Supplementing a Bacillus-based probiotic to high-risk stocker cattle.

This experiment evaluated performance, health, and physiological responses of high-risk steers receiving a Bacillus-based probiotic during a 90-d grazing period. A total of 240 Angus-influenced steers were used in this experiment that was replicated over 2 yr (120 steers/year). Each year, steers were obtained from an auction yard and transported to the experimental facility (120 km). Steer body weight (BW) was recorded at arrival (d -1), and this value was averaged with BW recorded on d 0 to represent initial BW (236.6 ± 1.5 kg). On d 0, steers were ranked by BW and allocated to 1 of 12 pastures with stockpiled native grass (4-ha pastures; 10 steers/pasture). Pastures were randomly assigned to receive daily supplementation with dried distillers' grains at 1% of BW containing either: 1) Bacillus subtilis + B. licheniformis probiotic (BOV; 2 g/steer daily of Bovacillus™; Novonesis, Horsholm, Denmark) or 2) no feed additive (CON). Cattle received treatments from d 0 to 90, in addition to free-choice access to water and mineral + vitamin mix without ionophore. Steers were assessed for bovine respiratory disease (BRD) signs daily. Blood samples were collected and full BW was recorded on d 0, 14, 28, 56, and 90. Shrunk BW was recorded on d 91 after 16 h of feed and water restriction, and a 4% pencil shrink was used to calculate final BW. Average daily gain (ADG) was calculated based on initial and final BW. No treatment effects were detected (P ≥ 0.73) for steer final BW and ADG. A treatment × day interaction was detected (P ≤ 0.05) for plasma haptoglobin concentration, which was greater for CON steers on d 14 and 28 (P ≤ 0.02). Incidence of BRD signs did not differ (P = 0.97) between treatments (51.7 and 51.3% for BOV and CON, respectively; SEM = 7.70). However, steer mortality + removals for health complications were greater (P = 0.01) in CON compared to BOV (0.00 vs. 5.04%, respectively; SEM = 1.41). Supplementing BOV improved (P ≤ 0.04) total pasture-based liveweight change (643 vs. 502 kg/pasture, respectively; SEM = 45) and final pasture-based total liveweight (3,007 vs. 2,869 kg/pasture, respectively; SEM = 46). Collectively, supplementation with a probiotic based on B. subtilis and B. licheniformis to high-risk stocker cattle did not alleviate incidence of BRD signs nor improved ADG, but decreased acute-phase protein response, reduced steer mortality + removal, and increased pasture-based productivity during a 90-d grazing period.

Maternal pre- and postpartum supplementation of a Bacillus-based DFM enhanced cow and calf performance.

This study evaluated the effects of maternal supplementation of a Bacillus-based direct-fed microbial (DFM) on the physiology and growth performance of Bos indicus-influenced cow-calf pairs. On day 0 (~139 d before expected calving date), 72 fall-calving, Brangus crossbred beef heifers (20 to 22 mo of age) pregnant with first offspring were stratified by their initial body weight (BW; 431 ±â€…31 kg) and body condition score (BCS; 6.0 ±â€…0.36; scale 1 to 9), and randomly allocated into 1 of 12 bahiagrass pastures (1 ha and six heifers per pasture). Treatments were randomly assigned to pastures (six pastures per treatment) and consisted of heifers supplemented with 1 kg/d of soybean hulls (dry matter, DM) that was added (BAC) or not (CON) with DFM containing Bacillus subtilis and B. licheniformis (Bovacillus; Chr. Hansen A/S, Hørsholm, Denmark). Treatments were provided from days 0 to 242 (139 ±â€…4 d prepartum to 104 ±â€…4 d postpartum). Calves were weaned on day 242 (96 ±â€…30 d of age) and then allocated into 1 of 16 drylot pens and fed the same concentrate at 3.25% of BW (DM) until day 319. Maternal treatment effects were not detected (P ≥ 0.29) for herbage allowance and forage chemical composition. Heifer BCS on days 39 and 63 tended (P ≤ 0.09) to be greater for BAC vs. CON heifers, whereas heifer BCS on day 91 was greater (P = 0.01) for BAC vs. CON heifers. Heifer BCS did not differ (P ≥ 0.20) between treatments on days 179 and 242. Plasma glucose concentration did not differ from days 0 to 63 (P ≥ 0.14) but were greater (P < 0.01) on day 179 and tended (P = 0.09) to be greater on day 242 for BAC vs. CON heifers. Calf BW at birth, ADG from birth to weaning, and BW at weaning did not differ (P ≥ 0.19) between treatments, but calf BW at drylot exit (day 319) was greater (P = 0.05) for BAC vs. CON calves. Maternal treatment effects were not detected (P ≥ 0.42) for calf serum concentration of IgG at birth and postvaccination plasma concentrations of glucose, cortisol, and haptoglobin. Serum titers against bovine respiratory syncytial virus (BRSV) were greater (P = 0.04) for BAC vs. CON calves on day 287, whereas seroconversion against parainfluenza-3 virus (PI-3) was greater (P < 0.01) for BAC vs. CON calves on day 271. Thus, maternal supplementation of a Bacillus-based DFM increased prepartum BCS gain and postpartum plasma glucose concentration of heifers and led to positive carryover effects on postweaning BW gain and humoral immune response in their offspring.

Direct-fed microbials (DFM), such as Bacillus spp., have been shown to produce a wide variety of enzymes related to nutrient digestion and to support gastrointestinal tract immune function and integrity, leading to increased nutrient digestibility and cattle performance. Nutritional management of beef cows during gestation and early lactation has been associated with enhanced future offspring growth performance and immune response following birth. The present study combined the use of Bacillus-based DFM for pregnant heifers during critical production stages (late gestation and early lactation) to promote the overall performance of heifers and their offspring. Heifers offered Bacillus-based DFM had greater body condition score at calving and postpartum plasma concentration of glucose, whereas their offspring had similar body weight at birth, but greater growth performance when fed relatively high amounts of protein and energy in drylot compared to cohorts born from heifers that did not receive Bacillus-based DFM supplementation.

Influence of BOVAMINE DEFEND Plus on growth performance, carcass characteristics, estimated dry matter digestibility, rumen fermentation characteristics, and immune function in finishing beef steers.

One hundred and eighty crossbred beef steers (406.0 ±â€…2.2 kg) were used to determine the impact of a novel direct-fed microbial (DFM) on growth performance, carcass characteristics, rumen fermentation characteristics, and immune response in finishing beef cattle. Steers were blocked by body weight (BW) and randomly assigned, within block, to 1 of 2 treatments (3 replicates/treatment: 30 steers/replicate). Treatments included: (1) no DFM (control) and (2) DFM supplementation at 50 mg ∙ animal-1 ∙ d-1 (BOVAMINE DEFEND Plus). All steers were fed a high-concentrate finishing diet and individual feed intake was recorded daily via the GrowSafe system. BWs were collected every 28 d. On day 55, 10 steers per pen were injected with ovalbumin (OVA). Jugular blood samples were collected from each steer on days 0, 7, 14, and 21 post injection. On day 112, the same steers were injected again with OVA and intramuscularly with a pig red blood cell solution. Jugular blood samples were collected from each steer on days 0, 7, 14, and 21 post injection. On day 124, rumen fluid was collected from 3 steers per treatment and used to estimate in vitro rumen fermentation characteristics. Equal numbers of steers per treatment were transported to a commercial abattoir on days 145, 167, and 185 of the experiment, harvested, and carcass data were collected. Initial BW was similar across treatments. On days 28 and 55, steers receiving DFM had heavier BW (P < 0.01) compared to controls. The average daily gain was greater in DFM-supplemented steers from days 0 to 28 (P < 0.01) and days 0 to 55 (P < 0.01) of the experiment compared to controls. Overall dry matter intake (DMI) was greater (P < 0.04) and overall feed efficiency was similar in DFM-supplemented steers compared to controls. Dressing percentage (P < 0.02) was greater in steers receiving DFM compared to controls. Antibody titers to injected antigens were similar across treatments. However, red blood cell superoxide dismutase activity was greater (P < 0.05) in DFM-supplemented steers compared to controls. In vitro molar proportions of isobutyric and butyric acid were greater (P < 0.01) and dry matter (DM) digestibility tended (P < 0.07) to be greater in rumen fluid obtained from steers supplemented with DFM. These data suggest that BOVAMINE DEFEND Plus supplementation improves growth performance during the initial period of the finishing phase, increases overall DMI and dressing percentage, and may impact antioxidant status in beef cattle.

Effects of supplementing direct-fed microbials on health and growth of pre-weaning Gyr × Holstein dairy calves.

This study aimed to evaluate the effects of direct-fed microbials (DFM) on health and growth responses of pre-weaning Bos indicus × B. taurus (Gyr × Holstein) crossbred calves. Ninety newborn heifer calves [initial body weight (BW) 35 ± 4.0 kg] were used. At birth, calves were ranked by initial BW and parity of the dam and assigned to: 1) whole milk without DFM supplementation (CON; n = 30), 2) whole milk with the addition of 1.0 g/calf per day of a Bacillus-based DFM (BAC; n = 30), or 3) whole milk with the addition of 1.0 g/calf per day of BAC and 1.2 g/calf per day of Enterococcus faecium 669 (MIX; n = 30). Milk was fed individually during the study (77 d) and the BAC and MIX treatments were offered daily throughout the 77-d pre-weaning period. All calves were offered a starter supplement and corn silage starting on d 1 and 60 of age, respectively. Milk and starter supplement intake were evaluated daily, and BW was recorded on d 0 and at weaning (d 77). Diarrhea and pneumonia were assessed daily, and fecal samples were collected on d 0, 7, 14, 21, and at weaning (d 77) for assessment of the presence of bacterial and protozoal pathogens via qPCR. All data were analyzed using SAS (v. 9.4) with calf as the experimental unit and using single-df orthogonal contrasts (BAC + MIX vs. CON; BAC vs. MIX). Daily feeding of DFM, regardless of type, improved weaning BW. Odds ratio for occurrence of pneumonia was lower for DFM-supplemented calves, but occurrence of both did not differ between BAC and MIX calves. No Salmonella spp. or E. coli F41 were detected in any of the calves. The proportion of calves positive for E. coli F17 was greater for DFM calves on d 7 (92 and 96% vs. 81% for BAC, MIX, and CON, respectively), 21 (13 and 26% vs. 7% for BAC, MIX, and CON, respectively), and at weaning (48 and 35% vs. 22% for BAC, MIX, and CON, respectively). For C. difficile, more DFM calves were positive on d 7 (65 and 30% vs. 35% for BAC, MIX, and CON, respectively) and 14 (20 and 28% vs. 7% for BAC, MIX, and CON, respectively), but also greater for BAC vs. MIX on d 7. More CON calves were positive for C. perfringens on d 14 (14% vs. 3 and 8% for CON, BAC, and MIX, respectively) compared with DFM-fed calves. Incidence of calves positive for C. perfringens was greater in BAC vs. MIX on d 7 (50 vs. 18%), and greater for MIX vs. BAC at weaning (9 vs. 0%). For protozoa occurrence, a lower proportion of DFM calves were positive for Cryptosporidium spp. on d 7 (58 and 48% vs. 76% for BAC, MIX, and CON, respectively), but opposite results were observed on d 21 for Cryptosporidium spp. (3 and 11% vs. 0% for BAC, MIX, and CON, respectively) and Eimeria spp. on d 14 (7 and 8% vs. 0% for BAC, MIX, and CON, respectively) and 21 (50 and 59% vs. 38% for BAC, MIX, and CON, respectively). In summary, DFM feeding alleviated the occurrence of pneumonia, improved growth rates, while also modulating the prevalence of bacteria and protozoa in pre-weaning Gyr × Holstein calves.

Short communication: a novel multispecies bacteria-based direct-fed microbial supports in vitro gut barrier integrity challenged with a pathogen or pro-inflammatory cytokines.

We conducted two experiments to evaluate the effects of a novel bacterial-based direct-fed microbial (DFM) on intestinal barrier integrity using the in vitro transepithelial electrical resistance (TEER) assay. In experiment 1, human-derived Caco-2 cells received or not (CON) a DFM containing Ligilactobacillus (formerly Lactobacillus) animalis 506, Propionibacterium freudenreichii 507, Bacillus paralicheniformis 809, and B. subtilis 597 (BDP; BOVAMINE DEFEND® Plus) at a rate of 1 × 108 CFU/transwell. Concurrently with treatment application (CON or BDP), a pathogenic challenge of Clostridium perfringens type A was added alone (PAT) or with BDP (PAT + BDP) at a rate of 2.8 × 107 CFU/transwell in a 2 × 2 factorial arrangement. In experiment 2, Caco-2 cells were also assigned in a 2 × 2 factorial design to CON or BDP and then, 2 h post-treatment administration (CON and BDP), a mixture of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) was added alone (CYT) or with BDP (CYT + BDP) at a 10:1 ratio, respectively. In both experiments, TEER was measured for 18 h. In experiment 1, a DFM × pathogen × hour interaction was observed for TEER (P < 0.0001). Adding the PAT alone initially tended to increase TEER vs. CON from 1.1 to 2.2 h (P ≤ 0.09), increased TEER at 3.2 h (P < 0.01), but reduced TEER from 5.4 to the end of the experimental period at 18.4 h (P ≤ 0.01). On the other hand, adding DFM, with or without the pathogenic challenge, yielded greater TEER vs. CON-CON and CON-PAT for most of the experimental period (P ≤ 0.04). A similar interaction was detected and reported in experiment 2 (P < 0.0001). The CYT challenge reduced mean TEER compared with all other treatments from 3.2 h to the remainder of the study (P ≤ 0.03). On the other hand, BDP-CYT was able to maintain the integrity of the epithelial cells when compared with CON-CON throughout the experimental period (P ≤ 0.03), the exception being at 3.2 h (P = 0.20). Moreover, BDP-CON increased (P ≤ 0.04) TEER when compared with CON-CON from 3.2 to 18.4 h, but also in comparison with BDP-CYT from 4.3 to 18.4 h post-DFM and challenge administration into the cells. In summary, C. perfringens type A and a pro-inflammatory cytokine cocktail compromised the integrity of intestinal epithelial cell monolayers in vitro, whereas adding a multispecies bacteria-based DFM counteracted these damaging effects.

Two experiments were designed to evaluate the effects of adding a bacterial-based direct-fed microbial (DFM) containing Lactobacillus animalis 506, Propionibacterium freudenreichii 507, Bacillus paralicheniformis 809, and Bacillus subtilis 597 on the integrity of intestinal epithelial cells challenged with Clostridium perfringens type A or a pro-inflammatory cytokine cocktail. Regardless of the challenge, the addition of the DFM maintained the integrity of the intestinal epithelial cells in vitro. These results help to elucidate the potential beneficial effects that the bacterial-based DFM containing L. animalis 506, P. freudenreichii 507, B. paralicheniformis 809, and B. subtilis 597 may bring to livestock species.

Bacillus paralicheniformis 809 and Bacillus subtilis 810 support in vitro intestinal integrity under hydrogen peroxide and deoxynivalenol challenges.

We designed and conducted two in vitro experiments to evaluate the effects of two Bacillus spp. probiotics on gut barrier integrity using the transepithelial electrical resistance (TEER) assay under two different challenge models. In Exp. 1, intestinal epithelial cells received or not (CON) B. paralicheniformis 809 (BLI) or B. subtilis 810 (BSU) at a rate of 1 × 108 colony forming units (CFU)/transwell. Two hours after treatment application (CON, BLI, or BSU), 5 mM of the reactive oxygen species hydrogen peroxide, mimicking mucosal oxidative stress, was added alone (HYP) or with each of the Bacillus spp. (HYP + BLI or HYP + BSU). In Exp. 2, cells were assigned to the same treatments as in Exp. 1 (CON, BLI, and BSU), or mycotoxin deoxynivalenol (DON), which was added alone or in combination with BLI or BSU, resulting in another two treatments (DON + BLI and DON + BSU). Transepithelial electrical resistance was measured for 14 h postchallenge. In Exp. 1, a treatment × hour interaction was observed for TEER (P < 0.0001). Adding BLI and BSU resulted in greater TEER values vs. CON for most of the experimental period (P < 0.02), whereas HYP reduced mean TEER and area under the curve (AUC), while increasing the amount of sugar that translocated through the monolayer cells (P < 0.001). A treatment × hour interaction was also observed in Exp. 2 (P < 0.0001), as DON led to an immediate and acute drop in TEER that lasted until the end of the experimental period (P < 0.0001). Both BLI and BSU alleviated the DON-induced damaging effects on the integrity of intestinal epithelial cells, whereas both Bacillus spp. alleviated the damage caused by DON alone and the proportion of sugar that translocated through the monolayer cells was not different between CON and DON + BLI (P = 0.14) and DON + BLI and DON + BSU (P = 0.62). In summary, both Bacillus spp. strains (B. paralicheniformis 809 and B. subtilis 810) were able to counteract the damaging effects of the challenge agents, hydrogen peroxide and deoxynivalenol, on gut barrier integrity.

Effects of supplementing a Bacillus-based direct-fed microbial on performance, nutrient digestibility, rumen fermentation characteristics, and metabolic responses of lactating dairy cows.

This experiment was conducted to evaluate the effects of feeding a Bacillus-based direct-fed microbial (DFM) on performance, nutrient digestibility, rumen fermentation, and metabolic response of lactating dairy cows. Sixty-eight lactating (50 ± 6 d in milk) Holstein-Friesian (n = 20) and Swedish Red (n = 48) cows were enrolled to a 15-wk experiment. Cows were blocked by breed, lactation number, and days in milk and, within blocks, assigned to 1 of the 2 treatments: (1) basal partial mixed ration (PMR) without DFM (n = 34; CON) or (2) basal PMR with the addition of 3 g/head per day of a DFM containing Bacillus licheniformis and Bacillus subtilis (n = 34; DFM). The DFM was mixed in a protein pellet, whereas the CON group was fed the same pellet without DFM (1 kg/cow per day). The PMR contained 53% clover grass silage and 47% compound feed plus 3 kg of a concentrate (dry matter basis) offered during milking. Milk yield and production efficiency were recorded daily, whereas milk samples were collected for 24 h every second week of the study for milk composition. During the experimental period, fecal, rumen fluid, and blood samples were collected from each cow for apparent nutrient digestibility, rumen fermentation, and metabolic responses, respectively. All data were analyzed using the MIXED procedure of SAS. No treatment effects were observed on cows final body weight and daily dry matter intake. However, cows fed DFM had a greater milk yield, milk production efficiency, lactose and total solids yield, and also tended to have a greater energy-corrected milk production efficiency and milk protein yield. No significant differences were observed on nutrient digestibility and total volatile fatty acids, but molar proportion of acetate was greater for cows fed DFM. In contrast, molar proportion of propionate was greater and butyrate tended to be greater for CON. Cows fed DFM had greater mean plasma insulin-like growth factor-I (IGF-I), but no differences were observed for plasma glucose and insulin. In summary, supplementing a Bacillus-based DFM benefited productive responses of lactating dairy cows, while also modulating rumen fermentation and serum IGF-I.

Design and Synthesis of Novel 3-Nitro-1H-1,2,4-triazole-1,2,3-triazole-1,4-disubstituted Analogs as Promising Antitrypanosomatid Agents: Evaluation of In Vitro Activity against Chagas Disease and Leishmaniasis.

A series of 28 compounds, 3-nitro-1H-1,2,4-triazole, were synthesized by click-chemistry with diverse substitution patterns using medicinal chemistry approaches, such as bioisosterism, Craig-plot, and the Topliss set with excellent yields. Overall, the analogs demonstrated relevant in vitro antitrypanosomatid activity. Analog 15g (R1 = 4-OCF3-Ph, IC50 = 0.09 µM, SI = >555.5) exhibited an outstanding antichagasic activity (Trypanosoma cruzi, Tulahuen LacZ strain) 68-fold more active than benznidazole (BZN, IC50 = 6.15 µM, SI = >8.13) with relevant selectivity index, and suitable LipE = 5.31. 15g was considered an appropriate substrate for the type I nitro reductases (TcNTR I), contributing to a likely potential mechanism of action for antichagasic activity. Finally, 15g showed nonmutagenic potential against Salmonella typhimurium strains (TA98, TA100, and TA102). Therefore, 3-nitro-1H-1,2,4-triazole 15g is a promising antitrypanosomatid candidate for in vivo studies.

Effects of feeding an inoculated corn silage with or without a direct-fed microbial on dry matter intake, milk production, and nutrient digestibility of high-producing lactating Holstein cows.

This study evaluated the effects of inoculating corn silage and/or feeding a direct-fed microbial (PRO) on performance and nutrient digestibility of lactating dairy cows. At harvesting, corn silage was treated either with water (culated or not [CON]) or Lactococcus lactis and Lentilactobacillus buchneri (INC; SiloSolve FC) at 1.5 × 105 cfu/g of corn silage. Ten mini silos and one farm-scale silo bunker per treatment were prepared for the laboratory and the lactating dairy cow trial, respectively. Five mini silos per treatment were opened on days 2 or 90 post-ensiling for pH measurement, as well as chemical analysis and aerobic stability, respectively. The farm-scale silo bunkers were opened 77 d post-ensiling for the beginning of the lactating cow trial. Eighty lactating Holstein cows were assigned in a 2 × 2 factorial design to: (1) CON silage without PRO (CON-CON; n = 20), (2) CON silage with PRO at 14 g/head/d (CON-PRO; n = 20), (3) INC silage without PRO (INC-CON; n = 20), and (4) INC silage with PRO at 14 g/head/d (INC-PRO; n = 20). Concurrently with the feeding trial, eight cows per treatment were chosen for nutrient digestibility. The pH of the corn silage was not affected by the silage inoculant (P ≥ 0.29), but INC yielded greater concentration of acetic acid and longer aerobic stability (P < 0.01). Dairy cows fed INC had a lower mean total dry matter intake (DMI), milk protein content, and somatic cell counts vs. CON (P ≤ 0.02). On the other hand, milk and fat- and protein-corrected milk (FPCM) production efficiency, milk urea-N, DM, crude protein, and starch digestibility were greater for INC-fed cows (P ≤ 0.03). Feeding direct-fed microbials (DFM) improved mean body weight, milk yield, and FPCM, as well as milk protein and lactose yield (P ≤ 0.05), but reduced milk fat and protein content (P = 0.02). A silage inoculant × DFM interaction was observed for milk production efficiency, milk protein and lactose content, and somatic cell count (P ≤ 0.05). Dairy cows fed INC-CON had a greater milk production efficiency and milk lactose content (P ≤ 0.04), but INC-PRO had lower milk protein content and SCC (P ≤ 0.03). In summary, inoculating L. lactis and L. buchneri increased acetic acid content and aerobic stability of corn silage, reduced DMI, but improved milk production efficiency and nutrient digestibility of lactating Holstein dairy cows. On the other hand, feeding PRO improved milk, protein, and lactose yield. Additionally, combining the feeding of an inoculated corn silage with PRO reduced milk somatic cell count.

Impact of healthcare inequities on survival in Mexican patients with metastatic renal cell carcinoma.

Introduction: The survival of patients with metastatic renal cell carcinoma (mRCC) has improved dramatically due to novel systemic treatments. However, mRCC mortality continues to rise in Latin America. Methods: A retrospective, multicenter study of patients diagnosed with mRCC between 2010-2018 in Mexico City was conducted. The aim of the study was to evaluate the impact of healthcare insurance on access to treatment and survival in patients with mRCC. Results: Among 924 patients, 55.4%, 42.6%, and 1.9% had no insurance (NI), social security, (SS) and private insurance (PI), respectively. De novo metastatic disease was more common in NI patients (70.9%) compared to SS (47.2%) and PI (55.6%) patients (p<0.001). According to IMDC Prognostic Index, 20.2% were classified as favorable, 49% as intermediate, and 30.8% as poor-risk disease. Access to systemic treatment differed by healthcare insurance: 36.1%, 99.5%, and 100% for the NI, SS, and PI patients, respectively (p<0.001). NI patients received fewer lines of treatment, with 24.8% receiving only one line of treatment (p<0.001). Median overall survival (OS) was 13.9 months for NI, 98.9 months for SS, and 147.6 months for NI patients (p<0.001). In multivariate analysis, NI status, brain metastases, sarcomatoid features, bone metastases, no treatment were significantly associated with worse OS. Conclusion: OS in mRCC was affected by insurance availability in this resource-limited cohort of Mexican patients. These results underscore the need for effective strategies to achieve equitable healthcare access in an era of effective, yet costly systemic treatments.

Utilizing intramammary Melaleuca alternifolia as an organic internal sealant for dry-off therapy in Murrah buffaloes.

The effects of intramammary dry cow therapy based on the administration of 5% Melaleuca alternifolia tea tree essential oil (TTO) as an internal teat sealant to Murrah cows were evaluated. A longitudinal prospective and retrospective negative control study was performed using 12 buffaloes from a total of 20 Murrah buffaloes on an organic farm, with the cow used as a control for herself. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for treatments with pure oil (TTO) and medication containing 5% TTO (O5) were determined. The buffaloes were clinically examined, and the teats were evaluated using thermography and ultrasound. Udder health was monitored during the first 100 days in milk (DIM) using milk somatic cell count (SCC) and California mastitis test (CMT). Laboratory tests against standard strains Staphylococcus aureus ATCC®25,923™, Escherichia coli ATCC®25,922™, and wild bacterial strains showed maximum MIC values of 50 µL/mL for the TTO and O5 treatments. One wild-type S. aureus strain showed no MBC. No adverse effects were observed after the intramammary application of TTO. The CMT and SCC values were similar (P > 0.05) for all observations. The medication containing 5% TTO was effective in vitro and compatible with the intramammary tissue in vivo of Murrah buffaloes. TTO was safe, not inducing inflammatory processes or other modifications of the teat detectable by thermography or ultrasound. It was able to protect buffaloes during the dry period under field conditions, demonstrating potential use as a teat sealant for organic farms.

A proposed model to evaluate how changes in body condition score and the fatty acid profile of a supplement affect physiology and metabolic responses of nonlactating females.

Two experiments were designed to evaluate the effects of altering body condition score (BCS) and the profile of a fatty acid (FA) supplement on the metabolism of Bos indicus Nellore females. In experiment 1, 16 and 24 B. indicus heifers and nonlactating cows, respectively, were assigned to (1) maintenance diet (MNT-MNT; n = 10), (2) maintenance diet and BCS loss (MNT-LSS; n = 10), (3) maintenance diet supplemented with calcium salts of soybean oil for 30 d and BCS loss for 40 d (MNT+CFA-LSS; n = 10), and (4) maintenance diet for 30 d and BCS loss for 40 d with a diet containing calcium salts of soybean oil (MNT-LSS+CFA; n = 10). Following the BCS loss period, MNT-LSS, MNT+CFA-LSS, and MNT-LSS+CFA were fed a diet to promote the gain of BCS. In experiment 2, 40 Bos indicus nulliparous heifers were assigned to (1) maintenance diet (MNT-MNT; n = 10), (2) BCS loss followed by a BCS gain (LSS-REM; n = 10), (3) BCS loss followed by a BCS gain diet with CFA of palm oil (LSS-REM+PLM; n = 10), and (4) BCS loss followed by a BCS gain diet with CFA of soybean oil (LSS-REM+SOY; n = 10). Blood samples were obtained for serum haptoglobin and fecal samples for pH (experiment 2 only). In experiment 1, a treatment × day interaction was observed for BCS during the 60-d BCS loss and gain period. Animals assigned to MNT-MNT had a greater BCS than the other treatment groups on d 40 and 60 of the experiment, but no other differences were observed. Moreover, a treatment × day interaction was observed for serum haptoglobin, as on d 60, MNT-LSS had a greater mean serum haptoglobin concentration. In experiment 2, a treatment × day interaction was also observed for BCS. From d -4 to 0, LSS-REM and LSS-REM+SOY had a reduced BCS versus MNT-MNT, but also lower for LSS-REM versus MNT-MNT on d 1, and LSS-REM+PLM versus MNT-MNT on d -1 and 0. For serum haptoglobin, no treatment or treatment × day interaction was observed. A treatment × day interaction was observed for fecal pH. From d -10 to 0, MNT often had a lower fecal pH, but during realimentation, LSS-REM heifers had a reduced fecal pH on d 1, 4, and 10. In summary, we failed to demonstrate an increase in serum haptoglobin due to a BCS loss. Still, supplementation with calcium salts of FA alleviated the increase in haptoglobin and maintained fecal pH at more stable values during realimentation, regardless of the FA profile of the supplement.

Design, synthesis and antitrypanosomatid activity of 2-nitroimidazole-3,5-disubstituted isoxazole compounds based on benznidazole.

Chagas disease and leishmaniasis are neglected diseases of high priority as a public health problem. Pharmacotherapy is based on the administration of a few drugs, which exhibit hazardous adverse effects and toxicity to the patients. Thus, the search for new antitrypanosomatid drugs is imperative to overcome the limitations of the treatments. In this work, 46 2-nitroimidazole 3,5-disubstituted isoxazole compounds were synthesized in good yields by [3 + 2] cycloaddition reaction between terminal acetylene (propargyl-2-nitroimidazole) and chloro-oximes. The compounds were non-toxic to LLC-MK2 cells. Compounds 30, 35, and 44 showed in vitro antichagasic activity, 15-fold, 12-fold, and 10-fold, respectively, more active than benznidazole (BZN). Compounds 30, 35, 44, 45, 53, and 61 acted as substrates for the TcNTR enzyme, indicating that this might be one of the mechanisms of action involved in their antiparasitic activity. Piperazine series and 4-monosubstituted compounds were potent against T. cruzi parasites. Besides the in vitro activity observed in compound 45, the in vivo assay showed that the compound only reduced the parasitemia levels by the seventh-day post-infection (77%, p > 0.001) compared to the control group. However, 45 significantly reduced the parasite load in cardiac tissue (p < 0.01) 11 days post-infection. Compounds 49, 52, and 54 showed antileishmanial activity against intracellular amastigotes of Leishmania (L.) amazonensis at the same range as amphotericin B. These findings highlight the antitrypanosomatid properties of 2-nitroimidazole 3,5-disubstituted isoxazole compounds and the possibility in using them as antitrypanosomatid agents in further studies.

Effects of supplementing a direct-fed microbial containing Enterococcus faecium 669 on performance, health, and metabolic responses of preweaning Holstein dairy calves.

This study aimed to evaluate the effects of Enterococcus faecium 669 supplementation on performance, health, parasitological, microbiological, and hematological responses of preweaning dairy calves. Forty-two newborn Holstein female calves (initial body weight [BW] 44 ± 4.5 kg) were used in the present study. At birth, calves were ranked by initial BW and assigned to 1 of 2 treatment groups: (1) whole milk (CON; n = 21) and (2) whole milk with the addition of direct-fed microbial Ent. faecium 669 (DFM; n = 21). During the entire experimental period (63 d), DFM was daily-fed at a rate of 2.5 × 109 cfu/head. All calves were offered a mixture of a starter feed and wheat straw for ad libitum consumption. Supplement intake was evaluated daily, whereas calves were weighed on a weekly basis from d 0 to weaning (d 63). Diarrhea was assessed once a day, and fecal and blood samples were collected for microbiological, parasitological, and hematological responses. All data were analyzed with SAS using calf as the experimental unit. A treatment × week interaction was observed for BW, as DFM-supplemented calves were heavier than CON cohorts on d 56 (+ 4.7 kg) and at weaning on d 63 (+ 4.8 kg). A similar interaction was observed for average daily gain (ADG) and dry matter intake (DMI), with greater ADG for DFM-supplemented calves from d 35 to 42, greater ADG and DMI from d 49 to 56, and greater DMI from d 56 to weaning. Moreover, diarrhea occurrence tended to be lower, whereas rectal temperature was 0.2°C lower for DFM-supplemented calves. Treatment × day interactions were observed for the occurrence and counts of Eimeria spp., as DFM-supplemented calves tended to have a reduced number of positive observations on d 42 of the study versus CON, and a significant reduction in positive animals from d 21 to 42 was observed in the DFM group but not in CON calves. For Cryptosporidium spp., no treatment effects were observed on overall occurrence (%), but DFM-supplemented calves had a greater count of oocyst per gram versus CON. No treatment × day interaction or main treatment effects were observed for any of the blood variables analyzed herein, exception being monocytes concentration. In summary, preweaning Ent. faecium 669 supplementation improved performance, diarrhea occurrence, and reduced the number of calves positively-detected for Eimeria spp.

Stability of Bacillus and Enterococcus faecium 669 Probiotic Strains When Added to Different Feed Matrices Used in Dairy Production.

Few data are available evaluating the stability of direct-fed microbials (DFM) following their inclusion in different feed matrices. Therefore, six Exp. evaluated the recovery of bacilli spores (BOVACILLUSTM; Exp. 1 to 3) and an Enterococcus faecium DFM (LACTIFERM®; Exp. 4 to 6) when included in different feed preparations. The Bacillus-based DFM was included into pelleted feed prepared in different temperatures (75 to 95 °C), whereas both DFM were assessed in premix and milk replacer preparations. Bacillus spores and E. faecium recovery was evaluated through standard methodologies and data were reported as log10 colony forming units/gram of feed. The recovery of Bacillus spores was within the expected range and was not impacted by the temperature of pellet preparation (Exp. 1). Bacilli recovery was also stable up to 12 months in the premix and was not impacted by the temperature of milk replacer preparation. Regarding the Exp. with E. faecium (Exp. 4 to 6), its recoveries in the mineral premix and milk powder did not differ from T0 and were not impacted by the conditions of milk replacer preparation. These data are novel and demonstrate the stability of a Bacillus-based and an E. faecium-based DFM when included in different feed matrices often used in dairy production.

Supplementation of direct-fed microbial Enterococcus faecium 669 affects performance of preweaning dairy calves.

Optimization and support of health and performance of preweaning dairy calves is paramount to any dairy operation, and natural solutions, such as probiotics, may help to achieve such a goal. Two experiments were designed to evaluate the effects of direct-fed microbial (DFM) Enterococcus faecium 669 on performance of preweaning dairy calves. In experiment 1, twenty 4-d-old Holstein calves [initial body weight (BW) 41 ± 2.1 kg] were randomly assigned to either (1) no probiotic supplementation (CON; n = 10) or (2) supplementation with probiotic strain E. faecium 669 during the preweaning period (DFM; n = 10) at 2.0 × 1010 cfu/kg of whole milk. Full individual BW was analyzed every 20 d for average daily gain (ADG) and feed efficiency (FE) determination. In experiment 2, thirty 4-d-old Holstein calves (initial BW 40 ± 1.9 kg) were assigned to the same treatments as in experiment 1 (CON and DFM). The DFM supplementation period was divided into period I (from d 0 to 21) and II (from d 22 to 63), with weaning occurring when animals were 67 d of age. During the entire experimental period, DFM was mixed into the whole milk at a rate of 1.5 × 1010 and 2.5 × 109 cfu/kg of whole milk/calf per day for periods I and II, respectively (6-time reduction). Full individual BW was taken every 21 d. As a routine of the experiment, calves were monitored daily, and diarrhea cases were evaluated using a daily 3-point fecal score. For both experiments, all data were analyzed using calf as the experimental unit. In experiment 1, DFM-supplemented calves were heavier on d 40 (+ 4.5 kg) and 60 (+ 6.5 kg) and had a greater ADG (+ 118 g) versus CON. In experiment 2, supplementation with DFM significantly tended to reduce diarrhea occurrence. Treatment × day and treatment × week interactions were observed for BW, ADG, and gain-to-feed ratio. Dairy calves supplemented with DFM were 1.8 and 3.5 kg heavier on d 42 and at weaning, respectively, and had a greater ADG from d 21 to 42 (+ 52 g) and 42 to 63 (+ 77 g) and gain-to-feed ratio from d 42 to 63 (+ 8.6%). In summary, supplementation of E. faecium 669 to dairy calves improved preweaning performance, even when the dose of the DFM was reduced by 6- to 8-times. Additionally, initial promising results were observed on diarrhea occurrence, but further studies are warranted.

Evaluation of a Bacillus-based direct-fed microbial probiotic on in vitro rumen gas production and nutrient digestibility of different feedstuffs and total mixed rations.

We evaluated the effects of a Bacillus-based direct-fed microbial (DFM) on total in vitro gas production, dry matter (DM), neutral detergent fiber (NDF), and starch disappearance of different feedstuffs and total mixed rations (TMR) in three different experiments. In experiment 1, six single fiber-based feedstuffs were evaluated: alfalfa hay, buffalo grass, beet pulp, eragrostis hay, oat hay, and smutsvinger grass. Experimental treatments were control (with no probiotic inoculation; CON) or incubation of a probiotic mixture containing Bacillus licheniformis and B. subtilis (3.2 × 109 CFU/g; DFM). The calculation of DFM dose under in vitro conditions was based on the assumption of a rumen capacity of 70 liter and the dose of 3 g of the DFM mixture/head/d (9.6 × 109 CFU). Total in vitro gas production, DM, and NDF disappearance were evaluated at 24- and 48 h posttreatment incubation. Mean treatment effects were observed at 24- and 48 h gas production (P < 0.0001), as DFM incubation increased in vitro gas production by 5.0% and 6.5%, respectively. For nutrient digestibility, mean DM digestibility was increased at 48 h (P = 0.05), whereas mean NDF digestibility increased at both timepoints by incubating DFM in vitro (P ≤ 0.02). In experiment 2, nine commercial dairy TMR were collected and evaluated for the same variables and treatments described in experiment 1, with the additional analysis of starch digestibility at 7 h post in vitro incubation. The only difference was the concentration of the DFM included, being representative for a dosage of 8.8 × 109 CFU/head/d. In vitro gas production was increased only at 48 h due to DFM incubation (P = 0.05), whereas DM and NDF digestibility were improved at 24 and 48 h (P ≤ 0.02). No treatment effects were observed on in vitro starch digestibility (P = 0.31). In experiment 3, a combined analysis of DM and NDF digestibility was performed by using quality values (NDF and crude protein or CP) of 16 substrates. Regardless of CP and NDF levels of the substrates, DFM improved in vitro 24 and 48 h DM and NDF digestibility (P ≤ 0.03). In summary, incubating a Bacillus-based DFM (B. licheniformis and B. subtilis; BOVACILLUS) improved mean in vitro gas production, DM, and NDF digestibility of single feedstuffs and commercial dairy TMR, highlighting the potential of this combination of Bacillus spp. to improve nutrient utilization, mainly fiber.

In Vivo Antileishmanial Effect of 3,5-Diaryl-isoxazole Analogues Based on Veraguensin, Grandisin, and Machilin G: A Glance at a Preclinical Study.

New treatment approaches targeting cutaneous leishmaniasis (CL) are required since conventional drugs exhibit limitations due to their several adverse effects and toxicity. In this study, we aimed to evaluate the in vivo intralesional treatment efficacy of five isoxazole derivatives previously synthesized and effective in vitro against intracellular amastigote forms of Leishmania (L.) amazonensis. Among the tested analogues, 7 exhibited relevant in vivo therapeutic effects. The in silico predictions provided interesting information about the toxicity, suggesting the safety of analogue 7. Experiments performed with Salmonella typhimurium strains (TA98, TA100, and TA102) showed a non-mutagenicity profile of 7. The treatment of Leishmania-infected BALB/c mice with isoxazole 7 showed remarkably smaller CL lesions and decreased the parasitism (by 98.4%) compared to the control group. Hence, analogue 7 is a promising drug candidate and alternative treatment for CL caused by L. amazonensis.

Productive and physiological responses of feedlot cattle receiving different sources of Ca salts of fatty acids in the finishing diet.

This study evaluated productive and physiological responses in feedlot cattle receiving a finishing diet that included Ca salts of palm oil (CSPALM), or a blend of Ca salts of palm, cottonseed, and soybean oils (CSMIX). Ninety yearling steers were housed in 15 pens equipped with Calan-gate feeders (6 steers/pen). Steers within each pen were stratified by shrunk body weight (BW; 410 ±â€…3.3 kg across pens) on d 0 and assigned to receive a total-mixed ration (TMR) containing (dry matter basis) 2.2% of CSPALM (n = 30), 2.2% of CSMIX (n = 30), or no supplemental fat (CON; n = 30). Individual TMR intake was evaluated weekly. Blood samples were collected on d 0, 28, 56, 91, 119, and 147. Samples of the Longissimus muscle (LM) were collected on d 84 via biopsy. Upon slaughter on d 148, hot carcass weight (HCW) was recorded to estimate final BW (63% dressing), and one LM steak sample (2.54 cm thickness) was removed from the right side of each carcass. Steer ADG was greater (P = 0.02) for CSMIX compared with CSPALM and tended to be greater (P = 0.09) for CSMIX compared with CON. The gain:feed ratio was greater (P ≤ 0.05) for CSMIX compared with CSPALM and CON, and carcass LM area was less (P = 0.01) for CSPALM compared with CSMIX and CON. No treatment effects were detected (P ≥ 0.21) for TMR intake, final BW, and other carcass merit traits including marbling. Mean plasma cholesterol concentrations were greater (P < 0.01) in CSMIX and CSPALM compared with CON, and mRNA expression of adipocyte fatty acid binding protein in the LM on d 84 was greater (P ≤ 0.04) in CSPALM compared with CSMIX and CON. No treatment effects were detected (P ≥ 0.15) for plasma concentrations of glucose, insulin, insulin-like growth factor I, and leptin, nor for other LM genes associated with marbling and muscle growth. Concentrations of total fatty acids (FA) in plasma and LM steak samples were greater (P < 0.01) in CSMIX compared with CSPALM and CON, and greater (P < 0.01) in the LM samples of CSPALM compared with CON. Steers receiving CSMIX had greater (P < 0.01) concentrations of polyunsaturated and ω-6 FA in plasma and LM steak samples compared with CSPALM and CON. Supplementing CSMIX improved gain efficiency and FA profile in the LM of feedlot steers compared with the CON diet, but the same responses were not observed when CSPALM was offered. Perhaps the advantages from CSMIX supplementation resulted from increasing the supply of polyunsaturated and ω-6 FA to the finishing diet.

Supplemental fat has been provided to feedlot cattle to increase energy density of their diets, and may yield nutraceutical advantages if includes polyunsaturated fatty acids (FA). Alternatively, carcass quality can be improved when the fat supplement is based on saturated and monounsaturated FA. Hence, this experiment evaluated a blend of saturated, monounsaturated, and polyunsaturated FA to improve both performance and carcass merit in feedlot cattle. Steers received a finishing diet that included this blend (CSMIX), a source of saturated and monounsaturated FA (CSPALM), or no supplemental fat (CON). Growth rate and gain efficiency were improved in steers that received CSMIX compared with CSPALM and CON, and these traits did not differ between the latter treatments. Inclusion of CSMIX increased FA concentrations in the circulation of steers throughout the 147-day study and in Longissimus muscle (LM) samples collected after slaughter. This increase in FA concentrations was associated with greater accumulation of polyunsaturated and ω-6 FA, suggesting that CSMIX resulted in LM with FA profile deemed more beneficial for human consumption. Collectively, supplementing CSMIX to feedlot steers improved gain efficiency and FA composition in the LM, and these advantages may be associated with increased supply of polyunsaturated ω-6 FA to the finishing diet.

Effects of feeding different probiotic types on metabolic, performance, and carcass responses of Bos indicus feedlot cattle offered a high-concentrate diet.

Two experiments were designed to evaluate the effects of different probiotic combinations on rumen fermentation characteristics, performance, and carcass characteristics of feedlot Bos indicus beef bulls offered a high-concentrate diet. In experiment 1, 30 rumen-fistulated Nellore steers were blocked by initial body weight (BW = 350 ± 35.0 kg) and within blocks (n = 10), animals were randomly assigned to receive: 1) high-concentrate diet without probiotic supplementation (n = 10; CONT), 2) CONT plus 1 g per head of a probiotic mixture containing three strains of Enterococcus faecium and one strain of Saccharomyces cerevisiae (3.5 × 109 CFU/g; n = 10; EFSC), and 3) CONT plus 2 g per head of a probiotic mixture containing Bacillus licheniformis and Bacillus subtilis (3.2 × 109 CFU/g; n = 10; BLBS). The experimental period lasted 35 d, being 28 d of adaptation and 7 d of sampling. From day 34 to day 35 of the experimental period, ruminal fluid and fecal samples were collected every 3 h, starting immediately before feeding (0 h) for rumen fermentation characteristics and apparent nutrient digestibility analysis, respectively. In experiment 2, 240 Nellore bulls were ranked by initial shrunk BW (375 ± 35.1 kg), assigned to pens (n = 4 bulls per pen), and pens randomly assigned to receive the same treatments as in experiment 1 (n = 20 pens per treatment). Regardless of treatment, all bulls received the same step-up and finishing diets throughout the experimental period, which lasted 115 d. In both experiments, data were analyzed as orthogonal contrasts to partition-specific treatment effects: 1) probiotic effect: CONT vs. PROB and 2) probiotic type: EFSC vs. BLBS (SAS Software Inc.). In experiment 1, no contrast effects were observed on nutrient intake, overall nutrient digestibility, and rumen fermentation analyses (P ≥ 0.13). Nonetheless, supplementation of probiotics, regardless of type (P = 0.59), reduced mean acetate:propionate ratio and rumen ammonia-N concentration vs. CONT (P ≤ 0.05). In experiment 2, no significant effects were observed for final BW and dry matter intake (P ≥ 0.12), but average daily gain and feed efficiency tended to improve (P ≤ 0.10) when probiotics were offered to the animals. Probiotic supplementation or type of probiotic did not affect carcass traits (P ≥ 0.22). In summary, supplementation of probiotics containing a mixture of E. faecium and S. cerevisiae or a mixture of B. licheniformis and B. subtilis reduced rumen acetate:propionate ratio and rumen ammonia-N levels and tended to improve the performance of feedlot cattle offered a high-concentrate diet.

Two experiments were designed to evaluate the effects of different probiotic combinations on rumen fermentation characteristics, performance, and carcass characteristics of feedlot Bos indicus beef bulls offered a high-concentrate diet. The two probiotics consisted of a mixture containing three strains of Enterococcus faecium and one strain of Saccharomyces cerevisiae or a mixture of Bacillus licheniformis and Bacillus subtilis. Supplementation of probiotics, regardless of type, reduced acetate:propionate ratio, and mean rumen ammonia-N concentration and tended to improve the performance of feedlot cattle offered a high-concentrate diet, demonstrating the potential of this technology to be used as a feed additive for beef cattle.