Application of behavior data to predictive exploratory models of metritis self-cure and treatment failure in dairy cows.

The objective was to evaluate the performance of exploratory models containing routinely available on-farm data, behavior data, and the combination of both to predict metritis self-cure (SC) and treatment failure (TF). Holstein cows (n = 1,061) were fitted with a collar-mounted automated-health monitoring device (AHMD) from -21 ± 3 to 60 ± 3 d relative to calving to monitor rumination time and activity. Cows were examined for diagnosis of metritis at 4 ± 1, 7 ± 1, and 9 ± 1 d in milk (DIM). Cows diagnosed with metritis (n = 132), characterized by watery, fetid, reddish/brownish vaginal discharge (VD), were randomly allocated to 1 of 2 treatments: control (CON; n = 62), no treatment at the time of metritis diagnosis (d 0); or ceftiofur (CEF; n = 70), subcutaneous injection of 6.6 mg/kg of ceftiofur crystalline-free acid on d 0 and 3 relative to diagnosis. Cure was determined 12 d after diagnosis and was considered when VD became mucoid and not fetid. Cows in CON were used to determine SC, and cows in CEF were used to determine TF. Univariable analyses were performed using farm-collected data (parity, calving season, calving-related disorders, body condition score, rectal temperature, and DIM at metritis diagnosis) and behavior data (i.e., daily averages of rumination time, activity generated by AHMD, and derived variables) to assess their association with metritis SC or TF. Variables with P-values ≤0.20 were included in the multivariable logistic regression exploratory models. To predict SC, the area under the curve (AUC) for the exploratory model containing only data routinely available on-farm was 0.75. The final exploratory model to predict SC combining routinely available on-farm data and behavior data increased the AUC to 0.87, with sensitivity (Se) of 89% and specificity (Sp) of 77%. To predict TF, the AUC for the exploratory model containing only data routinely available on-farm was 0.90. The final exploratory model combining routinely available on-farm data and behavior data increased the AUC to 0.93, with Se of 93% and Sp of 87%. Cross-validation analysis revealed that generalizability of the exploratory models was poor, which indicates that the findings are applicable to the conditions of the present exploratory study. In summary, the addition of behavior data contributed to increasing the prediction of SC and TF. Developing and validating accurate prediction models for SC could lead to a reduction in antimicrobial use, whereas accurate prediction of cows that would have TF may allow for better management decisions.

Effects of cashew nut shell extract and monensin on in vitro ruminal fermentation, methane production, and ruminal bacterial community.

The objective of this study was to evaluate the effects of cashew nut shell extract (CNSE) and monensin on ruminal in vitro fermentation, CH4 production, and ruminal bacterial community structure. Treatments were as follows: control (CON, basal diet without additives); 2.5 µM monensin (MON); 0.1 mg CNSE granule/g DM (CNSE100); and 0.2 mg CNSE granule/g DM (CNSE200). Each treatment was incubated with 52 mL of buffered ruminal content and 500 mg of total mixed ration for 24 h using serum vials. The experiment was performed as a complete randomized block design with 3 runs. Run was used as a blocking factor. Each treatment had 5 replicates, in which 2 were used to determine nutrient degradability, and 3 were used to determine pH, NH3-N, volatile fatty acids, lactate, total gas, CH4 production, and bacterial community composition. Treatment responses for all data, excluding bacterial abundance, were analyzed with the GLIMMIX procedure of SAS v9.4. Treatment responses for bacterial community structure were analyzed with a PERMANOVA test run with the R package vegan. Orthogonal contrasts were used to test the effects of (1) additive inclusion (ADD: CON vs. MON, CNSE100, and CNSE200); (2) additive type (MCN: MON vs. CNSE100 and CNSE200); and (3) CNSE dose (DOS: CNSE100 vs. CNSE200). We observed that pH, acetate, and acetate:propionate ratio in the CNSE100 treatment were lower compared with CNSE200, and propionate in the CNSE100 treatment was greater compared with CNSE200. Compared with MON, CNSE treatments tended to decrease total lactate concentration. Total gas production of CON was greater by 2.63% compared with all treatments, and total CH4 production was reduced by 10.64% in both CNSE treatments compared with MON. Also, compared with MON, in vitro dry matter degradabilities in CNSE treatments were lower. No effects were observed for NH3-N or in vitro neutral detergent fiber degradability. Finally, the relative abundances of Prevotella, Treponema, and Schwartzia were lower, whereas the relative abundances of Butyrivibrio and Succinivibrio were greater in all treatments compared with CON. Overall, the inclusion of CNSE decreased CH4 production compared with MON, making CNSE a possible CH4 mitigation additive in dairy cattle diets.

Induced endometritis in early lactation compromises production and reproduction in dairy cows.

Objectives of this experiment were to study the effect of infusing utero-pathogenic bacteria to induce endometrial inflammation on productive performance in early lactation and subsequent reproduction. Although endometritis is associated with perturbed reproduction, numerous factors may contribute to the observed association. It was hypothesized that induced endometrial inflammation, resulting in localized and systemic inflammatory responses, compromises production and reproduction. Holstein cows without clinical disease and with less than 18% polymorphonuclear leukocytes (PMN) in endometrial cytology on d 31 ± 3 postpartum had their estrous cycle synchronized. Cows were blocked by parity and genomic breeding value for cow conception rate and, within block, assigned randomly to remain as untreated controls (CON; n = 37) or to receive an intrauterine infusion of 5.19 × 108 cfu Escherichia coli and 4.34 × 108 cfu Trueperella pyogenes during the luteal phase to induce endometrial inflammation (INF; n = 48). Endometrial cytology was taken on d 2 and 7 after treatment to evaluate the proportion of PMN. Rectal temperature, dry matter intake, and yields of milk and components were measured in the first 7 d after treatment. Blood serum was analyzed for concentration of haptoglobin. Leukocytes were isolated from blood on d 2 and 7 after treatment and on d 19 after artificial insemination (AI) and mRNA was quantified for a select group of genes. Cows received AI and reproduction was followed for 300 d postpartum. Bacterial infusion induced endometrial inflammation with increased proportions of PMN in the endometrial cytology on d 2 (4.4 ± 0.7 vs. 26.3 ± 2.8%) and 7 (10.9 ± 1.7 vs. 17.4 ± 2.1%) after treatment, resulting in increased mean prevalence of subclinical endometritis (>10% PMN; 23.3 ± 6.3 vs. 80.9 ± 5.1%). Rectal temperature did not differ between CON and INF, but the concentration of haptoglobin in serum tended to increase in INF compared with CON (113 ± 14 vs. 150 ± 16 µg/mL). Induced endometrial inflammation reduced yields of milk (44.9 ± 0.8 vs. 41.6 ± 0.8 kg/d), protein (1.19 ± 0.03 vs. 1.12 ± 0.03 kg/d), and lactose (2.17 ± 0.04 vs. 2.03 ± 0.04 kg/d) and tended to reduce dry matter intake (20.7 ± 0.5 vs. 19.4 ± 0.6 kg/d) in the first 7 d after treatment. Indeed, the reduction in milk yield lasted 4 wk. However, treatment did not affect yields of energy-corrected milk or fat because treatment with INF increased the concentration of fat in milk (3.54 ± 0.10 vs. 3.84 ± 0.10%). Induced endometrial inflammation reduced pregnancy per AI at all inseminations (33.4 ± 5.1 vs. 21.6 ± 3.7%) and the hazard of pregnancy (0.61; 95% CI = 0.36-1.04), which extended the median days open by 24 d. Blood leukocytes from INF cows had increased mRNA expression of the pro-inflammatory gene IL1B on d 2 and 7 after treatment, but reduced expression of the IFN-stimulated genes ISG15 and MX2 on d 19 after AI. Induced endometrial inflammation depressed production and caused long-term negative effects on reproduction in lactating dairy cows.

Combinations of bacterial cultures, exogenous enzymes, and yeast-based feed additives and their impact on ruminal microbiome.

Our objective was to evaluate the effects of bacteria (Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus lichenformis, Bacillus subtilis, and Enterococcus faecium), enzymes (amylase, hemicellulose, and xylanase), and yeast as additives on the ruminal microbiome. We hypothesized that inclusion of bacteria, enzymes, and yeast would impact butyric bacterial populations. Eight fermenters were arranged in a duplicated 4 × 4 Latin square with the following treatments: 1) control without additives (CTRL); 2) bacterial culture and enzyme blend (EB); 3) bacterial culture and enzyme blend with a live yeast and yeast culture blend (EBY); and 4) double dose of bacterial culture and enzyme blend and the yeast products blend (2X). We conducted four fermentation periods of 10 d each, with the last 3 d for collection of samples. Overall, 64 solid and liquid samples were analyzed by amplification of the V4 region of bacterial 16S rRNA. Data were analyzed with R and SAS. The following orthogonal contrasts were used: 1) ADD-the control compared to all treatments with additives (CTRL vs. EB, EBY, and 2X); 2) YEAST-treatment without yeast compared to those with yeast (EB vs. EBY and 2X); and 3) DOSE-the single dose of enzymes, bacteria, and yeast compared to the doubled dose (EBY vs. 2X). Family Prevotellaceae was more abundant when additives were added (ADD). Additives (ADD) also increased relative abundance of Prevotellaceae Ga6A1 and YAB2003 in solid fraction, and of Prevotellaceae Ga6A1 and two members of Lachnospiracea family in liquid fraction. Yeast (YEAST) decreased relative abundance of Succinivibrionaceae UCG-001 and increased abundance of Ruminococcus and Prevotellaceae UCG-003 in solid fraction. Doubling the dose of enzymes and microbial additives (DOSE) decreased the abundance of Succiniclasticum in solid fraction and Selenomonadaceae in the liquid. Molar proportion of butyrate was highly correlated with abundance of Prevotellaceae Ga6A1 in solid (r = 0.68) and liquid fraction (r = 0.79), and with Unclassified Lachnospiraceae in liquid (r = 0.70). Our results demonstrate that YEAST decreases abundance of succinate synthesizing bacteria, while DOSE decreases abundance of bacteria that metabolize succinate into propionate. Combined bacteria, enzymes, and yeast increase the relative abundance of specific genera primarily within the Prevotellaceae family, which may explain the increase in butyrate molar proportion observed with ADD.

Effects of choline chloride on the ruminal microbiome at 2 dietary neutral detergent fiber concentrations in continuous culture.

Our objective was to evaluate the effects of unprotected choline chloride (Cho) on the ruminal microbiome at 2 dietary neutral detergent fiber (NDF) concentrations. We hypothesized that the effects of Cho on ruminal bacterial populations would depend on NDF. Eight dual-flow continuous-culture fermentors were arranged in a duplicated 4 × 4 Latin square as a 2 × 2 factorial with the following treatments: (1) 30% NDF-control (30% NDF diet, no supplemental choline); (2) 30% NDF-Cho (30% NDF diet plus 1.9 g of choline ion per kg of dry matter); (3) 40% NDF-control (40% NDF diet, no supplemental choline); and (4) 40% NDF-Cho (40% NDF diet plus 1.9 g of choline ion per kg of dry matter). We did 4 fermentation periods of 10 d each and used the last 3 d for collection of samples of solid and liquid digesta effluents for DNA extraction. Overall, 32 solid and 32 liquid samples were analyzed by amplification of the V4 variable region of bacterial 16S rRNA. Data were analyzed with R (R Project for Statistical Computing) and SAS (SAS Institute Inc.) to determine effects of Cho, NDF, and NDF × Cho on taxa relative abundance. The correlation of propionate molar proportion with taxa relative abundance was also analyzed. At the phylum level, relative abundance of Firmicutes in the liquid fraction tended to be greater when Cho was supplemented with a 30% NDF diet. At the order level, Cho increased Coriobacteriales in solid fraction and decreased Fibrobacterales in liquid fraction. Moreover, Cho decreased abundance of Clostridiales and increased Selenomonadales in the solid fraction, only with the 30% NDF diet. For genera, lower abundance of Pseudobutyrivibrio resulted from Cho in solid and liquid fractions. Greater abundance of Succinivibrio in solid and Selenomonas and Selenomonas 1 in liquid resulted from Cho with the 30% NDF diet. Propionate molar proportion was positively correlated with relative abundance of order Selenomonadales in solid and liquid fractions, and with genus Succinivibrio in solid and genera Selenomonas and Selenomonas 1 in liquid. Our results indicate that Cho primarily decreases abundance of bacteria involved in fiber degradation and increases abundance of bacteria mainly involved in nonstructural carbohydrate degradation and synthesis of propionate, particularly when a diet with 30% NDF is provided.

Effects of lactic acid-producing bacteria as direct-fed microbials on the ruminal microbiome.

The objective of this study was to evaluate ruminal microbiome changes associated with feeding Lactobacillus plantarum GB-LP1 as direct-fed microbials (DFM) in high-producing dairy cow diets. A dual-flow continuous culture system was used in a replicated 4 × 4 Latin square design. A basal diet was formulated to meet the requirements of a cow producing 45 kg of milk per day (16% crude protein and 28% starch). There were 4 experimental treatments: the basal diet without any DFM (CTRL); a mixture of Lactobacillus acidophilus, 1 × 109 cfu/g, and Propionibacterium freudenreichii, 2 × 109 cfu/g [MLP = 0.01% of diet dry matter (DM)]; and 2 different levels of L. plantarum, 1.35 × 109 cfu/g (L1 = 0.05% and L2 = 0.10% of diet DM). Bacterial samples were collected from the fluid and particulate effluents before feeding and at 2, 4, 6, and 8 h after feeding; a composite of all time points was made for each fermentor within their respective fractionations. Bacterial community composition was analyzed through sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform. Sequenced data were analyzed on DADA2, and statistical analyses were performed in R (RStudio 3.0.1, https://www.r-project.org/) and SAS 9.4 (SAS Institute Inc.); orthogonal contrasts were used to compare treatments. Different than in other fermentation scenarios (e.g., silage or beef cattle high-grain diets), treatments did not affect pH or lactic acid concentration. Effects were mainly from overall DFM inclusion, and they were mostly observed in the fluid phase. The relative abundance of the phylum Firmicutes, family Lachnospiraceae, and 6 genera decreased with DFM inclusion, with emphasis on Butyrivibrio_2, Saccharofermentans, and Ruminococcus_1 that are fibrolytic and may display peptidase activity during fermentation. Lachnospiraceae_AC2044_group and Lachnospiraceae_XPB1014_group also decreased in the fluid phase, and their relative abundances were positively correlated with NH3-N daily outflow from the fermentors. Specific effects of MLP and L. plantarum were mostly in specific bacteria associated with proteolytic and fibrolytic functions in the rumen. These findings help to explain why, in the previous results from this study, DFM inclusion decreased NH3-N concentration without altering pH and lactic acid concentration.

Economic comparison between ceftiofur-treated and nontreated dairy cows with metritis.

The objective was to investigate the economic effect of treating dairy cows with metritis using ceftiofur-free acid or leaving them untreated at the time of diagnosis. Cows with a fetid, watery, red-brownish vaginal discharge were diagnosed with metritis (d 0). Data from 875 dairy cows (506 primiparous and 369 multiparous) from 1 herd in northern Florida that had been part of a larger study evaluating different treatments for metritis were used for the economic analysis. Holstein cows with metritis had been randomly assigned to: Ceftiofur (CEF, n = 239) = subcutaneous injection of 6.6 mg/kg of ceftiofur crystalline-free acid in the base of the ear at d 0 and d 3; Untreated (UNT, n = 233) = no treatment applied at metritis diagnosis. Both groups could receive escape therapy if condition worsened. A group of nonmetritic healthy cows (NMET; n = 403) from the same cohort was randomly selected for comparison. Continuous outcomes such as 300-d milk production (kg/cow), milk sales ($/cow), cow sales ($/cow), treatment cost by 60 days in milk ($/cow), reproduction cost ($/cow), replacement cost ($/cow), feeding cost ($/cow), and gross profit per cow ($/cow) were analyzed using the ANOVA (MIXED procedure of SAS version 9.4). Dichotomous outcomes such as pregnancy and culling by 300 d were analyzed using logistic regression (GLIMMIX procedure of SAS). Models included the fixed effects of treatment, parity, and the interaction between treatment and parity. A stochastic analysis was performed with 10,000 iterations using the observed results from each group. The CEF treatment resulted in greater treatment cost by 60 DIM than UNT ($112 vs. $37), but resulted in a greater proportion of pregnant cows (71 vs. 61%) and decreased culling by 300 DIM (29 vs. 39%) compared with UNT. Gross profit was lesser for UNT than NMET ($2,969 vs. $3,426), and CEF was intermediate ($3,219). The stochastic analysis showed that the mean difference in gross profit between UNT and NMET was -$457; saleable milk (49%) and replacement cost (24%) accounted for most of the variation. The mean difference in gross profit between CEF and NMET group was -$207; saleable milk (82%) and initial metritis treatment cost (9%) accounted for most of the variation. The mean difference in gross profit between the UNT and the CEF group was -$250; replacement cost (41%) and cow sales (31%) accounted for most of the variation. In summary, metritis caused large economic losses when left untreated, and CEF reduced those losses by improving fertility, reducing culling and replacement cost, and reducing milk yield losses.

Using chitosan microparticles to treat metritis in lactating dairy cows.

The main objective of this study was to evaluate the efficacy of intrauterine administration of chitosan microparticles (CM) in curing metritis in dairy cows. A secondary objective was to evaluate the effects of metritis treatments on milk yield, survival, and reproductive performance. Cows with a fetid, watery, red-brownish vaginal discharge were diagnosed with metritis. Holstein cows (n = 826) with metritis from 3 dairies located in northern Florida were blocked by parity (primiparous or multiparous) and, within each block, randomly assigned to one of 3 treatments: CM (n = 276) = intrauterine infusion of 24 g of CM dissolved in 40 mL of sterile distilled water at the time of metritis diagnosis (d 0), 2 (d 2), and 4 (d 4) d later; ceftiofur (CEF; n = 275) = subcutaneous injection of 6.6 mg/kg ceftiofur crystalline-free acid in the base of the ear at d 0 and d 3; Control (CON; n = 275) = no treatment applied at metritis diagnosis. All groups could receive escape therapy if condition worsened. Cure was considered when vaginal discharge became mucoid and not fetid. A group of nonmetritic (NMET; n = 2,436) cows was used for comparison. Data were analyzed by generalized linear mixed and Cox's proportional hazard models. Cows in CM and CON had lesser risk of metritis cure on d 12 than cows in CEF (58.6 ± 5.0 vs. 61.9 ± 4.9% vs. 77.9 ± 3.9, respectively). The proportion of cows culled within 60 days in milk (DIM) was greater for cows in CM than for cows in CEF and CON (21.5 ± 2.7 vs. 9.7 ± 1.9 vs. 11.3 ± 2.0%, respectively). Treatment did not affect rectal temperature or plasma nonesterified fatty acids, ß-hydroxybutyrate, and haptoglobin concentrations. Milk yield in the first 60 DIM differed for all treatments, and it was lowest for CM (35.8 ± 0.3 kg/d), followed by CON (36.8 ± 0.3 kg/d) and CEF (37.9 ± 0.3 kg/d). The hazard of pregnancy up to 300 DIM was lesser for CM than CEF (hazard ratio = 0.62; 95% CI: 0.50-0.76), for CM than CON (hazard ratio = 0.77; 95% CI: 0.62-0.95) and for CON than CEF (hazard ratio = 0.80; 95% CI: 0.65-0.99). Culling was greater, and milk yield and fertility were lesser for CEF than NMET. In summary, CM did not improve the cure of metritis, and was detrimental to milk yield, survival, and fertility compared with CON. In contrast, CEF increased the cure of metritis, milk yield, and fertility compared with CM and CON. Finally, the negative effects of metritis on milk yield culling and fertility could not be completely reversed by CEF.

Effect of sequestering agents based on a Saccharomyces cerevisiae fermentation product and clay on the ruminal bacterial community of lactating dairy cows challenged with dietary aflatoxin B1.

This study was conducted to examine the effects of clay (CL) and Saccharomyces cerevisiae fermentation product (SCFP) on the ruminal bacterial community of Holstein dairy cows challenged with aflatoxin B1 (AFB1). A second objective was to examine correlations between bacterial abundance and performance measures. Eight lactating dairy cows stratified by milk yield and parity were randomly assigned to 4 treatments in a 4 × 4 Latin square design with 2 replicate squares, four 33-d periods, and a 5-d washout between periods. The treatments included (1) control (basal diet, no additive); (2) T (control + 63.4 µg/kg AFB1, oral dose); (3) CL (T + 200 g/head per day of sodium bentonite clay, top-dress); and (4) CL+SCFP [CL + 19 g/head per day Diamond V NutriTek (Diamond V Inc., Cedar Rapids, IA) + 16 g/head per day MetaShield (Diamond V Inc.), top-dress]. Cows were adapted to diets containing no AFB1 from d 1 to 25 (predosing period). From d 26 to 30 (dosing period), AFB1 was orally dosed and then withdrawn for d 31 to 33 (withdrawal period). During the predosing period, compared with the control, feeding CL and CL+SCFP increased the relative abundance of the most dominant phylum, Bacteroidetes (55.1 and 55.8 vs. 50.6%, respectively), and feeding CL+SCFP increased Prevotella abundance (43.3 and 43.6 vs. 40.0%, respectively). During the dosing period, feeding AFB1 did not affect the ruminal bacterial community, but the relative abundance of Fibrobacteraceae increased with CL+SCFP compared with T (1.45 vs. 0.97%); Fibrobacter abundance also tended to increase with CL+SCFP compared with T and control, respectively (1.45 vs. 0.97 and 1.05%, respectively). Feeding AFB1 with or without CL or CL+SCFP did not affect ruminal pH or concentrations of NH3-N, total volatile fatty acids, or individual volatile fatty acids. Milk yield and milk component yields were positively correlated with the relative abundance of unclassified Succinivibrionaceae, unclassified YS2, or Coprococcus. Feed efficiency was positively correlated (r ≥ 0.30) with the relative abundance of unclassified YS2, Coprococcus, or Treponema. Feeding aflatoxin at 63 µg/kg, a common contamination level on farms, did not affect the abundance of dominant bacteria or rumen fermentation. When aflatoxin was fed, CL+SCFP increased the abundance of Fibrobacter, a major fibrolytic bacteria genus. Milk yield and DMI were positively correlated with abundance of Succinivibrionaceae and Coprococcus. Feed efficiency was positively correlated with abundance of Coprococcus, Treponema, and YS2. Future studies should speciate culture and determine the functions of the bacteria to elucidate their roles in the rumen and potential contribution to increasing the performance of dairy cows.

Effects of lipopolysaccharide dosing on bacterial community composition and fermentation in a dual-flow continuous culture system.

The objectives of this study were to evaluate the effects of lipopolysaccharide (LPS) dosing on bacterial fermentation and bacterial community composition (BCC), to set up a subacute ruminal acidosis (SARA) nutritional model in vitro, and to determine the best sampling time for LPS dosing in a dual-flow continuous culture system. Diets were randomly assigned to 6 fermentors in a replicated 3 × 3 Latin square with three 11-d experimental periods that consisted of 7 d for diet adaptation and 4 d for sample collection. Treatments were control diet (CON), wheat and barley diet (WBD) to induce SARA, and control diet + LPS (LPSD). Fermenters were fed 72 g of dry matter/d. The forage:concentrate ratio of CON was 65:35. The WBD diet was achieved by replacing 40% of dry matter of the CON diet with 50% ground wheat and 50% ground barley. The LPS concentration in LPSD was 200,000 endotoxin units, which was similar to that observed in cows with SARA. The SARA inducing and LPS dosing started at d 8. The BCC was determined by sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform (Illumina Inc., San Diego, CA). The LPSD and CON maintained pH above 6 for the entire experimental period, and the WBD kept pH between 5.2 and 5.6 for 4 h/d, successfully inducing SARA. Digestibility of neutral detergent fiber and crude protein in LPSD were not different from WBD but tended to be lower than CON. Lipopolysaccharide dosing had no effect on pool of VFA concentrations and profiles but decreased bacterial N; the pattern changes of VFA and LPS in LPSD started to increase and be similar to WBD 6 h after LPS dosing. Pool of LPS concentration was around 11-fold higher in WBD and 4-fold higher in LPSD than CON. In the solid fraction, the BCC of LPSD was different from WBD and tended to be different from CON. In the liquid fraction, the BCC was different among treatments. The LPS dosing increased the relative abundance of Succinimonas, Anaeroplasma, Succinivibrio, Succiniclasticum, and Ruminobacter, which are main gram-negative bacteria related to starch digestion. Our results suggest that LPS dosing does not affect pH alone. However, LPS could drive the development of SARA by affecting bacteria and bacterial fermentation. For future studies, samples are suggested to be taken 6 h after LPS dosing in a dual-flow continuous culture system.

Effects of treatment of preweaning dairy calves with recombinant bovine somatotropin on immune responses and somatotropic axis.

Weaning may be associated with negative energy balance and body weight loss when calves are still immunologically immature, predisposing them to infectious diseases. The aim of the present experiment was to investigate the effects of treatment of preweaning dairy calves with recombinant bovine somatotropin (rbST) on the somatotropic axis, selected immune parameters, and hematology of calves around weaning. Thirty-six Holstein female calves were randomly assigned to receive 1.5 to 1.8 mg of rbST (Posilac, Elanco Animal Health, Greenfield, IN) per kilogram of body weight or to receive injections of saline (saline solution 0.9%, Valley Vet Supply, Marysville, KS) every 7 d from 21 to 63 d of life. Calves were fed milk replacer ad libitum from birth to 38 d of age (d -11), when progressive weaning started, and calves were weaned at 49 d of age (d 0). Calves were weighed at birth and weekly from 21 to 63 d of age, when wither height also was measured. Calves were vaccinated with 0.5 mg of ovalbumin on study d -28 and -7. Blood samples were collected on d -28, -25, -21, -11, 0, 3, 7, and 14. Polymorphonuclear leukocytes were isolated and challenged ex vivo with Escherichia coli to determine phagocytosis and oxidative burst capacity. Additionally, expression of cluster of differentiation (CD)62L and CD18 by granulocyte, lymphocyte, and CD14+ monocyte were determined. Blood samples were also used to determine hematological parameters and concentrations of growth hormone, insulin-like growth factor-1, insulin, glucose, fatty acids, ß-hydroxybutyrate, haptoglobin, and anti-ovalbumin IgG. Calves treated with rbST had greater concentrations of growth hormone and insulin-like growth factor-1 from d -25 to 14 than control calves, whereas insulin, fatty acid, and ß-hydroxybutyrate concentrations did not differ. On d -11, glucose concentration was greater for rbST-treated calves. Treatment did not affect polymorphonuclear lymphocyte phagocytosis and oxidative burst, but intensity of expression of CD62L and CD18 by granulocytes tended to be increased by rbST treatment. Treatment did not affect the concentration of anti-ovalbumin IgG in serum. Haptoglobin concentration was reduced in rbST treated calves on d 3 and we noted a tendency for hematocrit to be lower in rbST-treated calves. Treatment did not affect body weight, wither height, and average daily gain, despite the fact that rbST-treated calves had lower daily milk replacer intake. The relatively minor improvements in immune responses resulting from rbST treatment of weaning calves may not be sufficient to reduce the incidence of infectious diseases.

Bacterial diversity and composition of alfalfa silage as analyzed by Illumina MiSeq sequencing: Effects of Escherichia coli O157:H7 and silage additives.

The first objective of this study was to examine effects of adding Escherichia coli O157:H7 with or without chemical or microbial additives on the bacterial diversity and composition of alfalfa silage. The second objective was to examine associations between the relative abundance of known and unknown bacterial species and indices of silage fermentation quality. Alfalfa forage was harvested at 54% dry matter, chopped to a theoretical length of cut of 19 mm, and ensiled in quadruplicate in laboratory silos for 100 d after the following treatments were applied: (1) distilled water (control); (2) 1 × 105 cfu/g of E. coli O157:H7 (EC); (3) EC and 1 × 106 cfu/g of Lactobacillus plantarum (EC+LP); (4) EC and 1 × 106 cfu/g of Lactobacillus buchneri (EC+LB); and (5) EC and 0.22% propionic acid (EC+PA). After 100 d of ensiling, the silage samples were analyzed for bacterial diversity and composition via the Illumina MiSeq platform (Illumina Inc., San Diego, CA) and chemically characterized. Overall, Firmicutes (74.1 ± 4.86%) was the most predominant phylum followed by Proteobacteria (20.4 ± 3.80%). Relative to the control, adding E. coli O157:H7 alone at ensiling did not affect bacterial diversity or composition but adding EC+LP or EC+LB reduced the Shannon index, a measure of diversity (3.21 vs. 2.63 or 2.80, respectively). The relative abundance of Firmicutes (69.2 and 68.8%) was reduced, whereas that of Proteobacteria (24.0 and 24.9%) was increased by EC+LP and EC+PA treatments, relative to those of the control (79.5 and 16.5%) and EC+LB (77.4 and 18.5%) silages, respectively. Compared with the control, treatment with EC+LP increased the relative abundance of Lactobacillus, Sphingomonas, Pantoea, Pseudomonas, and Erwinia by 426, 157, 200, 194, and 163%, respectively, but reduced those of Pediococcus, Weissella, and Methylobacterium by 5,436, 763, and 250%, respectively. Relative abundance of Weissella (9.19%) and Methylobacterium (0.94%) were also reduced in the EC+LB silage compared with the control (29.7 and 1.50%, respectively). Application of propionic acid did not affect the relative abundance of Lactobacillus, Weissella, or Pediococcus. Lactate concentration correlated positively (r = 0.56) with relative abundance of Lactobacillus and negatively (r = -0.41) with relative abundance of Pediococcus. Negative correlations were detected between ammonia-N concentration and relative abundance of Sphingomonas (r = -0.51), Pantoea (r = -0.46), Pseudomonas (r = -0.45), and Stenotrophomonas (r = -0.38). Silage pH was negatively correlated with relative abundance of Lactobacillus (r = -0.59), Sphingomonas (r = -0.66), Pantoea (r = -0.69), Pseudomonas (r = -0.69), and Stenotrophomonas (r = -0.50). Future studies should aim to speciate, culture, and determine the functions of the unknown bacteria detected in this study to elucidate their roles in silage fermentation.

Environmental and health effects of the herbicide glyphosate.

The herbicide glyphosate, N-(phosphonomethyl) glycine, has been used extensively in the past 40years, under the assumption that side effects were minimal. However, in recent years, concerns have increased worldwide about the potential wide ranging direct and indirect health effects of the large scale use of glyphosate. In 2015, the World Health Organization reclassified glyphosate as probably carcinogenic to humans. A detailed overview is given of the scientific literature on the movement and residues of glyphosate and its breakdown product aminomethyl phosphonic acid (AMPA) in soil and water, their toxicity to macro- and microorganisms, their effects on microbial compositions and potential indirect effects on plant, animal and human health. Although the acute toxic effects of glyphosate and AMPA on mammals are low, there are animal data raising the possibility of health effects associated with chronic, ultra-low doses related to accumulation of these compounds in the environment. Intensive glyphosate use has led to the selection of glyphosate-resistant weeds and microorganisms. Shifts in microbial compositions due to selective pressure by glyphosate may have contributed to the proliferation of plant and animal pathogens. Research on a link between glyphosate and antibiotic resistance is still scarce but we hypothesize that the selection pressure for glyphosate-resistance in bacteria could lead to shifts in microbiome composition and increases in antibiotic resistance to clinically important antimicrobial agents. We recommend interdisciplinary research on the associations between low level chronic glyphosate exposure, distortions in microbial communities, expansion of antibiotic resistance and the emergence of animal, human and plant diseases. Independent research is needed to revisit the tolerance thresholds for glyphosate residues in water, food and animal feed taking all possible health risks into account.

Application of chitosan microparticles for mitigation of Salmonella in agricultural water.

AIM: The activity of chitosan microparticles (CM) was examined using a matrix of conditions in order to assess the efficacy of CM as a mitigation against various strains of Salmonella enterica in agricultural water. METHODS AND RESULTS: Different concentrations of CM (0, 0·01, 0·1, 0·2, 0·3% w/v) were examined for antimicrobial activity against log vs stationary phase cells of Salmonella and at different conditions of temperature, salinity and pH. Results showed greatest activity with 0·3% CM at pH 7, 25-37°C without additional of salt. Significant reductions in Salmonella levels were also achieved in natural pond water, although decreases were reduced compared to sterile water. All serotypes were sensitive to CM, with minimal inhibitory concentrations ranging from 0·0031 to 0·0250% w/v. Phylogenic analysis of Javiana strains showed increased resistance appeared in multiple genetic lineages. CONCLUSION: Conditions demonstrating greatest CM activity were compatible with agricultural practices. Although sensitivity to CM varied among Salmonella strains, all strains were sensitive under conditions examined in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This research indicated that CM, a natural compound with minimal environmental impact, could be an effective alternative for mitigating Salmonella in agricultural water applications.

The capacity of silage inoculant bacteria to bind aflatoxin B1 in vitro and in artificially contaminated corn silage.

The objectives were to examine the aflatoxin B1 (AFB1)-binding capacity of silage bacteria and factors affecting the responses. Experiments 1 and 2 examined the effects of bacterial strain and population on the AFB1-binding capacity of 10 bacteria. When applied at 106 cfu/mL to an in vitro medium, only Lactobacillus plantarum PT5B bound the AFB1 and the binding capacity was low (4%). When applied at 109 cfu/mL, all 10 bacteria bound AFB1, but L. plantarum R2014 (Lp) and EQ12, Lactobacillus buchneri R1102 (Lb), and Pediococcus acidilactici R2142 and EQ01 (Pa) had the greatest capacity (23.9 to 33%). Experiment 3 examined the AFB1-binding capacity of viable and nonviable (HCl-treated) forms of Lp, Lb, and Pa at different pH. Nonviable Lb and Lp, but not Pa, increased AFB1 binding. Binding of AFB1 was greatest at pH 2.5 and least at pH 8. As the nonviable Lb and Lp that bound AFB1 in experiment 3 would not be effective silage inoculants, experiment 4 examined effects of benign versus severe treatments (85 vs. 100°C; pH 2.5 vs. <1) on the viability of Lp, Lb, and Pa. The population of bacteria was reduced from 9 to 4 log cfu/mL by treatment with HCl at pH 2.5 and to 2 log cfu/mL by 85 or 100°C, whereas acidification at pH <1 eliminated the bacteria. Experiment 5 determined the effect of the ensiling duration and benign treatment methods [37 (viable cells) or 85°C (heated cells) or acidification with HCl at pH 2.5 (acid-treated cells)] on binding of AFB1 and silage quality during the fermentation of corn forage. Corn forage was ensiled after treatment with only deionized water (control), AFB1 (30 µg/kg of fresh forage), or a mixture of AFB1 and 109 cfu/g of each of the treated bacteria. Adding AFB1 alone to corn forage reduced the pH decline during the first 3 d of ensiling and increased or tended to increase butyric acid concentration and final pH after ensiling for 21 d. Bacterial inoculation inhibited these negative effects. The fermentation profile of silage treated with Lb and Pa did not differ from those of the control silage. In all silages treated with the toxin, the AFB1 concentration decreased linearly (from 30 to ≤0.35 µg/kg) within 3 d of ensiling. Certain silage bacteria can bind AFB1 but the efficacy depends on several factors.

Use of calcitriol to maintain postpartum blood calcium and improve immune function in dairy cows.

Our objectives were to determine the effects of an injectable formulation of calcitriol on mineral metabolism and immune function in postpartum Holstein cows that received an acidogenic diet prepartum to minimize hypocalcemia. In experiment 1, cows within 6 h of calving received calcitriol (0, 200, or 300 µg) to determine the dose needed to increase plasma concentrations of Ca; 300 µg was sufficient to sustain Ca for at least 3 d. In experiment 2, multiparous cows were assigned randomly to receive only vehicle (control, n = 25) or 300 µg of calcitriol (n = 25) subcutaneously within the first 6 h after calving. Blood was sampled before treatment and 12 h later, then daily until 15 d in milk (DIM), and analyzed for concentrations of ionized Ca (iCa), total Ca (tCa), total Mg (tMg), and total P (tP), metabolites, and hormones. Urine was sampled in the first 7 DIM and analyzed for concentrations of tCa, tMg, and creatinine. Neutrophil function was evaluated in the first week postpartum. Dry matter intake and production performance were evaluated for the first 36 DIM. Calcitriol administration increased concentrations of calcitriol in plasma within 12 h of application from 51 to 427 pg/mL, which returned to baseline within 5 d. Concentrations of iCa and tCa increased 24 h after treatment with calcitriol. Concentrations of iCa (control = 1.08 vs. calcitriol = 1.20 mM), tCa (control = 2.23 vs. calcitriol = 2.33 mM), and tP (control = 1.47 vs. calcitriol = 1.81 mM) remained elevated in cows treated with calcitriol until 3, 5, and 7 DIM, respectively, whereas concentration of tMg (control = 0.76 vs. calcitriol = 0.67 mM) was less in calcitriol cows than control cows until 3 DIM. Concentrations of parathyroid hormone decreased in calcitriol cows compared with control cows (control = 441 vs. calcitriol = 336 pg/mL). Calcitriol tended to increase plasma concentrations of ß-hydroxybutyrate and serotonin, but concentrations of glucose, nonesterified fatty acids, and C-telopeptide of type I collagen in plasma did not differ between treatments. Cows treated with calcitriol excreted more urinary tCa (control = 0.5 vs. calcitriol = 2.1 g/d) and tMg (control = 4.5 vs. calcitriol = 5.0 g/d) in the first 7 and 2 DIM, respectively, than control cows. Compared with control, calcitriol improved the proportion of neutrophils with oxidative burst (control = 31.9 vs. calcitriol = 40.6%), mean fluorescence intensity for oxidative burst (control = 90,900 vs. calcitriol = 99,746), and mean fluorescence intensity for phagocytosis (control = 23,887 vs. calcitriol = 28,080). Dry matter intake, yields of milk, and milk components did not differ between treatments. Administration of 300 µg of calcitriol at calving was safe and effective in increasing blood concentration of iCa and plasma concentrations of calcitriol, tCa, and tP for the first 6 d after treatment, and improved measures of innate immune function in early-lactation Holstein cows.

Fate of Escherichia coli O157:H7 and bacterial diversity in corn silage contaminated with the pathogen and treated with chemical or microbial additives.

Inhibiting the growth of Escherichia coli O157:H7 (EC) in feeds may prevent the transmission or cycling of the pathogen on farms. The first objective of this study was to examine if addition of propionic acid or microbial inoculants would inhibit the growth of EC during ensiling, at silo opening, or after aerobic exposure. The second objective was to examine how additives affected the bacterial community composition in corn silage. Corn forage was harvested at approximately 35% dry matter, chopped to a theoretical length of cut of 10 mm, and ensiled after treatment with one of the following: (1) distilled water (control); (2) 1 × 105 cfu/g of EC (ECCH); (3) EC and 1 × 106 cfu/g of Lactobacillus plantarum (ECLP); (4) EC and 1 × 106 cfu/g of Lactobacillus buchneri (ECLB); and (5) EC and 2.2 g/kg (fresh weight basis) of propionic acid, containing 99.5% of the acid (ECA). Each treatment was ensiled in quadruplicate in laboratory silos for 0, 3, 7, and 120 d and analyzed for EC, pH, and organic acids. Samples from d 0 and 120 were also analyzed for chemical composition. Furthermore, samples from d 120 were analyzed for ammonia N, yeasts and molds, lactic acid bacteria, bacterial community composition, and aerobic stability. The pH of silages from all treatments decreased below 4 within 3 d of ensiling. Escherichia coli O157:H7 counts were below the detection limit in all silages after 7 d of ensiling. Treatment with L. buchneri and propionic acid resulted in fewer yeasts and greater aerobic stability compared with control, ECCH, and ECLP silages. Compared with the control, the diversity analysis revealed a less diverse bacterial community in the ECLP silage and greater abundance of Lactobacillus in the ECLP and ECA silages. The ECLB silage also contained greater abundance of Acinetobacter and Weissella than other silages. Subsamples of silages were reinoculated with 5 × 105 cfu/g of EC either immediately after silo opening or after 168 h of aerobic exposure, and EC were enumerated after 6 or 24 h, respectively. All silages reinoculated with EC immediately after silo opening (120 h) had similar low pH values (<4.0) and EC counts were below the detection limit. The ECCH and ECLP silages reinoculated with EC after 168 h of aerobic exposure had relatively high pH values (>5.0) and EC counts (5.39 and 5.30 log cfu/g, respectively) 24 h later. However, those treated with L. buchneri or propionic acid had lower pH values (4.24 or 3.96, respectively) and lower EC counts (1.32 log cfu/g or none, respectively). During ensiling, EC was eliminated from all silages at pH below 4.0. During aerobic exposure, the growth of EC was reduced or prevented in silages that had been treated with L. buchneri or propionic acid at ensiling, respectively.

Clinical response after chitosan microparticle administration and preliminary assessment of efficacy in preventing metritis in lactating dairy cows.

The objectives were to evaluate the clinical response to intrauterine administration of chitosan microparticles (CM) and to assess efficacy for preventing metritis in dairy cows. Holstein cows (n=104; 40 primiparous and 64 multiparous) at increased risk for metritis (cows that had abortion, dystocia, twins, stillbirth, or retained placenta) were randomly assigned to 1 of 2 treatments at 1d in milk (DIM; 24h postpartum): CM group (n=52), daily intrauterine infusion of 8g of CM dissolved in 40mL of sterile water for 5d; control (CON) group (n=52), daily intrauterine infusion of 40mL of sterile water for 5d. Clinical response was assessed by evaluation of parameters associated with inflammation (rectal temperature and plasma haptoglobin concentration) and metabolism [plasma nonesterified fatty acid (NEFA) and ß-hydroxybutyrate (BHB) concentrations] up to 14 DIM, and daily milk yield up to 30 DIM. Uterine discharge pH was evaluated at 4, 7, 10, and 14 DIM as an indicator of bacterial load and acid byproduct production. The cumulative incidence of metritis was evaluated up to 4, 7, 10, and 14 DIM. Continuous and dichotomous outcomes were evaluated with mixed linear and logistic regression analysis, respectively. Treatment with CM did not affect rectal temperature (39.17±0.04 vs. 39.14±0.04°C), haptoglobin (1.10±0.05 vs. 1.07±0.05mg/mL), NEFA (0.64±0.04 vs. 0.63±0.04mmol/L), BHB (0.61±0.03 vs. 0.57±0.03mmol/L), or milk yield (30.3±0.92 vs. 30.1±0.97kg/d) compared with CON. An interaction between treatment and time showed that NEFA concentrations were lower for CM than CON at 10 DIM (0.46±0.06 vs. 0.64±0.06mmol/L). Treatment with CM resulted in greater uterine discharge pH than CON (6.91±0.03 vs. 6.83±0.02). Cows that developed metritis had increased concentrations of haptoglobin and BHB, and decreased uterine discharge pH and milk yield. Treatment with CM resulted in decreased incidence of metritis up to 7 DIM compared with CON (46.2 vs. 65.4%); however, no differences were found at 4 (11.5 vs. 17.3%), 10 (61.5 vs. 73.1%), and 14 DIM (63.5 vs. 73.1%) for CM versus CON, respectively. In conclusion, CM did not alter clinical parameters of cows at risk for metritis, and may merit further investigation for prevention of metritis. However, the duration of treatment may have to be extended to effectively reduce the incidence of metritis during the high-risk period.

Control of Escherichia coli O157:H7 in contaminated alfalfa silage: Effects of silage additives.

This study was conducted to examine if adding microbial inoculants or propionic acid to alfalfa silages contaminated with Escherichia coli O157:H7 would inhibit the growth of the pathogen during or after ensiling. Alfalfa forage was harvested at the early bloom stage, wilted to a dry matter concentration of 54%, chopped to 19-mm lengths, and ensiled after treatment with one of the following: (1) distilled water (control); (2) 1×10(5) cfu/g of E. coli O157:H7 (EC); (3) EC and 1×10(6) cfu/g of Lactobacillus plantarum (EC+LP); (4) EC and 1×10(6) cfu/g of Lactobacillus buchneri (EC+LB); and (5) EC and 2.2g/kg of propionic acid (EC+PA). Each treatment was ensiled in quadruplicate in laboratory silos for 0, 3, 7, 16, and 100d and analyzed for EC counts, pH, and organic acids. In addition, samples from d 100 were analyzed for chemical composition, ammonia-N, counts of yeasts and molds, and aerobic stability. Escherichia coli O157:H7 was detected in all silages until d 7, but by d 16 it was not detected in those treated with EC+LB and EC+LP, though it was still detected in EC and EC+PA silages. However, by d 100, the pathogen was not detected in any silage. The rate of pH decrease to 5.0 was fastest for the EC+LP silage (7d), followed by the EC+LB silage (16d). Nevertheless, all silages had attained a pH of or less than 5.0 by d 100. The rapid decrease in pH in EC+LP and EC+LB silages was observed due to higher lactate and acetate concentrations, respectively, relative to the other silages during the early fermentation phase (d 3-16). Propionic acid was only detected in the EC+PA silage. Yeast counts were lowest in EC+LB and EC+PA silages. Subsamples of all d-100 silages were reinoculated with 1×10(5) cfu/g of EC immediately after silo opening. When the pathogen was subsequently enumerated after 168h of aerobic exposure, it was not detected in silages treated with EC+PA, EC+LB, or EC+LP, which all had pH values less than 5.0. Whereas the EC silage had a pH value of 5.4 and 2.3 log cfu/g of the pathogen. Certain bacterial inoculants can hasten the inhibition of E. coli O157:H7 during ensiling, such as propionic acid, and they can also prevent its growth on silage contaminated with the pathogen after ensiling.

Effect of cooling during the dry period on immune response after Streptococcus uberis intramammary infection challenge of dairy cows.

Heat stress in the dry period affects the immune status of dairy cows in the subsequent lactation. We hypothesized that cooling during the dry period improves immune response to postpartum intramammary infection (IMI) by environmental pathogens such as Streptococcus uberis. Cows were dried off 46 d before expected calving and assigned to cooling (CL, n = 15) or heat stress (HT, n = 15). Cooled cows were housed with sprinklers, fans, and shade, whereas the HT group had only shade. All cows were cooled postpartum. Rectal temperature (RT) and respiration rate (RR) were recorded thrice weekly during the dry period. From −46 to 42 d relative to calving, dry matter intake was recorded daily, and both body weight (BW) and body condition score (BCS) weekly. Milk yield and composition were recorded daily after calving. Streptococcus uberis IMI was induced at 5 d postpartum in a subset of cows (CL, n = 5; HT, n = 5). Blood was collected at 0, 12, 18, 24, and 36 h after IMI. Hematological analysis was performed, and neutrophils isolated for RNA extraction. Immune response genes (TLR2, IL1-ß, IL6, IL8, IL10, and TNFα) were assessed by real-time, reverse transcription-PCR. Relative to HT cows, CL cows had lower RT and RR during the dry period. The CL cows also consumed more feed prepartum but not postpartum, gained more BW prepartum but lost more BW in lactation, and had higher BCS score prepartum and a lower BCS postpartum. During 40 wk of lactation, CL cows produced more milk (33.8 vs. 30.0 kg/d) than HT cows but milk composition was not affected. Cows in the CL group had greater white blood cell counts and more neutrophils than HT cows during IMI. From 0 to 36 h post-IMI, TNFα mRNA expression decreased, whereas that of IL6 and IL8 increased in both treatments. Additionally, CL cows had lower IL10 mRNA expression at 18 h post-IMI. Expression of TLR2 mRNA decreased over time in both treatments. However, CL cows had greater overall TLR2 mRNA expression than HT. No differences were detected for mRNA expression of IL1-ß, IL6, IL8, or TNFα. Cooling cows during the dry period alters immune function and neutrophil response to IMI in early lactation.