstein cattle II: Postpartum uterine health, ketosis, and milk production.

To evaluate the effect of recombinant bovine interleukin-8 (rbIL-8) on uterine health and milk production, 2 separate studies were conducted. For study 1, postpartum Holstein cows (n = 213) were randomly allocated into 1 of 3 intrauterine treatment groups: control (CTR, 250 mL of saline solution), low dose (L-IL8, 11.25 µg of rbIL-8 diluted in 250 mL of saline solution), and high dose (H-IL8, 1,125 µg of rbIL-8 diluted in 250 mL of saline solution). Intrauterine delivery of treatments was performed within 12 h of parturition. Cows were evaluated for retained fetal membranes, puerperal metritis, and clinical endometritis. Blood samples were collected immediately before treatment and 1, 2, and 3 d in milk for assessment of IL-8, haptoglobin, fatty acids, and ß-hydroxybutyrate concentrations. Treatment with rbIL-8 reduced the incidence of puerperal metritis in multiparous cows (CTR = 34.3, L-IL8 = 8.11, and H-IL8 = 6.35%). Both the L-IL8 and H-IL8 groups produced significantly more milk, fat-corrected milk, and energy-corrected milk yields when compared with placebo-treated controls. A second study was performed to confirm the effect of rbIL-8 on milk production. In study 2, 164 primiparous cows were randomly allocated into 1 of 4 treatment groups: control (CTR, 250 mL of saline solution), low dose (L-IL8, 0.14 µg of rbIL-8), medium dose (M-IL8, 14 µg of rbIL-8), and high dose (H-IL8, 1,400 µg of rbIL-8). Treatments were prepared and administered as described for study 1. Cows in the L-IL8, M-IL8, and H-IL8 groups produced significantly more milk, fat-corrected milk, and energy-corrected milk yields when compared with control cows. In conclusion, treatment with rbIL-8 decreased the incidence of puerperal metritis in multiparous cows. The administration of rbIL-8 was repeatedly associated with a dramatic and long-lasting improvement of lactation performance.

Effects of recombinant bovine interleukin-8 (rbIL-8) treatment on health, metabolism, and lactation performance in Holstein cattle IV: Insulin resistance, dry matter intake, and blood parameters.

We have shown in 2 independent studies that cows who received recombinant bovine interleukin-8 (rbIL-8) administered intrauterinely shortly after parturition have a significant and long-lasting increase in milk yield. In the present study, we hypothesized that the increased milk production associated with rbIL-8 treatment is a consequence of increased postpartum dry matter intake (DMI) and orchestrated homeorhetic changes that prioritize milk production. Cows were enrolled into 1 of 3 treatment groups: those assigned to the control group (CTR; n = 70) received an intrauterine (IU) administration of 500 mL of Dulbecco's phosphate-buffered saline (DPBS) solution and 1 mL of DPBS solution intravenously (IV; jugular vein), those assigned to the rbIL-8 IV group (rbIL8-IV, n = 70) received an IV injection of 167 µg of rbIL-8 and 500 mL of DPBS solution IU, and cows assigned to the rbIL-8 IU group (rbIL8-IU, n = 70) received an IU administration with 1,195 µg of rbIL-8 diluted in 499.5 mL of DPBS solution and 1 mL of DPBS solution IV. Animals were housed in a tiestall from calving to 30 d in milk (DIM) to measure DMI. Blood samples were collected daily from calving to 7 DIM and weekly until 28 DIM. Insulin resistance was evaluated using an intravenous glucose tolerance test and intravenous insulin challenge test (IVICT) in a subgroup of cows (n = 20/treatment) at 10 and 11 DIM, respectively. Additionally, liver biopsy samples were taken at 14 DIM from the same subgroup of cows to measure triglyceride levels and cell proliferation and apoptosis. Cows treated with rbIL8-IU produced more milk (CTR = 36.9 ± 1.5; rbIL8-IU = 38.5 ± 1.5; rbIL8-IV = 36.6 ± 1.5 kg/d), energy-corrected milk (CTR = 42.9 ± 0.9; rbIL8-IU = 46.1 ± 0.8; rbIL8-IV = 43.7 ± 0.9 kg/d), and fat-corrected milk (CTR = 44.3 ± 0.9; rbIL8-IU = 47.8 ± 0.9; rbIL8-IV = 45.2 ± 0.9 kg/d) yields when compared with CTR cows, and no differences were observed between rbIL8-IV and CTR cows. The administration of rbIL8-IU significantly increased DMI compared with CTR (CTR = 18.8 ± 0.3; rbIL8-IU = 19.9 ± 0.3; rbIL8-IV = 19.3 ± 0.3 kg/d). Recombinant bIL-8 treatment did not affect glucose, insulin, or fatty acids (i.e., IVICT only) concentrations or their area under the curve in response to an intravenous glucose tolerance test and IVICT when compared with CTR. Moreover, rbIL-8 treatment administered IU or IV increased liver triglyceride levels. Additionally, cows treated with rbIL8-IU tended to have lower odds of developing hyperketonemia (odds ratio = 0.46, 95% confidence interval: 0.19 to 1.10), lower odds of clinical ketosis and displaced abomasum combined (odds ratio = 0.17, 95% confidence interval: 0.03 to 0.89), and lower odds of diseases combined (odds ratio = 0.43, 95% confidence interval: 0.21 to 0.86) when compared with CTR. We conclude that the administration of rbIL8-IU increases DMI, milk production, fat-corrected milk, and energy-corrected milk while improving overall health during the postpartum period. This study supports the use of rbIL-8 administered IU shortly after calving to improve health and production responses in lactating cows.

Effects of recombinant bovine interleukin-8 (rbIL-8) treatment on health, metabolism, and lactation performance in Holstein cattle III: Administration of rbIL-8 induces insulin resistance in bull calves.

Our previous work has suggested that recombinant bovine interleukin-8 (rbIL-8) treatment might influence cow metabolism. Therefore, this study was conducted to initially assess the effects of systemic administration of rbIL-8 on response to a glucose challenge, blood metabolites, insulin, growth hormone, insulin-like growth factor-1, immune cell populations, and inflammatory parameters in Holstein bull calves. Calves from 30 ± 6 d of life were individually housed and randomly allocated to 1 of 2 treatment groups: rbIL-8 (rbIL-8, n = 10) and control (CTR, n = 8). Calves assigned to the rbIL-8 group received 1 s.c. injection (d 1, 0900 h) and 6 i.v. injections (d 1 at 1600 h, d 2 and 3 at 0900 h and 1600 h, and d 4 at 0900 h) of rbIL-8 (4 µg/kg of body weight), whereas the CTR group received 2 mL of sterile saline solution at each time point. Day of enrollment was considered as d 1, and the study duration was 10 d. Insulin concentrations and whole-body glucose disappearance were evaluated by an i.v. glucose tolerance test conducted at 12 h and 7 d following the last rbIL-8 injection. Rectal temperature and blood samples were collected on d 1, 2, 3, and 4 at -30 (before treatment, 0830 h), 30, 60, 120, 240, and 360 min relative to treatment, and daily at 0830 h for the rest of the study period. Serum was harvested, and the following parameters were measured: ß-hydroxybutyrate (BHB), nonesterified fatty acids, glucose, insulin, plasma urea nitrogen, haptoglobin, and differential blood count. Significant differences were considered when P ≤ 0.05 and a trend if 0.05

Effect of pegbovigrastim administration on the microbiome found in the vagina of cows postpartum.

The objective of this study was to evaluate the effect of pegbovigrastim (PEG) treatment of peripartum Holstein cows on the microbiome found in the vagina postpartum using sequencing of the 16S rRNA gene. A subset of cows was randomly sampled from a larger study where cows had been randomly assigned to 1 of 2 treatments: pegbovigrastim (PEG) or untreated control (CTR). The PEG-treated cows received a subcutaneous injection containing 15 mg of pegbovigrastim 7 d before expected calving and a second injection within 24 h of calving. Vaginal samples from 97 PEG-treated and 98 CTR cows were collected at calving, 7 ± 3, and 35 ± 3 d in milk (0, 7, and 35 DIM). Metritis was diagnosed at 7 ± 3 DIM and purulent vaginal discharge (PVD) at 35 ± 3 DIM. The PEG treatment did not alter the vaginal microbiome. Principal coordinate analysis (PCoA) showed that metritic cows had a dissimilar vaginal microbiome compared with cows that did not develop metritis, particularly at 7 but also at 35 DIM. This difference was characterized by higher relative abundance of Porphyromonas and Bacteroides and a lower relative abundance of Ureaplasma, Ruminococcaceae, and Clostridiales at 7 DIM, and a higher relative abundance of Ureaplasma and a lower relative abundance of Pasteurellaceae at 35 DIM. Based on PCoA, we observed that cows that developed PVD had a dissimilar vaginal microbiome compared with cows that did not develop PVD, particularly at 35 DIM but also at 7 DIM. This difference was characterized by a higher relative abundance of Bacteroides at 7 DIM and higher relative abundance of Fusobacterium and Bacteroides at 35 DIM. Cows that developed metritis and PVD also had higher relative abundance of Fusobacterium and Bacteroides at 0 DIM. Furthermore, the Chao1 and Shannon indices were decreased in metritic cows at 7 DIM and in PVD cows at 7 and 35 DIM. In summary, PEG treatment had no effect on the vaginal microbiome, and uterine disease was associated with major changes in the microbiome found in the vagina postpartum.

Association of peripartum plasma insulin concentration with milk production, colostrum insulin levels, and plasma metabolites of Holstein cows.

The main objective of this study was to assess associations between plasma insulin concentration around parturition and production in Holstein cows. Primiparous and multiparous cows (n = 267) were enrolled. Blood samples were collected within 12 h after parturition (d 0), and on d 3 and 10 after calving. In addition, blood samples were collected 7 d before (-7 d) the expected date of parturition and colostrum samples were collected within 8 h after parturition from a subset of cows to measure insulin concentration (n = 47). All samples were harvested from 0630 to 1100 h and were used to quantify insulin, nonesterified fatty acids (NEFA), and ß-hydroxybutyrate. The plasma concentrations of insulin on d -7 and 0 were not correlated with insulin levels in colostrum. Cows were grouped according to plasma insulin concentration based on the median as low insulin (L-INS) or high insulin (H-INS) on d 0 (median = 0.35 ng/mL; range 0.2 to 1.2), 3 (median = 0.32 ng/mL; range 0.2 to 1.6), and 10 (median = 0.30 ng/mL; range 0.2 to 0.8). We detected that cows in the L-INS group on d 0 (L-INS = 0.57 ± 0.02; H-INS = 0.49 ± 0.02 mmol/L), d 3 (L-INS = 0.56 ± 0.02; H-INS = 0.49 ± 0.02 mmol/L), and d 10 (L-INS = 0.61 ± 0.03; H-INS = 0.55 ± 0.03 mmol/L) had higher NEFA concentrations compared with cows in the H-INS group. Compared with H-INS cows, milk yield was higher for cows classified as L-INS on d 0 (L-INS = 40.75 ± 0.69; H-INS = 38.41 ± 0.64 kg) and d 10 (L-INS = 40.95 ± 0.74; H-INS = 38.66 ± 0.64 kg). Moreover, fat-corrected milk was higher for cows classified as L-INS on d 0 (L-INS = 40.59 ± 2.36; H-INS = 37.73 ± 2.31 kg) and d 10 (L-INS = 41.00 ± 2.42; H-INS = 38.65 ± 2.28 kg) compared with H-INS cows, and energy-corrected milk was higher for L-INS cows compared with H-INS cows regardless of the day (d 0, L-INS = 44.50 ± 0.70 vs. H-INS = 41.67 ± 0.64 kg; d 3, L-INS = 43.65 ± 0.74 vs. H-INS = 40.88 ± 0.72 kg; d 10, L-INS = 44.09 ± 0.73 vs. H-INS = 40.55 ± 0.68 kg). We conclude that low plasma insulin concentration during early lactation is associated with higher milk yield in the long term.

Effects of pegbovigrastim administration on periparturient diseases, milk production, and reproductive performance of Holstein cows.

The aim of this study was to evaluate the effects of treating Holstein cows with pegbovigrastim on periparturient diseases, milk production, and reproductive performance while exploring the mode of action of an immunomodulatory protein. Cows were randomly allocated to 1 of 2 treatments, untreated control (CTR, n = 423) and pegbovigrastim (PEG, n = 417). At 7 d from the anticipated calving date (d -7), cows allocated to PEG received a subcutaneous injection of 15 mg of pegylated recombinant bovine granulocyte colony stimulating factor (pegbovigrastim injection, Imrestor, Elanco Animal Health, Greenfield, IN). A second injection was administered within 24 h after calving (d 0). Blood samples were obtained from a subset of cows (CTR, n = 103; PEG, n = 102) at -7 and 0, 3, 7, and 14 d relative to parturition. Samples were used for hemogram and quantification of haptoglobin, nonesterified fatty acids, ß-hydroxybutyrate, and trace and macro minerals. Vaginal cytobrush was performed on the same subset cows at d 0, 7, and 14 to assess the relative neutrophil count. Additionally, colostrum samples were collected to measure IgG, IgM, IgA, and lactoferrin concentrations. Postpartum disease occurrence was recorded from calving until 30 d in milk (DIM). Weekly milk yield was recorded for the first 12 wk after calving. Cows treated with PEG had a 3- to 4-fold increase in circulating polymorphonuclear leukocyte, lymphocyte, and monocyte numbers, with a peak at 3 d after treatment followed by a gradual decline, but the counts remained significantly greater compared with CTR at 14 DIM. The administration of PEG did not affect the incidence of clinical and subclinical mastitis, retained fetal membranes, metritis, puerperal metritis, and endometritis. Primiparous cows treated with PEG tended to have lower odds of developing hyperketonemia than CTR [odds ratio (OR) = 0.57, 95% confidence interval (CI) = 0.23 to 1.42]. Cows treated with PEG had higher odds of being diagnosed with lameness within 30 DIM compared with CTR (OR = 1.79, 95% CI = 1.16 to 2.76); however, we found no significant differences by 60 DIM. Treatment with PEG increased the odds of displaced abomasum (OR = 8.27, 95% CI = 1.02 to 66.6). Cows treated with PEG had higher odds of being diagnosed with 1 or more clinical diseases compared with CTR cows (OR = 1.39, 95% CI = 1.02 to 1.90). We observed no differences in linear scores or milk composition between treatments. Furthermore, primiparous cows treated with PEG produced more milk than CTR primiparous cows during the first 12 wk postpartum (PEG = 37.51 ± 0.66; CTR = 35.91 ± 0.65 kg), but no differences were observed on energy-corrected milk. Treatment did not alter reproductive performance; additionally, cows diagnosed with metritis or puerperal metritis and treated with PEG tended to have higher proportion of neutrophils in the vaginal mucosa when compared with CTR metritic cows. Although PEG treatment increased circulating polymorphonuclear leukocyte, monocyte, and lymphocyte numbers, as expected, it was detrimental to cow health because it increased morbidity.

The effect of intrauterine infusion of dextrose on clinical endometritis cure rate and reproductive performance of dairy cows.

The main objective of this study was to evaluate the intrauterine administration use of 200 mL of 50% dextrose solution as a treatment against clinical endometritis (CE); CE cure rate and reproductive performance were evaluated. Additionally, the association of several relevant risk factors, such as retained placenta (RP), metritis, CE, anovulation, hyperketonemia, and body condition score with reproductive performance, early embryonic mortality, and CE were evaluated. A total of 1,313 Holstein cows housed on 4 commercial dairy farms were enrolled in the study. At 7±3 DIM cows were examined for metritis and had blood collected to determine serum ß-hydroxybutyrate concentration. To determine if cows had ovulated at least once before 44±3 DIM, the presence of a corpus luteum was evaluated by ovarian ultrasonography at 30±3 DIM and at 44±3 DIM. At 30±3 DIM, CE was diagnosed using the Metricheck device (SimcroTech, Hamilton, New Zealand); cows with purulent or mucopurulent vaginal discharge were diagnosed as having CE. Cows diagnosed with CE (n=175) were randomly allocated into 2 treatment groups: treatment (intrauterine infusion of 200 mL of 50% dextrose) or control (no infusion). Clinical endometritis cows were re-evaluated as described above at 44±3 DIM, and cows that were free of purulent or mucopurulent vaginal discharge were considered cured. Intrauterine infusion of dextrose tended to have a detrimental effect on CE cure rate, and treatment did not have an effect on first-service conception rate and early embryonic mortality. A multivariable Cox's proportional hazard model was performed to evaluate the effect of several variables on reproductive performance; the variables RP, CE, parity, anovulation, and the interaction term between parity and anovulation were associated with hazard of pregnancy. Cows that did not have RP or CE were more likely to conceive than cows that were diagnosed with RP or CE. Cows that had RP were at 3.36 times higher odds of losing their pregnancy than cows that did not have RP. In addition, cows diagnosed with CE were at 2.16 higher odds of losing their pregnancy than cows without CE. In conclusion, intrauterine infusion of 200 mL of 50% dextrose solution as a treatment for CE had a strong statistical tendency to decrease CE cure rate, did not improve first-service conception rate and early embryonic mortality, and did not decrease calving-to-conception interval.