A comparison of methods to detect low levels of Salmonella enterica in surface waters to support antimicrobial resistance surveillance efforts performed in multiple laboratories.

Developing effective and sensitive detection methods for antimicrobial resistant Salmonella enterica from surface water is a goal of the National Antimicrobial Resistance Monitoring System (NARMS). There are no specified methods for recovery of S. enterica in surface waters in the U.S. A multi-laboratory evaluation of four methods - bulk water enrichment (BW), vertical Modified Moore Swab (VMMS), modified Standard Method 9260.B2 (SM), and dead-end ultrafiltration (DEUF) - was undertaken to recover S. enterica from surface water. In Phase 1, one-liter volumes of water were collected from the same site on five different dates. Water was shipped and analyzed at four different laboratory locations (A, B, C, and D) for recovery of 1) inoculated fluorescent S. Typhimurium strain (ca. 30 CFU/L) and 2) Salmonella present in the water sampled. At each location, BW, VMMS, or SM recovery was performed on five separate 1 L water samples. Twenty 1 L water samples were subjected to each recovery method, and overall, sixty 1 L samples were assayed for Salmonella. Inoculated, fluorescent Salmonella Typhimurium and environmental Salmonella spp. were recovered from 65 % (39/60) and 45 % (27/60) of water samples, respectively. BW, VMMS, and SM recovered fluorescent S. Typhimurium from 60 %, 60 %, and 75 % of inoculated samples, respectively. Analysis by Chi-squared test determined laboratory location had a significant (p < 0.05) effect on fluorescent S. Typhimurium recovery compared to method or date of water collection. In Phase 2, recovery of inoculated fluorescent S. Typhimurium from 1 L samples by SM and DEUF was compared at laboratory locations B and D. SM and DEUF recovered fluorescent S. Typhimurium from 100 % (20/20) and 95 % (19/20) of inoculated water samples, respectively; laboratory location (p > 0.05) did not affect Salmonella recovery. Uniform laboratory methodology and training should be prioritized in conducting Salmonella recovery from surface water in laboratories.

CD97 is associated with mitogenic pathway activation, metabolic reprogramming, and immune microenvironment changes in glioblastoma.

Glioblastoma (GBM) is the most common primary brain tumor with a median survival under two years. Using in silico and in vitro techniques, we demonstrate heterogeneous expression of CD97, a leukocyte adhesion marker, in human GBM. Beyond its previous demonstrated role in tumor invasion, we show that CD97 is also associated with upregulation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathways in GBM. While CD97 knockout decreased Akt activation, CD97 targeting did not alter MAPK/Erk activation, did not slow GBM cell proliferation in culture, and increased levels of glycolytic and oxidative phosphorylation metabolites. Treatment with a soluble CD97 inhibitor did not alter activation of the MAPK/Erk and PI3K/Akt pathways. Tumors with high CD97 expression were associated with immune microenvironment changes including increased naïve macrophages, regulatory T cells, and resting natural killer (NK) cells. These data suggest that, while CD97 expression is associated with conflicting effects on tumor cell proliferative and metabolic pathways that overall do not affect tumor cell proliferation, CD97 exerts pro-tumoral effects on the tumor immune microenvironment, which along with the pro-invasive effects of CD97 we previously demonstrated, provides impetus to continue exploring CD97 as a therapeutic target in GBM.

Effects of Bacillus subtilis PB6 and/or chromium propionate supplementation on serum chemistry, complete blood count, and fecal Salmonella spp. count in high-risk cattle during the feedlot receiving and finishing periods.

The study objective was to determine the effects of Bacillus subtilis PB6 and/or chromium propionate supplementation on serum chemistry, complete blood count, and fecal Salmonella spp. count in high-risk beef cattle during a 56-d feedlot receiving period and the subsequent finishing period. Four truckload blocks of crossbred beef bulls (n = 300) and steers [n = 84; total n = 384; average initial body weight (BW) = 220 ± 16.2 kg] were sourced from regional auction markets and assigned randomly to treatments arranged in a 2 × 2 factorial. Blood samples were collected from two bulls nearest to the median BW on arrival in each pen (n = 96) and fecal samples were collected from cattle in block 3 (n = 96). The generalized complete block design consisted of 12 pen replications per treatment with pen as the experimental unit. Treatments were: 1) negative control (CON); 2) 13 g per animal daily of prepared B. subtilis PB6 product (CST); 3) 450 ppb dry matter (DM) chromium propionate (CHR); and 4) 13 g per animal daily of prepared B. subtilis PB6 product and 450 ppb DM chromium propionate (CST + CHR). Treatments were top dressed in feed bunks daily using 0.45 kg per animal ground corn carrier immediately following feed delivery. Data were analyzed using mixed models with repeated measures. Day affected all serum chemistry variables (P ≤ 0.03) except total CO2 (P = 0.34) and all complete blood count variables during receiving (P ≤ 0.02) except percentage basophils (P ≥ 0.12). During the overall receiving period, serum calcium was decreased (P = 0.02) by CHR. Cattle fed CHR had greater total leukocyte count (P = 0.04) and neutrophil count (P = 0.02) during the overall receiving period. Fecal Salmonella spp. count was markedly reduced in cattle fed CST on day 28 (P = 0.01) and overall (P = 0.07). Overall, these data provide metabolic and hematologic insight into the unique challenges presented by lightweight, high-risk feeder cattle. Notably, CST was found to be effective in mitigating fecal enumeration and presumably replication of Salmonella spp. in the gastrointestinal tract.

Layilin augments integrin activation to promote antitumor immunity.

Tumor-infiltrating CD8+ T cells mediate antitumor immune responses. However, the mechanisms by which T cells remain poised to kill cancer cells despite expressing high levels of inhibitory receptors are unknown. Here, we report that layilin, a C-type lectin domain-containing membrane glycoprotein, is selectively expressed on highly activated, clonally expanded, but phenotypically exhausted CD8+ T cells in human melanoma. Lineage-specific deletion of layilin on murine CD8+ T cells reduced their accumulation in tumors and increased tumor growth in vivo. Congruently, gene editing of LAYN in human CD8+ T cells reduced direct tumor cell killing ex vivo. On a molecular level, layilin colocalized with integrin αLß2 (LFA-1) on T cells, and cross-linking layilin promoted the activated state of this integrin. Accordingly, LAYN deletion resulted in attenuated LFA-1-dependent cellular adhesion. Collectively, our results identify layilin as part of a molecular pathway in which exhausted or "dysfunctional" CD8+ T cells enhance cellular adhesiveness to maintain their cytotoxic potential.

Scalable high throughput selection from phage-displayed synthetic antibody libraries.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

Development and model testing of antemortem screening methodology to predict required drug withholds in heifers.

A simple, cow-side test for the presence of drug residues in live animal fluids would provide useful information for tissue drug residue avoidance programs. This work describes adaptation and evaluation of rapid screening tests to detect drug residues in serum and urine. Medicated heifers had urine, serum, and tissue biopsy samples taken while on drug treatment. Samples were tested by rapid methods and high-performance liquid chromatography (HPLC). The adapted microbial inhibition method, kidney inhibition swab test, was useful in detecting sulfadimethoxine in serum, and its response correlated with the prescribed withdrawal time for the drug, 5 to 6 days posttreatment. The lateral flow screening method for flunixin and beta-lactams, adapted for urine, was useful in predicting flunixin in liver detected by HPLC, 96 h posttreatment. The same adapted methods were not useful to detect ceftiofur in serum or urine due to a lack of sensitivity at the levels of interest. These antemortem screening test studies demonstrated that the method selected, and the sampling matrix chosen (urine or serum), will depend on the drug used and should be based on animal treatment history if available. The live animal tests demonstrated the potential for verification that an individual animal is free of drug residues before sale for human consumption.

Severity of mastitis symptoms as a predictor of C-reactive protein in milk and blood during lactation.

OBJECTIVE: To investigate the presence of C-reactive protein (CRP) in breast milk and any relationship between changes in CRP in breast milk and blood, and the severity of systemic and breast symptoms experienced during mastitis. METHODS: Mothers (n = 26) were followed prospectively from day 5 postpartum to the end of their lactation. Milk from each breast, blood, 24-hour urine samples and data on breast and systemic pathologies were collected at reference intervals during the first 3 months postpartum, daily during the occurrence of any breast inflammation and at 7 days after resolution of symptoms. RESULTS: CRP in blood was significantly increased during mastitis (p < 0.001, df:1,81; F = 31) and severity of systemic symptoms was a significant predictor for changes of CRP in blood (p < 0.01; df:3,42; F = 9.6). During mastitis both the symptomatic breast (p < 0.001; df:1,79; F = 19) and the contralateral asymptomatic breast (p < 0.004; df:1,75; F = 8.7) had a significantly higher milk CRP when compared with women with no mastitis. CONCLUSIONS: Although an increasing severity of breast and systemic symptoms in mastitis was predictive of an increasing CRP in milk and blood, respectively, the presence of CRP in similar concentrations in the mastitis and asymptomatic breast suggests it is of little use in making a differential diagnosis between infective verses noninfective forms of mastitis.