Integrating uterine microbiome and metabolome to advance the understanding of the uterine environment in dairy cows with metritis.

BACKGROUND: Metritis is a prevalent uterine disease that affects the welfare, fertility, and survival of dairy cows. The uterine microbiome from cows that develop metritis and those that remain healthy do not differ from calving until 2 days postpartum, after which there is a dysbiosis of the uterine microbiome characterized by a shift towards opportunistic pathogens such as Fusobacteriota and Bacteroidota. Whether these opportunistic pathogens proliferate and overtake the uterine commensals could be determined by the type of substrates present in the uterus. The objective of this study was to integrate uterine microbiome and metabolome data to advance the understanding of the uterine environment in dairy cows that develop metritis. Holstein cows (n = 104) had uterine fluid collected at calving and at the day of metritis diagnosis. Cows with metritis (n = 52) were paired with cows without metritis (n = 52) based on days after calving. First, the uterine microbiome and metabolome were evaluated individually, and then integrated using network analyses. RESULTS: The uterine microbiome did not differ at calving but differed on the day of metritis diagnosis between cows with and without metritis. The uterine metabolome differed both at calving and on the day of metritis diagnosis between cows that did and did not develop metritis. Omics integration was performed between 6 significant bacteria genera and 153 significant metabolites on the day of metritis diagnosis. Integration was not performed at calving because there were no significant differences in the uterine microbiome. A total of 3 bacteria genera (i.e. Fusobacterium, Porphyromonas, and Bacteroides) were strongly correlated with 49 metabolites on the day of metritis diagnosis. Seven of the significant metabolites at calving were among the 49 metabolites strongly correlated with opportunistic pathogenic bacteria on the day of metritis diagnosis. The main metabolites have been associated with attenuation of biofilm formation by commensal bacteria, opportunistic pathogenic bacteria overgrowth, tissue damage and inflammation, immune evasion, and immune dysregulation. CONCLUSIONS: The data integration presented herein helps advance the understanding of the uterine environment in dairy cows with metritis. The identified metabolites may provide a competitive advantage to the main uterine pathogens Fusobacterium, Porphyromonas and Bacteroides, and may be promising targets for future interventions aiming to reduce opportunistic pathogenic bacteria growth in the uterus.

Evaluating differences in milk production, reproductive performance, and survival associated with vaginal discharge characteristics and fever in postpartum dairy cows.

The objective was to assess differences in productive and reproductive performance, and survival associated with vaginal discharge characteristics and fever in postpartum dairy cows located in Western and Southern states of the U.S.A. This retrospective cohort study included data from 3 experiments conducted in 9 dairies. Vaginal discharge was evaluated twice within 12 DIM and scored on a 5-point scale. The highest score observed for each cow was used for group assignment (VD group) as follows: VD 1 and 2 (VD 1/2; n = 1,174) = clear mucus/lochia with or without flecks of pus; VD 3 (n = 1,802) = mucopurulent with < 50% pus; VD 4 (n = 1,643) = mucopurulent with ≥50% of pus or non-fetid reddish/brownish mucous, n = 1,643; VD 5 = fetid, watery, and reddish/brownish, n = 1,800. All VD 5 cows received treatment according to each herd's protocol. Rectal temperature was assessed in a subset of VD 5 cows, and subsequently divided into Fever (rectal temperature ≥39.5°C; n = 334) and NoFever (n = 558) groups. A smaller proportion of cows with VD 5 (67.6%) resumed ovarian cyclicity compared with VD 1/2 (76.2%) and VD 4 (72.9%) cows; however, a similar proportion of VD5 and VD 3 (72.6%) cows resumed ovarian cyclicity. A smaller proportion of VD 5 (85.8%) cows received at least one artificial insemination (AI) compared with VD 1/2 (91.5%), VD 3 (91.0%), or VD 4 (91.6%) cows. Although we did not detect differences in pregnancy at first AI according to VD, fewer cows with VD 5 (64.4%) were pregnant at 300 DIM than cows with VD 1/2 (76.5%), VD 3 (76.2%), or VD 4 (74.7%). Hazard of pregnancy by 300 DIM was smaller for VD 5 compared with VD 1/2, VD 3, or VD 4 cows. A greater proportion of VD 5 cows were removed from the herd within 300 DIM compared with other VD groups. There was 760 kg lesser milk production within 300 DIM for VD 5 compared with VD 2, VD 3, and VD 4, whereas VD 2, VD 3, and VD 4 had similar milk production. We did not detect an association between fever at diagnosis of VD 5 and reproductive performance or milk production. A greater proportion of VD 5 cows without fever were removed from the herd by 300 DIM compared with VD 5 cows with fever. Differences in productive and reproductive performance, and removal of the herd were restricted to fetid, watery, and reddish/brownish vaginal discharge, which was independent of fever.

Blood metabolomics and impacted cellular mechanisms during transition into lactation in dairy cows that develop metritis.

The objective of this study was to identify metabolites associated with metritis and use them for identification of cellular mechanisms affected during transition into lactation. Holstein cows (n = 104) had blood collected in the prepartum period (d -14 ± 6 relative to calving), at calving (d 0), and at the day of metritis diagnosis (d 7 ± 2 after calving). Cows with reddish or brownish, watery, and fetid discharge were diagnosed with metritis (n = 52). Cows with metritis were paired with herdmates without metritis (n = 52) based on days in milk. The metabolome of plasma samples was evaluated using untargeted gas chromatography time-of-flight mass spectrometry. Univariate analyses included t-tests and fold change analyses. Metabolites with false discovery rate adjusted P ≤ 0.10 on t-tests were used for partial least squares discriminant analysis coupled with permutational analysis using 2,000 permutations. Metabolites with false discovery rate adjusted P ≤ 0.10 on t-tests were also used for enriched pathway analyses and identification of cellular processes. Cows that developed metritis had affected cellular processes associated with lower amino acid metabolism in the prepartum period, greater lipolysis, cell death, and oxidative stress at calving and at metritis diagnosis, and greater leukocyte activation at calving, but lower immune cell activation at metritis diagnosis. In summary, cows that developed metritis had plasma metabolomic changes associated with greater lipolysis, oxidative stress, and a dysregulated immune response which may predispose cows to metritis development.

Unraveling the immune and metabolic changes associated with metritis in dairy cows.

The objective was to unravel the peripartum immune and metabolic changes associated with metritis in Holstein cows. Holstein cows (n = 128) had blood collected at -14, 0, 3, and 7 d relative to parturition (DRP). Flow cytometry was used to evaluate blood leukocyte counts, proportions, and activation. Total cells, live cells, single cells, monocytes (CD172α+/CD14+), polymorphonuclears (CD172α+/CD14-/SSChigh), B-cells (CD21+/MHCII+), CD4+ T-cells (CD4+), CD8+ T-cells (CD8+), and γδ T-cells (γδTCR+) were evaluated. Both CD62L and CD11b were used as markers of cell activation. Major histocompatibility complex class II was used as a marker of antigen presentation in monocytes. A Milliplex Bovine Cytokine/Chemokine 08-plex kit was used to evaluate plasma concentrations of IFN-γ, IL-1α, IL-1ß, IL-4, IL-6, IL-8, IL-10, and tumor necrosis factor-α. The body weight (BW) change prepartum was calculated as the difference between calving BW and prepartum BW divided by the number of days between measurements. Plasma fatty acids (FA) were measured at -14 and 0 DRP using untargeted gas chromatography with time-of-flight mass spectrometry. Data were analyzed by ANOVA for repeated measures. Cows that developed metritis (n = 57) had greater prepartum BW, prepartum BW loss, and greater FA concentrations at calving. Plasma FA at calving was positively correlated with IL-1ß. Cows that developed metritis had persistent systemic inflammation, which was demonstrated by greater B-cell activation, greater pro-inflammatory cytokine concentrations, and greater cell damage pre- and postpartum. Postpartum, we observed greater polymorphonuclear cell activation and extravasation but lesser monocytes and CD4+ T-cells activation and extravasation, which suggests postpartum immune tolerance. Greater prepartum adiposity in cows that developed metritis may lead to systemic inflammation pre- and postpartum and immune tolerance postpartum, which may lead to failure to prevent bacterial infection, and development of puerperal metritis.

Combined effect of purulent vaginal discharge and anovulation on pregnancy status in a large multi-state population of Holstein cows.

The objective of this observational prospective cohort study was to evaluate the combined effect of purulent vaginal discharge (PVD) and anovulation (ANOV) on the reproductive performance of a large multi-state population of Holstein cows. Data were prospectively collected from 11,729 cows in 16 herds located in 4 regions in the United States [Northeast (4 herds), Midwest (6), Southeast (1), and Southwest (5)]. Cows were enrolled at calving and monitored weekly for disease occurrence, reproductive events, and survival. Prevalence of PVD was evaluated at 28 ± 3 d in milk and defined by the presence of mucopurulent to fetid vaginal discharge. Resumption of ovarian cyclicity was determined via transrectal ultrasonography at 40 ± 3 and 54 ± 3 d postpartum. Pregnancy diagnosis was performed by ultrasonography on d 32 ± 3 after artificial insemination (AI) and reconfirmed at d 60 ± 3 of gestation. Pregnancy loss (PL) was defined as a cow diagnosed pregnant at 32 ± 3 but nonpregnant at 60 ± 3 d after AI. The association of PVD and ANOV with pregnancy traits was analyzed using 4 PVD-cyclicity categories that considered the following combinations: NPVD-CYC = absence of PVD and cycling; PVD-CYC = presence of PVD and cycling; NPVD-ANOV = absence of PVD and anovular; and PVD-ANOV = presence of PVD and anovular. Multiple logistic regression and Cox proportional regression were used for the analysis of potential associations between PVD and cyclicity categories and pregnancy at first AI (PAI1), days from calving to pregnancy, and PL at first AI. The odds (95% confidence intervals) of pregnancy increased from cows in the PVD-ANOV category (reference category) to cows in NPVD-ANOV [2.09 (1.62-2.50)], PVD-CYC [2.52 (2.02-3.14)], and NPVD-CYC [3.46 (2.84-4.23)]. Similarly, days from calving to pregnancy were less for NPVD-CYC, followed by PVD-CYC, NPVD-ANOV, and PVD-ANOV (121.4, 137.2, 137.3, and 157.4 d, respectively). On the contrary, no clear association was identified between groups and PL. The results indicate that both PVD and ANOV had a negative impact on PAI1 and days from calving to pregnancy. The results indicated a variable magnitude in the negative impact on the reproductive traits analyzed when both conditions were combined.

Treatment with 2 commercial antibiotics reduced clinical and systemic signs of pneumonia and the abundance of pathogenic bacteria in the upper respiratory tract of preweaning dairy calves.

The aim of this study was to evaluate the effect of therapeutically administered tildipirosin or florfenicol + flunixin meglumine for the treatment of bovine respiratory disease (BRD) accompanied by fever in calves before weaning compared with diseased and untreated animals. As specific objectives, we evaluated the composition of the bacterial microbiota of the upper respiratory tract (URT) and blood and health parameters of the animals. Preweaning Holstein female calves diagnosed with naturally acquired pneumonia were randomly assigned to one of the following experimental groups on the day of diagnosis (d 0): (1) TLD (n = 36): single subcutaneous injection with 4 mg/kg tildipirosin; (2) FLF (n = 33): single subcutaneous injection with an antimicrobial (40 mg/kg florfenicol) combined with a nonsteroidal anti-inflammatory drug (2.2 mg/kg flunixin meglumine); and (3) NEG (n = 35): no treatment within the first 5 d following enrollment. The NEG treatment group was closely monitored for 5 d, and calves were removed from the study following a standardized late treatment protocol, when necessary, to minimize health concerns. Healthy untreated calves (CTR; n = 31) were also selected for the study and used as controls. Blood samples used for biochemical analysis and nasopharyngeal swabs used for evaluation of URT microbiota were collected daily from d 0 until d 5 and then weekly until weaning. Next-generation sequencing of the 16S rRNA gene was used to assess the URT microbiota at the phylum and genus levels. Clinical signs associated with pneumonia and otitis media were assessed daily, as was the need for antibiotic interventions. Calves in the TLD and FLF groups had faster recovery from fever within the first 5 d after enrollment. In addition, antibiotic-treated calves reached the same serum haptoglobin levels as healthy calves on d 2 after diagnosis, whereas calves in the NEG group had higher haptoglobin levels than the CTR group until at least d 5 after BRD diagnosis. Calves in the TLD and FLF groups had a lower risk of treatment for pneumonia (FLF = 22.8%; TLD = 27.7%) from d 5 to weaning than calves in the NEG group (54.7%). Furthermore, FLF treatment had a significantly lower risk of nasal discharge, otitis media, and treatment failure compared with the NEG group, but did not differ from the TLD group. Differences in the composition of the URT microbiota were found between groups, and the genus Mycoplasma was the most abundant in samples collected from the URT of calves with and without pneumonia. Both drugs were effective in reducing the mean relative abundance (MRA) of important genera associated with pneumonia (Mannheimia and Pasteurella), although an increase in Mycoplasma MRA was observed for tildipirosin-treated calves. In conclusion, both drugs were effective in reducing the inflammatory signs of pneumonia and the need for antimicrobial treatment after enrollment compared with no treatment. In addition, both TLD and FLF were effective in reducing the MRA of important bacterial genera associated with pneumonia; however, TLD treatment was associated with increased Mycoplasma MRA compared with healthy and untreated calves.

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.

Effect of body condition change and health status during early lactation on performance and survival of Holstein cows.

Body condition score (BCS) and disease records are commonly available in dairy operations. However, the effect of BCS changes (ΔBCS) considering specific health profiles has not been investigated extensively. The objective of this study was to assess the effects of different levels of ΔBCS on fertility, milk yield, and survival of Holstein cows diagnosed with reproductive disorders (REP; dystocia, twins, retained fetal membranes, metritis, and clinical endometritis), other health disorders (OTH; subclinical ketosis, left displaced abomasum, lameness, clinical mastitis, and respiratory disease), or with no disease events (HLT) within 40 days in milk (DIM). Data included lactation information from 11,733 cows calving between November 2012 and October 2014 in 16 herds across 4 geographical regions in the United States (Northeast, Midwest, Southwest, Southeast). Cows were evaluated for BCS at 5 ± 3 DIM (BCS5) and at 40 ± 3 DIM (BCS40) and the difference between BCS40 and BCS5 was classified as excessive loss of BCS (EL; ΔBCS ≤-0.75), moderate loss (ML; ΔBCS = -0.5 to -0.25), no change (NC; ΔBCS = 0), or gain of BCS (GN; ΔBCS ≥0.25). Multivariable logistic regression was used for assessing potential associations between the outcomes of interest and ΔBCS and health. The effect of the interaction term ΔBCS by health group was not statistically significant for any of the study outcomes. The odds of resumption of ovarian cyclicity (ROC), in GN, NC, and ML cows were 1.94 (95% CI: 1.57-2.40), 1.59 (1.28-1.97), and 1.27 (1.10-1.47) times greater than the odds of ROC in EL cows, respectively. The odds of pregnancy at 150 DIM (P150) in GN cows were 1.61 (1.20-2.17) times greater than the odds of P150 in EL cows. Cows with REP or OTH disorders had smaller odds of ROC compared with HLT cows [REP: OR = 0.65 (0.56-0.76) and OTH: OR = 0.79 (0.68-0.92)]. For pregnancy outcomes, REP cows had smaller odds of pregnancy at the first artificial insemination compared with HLT cows [0.70 (0.58-0.84)]. Similarly, REP cows had smaller odds of being diagnosed pregnant by 150 and 305 DIM compared with HLT cows [P150: 0.73 (0.59-0.87), P305: 0.58 (0.49-0.69)]. Overall, average daily milk within the first 90 DIM was greater in EL (39.5 ± 1.13 kg/d) and ML (38.9 ± 1.11 kg/d) cows than in NC (37.8 ± 1.12 kg/d) and GN (36.2 ± 1.12 kg/d) cows. On the other hand, average daily milk within the first 90 DIM was lower in REP (37.0 ± 1.11 kg/d) cows compared with OTH (38.7 ± 1.12 kg/d) and HLT cows (38.6 ± 1.11 kg/d). The magnitude of ΔBCS and the health status of early lactation cows should be considered when assessing subsequent cow performance and survival.

Effect of treatment of pneumonia and otitis media with tildipirosin or florfenicol + flunixin meglumine on health and upper respiratory tract microbiota of preweaned Holstein dairy heifers.

The objective of this randomized clinical study was to compare the effect of 2 antimicrobial interventions, tildipirosin or florfenicol + flunixin meglumine, used for treatment of pneumonia and extralabel treatment for otitis on health parameters and upper respiratory tract (URT) microbiota of preweaned Holstein calves. Housed preweaned Holstein heifers diagnosed with either otitis or pneumonia were assigned into 1 of 2 treatment groups, receiving a single subcutaneous injection of either 4 mg/kg of tildipirosin (TLD; n = 444) or 40 mg/kg of florfenicol combined with 2.2 mg/kg of a nonsteroidal anti-inflammatory, flunixin meglumine (FLF; n = 442). Calves were enrolled and treated on the day of diagnosis of the first case of pneumonia or otitis. If a calf had a recurrent case, the opposite drug was administered, respecting an interval of 5 d between drug injections. Blood samples for leukocyte counts were collected at 0, 2, 4, and 6 d after treatment, and rectal temperature was measured daily during the 5 d after treatment. Ear scores were observed from calves with otitis. Additionally, swabs of the URT were collected from a subset of 20 calves in each treatment group at d 0, 3, 6, 9, and 11 following enrollment for analysis of URT microbiota through next-generation sequencing of the 16S rRNA gene and quantitative PCR. Swabs were also collected from a comparative group of 20 healthy calves that did not receive any drug. No differences were observed between groups for recurrence risk of either pneumonia (TLD = 32.4%; FLF = 29.7%) or otitis (TLD = 72.7%; FLF = 73.6%). Similarly, no differences were observed for the total number of treatments for pneumonia (TLD = 1.45; FLF = 1.42) or otitis (TLD = 2.96; FLF = 3.07). On the other hand, both drugs reduced rectal temperature, ear scores, and leukocyte counts, with FLF calves having a greater reduction in rectal temperature within 4 d after treatment. Both TLD and FLF reduced the total bacterial load when compared with healthy untreated calves, but no differences were observed between treatment groups. Furthermore, compared with the untreated group, treated calves had lower mean relative abundances (MRA) of the genera Mannheimia, Moraxella, and Pasteurella within 11, 9, and 3 d after treatment, respectively; however, no significant differences were observed between TLD and FLF. On the other hand, MRA of Mycoplasma was not decreased by both treatments compared to untreated animals, and a higher MRA was observed in the TLD group during 11 d after treatment in comparison to FLF and untreated calves. Based on this data, we concluded that both drugs used in the study were effective in reducing rectal temperature, ear scores, leukocyte counts, and MRA of the genera Mannheimia, Pasteurella, and Moraxella in the URT, and calves treated with FLF had a greater reduction in rectal temperature.

Immunization with a novel recombinant protein (YidR) reduced the risk of clinical mastitis caused by Klebsiella spp. and decreased milk losses and culling risk after Escherichia coli infections.

The primary objective of this study was to evaluate the protective efficacy of a novel recombinant subunit vaccine containing the protein YidR (rYidR) against clinical mastitis (CM) caused by Klebsiella spp. and Escherichia coli. Given that E. coli infection is known to cause metritis, we also evaluated the effect of rYidR vaccination on the incidence of metritis and conception at the first artificial insemination. Retained placenta and abortion incidence, milk production and composition, and serological responses to specific antigens were also evaluated. In total, 3,107 cows were blocked by parity and randomly allocated into 1 of 3 treatment groups: experimental recombinant subunit vaccine containing the YidR protein (rYidR); commercial vaccine composed of Klebsiella pneumoniae siderophore receptors and porin protein (Kleb-SRP; KlebVax, Epitopix, Willmar, MN); and sterile water adjuvanted with aluminum hydroxide (20%; placebo). Vaccinations were performed at the dry-off for cows, and at 223 ± 3 d of pregnancy for pre-fresh heifers. A second administration was given at 21 ± 3 d after the first injection. Vaccination with rYidR significantly reduced the incidence of CM caused by Klebsiella spp. (3.2%) when compared with the placebo (5.1%) group. No difference was observed on risk of Klebsiella CM between Kleb-SRP (5.9%) and placebo groups. Cows in the rYidR group that experienced E. coli CM had a lower risk of death or culling (12.5%) compared with the Kleb-SRP (27.6%) and placebo groups (27.8%). Furthermore, among cows that developed E. coli CM, rYidR-immunized cows produced more milk than did cows in the placebo and Kleb-SRP groups. Regardless of CM occurrence, rYidR-immunized cows tended to have higher milk production up to the eighth month of lactation than cows in the other groups. No significant effect of treatment was observed on the overall incidence of abortion and metritis; however, the risk of retained placenta tended to be lower for the rYidR group (4.7%) compared with the placebo group (6.7%). In addition, primiparous cows in the rYidR group had the highest conception risk at the first artificial insemination (48.3%) compared with the placebo (39.5%) group, and no significant difference was observed when the Kleb-SRP (40.1%) group was compared with the placebo group. Generally, higher antibody serum titers (IgM and IgG) were observed for the immunized groups compared with the placebo. In conclusion, the rYidR vaccine reduced the risk of CM caused by Klebsiella spp. and the mortality or culling of cows with E. coli infections. Other benefits of the novel vaccine include maintenance of milk production after CM caused by E. coli, and higher conception risk at the first service in primiparous cows compared with cows in the placebo and Kleb-SRP groups.

The economic cost of metritis in dairy herds.

The objective of this study was to estimate the cost of metritis in dairy herds. Data from 11,733 dairy cows from 16 different farms located in 4 different regions of the United States were compiled for up to 305 d in milk, and 11,581 cows (2,907 with and 8,674 without metritis) were used for this study. Metritis was defined as fetid, watery, red-brownish vaginal discharge that occurs ≤21 d in milk. Continuous outcomes such as 305-d milk production, milk sales ($/cow), cow sales ($/cow), metritis treatment costs ($/cow), replacement costs ($/cow), reproduction costs ($/cow), feeding costs ($/cow), and gross profit per cow ($/cow) were analyzed using mixed effect models using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC). Gross profit was also compared using the Kruskal-Wallis test. Dichotomous outcomes such as pregnant and culling by 305 d in milk were analyzed using the GLIMMIX procedure of SAS. Time to pregnancy and culling were analyzed using the PHREG procedure of SAS. Models included the fixed effects of metritis, parity, and the interaction between metritis and parity, and farm as the random effect. Variables were considered significant when P ≤ 0.05. Metritis cost was calculated by subtracting the gross profit of cows with metritis from the gross profit of cows without metritis. A stochastic analysis was performed with 10,000 iterations using the observed results from each group. Milk yield and proportion of cows pregnant were lesser for cows with metritis than for cows without metritis, whereas the proportion of cows leaving the herd was greater for cows with metritis than for cows without metritis. Milk sales, feeding costs, residual cow value, and gross profit were lesser for cows with metritis than for cows without metritis. Cow sales and replacement costs were greater for cows with metritis than for cows without metritis. The mean cost of metritis from the study herds was $511 and the median was $398. The stochastic analysis showed that the mean cost of a case of metritis was $513, with 95% of the scenarios ranging from $240 to $884, and that milk price, treatment cost, replacement cost, and feed cost explained 59%, 19%, 12%, and 7%, respectively, of the total variation in cash flow differences. In conclusion, metritis caused large economic losses to dairy herds by decreasing milk production, reproduction, and survival in the herd.

Negatively controlled, randomized clinical trial comparing different antimicrobial interventions for treatment of clinical mastitis caused by gram-positive pathogens.

The general objective of this study was to evaluate the effect of 3 intramammary antibiotic interventions using 2 commercially available antibiotics with narrow- or broad-spectrum activity on cure rates of clinical mastitis (CM) caused by gram-positive bacteria. We also compared the efficacy of treatment protocols, including a negative control, on outcomes at the cow and mammary quarter level. Before the onset of the study, 5,987 animals more than 12 mo old were randomly preassigned to 1 of 4 protocols in the event of gram-positive CM (except for Staphylococcus aureus and Trueperella pyogenes) during lactation: 3 infusions with 62.5 mg of amoxicillin performed 12 h apart (AMOX-L); 5 infusions once a day with 62.5 mg of amoxicillin (AMOX-EL); 5 infusions once a day with 125 mg of ceftiofur hydrochloride (CEFT-L); or negative control, no treatment performed until 5 d after diagnosis (NEG-CTR). Randomization was performed to preassign 90% of cows to one of the antibiotic protocols (30% in each group) and 10% to the negative control. A total of 696 quarter cases of CM met the inclusion criteria and were evaluated in the study. Quarter-level outcomes were assessed based on 5 milk samples collected up to 14 ± 3 d following enrollment (i.e., first day of treatment), whereas variables at the cow level [composite somatic cell count (SCC), milk production, and survival in the herd] were assessed up to 90 d after CM diagnosis. Streptococcus uberis, followed by Streptococcus dysgalactiae, were the main causes of gram-positive CM. Overall, clinical cure was higher for CEFT-L than for AMOX-EL, and no difference was observed between CEFT-L and AMOX-L. Likewise, no significant differences were detected on overall bacteriological cure, although some treatment effects were observed at the species level. Compared with antibiotic-treated groups, quarters assigned to NEG-CTR had higher counts of colony-forming units (cfu), 16S rRNA gene copy numbers, and Streptococcus relative abundance (RA) until d 5 after enrollment. Quarters treated with AMOX-L had higher cfu counts on d 5, 8, and 14 after enrollment compared with the other antibiotic protocols. In addition, the RA of Streptococcus spp. was higher on d 14 after enrollment for AMOX-treated quarters compared with the CEFT-L group. Linear score of SCC was higher for AMOX-treated cows than for CEFT-L in the first test day after CM. However, cows assigned to AMOX-L had higher milk production than those submitted to the AMOX-EL and CEFT-L protocols. In conclusion, the 2-d protocol with 3 intramammary infusions of amoxicillin (narrow-spectrum antimicrobial) had similar overall clinical and bacteriological cures as 5 administrations (once a day) with ceftiofur hydrochloride (wide spectrum). No significant difference was observed on CM recurrence and cow survival. However, quarters treated with 5-d protocols were more effective at reducing milk cfu counts than quarters in the AMOX-L protocol. In addition, lower Streptococcus spp. RA was observed in ceftiofur-treated quarters compared with the amoxicillin protocols at d 14 after CM diagnosis. Based on results of microbiome and bacterial load (quantitative PCR and cfu count) up to 5 d after CM diagnosis, antibiotic use remains an indispensable strategy for treatment of CM caused by gram-positive bacteria.

Early-lactation diseases and fertility in 2 seasons of calving across US dairy herds.

The objective of this study was to characterize incidences of health disorders during early lactation in a large population of Holstein cows calving in 2 seasons across multiple US dairy herds. In addition, cumulative effects of combinations of health-related events on fertility and survival by season of calving and parity number were tested. Data were prospectively collected from a total of 11,729 cows in 16 herds located in 2 regions in the United States [north (7,820 cows in 10 herds) and south (3,909 cows in 6 herds)]. Cows were enrolled at parturition and monitored weekly for disease occurrence, reproductive events, and survival. Health-related events were grouped into reproductive disorders (REP; dystocia, twins, retained fetal membranes, metritis, and clinical endometritis) and other disorders (OTH; subclinical ketosis, mastitis, displaced abomasum, and pneumonia). Counts of health events within 50 d postpartum were added into each of the groups and categorized as 0, 1, 2, 3, and ≥4 for REP and 0, 1, 2, and ≥3 for OTH. Multivariable logistic regression was used for testing potential associations between categories of disease occurrence and outcome variables, including resumption of ovarian cyclicity, pregnancy per artificial insemination (AI), pregnancy loss, and survival up to and after 50 DIM. The incidence of disease varied with season of calving and parity, and these 2 variables were associated with the reproductive and survival outcomes. The size of the detrimental effect of disease incidence on reproduction and survival depended on disease group and varied for each specific outcome. Resumption of ovarian cyclicity decreased as incidences of disorders increased in both REP and OTH categories. Pregnancy at first AI also was smaller in greater number of REP categories, but the effect of number of OTH categories on pregnancy at first AI was not consistent. Similarly, pregnancy loss at first AI was not affected consistently by REP or OTH. Survival was reduced by REP and OTH. The magnitude of these negative effects was variable, depending on season of calving and parity, but consistently increased with the number of health events during early lactation.

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.

Associations of reproductive indices with fertility outcomes, milk yield, and survival in Holstein cows.

The study is part of a research effort investigating potential associations between genomic variation and fertility of Holstein cows. The objective was to compare the reproductive performance of Holstein cows in 3 categories of 2 reproductive indices (RI) that were developed for the allocation of cows in a ranking for potential fertility, based on the predicted probability of pregnancy. The associations between categories of the developed indices and multiple fertility variables in a large multistate population of Holstein cows were tested. In addition, we analyzed associations among the RI categories with milk yield and survival. Based on phenotypic information from individual cows, 2 reproductive indices (RI1 and RI2) were developed, representing a predicted probability that a cow will become pregnant at first artificial insemination postpartum, as a function of explanatory variables used in a logistic model. Data from a total of 11,733 cows calving in 16 farms located in 4 regions of the United States (Northeast, Midwest, Southeast, and Southwest) were available. Cows were enrolled at parturition and monitored weekly for reproductive events, health status, milk yield, and survival. To develop the indices, potential significant effects were initially tested by univariate analyses. Effects with P ≤ 0.05 were offered to the multivariate analysis, and the final models were determined through backward elimination, considering potentially significant interactions. The final model for RI1 included the random effect of farm and a complement of significant fixed effects as explanatory variables influencing a pregnancy outcome: (1) incidence of retained fetal membranes; (2) metritis; (3) clinical endometritis; (4) lameness at 35 days in milk (DIM); (5) resumption of postpartum ovulation by 50 DIM; (6) season of calving; and (7) parity number. The model for RI2 included (1) parity number; (2) body condition score at 40 DIM; (3) incidence of retained fetal membranes; (4) metritis; (5) resumption of postpartum ovulation by 50 DIM; (6) region; (7) subclinical ketosis; (8) mastitis; (9) clinical endometritis; and (10) milk yield at the first milk test after calving; as well as the interaction effects of postpartum resumption of ovulation by 50 DIM × region; mastitis × region; and milk yield at the first milk test after calving × parity number. Multivariate logistic regression, ANOVA, and survival analysis were used to test the correspondence between the resulting RI and individual fertility, milk yield, and survival from the population. To facilitate the analyses, the resulting RI values were categorized as low for cows in the lowest quartile, medium for cows within the interquartile range, or high for cows in the top quartile. We found consistent agreement between categories of the predicted RI and the measures of fertility and survival collected from individual cows. We conclude that the proposed RI represent a viable approach to refine the allocation of cows into potential low- and high-fertility populations.

Genome-enable prediction for health traits using high-density SNP panel in US Holstein cattle.

The objective of this study was to compare accuracies of different Bayesian regression models in predicting molecular breeding values for health traits in Holstein cattle. The dataset was composed of 2505 records reporting the occurrence of retained fetal membranes (RFM), metritis (MET), mastitis (MAST), displaced abomasum (DA), lameness (LS), clinical endometritis (CE), respiratory disease (RD), dystocia (DYST) and subclinical ketosis (SCK) in Holstein cows, collected between 2012 and 2014 in 16 dairies located across the US. Cows were genotyped with the Illumina BovineHD (HD, 777K). The quality controls for SNP genotypes were HWE P-value of at least 1 × 10-10 ; MAF greater than 0.01 and call rate greater than 0.95. The FImpute program was used for imputation of missing SNP markers. The effect of each SNP was estimated using the Bayesian Ridge Regression (BRR), Bayes A, Bayes B and Bayes Cπ methods. The prediction quality was assessed by the area under the curve, the prediction mean square error and the correlation between genomic breeding value and the observed phenotype, using a leave-one-out cross-validation technique that avoids iterative cross-validation. The highest accuracies of predictions achieved were: RFM [Bayes B (0.34)], MET [BRR (0.36)], MAST [Bayes B (0.55), DA [Bayes Cπ (0.26)], LS [Bayes A (0.12)], CE [Bayes A (0.32)], RD [Bayes Cπ (0.23)], DYST [Bayes A (0.35)] and SCK [Bayes Cπ (0.38)] models. Except for DA, LS and RD, the predictive abilities were similar between the methods. A strong relationship between the predictive ability and the heritability of the trait was observed, where traits with higher heritability achieved higher accuracy and lower bias when compared with those with low heritability. Overall, it has been shown that a high-density SNP panel can be used successfully to predict genomic breeding values of health traits in Holstein cattle and that the model of choice will depend mostly on the genetic architecture of the trait.

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.