Phylogeny of the family Pasteurellaceae based on rpoB sequences

Sequences of the gene encoding the beta-subunit of the RNA polymerase (rpoB) were used to delineate the phylogeny of the family Pasteurellaceae. A total of 72 strains, including the type strains of the major described species as well as selected field isolates, were included in the study. Selection of universal rpoB-derived primers for the family allowed straightforward amplification and sequencing of a 560 bp fragment of the rpoB gene. In parallel, 16S rDNA was sequenced from all strains. The phylogenetic tree obtained with the rpoB sequences reflected the major branches of the tree obtained with the 16S rDNA, especially at the genus level. Only a few discrepancies between the trees were observed. In certain cases the rpoB phylogeny was in better agreement with DNA-DNA hybridization studies than the phylogeny derived from 16S rDNA. The rpoB gene is strongly conserved within the various species of the family of Pasteurellaceae. Hence, rpoB gene sequence analysis in conjunction with 16S rDNA sequencing is a valuable tool for phylogenetic studies of the Pasteurellaceae and may also prove useful for reorganizing the current taxonomy of this bacterial family.

LakeMIP Kivu: evaluating the representation of a large, deep tropical lake by a set of one-dimensional lake models

The African great lakes are of utmost importance for the local economy (fishing), as well as being essential to the survival of the local people. During the past decades, these lakes experienced fast changes in ecosystem structure and functioning, and their future evolution is a major concern. In this study, for the first time a set of one-dimensional lake models are evaluated for Lake Kivu (2.28°S; 28.98°E), East Africa. The unique limnology of this meromictic lake, with the importance of salinity and subsurface springs in a tropical high-altitude climate, presents a worthy challenge to the seven models involved in the Lake Model Intercomparison Project (LakeMIP). Meteorological observations from two automatic weather stations are used to drive the models, whereas a unique dataset, containing over 150 temperature profiles recorded since 2002, is used to assess the model’s performance. Simulations are performed over the freshwater layer only (60 m) and over the average lake depth (240 m), since salinity increases with depth below 60 m in Lake Kivu and some lake models do not account for the influence of salinity upon lake stratification. All models are able to reproduce the mixing seasonality in Lake Kivu, as well as the magnitude and seasonal cycle of the lake enthalpy change. Differences between the models can be ascribed to variations in the treatment of the radiative forcing and the computation of the turbulent heat fluxes. Fluctuations in wind velocity and solar radiation explain inter-annual variability of observed water column temperatures. The good agreement between the deep simulations and the observed meromictic stratification also shows that a subset of models is able to account for the salinity- and geothermal-induced effects upon deep-water stratification. Finally, based on the strengths and weaknesses discerned in this study, an informed choice of a one-dimensional lake model for a given research purpose becomes possible.

Combining sedimentological, trace metal (Mn, Mo) and molecular evidence for reconstructing past water-column redox conditions: The example of meromictic Lake Cadagno (Swiss Alps)

Here, we present sedimentological, trace metal, and molecular evidence for tracking bottom water redox-state conditions during the past 12,500 years in nowadays sulfidic and meromictic Lake Cadagno (Switzerland). A 10.5 m long sediment core from the lake covering the Holocene period was investigated for concentration variations of the trace metals Mn and Mo (XRF core scanning and ICP-MS measurements), and for the presence of anoxygenic phototrophic sulfur bacteria (carotenoid pigment analysis and 16S rDNA real time PCR). Our trace metal analysis documents an oxic-intermediate-sulfidic redox-transition period beginning shortly after the lake formation similar to 12.5 kyr ago. The oxic period is characterized by low sedimentary Mn and Mo concentrations, as well as by the absence of any remnants of anoxygenic phototrophic sulfur bacteria. Enhanced accumulation/preservation of Mn (up to 5.6 wt%) in the sediments indicates an intermediate, Mn-enriched oxygenation state with fluctuating redox conditions during a similar to 2300-year long transition interval between similar to 12.1 and 9.8 kyr BP. We propose that the high Mn concentrations are the result of enhanced Mn2+ leaching from the sediments during reducing conditions and subsequent rapid precipitation of Mn-(oxyhydr) oxide minerals during episodic and short-term water-column mixing events mainly due to flood-induced underflows. At 9800 +/- 130 cal yr BP, a rapid transition to fully sulfidic conditions is indicated by the marked enrichment of Mo in the sediments (up to 490 ppm), accompanied by an abrupt drop in Mn concentrations and the increase of molecular biomarkers that indicate the presence of anoxygenic photosynthetic bacteria in the water column. Persistently high Mo concentrations >80 ppm provide evidence that sulfidic conditions prevailed thereafter until modern times, without any lasting hypolimnetic ventilation and reoxygenation. Hence, Lake Cadagno with its persistently stable chemocline offers a framework to study in great temporal detail over similar to 12 kyr the development of phototrophic sulfur bacteria communities and redox processes in a sulfidic environment, possibly depicting analogous conditions in an ancient ocean. Our study underscores the value of combining sedimentological, geochemical, and microbiological approaches to characterize paleo-environmental and -redox conditions in lacustrine and marine settings.

The evolution of host associations in the parasitic wasp genus Ichneumon (Hymenoptera: Ichneumonidae): convergent adaptations to host pupation sites

Background: The diversification of organisms with a parasitic lifestyle is often tightly linked to the evolution of their host associations. If a tight host association exists, closely related species tend to attack closely related hosts; host associations are less stable if associations are determined by more plastic traits like parasitoid searching and oviposition behaviour. The pupal-parasitoids of the genus Ichneumon attack a variety of macrolepidopteran hosts.They are either monophagous or polyphagous, and therefore offer a promissing system to investigate the evolution of host associations. Ichneumon was previously divided into two groups based on general body shape; however, a stout shape has been suggested as an adaptation to buried host pupation sites, and might thus not represent a reliable phylogenetic character. Results: We here reconstruct the first molecular phylogeny of the genus Ichneumon using two mitochondrial (CO1 and NADH1) and one nuclear marker (28S). The resulting phylogeny only supports monophyly of Ichneumon when Ichneumon lugens Gravenhorst, 1829 (formerly in Chasmias, stat. rev.) and Ichneumon deliratorius Linnaeus, 1758 (formerly Coelichneumon) are included. Neither parasitoid species that attack hosts belonging to one family nor those attacking butterflies (Rhopalocera) form monophyletic clades. Ancestral state reconstructions suggest multiple transitions between searching for hosts above versus below ground and between a stout versus elongated body shape. A model assuming correlated evolution between the two characters was preferred over independent evolution of host-searching niche and body shape. Conclusions: Host relations, both in terms of phylogeny and ecology, evolved at a high pace in the genus Ichneumon. Numerous switches between hosts of different lepidopteran families have occurred, a pattern that seems to be the rule among idiobiont parasitoids. A stout body and antennal shape in the parasitoid female is confirmed as an ecological adaptation to host pupation sites below ground and has evolved convergently several times. Morphological characters that might be involved in adaptation to hosts should be avoided as diagnostic characters for phylogeny and classification, as they can be expected to show high levels of homoplasy.

A METAGENOMIC STUDY OF THE TICK MIDGUT

A Metagenomic Study of the Tick Midgut Daniel Yuan, B.S. Supervisory Professor : Steven J. Norris, Ph.D. Southern tick–associated rash illness (STARI) or Master’s disease is a Lyme-like illness that occurs following bites by Amblyomma americanum, the lone-star tick. Clinical symptoms include a bull’s eye rash similar to the erythema migrans lesions of Lyme disease, as well as fever and joint pains. Lyme disease is caused by Borrelia burgdorferi and related spirochetes. However, B. burgdorferi has not been detected in STARI patients, or in ticks in the South Central U.S. The causative agent of STARI has not been identified, although it was once thought to be caused by another Borrelia species, Borrelia lonestari. Furthermore, while adult A. americanum have up to a 5.6% Borrelia lonestari infection rate, the prevalence of all Borrelia species in Texas ticks as a whole is not known. Previous studies indicate that 6%-30% of Northern Ixodes scapularis ticks are infected by Borrelia burgdorferi while only 10% of Northern A. americanum and I. scapularis ticks are infected by Borrelia species. The first specific aim of this project was to determine the bacterial community that inhabits the midgut of Texas and Northeastern ticks by using high throughput metagenomic sequencing to sequence bacterial 16S rDNA. Through the use of massively parallel 454 sequencing, we were able to individually sequence hundreds of thousands of 16S rDNA regions of the bacterial flora from 133 ticks from the New York, Missouri and Texas. The presence of previously confirmed endosymbionts, specifically the Rickettsia spp. and Coxiella spp., that are commonly found in ticks were confirmed, as well as some highly prevalent genera that were previously undocumented. Furthermore, multiple pathogenic genera sequences were often found in the same tick, suggesting the possibility of co-infection of multiple pathogenic species. The second specific aim was to use Borrelia specific primers to screen 344 individual ticks from Missouri, Texas and the Northeast to determine the prevalence of Borrelia species in ticks. To screen for Borrelia species, two housekeeping genes, uvrA and recG, were selected as well as the 16S-23S rDNA intergenic spacer. Ticks from Missouri, Texas and New York were screened. None of the Missouri or Texas ticks tested positive for Borrelia spp. The rate of I. scapularis infection by B.burgdorferi is dependent on tick feeding activity as well as reservoir availability. B. burgdorferi is endemic in the Northeast, sometimes reported as highly present in over 50% of all I. scapularis ticks. 11.6% of all New York ticks were positive for a species of Borrelia, however only 6.9% of all New York ticks were positive for B. burgdorferi. Despite being significantly lower than 50%, the results still fall in line with previous reports of about the prevalence of B. burgdorferi. 1.5% of all Texas ticks were positive for a Borrelia species, specifically B. lonestari. While this study was unable to identify the causative agent for STARI, 454 sequencing was able to provide a tremendous insight into the bacterial flora and possible pathogenic species of both the I. scapularis and the A. americanum tick.

A genomic perspective on a new bacterial genus and species from the Alcaligenaceae family, Basilea psittacipulmonis

BACKGROUND A novel Gram-negative, non-haemolytic, non-motile, rod-shaped bacterium was discovered in the lungs of a dead parakeet (Melopsittacus undulatus) that was kept in captivity in a petshop in Basel, Switzerland. The organism is described with a chemotaxonomic profile and the nearly complete genome sequence obtained through the assembly of short sequence reads. RESULTS Genome sequence analysis and characterization of respiratory quinones, fatty acids, polar lipids, and biochemical phenotype is presented here. Comparison of gene sequences revealed that the most similar species is Pelistega europaea, with BLAST identities of only 93% to the 16S rDNA gene, 76% identity to the rpoB gene, and a similar GC content (~43%) as the organism isolated from the parakeet, DSM 24701 (40%). The closest full genome sequences are those of Bordetella spp. and Taylorella spp. High-throughput sequencing reads from the Illumina-Solexa platform were assembled with the Edena de novo assembler to form 195 contigs comprising the ~2 Mb genome. Genome annotation with RAST, construction of phylogenetic trees with the 16S rDNA (rrs) gene sequence and the rpoB gene, and phylogenetic placement using other highly conserved marker genes with ML Tree all suggest that the bacterial species belongs to the Alcaligenaceae family. Analysis of samples from cages with healthy parakeets suggested that the newly discovered bacterial species is not widespread in parakeet living quarters. CONCLUSIONS Classification of this organism in the current taxonomy system requires the formation of a new genus and species. We designate the new genus Basilea and the new species psittacipulmonis. The type strain of Basilea psittacipulmonis is DSM 24701 (= CIP 110308 T, 16S rDNA gene sequence Genbank accession number JX412111 and GI 406042063).

Dynamics of the nasal microbiota in infancy: A prospective cohort study.

BACKGROUND Understanding the composition and dynamics of the upper respiratory tract microbiota in healthy infants is a prerequisite to investigate the role of the microbiota in patients with respiratory diseases. This is especially true in early life, when the immune system is in development. OBJECTIVE We sought to describe the dynamics of the upper respiratory tract microbiota in healthy infants within the first year of life. METHODS After exclusion of low-quality samples, microbiota characterization was performed by using 16S rDNA pyrosequencing of 872 nasal swabs collected biweekly from 47 unselected infants. RESULTS Bacterial density increased and diversity decreased within the first year of life (R(2) = 0.95 and 0.73, respectively). A distinct profile for the first 3 months of life was found with increased relative abundances of Staphlyococcaceae and Corynebacteriaceae (exponential decay: R(2) = 0.94 and 0.96, respectively). In addition, relative bacterial abundance and composition differed significantly from summer to winter months. The individual composition of the microbiota changed with increasing time intervals between samples and was best modeled by an exponential function (R(2) = 0.97). Within-subject dissimilarity in a 2-week time interval was consistently lower than that between subjects, indicating a personalized microbiota. CONCLUSION This study reveals age and seasonality as major factors driving the composition of the nasal microbiota within the first year of life. A subject's microbiota is personalized but dynamic throughout the first year. These data are indispensable to interpretation of cross-sectional studies and investigation of the role of the microbiota in both healthy subjects and patients with respiratory diseases. They might also serve as a baseline for future intervention studies.

DNA hypomethylation and oxidative stress-mediated increase in genomic instability in equine sarcoid-derived fibroblasts

It is widely accepted that equine sarcoid disease, the most common skin associated neoplasm in equids, is induced by bovine papillomavirus (BPV-1). Although BPV-1 DNA has been found in almost all examined sarcoids so far, its detailed impact on the horse's host cell metabolism is largely unknown. We used equine fibroblast cell lines originating from sarcoid biopsies to study BPV-1-associated changes on DNA methylation status and oxidative stress parameters. Sarcoid-derived fibroblasts manifested increased proliferation in vitro, transcriptional rDNA activity (NORs expression) and DNA hypomethylation compared to control cells. Cells isolated from equine sarcoids suffered from oxidative stress: the expression of antioxidant enzymes was decreased and the superoxide production was increased. Moreover, increased ploidy, oxidative DNA damage and micronuclei formation was monitored in sarcoid cells. We postulate that both altered DNA methylation status and redox milieu may affect genomic stability in BPV-1-infected cells and in turn contribute to sarcoid pathology.

Graphical Model-Based Vertebra Identification from X-Ray Image(s)

Automated identification of vertebrae from X-ray image(s) is an important step for various medical image computing tasks such as 2D/3D rigid and non-rigid registration. In this chapter we present a graphical model-based solution for automated vertebra identification from X-ray image(s). Our solution does not ask for a training process using training data and has the capability to automatically determine the number of vertebrae visible in the image(s). This is achieved by combining a graphical model-based maximum a posterior probability (MAP) estimate with a mean-shift based clustering. Experiments conducted on simulated X-ray images as well as on a low-dose low quality X-ray spinal image of a scoliotic patient verified its performance.

Comparative analysis of Tritrichomonas foetus (Riedmüller, 1928) cat genotype, T. foetus (Riedmüller, 1928) cattle genotype and Tritrichomonas suis (Davaine, 1875) at 10 DNA loci.

The parasitic protists in the genus Tritrichomonas cause significant disease in domestic cattle and cats. To assess the genetic diversity of feline and bovine isolates of Tritrichomonas foetus (Riedmüller, 1928) Wenrich and Emmerson, 1933, we used 10 different genetic regions, namely the protein coding genes of cysteine proteases 1, 2 and 4-9 (CP1, 2, 4-9) involved in the pathogenesis of the disease caused by the parasite. The cytosolic malate dehydrogenase 1 (MDH1) and internal transcribed spacer region 2 of the rDNA unit (ITS2) were included as additional markers. The gene sequences were compared with those of Tritrichomonas suis (Davaine, 1875) Morgan and Hawkins, 1948 and Tritrichomonas mobilensisCulberson et al., 1986. The study revealed 100% identity for all 10 genes among all feline isolates (=T. foetus cat genotype), 100% identity among all bovine isolates (=T. foetus cattle genotype) and a genetic distinctness of 1% between the cat and cattle genotypes of T. foetus. The cattle genotype of T. foetus was 100% identical to T. suis at nine loci (CP1, 2, 4-8, ITS2, MDH1). At CP9, three out of four T. suis isolates were identical to the T. foetus cattle genotype, while the T. suis isolate SUI-H3B sequence contained a single unique nucleotide substitution. Tritrichomonas mobilensis was 0.4% and 0.7% distinct from the cat and cattle genotypes of T. foetus, respectively. The genetic differences resulted in amino acid changes in the CP genes, most pronouncedly in CP2, potentially providing a platform for elucidation of genotype-specific host-pathogen interactions of T. foetus. On the basis of this data we judge T. suis and T. foetus to be subjective synonyms. For the first time, on objective nomenclatural grounds, the authority of T. suis is given to Davaine, 1875, rather than the commonly cited Gruby and Delafond, 1843. To maintain prevailing usage of T. foetus, we are suppressing the senior synomym T. suisDavaine, 1875 according to Article 23.9, because it has never been used as a valid name after 1899 and T. foetus is widely discussed as the cause of bovine trichomonosis. Thus bovine, feline and porcine isolates should all be given the name T. foetus. This promotes the stability of T. foetus for the veterinary and economically significant venereal parasite causing bovine trichomonosis.

Differential diagnosis of the honey bee trypanosomatids Crithidia mellificae and Lotmaria passim

Trypanosomatids infecting honey bees have been poorly studied with molecular methods until recently. After the description of Crithidia mellificae (Langridge and McGhee, 1967) it took about forty years until molecular data for honey bee trypanosomatids became available and were used to identify and describe a new trypanosomatid species from honey bees, Lotmaria passim (Evans and Schwarz, 2014). However, an easy method to distinguish them without sequencing is not yet available. Research on the related bumble bee parasites Crithidia bombi and Crithidia expoeki revealed a fragment length polymorphism in the internal transcribed spacer 1 (ITS1), which enabled species discrimination. In search of fragment length polymorphisms for differential diagnostics in honey bee trypanosomatids, we studied honey bee trypanosomatid cell cultures of C. mellificae and L. passim. This research resulted in the identification of fragment length polymorphisms in ITS1 and ITS1-2 markers, which enabled us to develop a diagnostic method to differentiate both honey bee trypanosomatid species without the need for sequencing. However, the amplification success of the ITS1 marker depends probably on the trypanosomatid infection level. Further investigation confirmed that L. passim is the dominant species in Belgium, Japan and Switzerland. We found C. mellificae only rarely in Belgian honey bee samples, but not in honey bee samples from other countries. C. mellificae was also detected in mason bees (Osmia bicornis and Osmia cornuta) besides in honey bees. Further, the characterization and comparison of additional markers from L. passim strain SF (published as C. mellificae strain SF) and a Belgian honey bee sample revealed very low divergence in the 18S rRNA, ITS1-2, 28S rRNA and cytochrome b sequences. Nevertheless, a variable stretch was observed in the gp63 virulence factor.

Experimental growth characteristics, cell size, yessotoxin values and bioassay data of Protoceratium reticulatum (Dinophyceae)

Harmful algal blooms are mainly caused by marine dinoflagellates and are known to produce potent toxins that may affect the ecosystem, human activities and health. Such events have increased in frequency and intensity worldwide in the past decades. Numerous processes involved in Global Change are amplified in the Arctic, but little is known about species specific responses of arctic dinoflagellates. The aim of this work was to perform an exhaustive morphological, phylogenetical and toxinological characterization of Greenland Protoceratium reticulatum and, in addition, to test the effect of temperature on growth and production of bioactive secondary metabolites. Seven clonal isolates, the first isolates of P. reticulatum available from arctic waters, were phylogenetically characterized by analysis of the LSU rDNA. Six isolates were further characterized morphologically and were shown to produce both yessotoxins (YTX) and lytic compounds, representing the first report of allelochemical activity in P. reticulatum. As shown for one of the isolates, growth was strongly affected by temperature with a maximum growth rate at 15 °C, a significant but slow growth at 1 °C, and cell death at 25 °C, suggesting an adaptation of P. reticulatum to temperate waters. Temperature had no major effect on total YTX cell quota or lytic activity but both were affected by the growth phase with a significant increase at stationary phase. A comparison of six isolates at a fixed temperature of 10 °C showed high intraspecific variability for all three physiological parameters tested. Growth rate varied from 0.06 to 0.19 per day, and total YTX concentration ranged from 0.3 to 15.0 pg YTX/cell and from 0.5 to 31.0 pg YTX/cell at exponential and stationary phase, respectively. All six isolates performed lytic activity; however, for two isolates lytic activity was only detectable at higher cell densities in stationary phase.

n-Alkane analyses from an transect of marine surface sediments off southwest Africa

An isobathic transect of marine surface sediments from 1°N to 28°S off southwest Africa was used to further evaluate the potential of the chain length distribution and carbon stable isotope composition of higher plant n-alkanes as proxies for continental vegetation and climate conditions. We found a strong increase in the n-C29-33 weighted mean average d13C values from -33 per mil near the equator to around -26 per mil further south. Additionally, C25-35n-alkanes reveal a southward trend of increasing average chain length from 30.0 to 30.5. The data reflect the changing contribution of plants employing different photosynthetic pathways (C3 and C4) and/or being differently influenced by the environmental conditions of their habitat. The C4 plant proportions calculated from the data (ca. 20% for rivers draining the rainforest, to ca. 70% at higher latitude) correspond to the C4 plant abundance in continental catchment areas postulated by considering prevailing wind systems and river outflows. Furthermore, the C4 plant contribution to the sediments correlates with the mean annual precipitation and aridity at selected continental locations in the postulated catchment areas, suggesting that the C4 plant fraction in marine sediments can be used to assess these environmental parameters.

Benthic foraminiferal Cd/Ca and isotope record from the South Atlantic

A depth transect of cores from 1268 to 3909 m water depth in the western South Atlantic are ideally situated to monitor the interocean exchange of deep water and variations in the relative strength of northern and southern sources of deep water production. Benthic foraminiferal Cd/Ca and d13C data suggest that Glacial North Atlantic Intermediate Water (GNAIW) extended at least as far south as 28°S in the western South Atlantic. The core of nutrient-depleted water was situated at ~1500 m, above and below water masses with higher nutrient concentrations. When examined in conjunction with published paired Cd/Ca and d13C from intermediate depth cores from other basins, it appears that the extent of GNAIW influence on the intermediate waters of the world's oceans was less than suggested previously. Differentiating among possible pathways for the glacial deep ocean (>3 km) requires a better understanding of the controls on Cd/Ca and d13C values of benthic foraminifera.

Svalbard 2010 mesocosm experiment: Protistan diversity

Hierarchical clustering. Taxonomic assignment of reads was performed using a preexisting database of SSU rDNA sequences from including XXX reference sequences generated by Sanger sequencing. Experimental amplicons (reads), sorted by abundance, were then concatenated with the reference extracted sequences sorted by decreasing length. All sequences, experimental and referential, were then clustered to 85% identity using the global alignment clustering option of the uclust module from the usearch v4.0 software (Edgar, 2010). Each 85% cluster was then reclustered at a higher stringency level (86%) and so on (87%, 88%,.) in a hierarchical manner up to 100% similarity. Each experimental sequence was then identified by the list of clusters to which it belonged at 85% to 100% levels. This information can be viewed as a matrix with the lines corresponding to different sequences and the columns corresponding to the cluster membership at each clustering level. Taxonomic assignment for a given read was performed by first looking if reference sequences clustered with the experimental sequence at the 100% clustering level. If this was the case, the last common taxonomic name of the reference sequence(s) within the cluster was used to assign the environmental read. If not, the same procedure was applied to clusters from 99% to 85% similarity if necessary, until a cluster was found containing both the experimental read and reference sequence(s), in which case sequences were taxonomically assigned as described above.