Epizootiology and management of feline leukemia virus in the Florida puma

Feline leukemia virus (FeLV) was not detected in Florida pumas (Puma concolor coryi) in almost 20 yr of surveillance; however, the finding of two FeLV antigen-positive pumas during the 2002-2003 capture season led to an investigation of FeLV in the population. Between January 1990 and April 2007, the proportion of pumas testing FeLV antibody positive increased, with antibody-positive pumas concentrated in the northern portion of puma range. Five of 131 (4%) pumas sampled between July 2000 and April 2007 were viremic, with all cases clustered in Okaloacoochee Slough (OKS). Clinical signs and clinical pathology at capture were absent or included lymphadenopathy, moderate-to-severe anemia, and lymphopenia. All viremic pumas died; causes of death were septicemia (n=2), intraspecific aggression (n=2), and anemia/dehydration (n=1). Outcome after FeLV exposure in pumas was similar to that in domestic cats, with evidence of regressive, latent, and persistent infections. Management of the epizootic included vaccination, and as of April 2007, 52 free-ranging pumas had received one or more inoculations. Vaccinations were concentrated in OKS and in a band between OKS and the remainder of the puma population. There have been no new cases since July 2004; however, the potential for reintroduction of the virus remains.     

Elongation factor 1Bgamma (eEF1Bgamma) expression during the molting cycle and cold acclimation in the crayfish Procambarus clarkii

Eukaryotic elongation factor 1Bγ (eEF1Bγ) is a subunit of elongation factor 1 (EF1), which regulates the recruitment of amino acyl-tRNAs to the ribosome during protein synthesis in eukaryotes. In addition to structural roles within eEF1, eEF1Bγ has properties which suggest sensory or regulatory activities. We have cloned eEF1Bγ from axial abdominal muscle of freshwater crayfish, Procambarus clarkii. The predicted amino acid sequence has 66% identity to Locusta migratoria eEF1Bγ and 65% identity to Artemia salina eEF1Bγ. We measured eEF1Bγ expression by real-time PCR, using the relative quantification method with 18s ribosomal RNA as an internal calibrator. eEF1Bγ expression was lowest in gill, axial abdominal muscle, and hepatopancreas, and was highest in the antennal gland (5.7-fold above hepatopancreas) and cardiac muscle (7.8-fold above hepatopancreas). In axial abdominal muscle, eEF1Bγ expression was 4.4-fold higher in premolt and 11.9 higher in postmolt compared to intermolt. In contrast, eEF1Bγ was decreased or unchanged in epithelial tissues during pre- and postmolt. eEF1Bγ expression in the hepatopancreas was 3.5-fold higher during intermolt compared to premolt and was unchanged in gill and antennal gland. No significant differences in eEF1Bγ were found after 1 week of acclimation to 4 °C. These results show that eEF1Bγ is regulated at the mRNA level with tissue-specific differences in expression patterns.     

Insights into the environmental resistance gene pool from the genome sequence of the multidrug-resistant environmental isolate Escherichia coli SMS-3-5

The increasing occurrence of multidrug-resistant pathogens of clinical and agricultural importance is a global public health concern. While antimicrobial use in human and veterinary medicine is known to contribute to the dissemination of antimicrobial resistance, the impact of microbial communities and mobile resistance genes from the environment in this process is not well understood. Isolated from an industrially polluted aquatic environment, Escherichia coli SMS-3-5 is resistant to a record number of antimicrobial compounds from all major classes, including two front-line fluoroquinolones (ciprofloxacin and moxifloxacin), and in many cases at record-high concentrations. To gain insights into antimicrobial resistance in environmental bacterial populations, the genome of E. coli SMS-3-5 was sequenced and compared to the genome sequences of other E. coli strains. In addition, selected genetic loci from E. coli SMS-3-5 predicted to be involved in antimicrobial resistance were phenotypically characterized. Using recombinant vector clones from shotgun sequencing libraries, resistance to tetracycline, streptomycin, and sulfonamide/trimethoprim was assigned to a single mosaic region on a 130-kb plasmid (pSMS35_130). The remaining plasmid backbone showed similarity to virulence plasmids from avian-pathogenic E. coli (APEC) strains. Individual resistance gene cassettes from pSMS35_130 are conserved among resistant bacterial isolates from multiple phylogenetic and geographic sources. Resistance to quinolones was assigned to several chromosomal loci, mostly encoding transport systems that are also present in susceptible E. coli isolates. Antimicrobial resistance in E. coli SMS-3-5 is therefore dependent both on determinants acquired from a mobile gene pool that is likely available to clinical and agricultural pathogens, as well, and on specifically adapted multidrug efflux systems. The association of antimicrobial resistance with APEC virulence genes on pSMS35_130 highlights the risk of promoting the spread of virulence through the extensive use of antibiotics.     

Late-stage polyribitol phosphate wall teichoic acid biosynthesis in Staphylococcus aureus

Wall teichoic acids are cell wall polymers that maintain the integrity of the cellular envelope and contribute to the virulence of Staphylococcus aureus. Despite the central role of wall teichoic acid in S. aureus virulence, details concerning the biosynthetic pathway of the predominant wall teichoic acid polymer are lacking, and workers have relied on a presumed similarity to the putative polyribitol phosphate wall teichoic acid pathway in Bacillus subtilis. Using high-resolution polyacrylamide gel electrophoresis for analysis of wall teichoic acid extracted from gene deletion mutants, a revised assembly pathway for the late-stage ribitol phosphate-utilizing enzymes is proposed. Complementation studies show that a putative ribitol phosphate polymerase, TarL, catalyzes both the addition of the priming ribitol phosphate onto the linkage unit and the subsequent polymerization of the polyribitol chain. It is known that the putative ribitol primase, TarK, is also a bifunctional enzyme that catalyzes both ribitol phosphate priming and polymerization. TarK directs the synthesis of a second, electrophoretically distinct polyribitol-containing teichoic acid that we designate K-WTA. The biosynthesis of K-WTA in S. aureus strain NCTC8325 is repressed by the accessory gene regulator (agr) system. The demonstration of regulated wall teichoic acid biosynthesis has implications for cell envelope remodeling in relation to S. aureus adhesion and pathogenesis.     

Assembly of heterohexameric trypanosome hexokinases reveals that hexokinase 2 is a regulable enzyme

Glycolysis is essential to Trypanosoma brucei, the protozoan parasite that causes African sleeping sickness in humans and nagana in cattle. Hexokinase (HK), the first enzyme in glycolysis, catalyzes the phosphorylation of glucose to form glucose 6-phosphate. T. brucei harbors two HKs that are 98% identical at the amino acid level, T. brucei hexokinase 1 (TbHK1) and TbHK2. Recombinant TbHK1 (rTbHK1) has HK activity, whereas rTbHK2 does not. Unlike other eukaryotic HKs, TbHK1 is not subject to inhibition by ADP and glucose 6-phosphate. However, TbHK1 is inhibited by myristate, a critical fatty acid in T. brucei biology. We report here that rTbHKs, similar to authentic TbHK, form oligomers. Myristate dissociated these assemblies when incubated with either ATP or glucose. Furthermore, oligomer disruption was reversible by removal of myristate. Mixing of rTbHK1 and rTbHK2 monomers followed by reassembly yielded enzyme with an approximately 3-fold increase in specific activity compared with similarly treated rTbHK1 alone. Surprisingly, reassembly of rTbHK2 with an inactive rTbHK1 variant yielded an active HK, revealing for the first time that rTbHK2 is competent for HK activity. Finally, pyrophosphate inhibits active reassembled rTbHK2 oligomers but not oligomeric rTbHK1, suggesting that the two enzymes have distinct regulatory mechanisms.     

Probing mechanical properties of fully hydrated gels and biological tissues

A longstanding challenge in accurate mechanical characterization of engineered and biological tissues is maintenance of both stable sample hydration and high instrument signal resolution. Here, we describe the modification of an instrumented indenter to accommodate nanomechanical characterization of biological and synthetic tissues in liquid media, and demonstrate accurate acquisition of force-displacement data that can be used to extract viscoelastoplastic properties of hydrated gels and tissues. We demonstrate the validity of this approach via elastoplastic analysis of relatively stiff, water-insensitive materials of elastic moduli E>1000 kPa (borosilicate glass and polypropylene), and then consider the viscoelastic response and representative mechanical properties of compliant, synthetic polymer hydrogels (polyacrylamide-based hydrogels of varying mol%-bis crosslinker) and biological tissues (porcine skin and liver) of E<500 kPa. Indentation responses obtained via loading/unloading hystereses and contact creep loading were highly repeatable, and the inferred E were in good agreement with available macroscopic data for all samples. As expected, increased chemical crosslinking of polyacrylamide increased stiffness (E40 kPa) and decreased creep compliance. E of porcine liver (760 kPa) and skin (222 kPa) were also within the range of macroscopic measurements reported for a limited subset of species and disease states. These data show that instrumented indentation of fully immersed samples can be reliably applied for materials spanning several orders of magnitude in stiffness (E=kPa-GPa). These capabilities are particularly important to materials design and characterization of macromolecules, cells, explanted tissues, and synthetic extracellular matrices as a function of spatial position, degree of hydration, or hydrolytic/enzymatic/corrosion reaction times.     

Simple sequence repeat analysis of a clonally propagated species: a tool for managing a grape germplasm collection.

The USDA germplasm repositories help to preserve the genetic variability of important crop species by collecting and maintaining representative cultivars and related germplasm. Simple sequence repeat markers with high allelic diversity were used to type 41 grapevines from 40 accessions. All vines were either seedless table grape cultivars or cultivars with names similar to table grape cultivars. The proportion of shared alleles was selected as the most appropriate statistical measure of genetic distance for this population. In conjunction with morphological traits, known synonyms were confirmed and a previously unknown synonym was discovered. An alleged synonym in the literature was disproved by the DNA data. The data were consistent with known parentage, where such data were available. Two mislabeled vines in the USDA collection were identified. UPGMA grouped the cultivars loosely into three groups: a group of nine mostly Middle Eastern cultivars, a group of 22 accessions mostly from Russia and Afghanistan that were morphologically similar to 'Thompson Seedless', and a third very loose group of 11 accessions consisting mostly of eastern European wine grape cultivars. The limitations and usefulness of this type of analysis are discussed.