Short communication: Comparison of the fecal bacterial communities in diarrheic and nondiarrheic dairy calves from multiple farms in southeastern Pennsylvania.

Diarrhea is a major cause of illness and death in preweaned calves and causes significant economic losses to producers. A better understanding of the fecal microbiota in diarrheic and nondiarrheic calves could lead to improved treatment and prevention strategies. The purpose of this study was to compare the fecal microbiota of diarrheic and nondiarrheic calves to improve our understanding of what constitutes a healthy fecal microbiota in preweaned calves. At each of 7 farms, fecal samples were obtained from 1 to 3 diarrheic Holstein dairy calves (2 to 17 d old at sampling time) and age-matched (within 5 d) nondiarrheic controls for a total of 20 samples. Calves were fed either acidified bulk milk, pasteurized or unpasteurized waste milk, or milk replacer depending on farm. Fecal samples were extracted for genomic DNA, PCR-amplified for the V1-V2 region of the 16S rRNA bacterial gene, sequenced on the Illumina MiSeq (Illumina Inc., San Diego, CA) platform, and analyzed using QIIME2. Firmicutes and Bacteroidetes were the most abundant phyla in both groups; Fusobacteria was numerically more abundant in the diarrheic group, whereas Proteobacteria and Actinobacteria were numerically more abundant in the nondiarrheic group. At the genus level, Bacteroides was the most abundant genus in both groups and was numerically more abundant in the nondiarrheic group. Results from the mixed-effects regression model showed that Faecalibacterium and Butyricimonas were more abundant in the nondiarrheic calves, whereas Clostridium and Peptostreptococcus were more abundant in the diarrheic calves. Our results indicate that commensal bacteria acquired in the neonatal period may have been replaced with potential pathogens in diarrheic calves, which may have contributed to the incidence of diarrhea either directly or indirectly.

Pointwise Bounds for Joint Eigenfunctions of Quantum Completely Integrable Systems.

Let (M, g) be a compact Riemannian manifold of dimension n and P 1 : = - h 2 Δ g + V ( x ) - E 1 so that d p 1 ≠ 0 on p 1 = 0 . We assume that P 1 is quantum completely integrable (ACI) in the sense that there exist functionally independent pseuodifferential operators P 2 , ⋯ P n with [ P i , P j ] = 0 , i , j = 1 , ⋯ n . We study the pointwise bounds for the joint eigenfunctions, u h of the system { P i } i = 1 n with P 1 u h = E 1 u h + o ( 1 ) . In Theorem 1, we first give polynomial improvements over the standard Hörmander bounds for typical points in M. In two and three dimensions, these estimates agree with the Hardy exponent h - 1 - n 4 and in higher dimensions we obtain a gain of h 1 2 over the Hörmander bound. In our second main result (Theorem 3), under a real-analyticity assumption on the QCI system, we give exponential decay estimates for joint eigenfunctions at points outside the projection of invariant Lagrangian tori; that is at points x ∈ M in the "microlocally forbidden" region p 1 - 1 ( E 1 ) ∩ ⋯ ∩ p n - 1 ( E n ) ∩ T x ∗ M = ∅ . These bounds are sharp locally near the projection of the invariant tori.

Metagenomic Evidence of the Prevalence and Distribution Patterns of Antimicrobial Resistance Genes in Dairy Agroecosystems.

Antimicrobial resistance (AR) is a global problem with serious implications for public health. AR genes are frequently detected on animal farms, but little is known about their origin and distribution patterns. We hypothesized that AR genes can transfer from animal feces to the environment through manure, and to this end, we characterized and compared the resistomes (collections of AR genes) of animal feces, manure, and soil samples collected from five dairy farms using a metagenomics approach. Resistomes constituted only up to 1% of the total gene content, but were variable by sector and also farm. Broadly, the identified AR genes were associated with 18 antibiotic resistances classes across all samples; however, the most abundant genes were classified under multidrug transporters (44.75%), followed by resistance to vancomycin (12.48%), tetracycline (10.52%), bacitracin (10.43%), beta-lactam resistance (7.12%), and MLS efflux pump (6.86%) antimicrobials. The AR gene profiles were variable between farms. Farm 09 was categorized as a high risk farm, as a greater proportion of AR genes were common to at least three sectors, suggesting possible horizontal transfer of AR genes. Taxonomic characterization of AR genes revealed that a majority of AR genes were associated with the phylum Proteobacteria. Nonetheless, there were several members of Bacteroidetes, particularly Bacteroides genus and several lineages from Firmicutes that carried similar AR genes in different sectors, suggesting a strong potential for horizontal transfer of AR genes between unrelated bacterial hosts in different sectors of the farms. Further studies are required to affirm the horizontal gene transfer mechanisms between microbiomes of different sectors in animal agroecosystems.

Proof of concept for developing novel feeds for cattle from wasted food and crop biomass to enhance agri-food system efficiency.

Modern agri-food systems generate large amounts of crop-based biomass that are unfit for direct human consumption but potentially suitable for livestock feeding in production of meats, milk, and eggs. This study aims to develop novel feeds for cattle from some of those biomass materials through the natural microbial-driven processes of ensiling. Fruit and vegetables resembling supermarket discards were ensiled alone or co-ensiled with corn crop residues, mushroom wastes, etc. via laboratory experiments. Longitudinal sample analyses showed that (co-)ensiling was successful, with pH and fermentation acids changing rapidly into desirable ranges (pH < 4.5, the acids 5-13% DM with lactic acid dominating). The (co-)ensiled products had key nutritional parameters comparable to those of good quality forages commonly used on dairy farms. Additionally, in vitro incubation experiments indicated that the ensiled products could substitute certain conventional feeds while maintaining diet digestibility. Findings from this pilot study provide a proof of principle that quality novel feeds for cattle can be generated by co-ensiling food discards and low-value crop residues. Future research and animal feeding trials to demonstrate the utility of this approach can help societies more effectively utilize untapped biomass resources, strengthening the regenerative capacity of agri-food systems towards a more sustainable food future.

Cdk1 activity is required for mitotic activation of aurora A during G2/M transition of human cells.

In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G(2)/M transition. We find that activation of Aurora A first occurs at centrosomes at late G(2) and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.

Temporal changes in the fecal bacterial community in Holstein dairy calves from birth through the transition to a solid diet.

The development of a robust microbiome is critical to the health of dairy calves, but relatively little is known about the progression of the microbiome through the weaning transition. In this study, fecal samples were obtained from ten female Holstein calves at 6 timepoints between 2-13 weeks of age. Calves were fed acidified milk until weaning at 8 weeks old and had access to starter grain throughout the study. Fecal samples were extracted for genomic DNA, PCR-amplified for the V1-V2 region of the 16S rRNA bacterial gene, sequenced on the Illumina MiSeq platform, and analyzed using the QIIME2 pipeline. Bacterial richness, estimated by number of observed species, and bacterial diversity, estimated by Shannon diversity index, both differed significantly between timepoints and both increased over time (P <0.05), with the largest increases occurring during weaning. Weighted and unweighted UniFrac analysis showed significant differences (P <0.05) between bacterial communities across timepoints; betadisper analysis revealed that the microbiomes of individual calves became more similar with time. Throughout the study, Firmicutes was the dominant phylum, followed by Bacteroidetes. Thirteen bacterial genera were found to be significantly influenced by time, including Faecalibacterium, Clostridium, unclassified S24-7, Collinsella, Sharpea, and Treponema. Unclassified Ruminococcaceae was the most prevalent genus at timepoints 1, 3, 5, and 6, but different amplicon sequence variants were detected at each timepoint suggesting the presence of different species of Ruminococcaceae at different times. Bacteroides was the most prevalent genus at timepoint 2, and Prevotella was most prevalent at timepoint 4. Our results indicate that there is considerable variation in the calf microbiome pre-weaning, but the microbial community stabilizes and becomes similar to the adult microbiome at weaning. Further studies to describe the phylogeny and functionality of core microbiota through the weaning transition are needed to improve health and reduce diarrhea in the neonatal period.

The distribution of microbiomes and resistomes across farm environments in conventional and organic dairy herds in Pennsylvania.

BACKGROUND: Antimicrobial resistance is a serious concern. Although the widespread use of antimicrobials in livestock has exacerbated the emergence and dissemination of antimicrobial resistance genes (ARG) in farm environments, little is known about whether antimicrobial use affects distribution of ARG in livestock systems. This study compared the distribution of microbiomes and resistomes (collections of ARG) across different farm sectors in dairy herds that differed in their use of antimicrobials. Feces from heifers, non-lactating, and lactating cows, manure storage, and soil from three conventional (antimicrobials used to treat cows) and three organic (no antimicrobials used for at least four years) farms in Pennsylvania were sampled. Samples were extracted for genomic DNA, processed, sequenced on the Illumina NextSeq platform, and analyzed for microbial community and resistome profiles using established procedures. RESULTS: Microbial communities and resistome profiles clustered by sample type across all farms. Overall, abundance and diversity of ARG in feces was significantly higher in conventional herds compared to organic herds. The ARG conferring resistance to betalactams, macrolide-lincosamide-streptogramin (MLS), and tetracyclines were significantly higher in fecal samples of dairy cows from conventional herds compared to organic herds. Regardless of farm type, all manure storage samples had greater diversity (albeit low abundance) of ARG conferring resistance to aminoglycosides, tetracyclines, MLS, multidrug resistance, and phenicol. All soil samples had lower abundance of ARG compared to feces, manure, and lagoon samples and were comprised of ARG conferring resistance to aminoglycosides, glycopeptides, and multi-drug resistance. The distribution of ARG is likely driven by the composition of microbiota in the respective sample types. CONCLUSIONS: Antimicrobial use on farms significantly influenced specific groups of ARG in feces but not in manure storage or soil samples.

Imidazolyl benzimidazoles and imidazo[4,5-b]pyridines as potent p38alpha MAP kinase inhibitors with excellent in vivo antiinflammatory properties.

Herein we report investigations into the p38alpha MAP kinase activity of trisubstituted imidazoles that led to the identification of compounds possessing highly potent in vivo activity. The SAR of a novel series of imidazopyridines is demonstrated as well, resulting in compounds possessing cellular potency and enhanced in vivo activity in the rat collagen-induced arthritis model of chronic inflammation.

Viscosupplementation for Osteoarthritis of the Knee: A Key Opinion Leader Panel Discussion.

With a sharp rise in the prevalence of osteoarthritis of the knee (OAK) in a younger population, new management strategies are needed to preserve mobility, improve patients' quality of life, and reduce the effects of potential disease-related comorbidities. Viscosupplementation with the use of hyaluronic acid (HA) injection is a treatment option for OAK that can provide lubrication and elastic shock absorption, leading to potential pain relief, improved function, and reduced stiffness. A key opinion leader (KOL) panel discussion was held December 3, 2016, with the objective of sharing opinions, ideas, information, and trends regarding OAK and the potential treatment and management offered by viscosupplementation. The panel concluded that viscosupplementation with HA injections presents a viable, cost-effective, and safe alternative for the treatment of OAK. DISCLOSURES: This panel discussion and report was facilitated by Magellan Rx Manage-ment and funded by Sanofi. Bert and Ruane report fees from Sanofi outside of this project. Sgaglione reports royalty payments from Zimmer Biomet and Wolters Kluwer. Dasa has received fees from Bioventus and Myoscience. All the authors received an honorarium for work on this project. Lopes is employed by Magellan Rx Management.

Selection of a water-extractable phosphorus test for manures and biosolids as an indicator of runoff loss potential.

The correlation of runoff phosphorus (P) with water-extractable phosphorus (WEP) in land-applied manures and biosolids has spurred wide use of WEP as a water quality indicator. Land managers, planners, and researchers need a common WEP protocol to consistently use WEP in nutrient management. Our objectives were to (i) identify a common WEP protocol with sufficient accuracy and precision to be adopted by commercial testing laboratories and (ii) confirm that the common protocol is a reliable index of runoff P. Ten laboratories across North America evaluated alternative protocols with an array of manure and biosolids samples. A single laboratory analyzed all samples and conducted a separate runoff study with the manures and biosolids. Extraction ratio (solution:solids) was the most important factor affecting WEP, with WEP increasing from 10:1 to 100:1 and increasing from 100:1 to 200:1. When WEP was measured by a single laboratory, correlations with runoff P from packed soil boxes amended with manure and biosolids ranged from 0.79 to 0.92 across all protocol combinations (extraction ratio, filtration method, and P determination method). Correlations with P in runoff were slightly lower but significant when WEP was measured by the 10 labs (r=0.56-0.86). Based on laboratory repeatability and water quality evaluation criteria, we recommend the following common protocol: 100:1 extraction ratio; 1-h shaking and centrifuge 10 min at 1500xg (filter with Whatman #1 paper if necessary); and determining P by inductively coupled plasma-atomic emission spectrometry or colorimetric methods.

Nitrogen- vs. phosphorus-based dairy manure applications to field crops: nitrate and phosphorus leaching and soil phosphorus accumulation.

Management of animal manures to provide nutrients for crop growth has generally been based on crop N needs. However, because manures have a lower N/P ratio than most harvested crops, N-based manure management often oversupplies the crop-soil system with P, which can be lost into the environment and contribute to eutrophication of water bodies. We examined the effects of N- vs. P-based manure applications on N and P uptake by alfalfa (Medicago sativa L.), corn (Zea mays L.) for silage, and orchardgrass (Dactylis glomerata L.), leaching below the root zone, and accumulation of P in soil. Treatments included N- and P-based manure rates, with no nutrient input controls and inorganically fertilized plots for comparison. Nitrate concentrations in leachate from inorganic fertilizer or manure treatments averaged 14 mg NO(3)-N L(-1), and did not differ by nutrient treatment. Average annual total P losses in leachate did not exceed 1 kg ha(-1). In the top 5 cm of soil in plots receiving the N-based manure treatment, soil test P increased by 47%, from 85 to 125 mg kg(-1). Nitrogen- and P-based manure applications did not differ in ability to supply nutrients for crop growth, or in losses of nitrate and total P in leachate. However, the N-based manure led to significantly greater accumulation of soil test P in the surface 5 cm of soil. Surface soil P accumulation has implications for increased risk of off-field P movement.

Mitotic requirement for aurora A kinase is bypassed in the absence of aurora B kinase.

We investigated why treatment of cells with dual aurora A and B kinase inhibitors produces phenotypes identical to inactivation of aurora B. We found that dual aurora kinase inhibitors in fact potently inhibit cellular activities of both kinases, indicating that inactivation of aurora B bypasses aurora A in mitosis. RNAi experiments further established that inactivation of aurora B indeed bypasses the requirement for aurora A and leads to polyploidy. Inactivation of aurora A activates checkpoint kinase BubR1 in an aurora B-dependent manner. Our results thus show that aurora B is responsible for mitotic arrest in the absence of aurora A.

Synthesis and in vitro antimicrobial activity of 3-keto 16-membered macrolides derived from tylosin.

Several series of 14-membered ketolides derived from erythromycin exhibit useful antimicrobial activity against macrolide-resistant bacteria. To determine if 16-membered ketolides may possess analogous activity, 3-keto derivatives of 5-O-mycaminosyl-23-O-acetyltylonolide and desmycosin were synthesized by protection of susceptible functional groups, oxidation of the 3-hydroxyl group under modified Moffatt-Pfitzner conditions, and subsequent deprotection. The resulting 3-keto products unexpectedly adopted the 2,3-trans enol rather than the 3-keto tautomer. The trans configuration of the 2,3-double bond in the macrolide chain is most likely the result of hydrogen bond stabilization between the enol hydroxyl and lactone carbonyl, which places these two groups in a cis relationship. This preference for the enol tautomer in 16-membered macrolides is not seen with 14-membered ketolides. The in vitro antimicrobial activity of the enol derivatives was greatly reduced compared to their unoxidized parent compounds, but the reduced antimicrobial activity of the enol derivatives paralleled results from corresponding 2,3-anhydro derivatives of 16-membered macrolides, which also have 2,3-trans stereochemistry. These results are in contrast to those from 14-membered-ring macrolides in which 3-keto and 2,3-anhydro derivatives exhibit greater activity than 3-hydroxy compounds.

Phosphorus characteristics of dairy feces affected by diets.

Phosphorus (P) surplus on dairy farms, especially confined operations, contributes to P buildup in soils with increased potential for P loss to waters. One approach to reduce P surplus and improve water quality is to optimize P feeding and improve P balance on farms. Here we report how varying P concentrations in lactating cow diets affects the amount as well as the chemical forms and fraction distribution of P in fecal excretion, and the environmental implications of this effect. Analysis of fecal samples collected from three independent feeding trials indicates that increasing dietary P levels through the use of P minerals not only led to a higher concentration of acid digest total phosphorus (TP) in feces, but more importantly increased the amount and proportion of P that is water soluble and thus most susceptible to loss in the environment. For instance, with diets containing 3.4, 5.1, or 6.7 g P kg(-1) feed dry matter (DM), the water-soluble fraction of fecal P was 2.91, 7.13, and 10.46 g kg(-1) fecal DM, respectively, accounting for 56, 77, and 83% of acid digest TP. The other fecal P fractions (those soluble in dilute alkaline and acid extractants) remained small and were unaffected by dietary P concentration. Excess P in the P supplemented diets was excreted in feces as water-soluble forms. A simple measure of inorganic phosphorus (Pi) in a single water extract is highly responsive to changes in diet P concentrations and hence can be indicative of dietary P status. A fecal P indicator concept is proposed and discussed.

The role of lateral retinacular release in the treatment of patellar instability.

In the last 2 decades many authors have described the use of an isolated lateral retinacular release for the treatment of patellar instability. This review analyzes the published long-term results of this procedure for the treatment of patellar instability. The isolated use of a lateral retinacular release of the patella has not proven to be of long-term benefit for the treatment of patellar instability. It may be used as an adjunct procedure to a proximal or distal realignment of the extensor mechanism. Various pitfalls of a lateral release for patellar instability are discussed.

A novel test for measuring and managing potential phosphorus loss from dairy cattle feces.

Pollution of waters resulting from phosphorus (P) runoff from agricultural land receiving long-term manure application is one of the most serious threats to water quality in many regions of the world. Of various approaches to alleviate the problem, reducing P surplus on animal farms through optimizing P intake and minimizing P excretion in manure offers a great opportunity. Here, we present a fecal P test method that has the potential to identify over-feeding of P in dairy cattle. Previous research has suggested that water-extractable P in dairy cow feces closely reflects dietary P changes and may indicate the animal's P status (adequate vs excessive). However, the notion was somewhat confounded when a subsequent study found other factors (pH and Ca content as well as sample handling method) also affecting P extractability in water. In the present work, we hypothesize that the impact of those factors on P extractability can be overcome by selecting dilute acid solutions to replace deionized water as the extractant. Using samples from 25 commercial dairy farms, we tested an array of acid solutions (including HCI, citric acid, and acetic acid) and found that 0.1% HCI is the most suitable extractant. Inorganic P (P(i)) released in 0.1% HCl closely reflected dietary P changes among the farms (R2 = 0.69) and was independent of pH, Ca, or sample handling method. Knowledge of P metabolism and partitioning in dairy cows and our experimental data suggest that excess P intake by the animal leads to greater amounts of bioavailable but unabsorbed P, which is excreted in feces. Its relative magnitude may be estimated by measuring P(i) extractable in 0.1% HCl. This novel and simple fecal P test could potentially be used as an indicator of the animal's P supply utilization status and thus serve as a screening tool for the presence of P over-feeding on dairy farms.

Survival of Salmonella enterica serovar Newport in manure and manure-amended soils.

Salmonella enterica serovar Newport has undergone a rapid epidemic spread in dairy cattle. This provides an efficient mechanism for pathogen amplification and dissemination into the environment through manure spreading on agricultural land. The objective of this study was to determine the survival characteristics of Salmonella serovar Newport in manure and manure-amended soils where the pathogen may be amplified. A multidrug-resistant (MDR) Salmonella serovar Newport strain and a drug-susceptible (DS) strain, both bovine isolates, were inoculated into dairy manure that was incubated under constant temperature and moisture conditions alone or after being mixed with sterilized or nonsterilized soil. Salmonella serovar Newport concentrations increased by up to 400% in the first 1 to 3 days following inoculation, and a trend of steady decline followed. With manure treatment, a sharp decline in cell concentration occurred after day 35, possibly due to microbial antagonism. For all treatments, decreases in Salmonella serovar Newport concentrations over time fit a first-order kinetic model. Log reduction time was 14 to 32 days for 1 log(10), 28 to 64 days for 2 log(10), and 42 to 96 days for 3 log(10) declines in the organisms' populations from initially inoculated concentrations. Most-probable-number monitoring data indicated that the organisms persisted for 184, 332, and 405 days in manure, manure-amended nonsterilized soil, and manure-amended sterilized soil, respectively. The MDR strain and the DS strain had similar survival patterns.

Complete inhibition of anisomycin and UV radiation but not cytokine induced JNK and p38 activation by an aryl-substituted dihydropyrrolopyrazole quinoline and mixed lineage kinase 7 small interfering RNA.

Mixed lineage kinase 7 (MLK7) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the pro-apoptotic signaling pathways p38 and JNK. A library of potential kinase inhibitors was screened, and a series of dihydropyrrolopyrazole quinolines was identified as highly potent inhibitors of MLK7 in vitro catalytic activity. Of this series, an aryl-substituted dihydropyrrolopyrazole quinoline (DHP-2) demonstrated an IC50 of 70 nM for inhibition of pJNK formation in COS-7 cell MLK7/JNK co-transfection assays. In stimulated cells, DHP-2 at 200 nM or MLK7 small interfering RNA completely blocked anisomycin and UV induced but had no effect on interleukin-1beta or tumor necrosis factor-alpha-induced p38 and JNK activation. Additionally, the compound blocked anisomycin and UV-induced apoptosis in COS-7 cells. Heart tissue homogenates from MLK7 transgenic mice treated with DHP-2 at 30 mg/kg had reduced JNK and p38 activation with no apparent effect on ERK activation, demonstrating that this compound can be used to block MLK7-driven MAPK pathway activation in vivo. Taken together, these data demonstrate that MLK7 is the MAPKKK required for modulation of the stress-activated MAPKs downstream of anisomycin and UV stimulation and that DHP-2 can be used to block MLK7 pathway activation in cells as well as in vivo.