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Twelve 5-year-old beef steers, with an average weight of 2000 lbs., fitted with rumen canulae were used in a 4 × 4 incomplete Latin square design to examine the impact of the direct fed microbial Propionibacterium acidipropionici CP 88 (PA) on rumen fermentation characteristics, in vitro CH4, CO2, and N2 production, and in vivo CH4 and CO2 production. All steers were housed in the same pen equipped with eight GrowSafe feeding stations to monitor individual animal feed intake and one GreenFeed System to estimate individual animal CH4 and CO2 production. Steers were fed a corn-silage-based diet throughout the experiment. Treatments consisted of PA administered at: (1) control (0.0); (2) 1.0 × 108; (3) 1.0 × 109; and (4) 1.0 × 1010 cfu∙animal−1∙day (d)−1. Treatments were administered directly into the rumen as a single bolus dose daily. On day 7 and 14 of each period, rumen fluid was collected from each steer 2 h post treatment administration for VFA analysis and in vitro DM digestibility determination. Following a 14 d washout period, animal treatments were switched and the experiment repeated until the 4 × 4 Latin square was complete. In vivo propionic acid molar proportions and total VFA concentrations were greater (p < 0.05) in steers receiving PA when compared with controls. All other in vivo rumen fermentation characteristics were similar across treatments. In vitro DM disappearance (p < 0.05) and total VFA (p < 0.05) were greater and CH4 lesser (p < 0.04) in fermentation vessels incubated with rumen fluid from animals receiving PA when compared with controls. Dry matter disappearance (p < 0.03) and propionic acid molar proportions increased (p < 0.04) linearly as the dose of PA increased. In vitro total VFA tended (p < 0.08) to increase linearly and CH4 production per unit of DM digested tended (p < 0.09) to decrease quadratically in response to PA dose. All other in vitro rumen fermentation characteristics were similar across treatments. These data indicate that PA impacts in vivo and in vitro rumen fermentation characteristics.

Clusters of the 37 genes involved in the cell cycle and differentially expressed in malignant hepatocytes derived from HDV-HCC.

Davide De Battista,1 Fausto Zamboni,2 Hannah Gerstein,1 Shinya Sato,1 Tovah E Markowitz,3,4 Justin Lack,3,4 Ronald E Engle,1 Patrizia Farci1 1Hepatic Pathogenesis Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA; 2Liver Transplantation Center, Azienda Ospedaliera Brotzu, Cagliari, Italy; 3NIAID Collaborative Bioinformatics Resource, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA; 4Advanced Biomedical Computational Science, Frederick National Laboratory for Cancer Research Sponsored by the National Cancer Institute, Frederick, MD, 21702, USACorrespondence: Patrizia FarciHepatic Pathogenesis Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USATel +1 240-669-5921Fax +1 301-402-0524Email pfarci@niaid.nih.govIntroduction: HCC is the third leading cause of cancer-related death worldwide, with chronic viral hepatitis accounting for more than 70% of the cases. Therapeutic options are limited and ineffective. The increasing use of immune-based therapies in solid tumors highlights the need to expand our knowledge on the immunologic microenvironment of HCC.Methods: Access to liver samples from 20 well-characterized patients with HCC associated with HCV (n = 9) or HBV (n = 11) gave us the opportunity to study the immunologic landscape in these tumors. For each patient, RNA-sequencing was performed on the tumor and surrounding nontumorous tissue.Results: We found that both HCV- and HBV-HCC are associated with a predominance of downregulated genes (74% and 67%, respectively). Analysis of the immune landscape using a curated gene list showed 216 of 2481 (9%) immune genes in HCV-HCC and 164 of 2560 (6%) in HBV-HCC. However, only 8 immune genes (4%) were upregulated in HCV-HCC and 27 (16.5%) in HBV-HCC. HCV-HCC was characterized by an enrichment of downregulated genes related to T-cell activation and oxidative stress. The dramatic downregulation of immune genes related to T-cell activation in HCV-HCC prompted us to perform an extensive immunohistochemistry analysis on paraffin-embedded liver specimen. Interestingly, we found a significant reduction of immune-cell infiltration (CD3, CD8 and CD20 positive cells) within the tumor. Moreover, we observed that HCV-HCC is characterized by an enrichment of M2-like CD68-positive cells. These data are consistent with the dramatic downregulation of immune-cell infiltration seen in HCV-HCC. Conversely, HBV-HCC was characterized by upregulation of genes related to monocyte/macrophage activation and cell cycle control, and downregulation of genes involved in various cell metabolisms.Conclusion: This study demonstrates a distinctive molecular signature and immune landscape in HCC of different viral etiology, which could provide new insights into pathogenesis and lead to the development of novel immune-based therapies.Keywords: hepatocellular carcinoma, hepatitis C virus, hepatitis B virus, RNA-sequencing, molecular signature, immune landscape

Primary cilia are small immotile cellular appendages which mediate diverse types of singling and are found on most mammalian cell types including throughout the central nervous system. Cilia are known to localize certain G protein-coupled receptors (GPCRs) and are critical for mediating the signaling of these receptors. Several of these neuronal GPCRs have recognized roles in feeding behavior and energy homeostasis. Heterologous cell line and model systems likeC. elegansandChlamydomonashave implicated both dynamic GPCR cilia localization and cilia length and shape changes as key for signaling. However, it is unclear if mammalian ciliary GPCRs utilize similar mechanismsin vivoand under what physiological conditions these processes may occur. Here, we use the ciliary GPCRs, melanin concentrating hormone receptor 1 (MCHR1) and neuropeptide-Y receptor 2 (NPY2R) as model ciliary receptors to determine if dynamic localization to cilia occurs. We tested physiological conditions in which these GPCRs have been implicated such as feeding behavior, obesity, and circadian rhythm. Cilia were imaged using confocal microscopy and analyzed with a computer assisted approach allowing for unbiased and high throughput analysis of cilia. We analyzed GPCR positive cilia, cilia frequency as well as cilia length and receptor occupancy. Interestingly we observed changes in ciliary length, receptor occupancy, and cilia frequency under different conditions, but no consistent theme across GPCRs or brain nuclei was observed. A better understanding of the subcellular localization dynamics of ciliary GPCRs could reveal unrecognized molecular mechanisms regulating behaviors like feeding.Significance StatementOften, primary cilia localize specific G protein-coupled receptors (GPCRs) for subcellular signaling. Cell lines and model systems have indicated that cilia deploy dynamic GPCR localization and change their shape or length to modulate signaling. We used mice to assess neuronal cilia GPCRs under physiological conditions associated with both the receptors’ known functions and ciliopathy clinical features like obesity. We show that certain cilia with specific GPCRs appear to dynamically alter their length while others appear relatively stable under these conditions. These results implicate multiple themes across cilia GPCR mediated signaling and indicate that not all cilia modulate GPCR signaling using the same mechanisms. These data will be important for potential pharmacological approaches to target cilia GPCR-mediated signaling.

Fifty-four multiparous beef cows with calves were used to evaluate the effects of Mo source (feed or water) on reproduction, mineral status, and performance over two cow-calf production cycles (553 d). Cows were stratified by age, body weight, liver Cu and Mo status, and were then randomly assigned to one of six treatment groups. Treatments were: 1) Negative control (NC; basal diet with no supplemental Mo or Cu); 2) Positive control (NC + Cu; 3 mg of supplemental Cu/kg DM); 3) NC + 500 µg Mo/L from Na2MoO4·2H2O supplied in drinking water; 4) NC + 1000 µg Mo/L of Na2MoO4·2H2O supplied in drinking water; 5) NC + Mo 1000-water + 3 mg of supplemental Cu/kg DM; and 6) NC + 3.0 mg of supplemental Mo/kg diet DM from Na2MoO4·2H2O. Animals were allowed ad libitum access to both harvested grass hay (DM basis: 6.6% crude protein; 0.15% S, 6.7 mg Cu/kg, 2.4 mg Mo/kg) and water throughout the experiment. Calves were weaned at approximately 6 months of age each year. Dietary Cu concentration below 10.0 mg Cu/kg DM total diet reduced liver and plasma Cu concentrations to values indicative of a marginal Cu deficiency in beef cows. However, no production parameters measured in this experiment were affected by treatment. Results suggest that Mo supplemented in water or feed at the concentrations used in this experiment had minimal impact on Cu status and overall performance.

Fifty-four multiparous beef cows with calves were used to evaluate the effects of Mo source (feed or water) on reproduction, mineral status, and performance over two cow-calf production cycles (553 days). Cows were stratified by age, body weight, liver Cu, and Mo status and were then randomly assigned to one of six treatment groups. Treatments were (1) negative control (NC; basal diet with no supplemental Mo or Cu), (2) positive control (NC + Cu; 3 mg of supplemental Cu/kg DM), (3) NC + 500 µg Mo/L from Na2MoO4·2H2O supplied in drinking water, (4) NC + 1000 µg Mo/L of Na2MoO4·2H2O supplied in drinking water, (5) NC + Mo 1000-water + 3 mg of supplemental Cu/kg DM, and (6) NC + 3.0 mg of supplemental Mo/kg diet DM from Na2MoO4·2H2O. Animals were allowed ad libitum access to both harvested grass hay (DM basis: 6.6% crude protein; 0.15% S, 6.7 mg Cu/kg, 2.4 mg Mo/kg) and water throughout the experiment. Calves were weaned at approximately 6 months of age each year. Dietary Cu concentration below 10.0 mg Cu/kg DM total diet reduced liver and plasma Cu concentrations to values indicative of a marginal Cu deficiency in beef cows. However, no production parameters measured in this experiment were affected by treatment. Results suggest that Mo supplemented in water or feed at the concentrations used in this experiment had minimal impact on Cu status and overall performance.

Objective The objectives of these experiments were to evaluate (1) if inclusion of inedible tallow containing ractopamine (RAC) in the cattle diet influences feedlot performance, carcass characteristics, and results in detectable RAC in tissues and (2) the effect of withdrawal time on RAC residues in the gastrointestinal-tract digesta of cattle. Materials and Methods In Exp. 1, beef steers (n = 28) were assigned to dietary treatments formulated with feed tallow ranging from 100% inedible tallow to 100% technical tallow. After 91 d, steers were transported to a commercial abattoir and slaughtered, and tissues were collected for RAC analysis. In Exp. 2, steers fitted with ruminal and duodenal cannulas were assigned to the control group (no RAC) or a group in which RAC was supplemented at 400 mg of RAC∙animal−1∙d−1 for 28 d. On d 29, RAC supplementation was withdrawn. Daily ruminal, duodenal, and fecal samples were obtained for RAC analysis beginning 3 d before RAC withdrawal and continuing to d 13 after withdrawal. Results and Discussion No treatment effects of dietary inedible tallow inclusion was observed for final BW (P Implications and Applications The results indicate that RAC residue in feed tallow does not affect animal performance or carcass characteristics. Detectable RAC in sampled tissues and gastrointestinal digesta suggests a need for further investigation of RAC residues to maintain compliance for exported beef products.

The objectives of these experiments were to investigate (1) the relative abundance of transcripts for Cu-responsive genes in whole bovine liver vs. cultured hepatocytes and (2) the influence of Cu dose on the relative abundance of transcripts for Cu-responsive genes in cultured bovine hepatocytes. Experiment 1: Liver samples were obtained immediately post-mortem from one healthy Angus steer. Half of the tissue samples were placed in RNAlater solution; the remaining half was used to isolate hepatocytes. Experiment 2: A subset of cultured hepatocytes was incubated in media containing: 0 mg/L, 0.10 mg/L, 1.0 mg/L, 10.0 mg/L, and 100 mg/L Cu for 1 h. Transcripts analyzed were aldehyde dehydrogenase (ALDH2), apolipoprotein A-1 (APOA1), antioxidant 1 (ATOX1), ATPase copper transporting alpha (ATP7A), ATPase copper transporting beta (ATP7B), betaine homocysteine methyltransferase (BHMT), flavin reductase (BLVRB), carbonic anhydrase II (CA2), copper chaperone for superoxide dismutase (CCS), cytochrome c oxidase copper chaperone (COX17), Cu transporter 1 (CTR1), glutamate dehydrogenase (GLUD1), glutathione synthetase (GSS), protein disulfide isomerase A3 (PDIA3), and superoxide dismutase (Cu–Zn) (SOD1). Β-Actin (ACTB) was selected as the endogenous control in both experiments. Experiment 1: Whole liver had greater (P

The objective of this study was to characterize flavor, fatty acid composition, and volatile compounds of beef treated with common antimicrobial interventions in beef processing facilities. The effect of 3 prechilling antimicrobial interventions (4.5% lactic acid [LA]; 400 ppm peroxyacetic acid acidified to pH 1.2 with a sulfuric acid and sodium sulfate blend [aPAA]; or untreated control [CON]) and 4 postchilling treatments (CON; LA; aPAA; or a 2.5% solution of a commercial blend of lactic and citric acid [LAC]) were analyzed. Briskets (n = 30/treatment) were treated before and after chilling using a custom-built pilot-sized spray cabinet, ground twice, and formed into patties. Cooked patties were analyzed by a trained sensory panel, and a subset of raw samples (n = 6) were analyzed for fatty acid composition and volatile compounds. Samples treated with LA before and after chilling were more intense in sourness than the CON (P < 0.05). Fatty acid analysis showed no differences (P > 0.05) due to the use of chemical interventions. Only postchilling treatments had an effect on volatile compounds. The relative abundances of pentanal and pentanol were greater (P < 0.05) in LA-treated postchilling intervention samples than CON and LAC, hexanoic acid was greater (P < 0.05) in aPAA than CON and LAC, and acetic acid was greater (P < 0.05) in aPAA than LAC. Overall, these results demonstrated that LA pre- and postchilling antimicrobial interventions only impact the sourness of ground beef but did not affect the fatty acid composition, while postchilling antimicrobial treatments had a minimal impact on volatile compounds.

An emerging number of rare genetic disorders termed ciliopathies are associated with pediatric obesity. It is becoming clear that the mechanisms associated with cilia dysfunction and obesity in these syndromes are complex. In addition to ciliopathic syndromic forms of obesity, several cilia-associated signaling gene mutations also lead to morbid obesity. While cilia have critical and diverse functions in energy homeostasis including their roles in centrally mediated food intake as well as in peripheral tissues, many questions remain. Here, we briefly discuss the syndromic ciliopathies and monoallelic cilia signaling gene mutations associated with obesity. We also describe potential ways cilia may be involved in common obesity. We discuss how neuronal cilia impact food intake potentially through leptin signaling and changes in ciliary G protein-coupled receptor (GPCR) signaling. We highlight several recent studies that have implicated the potential for cilia in peripheral tissues such as adipose and the pancreas to contribute to metabolic dysfunction. Then we discuss the potential for cilia to impact energy homeostasis through their roles in both development and adult tissue homeostasis. The studies discussed in this review highlight how a comprehensive understanding of the requirement of cilia for the regulation of diverse biological functions will contribute to our understanding of common forms of obesity.

Primary cilia are critical sensory and signaling compartments present on most mammalian cell types. These specialized structures require a unique signaling protein composition relative to the rest of the cell to carry out their functions. Defects in ciliary structure and signaling result in a broad group of disorders collectively known as ciliopathies. One ciliopathy, Bardet–Biedl syndrome (BBS; OMIM 209900), presents with diverse clinical features, many of which are attributed to defects in ciliary signaling during both embryonic development and postnatal life. For example, patients exhibit obesity, polydactyly, hypogonadism, developmental delay and skeletal abnormalities along with sensory and cognitive deficits, but for many of these phenotypes it is uncertain, which are developmental in origin. A subset of BBS proteins assembles into the core BBSome complex, which is responsible for mediating transport of membrane proteins into and out of the cilium, establishing it as a sensory and signaling hub. Here, we describe two new mouse models for BBS resulting from a targeted LacZ gene trap allele (Bbs5−/−) that is a predicted congenital null mutation and conditional (Bbs5flox/flox) allele of Bbs5. Bbs5−/− mice develop a complex phenotype consisting of increased pre-weaning lethality craniofacial and skeletal defects, ventriculomegaly, infertility and pituitary anomalies. Utilizing the conditional allele, we show that the male fertility defects, ventriculomegaly and pituitary abnormalities are only present when Bbs5 is disrupted prior to postnatal day 7, indicating a developmental origin. In contrast, mutation of Bbs5 results in obesity, independent of the age of Bbs5 loss.

Objective This experiment evaluated the effects of Cu, Mn, and Zn source on mineral status, reproduction, and performance of grazing beef cattle in eastern Colorado. Materials and Methods Crossbred (Angus and Angus × Hereford; n = 261) 3-yr-old beef cows were stratified by expected calving date, BW, BCS, and liver Cu status and randomly assigned to 1 of 6 replicates. Replicates were then assigned to 1 of 2 treatments (n = 40–45 cows per replicate), resulting in 3 replicates per treatment for the 2-yr experiment. Treatments consisted of (1) inorganic–organic trace mineral combination (IOC; 75% of Cu, Mn, and Zn from sulfate forms and the remaining 25% from organic AA complexes) and (2) hydroxy trace minerals (HTM; 100% from hydroxychloride forms). Replicates were rotated among pastures approximately every 28 d to minimize pasture effects. Free-choice mineral feeders were used to provide NASEM (2000) recommended concentrations of Cu, Mn, and Zn continuously for 2 yr. Blood samples and liver biopsies were obtained from every cow before the initiation of the experiment and then from a subgroup of animals (20 random animals per replicate) at the end of yr 1 (d 335 of the experiment) and 2 (d 638 of the experiment). Results and Discussion Over the 2-yr experiment, mineral intake, cow BW, BCS, pregnancy rate to AI, and overall pregnancy rate did not differ across treatments. Calf weaning weights were also not affected by trace mineral source. At the end of yr 1 and yr 2, liver Cu concentrations were greater (P ≤ 0.05) in HTM- compared with IOC-supplemented cows. Liver Zn concentrations were greater (P 0.05) by trace mineral source. Implications and Applications Overall productivity of grazing beef cows and their calves over a 2-yr period were similar in cattle supplemented with hydroxy or a combination of sulfate and organic trace minerals. Liver Cu and Zn concentrations were greater in cows fed HTM.

Objective Production diseases, such as liver abscesses, can affect cattle performance; however, the well-being implications are not well understood. The purpose of this study was to investigate the effect of liver abscess presence on stress-related parameters in beef breed feedlot cattle. Materials and Methods A total of 363 Bos taurus crossbred beef feedlot steers (675 ± 2.3 kg) were assigned to 1 of 3 groups in accordance with the liver abscess score assigned after slaughter: no liver abscess presence (n = 316), mild liver abscess presence (n = 21), and severe liver abscess presence (n = 24). Hair cortisol concentrations were obtained from hair samples collected from each animal 2 d before slaughter. A maximum eye temperature using infrared thermography and a mobility score were assigned to each animal. Serum cortisol concentrations were collected via exsanguination blood for 115 of the 363 animals. Analyses of variance were used to determine differences in measured well-being variables between liver abscess score groups. Results and Discussion All measured parameters, including infrared thermography (P = 0.55), hair cortisol (P = 0.96), and serum cortisol (P = 0.21), showed no effect of liver abscess scores. No statistical analysis was performed with mobility data because only scores of normal locomotion were assigned. Implications and Applications Under the conditions of this experiment, liver abscesses did not affect measured stress-related outcomes. Further research is warranted to improve our understanding of the relationship between liver abscess presence and other stress-related parameters associated with well-being in cattle.

Hepatitis B virus (HBV) is a major cause of acute liver failure (ALF) worldwide. While liver damage in classic acute hepatitis B is believed to be T-cell mediated, the pathogenesis of HBV-associated ALF remains largely unknown. Access to liver specimens from well-characterized patients with HBV-associated ALF provided us with the opportunity to perform next-generation sequencing (NGS) of the entire VH repertoires of IgM and IgG from the livers of four ALF patients, a control liver donor and a patient with chronic HBV infection. We found that ALF is not associated with expansion of specific B-cell lineages. However, NGS showed that the intrahepatic VH repertoires from ALF patients were characterized by the abundant presence of antibodies in germline configuration in contrast to their marginal prevalence in controls. Moreover, NGS identified a large number of VH genes in germline configuration with identical VDJ sequences in the IgM and IgG repertoires in all four ALF patients, indicating that isotype switch from IgM to IgG had occurred without somatic hypermutation. The results of this study indicate that the presence of intrahepatic antibodies in unmutated germline configuration is a broad phenomenon in the global antibody repertoire generated from total RNA derived from whole-liver tissue that is strongly associated with ALF, suggesting a major role of T cell-independent humoral immunity in the pathogenesis of ALF.

Liver abscess etiology in feedlot steers involves the escape of bacteria from the digestive tract to form a polymicrobial abscess within or on the external surface of the liver. However, little is known about the effects of feedlot finishing systems on the microbial composition of the liver abscess purulent material. Liver abscesses were collected at the time of harvest from steers originating from a single feedlot managed in either a traditional program (which included tylosin phosphate supplementation) or a natural program (without tylosin phosphate supplementation). The purulent material of liver abscesses from traditionally managed steers (N = 53 abscesses) and that of naturally managed steers (N = 62 abscesses) was characterized using the V4 region of the 16S rRNA gene. Two phyla and three genera were found in greater than 1% relative abundance across all abscesses. The genus Fusobacterium was identified in all liver abscess samples and accounted for 64% of sequencing reads. Bacteroides and Porphyromonas genera accounted for 33% and 1% of reads, respectively. Trueperella was more likely to be found in the liver abscesses of naturally managed steers than traditionally managed steers (P = 0.022). Over 99% of the genus-level bacterial sequences observed across all liver abscesses belonged to Gram-negative genera. Bacteria known to colonize both the rumen and hindgut were identified within liver abscesses. No differences in alpha diversity or beta diversity were detected between liver abscess communities (between the two management programs or individual pens) when tested as richness, Shannon Diversity Index, or weighted UniFrac distances (P0.05). These results were consistent with previous identification of Fusobacterium necrophorum as the primary bacteriologic agent within liver abscesses and emphasized the relationship between the gastrointestinal microbiota and liver abscess formation. Though the microbiota of the liver abscess purulent material was similar between steers fed an antibiotic-free diet and those fed an antibiotic-containing diet from the same feedlot, divergence was detected in liver abscess communities with some being dominated by Fusobacterium and others being dominated by Bacteroides.As feedlot cattle consume grain, the rumen becomes more acidic. If the lining of the digestive tract is damaged, bacteria that normally remain in the digestive tract can enter the body. Certain bacteria like Fusobacterium necrophorum are involved in the formation of liver abscesses. Feedlot cattle are commonly fed an antibiotic (tylosin phosphate) to reduce the occurrence of liver abscesses, but increasing scrutiny is placed on the antibiotic use. However, the effect of eliminating the antibiotic used to prevent liver abscesses on the bacterial communities involved in liver abscess formation is unknown. This study compared the bacteria found within liver abscesses of cattle fed tylosin phosphate with that of cattle not fed tylosin phosphate. All liver abscesses contained F. necrophorum, and Bacteroides was the second most commonly identified bacterium. Trace amounts of bacteria known to colonize the mouth and digestive tract were observed. Trueperella, a bacteria targeted by tylosin phosphate, was found more frequently in liver abscesses from cattle that received no antibiotic. While the core bacterial composition of the liver abscess was unaffected by antibiotic supplementation to feedlot steers, reduced Trueperella in liver abscesses from cattle-fed tylosin phosphate could be related to a reduction in liver abscess prevalence.

Cattle are stoic by nature and have long been subject to evolutionary pressure to mask pain to avoid becoming prey. Quantifying pain via biomarkers allows researchers to capture changes that are not easily detected by the human eye. Negative public perception of routine husbandry practices such as dehorning and castration is growing, increasing the need for the development of practices to relieve pain and suffering in cattle. The objective was to quantify pain associated with induced bacterial pneumonia, scoop and hot-iron dehorning, surgical castration, and hot-iron branding, and to investigate analgesic effectiveness. Calves were experimentally inoculated with Mannheimia haemolytica, scoop or cautery dehorned, surgically castrated, or hot-iron branded. Outcome variables were collected at baseline and following husbandry procedures and included serum cortisol; infrared thermography (IRT); mechanical nociceptive threshold (MNT); substance P; kinematic gait analysis; a visual analog scale; chute defense and behavior scoring; clinical illness score; computerized lung score; average activity and rumination; prostaglandin E2 metabolite; plasma serum amyloid A and rectal temperature. Responses were analyzed using repeated measures with calf nested in treatment designated as a random effect, and treatment, time, and their interaction designated as fixed effects. A combination of reduced activity levels, decreased force on calves’ right front limb, and increased visual analog scale pain scores all support that bacterial pneumonia in cattle is painful (P ≤ 0.01). Differences in right front force were observed in calves challenged with M. haemolytica and treated with flunixin transdermal (96.5 kg) and those given a placebo (85.5 kg), indicating that flunixin transdermal may attenuate specific pain biomarkers in cattle with respiratory disease (P < 0.01). Administration of a bupivacaine liposome suspension block at the time of dehorning and castration was as effective at controlling pain as a multi-modal approach of lidocaine and meloxicam (as evidenced by treatment differences in MNT, cortisol and gait analysis (P ≤ 0.04)). Oral meloxicam administration at branding reduced IRT differences from the branding and control site and reduced lying bouts (P < 0.01). Breed and sex effects were observed across a wide range of biomarkers and should be investigated in future pain studies. The need for long-acting analgesic options for cattle that demonstrate pain alleviation across multiple biomarkers is apparent and would be beneficial to alleviating pain from routine husbandry procedures like dehorning, castration, and branding, as well as painful disease conditions such as bovine respiratory disease.

Eighty-three American Wagyu steers (725 ±10.7 kg) were used to evaluate the effects of olive byproduct supplementation on feedlot performance and carcass characteristics. We hypothesized that with supplementation of olive byproduct would improve feedlot performance and longissimus muscle intramuscular fat composition. Steers were blocked by initial body weight (BW) and randomly assigned within block to one of two treatments. Treatments consisted of: 1) Control diet (basal ration with no olive byproduct) + 1 kg of supplemental cracked corn per animal per day, or 2) Control diet + 1 kg of supplemental olive byproduct per animal per day. Steers were housed in feedlot pens (n=4 steers/pen; 11 replicates/treatment) and fed a traditional American Wagyu finishing diet (DM basis: 68.4% DM, 14.3% CP; 74.8% TDN, 1.16 Mcal/kg NEg, 5.3% crude fat). Diets were delivered to pens, once daily, in the morning in amounts to allow ad libitum access to feed over a 24 h period. Olive byproduct and cracked corn were top-dressed to the appropriate treatment pens immediately after delivery of the basal ration. Steers were individually weighed on d -1 and 0, and approximately every 28 d throughout the 177 d experiment. Equal numbers of steers per treatment were slaughtered throughout the experiment and carcass data were collected. Data were analyzed using a mixed effects model of SAS (SAS Inst. Inc.) for a randomized complete block design. Steers receiving olive byproduct had a lower final BW (P < 0.01) when compared to steers receiving the control diet. Longissimus muscle long chain fatty acids C18:1 and C:22:0 were greater (P < 0.05) and C18:0 lesser (P < 0.05) in controls when compared to steers supplemented with olive byproduct. Under the conditions of this experiment, feeding olive byproduct reduced final BW and had minimal impacts on longissimus muscle fatty acid composition.

Twelve steers, fitted with rumen canulae were used in a 4 x 4 Latin square design to examine the impact of the direct fed microbial Propionibacteria acidipropionici (PA) on rumen fermentation characteristics. All steers were housed together in one pen equipped with GrowSafe feed intake monitoring stations and one Greenfeed system used to estimate in vivo methane production. Steers were fed a corn silage-based diet throughout the experiment. Treatments consisted of PA administered at: 1) 0.0; 2) 1.0 x 108; 3)1.0 x 109; and 4) 1.0 x 1010 CFU∙animal-1∙day-1. Treatments were administered directly into the rumen as a single bolus dose daily. On day 7 and 14 of each period, rumen fluid was collected from each steer 2 h post treatment administration for VFA analysis and for determining in vitro fermentation characteristics. Following a 14d washout period, animal treatments were switched, and the experiment repeated. Data were analyzed as a 4 x 4 Latin square design. In vivo propionic acid molar proportions (25.4 vs 23.6±0.24) and total VFA concentrations (125.2 vs 121.3±1.87) were greater (P < 0.05) in steers receiving PA compared with controls. In vitro DM disappearance (P < 0.05; 63.3% vs 59.2%±1.12) and total VFA (P < 0.05; 147.9 vs 145.2±1.76) were greater and methane (ml/g DMD) lesser (P < 0.04; 13.1 vs 15.6±0.11) in fermentation vessels incubated with rumen fluid from animals receiving PA compared with controls. Dry matter disappearance (P < 0.03) and propionic acid (P < 0.04) increased linearly as dose of PA increased. In vitro Total VFA tended (P < 0.08) to increase linearly and methane production tended (P < 0.09) to decrease quadratically in response PA dose. In vivo methane production was similar across treatments. In conclusion, PA addition improved in vivo and in vitro rumen fermentation characteristics.

This study was designed to evaluate the effects of açaí seed (AS) as a fiber source on intake, digestibility, and ruminal fermentation parameters of beef cattle fed a high concentrate diet. Five rumen-fistulated Zebu bulls were used in a 5x5 Latin-square design. Treatments consisted of five diets with different inclusion rates of AS: 0, 5, 10, 15, and 20% of the diet dry matter (DM). The total mixed ration contained 75% of concentrate and 25% of roughage. Mombasa grass silage and AS were used as the roughage sources. The concentrate portion of the diet contained cracked corn (61.8%), soybean meal (8%) and vitamin/mineral premix (5.2% of DM). Each experimental period lasted 25 d and the last 11 d were used for data collection. Total fecal collections were performed over three days. Titanium dioxide was used to estimate the omasal outflow. Statistical significance was declared at P < 0.05, and tendency at 0.05 > P < 0.10. A linear increase was observed for DM (P= 0.07), organic matter (P < 0.05), crude protein (CP, P < 0.05), and neutral detergent fiber (NDF) intake with the inclusion rate of AS. Intake was greatest in bulls receiving diets containing 20% AS. A linear decrease (P< 0.05) in NDF digestibility was detected with the inclusion rate of AS. DM, CP digestibility, and metabolic energy were similar across treatments (P > 0.10). Ruminal and intestinal DM and nutrient digestibility were similar between the treatments (P > 0.10). Rumen concentrations of acetic acid increased (P < 0.05), and total short-chain fatty acids (P = 0.07) and butyrate (P = 0.08) tended to increase linearly with the inclusion of AS. Açaí seed as a source of fiber in confined Zebu bulls diets alters DM intake and rumen fermentation but has no impact on total tract DM digestibility.

Hepatitis E virus (HEV) infection rarely causes icteric hepatitis, yet 10%-40% of adult Americans have serological evidence of previous infection. The aim of this study was to investigate the incidence, presentation, and outcome of acute and previous HEV infection in a large cohort of patients with suspected drug-induced liver injury (DILI).Serum samples from 2012 patients enrolled in the DILI Network were tested for anti-HEV immunoglobulin G (IgG). Those with detectable anti-HEV IgG underwent testing for anti-HEV IgM; those with detectable anti-HEV immunoglobulin m (IgM) were tested for HEV RNA.Anti-HEV IgG was detected in 407 (20%) patients and associated with increasing subject age and earlier year of enrollment. The median age of seropositive subjects was more than a decade higher than seronegative subjects (59.8 vs 48.7 years). The overall prevalence of anti-HEV declined from 22% (2004-2011) to 18% (2012-2019), suggestive of a cohort effect. The frequency of acute hepatitis E (median ALT = 1231 IU/L) also decreased from 3% (2004-2008) to 1.2% (2009-2013) to 0.6% (2014-2019). These results suggest that acute HEV infection is usually subclinical and was much more frequent in this cohort before 2004.Acute HEV infection accounts for less than 1% of suspected American DILI cases and is more frequent in older men. Previous HEV infection is also most commonly seen in older individuals. Clinicians should consider testing for unsuspected acute HEV infection in older adult patients with acute hepatocellular DILI and jaundice.

Hot-iron branding uses thermal injury to permanently identify cattle causing painful tissue damage. The primary objective was to examine the physiological and behavioral effects of oral meloxicam (MEL), compared to a control, administered at the time of hot-iron branding in Angus and Hereford steers and heifers. The secondary objectives were to investigate breed and sex effects on pain biomarkers. A total of 70 yearlings, consisting of 35 heifers and 35 steers (Angus, Hereford, or Angus × Hereford), were enrolled in the study. Animals were blocked by sex, randomized across weight, and assigned to receive MEL (1 mg/kg) or a placebo (CON). Biomarkers were assessed for 48 h after branding and included infrared thermography (IRT), mechanical nociceptive threshold (MNT), accelerometry and a visual analog scale (VAS), and serum cortisol and prostaglandin E2 metabolites (PGEM). Wound healing was assessed for 12 wk. Hair samples to quantify cortisol levels were taken prior to and 30 d post-branding. Responses were analyzed using repeated measures with calf nested in treatment as a random effect, and treatment, time, treatment by time interaction, breed, and sex as fixed effects. There was a treatment by time interaction for PGEM (P0.01) with MEL having lower values than CON at 6, 24, and 48 h (MEL: 18.34 ± 3.52, 19.61 ± 3.48, and 22.24 ± 3.48 pg/mL, respectively; CON: 32.57 ± 3.58, 37.00 ± 3.52, and 33.07 ± 3.48 pg/mL; P0.01). MEL showed less of a difference in maximum IRT values between the branded (2.27 ± 0.29 °C) and control site (3.15 ± 0.29 °C; P0.01). MEL took fewer lying bouts at 0-12 h (4.91 bouts ± 0.56) compared with CON (6.87 bouts ± 0.55; P0.01). Compared with Hereford calves, Angus calves exhibited greater serum but lower hair cortisol, greater PGEM, more lying bouts, and less healed wound scores at 3, 4, and 5 wk. Compared with heifers, steers exhibited lower PGEM, lower branding site and ocular IRT, higher MNT, and lower plasma meloxicam levels. Steers spent more time lying, took more lying bouts and had greater VAS pain, and more healed wound scores at 5 wk than heifers. Meloxicam administration at branding reduced branding and control site temperature differences and reduced lying bouts for the first 12 h. Breed and sex effects were observed across many biomarkers. Changes from baseline values for IRT, MNT, lying time, step count, VAS pain, and wound scoring all support that branding cattle is painful.Hot-iron branding uses thermal injury to permanently identify cattle causing painful tissue damage. The primary objective was to examine the effects of oral meloxicam (MEL), compared with a control, administered at the time of hot-iron branding in Angus and Hereford steers and heifers. The secondary objectives were to investigate breed and sex effects on pain biomarkers. A total of 70 yearlings, consisting of 35 heifers and 35 steers (Angus, Hereford, or Angus × Hereford), were enrolled. Animals were assigned to receive MEL or a placebo. Changes from baseline values for infrared thermography (IRT), mechanical nociceptive threshold, lying time, step count, visual analog scale score, and wound scoring all support that hot-iron branding cattle is painful and investigation into analgesic strategies is needed. MEL administration reduced IRT differences from the branding and control site and reduced lying bouts. Breed and sex effects were observed across a wide range of biomarkers and should be considered in future pain studies. The practicality of administering a nonsteroidal anti-inflammatory drug once at the time of branding is attractive. However, a multimodal approach using a combination of analgesics or longer acting analgesic option warrants further investigation to alleviate pain and discomfort caused by hot-iron branding.

Primary cilia are cellular appendages critical for diverse types of Signaling. They are found on most cell types, including cells throughout the CNS. Cilia preferentially localize certain G-protein-coupled receptors (GPCRs) and are critical for mediating the signaling of these receptors. Several of these neuronal GPCRs have recognized roles in feeding behavior and energy homeostasis. Cell and model systems, such asCaenorhabditis elegansandChlamydomonas, have implicated both dynamic GPCR cilia localization and cilia length and shape changes as key for signaling. It is unclear whether mammalian ciliary GPCRs use similar mechanismsin vivoand under what conditions these processes may occur. Here, we assess two neuronal cilia GPCRs, melanin-concentrating hormone receptor 1 (MCHR1) and neuropeptide-Y receptor 2 (NPY2R), as mammalian model ciliary receptors in the mouse brain. We test the hypothesis that dynamic localization to cilia occurs under physiological conditions associated with these GPCR functions. Both receptors are involved in feeding behaviors, and MCHR1 is also associated with sleep and reward. Cilia were analyzed with a computer-assisted approach allowing for unbiased and high-throughput analysis. We measured cilia frequency, length, and receptor occupancy. We observed changes in ciliary length, receptor occupancy, and cilia frequency under different conditions for one receptor but not another and in specific brain regions. These data suggest that dynamic cilia localization of GPCRs depends on properties of individual receptors and cells where they are expressed. A better understanding of subcellular localization dynamics of ciliary GPCRs could reveal unknown molecular mechanisms regulating behaviors like feeding.