Enterococci isolated from a bison population on a native tall-grass prairie preserve in Kansas were characterized and compared to enterococci isolated from pastured cattle. The species diversity was dominated by Enterococcus casseliflavus in bison (62.4%), while Enterococcus hirae was the most common isolate from cattle (39.7%). Enterococcus faecalis was the second most common species isolated from bison (16%). In cattle, E. faecalis and Enterococcus faecium were isolated at lower percentages (3.2% and 1.6%, respectively). No resistance to ampicillin, chloramphenicol, gentamicin, or high levels of vancomycin was detected from either source. Tetracycline and erythromycin resistance phenotypes, encoded by tetO and ermB, respectively, were common in cattle isolates (42.9% and 12.7%, respectively). A significant percentage of bison isolates (8% and 4%, respectively) were also resistant to these two antibiotics. The tetracycline resistance genes from both bison and cattle isolates resided on mobile genetic elements and showed a transfer frequency of 10 ؊6 per donor, whereas erythromycin resistance was not transferable. Resistance to ciprofloxacin was found to be higher in enterococci from bison (14.4%) than in enterococci isolated from cattle (9.5%). The bison population can serve as a sentinel population for studying the spread and origin of antibiotic resistance.Enterococci, in particular Enterococcus faecalis and Enterococcus faecium, are among the top three types of organisms isolated from nosocomial infections worldwide (15), and the prevalence of vancomycin-resistant enterococci has been on the rise in the last decades. Enterococci host a wide variety of mobile genetic elements and are considered a reservoir for acquisition and distribution of antibiotic resistance genes among gram-positive bacteria (5). The discussion about the use of antibiotics in animal husbandry and the potential for spread of resistant strains into the human community was highlighted by experiences in the European Union (EU) with the use of the growth promoter avoparcin. The link between avoparcin use and occurrence of vancomycin-resistant enterococci in the EU resulted in the eventual ban of avoparcin in animal feed in 1996 by Denmark, followed by an EU-wide ban in 1998. In the United States, over 50% of antibiotic use is estimated to occur in the animal industry (21). Consequently, antibiotic-resistant bacterial strains are routinely isolated from farm animals (12). While many studies have been conducted on antibiotic resistance among human populations and farm animals, less is known about the occurrence, distribution, and spread of antibiotic resistance in wild animal populations. It is expected that environments with no antibiotic pressure, such as nature preserves, harbor few antibiotic-resistant bacteria. Antibiotic resistance, however, could arise as consequence of inherent environmental pressure by antibiotic-producing bacteria or the spread of antibiotic-resistant bacteria to wild animal populations.In this study, an American bis...

Positive selection of T-cell precursors is the process by which a diverse T-cell repertoire is established. Positive selection begins at the CD4 1 CD8 1 double positive (DP) stage of development and involves at least two steps. First, DP thymocytes down-regulate CD8 to become transitional single positive (TSP) CD4 1 thymocytes. Then, cells are selected to become either mature single positive CD4 1 or mature single positive CD8 1 thymocytes. We sought to define the function of Gads during the two steps of positive selection by analyzing a Gads-deficient mouse line. In Gads 1/1 mice, most TSP CD4 1 thymocytes are TCR hi Bcl-2 hi CD69 1 , suggesting that essential steps in positive selection occurred in the DP stage. Despite that Gads À/À mice could readily generate TSP CD4 1 thymocytes, many Gads À/À TSP CD4 1 cells were TCR lo Bcl-2 lo CD69 À , suggesting that Gads À/À cells proceeded to the TSP CD4 1 stage prior to being positively selected. These data suggest that positive selection is not a prerequisite for the differentiation of DP thymocytes into TSP CD4 1 thymocytes. We propose a model in which positive selection and differentiation into the TSP CD4 1 stage are separable events and Gads is only required for positive selection.

Factor V Leiden (FVL) is an inherited disorder associated with blood clotting. Approximately 5% of Caucasians have FVL. We have developed a real‐time PCR assay for detection of FVL from buccal swabs or blood samples. A HyBeacons probe is used to detect FVL during melt curve analysis. HyBeacons probes are hybridization probes that allow simultaneous identification of single nucleotide polymorphisms (SNPs) via melt curve analysis. The probe is designed to hybridize to a region around a SNP associated with FVL. This allows the probe to bind to wild type and mutant forms of Factor V (FV). A normal amplification curve is seen for FV and FVL, however, during melt curve analysis a melt peak temperature shift is observed between FV and FVL. To optimize the assay, plasmids were constructed to both FV and FVL sequences. The assay was then run against each individual plasmid and a mixture of both to determine melt peaks for FV, FVL, and heterozygous samples. The melts peaks were approximately 52.3°C for FV, 57.5°C for FVL, and heterozygous samples had peaks at 52.3°C and 57.5°C. Twenty blood and buccal samples were obtained. DNA was extracted from the samples using Evogen ONE‐B and Evogen ONE, respectively and used in the FVL assay. All samples showed amplification but were negative for FVL. This newly developed assay is able to detect FV, FVL, and heterozygote from blood and buccal samples using a single HyBeacons probe.

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Stool samples are a common specimen used in a diagnostic setting. Due to the nature of stool samples, which have a large number of PCR inhibitors, extracting PCR‐ready DNA can be difficult. We have developed a rapid, simple protocol using Evogen ONE chemistry to extract DNA from stools. In this protocol, either 100ul of liquid stool was pipetted or a sterile swab was immersed in formed stool to gather sample which was washed with phosphate‐buffered saline. The supernatant was removed. 250ul of Evogen ONE was added to the sample, vortexed and then incubated for 5 minutes at 95°C. Then the sample was centrifuged and the lysate was removed. 1ul of the lysate containing the DNA was used in the following PCR reactions. A universal bacterial primer set was used with SYBR Green (Bio‐Rad) to show a wide variety of bacterial DNA present. Stool samples that were initially spiked with Salmonella enterica to show specific target recovery were amplified with the Salmonella ttr primer set and a ttr specific HyBeacons probe. All DNA extracts demonstrated amplification products. DNA concentration was measured on a NanoDrop (ThermoFisher). The crossing threshold (Ct) for the generic bacterial primer set was on average 13 cycles, and the Ct for Salmonella‐spiked stools was 27. DNA concentration varied from 150 to 600ng/ul which is typical of the sample type. We have shown that the above protocol is fast, easy to use, and produces high‐quality DNA.