Nucleotide sequences representing nine genes and five presumptive genetic loci were used to infer phylogenetic relationships among seven Baylisascaris species, including one species with no previously available molecular data. These genes were used to test the species status of B. procyonis and B. columnaris using a coalescent approach. Phylogenetic analysis based on combined analysis of sequence data strongly supported monophyly of the genus and separated the species into two main clades. Clade 1 included B. procyonis, B. columnaris, and B. devosi, species hosted by musteloid carnivores. Clade 2 included B. transfuga and B. schroederi from ursids, B. ailuri, a species from the red panda (a musteloid), and B. tasmaniensis from a marsupial. Within clade 2, geographic isolates of B. transfuga, B. schroederi (from giant panda), and B. ailuri formed a strongly supported clade. In certain analyses (e.g., some single genes), B. tasmaniensis was sister to all other Baylisascaris species rather than sister to the species from ursids and red panda. Using one combination of priors corresponding to moderate population size and shallow genetic divergence, the multispecies coalescent analysis of B. procyonis and B. columnaris yielded moderate support (posterior probability 0.91) for these taxa as separate species. However, other prior combinations yielded weak or no support for delimiting these taxa as separate species. Similarly, tree topologies constrained to represent reciprocal monophyly of B. columnaris and B. procyonis individuals (topologies consistent with separate species) were significantly worse in some cases, but not others, depending on the dataset analyzed. An expanded analysis of SNPs and other genetic markers that were previously suggested to distinguish between individuals of B. procyonis and B. columnaris was made by characterization of additional individual nematodes. The results suggest that many of these SNPs do not represent fixed differences between nematodes derived from raccoon and skunk hosts.

Larval Baylisascaris nematodes (L3), resulting from transuterine infection and neural migration, were discovered in the cerebrum of sibling moose calves (Alces alces gigas) near 1-3 days in age from Alaska. We provide the first definitive identification, linking morphology, biogeography, and molecular phylogenetics, of Baylisascaris transfuga in naturally infected ungulates. Life history and involvement of paratenic hosts across a broader assemblage of mammals, from rodents to ungulates, in the transmission of B. transfuga remains undefined. Neural infections, debilitating young moose, may seasonally predispose calves to predation by brown bears, facilitating transmission to definitive hosts. Discovery of fatal neurological infections by L3 of B. transfuga in mammalian hosts serves to demonstrate the potential for zoonotic infection, as widely established for B. procyonis, in other regions and where raccoon definitive hosts are abundant. In zones of sympatry for multi-species assemblages of Baylisascaris across the Holarctic region presumptive identification of B. procyonis in cases of neurological larval migrans must be considered with caution. Diagnostics in neural and somatic larval migrans involving species of Baylisascaris in mammalian and other vertebrate hosts should include molecular-based and authoritative identification established in a phylogenetic context.

Background: Extremophiles are key for remediation of groundwater contaminants and for biofuels development. Standard fermenters are not equipped for anaerobic growth conditions, nor can the stainless steel withstand the reactivity of metal-reducing organisms and their metabolic by-products, such as hydrogen sulfide. We have developed methods to grow sulfate-reducing bacteria (Desulfovibrio vulgaris Hildenborough, DvH) under various conditions for detailed physiological and molecular analyses, using custom-built fermenters. Methods: Five-liter-volume fermenters have PEEK headplates and agitators. During culture growth, optical density and redox potential are continuously measured, and discrete samples for substrate utilization and metabolite concentrations, phospholipid fatty acid (PLFA), cell counts, total cell proteins and whole cell protein analysis are taken. DvH is grown in batch and continuous flow modes using defined lactate-sulfate media. DvH batch cultures have also been grown under stressed conditions, including pyruvate fermentation. Results: The growth rate constant for DvH in the 5 L fermenter is 0.11 1/hr. At the onset of deceleration phase, all lactate (60 mM) is depleted, and 30 mM of the initial 50 mM sulfate is utilized. The dilution rates to maintain the culture at mid-log and late-log phase during continuous flow are 0.15 and 0.13 1/hr, respectively. Cell densities and total proteins at late mid-log phase range from 5-10 x 108 cells/mL, and 80 to 120 ?mu g/mL, respectively. PLFA profiles for DvH are sensitive to the growth conditions and growth phase, while preliminary results show consistent whole-cell protein patterns when visualized using SDS-PAGE. Conclusion: Detailed characterization during DvH growth provides insights into physiological changes during different growth phases and has resulted in the development of protocols for controlled and reproducible growth conditions for DvH. These protocols are essential in evaluating effects of stressors on growth, which may influence efficiency during subsurface remediation of heavy metals and radionuclides.