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Francisella tularensis is the causative agent of tularemia and is a category A select agent. Francisella novicida, considered by some to be one of four subspecies of F. tularensis, is used as a model in pathogenesis studies because it causes a disease similar to tularemia in rodents but is not harmful to humans. F. novicida exhibits a strong restriction barrier which reduces the transformation frequency of foreign DNA up to 106-fold. To identify the genetic basis of this barrier, we carried out a mutational analysis of restriction genes identified in the F. novicida genome. Strains carrying combinations of insertion mutations in eight candidate loci were created and assayed for reduced restriction of unmodified plasmid DNA introduced by transformation. Restriction was reduced by mutations in four genes, corresponding to two type I, one type II, and one type III restriction system. Restriction was almost fully eliminated in a strain in which all four genes were inactive. The strongest contributor to the restriction barrier, the type II gene, encodes an enzyme which specifically cleaves Dam-methylated DNA. Genome comparisons show that most restriction genes in the F. tularensis subspecies are pseudogenes, explaining the unusually strong restriction barrier in F. novicida and suggesting that restriction was lost during evolution of the human pathogenic subspecies. As part of this study, procedures were developed to introduce unmodified plasmid DNA into F. novicida efficiently, to generate defined multiple mutants, and to produce chromosomal deletions of multiple adjacent genes. [ABSTRACT FROM AUTHOR]

We report the development and application of a method using domestic dogs (Canis familiaris Linnaeus, 1758) to systematically locate wildlife scat over large remote areas. Detection dogs are chosen for their strong object orientation, high play drive, and willingness to strive for a reward. Dogs were trained to detect grizzly bear (Ursus arctos Linnaeus, 1758) and black bear (Ursus americanus Pallas, 1780) scats over a 5200-km2 area of the Yellowhead Ecosystem, Alberta, Canada. DNA from scat provided the species and (for grizzly bears only) sex and individual identities of the animal at each location. Concentrations of fecal cortisol and progesterone metabolites from these same grizzly bear scats provided indices of physiological stress and reproductive activity (in females), respectively. Black and grizzly bears were most concentrated in the northern portion of the multiuse study area, where food is most abundant yet poaching-related mortality appears to be heaviest. Physiologic stress was also lowest and female reproductive activity correspondingly highest for grizzly bears in the north. The scat-based distributions corresponded to concurrently collected hair-snag data in 1999 and global positioning system radiotelemetry data (of grizzly bears) in 1999 and 2001. Results suggest that the scat dog detection methodology provides a promising tool for addressing a variety of management and research questions in the wildlife sciences. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei, the etiologic agent of human melioidosis, is capable of causing severe acute infection with overwhelming septicemia leading to death. A high rate of recurrent disease occurs in adult patients, most often due to recrudescence of the initial infecting strain. Pathogen persistence and evolution during such relapsing infections are not well understood. Bacterial cells present in the primary inoculum and in late infections may differ greatly, as has been observed in chronic disease, or they may be genetically similar. To test these alternative models, we conducted wholegenome comparisons of clonal primary and relapse B. pseudomallei isolates recovered six months to six years apart from four adult Thai patients. We found differences within each of the four pairs, and some, including a 330 Kb deletion, affected substantial portions of the genome. Many of the changes were associated with increased antibiotic resistance. We also found evidence of positive selection for deleterious mutations in a TetR family transcriptional regulator from a set of 107 additional B. pseudomallei strains. As part of the study, we sequenced to base-pair accuracy the genome of B. pseudomallei strain 1026b, the model used for genetic studies of B. pseudomallei pathogenesis and antibiotic resistance. Our findings provide new insights into pathogen evolution during long-term infections and have important implications for the development of intervention strategies to combat recurrent melioidosis. [ABSTRACT FROM AUTHOR]